Luis Roberto González-Torres

Alejandro Palmarola

Majela Hernández

Emily Veltjen

Arlet Marie-Stephanie Samain

Rodríguez Meno

Randy Simón

Pieter Asselman

Isabel Larridon

Salvador Guzmán Díaz

Fabián A Aldaba- Núñez

Jhonny Quintana

Gabriel Martínez

Yaniela Infante

Rosalina Berazaín

Carlos Mancina

Georgina Espinosa

Marlyn Valdéz

Daymara Rodríguez

Jacqueline Pérez Camacho

Banessa Falcón

Carlos Sánchez

Loiret Fernández

Jorge Gutiérrez

Annele Roque

Dubiel Alfonso

Juan Fernández

Ángel Motito

Roberto Alonso

Leneidy Pérez

Karine Alix

Lilian Eggers

Yann Dussert

Odalys García

Diana Rodríguez Cala

Dennis Denis

José Angel

García Beltrán

Duniel Barrios

Enma M Torres

Edgardo Díaz

Ignacio D Díaz

Rosa Rankin

Wilder Alonso

Damaris Espósito

Sergio Hernández

José Luis Gómez

Adonis Sosa

Yenia Molina

Luis Manuel Leyva

Béatrice Albert

Xavier Aubriot

Olivier Chauveau

Sophie Nadot

Najat Takvorian

Sonia Yakovlev

Aisel García

Alexander Cantero

Leandro Galano

Jorge Loynaz

Sandra Díaz

Lafargue

BC-Oph: BC-Oph: Backcross to Backcross to Ophiticola Ophiticola

Keywords: Integrative taxonomic analysis of Magnolia subsect. Talauma (Magnoliaceae) in Cuba species concept, species delimitation, genetic structure, phylogeny, gene flow, hybridization, genetic markers, microsatellites, ecological niche models, geometric morphometrics

La phylogénie du plastome valide la monophylie de Magnolia subsect. Talauma à Cuba, mais ne résout pas la délimitation taxonomique des groupes du nord-est de Cuba. Orbiculata a été clairement distingué des autres groupes de la sous-section sur la base des différentes sources de preuves qui soutiennent fortement leur statut d'espèce valide. Les principaux problèmes taxonomiques concernent les populations du nord-est de Magnolia subsect. Talauma à Cuba. Les données morphologiques et génétiques ont soutenu l'existence de deux groupes : correspondant principalement à Minor-Oblongifolia et Ophiticola.

Des échantillons pour les analyses écologiques (858), morphologiques (243), génétiques (461) et phylogénétiques (7) ont été collectés sur l'ensemble de l'aire de répartition. Pour toutes les analyses, les résultats et les représentations graphiques ont été construits en utilisant le système de classification à quatre taxons. Ce système a divisé la variabilité morphologique en le plus grand nombre d'unités et a été utilisé sur le terrain pour identifier chaque taxon. Afin d'éviter toute confusion avec les noms scientifiques des espèces et les différents traitements taxonomiques effectués sur le groupe, les taxons ont été traités comme des groupes morphologiques (Orbiculata, Minor, Oblongifolia, Ophiticola).

La variabilité de chaque groupe a été analysée à travers des modèles de niche écologique, des morphométries multivariées et géométriques, 21 marqueurs génétiques et un plastome entièrement séquencé. Orbiculata avait les plus petites zones de convenance de niche, et les groupes de la région nord-est ont montré un niveau élevé de similarité écologique. La variabilité morphologique observée des feuilles était différente entre les groupes, et de celle décrite par les études précédentes. Les résultats génétiques ont montré trois comme le nombre le plus probable de groupes génétiques, avec un niveau élevé d'admixture génétique entre les groupes du nord-est de Cuba. L'existence d'individus purs, de rétrocroisements vers Minor et de F2 dans les groupes du nord-est de Cuba a été détectée.

Samples for the ecological (858), morphological (243), genetic (461), and phylogenetic (7) analyses were collected throughout the entire distribution range. For all the analyses, the results and graphical representations were built using the four taxa classification system. This system divided the morphological variability into the highest number of units and was used in the field to identify each taxon. In order to avoid confusion with the scientific names of the species and the different taxonomic treatments carried out on the group, the taxa were treated as morphological groups (Orbiculata, Minor, Oblongifolia, Ophiticola).

The variability of each group was analysed through ecological niche models, multivariate and geometric morphometrics, 21 genetic markers, and a fully-sequenced plastome. Orbiculata had the smallest areas of niche suitability, and the groups from northeastern region showed a high level of ecological similarity. The observed leaf morphological variability was different between groups, and from that described by previous studies. The genetic results showed three as the most probable number of genetic groups, with a high level of genetic admixture between the groups from northeastern Cuba. The existence of pure individuals, backcrosses to Minor, and F2 in the groups from northeastern Cuba were detected.

The plastome phylogeny validates the monophyly of Magnolia subsect. Talauma in Cuba, but does not solve the taxonomic delimitation of the groups from northeastern Cuba. Orbiculata was clearly distinguished from the other groups of the subsection based on the different sources of evidence that strongly supported their status as a valid species. The main taxonomic issues concern northeastern populations of Magnolia subsect. Talauma in Cuba. The morphological and genetic data supported the existence of two groups: corresponding mainly to Minor-Oblongifolia and Ophiticola.

Título : Análisis taxonómico integrativo de Magnolia subsect. Talauma (Magnoliaceae) en Cuba Palabras clave : concepto de especie, delimitación de especies, estructura genética, filogenia, flujo genético, hibridación, marcadores genéticos, microsatélites, modelos de nicho ecológico, morfometría geométrica Resumen : Una delimitación de taxones más precisa, basada en una comprensión completa de los procesos evolutivos involucrados en la diferenciación de los taxones, se puede obtener a partir de una combinación de enfoques ecológicos, morfológicos y moleculares. La taxonomía de Magnolia subsect. Talauma en Cuba ha sido ampliamente debatida, y se ha basado exclusivamente en estudios morfológicos tradicionales de un número limitado de individuos. El objetivo del presente estudio es delimitar los taxones cubanos de Magnolia subsect. Talauma para actualizar su tratamiento taxonómico mediante la integración de criterios ecológicos, morfológicos, genéticos y filogenéticos.

Se colectaron muestras para los análisis ecológicos (858), morfológicos (243), genéticos (461) y filogenéticos (7) en todo el rango de distribución del grupo. Para todos los análisis, los resultados y las representaciones gráficas se construyeron utilizando el sistema de clasificación de cuatro taxones. Este sistema divide la variabilidad morfológica en el mayor número de unidades y se utilizó en campo para identificar cada taxón. Para evitar confusiones con los nombres científicos de las especies y los diferentes tratamientos taxonómicos realizados sobre el grupo, los taxones fueron tratados como grupos morfológicos (Orbiculata, Minor, Oblongifolia, Ophiticola).

La variabilidad de cada taxón se analizó a través de modelos de nicho ecológico, morfometría multivariada y geométrica, 21 marcadores genéticos y la secuencia completa del cloroplasto. Orbiculata presentó las áreas más pequeñas de idoneidad climática, mientras que los taxones del norte de oriente mostraron altos niveles de similitud ecológica. La variabilidad morfológica de la hoja fue diferente entre grupos y con respecto a la descrita por estudios previos. Los resultados genéticos mostraron a tres como el número más probable de grupos genéticos, con un alto nivel de mezcla genética entre los taxones del norte de oriente. Se detectó la existencia de individuos puros, retrocruzamientos con Minor y F2 en los taxones del norte de Cuba oriental.

La filogenia del cloroplasto valida la monofilia de Magnolia subsect. Talauma, pero no resuelve la delimitación taxonómica de los grupos del norte de oriente. Orbiculata se distinguió claramente de los otros taxones de la subsección en función de las diferentes fuentes de evidencia, lo que respaldó su condición de especie válida. Las principales cuestiones taxonómicas se refieren a las poblaciones de Magnolia subsect. Talauma del norte de oriente. Los datos morfológicos y genéticos apoyaron la existencia de dos grupos correspondientes principalmente a Minor-Oblongifolia y Ophiticola.

RÉSUMÉ DÉTAILLÉ DE LA THÈSE

Définir ce qu'est une espèce a fait l'objet de longs débats dans l'histoire de la biologie, débats qui ont produit de multiples concepts d'espèces au fil du temps. Au cours des deux dernières décennies, l'idée selon laquelle les espèces peuvent être définies comme des lignées de métapopulations évoluant séparément (SC unifié) s'est de plus en plus répandue au sein de la communauté scientifique. Délimiter les espèces nécessite ainsi d'accumuler des preuves que les taxons considérés évoluent actuellement de manière indépendante. Il est maintenant largement reconnu qu'une délimitation précise des taxons, basée sur une compréhension complète des processus évolutifs impliqués dans la différenciation des taxons, peut être obtenue à partir d'une combinaison d'approches écologiques, morphologiques et moléculaires pour évaluer la diversité des taxons et leur différenciation sur l'ensemble de leur aire de répartition géographique. Cette approche multi-niveaux est connue sous le nom de taxonomie intégrative.

Le genre Magnolia est un bon modèle pour explorer l'efficacité d'une approche intégrative dans des problèmes taxonomiques complexes. Les données moléculaires récentes remettent en question la délimitation des espèces basée sur la morphologie. La taxonomie de Magnolia subsect. Talauma à Cuba a longtemps fait débat avec de nombreux changements dans le nombre, et les limites des espèces au cours du siècle dernier. Toutes les révisions taxonomiques étaient exclusivement basées sur les descripteurs morphologiques traditionnels des feuilles, et sur seulement quelques individus, limitant leur capacité à élucider les limites des taxons. La présente étude propose l'objectif suivant: délimiter les taxons cubains de Magnolia subsect. Talauma afin de mettre à jour leur traitement taxonomique par l'intégration de critères écologiques, morphologiques, génétiques et phylogénétiques.

Les échantillons pour les analyses écologiques, morphologiques et génétiques ont été collectés (2015)(2016)(2017)(2018)(2019)(2020) sur des individus représentant tous les taxons cubains de Magnolia subsect. Talauma, dans toute leur aire de répartition dans les montagnes de Nipe-Sagua-Baracoa et Sierra Maestra à l'Est de Cuba.

Sur le terrain, les individus ont été identifiés en fonction de la forme des arbres et des feuilles selon les critères morphologiques qui a défini le plus grand nombre d'unités taxonomiques. Pour toutes les analyses, les résultats et les représentations graphiques ont été construits en utilisant le système de classification de quatre espèces. Afin d'éviter toute confusion avec les noms scientifiques des espèces et les différents traitements taxonomiques effectués sur le groupe, les taxons ont été considérés comme des groupes morphologiques (Orbiculata, Minor, Oblongifolia, Ophiticola), et ils ont été traités comme tels dans toutes les analyses suivantes.

Pour l'analyse écologique, les coordonnées GPS ont été enregistrées pour chaque individu. Un ensemble des variables bioclimatiques a été choisi comme prédicteur de niche écologique. Après un processus de calibration du modèle, les modèles finaux ont été exécutés dans Maxent v. 3.4.1. Un test d'identité a été utilisé afin d'examiner la divergence de niche entre les groupes. Les valeurs de pixel pour chaque couche environnementale ont été extraites à chaque point d'occurrence du Magnolia subsect.

Talauma, et utilisées pour évaluer les différences de niche entre les groupes à l'aide d'une analyse en composantes principales (ACP). Pour les analyses morphologiques, 243 individus ont été analysé. Deux types d'analyses morphologiques ont été effectuées sur trois ensemble de variables indépendantes, permettant d'appréhender la forme de la feuille. La signification statistique des différences entre les groupes pour chaque variable mesurée a été évaluée. La variabilité de l'ensemble de l'échantillon a été décrite à l'aide d'une ACP. Une approche de classification bayésienne basée sur des modèles de mélanges de distributions a été réalisée en utilisant chacun des trois ensembles de données de variables morphologiques.

Pour les analyses génétiques, l'ADN des échantillons de jeunes feuilles saines d'un total de 461 individus a été extrait. Les individus ont été génotypés à l'aide de 21 marqueurs microsatellites. Les valeurs de diversité génétique et la différenciation entre les groupes ont été estimées. L'identification des clusters génétiques et l'attribution des individus ont été réalisées à l'aide de STRUCTURE et de l'analyse DAPC.

La présence ou l'absence d'individus hybrides a été estimée avec NEWHYBRIDS. La reconstruction phylogénétique a été réalisée avec un plastome entièrement séquencé (deux individus pour: Minor, Oblongifolia et Ophiticola; un individu pour Orbiculata). En outre, un échantillon de chaque taxon des différentes régions des Caraïbes et des sous-sections Magnolia ont été inclus dans l'analyse.

Presque tous les modèles de niche écologique réalisés pour chaque groupe ont montré des régions potentiellement appropriées dans de vastes zones des montagnes de Nipe-Sagua-Baracoa et de la Sierra Maestra. Orbiculata était le groupe avec la plus petite zone d'aptitude potentielle, ce qui pourrait s'expliquer par le faible nombre d'individus connus et leur répartition géographique limitée. L'analyse du test d'identité de niche a indiqué des niveaux élevés de chevauchement de niche entre les différents groupes. L'ACP a montré que le groupe Orbiculata possède la niche environnementale distincte la plus petite sans chevauchement avec celle des autres groupes. Les groupes du nord-est de Cuba vivent dans des conditions environnementales similaires.

Pour la plupart des douze variables, des différences significatives entre chaque paire de groupes ont été trouvées. Sur la base de l'ACP pour les trois ensembles de variables, les différents groupes taxonomiques ont montré peu de chevauchement dans l'espace d'ordination, ainsi que des moyennes statistiquement différentes les unes des autres. La variabilité morphologique des feuilles observée pour les magnolias de Cuba était plus forte que celle décrite par les études précédentes, probablement en raison de la plus grande taille de l'échantillon utilisé dans le présent travail et de sa plus large représentativité géographique.

Les variables linéaires et angulaires ont permis une discrimination claire entre Orbiculata, Ophiticola et Minor, ce dernier groupe étant divisé en deux sous-groupes. L'un deux était commun avec la majorité des individus d'Oblongifolia. Les contour de la feuille ont permis d'obtenir une distinction très claire entre Orbiculata et les trois autres groupes. L'analyse effectuée sur la matrice de Landmark a montré un schéma similaire à celui obtenu avec les variables linéaires et angulaires pour Minor, Ophiticola et Orbiculata. Le niveau élevé de différenciation morphologique entre les taxons dans cette étude renforce la valeur des caractéristiques foliaires dans les études taxonomiques des magnolias cubains.

Les groupes ont montré des valeurs de diversité génétique similaires, avec les valeurs les plus basses chez Orbiculata. Son aire de distribution plus réduite que celle des autres groupes, ainsi que son relatif isolement géographique permet de supposer que la variabilité génétique plus faible chez Orbiculata pourrait traduire un effectif efficace plus faible que ceux des autres groupes. La différenciation génétique entre les taxons était relativement élevée (F ST global = 0,10, D JOST = 0,23), Orbiculata contribuant principalement à ce résultat, tandis que Minor et Oblongifolia étaient les deux taxons les moins différenciés l'un de l'autre. L'analyse de classification bayésienne a permis d'identifier sans ambiguïté trois groupes génétiques, correspondant majoritairement à Orbiculata, Minor (comprend Oblongifolia)

INTRODUCTION

Defining what a species is has been the subject of long debates in the history of biology, debates that have produced multiple species concepts (SC) over time (e.g., Genetic SC, Morphological SC, Phylogenetic SC, Ecological SC, Biological SC, among others [START_REF] Mayr | What is a species and what is not?[END_REF][START_REF] De Queiroz | Species Concepts and Species Delimitation[END_REF]. However, as [START_REF] Hey | On the failure of modern species concepts[END_REF] pointed out, this theoretical dilemma should not hinder the fact that biologists agree about general and straightforward ideas such as species are fundamental units in biology and that individuals belonging to the same species share a higher co-ancestry than individuals from other species. As this author states, the problem arises when one defines criteria for defining a species because these criteria are linked to methodologies used to delineate species, therefore leading to different ways to define species.

In the last two decades, there has been an ever-growing shared idea that species can be defined as separately evolving metapopulation lineages (Unified SC) [START_REF] De Queiroz | The general lineage concept of species, species criteria, and the process of speciation: A conceptual unification and terminological recommendations[END_REF][START_REF] De Queiroz | Species Concepts and Species Delimitation[END_REF].

Therefore, delimiting species boundaries calls for accumulating evidence that the considered taxa are currently evolving independently. This task is especially challenging for taxa that have recently diverged due to several evolutionary and genetic factors previously described (see

Naciri & Linder 2015, for a review).

It is now largely recognized that an accurate taxon delimitation, based on a full understanding of the evolutionary processes involved in taxon differentiation, can be gained from, and even should rely on, a combination of ecological, morphological, and molecular approaches to assess within-taxon diversity and among-taxa differentiation, across their whole geographical range (de Queiroz 2007; [START_REF] Padial | The integrative future of taxonomy[END_REF]. The combination of several species concepts to broadly support species limits is known as integrative taxonomy [START_REF] Padial | The integrative future of taxonomy[END_REF]. [START_REF] Dayrat | Towards integrative taxonomy[END_REF] and [START_REF] Dayrat | Towards integrative taxonomy[END_REF] recommended that species should only be named when their limits are supported by multiple lines of evidence. Integrative taxonomy does not replace traditional taxonomy but uses complementarity among disciplines to improve accuracy [START_REF] Schlick-Steiner | Integrative Taxonomy: A Multisource Approach to Exploring Biodiversity[END_REF][START_REF] Yeates | Integrative taxonomy, or iterative taxonomy?[END_REF]. The potential for such integrative taxonomic approaches has not yet been fully embraced in botany, particularly in the tropics [START_REF] Damasco | Reestablishment of Protium cordatum (Burseraceae) based on integrative taxonomy[END_REF]. The works of [START_REF] Zheng | Species Delimitation and Lineage Separation History of a Species Complex of Aspens in China[END_REF][START_REF] Alvarado-Sizzo | Species delimitation in the Stenocereus griseus (Cactaceae) species complex reveals a new species, S. huastecorum[END_REF], [START_REF] Damasco | Reestablishment of Protium cordatum (Burseraceae) based on integrative taxonomy[END_REF], [START_REF] Denham | Using integrative taxonomy and multispecies coalescent models for phylogeny reconstruction and species delimitation within the "Nastanthus-Gamocarpha" clade (Calyceraceae)[END_REF], [START_REF] Moein | An integrating study of genetic diversity and ecological niche modelling in Salvia aristata (Lamiaceae)[END_REF], [START_REF] Yang | Different species or genetically divergent populations? Integrative species delimitation of the Primulina hochiensis complex from isolated karst habitats[END_REF] are good examples of the use of an integrative approach carried out to solve the taxonomic problems in different plant families.

The genus Magnolia L. is a good model for applying an integrative taxonomic approach. It is the largest genus of the family Magnoliaceae Juss. It includes three subgenera, 13 sections, and an equal number of subsections [START_REF] Figlar | Notes on Magnoliaceae IV[END_REF][START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF]. Magnolia section Talauma Baill., with a Neotropical distribution, includes around 120 species distributed in four subsections: Dugandiodendron Lozano, Chocotalauma A. Vázquez, Á.J. Pérez and F.

Arroyo, Cubenses Imkhan., and Talauma Juss. [START_REF] Figlar | Notes on Magnoliaceae IV[END_REF][START_REF] Vázquez-García | Magnolia mindoensis (subsect. Talauma, Magnoliaceae): Una especie nueva del Chocó biogeográfico premontano en Colombia y Ecuador[END_REF]. The 85 taxa of the subsection Talauma, the most species-rich of all Magnolia subsections, occur in lowlands and mountainous areas (0 -3 300 m.a.s.l.) of Central and South America and the Caribbean Islands [START_REF] Vázquez-García | Magnolia mindoensis (subsect. Talauma, Magnoliaceae): Una especie nueva del Chocó biogeográfico premontano en Colombia y Ecuador[END_REF]. The genus Magnolia includes species that could be distinguished morphologically and genetically conservative [START_REF] Treseder | Magnolias[END_REF][START_REF] Callaway | Magnolias[END_REF][START_REF] Chappell | Biochemical and Genomic Characterization of Terpene Synthases in Magnolia grandiflora[END_REF][START_REF] Li | Complete chloroplast genome sequence of Magnolia grandiflora and comparative analysis with related species[END_REF][START_REF] Shen | Comparative and phylogenetic analyses of 26 Magnoliaceae species based on complete chloroplast genome sequences[END_REF][START_REF] Sun | The complete chloroplast genome of Magnolia polytepala: Comparative analyses offer implication for genetics and phylogeny of Yulania[END_REF]. Recent molecular research is challenging species delimitation based on morphology [START_REF] Azuma | Intraspecific Sequence Variation of cpDNA Shows Two Distinct Groups within Magnolia virginiana L. of Eastern North America and Cuba[END_REF]Rico & Gutiérrez-Becerril 2019;[START_REF] Aldaba-Núñez | Disentangling Species Delineation and Guiding Conservation of Endangered Magnolias in Veracruz, Mexico[END_REF]). The majority of these studies conclude that more evidence from ecology and morphology is needed to understand the discrepancies with molecular data.

Cuba has the highest diversity of magnolias among the Caribbean islands, with seven endemic taxa [START_REF] Veltjen | Genetic patterns in Neotropical Magnolias (Magnoliaceae) using de novo developed microsatellite markers[END_REF]. The Cuban taxa of Magnolia belong to two sections, Magnolia and Talauma. The section Talauma, the most diverse with six taxa, is represented by two subsections in Cuba: Cubenses and Talauma. The taxonomy of Magnolia subsect. Talauma in Cuba has long been debated, although based until now only on leaf morphology. The first taxon described was Talauma minor Urb. followed by T. orbiculata Britton & P. Wilson, in 1912INTRODUCTION and 1923, respectively (Urban 1912;[START_REF] Britton | Studies of West Indian Plants-XI[END_REF]. [START_REF] Howard | The Morphology and Systematics of the West Indian Magnoliaceae[END_REF] recognized two species of Talauma, T. minor Urb., and T. truncata (Moldenke) R.A. Howard, previously described by [START_REF] Moldenke | Nomenclatural notes III[END_REF] as Svenhedinia truncata Moldenke. Two years later, [START_REF] León | Novedades de la Flora Cubana (II)[END_REF] described a variety of Talauma minor with extremely oblong leaves that they named T. minor var. oblongifolia León. In the taxonomic treatment for the "Flora of Cuba", [START_REF] León | Flora de Cuba II[END_REF] mentioned four taxa of Talauma: T. minor var. minor, T. minor var. oblongifolia, T. orbiculata, and T. truncata. These authors distinguished T. orbiculata and T. truncata based on the largely truncate leaves of the latter, which inhabits only areas around Pico Turquino [START_REF] León | Flora de Cuba II[END_REF]. However, due to the large variation of leaf-base shape observed in these two taxa, they

were not recognized as separated entities in the subsequent taxonomic reviews of the group (e.g., [START_REF] Bisse | Árboles de Cuba[END_REF][START_REF] Imkhanitzkaja | Genus Talauma A.L. Juss. (Magnoliaceae) in flora Cubae[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]).

In the Supplement for the "Flora of Cuba" published by [START_REF]Flora de Cuba[END_REF], he suggested that all previously described taxa of Talauma from Cuba could be considered as one single taxon. Later, [START_REF] Borhidi | New plants in Cuba I[END_REF] [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF], Based on anatomical and morphological (vegetative and reproductive) traits described by [START_REF] Nooteboom | Magnoliaceae[END_REF], [START_REF] Frodin | World Checklist and Bibliography of Magnoliaceae[END_REF] has leaves with the characteristics used by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] to define both taxa.

was
All above-mentioned taxonomic revisions (e.g., [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF][START_REF] Borhidi | New plants in Cuba I[END_REF][START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF][START_REF] Imkhanitzkaja | Genus Talauma A.L. Juss. (Magnoliaceae) in flora Cubae[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] were exclusively based on traditional leaf morphological descriptors and only a few individuals, limiting their ability to elucidate taxon boundaries. Therefore, our research questions are:

• It is the current delimitation of the Cuban taxa of Magnolia subsect. Talauma, based on morphological criteria, consistent with other criteria to delimit the species?

• What is the ecological, morphological, and genetic variability of the Cuban taxa of Magnolia subsect. Talauma?

The present work is based on the following hypothesis: For the Cuban members of Magnolia subsect. Talauma, the number and limits of species defined by the genetic, ecological, and phylogenetic criteria are the same as that defined by the morphological species criteria.

In order to test the research hypothesis, the present study proposes the goal: to delimit the Cuban taxa of Magnolia subsect. Talauma in order to update their taxonomic treatment through the integration of ecological, morphological, genetic, and phylogenetic criteria.

INTRODUCTION Tasks 1. To analyze the ecological limits of the Cuban taxa of Magnolia subsect. Talauma in Cuba throughout their ecological niche.

2. To evaluate the morphological limits of the Cuban taxa of Magnolia subsect. Talauma using multivariate and geometric morphometrics of the leaves.

3.

To describe the genetic structure of the Cuban taxa of Magnolia subsect. Talauma using microsatellite markers.

4.

To analyze the phylogenetic relationships of the Cuban taxa of Magnolia subsect. Talauma, using the complete plastome sequence.

5. To integrate the ecological, morphological, genetic, and phylogenetic criteria in the taxonomic delimitation of Magnolia subsect. Talauma in Cuba.

6.

To update the taxonomy and nomenclature of Magnolia subsect. Talauma in Cuba.

SCIENTIFIC NOVELTIES OF THE THESIS

1. The delimitation of the taxa of Magnolia subsect. Talauma through the integration of the ecological, morphological, genetic, and phylogenetic criteria. This is the first research of this type in Magnoliaceae family and in Cuban species.

2. The analysis of hybridization in Magnolia subsect. Talauma. The first research on population genetics applied to natural hybridization in Cuban plants.

3. The application of the ecological and genetic species concept in Magnolia subsect.

Talauma.

4. The evaluation of ecological differences using two niche identity tests in Cuban plants.

5. The morphological characterization of Magnolia subsect. Talauma in Cuba, using multivariate and geometric morphometrics.

6. The description of specific microsatellite markers for the Cuban taxa of Magnolia subsect.

Talauma.

7. The genetic characterization of Magnolia subsect. Talauma in Cuba.

8. The complete plastome phylogeny of Magnolia sect. Talauma in Cuba.

9. The definition of Significant Evolutionary and Conservation units of Magnolia subsect Talauma in Cuba.

The theoretical importance is the delimitation of the different taxa of Magnolia subsect. Talauma in Cuba using four different approaches (ecological, morphological, population genetics, and phylogenetic). The genetic characterization of the Cuban taxa of Magnolia subsect. Talauma, using microsatellite markers and chloroplast genome, will allow the analyses of the evolutionary history of this group and their conservation. The ecological and morphological data will help to understand the variability of the group and the possible effect of the environment on morphology and genetics.

The practical importance is reflected in the methodological value of the thesis for future taxonomic studies in Cuban species and magnolias since it is the first work in Cuba that analyses the limits of species through an integrative approach. In addition, it serves from the methodological point of view in the analysis of ecological niche, leaf morphology, population genetics, and phylogeny of other species of Cuban flora. Three articles related to the thesis have been published in indexed journals (Revista del Jardín Botánico Nacional-Universidad de La Habana, Botanical Sciences, and PhytoKeys), and three other manuscripts are in process (see page 120). The results of this thesis have been presented

in five scientific event.

BIBLIOGRAPHIC REVISION

Species concept

The species concept (SC), also called the species problem, is one of the most controversial and debated issues within biology [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF][START_REF] Zachos | Species Concepts in Biology: Historical Development, Theoretical Foundations and Practical Relevance[END_REF][START_REF] Aldhebiani | Species Concept and Speciation[END_REF]. One of the reasons for this is that the term species has been poorly defined since its beginning more than three centuries ago [START_REF] Roberts | Reconciling molecules and morphology in benthic foraminifera: A morphometric study of Ammonia and Elphidiidae in the NE Atlantic[END_REF][START_REF] Aldhebiani | Species Concept and Speciation[END_REF]. A species is one of the fundamental units of biology, comparable in importance to genes, cells, and organisms (de Queiroz 2007). Defining species is central in biodiversity sciences because changes in the number of species can directly affect fields such as taxonomy, biogeography, ecology, agriculture, and macroevolution [START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF][START_REF] Denham | Using integrative taxonomy and multispecies coalescent models for phylogeny reconstruction and species delimitation within the "Nastanthus-Gamocarpha" clade (Calyceraceae)[END_REF][START_REF] Yang | Different species or genetically divergent populations? Integrative species delimitation of the Primulina hochiensis complex from isolated karst habitats[END_REF]. Species constitute the basic unit of conservation [START_REF] Prebble | Species delimitation and the population genetics of rare plants: A case study using the New Zealand native pygmy forget-me-not group (Myosotis; Boraginaceae)[END_REF], and are the basis for the creation and implementation of policies [START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF][START_REF] Zachos | Species Concepts in Biology: Historical Development, Theoretical Foundations and Practical Relevance[END_REF]. The term species has two basic functions: (1) the species category as a rank in the Linnaean hierarchy created by taxonomists for grouping organisms and

(2) the species as a taxon with a location in space and time and referring to objective, observable entities, to living objects perceptible by touch [START_REF] Mayr | What is a species and what is not?[END_REF].

Species are an artificial category created to organize information [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF]. There are different arguments on how a species should be defined, which has resulted in more than 30 concepts [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF][START_REF] Zachos | Taxonomic inflation, the Phylogenetic Species Concept and lineages in the Tree of Life-a cautionary comment on species splitting[END_REF][START_REF] Haider | A Brief Review on Species Concepts with Emphasis on Plants[END_REF]. Most species concepts have marked incompatibilities, which leads to different interpretations of species boundaries. The incompatibility between the different species concepts is given by the different biological properties on which each of the concepts is based [START_REF] De Queiroz | The general lineage concept of species, species criteria, and the process of speciation: A conceptual unification and terminological recommendations[END_REF][START_REF] De Queiroz | Species Concepts and Species Delimitation[END_REF], which do not escape criticism due to their limitations and counterexamples. The problem of the definition of species cannot be solved easily, given that the problem is philosophical and not a simple question of science [START_REF] Rusmana | Probing high school students´ perceptions of the concept of species: a semantic network analysis approach[END_REF]. Natural populations constitute a continuum of variability (morphological, genetic, reproductive, ecological, etc.), composed of phenomena such as speciation, introgression, and hybridization that contribute to unclear boundaries between groups of individuals [START_REF] Nyléhn | The "Species Concept" as a Gateway to Nature of Science, Species Concepts in Norwegian Textbook[END_REF]. This complexity becomes more relevant in groups like trees, where factors such as generation length and effective population sizes can delay lineage differentiation [START_REF] Zheng | Species Delimitation and Lineage Separation History of a Species Complex of Aspens in China[END_REF].

The ecological SC, the morphological SC, the genetic SC, and the phylogenetic SC have been widely used by scientists. All these concepts have advantages and limitations (explained below) in establishing limits between species.

Ecological Species Concept (ESC)

The ecological niche is considered a central concept in modern biology [START_REF] Hutchinson | Concluding remarks[END_REF][START_REF] Qiao | NicheA: creating virtual species and ecological niches in multivariate environmental scenarios[END_REF]. The ecological niche is the set of environmental conditions and requirements that allow the species to maintain stable populations without the occurrence of immigration [START_REF] Peterson | Predicting the geography of species invasions via ecological niche modeling[END_REF][START_REF] Pearman | Niche Dynamics in space and time[END_REF][START_REF] Soberón | Niches and distributional areas: Concepts, methods, and Assumptions[END_REF]. It can also be defined as an N-dimensional hyper-volume where each dimension represents the range of environmental conditions and resources necessary for the survival, stability, and reproduction of a population [START_REF] Hutchinson | Concluding remarks[END_REF]. There are two approaches to the niche concept: the Eltonian and the Grinnellian. The Eltonian niche is spatially fine, focuses on the functional role of the species and its place in the biotic environment, and takes into account biological interactions between species [START_REF] Peterson | Predicting the geography of species invasions via ecological niche modeling[END_REF]. The niche from the Grinnellian approach is understood as the range of conditions defined in terms of variables that are not consumed or affected by the presence of the species [START_REF] Soberón | Niches and distributional areas: Concepts, methods, and Assumptions[END_REF]. This approach, unlike the previous one, does not take biological interactions into account and works on a large scale [START_REF] Jiménez-Valverde | Use of niche models in invasive species risk assessments[END_REF]. The concept of ecological niche is vital for understanding patterns of distribution and biological diversity, the identification of critical habitats to mitigate the effect of climate change, and the reconstruction of evolutionary trajectories [START_REF] Smith | Niche Estimation Above and Below the Species Level[END_REF].

The ESC, proposed by [START_REF] Van Valen | Ecological species, multispecies, and oaks[END_REF], emphasizes the importance of natural selection based on ecology for the long-term survival of species. A species is a lineage (or a group of related lineages) that occupies a minimally different adaptive zone from other lineages in its range and that has evolved separately from all lineages outside its range. The ESC is based on the differential occupation of the ecological niche or an adaptive zone [START_REF] De Queiroz | The general lineage concept of species, species criteria, and the process of speciation: A conceptual unification and terminological recommendations[END_REF][START_REF] Aldhebiani | Species Concept and Speciation[END_REF][START_REF] Haider | A Brief Review on Species Concepts with Emphasis on Plants[END_REF]. In this sense, each species has a specific ecological niche that is different from other species, including related and sympatric species [START_REF] Soberón | Interpretation of models of fundamental ecological niches and species' distributional areas[END_REF][START_REF] Kurnaz | Ecological niche divergence between Darevskia rudis and D. bithynica (Lacertidae) in Turkey[END_REF]. The existence of two or more groups of related individuals occupying different niches can be seen as evidence of genetic differences between them.

Ecological Niche Models (ENMs)

The ENMs are a set of analytical tools that allows identifying, in a quantitative way, the ideal environmental conditions for the maintenance of species or natural populations [START_REF] Peterson | Ecological Niches and Geographic Distributions[END_REF][START_REF] Smith | Niche Estimation Above and Below the Species Level[END_REF]. From this analysis, the Grinnellian ecological niche of the species is characterized and projected into geographic space, generating probability maps of favorable conditions for the presence and development of populations [START_REF] Felicísimo | Modelos de distribución de especies: Una revisión sintética[END_REF][START_REF] Peterson | Species Distribution Modeling and Ecological Niche Modeling: Getting the Concepts Rigth[END_REF]). One of the main assumptions of the ENMs is that environmental adaptation and stress tolerance are the primary determinants of the current distribution of species [START_REF] Rödder | Predicting the potential distributions of two alien invasive Housegeckos (Gekkonidae: Hemidactylus frenatus, Hemidactylus mabouia)[END_REF]) and that the ecological niche is quite conserved (stable in time and space) [START_REF] Thuiller | Niche-based modelling as a tool for predicting the risk of alien plant invasions at a global scale[END_REF][START_REF] Rödder | Predicting the potential distributions of two alien invasive Housegeckos (Gekkonidae: Hemidactylus frenatus, Hemidactylus mabouia)[END_REF][START_REF] Felicísimo | Modelos de distribución de especies: Una revisión sintética[END_REF]. ENMs are a complex tool that manages to integrate elements of biology, statistics, cartography, databases, computing, remote sensing, and climatology [START_REF] Mateo | Modelos predictivos de riqueza de diversidad vegetal. Comparación y optimización de métodos de modelado ecológico[END_REF]). This has meant that ENMs have been integrated into a wide variety of fields within the natural sciences, such as geography, species conservation, invasive species, evolution, and systematics [START_REF] Guisan | Predicting species distribution: offering more than simple habitat model[END_REF][START_REF] Peterson | Ecological Niches and Geographic Distributions[END_REF][START_REF] Moein | An integrating study of genetic diversity and ecological niche modelling in Salvia aristata (Lamiaceae)[END_REF].

There are several statistical modeling algorithms [START_REF] Phillips | Maximum entropy modeling of species geographic distribution[END_REF][START_REF] Wolmarans | Predicting invasive alien plant distributions: how geographical bias in occurrence records influence model performance[END_REF][START_REF] Escobar | Ecological niche modeling re-examined: A case study with the Darwin's fox[END_REF]; among the most used are GARP [START_REF] Stockwell | The GARP modeling system: Problems and solutions to automated REFERENCES spatial prediction[END_REF], BIOCLIM [START_REF] Busby | Nature conservation: cost effective biological surveys and data analysis[END_REF], BIOMOD [START_REF] Thuiller | BIOMOD-a platform for ensemble forecasting of species distributions[END_REF], and Maxent [START_REF] Phillips | Maximum entropy modeling of species geographic distribution[END_REF]. Maxent, which implements a learning machine-based algorithm, is based on the principle of maximum entropy and assumes that the best approximation to an unknown distribution is the one that will maximize the entropy of the probability distribution [START_REF] Phillips | Maximum entropy modeling of species geographic distribution[END_REF][START_REF] Mateo | Modelos predictivos de riqueza de diversidad vegetal. Comparación y optimización de métodos de modelado ecológico[END_REF][START_REF] Elith | A statistical explanation of Maxent for Ecologists[END_REF]. A certain probability of maximum entropy is subject to restrictions that represent our incomplete information; this is what allows us to justify a distribution as an inference [START_REF] Phillips | Maximum entropy modeling of species geographic distribution[END_REF].

Maxent is a robust method which has a high performance with few data and generates stable results, despite not being easy to interpret [START_REF] Mateo | Modelos predictivos de riqueza de diversidad vegetal. Comparación y optimización de métodos de modelado ecológico[END_REF][START_REF] Escobar | Ecological niche modeling re-examined: A case study with the Darwin's fox[END_REF]. Furthermore, it only requires records documenting the presence or the absence of the studied species [START_REF] Phillips | Maximum entropy modeling of species geographic distribution[END_REF][START_REF] Elith | A statistical explanation of Maxent for Ecologists[END_REF]. Thus, information from databases, herbaria, and other collections can be used [START_REF] Felicísimo | Modelos de distribución de especies: Una revisión sintética[END_REF].

Ecological niche modeling can be used as a taxonomic framework that helps define or delimit species based on the differences in their ecological niches [START_REF] Wiens | Niche conservatism: integrating evolution, ecology, and conservation biology[END_REF][START_REF] Raxworthy | Applications of Ecological Niche Modeling for Species Delimitation: A Review and Empirical Evaluation Using Day Geckos (Phelsuma) from Madagascar[END_REF][START_REF] Kurnaz | Ecological niche divergence between Darevskia rudis and D. bithynica (Lacertidae) in Turkey[END_REF][START_REF] Hosseinian-Yousefkhani | Evidences for ecological niche differentiation on the Anatolian lizard (Apathya cappadocica ssp.) (Reptilia: Lacertidae) in western Asia[END_REF].

Niche models can also provide evidence of geographic isolation between populations (based on divergence or conservation of the ecological niche). In addition, they provide elements that support that these populations are separate lineages when gene flow is unlikely due to unsuitable regions [START_REF] Raxworthy | Applications of Ecological Niche Modeling for Species Delimitation: A Review and Empirical Evaluation Using Day Geckos (Phelsuma) from Madagascar[END_REF]).

Morphological Species Concept (MSC)

The comparison of the morphological characteristics of organisms has been a central element in biology for centuries [START_REF] Remagnino | Computational Botany: Methods for Automated Species Identification[END_REF]. The taxonomic classification of organisms and the understanding of biological diversity have historically been based on morphological forms [START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF]). The MSC defines species as the smallest group that is consistently and persistently different and distinguishable by ordinary measures [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF][START_REF] Nyléhn | The "Species Concept" as a Gateway to Nature of Science, Species Concepts in Norwegian Textbook[END_REF][START_REF] Haider | A Brief Review on Species Concepts with Emphasis on Plants[END_REF]. Species can also be defined as a group or related groups that have different morphological characters. In the opinion of the taxonomist, these characters are enough to delimit them [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF]. Species are categorized based on observable physical characteristics, such as structures, sexual organs, sizes, and shapes [START_REF] Rusmana | Probing high school students´ perceptions of the concept of species: a semantic network analysis approach[END_REF].

The MSC, also known as Linnean, Phenetic, Classic, Typological, Essentialist [START_REF] Haider | A Brief Review on Species Concepts with Emphasis on Plants[END_REF], is one of the most used species concepts [START_REF] Soltis | The Role of Hybridization in Plant Speciation[END_REF][START_REF] Padial | The integrative future of taxonomy[END_REF][START_REF] Karbstein | Phylogenomics supported by geometric morphometrics reveals delimitation of sexual species within the polyploid apomictic Ranunculus auricomus complex (Ranunculaceae)[END_REF]. The delimitation of species based on the MSC has been the basis of taxonomy since its origin and has been used in almost all classification systems [START_REF] Jensen | The conundrum of morphometrics[END_REF]. The MSC can be applied in sexual and asexual organisms and is very useful for fossil records. However, morphological characteristics are sometimes quite subjective and depend on specialized opinions for some key features. MSC has shortcomings when working with natural hybrids and does not consider the evolutionary origin of organisms [START_REF] Haider | A Brief Review on Species Concepts with Emphasis on Plants[END_REF].

As previously mentioned, MSC is based on morphological differences between groups of individuals. It is at this point that morphometrics (from the Greek morphé -shape; metricsmeasurement) becomes relevant, which focuses on the statistical analysis of quantitative morphological variations within and between species [START_REF] Remagnino | Computational Botany: Methods for Automated Species Identification[END_REF]. Comparing the morphological characteristics of organisms and understanding how these characteristics vary in association with other traits are of great interest to ecologists, taxonomists, and evolutionists [START_REF] Adams | Geomorph: an R package for the collection and analysis of geometric morphometric shape data[END_REF]. At present, morphometrics remains an invaluable tool for the delimitation of species. However, it is necessary to reexamine the classical concept of morpho-species and their limits in light of new molecular evidence [START_REF] Roberts | Reconciling molecules and morphology in benthic foraminifera: A morphometric study of Ammonia and Elphidiidae in the NE Atlantic[END_REF]. There are two morphometrics methods to assess the morphological limits between species: multivariate morphometrics (MM) and geometric morphometrics (GM).

Multivariate Morphometrics (MM)

Morphological measurements have been used for a long time in the description and characterization of morphological groups. However, it was not until 1960 that the use of traditional or multivariate morphometrics began in quantitative description and analysis of shape variation [START_REF] Marcus | Traditional morphometrics[END_REF][START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF]). Multivariate morphometrics is based on linear and angular measurements and their relationships [START_REF] Rohlf | A revolution in morphometrics[END_REF][START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF][START_REF] Slice | Geometric Morphometrics[END_REF][START_REF] Portillo | Advances in Morphometrics in Archaeobotany[END_REF], which can be compared independently or together using multivariate analysis [START_REF] Remagnino | Computational Botany: Methods for Automated Species Identification[END_REF]. Multivariate morphometrics plays a fundamental role in establishing the limits of taxa and in evaluating the relationships between them [START_REF] Jensen | The conundrum of morphometrics[END_REF]. Although there has been significant

development of new morphometrics techniques, traditional measurements (linear and angular) continue to be present in descriptions, comparisons, and exploration of patterns of variation at different taxonomic levels [START_REF] Van Der Niet | Three-dimensional geometric morphometrics for studying floral shape variation[END_REF][START_REF] Remagnino | Computational Botany: Methods for Automated Species Identification[END_REF].

Unfortunately, the use of MM in the analysis of structures does not guarantee a complete understanding of the shape of objects [START_REF] Dryden | Statistical shape analysis[END_REF][START_REF] Karbstein | Phylogenomics supported by geometric morphometrics reveals delimitation of sexual species within the polyploid apomictic Ranunculus auricomus complex (Ranunculaceae)[END_REF]). Multivariate morphometrics does not have a high statistical value for defining shapes because different shapes can be obtained for the same set of distance measurements, and in general, it is not possible to generate graphic representations of the shape based on linear distances [START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF][START_REF] Slice | Geometric Morphometrics[END_REF]). According to [START_REF] Jensen | The conundrum of morphometrics[END_REF], if a researcher is really interested in focusing on shape rather than only on size, traditional approaches are not the most appropriate.

Geometric Morphometrics (GM)

The development of GM techniques at the beginning of the 1980s, based on the spatial relationship between homologous points, has revolutionized the studies of the variation of shape in objects and biological structures [START_REF] Bookstein | Morphometric tools for landmark data: geometry and biology[END_REF][START_REF] Rohlf | A revolution in morphometrics[END_REF][START_REF] Jensen | The conundrum of morphometrics[END_REF][START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF][START_REF] Chen | A study of floral shape variation in Delphinieae (Ranunculaceae) using geometric morphometrics on herbarium specimens[END_REF]. MG is defined as the set of methods and procedures with a geometric basis for the analysis of shape patterns, where the measurements are usually derived from contours and landmarks [START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF][START_REF] Viscosi | Leaf shape variation and differentiation in three sympatric white oak species revealed by elliptic Fourier analysis[END_REF][START_REF] Karbstein | Phylogenomics supported by geometric morphometrics reveals delimitation of sexual species within the polyploid apomictic Ranunculus auricomus complex (Ranunculaceae)[END_REF]. [START_REF] Bookstein | A statistical method for biological shape change[END_REF], who established the theoretical basis of the GM, proposed the description of the shape using

Cartesian coordinates that allowed the visualization of differences between individuals and the possibility of applying statistical tests. [START_REF] Kendall | Shape-manifolds, Procrustean metrics and complex projective spaces[END_REF] theorized the existence of a space of the form (or Kendall's space) by demonstrating how the work of [START_REF] Bookstein | A statistical method for biological shape change[END_REF] was interpreted in this space. MG allows obtaining a more useful and consistent definition of shape and size [START_REF] Bookstein | Morphometric tools for landmark data: geometry and biology[END_REF][START_REF] Rohlf | NTSYSpc. Numerical Systematics and Multivariate Analysis System[END_REF], which makes it possible to analyse their variations separately. The first step in GM is to remove the effect of size through uniform transformations, which includes isotropic underlining (equal size), rotations, and translations of the analysed structure [START_REF] Kuhly | Elliptic Fourier Features of a Closed Contour[END_REF]Klingenberg & Mclntyre 1998;[START_REF] Remagnino | Computational Botany: Methods for Automated Species Identification[END_REF].

Contour methods were the first to be used in MG analyses. These consist of digitizing points that quantify the periphery of an object through the use of sequences of coordinates (x, y) or (x, y, z) [START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF]). Contours can be opened (start and end at different points) or closed (start and end at the same point). Open contours are studied using polynomial equations, cubic splines, and Bezeir curves [START_REF] Evans | Parametric cubic splines and geological shape descriptions[END_REF][START_REF] Engel | A least squares method for estimation of Bezier curves and surfaces and its applicability to multivariate analysis[END_REF], while closed contours usually use elliptical Fourier descriptors, elliptical Fourier analysis, and eigenshapes [START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF].

Elliptical Fourier descriptors (EFDs) were proposed by Kuhl & Giardina (1982) and allow any closed and bi-dimensional contour to be described [START_REF] Iwata | SHAPE: A Computer Program Package for Quantitative Evaluation of Biological Shapes Based on Elliptic Fourier Descriptors[END_REF], starting from a sequence of coordinates (X, Y). The set of coordinates allows the formation of mathematical functions (sins and cosines) known as Fourier harmonics, from which the coefficients are calculated, which will form the EFDs. These coefficients present similar values for similar shapes [START_REF] Viscosi | Leaf shape variation and differentiation in three sympatric white oak species revealed by elliptic Fourier analysis[END_REF]. The higher the number of harmonics, the greater the precision in the descriptors [START_REF] Cope | Plant species identification using digital morphometrics: a review[END_REF], but there is a number beyond which the description is not more efficient. [START_REF] Chuanromanee | Morphological Analysis of Size and Shape (MASS): An integrative software program for morphometric analyses of leaves[END_REF] suggest that between 15 and 20 Fourier harmonics is sufficient to find differences in leaf shape in various plant species.

The landmark methods allow characterizing the shape through coordinates of a set of points that can be unequivocally located in each of the specimens under study [START_REF] Klingenberg | Shape analysis of symmetric structures: quantifying variation among individuals and asymmetry[END_REF][START_REF] Slice | Geometric Morphometrics[END_REF]. Landmarks are points in space that correspond to the position of a particular feature on an object of interest, which allow the shape of each specimen to be quantified [START_REF] Adams | Geometric morphometrics: Ten years of progress following the 'revolution[END_REF]). There are three types of key points: i) type I or morphological landmarks, which are points whose presumed homology between taxa is supported by biological significance; ii)

type II or mathematical landmarks are points whose presumed homology between individuals is supported only by geometry and not by anatomical evidence, and iii) type III or semi landmarks which are extreme points and allow the form to be analysed to be fully covered [START_REF] Bookstein | Size and shape spaces for landmark data in two dimensions (with discussion and rejoinder)[END_REF].

The landmark methods are superior to the contour methods, especially when the landmarks represent biologically defined and homologous points [START_REF] Jensen | The conundrum of morphometrics[END_REF].

In a general sense, GM has proven to be a powerful tool to capture the shape of biological structures [START_REF] Rohlf | A revolution in morphometrics[END_REF]) and helps to increase the rigor of scientific descriptions [START_REF] Viscosi | Leaf shape variation and differentiation in three sympatric white oak species revealed by elliptic Fourier analysis[END_REF]. The GM techniques have been used in the delimitation of plants species in many studies: [START_REF] Jensen | Lines, outlines, and landmarks: Morphometric analyses of leaves of Acer rubrum, Acer saccharinum (Aceraceae) and their hybrid[END_REF] in Acer (Sapindaceae), [START_REF] Viscosi | Leaf morphological analyses in four European oak species (Quercus) and their hybrids: A comparison of traditional and geometric morphometric methods[END_REF] in Quercus (Fagaceae), been studied using traditional morphometrics. However, in recent years has been an increasing interest in using geometric morphometrics to study the form of leaves because this structure is particularly suitable for outline and landmark based description.

Genetic Species Concept (GSC)

Like many other fields in the life sciences, systematics has advanced significantly with genomics and evolutionary modeling. The development of coalescent modeling, Bayesian approaches, and capacities to generate large datasets, thanks to progress in sequencing technologies, allow us to assess patterns of genetic diversity and infer evolutionary processes that underlie them [START_REF] Funk | Harnessing genomics for delineating conservation units[END_REF]. Genetic data provide a great power to identify differentiation and divergence within and between species, requiring further consideration of how this affects the delineation of taxonomic units [START_REF] Coates | Genetic Diversity and Conservation Units: Dealing With the Species-Population Continuum in the Age of Genomics[END_REF]. Genetic data can contribute to the delimitation of taxonomic groups, whose identity can be masked by other types of information such as morphology or environment [START_REF] Postaire | Molecular species delimitation methods and population genetics data reveal extensive lineage diversity and cryptic species in Aglaopheniidae (Hydrozoa)[END_REF]Rico & Gutiérrez-Becerril 2019); these groups can be identified as independent evolutionary lineages.

From the genetic point of view, a species can be defined as the largest and most inclusive reproductive community of individuals who share a common gene pool [START_REF] Mayr | Principles of systematic zoology[END_REF][START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF]). The GSC is quite similar to the MSC, except that the method used to delimit species is based on genetic distances [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF]. The main problem with this concept is that there is no genetic information for most of the diversity, and obtaining it is not a simple or cheap process.

Furthermore, most species are not genetically uniform, as processes such as divergence (genetic drift and geographic isolation) and gene flow can shape geographic and genomic patterns of variation within species [START_REF] Coates | Genetic Diversity and Conservation Units: Dealing With the Species-Population Continuum in the Age of Genomics[END_REF].

Despite all these limitations, GSC is very useful when working with cryptic species and natural hybrids with unclear morphological limits. Furthermore, it provides a good level of resolution in the study of the number of species and their limits [START_REF] Groves | Species definitions and conservation: a review and case studies from African mammals[END_REF][START_REF] Ramírez-Barahona | The influence of climatic niche preferences on the population genetic structure of a mistletoe species complex[END_REF]Rico & Gutiérrez-Becerril 2019;[START_REF] Muñiz-Castro | Population genetics, species boundaries, and conservation in the Magnolia pacifica species complex along a continentally and moisture gradient in western Mexico[END_REF].

Population genetics

Population genetics studies the origin, quantity, and distribution of the variation of genes present in populations, as well as the fate of this variation through space and time [START_REF] Templeton | Population genetics and microevolutionary theory[END_REF].

Population genetics is based on the measurement and characterization of genetic variation within and between populations. This analysis allows making inferences about processes like genetic drift, mutation rates, gene flow, and natural selection, among others [START_REF] Hartl | Principles of Population Genetics[END_REF]. Withinpopulation, genetic variability and the population genetic structure of a species is determined by habitat, reproductive system, pollen and seed dispersal range, and evolutionary history, as well as by the spatial and temporal stability of the environment used by the species to complete its life cycle (selection) [START_REF] Templeton | Population genetics and microevolutionary theory[END_REF][START_REF] Hirsch | Historical range contraction, and not taxonomy, explains the contemporary genetic structure of the Australian tree Acacia dealbata Link[END_REF]. There is a wide variety of methods and statistics to describe the genetic structure and diversity of populations and to summarize the information brought by dozens to billions of genetic markers in manageable terms.

Population genetic structure describes the composition and distribution of genetic variability (genes and genotypes) among populations of a given species. The methods for evaluating population genetic structure aim at identifying the number of different genetic groups (species/ populations) in a sample and classify individuals or fractions of individual genomes into the inferred groups [START_REF] Godoy | La genética, los marcadores moleculares y la conservación de especies[END_REF][START_REF] Grünwald | Best Practices for Population genetic Analyses[END_REF]. Prior geographical or ecological information, when available, can be included in the clustering methods. Genetic diversity is the variation in the DNA sequence (in terms of the number and frequency of alleles and genotypes) between different individuals of a population or species [START_REF] Futuyma | Evolution[END_REF].

Genetic diversity contributes to the ability of species to respond to environmental changes [START_REF] Frankham | Introduction to Conservation Genetics[END_REF], which has implications for the development and maintenance of natural populations in the long term. The levels of genetic diversity in populations are tightly linked to the population genetic effective size (Ne), a parameter that is strongly influenced by different so-called neutral mechanisms such as demography, genetic structure, migration, and their adaptation to the environment (selection) [START_REF] Kim | Population genetic structure of eelgrass (Zostera marina) on the Korean coast: Current status and conservation implications for future management[END_REF]. Because each species has its own evolutionary history, the genetic diversity and structure vary considerably between species [START_REF] Ellegren | Determinants of genetic diversity[END_REF].

Molecular markers: microsatellites

A molecular marker (also known as genetic marker) is any molecular phenotype without any contribution to the environment that can be used to infer or obtain genetic information about the organism or population to which it belongs [START_REF] Schlötterer | The evolution of molecular markers -just a matter of fashion?[END_REF]). Molecular markers provide information about allelic variation and allow the description of genetic patterns in natural populations at scales ranging from individuals to species [START_REF] Schlötterer | The evolution of molecular markers -just a matter of fashion?[END_REF][START_REF] Godoy | La genética, los marcadores moleculares y la conservación de especies[END_REF].

Molecular markers can be classified into three general classes: protein variants (allozymes, no longer used by now), polymorphisms at the non-repetitive DNA sequence level (RFLPs:

Restriction Fragment Length Polymorphisms -no longer used by now-, AFLPs: Amplified

Fragment Length Polymorphisms, SNPs: Single Nucleotide Polymorphism), and DNA variation with repetitive motifs (minisatellites and microsatellites) [START_REF] Schlötterer | The evolution of molecular markers -just a matter of fashion?[END_REF][START_REF] Arif | Molecular markers for biodiversity analysis of wildlife animals: a brief review[END_REF].

Molecular markers have been applied to various biological issues, from gene mapping, phylogenies, population genetics, paternity testing, forensic analysis, and conservation genetics, among others [START_REF] Schlötterer | The evolution of molecular markers -just a matter of fashion?[END_REF][START_REF] Godoy | La genética, los marcadores moleculares y la conservación de especies[END_REF].

Microsatellites or simple sequence repeat (SSR) are short motifs of DNA sequences (1-10 base pairs) tandemly repeated [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF]. These markers are generally highly polymorphic (mostly due to the variation in the number of tandem repeats), abundant, and widely distributed within the genomes of most eukaryotic species. They are found in gene sequences (exons, introns, regulatory regions) and in non-functional DNA sequences as well [START_REF] Schlötterer | The evolution of molecular markers -just a matter of fashion?[END_REF]. Apart from their level of polymorphism, another advantage of microsatellites is that they are easily amplified by PCR, which makes them highly useful markers for evaluating genetic diversity [START_REF] Arif | Molecular markers for biodiversity analysis of wildlife animals: a brief review[END_REF]. Microsatellite markers have proven to be efficient and widely used in the evaluation of the population genetic structure and diversity in several species of the Magnoliaceae family [START_REF] Hirayama | Reduced seed production, inbreeding, and pollen shortage in a small population of a threatened tree, Magnolia stellata[END_REF][START_REF] Ishida | Population differentiation and gene flow within a metapopulation of a threatened tree, Magnolia stellata (Magnoliaceae)[END_REF][START_REF] Tamaki | Genetic variation and differentiation in populations of a threatened tree, Magnolia stellata: factors influencing the level of within-population genetic variation[END_REF][START_REF] He | Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae)[END_REF][START_REF] Yu | Genetic diversity and relationship of endangered plant Magnolia officinalis (Magnoliaceae) assessed with ISSR polymorphisms[END_REF]Rico & Gutiérrez-Becerril 2019;Aldaba-Nuñez et al. 2021;[START_REF] Hernández | Effect of the landscape on functional and spatial connectivity in Magnolia cubensis (Magnoliaceae) in two mountain massifs of Cuba[END_REF]. In all these works, between 10-20 microsatellite loci were used.

Phylogenetic Species Concept (PSC)

According to the PSC, a species is the smallest aggregation of populations (sexual) or lineages (asexual) with a specific common ancestry (monophyly) diagnosable by unique combinations of character states [START_REF] Mayden | A hierarchy of species concepts: The denouement in the saga of the species problem[END_REF][START_REF] Wheeler | The phylogenetic species concept[END_REF][START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF][START_REF] Soltis | The Role of Hybridization in Plant Speciation[END_REF][START_REF] Haider | A Brief Review on Species Concepts with Emphasis on Plants[END_REF]. [START_REF] Wheeler | Why the phylogenetic species concept?[END_REF] stated that the phylogenetic concept is simple, broadly applicable, and testable. The PSC is more widely applicable in practice than other species concepts because it can be applied to asexual organisms and allopatric populations.

Phylogenetic analysis is a powerful approach that provides new lines of taxonomic evidence for species identifications and biodiversity assessments [START_REF] Roberts | Reconciling molecules and morphology in benthic foraminifera: A morphometric study of Ammonia and Elphidiidae in the NE Atlantic[END_REF].

It has been argued that the PSC is more objective than other definitions of species and may reveal morphologically unremarkable but significant populations [START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF]). Within the PSC, there is no privileged phylogenetic level that corresponds to a species other than "the smallest aggregate". Where the PSC is used to delimit species, sufficient individuals and characters should be sampled to ensure an adequate resolution of the species status across clades [START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF]. It is important to remark that all species phylogenies are, in reality, genes genealogies. The evolutionary history of a species and its genes may not match because identifying truly orthologous genes is not always obvious, the existence of long branches issues, and incomplete lineage sorting [START_REF] Mindell | Homology evolving[END_REF]. Thus, it is widely recognized that the PSC will often split a species determined under non-phylogenetic criteria with two or more "new" species [START_REF] Agapow | The impact of species concept on biodiversity studies[END_REF][START_REF] Sukumaran | Multispecies coalescent delimits structure, not species[END_REF].

The chloroplast, an important plant organelle, is essential for photosynthesis and several metabolic pathways [START_REF] Zoschke | Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation[END_REF][START_REF] Liu | The complete chloroplast genome sequence of Magnolia Mexicana DC. (Magnoliaceae) from Central America[END_REF]. The chloroplast genome has a highly conserved circular DNA arrangement ranging from 75 to 250 kb (kilo base pair) [START_REF] Song | Development of Chloroplast Genomic Resources for Oryza Species Discrimination[END_REF]. This molecule has four parts: a large single-copy (LSC) region, a small singlecopy (SSC) region and two inverted repeat (IRa and IRb) regions [START_REF] Liu | The complete chloroplast genome sequence of Magnolia Mexicana DC. (Magnoliaceae) from Central America[END_REF][START_REF] Dong | Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)[END_REF]). Chloroplast genomes have often been used for phylogenetic studies and species identification, especially in difficult complexes of morphologically homogeneous species [START_REF] Kim | Complete sequence and organization of the cucumber (Cucumis sativus L. cv. Baekmibaekdadagi) chloroplast genome[END_REF][START_REF] Sun | The complete chloroplast genome of Magnolia polytepala: Comparative analyses offer implication for genetics and phylogeny of Yulania[END_REF][START_REF] Dong | Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)[END_REF]. Genetic information from the chloroplast genome is usually maternal inheritance in angiosperms, making the chloroplast genome a good indicator of maternal ancestry [START_REF] Song | Development of Chloroplast Genomic Resources for Oryza Species Discrimination[END_REF][START_REF] Li | The Complete Plastid Genome of Magnolia zenii and Genetic Comparison to Magnoliaceae species[END_REF]).

The complete chloroplast genome in Magnoliaceae family has been found to have the typical structure with approximately 160 000 bp (base pair) in length [START_REF] Liu | The complete chloroplast genome sequence of Magnolia Mexicana DC. (Magnoliaceae) from Central America[END_REF]. Comparative analysis of full plastome provides greater insight into the evolutionary history and interspecies relationships in the family, with many examples in the last years [START_REF] Kuang | Complete chloroplast genome sequence of Magnolia kwangsiensis (Magnoliaceae): implication for DNA barcoding and population genetics[END_REF][START_REF] Li | The Complete Plastid Genome of Magnolia zenii and Genetic Comparison to Magnoliaceae species[END_REF][START_REF] Liu | The complete chloroplast genome sequence of Magnolia Mexicana DC. (Magnoliaceae) from Central America[END_REF][START_REF] Sun | The complete chloroplast genome of Magnolia polytepala: Comparative analyses offer implication for genetics and phylogeny of Yulania[END_REF][START_REF] Guzmán-Díaz | Comparison of Magnoliaceae Plastomes: Adding Neotropical Magnolia to the Discussion[END_REF][START_REF] Wang | Major clades and a revised classification of Magnolia and Magnoliaceae based on whole plastid genome sequences via genome skimming[END_REF][START_REF] Dong | Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)[END_REF]).

Integrative taxonomy

Integrative taxonomy is a relatively young discipline, which was introduced independently into the literature by [START_REF] Dayrat | Towards integrative taxonomy[END_REF] and [START_REF] Will | The perils of DNA barcoding and the need for integrative taxonomy[END_REF]. [START_REF] Will | The perils of DNA barcoding and the need for integrative taxonomy[END_REF] used the term integrative taxonomy to refer to a taxonomic process that was inclusive of all available data sources to delimit, discover, and identify species at all levels. [START_REF] Dayrat | Towards integrative taxonomy[END_REF] used the term integrative taxonomy in a set of guidelines that taxonomists should follow when describing new species.

Guideline 5: 'species should only be named when their limits are supported by multiple lines of evidence'. Species are always hypothetical, even when they are 'broadly supported', but our level of confidence is much higher for entities supported by different kinds of data than for entities supported by only one kind [START_REF] Dayrat | Towards integrative taxonomy[END_REF]. Both papers focused on a taxonomy that integrated all available sources of data to develop hypotheses of species-level taxa.

Ancient and more divergent species can usually be easily recognized by considering any of the different species concepts [START_REF] Prebble | Species delimitation and the population genetics of rare plants: A case study using the New Zealand native pygmy forget-me-not group (Myosotis; Boraginaceae)[END_REF][START_REF] Denham | Using integrative taxonomy and multispecies coalescent models for phylogeny reconstruction and species delimitation within the "Nastanthus-Gamocarpha" clade (Calyceraceae)[END_REF]. However, difficulties arise during the early stages of lineage divergence, when lineages may have diverged and formed new species but may not have acquired all of the different properties on which different species concepts are based (de Queiroz 2007; [START_REF] Denham | Using integrative taxonomy and multispecies coalescent models for phylogeny reconstruction and species delimitation within the "Nastanthus-Gamocarpha" clade (Calyceraceae)[END_REF]. In those cases, the use of a single taxonomic data source (morphological, ecological, genetic, phylogenetic, reproductive, etc.) to delimitate species would fail to resolve the species limits. In complex cases, the integrative taxonomy approach that integrates lines of evidence from morphology, genetics, phylogeny, and ecological traits is often required to reliably delineate, identify, and help reveal evolutionary relationships among taxa [START_REF] Padial | The integrative future of taxonomy[END_REF][START_REF] Schlick-Steiner | Integrative Taxonomy: A Multisource Approach to Exploring Biodiversity[END_REF][START_REF] Yeates | Integrative taxonomy, or iterative taxonomy?[END_REF].

The integrative approach is especially useful when using a single line of evidence fails, which is common in polymorphic, cryptic groups, and species complexes [START_REF] Masonick | Integrative species delimitation in Nearctic ambush bugs (Heteroptera: Reduviidae: Phymatinae): insights from molecules, geometric morphometrics and ecological associations[END_REF].

Evaluating multiple criteria not only increases our ability to detect recently separated lineages but also can provide more robust support for lineage separation [START_REF] Prebble | Species delimitation and the population genetics of rare plants: A case study using the New Zealand native pygmy forget-me-not group (Myosotis; Boraginaceae)[END_REF] 3) exhibit conserved features that enable their recognition. A pragmatic way to approach the question of species delimitation is to start with the null hypothesis that two or more individuals belong to the same species and then aim to falsify this null hypothesis by one or more sources of evidence (the more, the better). Therefore if one piece of evidence does not falsify the null hypothesis, it is not proof that there is only one species present; it simply indicates that that particular piece of evidence is not informative, yet others might be [START_REF] Prebble | Species delimitation and the population genetics of rare plants: A case study using the New Zealand native pygmy forget-me-not group (Myosotis; Boraginaceae)[END_REF].

There is probably no magic solution for species discovery and delimitation, but an integrative and evolutionary framework provides taxonomists with a larger arsenal to face the underestimated [START_REF] Damasco | Reestablishment of Protium cordatum (Burseraceae) based on integrative taxonomy[END_REF] in Burseraceae, [START_REF] Denham | Using integrative taxonomy and multispecies coalescent models for phylogeny reconstruction and species delimitation within the "Nastanthus-Gamocarpha" clade (Calyceraceae)[END_REF] in Calyceraceae, [START_REF] Moein | An integrating study of genetic diversity and ecological niche modelling in Salvia aristata (Lamiaceae)[END_REF] in Lamiaceae, [START_REF] Yang | Different species or genetically divergent populations? Integrative species delimitation of the Primulina hochiensis complex from isolated karst habitats[END_REF] in Gesneriaceae, [START_REF] Karbstein | Phylogenomics supported by geometric morphometrics reveals delimitation of sexual species within the polyploid apomictic Ranunculus auricomus complex (Ranunculaceae)[END_REF] in Ranunculaceae, [START_REF] Giorgi | An Integrated Taxonomic Approach Points towards a Single-Species Hypothesis for Santolina (Asteraceae) in Corsica and Sardinia[END_REF] in Asteraceae, [START_REF] Meng | Integrative Taxonomy Supports Two New Species of Rhodiola (Crassulaceae) in Xizang, China[END_REF] in Crassulaceae, showed a rise in the number of works that applied the integrative taxonomy approach to plants. These works have contributed to a better understanding species limits, evolution, and diversity.

Inter-specific hybridization and genetic introgression in plants

Hybridization is the crossing between two individuals, whatever they belong to the same variety, The levels of hybridization and introgression have increased dramatically on a global scale, caused mainly by the introduction of exotic species, fragmentation, and habitat modifications [START_REF] Grabenstein | Breaking Barriers: Causes, Consequences, and Experimental Utility of Human-Mediated Hybridization[END_REF][START_REF] Ottenburghs | The genic view of hybridization in the Anthropocene[END_REF]). According to [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF], hybridization is a serious problem for conservation, whose impact has been underestimated by conservation biologists and, in the future, will become even more problematic. Hybridization and genetic introgression can contribute to the decline and eventual extinction of species.

In the case of outbreeding depression, the hybrid progeny has lower performance or fitness than either parent [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF][START_REF] Becker | Hybridization may facilitate in situ survival of endemic species through periods of climate change[END_REF]. Also, the sterile or partially sterile hybrids could result in a loss of reproductive potential and may reduce the population growth rate. In the case of fertile hybrids, genetically distinct populations may be lost through genetic mixing [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF][START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF]An et al. 2017). On the other hand, hybridization and introgression may have a wide variety of effects on fitness. In the case of heterosis, or hybrid vigor, hybrids have enhanced performance or fitness relative to either parental taxa. Hybrids may also have a fitness advantage because they possess advantageous traits from both parental populations.

The assessment of the inter-population or inter-specific gene flow has therefore been the focus of interest of a huge number of evolutionary studies. Interspecific hybridization and introgression seem to be a common and widespread phenomenon in plants and even more in angiosperms and ferns [START_REF] Soltis | The Role of Hybridization in Plant Speciation[END_REF][START_REF] Burgarella | Detection of hybrids in nature: application to oaks (Quercus suber and Q. ilex)[END_REF]. Estimates of several well-studied floras suggest that about 11% of plant species hybridize, and about a quarter of them are rare or endangered [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF]. Species with strong premating barriers, for example, or that have coexisted naturally for thousands of generations, are unlikely to be threatened by hybridization. However, plants from islands are particularly vulnerable to hybridization due to the premating barriers often being weak and their smallest geographic ranges. In the specific cases of Magnoliaceae, hybridization is likely to occur and may have played an important role in speciation [START_REF] Nie | Phylogenetic and biogeographic complexity of Magnoliaceae in the Northern Hemisphere inferred from three nuclear data sets[END_REF][START_REF] Wang | Major clades and a revised classification of Magnolia and Magnoliaceae based on whole plastid genome sequences via genome skimming[END_REF]. Studies of natural hybridization in Magnoliaceae are not common [START_REF] Muranishi | Identification of natural hybrids between Magnolia stellate and M. salicifolia[END_REF][START_REF] Muranishi | Asymmetric introgression between Magnolia stellate and M. salicifolia at a site where the two species grow sympatrically[END_REF]). Yet, many interspecific hybrids have been created by artificial crosses for ornamental use [START_REF] Treseder | Magnolias[END_REF][START_REF] Callaway | Magnolias[END_REF][START_REF] Muranishi | Asymmetric introgression between Magnolia stellate and M. salicifolia at a site where the two species grow sympatrically[END_REF][START_REF] Xu | Interspecific Hybridization Processes between Michelia yunnanensis and M. crassipes and Embryogenesis of the Heterozygote[END_REF]; suggesting that the involved species have diverged only recently.

Genus Magnolia

The family Magnoliaceae Juss. is included in the order Magnoliales and belongs to the clade of Magnoliides (APG IV 206). The family is divided into two genera: Magnolia L. and Liriodendron L. [START_REF] Figlar | Notes on Magnoliaceae IV[END_REF]). The genus Magnolia, the largest within the family, are trees and shrubs that could be evergreen or deciduous [START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF]. Its leaves are simple and alternate, with stipules surrounding the terminal bud. The flowers are solitary and large with numerous tepals (sometimes sepals and petals), bisexual with an elongated receptacle, and numerous stamens and carpels [START_REF] Treseder | Magnolias[END_REF][START_REF] Judd | Plant systematics: a phylogenetic approach[END_REF]. The fruit is an aggregate of follicles, and the seeds have a fleshy aril. Pollination in the family is mainly mediated by beetles and dispersal by birds and small mammals [START_REF] Callaway | Magnolias[END_REF][START_REF] Judd | Plant systematics: a phylogenetic approach[END_REF] [START_REF] Callaway | Magnolias[END_REF].

Magnolias have attracted the interest of many botanists due to their ancestral evolutionary history and the retention of primitive characters [START_REF] Treseder | Magnolias[END_REF][START_REF] Callaway | Magnolias[END_REF][START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF]. This group has also been relevant from a commercial point of view since most of its species are globally appreciated as ornamentals due to their attractive flowers and foliage (Vázquez-García 1994).

These plants are also valued for their medicinal properties and the quality of their wood [START_REF] Cires | Gap analyses to support ex situ conservation of genetic diversity in Magnolia, a flagship group[END_REF][START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF][START_REF] Wang | Major clades and a revised classification of Magnolia and Magnoliaceae based on whole plastid genome sequences via genome skimming[END_REF]. Due to the high economic value of magnolias, their conservation status has been recognized as a priority by international organizations [START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF]. Approximately 48 % of the species in the family are threatened. Among the main threats faced by the species of the family are logging, agriculture and livestock, anthropization, and climate change, among others [START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF].

Seven taxa of

Magnolia sect. Talauma

The members of Magnolia section Talauma are evergreen trees with a stamen connective with a long appendix and embedded in the gynoecium (except in the subsection Talauma). The carpels present circumscissile dehiscence (except in the subsection Cubenses) and two ovules per carpel [START_REF] Figlar | Notes on Magnoliaceae IV[END_REF]). The section Talauma occurs in Central America, South America, and the Caribbean Islands [START_REF] Pérez | Magnolia chiguila and M. mashpi (Magnoliaceae): two new species and a new subsection (Chocotalauma, sect. Talauma) from the Chocó biogeographic region of Colombia and Ecuador[END_REF]). This section is divided into four subsections (following R.A Howard. The main criterion used by [START_REF] Howard | The Morphology and Systematics of the West Indian Magnoliaceae[END_REF] to differentiate both taxa was the truncated base of the leaves present in T. truncata, against rounded or acute base leaves in T.

minor. However, he pointed out that the material used to describe T. truncata may have been a

vegetative branch of T. minor.

In 1950 Brother León and Brother Alain, described Talauma minor var. oblongifolia León [START_REF] León | Novedades de la Flora Cubana (II)[END_REF]. They recognized that the shape of the leaves, the shorter and more widely petiole, as well as the carpels and smaller seeds, seem to indicate a new species; however, in the absence of flowers, only the range of variation was given. These authors state that type specimens have been collected on the coast of Moa, as well as intermediate forms with oblong leaves mixed with elliptical leaves. The shared distribution area was the reason to consider it as a variety of T.

minor. Also, [START_REF] León | Novedades de la Flora Cubana (II)[END_REF] revalidated the name Talauma orbiculata Britton & P. Wilson, considered by [START_REF] Urban | Sertum antillanum[END_REF] and [START_REF] Howard | The Morphology and Systematics of the West Indian Magnoliaceae[END_REF] as synonymous of Talauma minor Urb. [START_REF] León | Flora de Cuba II[END_REF], in the treatment of Magnoliaceae for the "Flora de Cuba", recognized four taxa of Magnolia subsect. Talauma: (1) T. minor Urb. var. minor, (2) T. minor var. oblongifolia León,

(3) T. orbiculata Britton & P. Wilson, and (4) T. truncata (Moldenke) R.A. Howard. [START_REF] León | Flora de Cuba II[END_REF] report that T. truncata is exclusive to the Pico Turquino region, while T. orbiculata is found throughout the Sierra Maestra.

In the Supplement for the "Flora of Cuba" published by [START_REF]Flora de Cuba[END_REF], he suggested that all previously described taxa of Talauma from Cuba could be considered as one single taxon. Later, [START_REF] Borhidi | New plants in Cuba I[END_REF] considered T. minor as the only species of Talauma in Cuba and defined two subspecies: T. minor subsp. oblongifolia (León) Borhidi and T. minor subsp. orbiculata (Britton by [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF] and [START_REF] Borhidi | New plants in Cuba I[END_REF], into four species. For T. minor Urb.

and T. orbiculata Britton & P. Wilson considered the criteria of [START_REF] Urban | Fundamenta Florae Indiae Occidentalis[END_REF] and [START_REF] Britton | Studies of West Indian Plants-XI[END_REF], respectively. [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF]

MATERIALS AND METHODS

Data sampling: occurrence, leaf and DNA records

The samples for the ecological, morphological, and genetic analyses were collected (2015)(2016)(2017)(2018)(2019)(2020) from individuals representing all the Cuban taxa of Magnolia subsect. Talauma throughout their entire distribution range in the mountains of Nipe-Sagua-Baracoa and Sierra Maestra in eastern Cuba (Fig. 1). In the field, individuals were identified based on tree and leaf shape according to the morphological criteria outlined by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] because this author defined the highest number of species units (see Table 1). To confirm species identity, 37 herbarium vouchers were collected or consulted (Table 2). All herbarium vouchers were deposited in the Herbarium Johannes Bisse (HAJB, herbarium codes follow Thiers 2023+) at the National Botanic Garden, University of Havana. The number of samples per species and localities is shown in Table 2. For the ecological and morphological analyses, data from herbarium specimens (deposited in HAC, HAJB, NY, and B) were included to cover not visited localities. Herbarium specimens reported before 1950 were not considered for niche analysis.

For ecological analysis, the GPS coordinates and the type of soil (serpentine derivative, limestone derivative, volcanic, and limestone derivate) were recorded for each individual. For the morphological analyses, 4-8 adult healthy leaves from 200 individuals were randomly collected and herborized. A leaf was considered healthy if the full outline of the leaf was undamaged.

Leaves were photographed with a Nikon camera on a white background with a fixed ruler. The Herbario de la Flora de Cuba (HFC), Herbarium "Instituto de Ecología y Sistemática" (HAC), Herbarium "Jardín Botánico Nacional, Universidad de La Habana" (HAJB), Herbarium "The New York Botanical Garden" (NY), Herbarium "Botanischer Garten, Freie Universität Berlin" (B), National Park (NP), Ecological Reserve (ER), Natural Outstanding Element (NOE), Protected Area of Management Resources (PAMR), Extinct in the locality (*), No field data available (?), no voucher (NV), Abreviature (Abrev.), Number of individuals (Indiv.).

32

MATERIALS AND METHODS

For all the following analyses (ecological, morphological, genetic, and phylogenetic), the results and graphical representations were built using the classification system proposed by Bisse (1974,1988). This system divided the morphological variability into the highest number of units (M. minor, M. orbiculata, M. oblongifolia, and T. ophiticola), and was used in the field to identify each taxon. For the three taxa CS: M. oblongifolia = M. oblongifolia + Talauma ophiticola; and for the Two taxa CS: M. minor = M. minor + M. oblongifolia + T. ophiticola. However, in order to avoid confusion with the scientific names of the species and the different taxonomic treatments carried out on the group (see table 1), the taxa were considered morphological groups (Orbiculata, Minor, Oblongifolia, Ophiticola), and they will be treated as such in the following analyses.

Ecological analysis

Ecological Niche Models (ENMs)

In order to avoid autocorrelation and model overfitting [START_REF] Anderson | Harnessing the world's biodiversity data: promise and peril in ecological niche modelling of species distributions[END_REF], a spatial thinning of the occurrences of Magnolia subsect. Talauma was performed, leaving gaps of 3 km between records. This "gap distance" was based on the spatial resolution of the environmental variables (~1 km) and the environmental heterogeneity of Cuban mountain habitats. The final occurrence data used for the ENMs are in Considering the distribution in sympatry of the population from northeastern Cuba (see results),

we treat these as only one ecological group for the calibration process. We built 1116 candidate models for the northeastern group and Orbiculata. These models integrated 12 values of regularization multiplier (0.5 to 6, at intervals of 0.5), all combinations of the five features classes (linear = l; quadratic = q; product = p; threshold = t; hinge = h), and the three set of environmental variables. The background extent selected for the models was based on a minimum convex polygon with a buffer distance of 0.5 degrees, and the models were limited to the calibration area.

The performance of the candidate models was evaluated by considering the significance (partial ROC), omission rates, and model complexity (AICc). The selected best models were the significant models with omission rates ≤5 % and delta AICc ≤2. The model calibration process was done using kuenm [START_REF] Cobos | Kuenm: An R package for detailed development of ecological niche models using Maxent[END_REF], an R v. 4.2.0 (R Core Team 2022) package for detailed development of ENMs using Maxent v. 3.4.1.

The parameter combination of the best model was used for the niche modeling. The final models were created with the full set of thinned records (Table 3) and the selected sets of variables (

Niche Identity Test

A Niche Identity Test was used in order to examine the niche divergence within Magnolia subsect.

Talauma. This test is used to ask whether ENMs generated from two or more species are more different than expected if they are drawn from the same underlying distribution [START_REF] Warren | Environmental niche equivalency versus conservatism: Quantitative approaches to niche evolution[END_REF]. Two indices for niche identity (Schoener's D and Hellinger's-based I) were computed with ENMTools package with 1 000 replicates in R v. 4.2.0 (R Core Team 2022). Schoener's D calculates the suitable range for a given species based on the probability distributions for inhabiting particular regions and assumes that the suitability scores produced by Maxent are proportional to the species abundance. Hellinger's-based also estimates habitat suitability from probability distributions but without the assumption of Schoener's D [START_REF] Warren | ENMTools: A toolbox for comparative studies of environmental niche models[END_REF]. Both indices range from zero (no overlap) to one (complete overlap) [START_REF] Warren | Environmental niche equivalency versus conservatism: Quantitative approaches to niche evolution[END_REF].

Point-based and cluster analyses

Using the best suitable dataset of variables (from ENMs analysis), pixel values for each environmental layer were extracted at each point of occurrence of Magnolia subsect. Talauma, and used to assess niche differences between groups. A Principal Component Analysis (PCA) was performed in R v. 4.2.0 (R Core Team 2022) to explore the relation across the climate variables. To test the differences in elevation over sea level between groups, a MonteCarlo analysis in add-ins PopTools v. 3.23 MSExcel [START_REF] Hood | PopTools version 3[END_REF]) with 10 000 random permutations was made. We used the total number of geographical records (before spatial thinning) for Magnolia subsect. Talauma (858 records).

A Bayesian clustering approach based on Gaussian finite mixture models was carried out using the best suitable dataset of variables using the "mclust" R package [START_REF] Fraley | mclust Version 4 for R: Normal Mixture Modeling for Model-Based Clustering, Classification, and Density Estimation[END_REF][START_REF] Scrucca | mclust 5: Clustering, Classification and Density Estimation Using Gaussian Finite Mixture Models[END_REF]. The method tests the number of clusters and different mixture models that best fit the data according to the number of clusters (Cl) chosen a priori. The method allows comparing the quality of the discrimination among clusters based on the Bayesian Information Criteria (BIC), allowing one to choose the best value(s) of Cl without any information about individual assignation to the different clusters. The default "mclust" setting was used to assess the 14 types of models, which all differ in the covariance matrix landscape (see [START_REF] Scrucca | mclust 5: Clustering, Classification and Density Estimation Using Gaussian Finite Mixture Models[END_REF];

Zhang & Di 2020, for further details about the models). We varied Cl values between 1 and 12.

Because the analysis sometimes provided clusters having only one individual, those clusters were therefore considered "ghost" clusters and hence not considered true clusters.

Foliar morphological analyses

Multivariate and Geometric morphometrics

Two types of morphological analyses were carried out on three independent datasets: 1) multivariate morphometrics analyses: a linear and angular measures dataset, 2) geometric morphometrics analyses: an outline dataset, and 3) a landmark coordinates dataset.

In the multivariate morphometrics analyses, linear and angular measures of leaf characters were automatically taken from the digital photographs using the R v. 4.2.0 (R Core Team 2022) package In the geometric morphometrics analyses, the outline dataset was obtained through a semiautomated shape analysis performed in FoliometriK v. 0.2.2 (Ramírez-Arrieta & Denis 2020).

We set the program outputs to the elliptic Fourier descriptors (EFDs) [START_REF] Jensen | The conundrum of morphometrics[END_REF], to obtain the first 25 harmonics [START_REF] Chuanromanee | Morphological Analysis of Size and Shape (MASS): An integrative software program for morphometric analyses of leaves[END_REF]. The harmonics were normalized to eliminate the differences in size, position, rotation, and starting point. This allowed removing the undesired experimental source of random variation and analysing true differences of leaf shape between individual measurements [START_REF] Jensen | The conundrum of morphometrics[END_REF]. The landmark coordinates dataset was obtained as follows. The positions of landmarks were determined by placing a quadratic grid with six lines on each leaf. In between the intersections of the grid and the border of the leaf, we set 14 landmarks, two of them anatomical (type 1, i.e. apex and base) and the other 12 mathematically defined (type 2) (Fig. 2B). The landmarks X and Y coordinates were standardized using a Generalized

Procrustes Analysis in Past v. 2.14 [START_REF] Hammer | PAST: Paleontological Statistics software package for education and data analysis[END_REF]. Next, two variables: the Sum EDMA (Euclidean Distance Matrix Analysis) and centroid size, were calculated.

The statistical significance of the differences among groups (Orbiculata, Minor, Oblongifolia, and Ophiticola) for each measured variable (linear and angular variables, Sum EDMA, and centroid size) was assessed by a MonteCarlo analysis in PopTools v. 3.23 [START_REF] Hood | PopTools version 3[END_REF]) with 10 000 random permutations. The variability of the whole sample was described by using a normalized

Principal Component Analysis (PCA). Differences among groups were tested according to a one-way nonparametric MANOVA, using Euclidian distance, with 10 000 random permutations.

Correction of p-values for multiple testing was done using the Bonferroni method. The multivariate comparisons were made independently for each dataset (linear and angular measures dataset, outline datasets, and landmark coordinates dataset). All statistical analyses were conducted in R v. 4.2.0 (R Core Team 2022) and Past v. 2.14 [START_REF] Hammer | PAST: Paleontological Statistics software package for education and data analysis[END_REF], and the threshold used to decide for statistical significance was a p-value of 0.001.

Clustering Analysis based on morphological variability

A Bayesian clustering approach based on Gaussian finite mixture models was carried out using each of the three datasets of morphological variables using the "mclust" R package [START_REF] Scrucca | mclust 5: Clustering, Classification and Density Estimation Using Gaussian Finite Mixture Models[END_REF]). The default "mclust" setting was used to assess the 14 types of models and varied

Cl (number of clusters) between 1 and 9 (default option). Three independent analyses were performed using the three datasets; to compare the power of those three sets of variables to discriminate among groups. The clusters with only one individual were considered "ghost" clusters and hence not considered true clusters.

Genetic analyses

DNA extraction and PCR

DNA was extracted from dried leaf tissue using a modified cetyltrimethylammonium bromide Subsequently, 500 μl of chloroform-isoamylalcohol was added. The mix was centrifuged for 8 minutes at maximum speed (15 000 rpm) and 4ºC, and the supernatant was later transferred to a new 1.5 mL tube to which the precipitation buffer was added (7.5 M ammonium acetate) and isopropanol. The tubes were kept at room temperature (RT) for 5 minutes, and after centrifuging again at 15 000 rpm and 4ºC for 30 minutes, the supernatant was removed by decantation.

The DNA was washed with 1 ml of 70 % ethanol (inverting the tube 2-3 times to clean the inner surface), and after centrifuging for 5 min at maximum speed (15 000 rpm), the fluid was removed with a pipette. The tubes were allowed to dry at 37ºC for 7 min, and the DNA was subsequently resuspended in 100 μl of high-salt TBE buffer (10mM Tris pH 8.0, 2mM EDTA, 1M NaCl). The samples were incubated at 60ºC for 30 minutes and subsequently left at RT overnight. Finally, 5 μl of MagAttract Suspension G solution was added to the bottom of each tube.

A total of 120 μl of 100 % ethanol was added, mixed by pipetting, and incubated at RT for 5 Multiplex-PCRs were designed with microsatellite markers that showed clear peaks, amplification quality, and more than one allele in the Multiplex Manager program [START_REF] Holleley | Multiplex Manager 1.0: a cross-platform computer program that plans and optimizes multiplex PCR[END_REF].

This program considers the range of each marker, the fluorescent probe attached to it, the base sequence, and the possibility of hybridization between markers and shows possible multiplex-PCR options. The variants obtained in the Multiplex Manager were modified and refined until the desired marker combinations were achieved.

For the multiplex-PCRs execution, the forward primers were attached to a universal tail at the 5' position [START_REF] Vartia | Multiplexing with three-primer PCR for rapid and economical microsatellite validation[END_REF]. The labels of the tails were the following: T3: 5′ AATTAACCCTCACTAAAGGG 3′, Hill: 5′ TGACCGGCAGCAAAATTG 3′, Neomycin inverse: 5′ AGGTGAGATGACAGGAGATC 3′ and M13(-20): 5′ GTAAAACGACGGCCAGT 3′ [START_REF] Tozaki | Characterization of equine microsatellites and microsatellite-linked repetitive elements (eMLREs) by efficient cloning and genotyping methods[END_REF]. The forward oligonucleotide with the universal tail was used in combination with a primer with the same 5' sequence labeled with a fluorescent probe. FAM (blue), NED (yellow), PET (red), and VIC (green) fluorescent probes were ligated to the T3, Hill, Neo, and M13 tails, respectively.

The combination and parameters of the four multiplex reactions are given in Table 5.

The lengths of the DNA fragments were detected using an ABI 3130XL fragment analyser, quantified with a GeneScanTM 500 LIZTM size standard (Thermo Fisher Scientific), and analysed in Geneious v. 8.0.5 [START_REF] Kearse | Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF] with the microsatellite plugin. (-20): 5′ GTAAAACGACGGCCAGT 3', Hill: 5' TGACCGGCAGCAAAATTG 3′ [START_REF] Tozaki | Characterization of equine microsatellites and microsatellite-linked repetitive elements (eMLREs) by efficient cloning and genotyping methods[END_REF]) and Neomycin reverse: 5′ AGGTGAGATGACAGGAGATC 3'. The reverse primers had a PIG-tail [START_REF] Brownstein | Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping[END_REF]. Tm: Primer Melting Temperature (temperature at which one-half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability).

Population Genetic structure at subsection level

Genetic diversity values per group (Orbiculata, Minor, Oblongifolia, and Ophiticola) were calculated using GeneAlex v. 6.5 [START_REF] Peakall | GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research -an update[END_REF], Genepop v. 4.7.5 [START_REF] Rousset | Genepop'007: a complete re-implementation of the genepop software for Windows and Linux[END_REF]FSTAT v. 2.9.4 (Goudet 2001). Genetic differentiation between groups was estimated through pairwise comparisons of F ST [START_REF] Weir | Estimating F ST statistics for the Analysis of Population Structure[END_REF] and D JOST values [START_REF] Jost | G ST and its Relatives do not Measure Differentiation[END_REF]) using the fastDivPart function of the R package diveRsity [START_REF] Keenan | DiveRsity: An R Package for the Estimation and Exploration of Population Genetics Parameters and Their Associated Errors[END_REF]. The identification of genetic clusters and the assignment of individuals was performed in STRUCTURE v. 2.3.4 [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF], which uses a Bayesian clustering approach using MCMC (Markov Chain Monte Carlo) for posterior distribution sampling. STRUCTURE analyses were conducted by selecting a model that assumes admixture, correlated allele frequencies, and without prior population information.

First, 10 replicates were run for each number of genetic clusters considered (K), with K varying between 1 to 20 and a burn-in period of 50 000 iterations followed by a run-length of 150 000 iterations of the Markov Chain. The model (value of K) choice was made according to the method of [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: A simulation study[END_REF] as implemented in STRUCTURE HARVESTER v. 0.6.94 (http://taylor0. biology.ucla.edu/structureHarvester) [START_REF] Earl | Structure harvester: A website and program for visualizing Structure output and implementing the Evanno method[END_REF]. Then, 100 new repetitions of the MCMC method were run for the best K value. CLUMPP v. 1.1.2 [START_REF] Jakobsson | CLUMPP: a cluster matching and permutation program for dealing with label switching and multimodality in analysis of population structure[END_REF] was used to estimate similarities between runs and to average the membership probabilities.

Final bar plots displaying individual admixture coefficients were obtained thanks to STRUCTURE PLOT v. 2.0 [START_REF] Ramasamy | STRUCTURE PLOT: A program for drawing elegant STRUCTURE bar plots in user friendly interface[END_REF]). An individual was considered as a member of a genetic cluster when its probability of belonging to that group was higher than or equal to 0.9. A second STRUCTURE analysis was executed (using the same configuration) without considering the individuals of Oblongifolia (see the result section for rationale).

Because the MCMC method implemented in STRUCTURE is based on a population genetic model (assuming Hardy-Weinberg genotypic proportions and no linkage disequilibrium within each inferred genetic cluster), the results of genetic clusters and assignment of individuals may be affected by the potential low model fit to data. Thus, a non-model-based multivariate

Phylogenetic analysis

The complete chloroplast sequence with the annotations for Magnolia subsect. Talauma in Cuba and the outgroup was obtained from the research group of Marie-Stéphanie Samain PhD. (Instituto de Ecología, Red de Diversidad Biológica del Occidente Mexicano, Pátzcuaro, Mexico). The phylogenetic reconstruction was performed with a fully-sequenced plastome (two samples: Minor, Oblongifolia, Ophiticola; one sample: Orbiculata). Based on the result of Veltjen These plastomes were trimmed to include only one copy of the IR (inverted repeat region), as proposed by [START_REF] Dong | Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)[END_REF]. The aligned was made using MAFFT v. 7.272 [START_REF] Katoh | MAFFT: multiple sequence alignment software version 7: improvements in performance and usability[END_REF] in its default settings. The resulting alignment was visually inspected in Geneious v. 8.0.5 [START_REF] Kearse | Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF]. The length of the plastome sequence after the alignment was 133 450 pb. ML (Maximum Likelihood) analyses were run under the GTR + G substitution model using the software RAxML-HPC v. 8 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and postanalysis of large phylogenies[END_REF]. The analysis was run on XSEDE (www.sxede.org) in the CIPRES Science Gateway v. 3.1 [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF]. The options "searching the best ML tree" and "performing a rapid bootstrapping (1 000 replicates)" were selected. Branches were considered statistically supported for bootstrap values > 70 % [START_REF] Hillis | An Empirical Test of Bootstrapping as a Method for Assessing Confidence in Phylogenetic Analysis[END_REF]. The model GTRGAMMA was selected for bootstrapping. MrBayes v. 3.2.7a [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF] was used to estimate Bayesian trees, which were conducted on XSEDE in the CIPRES Science Gateway v. 3.1 [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF], under the following settings: starting from random trees, Markov Chain Monte Carlo (MCMC) simulations for 10 million generations, sampling every 1 000 generations. The first 10 % of the trees were discarded as burn-in. Output trees were visualized and exported in TreeGraph v. 2.15.0-887 beta [START_REF] Stöver | TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses[END_REF].

For the ancestral range reconstruction, the plastome sequence alignment was used to infer a calibrated phylogeny using BEAST 2.6.7 [START_REF] Bouckaert | BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis[END_REF]. Substitution model was estimated using the bModelTest 1.2.1 package [START_REF] Bouckaert | bModelTest: Bayesian phylogenetic site model aver-aging and model comparison[END_REF]. A relaxed clock Log Normal and a Yule prior were used. Due to the lack of fossil data for the species included in our analysis, we calibrated our tree using the dates presented by [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF] for Magnolia sect. Talauma. A uniform prior was set with a minimum bound of 24 and a maximum of 36 mya for the section. BEAST analysis was set to run for 100 million generations with 10 % as burn-in. We checked the convergence values using Tracer 1.7 [START_REF] Rambaut | Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7[END_REF] to ensure an effective sample size (ESS) > 200. Treeannotator 2.6.7 [START_REF] Bouckaert | BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis[END_REF]) was used to create a maximum clade credibility tree with node height representing the mean heights.

The ancestral range reconstruction was estimated using R 4.2.2 (R Core Team 2022) and the BioGeoBEARS package [START_REF] Matzke | Model Selection in Historical Biogeography Reveals that Founder-Event Speciation Is a Crucial Process in Island Clades[END_REF][START_REF] Matzke | Probabilistic historical biogeography: new models for founder-event speciation, imperfect detection, and fossils allow improved accuracy and model-testing[END_REF]. Six regions were defined: Mesoamerica, Puerto Rico, Hispaniola, and the three Cuban regions of Guamuhaya, Nipe-Sagua-Baracoa, and Sierra Maestra. The calibrated tree was used to test a total of six models that included three different base models (DEC, DIVALIKE, and BAYAREALIKE) along with their jump dispersal (+J) variants.

The models were compared using the AICc criterion (Burnham & Anderson 2002).

Integrative taxonomy

The values of probabilities of cluster assignation (mclust and STRUCTURE) based on morphological, ecological, and genetic data were related. For this analysis, a filter was made to ensure that the same individual had the three types of data (ecological, morphological, and genetic), resulting in 138 individuals. A Chi 2 Contingency Table was computed, in Past v. 2.14 [START_REF] Hammer | PAST: Paleontological Statistics software package for education and data analysis[END_REF], to assess the agreement between ecological, morphological, and genetic classification. A heatmap was made to analyse the variation in the cluster assignation inside each group; this analysis was only done for the morphological and genetic classification.

Three independent Mantel paired tests, with 10 000 random permutations, were made in order to prove the existence of a correlation between the matrix of ecological, genetic, and geographic distances. The test ecological vs. morphological variables was made based on 222 individuals, ecological vs. genetic variables: 461 individuals, and morphological vs. genetic: 138 individuals.

A partial Mantel test was made to correlate, at the same time, the three sets of data: ecological, morphological, and genetic. In this case, the analysis was made based on the 138 individuals, which are the only individuals with values for the three sets of data. For the Mantel tests, a statistical significance probability value of 0.05 was chosen, and in the partial Mantel test, a Bonferroni correction was made, and the significance probability was 0.02. The group´s limits based on ecological, morphological, genetic, and phylogenetic data were combined to analyse their similarities.

The genetic distances between individuals were estimated from the Codom-Genotypic distances and the linear genetic distances, calculated according to the method proposed by [START_REF] Smouse | Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure[END_REF] with interpolation of the missing data. When data from a locus have been lost in an individual, this procedure allows interpolating the average of the genetic distances (calculated across all pairwise comparisons of the individual distances from the non-missing data).

Taxonomic treatment

Protologues of all Cuban names of Magnolia subsect. Talauma as well as all relevant taxonomic literature were consulted [START_REF] Urban | Fundamenta Florae Indiae Occidentalis[END_REF][START_REF] Britton | Studies of West Indian Plants-XI[END_REF][START_REF] Urban | Sertum antillanum[END_REF][START_REF] Moldenke | Nomenclatural notes III[END_REF][START_REF] Howard | The Morphology and Systematics of the West Indian Magnoliaceae[END_REF][START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF][START_REF] Borhidi | New plants in Cuba I[END_REF][START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF]Imchanickaja 1993;[START_REF] Frodin | World Checklist and Bibliography of Magnoliaceae[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]. The present revision was based on the study of nomenclatural types and other specimens. High-resolution images of 134 specimens from the herbaria: B, BM, BR, G, GH, HAC, HAJB, JE, K, LE, NY, P, S, US, were included in the analysis.

Herbarium acronyms were cited according to Thiers (2023+), while the series of specimens were cited according to the recommendations of [START_REF] Regalado | Revisión histórica de los herbarios cubanos con énfasis en las series de especímenes[END_REF]. The digital images were consulted online on the websites of the respective herbaria or via http://plants.jstor.org. Lectotypes were designated in accordance with the relevant articles of the Shenzhen Code (International Code of Nomenclature; [START_REF] Turland | International Code of Nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress[END_REF]. For the types of names based on collections by Charles Wright, the considerations proposed by [START_REF] Howard | Toward a General Taxonomy of Mis Research: A Progress Report on Defining the Discipline[END_REF] were followed. Due to the uncertainty that "duplicates" of a Charles Wright numbers correspond to the same collection (since most do not correspond to the same location and date), such specimens were designated as possible duplicates of the type and indicated with the legend "isolectotypes?". The growth type of each group was registred during the field work. The size of the petioles was measured on the images of the herbarium materials with the TPSdig program of the TPS series [START_REF] Rohlf | The tps series of software[END_REF].

48

RESULTS

RESULTS

Ecological limits

ENMs and identity test

For the northeastern groups, the best model resulted from the combination of 1.5 regularization multipliers, feature class threshold, and set 3 of variables (Bio5, Bio7, Bio12, Bio15, and DEM). For Orbiculata group, the best model resulted from the combination of 3.5 regularization multipliers, feature class quadratic, and set 3 of variables. Annual precipitation (Bio12) and precipitation seasonality (Bio15) were the variables with the highest contribution to the models for all the groups, except for Orbiculata, in which maximum temperature (Bio5) and elevation (DEM) contributed the most to the model. Almost all ENMs performed for each group showed potentially suitable regions in large areas of the mountains of Nipe-Sagua-Baracoa and the Sierra Maestra (Fig. 3). Orbiculata was the group with the smallest potential area of suitability (Fig. 3D), which was restricted to the Sierra Maestra and a many places in northeastern Cuba. Based on the MTP (Minimum training presence) suitability area, Oblongifolia and Minor were the groups with the largest predicted area, and Orbiculata had the lowest value of predicted area (Table 6). Niche identity test analysis indicated high levels of niche overlap between the different groups (Table 7).

Orbiculata had the lowest values for both indexes. Based on the comparison with the replicates (Supplementary material 1), it is clear that all groups are more similar than expected by chance.

Groups

MTP Suitability area (km

Point based and cluster analysis

Principal Component Analysis showed that Orbiculata group has the smallest and the clearest distinct environmental niche with no overlap (p<0.001) with the other group (Fig. 4A). The groups from northeastern Cuba, which showed the highest space of occupied environmental conditions, live under similar environmental conditions. In the PCA, the DEM was the variable with the higher contribution to the analysis. Figure 4B shows the clear disjointed geographic distribution of Orbiculata compared with the groups from the northeastern, with a distance of 83 km between the closest localities. The other groups of Magnolia subsect. Talauma seem to live in sympatry.

Orbiculata was the only group distributed at higher elevations (795-1 200 m.a.s.l.), while

Ophiticola was the group with the highest variability in its elevation range (34-987 m.a.s.l.) (Fig. 5). Individuals of the groups Minor and Oblongifolia were found to be present at the same altitudes (p=0.033), and similar results were found between the groups Minor and Ophiticola (p=0.109). All the individuals of Ophiticola and Orbiculata were found on serpentine derivate and volcanic-limestone derivate soils, respectively. In the case of Minor, 43 % of individuals were found on limestone derivative, and the rest on serpentine. In Oblongifolia, only one individual was found on limestone derivative.

The clustering analysis based on ecological data showed Cl=9 as the number of groups inferred by the best models (Table 8; Supplementary material 2). The individuals of Oblongifolia were included in all cases with the individuals of Minor; in this last group, almost all individuals were organized in seven clusters. 57.26 % of the individuals of Ophiticola were included in the same groups of Minor and Oblongifolia, but 37.75 % of the individuals of this group were organized in an independent cluster, only shared with one individual of Minor. In the case of Orbiculata, 95 % of its individuals were grouped in a unique cluster (Cl 7 on Table 8), not shared with another group. An apparent correspondence between the assigned clusters and the geographic distribution was found due to the existence of many individuals of the same 51

RESULTS

Groups

Cl-1 Cl- group/locality who were assigned to the same clusters. However, more analyses are needed to prove this association. The probabilities of assignment of each individual were higher than 0.9 in all cases. Talauma in Cuba.

53

RESULTS

Foliar morphological limits Foliar morphological limits

Multivariate and Geometric morphometrics Multivariate and Geometric morphometrics

For most of the twelve varia For most of the twelve variables, significant differences between each pair of groups were found (Fig. 6). There were three exceptions: the leaf perimeter did not show significant differences between Minor and Ophiticola (p=0.211), and between Oblongifolia and Ophiticola (p=0.132).

Ophiticola showed higher values for the length and a lower one for the width. On the other hand, Orbiculata showed higher values for the width and lower for the length of the leaf. The relation width-length was close to 1 in Orbiculata; the minimum value for this relation was found in Ophiticola (Supplementary material 3).

Despite the clear morphological differentiation between groups, overlap in the multivariate distributions of leaf morphology variables was observed (Fig. 7). The internal angle of the base (-0.324) and the leaf perimeter (0.5627) displayed the highest weight in the first two principal components, respectively (Supplementary material 4). It is possible to observe a clear differentiation between Minor and Ophiticola (Fig. 7). The variability of the individuals considered as Oblongifolia is distributed between Minor and Ophiticola. The morphological variation of Orbiculata and Minor (Fig. 7) showed some levels of superposition. The individuals of Orbiculata showed the highest morphological variation. The NPMANOVA showed significant statistical differences (p<0.0001) between groups.

The comparison between groups, based on Sum EDMA and centroid size, showed significant differences for most comparisons (Fig. 8). The exceptions were: the Sum EDMA between Minor and Oblongifolia (p=0.316) and between Orbiculata and Ophiticola (p=0.406). Based on PCA for elliptic Fourier descriptors (Fig. 9A) and matrix of Landmark (Fig. 9B), the different groups had little overlap in the ordination space (Fig. 9). However, a clearer distinction among groups was obtained with landmark positions than with other quantitative variables. This was especially obvious with Orbiculata, which was strongly differentiated from other groups when using landmark 54

RESULTS

positions. In all cases, the NPMANOVA showed significant statistical differences (p<0.001) between the groups. The shape reconstruction based on the elliptic Fourier descriptors and the matrix of Landmark is shown in Figures 10 and11.

Clustering analysis

The clustering analysis based on morphological variability showed differences in the number of groups inferred by the best models according to the different datasets (Fig. 12; Supplementary material 5). The highest BIC scores were retrieved for Cl=4 for the linear and angular dataset, Cl=2

for the EDFs dataset (with other three ghost clusters), and Cl=6 for the matrix of Landmark (with another ghost clusters) (Fig. 12; Supplementary material 5). The probabilities of assignment of each individual were higher than 0.9 based on the EDFs. In the case of the linear and angular variables and the matrix of Landmark, only 5 and 22 individuals showed probabilities of an assignment less than 0.9, respectively (data not shown). The linear and angular variables allowed a clear discrimination between Orbiculata, Ophiticola, and Minor, the latter group being split into two clusters. One of these two clusters was shared only with the majority of Oblongifolia individuals.

The clustering analysis based on EFDs provided only two clusters (Fig. 12). The assignment of individuals was, therefore, different from that obtained with linear and angular variables. Indeed, all individuals of Oblongifolia, and most individuals of Ophiticola and Minor, were assigned to the same cluster (cluster 1), while most individuals of Orbiculata were assigned to a different cluster (cluster 4). Therefore, EFDs were efficient in discriminating between Orbiculata on the one hand and the three other groups on the other hand. Finally, the analysis carried out on the matrix of Landmark showed a similar pattern to that obtained with the linear and angular variables for Minor,

Ophiticola, and Orbiculata. The main difference between these two analyses (matrix of Landmark and linear and angular dataset) was that in the first one, Oblongifolia was split into two clusters, one of which is shared with Minor and the other one with Ophiticola (Fig. 12). Thus, despite a continuous variation of leaf morphology across groups, a clear delimitation of Orbiculata is shown by our analysis whatever the dataset used. In cases where individuals of the same group were assigned to different clusters, no obvious correspondence between the assigned clusters and the geographic origin of those individuals was found. Indeed, many individuals of the same group/locality were assigned to different clusters (data not shown). The groups showed similar genetic diversity values, with the lowest values in Orbiculata (Table 9). The genetic differentiation among groups was relatively high (global F ST =0.10, D JOST =0.23).

Orbiculata contributed mainly to this result since it was highly differentiated from the three other groups, while Minor and Oblongifolia were the less differentiated groups (Table 10). The number of alleles and the diversity values per locus are resumed in supplementary material 6-7. Minor showed the highest private allele values, followed by Ophiticola (Fig. 13). In both groups, the total number of individuals with private alleles represents 30.7 % and 26.7 % of the total number of known individuals, respectively. In Minor, 81 % of the markers showed private alleles. 10. Pairwise genetic differentiation and distances measures: fixation indices (F ST ) (below diagonal) and allelic differentiation index (D JOST ) (above diagonal) calculated for the groups of Magnolia subsect. Talauma in Cuba. In all case significant differences were found (p˂0.001). The Bayesian clustering analysis clearly provided three genetic clusters as the unambiguously best solution in the two analyses (with and without Oblongifolia) (Fig. 14). In the following, an individual was considered to be correctly assigned to a unique genetic cluster if the ancestry coefficient of this individual to this cluster was higher than or equal to 0.9. One of those clusters corresponded obviously to Orbiculata (red cluster in Figure 15A). The 88.8 % (32/36) of individuals from Orbiculata were assigned to this cluster, while only 4/36 Orbiculata individuals displayed genome admixture (see hereafter). The second genetic cluster (green cluster in Figure 15A) consisted mainly of the majority of Minor (171) and Oblongifolia ( 16) individuals but also included some individuals (14) of Ophiticola (Fig. 15A). The third genetic cluster (blue cluster on Figure 15A) was predominantly composed of Ophiticola with only one individual of Minor. We will therefore refer hereafter to the "orbiculata", "minor-oblongifolia", and "ophiticola" genetic clusters, keeping in mind that ancestry coefficients within each group to these genetic clusters still varied.

Groups

Indeed, despite a clear delimitation between the three genetic clusters, a significant proportion of individuals (130/461) displayed genetic admixture. Based on these individuals, the level of genetic admixture varied according to groups; the mean value of probability to belong to each cluster was 0.624 (±0.104) for Orbiculata, 0.678 (±0.172) for Minor (including Oblongifolia), and 0.477 (±0.262) for Ophiticola.

Orbiculata showed strongly homogeneous ancestry coefficient values, with only four individuals displaying genome admixture with the "minor-oblongifolia" cluster (Fig. 15A). Minor and

Oblongifolia displayed a high level of genome admixture with the "ophiticola" cluster. Thirtyfive individuals (16.1 %) of Minor showed genome admixture with the "ophiticola" cluster. In Oblongifolia, 51 % of the individuals exhibited an ancestry coefficient over 0.9 to belong to the "minor-oblongifolia" cluster; the rest showed high admixture levels. Moreover, it is noticeable that one individual of Oblongifolia displayed a very high ancestry to Orbiculata. It was noticeable that the level of admixture within the "minor-oblongifolia" cluster was not evenly distributed among sites. The localities of Cupeyal del Norte (CN), Monte Fresco (MF), Piedra la Vela (PV), and

Cayo Guam (CG) show the highest levels of "misclassification" in the "minor-oblongifolia" cluster.

For Ophiticola, 56.4 % (97/172) of individuals could be assigned to the third (blue in Figure 15A) genetic cluster, while 8.14 % (14/172) could be assigned to the "minor-oblongifolia" genetic cluster (referred to as "misclassified" individuals hereafter). Similar to Minor and Oblongifolia, many individuals of Ophiticola (61/172) also displayed signals of genetic admixture, mainly with the "minor-oblongifolia" cluster, but also, for a few of them, with the "orbiculata" cluster. The localities of Subida a la Melba (SM), Mina Iberia (MI), and Sur de las Delicias del Duaba (SDD)

showed the lowest levels of misclassification. Most individuals from La Melba (MEL), Pico Cristal (PC), and Monte Fresco (MF) showed an ancestry coefficient similar to the "minor-oblongifolia cluster". The clustering analysis without individuals of Oblongifolia also provided K=3 as the best solution (Fig. 14,15B), reinforcing the conclusion of the analysis carried out on the whole sample. Moreover, it was very striking that this analysis provided an ancestry pattern very similar (Fig. 15B) to the analysis including this taxon (Fig. 15A).

The PCA analysis on the whole SSR dataset showed that the 200 first principal components explained 99.3 % of the variation, which were therefore kept for the discriminant analyses.

Based on the number of groups that have been defined across the history of Cuban talaumas taxonomy, but also on the STRUCTURE results, two solutions for the number of genetic clusters were considered in the following: K=3 and K=4. When K=3, individual assignment displayed a pattern very similar to that found with the Bayesian clustering approach; many individuals "misclassified" in the DAPC analysis were also "misclassified" based on the STRUCTURE analysis. The DAPC result confirmed the correspondence of Orbiculata to a unique genetic cluster and also showed that Orbiculata is more genetically differentiated from the three other defined groups (Fig. 16). For K=3 only one individual of Ophiticola showed an assignment probability value less than 0.9 to that genetic cluster. K=4 (Fig. 16) seems to be a less meaningful solution. In this case three clusters were predominantly composed of Minor, Ophiticola, Orbiculata, respectively, confirming the main pattern found with K=3, with the difference that a higher proportion of Minor and Ophiticola, but also a majority of Oblongifolia,

were not assigned to unique clusters. When K=4, seven and three individuals of Minor and Oblongifolia, respectively, showed probabilities values under 0.9. As for STRUCTURE, the analysis without considering Oblongifolia with K=3 displayed very similar results to the analysis including this taxon (Fig. 16); in this case, only one individual of Ophiticola showed probabilities values under 0.9. The genetic characterization of Magnolia subsect. Talauma represents the first work made in the group in Cuba.

Testing hybrids

The NEWHYBRIDS analysis with and without prior information showed very similar results; the following results are based only on the data with prior information. 100 % of the individuals used to validate the analysis were correctly classified as pure Minor and pure Ophiticola. The pure individuals of Minor and Ophiticola were the most abundant genetic groups (Fig. 17; Table 11). The confusion matrix, based on a probability threshold of 0.9, showed a high number of individuals as non-classified; because this 0.7 was also used as a second threshold. Based on 0.7, no individuals were classified as F1 or backcrossed with Ophiticola. Among Oblongifolia individuals, 14 were classified as pure Minor and only as pure Ophiticola. Ophiticola was the group with the higher level of confusion (Table 11) since a high proportion of individuals were classified either as pure Minor, F2, or backcrosses with Minor. 23 % of individuals of Ophiticola were considered by the analysis as being F2 individuals, the second more frequent group inside Ophiticola, after pure Ophiticola with 51 %. Also, 87 (20.5 %) individuals were considered nonclassified. The hybridization analysis represents the first research on population genetics applied to natural hybridization in Cuban plants.

Integrative taxonomy

An obvious association between genetic vs. morphological classification (using the three sets of data), genetic vs. niche classification, and niche vs. morphological classification were found based on Chi 2 test (values probabilities less than 0.0001). In addition, the heatmap analysis showed a high concordance between morphological and genetic classification for Orbiculata and Minor; and to a lesser extent for Oblongifolia and Ophiticola (Fig. 20). In this last group, several individuals with different genetic characteristics whose leaf morphology was the same were found. In Minor (Fig. 20), only a few individuals showed different morphology having a similar genetic pattern.

A relation between the three datasets of variables (ecological, morphological, and genetic) was Talauma using the DIVALIKE+J model. Colors represent geographic regions. GM: Guamuhaya, NSB: Nipe-Sagua-Baracoa, SM:

Sierra Maestra.

75

RESULTS

found based on the bi-dataset and multiple comparisons (ecological vs. morphological vs. genetic).

The mantel correlation values were moderate in the multiple comparisons (r=0.402) and between morphological vs. genetic distances (r=0.41); and low between ecological vs. morphological (r=0.115) and ecological vs. genetic distances (r=0.118). In all cases, the probability values were less than 0.0001.

Figure 21 shows the group´s limits based on the results obtained in this thesis. Orbiculata was the only group with consistent limits based on morphology, ecology, genetics, and phylogenetics data. Concerning the group of northeastern Cuba, the different sets of data showed different limits.

Based on the ecological and EFDs dataset (morphology), everything could be considered as only one species. The linear and angular dataset (morphology) considered Minor and Oblongifolia as the same species, different from Ophiticola. These same limits were shown by the matrix of Landmark and the genetic data. In these last two datasets, the boundaries between both groups (Minor-Oblongifolia and Ophiticola) were more confused.

Even when the limits in the groups from northeastern Cuba were not totally clear, based on the result of the thesis, it is possible to define three groups of species of Magnolia subsect.

Talauma in Cuba: (1) Orbiculata, an endemic tree from Sierra Maestra range with leaves as long as wide; (2) Minor (including the individuals considered as Oblongifolia), an endemic tree from Nipe-Sagua-Baracoa range, with leaves elliptic-rounded (equal or twice larger than wide); and

(3) Ophiticola, a endemic small tree or decumbent shrub from Nipe-Sagua-Baracoa range, with leaves oblong-elliptic (two to four time larger than wider). -Talauma oblongifolia sensu [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] pro parte (excludes the type, LS 22588 HAC ex LS!.

Taxonomic treatment

Magnoliaceae

-Magnolia oblongifolia sensu [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] pro parte (excludes the type, LS 22588

HAC ex LS!)
Trees, up to 30 m tall and 76.8 cm in trunk diameter, leaves with petiole of 1.5 -5 cm long, leaf blade de 8 -17.7 × 4.8 -10.7 cm, elliptic, widely elliptic, width-length Small trees or decumbent shrubs, up to 14 m tall and 23 cm in trunk diameter, leaves with petiole of 0.8 -3.4 cm long, leaf blade de 7.9 -19.7 × 2.5 -6.4 cm, oblong-elliptic or oblanceolate, width-length relation between 0.22 to 0.46; apex rounded, retuse-emarginate or acute; base cuneate or narrowly cuneate; adaxial surface dark green, abaxial surface light green; glabrous and reticulate-veined in both sides, the midrib flat above, prominent beneath.

Talauma ophiticola was described based on the gathering Bisse & Kholer HFC 6519 (Fig. 22A), which is strongly similar to the holotype of Talauma minor var. oblongifolia (León, Alain, Clemente, Crisógono LS 22588) (Fig. 22B). The types of Talauma minor var. oblongifolia León,

and Talauma ophiticola Bisse have the same morphological variability (Fig. 22). Based on this, the correct name for the individuals considered as Ophiticola group, is Magnolia oblongifolia (León) Palmarola, which was the first used name.

DISCUSSION

Ecological Niche

Two precipitation variables, annual rainfall and precipitation seasonality, presented the highest contribution to the ENMs of Magnolia subsect. Talauma in Cuba, except for Orbiculata, which was more influenced by extreme temperature. Rainfall and temperature are among the factors that influence the most the distribution of plants because both climate parameters are the primary control of biomes and are closely correlated with terrestrial net productivity [START_REF] Hull | Plant Ecology[END_REF][START_REF] Zhao | Climatic and hydrologic controls on net primary production in a semiarid loess watershed[END_REF]). In the specific case of magnolias, some authors (Song et al. 2019;Song & Liu 2019;[START_REF] Rodríguez-Ramírez | Plastic responses of Magnolia schiedeana Schltdl., a relict-endangered Mexican cloud forest tree, to climatic events: Evidences from leaf venation and wood vessel anatomy[END_REF] have already report the importance of these bioclimatic variables in the distribution of this group of plants. The differences observed between Orbiculata and the remaining groups in the contributions of the variables used for the niche model reflect the differences that exist between the Sierra Maestra and the Nipe-Sagua-Baracoa range. The latter is the region with the highest annual rainfall values in Cuba, above 3 000 mm per year [START_REF] Borhidi | Phytogeography and Vegetation Ecology of Cuba[END_REF], and the Sierra Maestra is the one with the highest elevation in Cuba, 1 974 m.a.s.l.

According to [START_REF] Borhidi | Phytogeography and Vegetation Ecology of Cuba[END_REF], in the highest mountains, the vertical gradients of temperature are more abrupt when the mountains are closest to the south coast. This author reports in the Sierra Maestra a change of 0.99˚C every 100 m, and for Nipe-Sagua-Baracoa 0.66˚C. Song & Liu (2019) report a high correlation between elevation and maximum temperature of the warmest month, the most influencing variables for Orbiculata. This correlation was not detected in our dataset, but a possible association between both variables could explain the strong influence on the model of Orbiculata.

The significant contribution of annual precipitation, precipitation seasonality, and maximum temperature of the warmest month to the distribution of Magnolia subsect. Talauma in Cuba is indicative of the sensitivity of this taxon to Climate Change. Global warming is likely to reach between 1-5ºC in 2100, depending on the model, and the precipitation could likely decrease (Stocker et al. 2013). Climate change could exceed the adaptive capacity of these trees, causing geographic displacement and local extinctions. The individuals of Orbiculata, with a constrained distribution to the higher elevations in Sierra Maestra, will have a higher risk of extinction.

However, other groups of the subsection may also be affected by climate change, as proposed by [START_REF] Fuentes-Marrero | Efecto potencial del cambio climático sobre la distribución de plantas asociadas a bosques húmedos del oriente de Cuba[END_REF]. These authors report that by 2050, under different climatic scenarios, the suitable areas of distribution of Magnolia minor (sensu [START_REF] Bisse | Árboles de Cuba[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] will be considerably reduced.

The small areas found to be suitable for Orbiculata could be explained by the low numbers of known individuals and their limited geographic distribution (see Figure 3). In the case of the magnolias from northeastern Cuba, their ecological niche has large extensions, which is in concordance with their higher values of extent of occurrence and area of occupancy. According to the results of this thesis, all the groups exclusive to this area (northeastern Cuba) showed large areas of environmental suitability in the region of the Sierra Maestra. The absence of individuals of Minor, Oblongifolia, and Ophiticola in this last area could be due to large unsuitable areas between both mountain systems. Another possible explanation may be that the common ancestor of Magnolia subsect. Talauma in Cuba lived in the Sierra Maestra, and the extant taxa that diverged from it maintain similar niche characteristics. According to [START_REF] Wiens | Niche conservatism: integrating evolution, ecology, and conservation biology[END_REF], the species tends to retain ancestral ecological aspects of their fundamental niche over time.

Another result was the absence of Magnolia subsect. Talauma in the region of Gran Piedra, in the Sierra Maestra. This locality showed climatic conditions for the presence of individuals of the subsection. Also in Gran Piedra lives Magnolia cubensis subsp. cubensis, a Cuban endemic magnolia from subsection Cubenses [START_REF] Hernández | Evaluación de patrones genético-espaciales en poblaciones de Magnolia subsect[END_REF].

The individuals of Magnolia subsect. Talauma from northeastern Cuba can live on serpentine and limestone soils, Ophiticola´s individuals being exclusive of serpentine. According to [START_REF] Borhidi | Phytogeography and Vegetation Ecology of Cuba[END_REF], the serpentine areas in Cuba have been proven to be extreme and successful places for plant speciation and diversification. In this sense, the extreme conditions in this area could have influenced the diversification of this botanical group. It is true that many individuals of Minor occur on serpentine too, but in this case, all of them were found closer to rivers and streams (pers.

obs.). More analyses about the influence of the type of soil over the morphology and the genetic differentiation due to the effect of local adaptation (soil type) are needed.

Morphological variability

The observed leaf morphological variability for Cuban magnolias was different from that described by previous studies. The values of leaf length and width were higher than those reported by [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF] for Magnolia minor and M. orbiculata. Likewise, these values were higher than what was previously reported by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF], except for M. oblongifolia. These values were similar to those of [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] for M. orbiculata and M. oblongifolia. For the individuals considered in this research as Minor and Ophiticola, the average values of B CI are slightly lower and slightly higher, respectively, than those reported by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] The use of Geometric Morphometrics methodology is not frequent in magnolias, even when [START_REF] Jensen | The conundrum of morphometrics[END_REF] recommends their uses in the family in which all the species have simple leaves.

Only the works from Hernández (2014), [START_REF] Tamaki | Morphological and genetic divergence between two lineages of Magnolia salicifolia (Magnoliaceae) in Japan[END_REF][START_REF] Tamaki | Population genetic structure and demography of Magnolia kobus: variety borealisis not supported genetically[END_REF] use this approach in order to clarify the delimitation among taxa. The landmark methods allow a better visualization and characterization of the differences in the leaves in Magnolia subsect. Talauma in Cuba, compared with the method based on EFDs (see Figure 9). Also, the landmark methods are better for discriminating the individuals based on blind analysis, just like clustering methods (see Figure 12). [START_REF] Hernández | Identificación de las subespecies de Magnolia cubensis (Magnoliaceae) mediante análisis digital de imágenes de las hojas[END_REF] recommends the use of both methods in the delimitation of Magnolia cubensis, a Cuban sister species from Magnolia subsect. Cubenses. The high level of morphological differentiation between taxa in this study reinforces the value of leaf characteristics in taxonomic studies of Cuban magnolias [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF][START_REF]Flora de Cuba[END_REF][START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF][START_REF] Imkhanitzkaja | Genus Magnolia L. (Magnoliaceae) in flora Cubae[END_REF][START_REF] Imkhanitzkaja | Genus Talauma A.L. Juss. (Magnoliaceae) in flora Cubae[END_REF][START_REF] Hernández | Identificación de las subespecies de Magnolia cubensis (Magnoliaceae) mediante análisis digital de imágenes de las hojas[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]. Leaf morphology is a key to species delimitation [START_REF] Jensen | Lines, outlines, and landmarks: Morphometric analyses of leaves of Acer rubrum, Acer saccharinum (Aceraceae) and their hybrid[END_REF][START_REF] Jensen | The conundrum of morphometrics[END_REF], and this study confirms that they are highly relevant in groups like Magnolia.

It is widely recognized that the variation in the leaf shape is used in taxonomy since it varies between species, subspecies, and populations in plants; it is also a highly sensitive trait to the influence of biotic and abiotic factors. Numerous investigations have shown that the shape of angiosperm leaves is strongly affected by the availability of water [START_REF] Picotte | Leaf morphological responses to variation in water availability for plants in the Piriqueta caroliniana complex[END_REF][START_REF] Reichgelt | Geographic variation of leaf form among indigenous woody angiosperms in New Zealand[END_REF], temperature [START_REF] Royer | Phenotypic Plasticity of Leaf Shape along a Temperature Gradient in Acer rubrum[END_REF][START_REF] Walter | Environmental Effects on Spatial and Temporal Patterns of Leaf and Root Growth[END_REF][START_REF] Reichgelt | Geographic variation of leaf form among indigenous woody angiosperms in New Zealand[END_REF] or lighting [START_REF] Xu | Leaf morphology correlates with water and light availability: What consequences for simple and compound leaves?[END_REF][START_REF] Fan | Effects of light intensity on the growth and leaf development of young tomato plants grown under a combination of red and blue light[END_REF]. According to [START_REF] Reichgelt | Geographic variation of leaf form among indigenous woody angiosperms in New Zealand[END_REF], environmental changes can frequently produce alterations in leaf morphology, even for the same species growing in different habitats. The analysis of the influence of the environment on the leaf morphology should be carried on in order to understand its effect on the variation among populations.

Not including flowers and fruits in the morphological analysis of Magnolia subsect. Talauma in Cuba was given by the fact that finding both structures is very rare. However, field observations and literature revision [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF][START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]) confirm the differences in the fruit shape and size between the groups of the subsection. Orbiculata and Minor display spherical fruits but with differences in size, the fruit of Orbiculata being larger.

In the case of Ophiticola, fruits have a rhombic shape, with a size more similar to those of Orbiculata.

Genetic Structure

Some of the genetic markers used in the present work are used by [START_REF] Hernández | Evaluación de patrones genético-espaciales en poblaciones de Magnolia subsect[END_REF] in the genetic characterization of Magnolia cubensis subsp. acunae (five markers in common), Magnolia cubensis subsp. cubensis and Magnolia cristalensis (both with six markers in common); and by [START_REF] Veltjen | The Caribbean Magnolia species (Magnoliaceae): Assessment of the genetic diversity and the underlying evolutionary history[END_REF] in Magnolia dodecapetala (Lam.) Govaerts (eleven markers in common). The first three taxa are from Cuba and belong to Magnolia subsect. Cubenses, while the other taxon belongs to the subsection Talauma [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF]. According to [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF] [START_REF] Hernández | Evaluación de patrones genético-espaciales en poblaciones de Magnolia subsect[END_REF] between the populations of Magnolia cubensis subsp. cubensis and Magnolia cristalensis, and by [START_REF] Veltjen | The Caribbean Magnolia species (Magnoliaceae): Assessment of the genetic diversity and the underlying evolutionary history[END_REF] in Magnolia dodecapetala between the different islands of the Less Antilles, are higher or equal to the values found between the groups of Magnolia subsect. Talauma in Cuba in the present work.

The STRUCTURE results (see Figure 15), with and without Oblongifolia individuals, showed the same genetic structure and admixture pattern. These results showed the excellent stability of inferences made on individual assignment and genetic admixture between clusters, probably because of the strong genetic differentiation between the genetic clusters. Also, this is an indicator that Oblongifolia does not represent an independent genetic group, as proposed by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF]. The algorithm implemented in STRUCTURE is very useful for the delimitation The STRUCTURE results showed that some individuals of each group displayed genetic admixture with the other groups, being more evident in the groups of northeastern Cuba. According to [START_REF] Hey | Population genetics and objectivity in species diagnosis[END_REF], the levels of genetic differentiation among species are influenced by the time of separation and the amount of gene exchange. According to [START_REF] Zhou | Importance of incomplete lineage sorting and introgression in the origin of shared genetic variation between two closely related pines with overlapping distributions[END_REF], genetic variation shared between closely related species may be due to the retention of ancestral polymorphisms because of incomplete lineage sorting (ILS) and/or introgression following secondary contact.

Distinguishing between those two causes from observed patterns is challenging, although coalescence modeling can help (e.g. [START_REF] Zhou | Importance of incomplete lineage sorting and introgression in the origin of shared genetic variation between two closely related pines with overlapping distributions[END_REF][START_REF] Meleshko | Extensive genome-wide phylogenetic discordance is due to incomplete lineage sorting and not ongoing introgression in a rapidly radiated bryophyte genus[END_REF].

It seems more likely that the admixture signal between Orbiculata and the other groups could be explained by the common origin and recent diversification of the subsection in Cuba, which is less than five mya according to [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF], and the recent separation of Orbiculata from the other groups. However, the possibility of rare events of inter-taxa hybridization involving

Orbiculata´s individuals as one parent cannot be totally ruled out, especially because individuals that have displayed admixed genomes involving this group have intermediate ancestry coefficients, which is compatible with a hypothetical first-or early-generation hybrid status. The ecological differentiation between Orbiculata and the other groups of the subsection may suggest that selection against first-or early-generation hybrids due to local adaptation could also contribute to preventing genetic exchanges with the other groups. In the groups from northeastern Cuba, the level of genetic admixture could be explained by a recent diversification that led to numerous genetic loci with incomplete lineage sorting and overlaps in the distribution of morphological traits.

In trees, factors such as long generation time, and large effective population sizes, increase the opportunity of sharing ancestral polymorphisms through incomplete lineage sorting, which makes species identification based on neutral markers even more problematic [START_REF] Zhou | Importance of incomplete lineage sorting and introgression in the origin of shared genetic variation between two closely related pines with overlapping distributions[END_REF].

The individual assignment in the solution with three genetic clusters in the DAPC analysis (see Figure 16) was strongly similar to the previously obtained using STRUCTURE. This result strongly supported these three genetic groups corresponding to: Orbiculata, Minor (includes Oblongifolia), and Ophiticola. However, a second solution with four genetic clusters was also likely. DAPC analysis is not based on a population genetic model, which is the case of STRUCTURE [START_REF] Jombart | Discriminant Analysis of Principal Components: A New Method for the Analysis of Genetically Structured Populations[END_REF]; this makes that the results of genetic clusters and assignment of individuals obtained using this analysis (DAPC) are not affected by the potential low model fit to data. The fact that two different analyses get the same results validates the inference made in the number of genetic groups and individuals assignment of Magnolia subsect. Talauma in Cuba.

In the future, a complete analysis of the population genetic structure of each species would be needed in order to obtain a better insight into the link between spatial distance, ecological differentiation, and genetic diversity. Such a landscape genetic approach would be necessary to deeply understand environmental parameters that influence the genetic connectivity between populations and gene flow between species in Magnolia subsect. Talauma in Cuba. Such knowledge is necessary to define conservation units and propose management measures accordingly. However, this analysis goes beyond the objectives of the present work.

Testing hybrids

The foliar phenotype observed in the individuals of Oblongifolia appears to be intermediate between Minor and Ophiticola. A feasible explanation is a recent diversification process of these two groups, opening the possibility that reproductive isolation would not be completed. Natural hybridization might explain the intermediate characteristics of Oblongifolia, as has been observed

for Quercus species [START_REF] Burgarella | Detection of hybrids in nature: application to oaks (Quercus suber and Q. ilex)[END_REF]An et al. 2017) and for the genus Rhizophora [START_REF] Francisco | Population genetic structure, introgression, and hybridization in the genus Rhizophora along the Brazilian coast[END_REF]. According to [START_REF] Lihová | Hybridization between Two Polyploid Cardamine (Brassicaceae) Species in North-western Spain: Discordance Between Morphological and Genetic Variation Patterns[END_REF]), An et al. (2017), and Teixeira et al.

(2019), morphological data have been widely used in the detection of hybrid individuals. In Magnolia subsect. Talauma, genetic data support rather the fact the two groups (Minor and Ophiticola) are totally genetically compatible and showed that they could be considered as a single genetic group. However, not all morphological variation has a genetic basis. Also, the assumption that hybrid individuals are phenotypically intermediate to parents is not necessarily true because of dominance effects or because of recombination from first-generation hybrids, leading to interspecific hybrids displaying a mosaic of parental phenotypes [START_REF] Allendorf | The problems with hybrids: setting conservation guidelines[END_REF]). In the specific case of Oblongifolia individuals, even when their foliar phenotype looks intermediate (between Minor and Ophiticola), the genetic data showed unambiguously that this group is closer to Minor.

Yet, backcrosses between first-generation hybrids and parental species and crosses between hybrids may occur. Morphological characters do not allow us to determine whether an individual is a first-generation hybrid (F1), a backcross, or a later generation hybrid, which reduces our capacity to assess the extent of interspecific gene flow [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF][START_REF] Soltis | The Role of Hybridization in Plant Speciation[END_REF]. The previous elements raise the importance of using molecular markers as an indicator for hybrid origins and subsequently comparing them with morphology. According to [START_REF] Lihová | Hybridization between Two Polyploid Cardamine (Brassicaceae) Species in North-western Spain: Discordance Between Morphological and Genetic Variation Patterns[END_REF], in plants, hybridization processes can cause discordance between morphological and molecular data.

The NEWHYBRIDS analysis (see Figure 17 and Table 11) detected the existence of pure individuals, backcrosses to Minor, and F2 in the groups from northeastern Cuba. Some individuals of Ophiticola were identified as pure Minor, which supports the existence of genetic introgression between those species. Detection of backcross hybrids and evidence of historic introgression suggest hybrid zones between Minor and Ophiticola displayed a complex genetic structure, which is not limited to the first generation of hybrids. The occurrence of F2 hybrids indicates that F1 hybrids can mate with each other and are fertile.

In any case, F1 and backcross to Ophiticola were found, even when a 0. The absence of F1 individuals in hybrid populations could be due, in some degree, to fairly strong pre-zygotic reproductive isolation [START_REF] Arnold | Asymmetric Introgressive Hybridization Among Louisiana Iris Species[END_REF], minimizing initial F1 formation events. Once an F1 is formed, all of its descendants will be hybrid derivatives, and these can hence rapidly multiply in number, producing later-generation hybrids, from which the transfer of novel alleles between hybridizing lineages via repeated backcrossing (introgression) becomes possible [START_REF] Yan | Asymmetrical natural hybridization varies among hybrid swarms between two diploid Rhododendron species[END_REF]. Further studies are required to understand the causes of this observed asymmetric introgression, and it will be helpful for conservation to determine the contribution of each hybrid class on the population diversity.

Without analysing young individuals in these groups, it is impossible to confirm that the hybridization process still occurs. However, the high admixture level observed using SSR markers, as well as the few cases of reciprocal "miss-assignment", suggest that gene flow between the groups of northeastern Cuba has occurred recently and must still be occurring. The latter hypothesis is reinforced by the observation that reciprocal genetic admixture between the two genetic clusters mainly corresponds to Minor and to Ophiticola, respectively, as it is more frequent in the localities where both groups occur (see Figures 15,16,17). This hypothesis is well-supported because these groups live in the same habitats and in similar ecological conditions (see Figure 4), a situation that is not favorable for the emergence of reproductive barriers. The phylogenetic closeness between those three groups (Minor, Oblongifolia, and Ophiticola) and the high overlap detected in the ecological conditions are parameters that may favor hybridization between them. According to [START_REF] Callaway | Magnolias[END_REF], hybridization is a common process in magnolias and is obviously more common when the distribution ranges of two or more highly related taxa overlap [START_REF] Soltis | The Role of Hybridization in Plant Speciation[END_REF]).

According to [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF] and Becker According to [START_REF] Allendorf | Conservation and the Genetics of Populations[END_REF] and [START_REF] Becker | Hybridization may facilitate in situ survival of endemic species through periods of climate change[END_REF], hybridization may have a wide . ( 2013), hybridization may have a wide variety of effects on fitness, whether positiv variety of effects on fitness, whether positive (heterosis or adaptive introgression) e (heterosis or adaptive introgression) or negative or negative (outbreeding depression). Also, hybridization could lead the introgressed species to become (outbreeding depression). Also, hybridization could lead the introgressed species to become extinct due to the dilution of the genetic pool of one species involved. Hybridization is an extinct due to the dilution of the genetic pool of one species involved. Hybridization is an important evolutionary phenomenon, and therefore a detailed understanding of the processes important evolutionary phenomenon, and therefore a detailed understanding of the processes involved in hybridization is of widespread interest. Many plant species have hybrid origins, and involved in hybridization is of widespread interest. Many plant species have hybrid origins, and hybridization is a key mechanism for the production of new species and novel adaptations. The hybridization is a key mechanism for the production of new species and novel adaptations. The loss of a species by hybridization is not necessarily a conservation problem as long as its origin loss of a species by hybridization is not necessarily a conservation problem as long as its origin is natural and never provoked by human intervention. In the present case, it is hard to say if is natural and never provoked by human intervention. In the present case, it is hard to say if hybridization is natural or human induced because the mountains of Nipe-Sagua-Baracoa have hybridization is natural or human induced because the mountains of Nipe-Sagua-Baracoa have been under the influence of human activity for many years [START_REF] Carabia | The Vegetation of Sierra de Nipe, Cuba[END_REF]; González-Torres been under the influence of human activity for many years [START_REF] Carabia | The Vegetation of Sierra de Nipe, Cuba[END_REF][START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]. This problem remains in our days but to a lesser extent. Knowing if hybridization has . 2016). This problem remains in our days but to a lesser extent. Knowing if hybridization has been promoted by human impact is very complicated. Researches focusing on incompatibility, been promoted by human impact is very complicated. Researches focusing on incompatibility, phenology, pollination, dispersion, seed production and viability, local adaptability, and response phenology, pollination, dispersion, seed production and viability, local adaptability, and response to the environment and climatic change are necessary in order to understand the possible effect to the environment and climatic change are necessary in order to understand the possible effect (negative or positive) of hybridization on the fitness of the Cuban species of (negative or positive) of hybridization on the fitness of the Cuban species of Magnolia Magnolia subsect. subsect.

Talauma Talauma. .

Plastome phylogeny

The previous molecular phylogeny of the group in Cuba [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF]) is based on eleven genome regions (five partial nuclear genes, three partial chloroplast genes, and three chloroplast intergenic spacers). Unfortunately, the work of [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF] does not solve, just like in the present case, the taxonomic delimitation of the species from northeastern Cuba.

According to [START_REF] Wang | Major clades and a revised classification of Magnolia and Magnoliaceae based on whole plastid genome sequences via genome skimming[END_REF] and [START_REF] Dong | Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)[END_REF], using a few plastids or nuclear loci in the reconstruction of the phylogenetic relations inside Magnoliaceae family has recovered some well-defined clades which are in concordance with relevant morphological circumscriptions.

However, when using only a few loci, some deep nodes are still unresolved, possibly due to the slow nucleotide substitution rates, incomplete lineage sorting, hybridization, or a combination of these factors [START_REF] Dong | Plastid and nuclear phylogenomic incongruences and biogeographic implications of Magnolia s.l. (Magnoliaceae)[END_REF]. Our results showed that the Cuban species of Magnolia sect. Talauma did not constitute a monophyletic group, similar to [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF] and [START_REF] Guzmán-Díaz | Comparison of Magnoliaceae Plastomes: Adding Neotropical Magnolia to the Discussion[END_REF].

The plastome phylogeny in Magnolia subsect. Cubenses showed the proximity of the Cuban taxa with those from Hispaniola and Puerto Rico. The present research is in concordance with [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF], in the state that the migration of Magnolia species across the Caribbean could have occurred from South America to Puerto Rico, from there to Hispaniola, and finally to Cuba (around 5 mya). The time of arrival in Cuba corresponds to the Pliocene and Pleistocene, according to [START_REF] Iturralde-Vinent | Meso-Cenozoic Caribbean paleogeography: Implications for the historical biogeography of the region[END_REF].

The plastome phylogeny validates the statement of [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF] that Magnolia subsect.

Talauma from Cuba represents a monophyletic group, more related to the Mexican species from the same subsection than to the other magnolias of Cuba (see Figures 18,19). The phylogeny showed the proximity between Magnolia subsect. Talauma from Cuba and Central America.

According to our result, and the one found by [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF], the ancestor of Magnolia subsect. Talauma in Cuba came from Mesoamerica, with a colonization time of Cuba less than 10 mya ago. According to [START_REF] Iturralde-Vinent | Meso-Cenozoic Caribbean paleogeography: Implications for the historical biogeography of the region[END_REF], during this period of time, the mountains of Sierra Maestra and Nipe-Sagua-Baracoa had emerged for at least 16 mya. During the Upper Miocene (9-7 mya) and Miocene-Pliocene (7-3.7 mya), there were shallow marine areas (Nicaragua Rise, Pedro Bank, and Western Jamaica) between Central America and the Sierra Maestra that could have served as migration pathway for the ancestor, probably mediated by birds.

Taking into account the results, Sierra Maestra could be the first population founded by the ancestor Taking into account the results, Sierra Maestra could be the first population founded by the ancestor of the subsection (assuming a unique colonization event); and, from them, another migration of the subsection (assuming a unique colonization event); and, from them, another migration event to northeastern Cuba. This hypothesis is supported by the statement that Orbiculata is event to northeastern Cuba. This hypothesis is supported by the statement that Orbiculata is genetically the most different group of the subsection, probably reflecting more time of isolation genetically the most different group of the subsection, probably reflecting more time of isolation and divergence. Concerning the group from northeastern Cuba, probably the plastome genome and divergence. Concerning the group from northeastern Cuba, probably the plastome genome divergence is still low because of the recent differentiation of this group. According to Song divergence is still low because of the recent differentiation of this group. According to [START_REF] Song | Development of Chloroplast Genomic Resources for Oryza Species Discrimination[END_REF], the chloroplast genome is highly conserved, which makes it, in some cases, very hard to (2017), the chloroplast genome is highly conserved, which makes it, in some cases, very hard to find differences, especially in poorly evolved groups. find differences, especially in poorly evolved groups.

The retention of ancient features characterizes the

The retention of ancient features characterizes the Magnoliaceae Magnoliaceae family [START_REF] Treseder | Magnolias[END_REF]; family [START_REF] Treseder | Magnolias[END_REF][START_REF] Callaway | Magnolias[END_REF]Rivers Callaway 1994;[START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF]. Therefore, the ancestor may have displayed the current red-. 2016). Therefore, the ancestor may have displayed the current redorange fleshy aril characteristic of orange fleshy aril characteristic of Magnoliaceae Magnoliaceae seeds [START_REF] Callaway | Magnolias[END_REF]Judd seeds (Callaway 1994;[START_REF] Judd | Plant systematics: a phylogenetic approach[END_REF], which . 2016), which is easily visible and ingested by birds. As a result, the seeds can be widely dispersed through is easily visible and ingested by birds. As a result, the seeds can be widely dispersed through long-distance dispersal, and as a consequence these characteristics may have facilitated the long-distance dispersal, and as a consequence these characteristics may have facilitated the colonization of new areas by colonization of new areas by Magnolia Magnolia species. species.

An integrative classification of Magnolia subsect. Talauma in Cuba

In the present study, Orbiculata was clearly distinguished from the other groups of Magnolia subsect. Talauma in Cuba based on ecological, morphological, genetic markers, and phylogenetic data. The previously observed large variation of leaf morphology across Magnolia subsect. Talauma in Cuba, although based on the observation of only a few specimens, has been the basis for several authors to consider a unique species in this subsection, therefore including Orbiculata within Minor [START_REF] Howard | The Morphology and Systematics of the West Indian Magnoliaceae[END_REF][START_REF]Flora de Cuba[END_REF][START_REF] Borhidi | New plants in Cuba I[END_REF][START_REF] Lozano-Contreras | Dugandiodendron y Talauma (Magnoliaceae) en el Neotrópico[END_REF]. In contrast, the present study and the line of evidence already brought by the molecular phylogeny of Magnolia subsect. Talauma [START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF]) strongly support that Orbiculata may be considered a well-delineated species (Magnolia orbiculata (Britton & P.

Wilson) Palmarola, from now). However, in our study, a few cases of confusion with Minor on the basis of leaf morphology traits were observed. This confusion could be explained by the similar rounded shape and relation width-length present in both taxa. Different specialists have already erroneously identified some herbarium specimens of M. orbiculata as Minor in the past (personal observation on herbarium records).

Regular gene flow seems unlikely between M. orbiculata individuals with respect to other groups.

The clear morphological and genetic differentiation of M. orbiculata (see Figures 12,15,16) from other groups of Magnolia subsect. Talauma in Cuba strongly suggested that, at least, the lowland between the Sierra Maestra (habitat of M. orbiculata) and Nipe-Sagua-Baracoa (habitat of the other groups) may have acted and still acts as a barrier to gene flow by limiting pollination and seed dispersal. This is strongly supported by the differentiation in the ecological niche of M. orbiculata (see Figure 4), respecting the other groups of the subsection in Cuba. However, the genetic data of our study do not allow us to reject the possibility of rare events of gene flow. It is possible that post-zygotic reproductive barriers also contribute to the genetic isolation between the two groups (M. orbiculata and northeastern populations) by counter-selection of hybrids. [START_REF] Hernández | Evaluación de patrones genético-espaciales en poblaciones de Magnolia subsect[END_REF] reports high levels of genetic differentiation between Magnolia cubensis subsp. cubensis (from the Sierra Maestra) and Magnolia cristalensis (from Nipe-Sagua-Baracoa), both from Magnolia subsect. Cubenses. [START_REF] Vázquez-García | Magnolias de Ecuador: en riesgo de extinción[END_REF] state that allopatric speciation seems to be a major driver of Magnolia diversification in the Neotropics.

Undoubtedly, our data confirmed the main taxonomic issues concerning the northeastern Cuban populations of Magnolia subsect. Talauma. [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF]) considered all the variations of this group in a single species: Magnolia minor, with two varieties (Magnolia minor var. minor and Magnolia minor var. oblongifolia). [START_REF] León | Novedades de la Flora Cubana (II)[END_REF] point out that individuals with more oblong leaves, considered by them as Talauma minor var. oblongifolia, may belong to a different species. However, these authors do not assign the species rank to this group because of the absence of reproductive structures in the available specimens. The absence of flowers and fruits is still a major concern in this group because it is tough to find these structures in the field. The existence of a single species (Magnolia minor), including the three groups considered in the present thesis (Minor, Oblongifolia, and Ophiticola) was supported neither by our morphological results (based on linear and angular variables and the matrix of Landmark) nor by genetic markers, which both showed differentiation between the individuals of Minor and Ophiticola.

On the other hand, [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] proposes to divide Magnolia minor (sensu [START_REF] León | Novedades de la Flora Cubana (II)[END_REF][START_REF] León | Flora de Cuba II[END_REF] The results of the thesis do not support either the combination of Talauma ophiticola and Magnolia oblongifolia (sensu [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] minor (Minor group) than to T. ophiticola (Ophiticola group). Nevertheless, the delimitation of the individuals of Ophiticola group is still challenging. In the present study, a significant proportion of individuals that were assigned to that group based on leaf morphology were unambiguously assigned to the "minor-oblongifolia" genetic cluster, while only one individual from Minor group was assigned to the "ophiticola" genetic cluster. This could be explained by a recent and/or continued asymmetrical gene flow among both species. This statement could be possible taking into consideration the sympatric distribution and their ecological similarities (not favorable for the emergence of reproductive barriers).

After analysing the results and taking into account, the differences found for the evaluated taxa, the hypothesis of this thesis ("For the Cuban members of Magnolia subsect. Talauma, the number and limits of species defined by the genetic, ecological, and phylogenetic criteria are the same as that defined by the morphological species criteria") is partially refuted. According to the results, not all taxa of Magnolia subsect. Talauma in Cuba are consistent in their morphological, ecological, genetic, and phylogenetic limits. Only for Magnolia orbiculata (Orbiculata group in the thesis), the different sources of evidence support this group as independent lineages and, consequently, a valid species. In the case of northeastern groups, each approach used showed different species limits.

Based on the absence of solid evidence of species delimitation, two possible taxonomic solutions could be considered 1) the population of northeastern Cuba could be considered as one single and highly variable species: M. minor (Urb.) Govaerts, the oldest name, which includes all the sister species, such as morphological differentiation, ecological adaptation to new niches and reproductive isolation among other criteria (de Queiroz 2007; [START_REF] Prata | Towards integrative taxonomy in Neotropical botany: Disentangling the Pagamea guianensis species complex (Rubiaceae)[END_REF].

It is possible that Magnolia minor (includes Minor and Oblongifolia groups) and Magnolia oblongifolia (includes Ophiticola group) are still in a speciation process. According to de Queiroz (2007), both taxa could be in a grey zone of speciation, in which they have not yet acquired all the necessary properties to be two completely differentiated species. This could explain why the different species concepts evaluated here come into conflict. Even when there is not a complete differentiation, the found differences between both taxa concerning morphology and genetics seem to support two different evolutionary lineages. Based on the Unified SC and the Integrative Approach, both taxa could be considered as two independent species.

Taxonomic treatment

The taxonomic circumscription for Magnolia orbiculata (Britton & P. Wilson) Palmarola remains similar to the one proposed by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF], 1988), Imchanickaja (1993[START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]. However, [START_REF] Britton | Studies of West Indian Plants-XI[END_REF] only mentions the material León LS 10955 in the description of the species without specifying the herbarium where it was deposited. Because of this, all duplicates of such a collection in all herbaria are syntypes (Turland et al. 2018: Art. 40 Note [START_REF]To analyse the venation patterns of the leaves in Magnolia subsect[END_REF]. However, [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] recognize the NY specimen as a holotype, but such designation cannot be considered effective lectotypification (inadvertent lectotypification) since, as of January 1 st , 2001, the phrase "designated here" or an equivalent is required (Turland et al. 2018: Art. 7.11). For this reason, Talauma orbiculata Britton & P. Wilson is lectotypified here with the material (León LS 10955, NY #73699) suggested by [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF].

In the case of In the case of Magnolia minor Magnolia minor (Urb.) Govaerts and (Urb.) Govaerts and Magnolia oblongifolia Magnolia oblongifolia (León) Palmarola, (León) Palmarola, the results of this research change the limits of these species. the results of this research change the limits of these species. Magnolia minor Magnolia minor extends its extends its species limits from the proposed by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] and Palmarola species limits from the proposed by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] and [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] to include . ( 2016) to include the individuals considered by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] as the individuals considered by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] as Talauma oblongifolia Talauma oblongifolia (except the type).

(except the type). The individuals considered by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] as The individuals considered by [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF][START_REF] Bisse | Árboles de Cuba[END_REF] as Talauma ophiticola Talauma ophiticola ( (considered as M.

oblongifolia in the present taxonomic treatment) are an independent species (Fig. 23). In this part, it is important to clarify the confusion in the names and types that [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF] makes for akes for

Talauma oblongifolia

Talauma oblongifolia (León) Bisse and (León) Bisse and Talauma ophiticola Talauma ophiticola Bisse. This confusion is not cleared Bisse. This confusion is not cleared up by Palmarola up by [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] in the revision of . ( 2016) in the revision of Magnolia Magnolia subsect. subsect. Talauma Talauma of Cuba. of Cuba.

In the species description of Talauma ophiticola Bisse [START_REF] Bisse | Nuevos árboles de la flora de Cuba I[END_REF], the type material (Bisse & Köhler HFC 6519) is referred in HAJB, without mention of which of the two specimens (HAJB #634, #635) is the holotype. [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF] recognize the specimen HAJB #634 as the holotype, but such designation cannot be considered effective lectotypification (inadvertent lectotypification) since, as of January 1 st , 2001, the phrase "designated here" or an equivalent is required (Turland et al. 2018: Art. 7.11). For this reason, Talauma ophiticola Bisse is lectotypified here with the material suggested by [START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF].

CONCLUSIONS

1. Magnolia orbiculata has the most restricted potential suitability area and is the only taxon with a different ecological niche, supporting its species status. The taxa from northeastern Cuba are sympatric and show high ecological similarity, making it impossible to determine their ecological limits.

2. Three morphological groups are possible to delimited in Magnolia subsect. Talauma in Cuba, being Magnolia orbiculata the most differentiated one. Also, these taxa showed a high interspecific leaf morphological variability, which reinforces the value of leaf characteristics in taxonomic studies of Cuban magnolias.

3.

In Magnolia subsect. Talauma in Cuba it is possible to differentiate three genetic groups, with

Magnolia orbiculata the clearest one. The taxa from northeastern Cuba showed high levels of genetic admixture given by a recent or still going asymmetric hybridization process.

4. Magnolia subsect. Talauma in Cuba is a monophyletic group, closer to the species from Central America than to the other species of Magnolia in Cuba. Magnolia orbiculata represent an independent lineage and the group from northeastern Cuba is a well-supported clade with no resolution between their different taxa.

5. Magnolia orbiculata appeared to be an evolutionary lineage separated from other Cuban taxa of Magnolia subsect. Talauma , in which the morphological limits are consistent with the ecological, genetic, and phylogenetic limits. Therefore, this taxon can thus be considered a valid species. The morphological limits of Magnolia minor and Magnolia oblongifolia, are not consistent with the ecological, genetic, and phylogenetic limits. Each approach used showed different species limits. 223,225,227,229,231,233 MA39_191 192,194,196,198,200,202,204,206,208,210 MA39_259 121,123,125 MA39_280 160,162,164,166,172,180,184,188,198,200,204,210,218,220 MA39_342 168,170,172,174,176,178,180,182,186,188 MA39_442 140 MA40_045 255,259,261,265,267,269,271,275,277,285,287,289,301,311,323 MA41_120 163,169,171,173,177,183,185,189,191,193,195,197,199,201,203,205,211,213 MA41_373 176 MA42_028 127,129,131 MA42_072 268,269,270 MA42_130 123,127 MA42_150 144,146,148,150,152,154,156 MA42_231 145,147,153,159,163 MA42_255 154,156,158,160,162,164,166,170,172 MA42_421 307,347,349,351,353 MA42_441 327 MA42_471 142,168,170,172,174,176,178,180,186,188 MA42_495 101,103,107,109,111,115,117,119,123,125,129,131,133,135,139

  Analyse taxonomique intégrative des espèces cubaines de Magnolia subsect. Talauma (Magnoliaceae) Mots clés : concept d'espèce, délimitation des espèces, structure génétique, phylogénie, flux génétique, hybridation, marqueurs génétiques, microsatellites, modèles de niche écologique, morphométrie géométrique Résumé : Une délimitation taxonomique précise, basée sur une compréhension complète des processus évolutifs impliqués dans la différenciation des taxons, peut être obtenue à partir d'une combinaison d'approches écologiques, morphologiques et moléculaires. La taxonomie de Magnolia subsect. Talauma à Cuba a longtemps été débattue et exclusivement basée sur une étude morphologique traditionnelle d'un nombre limité d'individus. L'objectif de la présente étude est de délimiter les taxons cubains de Magnolia subsect. Talauma afin de mettre à jour leur traitement taxonomique par l'intégration de critères écologiques, morphologiques, génétiques et phylogénétiques.

  et Ophiticola, respectivement. Elle montre aussi l'existence d'admixtion génétique entre ces groupes, essentiellement au sein des groupes du nord-est de Cuba. Ces résultats ont été confirmés dans une analyse de classification multivariée (méthode DAPC). L'analyse NEWHYBRIDS a permis de préciser que les individus montrant des profils génétiques admixés au sein des taxons du nord-est Cuba sont vraisemblablement des individus issus de rétrocroisements sur Minor, à partir d'hybrides (Minor x Ophiticola) et des individus de type F2. Par ailleurs, certains individus d'Ophiticola avaient une assignation génétique purement Minor, ce qui appuie l'existence d'introgressions génétiques entre ces espèces. Les données génétiques démontrent ainsi l'existence de flux de gênes dans les zones hybrides entre Minor et Ophiticola. INDEX INTRODUCTION .............................................................................................................

Figure 2 .

 2 Figure 2 ...........................................................................................................................

Figure 3 .

 3 Figure 3 ...........................................................................................................................

Figure 4 .

 4 Figure 4 ...........................................................................................................................

Figure 5 .

 5 Figure 5 ...........................................................................................................................

Figure 6 .

 6 Figure 6 ........................................................................................................................... 54-55 Figure 7 ...........................................................................................................................Figure 8 ...........................................................................................................................Figure 9 ...........................................................................................................................Figure 10 .........................................................................................................................Figure 11 .........................................................................................................................Figure 12 .........................................................................................................................

Figure 13 .

 13 Figure 13 .........................................................................................................................Figure 14 .........................................................................................................................

Figure 15 .

 15 Figure 15 .........................................................................................................................

Figure 16 .

 16 Figure 16 .........................................................................................................................

Figure 17 .

 17 Figure 17 .........................................................................................................................

Figure 18 .

 18 Figure 18 .........................................................................................................................

Figure 19 .

 19 Figure 19 .........................................................................................................................

Figure 20 .

 20 Figure 20 ......................................................................................................................... 76-78 Figure 21 .........................................................................................................................Figure 22 .........................................................................................................................

Figure 23

 23 Figure 23 ......................................................................................................................... 100

  mainly supported, as in previous works, by leaf morphological characters. Imchanickaja (1993) recognized T. minor and considered their variability as subspecies: T. minor Urb. subsp. minor and T. minor subsp. oblongifolia (León) Borhidi. Talauma orbiculata was still considered by Imchanickaja (1993) as a different valid species.

Chen

  et al. (2018) in Delphinieae (Ranunculaceae),[START_REF] Renner | Geometric morphometric methods achieve type specimen assignment in the cryptic Plagiochila arbuscula complex (Plagiochilaceae: Jungermanniopsida) with the minimum of morphological evidence[END_REF] in Plagiochila arbuscula (Brid. ex Lehm. & Lindenb.) Lindenb. complex (Plagiochilaceae), Menini-Neto et al. (2019) in Pseudolaelia vellozicola (Hoehne) Porto & Brade complex (Orchidaceae), and Nery & Fiaschi (2019) in Hydrocotyle quinqueloba Ruiz & Pav. (Araliaceae). Almost of these works are based on leaves. Without question, leaf morphology is central to plant taxonomy, and it has mainly

  Magnoliaceae occur in Cuba, all of which belong to the genus Magnolia. Magnolia virginiana subsp. oviedoae Palmarola, M.S. Romanov & A.V. Bobrov is the only species within the section Magnolia L. The remaining six taxa are part of the section Talauma Baill., which has three taxa in the subsection Cubenses Imkhan.: Magnolia cristalensis Bisse, Magnolia cubensis subsp. acunae Imkhan., and Magnolia cubensis Urb. subsp. cubensis. The other three taxa belong to the subsection Talauma Juss.: Magnolia minor (Urb.) Govaerts, Magnolia oblongifolia (León) Palmarola, and Magnolia orbiculata (Britton & P. Wilson) Palmarola[START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF][START_REF] Greuter | Plantas Vasculares de Cuba -Inventario preliminar. Segunda edición, actualizada, de Espermatófitos de Cuba con inclusión de los Pteridófitos. Berlín & La Habana[END_REF][START_REF] Veltjen | The evolutionary history of the Caribbean Magnolias (Magnoliaceae): testing species delimitations and biogeographical hypotheses using molecular data[END_REF]).

Figlar&

  Figlar & Nooteboom 2004; Veltjen et al. 2022): 1) Magnolia subsect. Cubenses Imkhan.: from the Antilles, 2) Magnolia subsect. Dugandiodendron Lozano: from the middle elevations in the Andes and Guyana; 3) Magnolia subsect. Chocotalauma A. Vázquez, Á.J. Pérez & F. Arroyo: from the Chocó biogeographical region, and 4) Magnolia subsect. Talauma: from the lowland of the Caribbean and Continental Neotropical zone to low montane areas (Vázquez-García et al.2018; Domínguez-Yescas & Vázquez-García 2019). The subsection Talauma is the group with the largest number of species, characterized by the presence of early deciduous stipules adnate to the petioles, leaving a scar of variable length depending on the species on both edges of the adaxial side of petioles and converging apically[START_REF] Figlar | Notes on Magnoliaceae IV[END_REF][START_REF] Pérez | Magnolia chiguila and M. mashpi (Magnoliaceae): two new species and a new subsection (Chocotalauma, sect. Talauma) from the Chocó biogeographic region of Colombia and Ecuador[END_REF][START_REF] Vázquez-García | Magnolia mindoensis (subsect. Talauma, Magnoliaceae): Una especie nueva del Chocó biogeográfico premontano en Colombia y Ecuador[END_REF].

  petiole of the leaf was removed before taking pictures, and the camera was mounted on a tripod to standardize the angle and distance of the photographs. To expand the geographic scope of the study, leaf samples from 43 herbarium specimens were included (deposited in HAC, HAJB, NY, and B). Hence, 243 individuals of Magnolia sect. Talauma subsect. Talauma in Cuba were morphologically analysed. For the genetic analyses, healthy young leaf samples of a total of 461 individuals belonging to 26 of 31 known localities were stored in self-sealed bags with silica gel for DNA to be extracted from. The resulting number of DNA samples represented 52 % of the known individuals of Magnolia subsect. Talauma in Cuba (close to 900 individuals, data unpublished).

Figure 1 .

 1 Figure 1. Geographic distribution of the sampled individuals of Magnolia subsect. Talauma in Cuba. Calizas de Mucaral (MUC), Camarones-Río Báez (CRB), Cañón del Río Yumurí (CRY), Cayo Guam (CG), Cayo Mujeres (CM), Cupeyal del Norte (CN), El Recreo (REC), El Gigante (GIG), Loma del Gato (GAT), Pico Caracas (CAR), La Melba (MEL), Mina Iberia (MI), Mina la Hoya (MH), Monte Fresco (MF), Monte Verde (MV), Naranjo del Toa (NT), Pico Cristal (PC), Pico La Bayamesa (BAY), Piedra La Vela (PV), Pinares de Montecristo (PM), Presa de Mayarí (MAY), Región del Toa (RT), Río Yamanigüey (YAM), Sierra de Imías (IMI), Subida a La Melba (SM), Sur de las Delicias del Duaba (SDD), Turquino (TUR), Yumurí del Sur (YS), Yunque de Baracoa (YB).

FoliometriK v. 0. 2 . 2 (

 22 [START_REF] Ramírez-Arrieta | FoliometriK: aplicación en lenguaje R para mediciones morfométricas automatizadas de hojas a partir de fotografías digitales[END_REF]. Eleven leaf variables were measured for each leaf: central axis length (Length), maximum width, width at the three main quartiles (25, 50, and 75 quartiles), the perimeter of the contour (Perimeter), surface area (Area), and internal angles (v1 = angle of the base, v2 = angle of the apex; m1 and m2 = lateral angles at the maximum width) (Fig.2A). Additional to the eleven measured variables, the maximum width/ length ratio, named Calculated Index of Bisse (B CI ), was calculated for each leaf. The twelve variables were averaged per individual for the 4-8 leaves available per individual. The averages of the twelve variables were used for all the subsequent statistical analyses.

Figure 2 .

 2 Figure 2. A: The 11 morphological variables measured on leaves of Magnolia subsect. Talauma in Cuba; v1 = angle of the base, v2 = angle of the apex; m1 and m2 = lateral angles in the maximum wide. B: Quadratic grid with six lines and the position of the 14 landmarks (type 1: points 1 and 8; type 2: the other 12 points), placed on the leaves of Magnolia subsect. Talauma in Cuba.

(

  CTAB) extraction protocol[START_REF] Doyle | Isolation of plant DNA from fresh tissue[END_REF] with MagAttract Suspension G solution-mediated cleaning[START_REF] Xin | A high throughput DNA extraction method with high yield and quality[END_REF]. For each sample, between 50 and 70 mg (≈ 1cm2) of plant tissue was used and placed in 2 ml Eppendorf tubes. To the fragmented tissue, 700 μl of CTAB lysis buffer, 5 μl of B-mercaptoethanol, 5 μl of RNAase, and 5 μl of proteinase k were added and mixed by vigorous vortexing and left to incubate at 60ºC for at least one hour or for all night.

  minutes. The tubes were placed in the Dynamag, which attracts the MagAttract particles and allows for easier extraction of the ethanol solution. Samples were washed twice with 200 μl of 70 % ethanol and allowed to dry at 37ºC for 7 minutes. To resuspend the DNA, 100 µl of TBE buffer (10 mM Tris, 1 mM EDTA, pH 8) was added to each tube and mixed by light vortexing. The samples were incubated at 60ºC for 5 minutes and placed back in the Dynamag to transfer the TE-DNA solution to a new tube. DNA quality was assessed by reading the Optical Density values at the wavelengths of 230 nm, 260 nm, and 280 nm using a spectrophotometer NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, U.S.A). A total of 174 microsatellite markers (SSR) developed on four Neotropical Magnolia species: M. lacandonica A. Vázquez, Pérez-Farr. & Mart.-Camilo (MA39), M. mayae Vázquez & Pérez-Farrera (MA40), M. dealbata Zucc. (MA41) and M. cubensis subsp. acunae Imkhan. (MA42) (Veltjen et al. 2019), were tested. The individuals of Magnolia subsect. Talauma were finally genotyped using 21 microsatellite markers (the only ones which amplified for all the groups) (Table5). Simplex-PCRs were performed with the 21 selected microsatellite markers. Samples with high DNA purity and quality were chosen, with ratios 260/230 and 260/280 Optical Density close to 2. These samples were diluted in distilled water in a ratio of 1 DNA: 10 H 2 O. Two samples per group were selected.PCR reactions were performed with 2 μl of microsatellite marker (in ratio 1 forward primer: 4 reverse primer: 4 fluorescent probe), 1 μl of DNA, and 2 μl of master mix for a final volume of 5 μl. The master mix consisted of 1×TrueStart Taq Buffer (Thermo Fisher Scientific), 1.5 µM MgCl2 (Thermo Fisher Scientific), 0.125 µM dNTP, 5U of True Start Hot Start DNA polymerase (Thermo Fisher Scientific), and 0.4 mg /ml BSA (bovine serum albumin) per reaction. The amplification program used was as follows: 95ºC for 4 minutes, 35 cycles of 95ºC for 30 seconds, 52ºC for 30 seconds, 72ºC for 1 minute, and a final extension step at 72ºC for 15 minutes. To the products of the PCR reactions were added 20 µl distilled water for a final volume of 25 µl and plated in 96-well plates. Identification by sequencing of amplified allelic variants was performed by Macrogen Inc. (Seoul, South Korea). The results were analyzed in the Geneious Prime program (http://www.geneious.com,[START_REF] Kearse | Geneious basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF].

  et al. (2022), as outgroups, one sample of each taxon from different Caribbean regions (Mexico, Puerto Rico, Dominican Republic, and Cuba) and subsections of Magnolia were selected. Magnolia sect. Talauma subsect. Cubenses: M. cubensis Urb. subsp. cubensis (Cuba), M. cubensis subsp. acunae Imkhan. (Cuba), M. cristalensis Bisse (Cuba), M. domingensis Urb. (Dominican Republic), M. portoricensis Bello (Puerto Rico); Magnolia sect. Talauma subsect. Talauma: M. lacandonica A. Vázquez, Pérez-Farr. & Mart.-Camilo (Mexico), and M. sinacacolinii A. Vázquez (Mexico); Magnolia sect. Macrophylla: M. dealbata Zucc.

Figure 3 .

 3 Figure 3. Areas of climatic suitability for Magnolia subsect. Talauma in Cuba.

Figure 5 .

 5 Figure 5. Graphical representation of digital elevation model values extracted from the geographic position of Magnolia subsect. Talauma in Cuba.

Figure 4 .

 4 Figure 4. A: Results of the Principal Component Analysis (PCA) performed with five bioclimatic variables (Bio5, Bio7, Bio12, Bio15, and DEM) used in the description of the ecological niche of Magnolia subsect. Talauma in Cuba. B: Current geographic distribution of Magnolia subsect.

Figure 6 .

 6 Figure 6. Graphic representation of the leaf´s morphological variables measured in the individuals of Magnolia subsect. Talauma in Cuba. The MonteCarlo probabilities were less than 0.001; except in the perimeter for Minor-Ophiticola (p=0.211) and Oblongifolia-Ophiticola (p=0.132).

Figure 6 (

 6 Figure 6 (cont.). Graphic representation of the leaf´s morphological variables measured in the individuals of Magnolia subsect. Talauma in Cuba. The MonteCarlo probabilities were less than 0.001; except in the perimeter for Minor-Ophiticola (p=0.211) and Oblongifolia-Ophiticola (p=0.132).

Figure 7 .RESULTSFigure 8 .

 78 Figure 7. Principal Component Analysis for the multivariate morphometrics variables measured in the individuals of Magnolia subsect. Talauma in Cuba.

Figure 9 .BFigure 10 .

 910 Figure 9. Principal Component Analysis for the elliptic Fourier descriptors (A) and matrix of Landmark (B) which characterized the leaves of Magnolia subsect. Talauma in Cuba.

Figure 11 .

 11 Figure 11. Shape reconstruction of the leaves of Magnolia subsect. Talauma in Cuba based on the matrix of Landmark. The extremes shapes are based on the Principal Components, from the Principal Component Analysis.

Figure 12 .

 12 Figure 12. Graphic representation and classification matrix obtained after the cluster analysis using the morphological data of Magnolia subsect. Talauma in Cuba. A: Linear and angular variables, B: elliptic Fourier descriptors, C: matrix of Landmark. * Ghost Cluster.

Figure 13 .

 13 Figure 13. Privates alleles by taxa, locus, and individuals in Magnolia subsect. Talauma in Cuba.

Figure 14 .

 14 Figure 14. Delta K plot (A) and mean Ln(K) plot (B) for the complete dataset of Magnolia subsect. Talauma in Cuba. Delta K plot (C) and mean Ln(K) plot (D) for the dataset without the individuals of Oblongifolia.

Figure 15 .

 15 Figure 15. STRUCTURE results of Magnolia subsect. Talauma in Cuba. A: Representative bar plot (out of 100 en replicates) for K=3 based on the complete dataset; B: Representative bar plot (out of 100 en replicates) for K=3, without Oblongifolia. Calizas de Mucaral (MUC), Camarones-Río Báez (CRB), Cañón del Río Yumurí (CRY), Cayo Guam (CG), Cayo Mujeres (CM), Cupeyal del Norte (CN), El Gigante (GIG), El Recreo (REC), La Bayamesa (BAY), La Melba (MEL), Mina Iberia (MI), Mina la Hoya (MH), Monte Fresco (MF), Naranjo del Toa (NT), Pico Caracas (CAR), Pico Cristal (PC), Piedra La Vela (PV), Pinares de Montecristo (PM), Región del Toa (RT), Río Yamanigüey (YAM), Subida a la Melba (SM), Sur de las Delicias del Duaba (SDD), Turquino (TUR), Yumurí del Sur (YS), Yunque de Baracoa (YB).

Figure 16 .

 16 Figure 16. Discriminant Analysis of Principal Components (DAPC) of Magnolia subsect. Talauma in Cuba. The axes represent the first two linear discriminant functions. The upper left graph (Principal Component Analysis -PCA-eigenvalues) inset displays the variance explained by the principal component axes used for DAPC and the bottom-right inset (DA eigenvalues) displays in relative magnitude the variance explained by the two discriminant axes plotted. DAPC graph of the all the taxa, 200 principal components (PCs) retained. A: K=3, B: K=4, C: K=3 without Oblongifolia.

Figure 17 .

 17 Figure 17. Genotype assignment of the individuals of Magnolia subsect. Talauma from northeastern Cuba based on the program NEWHYBRIDS using microsatellite data and location prior information: Minor pure (green); Ophiticola pure (blue); F1 (black); F2 (red); Backcross to Minor (gray); Backcross to Ophiticola (yellow). Calizas de Mucaral (MUC), Camarones-Río Báez (CRB), Cañón del Río Yumurí (CRY), Cayo Guam (CG), Cayo Mujeres (CM), Cupeyal del Norte (CN), El Recreo (REC), La Melba (MEL), Mina Iberia (MI), Mina la Hoya (MH), Monte Fresco (MF), Naranjo del Toa (NT), Pico Cristal (PC), Piedra La Vela (PV), Pinares de Montecristo (PM), Región del Toa (RT), Río Yamanigüey (YAM), Subida a la Melba (SM), Sur de las Delicias del Duaba (SDD), Yumurí del Sur (YS), Yunque de Baracoa (YB).

Figure 18 .

 18 Figure 18. Phylogenetic relationships inferred for Magnolia subsect. Talauma from Cuba based on full plastome sequences. Tree constructed by the best tree based on maximum likelihood (ML). Support values marked above the branches follow the order bootstrap support/bayesian posterior probability. Cuba (Cu), Mexico (Mex), Puerto Rico (PR), Dominican Republic (DR); Cañón del Río Yumurí (CRY), Cupeyal del Norte (CN), Mina Iberia (MI), Pico Caracas (CAR), Pico Cristal (PC).

Figure 19 .

 19 Figure 19. Ancestral range reconstruction resulting from the BioGeoBEARS analysis of the Cuban groups of Magnolia subsect.

Figure 20 .

 20 Figure 20. Heatmap with the congruence between morphological (MT: Linear and angular variables, OUT: elliptic Fourier descriptors, LM: matrix of Landmark) and genetic (STRUCTURE) cluster probabilities, inside each group of Magnolia subsect. Talauma in Cuba. Orbiculata (A-E-I); Minor (B-F-J); Oblongifolia (C-G-K); Ophiticola (D-H-L); The blue color represents the number of individuals (less individuals: light blue; more individuals: dark blue).

Figure 20 .

 20 Figure 20. Heatmap with the congruence between morphological (MT: Linear and angular variables, OUT: elliptic Fourier descriptors, LM: matrix of Landmark) and genetic (STRUCTURE) cluster probabilities, inside each group of Magnolia subsect. Talauma in Cuba. Orbiculata (A-E-I); Minor (B-F-J); Oblongifolia (C-G-K); Ophiticola (D-H-L); The blue color represents the number of individuals (less individuals: light blue; more individuals: dark blue).

Figure 20 .

 20 Figure 20. Heatmap with the congruence between morphological (MT: Linear and angular variables, OUT: elliptic Fourier descriptors, LM: matrix of Landmark) and genetic (STRUCTURE) cluster probabilities, inside each group of Magnolia subsect. Talauma in Cuba. Orbiculata (A-E-I); Minor (B-F-J); Oblongifolia (C-G-K); Ophiticola (D-H-L); The blue color represents the number of individuals (less individuals: light blue; more individuals: dark blue).

Figure 21 .

 21 Figure 21. Integration of the morphological, ecological, genetic, and phylogeny limits of Magnolia subsect. Talauma in Cuba. Linear and angular variables (MT); elliptic Fourier descriptors (OUT); matrix of Landmark (LM). Discontinues lines represent a not clear differentiation between the groups.

Figure 22 .

 22 Figure 22. Lectotype of Talauma ophiticola Bisse (A) and Talauma minor var. oblongifolia León (B).

  differences with previous studies are due to the larger sample size used in the present work and its wider geographic representativeness. Palmarola et al. (2016) report similar values of length and width for M. minor and lower values for M. oblongifolia and M. orbiculata. According to Padial et al. (2010), one of the disadvantages of the MSC is that demonstrating the fixation of a state requires large sample sizes. Based on the Calculated Index of Bisse (B CI ), the average

  for M. minor and T. ophiticola. The other nine linear and angular variables are not possible to compare with previously published papers because this work represents their first use in Magnoliaceae family.

in

  Neotropical Magnolia, with good results as demonstrated by Rico & Gutiérrez-Becerril (2019), Muñiz-Castro et al. (2020), Veltjen (2020), Aldaba-Nuñez et al. (2021), Chávez-Cortázar et al. (2021), and Hernández et al. (2021).

  5 threshold was used (data not shown). The absence of BC-Oph and the existence of Ophiticola´s individuals considered pure Minor seem to indicate asymmetric introgression from Ophiticola to Minor. According to some authors[START_REF] Arnold | Asymmetric Introgressive Hybridization Among Louisiana Iris Species[END_REF][START_REF] Hu | Hybridization and asymmetric introgression between Cypripedium tibeticum and C. yunnanense in Shangrila County, Yunnan Province, China[END_REF][START_REF] Muranishi | Asymmetric introgression between Magnolia stellate and M. salicifolia at a site where the two species grow sympatrically[END_REF], asymmetric introgression has been reported in several species (including magnolias), and relevant explaining mechanisms have been explored from the perspective of prezygotic (gamete competition, spatial, temporal, mechanical and gametic isolation, and pollination behavior) and postzygotic (hybrid zygote abnormality, hybrid infertility, and low hybrid viability) barriers. Based on the results, the BC-Oph individuals could have low fitness, or crosses between F1/F2 with pure Ophiticola may have some prezygotic or postzygotic barriers, which does not allow the viability of BC-Oph individuals.

  in a single taxon, as recently proposed by Palmarola et al. (2016) based on the gathering Bisse & Kohler HFC 5358 (HAJB, JE) from the coast of Moa, which shows both oblong and elliptical leaves. In fact, the individuals considered as M. oblongifolia (Oblongifolia group) are, morphologically and genetically, more similar to M.

Figure 23 .

 23 Figure 23. The main classification systems of Magnolia subsect. Talauma in Cuba, with the taxonomic results of this research. The lines and points on the right of the blocks represents the leaf shape variability obtained from the morphological analyses.

6.

  Magnolia subsect. Talauma in Cuba is integrated by three species: Magnolia orbiculata (Britton & P. Wilson) Palmarola, Magnolia minor (Urb.) Govaerts, and Magnolia oblongifolia (León) Palmarola. Only the species limits of Magnolia orbiculata remain similar to the proposed by the last taxonomic revision of the group in Cuba.

1

  BIBLIOGRAPHIC REVISION ......................................................................................... 7 2.1 Species concept .................................................................................................... 7 2.1.1 Ecological Species Concept (ESC) ................................................................. 8 2.1.1.1 Ecological Niche Models (ENMs) ............................................................. 9 2.1.2 Morphological Species Concept (MSC) .......................................................... 2.1.2.1 Multivariate Morphometrics (MM) ............................................................. 2.1.2.2 Geometric Morphometrics (GM) ............................................................... 2.1.3 Genetic Species Concept (GSC) .................................................................... 2.1.3.1 Population genetics .................................................................................. Ecological limits ..................................................................................................... 4.1.1 ENMs and identity test .................................................................................... 4.1.2 Point based and cluster analysis .................................................................... 4.2 Foliar morphological limits ..................................................................................... 4.2.1 Multivariate and Geometric morphometrics .................................................... 4.2.2 Clustering analysis .......................................................................................... 4.3 Genetic limits ......................................................................................................... 4.3.1 Genetic Structure in Magnolia subsect. Talauma ............................................ 4.3.2 Testing hybrids ................................................................................................ 4.4 Phylogenetic limits ................................................................................................. 4.5 Integrative taxonomy .............................................................................................

2.1.3.2 Molecular markers: microsatellites ........................................................... 2.1.4 Phylogenetic Species Concept (PSC) ............................................................. 2.2 Integrative taxonomy ............................................................................................. 2.3 Inter-specific hybridization and genetic introgression in plants .............................. 2.4 Genus Magnolia ..................................................................................................... 2.4.1 Magnolia sect. Talauma .................................................................................. 2.4.2 Magnolia subsect. Talauma in Cuba: taxonomic history ................................. MATERIALS AND METHODS ........................................................................................ 3.1 Data sampling: occurrence, leaf and DNA records ................................................ 3.2 Ecological analysis ................................................................................................ 3.2.1 Ecological Niche Models (ENMs) .................................................................... 3.2.2 Niche Identity Test ........................................................................................... 3.2.3 Point-based and cluster analyses ................................................................... 3.3 Foliar morphological analyses ............................................................................... 3.3.1 Multivariate and Geometric morphometrics .................................................... 3.3.2 Clustering Analysis based on morphological variability ................................... 3.4 Genetic analyses ................................................................................................... 3.4.1 DNA extraction and PCR ................................................................................. 3.4.2 Population Genetic structure at subsection level ............................................ 3.4.3 Testing hybrids ................................................................................................ 3.5 Phylogenetic analysis ............................................................................................ 3.6 Integrative taxonomy ............................................................................................. 3.7 Taxonomic treatment ............................................................................................. RESULTS ........................................................................................................................ 4.1 4.6 Taxonomic treatment ............................................................................................. DISCUSSION .................................................................................................................. 5.1 Ecological Niche .................................................................................................... 5.2 Morphological variability ........................................................................................ 5.3 Genetic Structure ................................................................................................... 5.4 Testing hybrids ....................................................................................................... 5.5. Plastome phylogeny ............................................................................................. 5.6. An integrative classification of Magnolia subsect. Talauma in Cuba ..................... 5.7 Taxonomic treatment ............................................................................................. CONCLUSIONS .............................................................................................................. 101 RECOMENDATIONS ...................................................................................................... 102 REFERENCES ................................................................................................................ 103 Author's articles related to the thesis ......................................................................... 120 Scientific events related to the topic of the thesis .................................................... 120 Author's articles related to the Magnoliaceae family ................................................ 121 Thesis supervision related to Magnoliaceae family ................................................... 122 SUPPLEMENTARY MATERIALS ............................................................................... 123 INDEX OF TABLES

Table 1 .

 1 ...........................................................................................................................

Table 2 .

 2 ........................................................................................................................... 30-31

Table 3 .

 3 ........................................................................................................................... Table 4 ............................................................................................................................ Table 5 ............................................................................................................................ Table 6 ............................................................................................................................ Table 7 ............................................................................................................................ Table 8 ............................................................................................................................ Table 9 ........................................................................................................................... Table 10 .......................................................................................................................... Table 11 ..........................................................................................................................

INDEX OF FIGURES

Figure 1 ...........................................................................................................................

  made the combination of Talauma minor to Magnolia minor (Urb.) Govaerts, and considered all the other names of the Cuban Talauma species as synonyms of M. minor[START_REF] Acevedo-Rodríguez | Catalogue of seed plants of the West Indies[END_REF][START_REF] Rivers | The Red List of Magnoliaceae, revised and extended[END_REF]. The latest taxonomic review of Magnolia subsect. Talauma in Cuba was published by[START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF], who made the new combinations from Talauma to Magnolia.[START_REF] Palmarola | Las magnolias de Cuba: Talauma -taxonomía y nomenclatura[END_REF]

	recognize three species: Magnolia orbiculata (Britton & P. Wilson) Palmarola, Magnolia minor,
	and Magnolia oblongifolia (León) Palmarola (Palmarola et al. 2016). In the absence of additional
	evidence, these authors considered T. ophiticola (without name under Magnolia) a synonym of
	M. oblongifolia due to the existence of one specimen (Bisse & Kohler HFC 5358 HAJB, JE) that

  . Integrative taxonomy does not replace traditional taxonomy but uses complementarity among disciplines to improve rigor[START_REF] Schlick-Steiner | Integrative Taxonomy: A Multisource Approach to Exploring Biodiversity[END_REF][START_REF] Yeates | Integrative taxonomy, or iterative taxonomy?[END_REF]).

Since taxonomy has moved towards being an integrative science,

[START_REF] Damasco | Reestablishment of Protium cordatum (Burseraceae) based on integrative taxonomy[END_REF]

believe that formal species should represent evolutionarily diverged populations that: (1) form highly supported monophyletic clades according to molecular evidence,

[START_REF]To study the reproductive structures (flowers and fruits) of the Cuban taxa of Magnolia subsect[END_REF]

have not (natural) gene flow among closely related lineages or sister groups, and (

  . The genusMagnolia includes approximately 350 species[START_REF] Vázquez-García | Corncob flower, Magnolia mixteca (M. sect. Macrophylla, Magnoliaceae) a new species endemic to the Alto Balsas Basin (Baja Mixteca), in the Pacific slopes of Oaxaca, Mexico[END_REF], distributed in the temperate and tropical regions of East and Southeast Asia and America[START_REF] Azuma | Molecular phylogeny of the Magnoliaceae: the biogeography of tropical and temperate disjunctions[END_REF][START_REF] Cires | Gap analyses to support ex situ conservation of genetic diversity in Magnolia, a flagship group[END_REF], although they are known from fossil records in Europe

Table 1 .

 1 accepted T. oblongifolia (León) Bisse as a species and described Talauma ophiticola Bisse as a new species. Imchanickaja (1993) recognized T. orbiculata Britton & P. Wilson as a different valid species and considered the other taxa as subspecies of T. minor: T. minor Urb. subsp. minor and T. minor subsp. oblongifolia (León) Borhidi. Historical classification of the Cuban taxa of Magnolia subsect. Talauma.

	Based on anatomical and morphological (vegetative and reproductive) traits described by
	Nooteboom (1993), Frodin & Govaerts (1996) made the combination of T. minor to Magnolia

minor (Urb.) Govaerts and considered all the other names of the Cuban Talauma synonyms of M. minor (Acevedo-Rodríguez & Strong 2012; Rivers et al. 2016). The last taxonomic treatment of Magnolia subsect. Talauma in Cuba was published by Palmarola et al. (2016), who made the new combinations from Talauma to Magnolia. Palmarola et al. (2016) recognize Magnolia minor (Urb.) Govaerts and Magnolia orbiculata (Britton & P. Wilson) Palmarola in the same sense as Urban (1912) and Britton (1923), respectively. Palmarola et al. (2016) considered Magnolia oblongifolia (León) Palmarola, including Talauma ophiticola Bisse (without name under Magnolia), as a synonym, based on the existence of the collection Bisse & Kohler HFC 5358 in the herbaria HAJB and JE, that combines oblong and elliptical leaves in a presumably unique individual.

[START_REF] León | Novedades de la Flora Cubana (II)[END_REF]

had already mentioned this situation when they described T. minor var. oblongifolia: "on the coast of Moa, typical specimens have been collected as well as intermediate forms with oblong leaves mixed with elliptical leaves". The different taxonomic classification criteria of Magnolia subsect. Talauma in Cuba are shown in Table

1

.

Table 2 .

 2 Recorded localities, demographic information, DNA samples and herbarium vouchers of Magnolia subsect. Talauma in Cuba. Serie Herbario de la Flora de Cuba (HFC), Herbarium "Instituto de Ecología y Sistemática" (HAC), Herbarium "Jardín Botánico Nacional, Universidad de La Habana" (HAJB), Herbarium "The New York Botanical Garden" (NY), Herbarium "Botanischer Garten, Freie Universität Berlin" (B), National Park (NP), Ecological Reserve (ER), Natural Outstanding Element (NOE), Protected Area of Management Resources (PAMR), Extinct in the locality (*), No field data available (?), no voucher (NV), Abreviature (Abrev.), Number of individuals (Indiv.).

	Group	Localities (Proteced Area)	Abrev. Indiv. Leaves DNA	Vouchers (Herbarium)
	Minor	Río Yamanigüey (NP Alejandro de Humboldt)	YAM	72	5	28	Bécquer E.R. et al. HFC 89449 (HAJB)
	Minor	Sierra de Imías (PAMR Cuchillas del Toa)	IMI	?	2	0	Álvarez A. et al. HFC 27534 (HAJB)
	Minor	Sur de las Delicias del Duaba (PAMR Cuchillas del Toa)	SDD	2	1	2	Díaz J. et al. HFC 89435 (HAJB)
	Minor	Yumurí del Sur	YS	8	5	5	Bécquer E.R. et al. HFC 89510 (HAJB)
	Oblongifolia Calizas de Mucaral (NP Alejandro de Humboldt)	MUC	1	1	1	Bécquer E.R. et al. HFC 90655 (HAJB)
	Oblongifolia Cayo Guam	CG	11	11	3	Palmarola A. et al. HFC 89249 (HAJB)
	Oblongifolia Cupeyal del Norte (NP Alejandro de Humboldt)	CN	31	12	15	Falcón B. et al. HFC 88959 (HAJB)
	Oblongifolia La Melba (NP Alejandro de Humboldt)	MEL	5	0	2	Palmarola A. et al. HFC 89589 (HAJB)
	Oblongifolia NOE Yunque de Baracoa	YB	2	2	1	Bécquer E.R. et al. HFC 89531 (HAJB)
	Oblongifolia Pico Cristal (NP Pico Cristal)	PC	5	4	3	Bécquer E.R. et al. HFC 89933 (HAJB)
	Oblongifolia Piedra La Vela (NP Alejandro de Humboldt)	PV	4	3	3	Bécquer E.R. et al. HFC 90543 (HAJB)
	Oblongifolia Río Yamanigüey (NP Alejandro de Humboldt)	YAM	6	6	6	Bécquer E.R. et al. HFC 89452 (HAJB)
	Oblongifolia Sur de las Delicias del Duaba (PAMR Cuchillas del Toa)	SDD	1	1	1	Díaz J. et al. HFC 89435 (HAJB)
	Ophiticola	Cayo Guam	CG	130	15	33	Bécquer E.R & Testé E. HFC 89439 (HAJB)
	Ophiticola	Cupeyal del Norte (NP Alejandro de Humboldt)	CN	82	23	39	Falcón B. et al. HFC 88950 (HAJB)
	Ophiticola	La Melba (NP Alejandro de Humboldt)	MEL	12	5	12	Palmarola A. et al. HFC 89587 (HAJB)
	Ophiticola	Mina Iberia (NP Alejandro de Humboldt)	MI	77	16	45	Palmarola A. et al. HFC 89261 (HAJB)
	Ophiticola	Monte Fresco (NP Alejandro de Humboldt)	MF	11	0	8	NV
	Ophiticola	NOE Yunque de Baracoa	YB	19	2	8	Bécquer E.R. et al. HFC 89529 (HAJB)
	Ophiticola	Pico Cristal (NP Pico Cristal)	PC	8	6	7	Bécquer E.R. et al. HFC 89917 (HAJB)
	Ophiticola	Piedra La Vela (NP Alejandro de Humboldt)	PV	4	0	3	Bécquer E.R. et al. HFC 90531 (HAJB)
	Ophiticola	Sierra de Imías (PAMR Cuchillas del Toa)	IMI	?	0	0	Bisse J. et al. HFC 53300 (HAJB)
	Ophiticola	Subida a la Melba (km 10)	SM	7	0	7	Álvarez A. et al. HFC 42531 (HAJB)
	Ophiticola	Sur de las Delicias del Duaba (PAMR Cuchillas del Toa)	SDD	12	8	10	Bécquer E.

R. et al. HFC 89556 (HAJB)

Table 2 .

 2 Recorded localities, demographic information, DNA samples and herbarium vouchers of Magnolia subsect. Talauma in Cuba. Serie

Table 3 .

 3 As niche predictors, a set of 15 bioclimatic variables (spatial resolution: 0.86 km 2 ) and the digital elevation model (DEM) were chosen from the WorldClim database (http://www.worldclim.com;[START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]. Four of the bioclimatic variables (Bio8: Mean Temperature of Wettest Quarter, Bio9: Mean Temperature of Driest Quarter, Bio18: Precipitation of Warmest Quarter, Bio19: Precipitation of Coldest Quarter) were excluded from all analyses because they show odd spatial anomalies in the form of discontinuities between neighboring pixels in the absence of environmental gradients on the ground(Escobar et al. 2014). The variables were trimmed to the eastern Cuba region (extent of accessible area -M)

	Taxa	Initial number	Number of records after
		of records	3 km spatial thinning
	Minor	371	28
	Orbiculata	78	6
	Oblongifolia	58	10
	Ophiticola	351	15

using ArcGis v. 10.

2 (ESRI 2014)

. A pre-model using Maxent v.

3.4.1 (Phillips et al. 2006)

was built. For model calibration, the Magnolia subsect. Talauma individuals' thinned occurrence data and the bioclimatic variables were used, excluding the ones with the lower contribution of any

Table 3 .

 3 Number of original records and thinning records (after 3 km spatial thinning) used to create the ecological niche model of Magnolia subsect. Talauma in Cuba.

	Climatic variables	Code Set 1 Set 2 Set 3
	Annual precipitation	Bio12	x	x	x
	Precipitation seasonality (Coefficient of variation)	Bio15	x	x	x
	Maximum temperature of warmest month	Bio5	x	x	x
	Digital elevation model	DEM	x	x	x
	Temperature annual range (BIO5-BIO6)	Bio7	x	x	x
	Temperature seasonality (standard deviation *100)	Bio4	x	x	-
	Isothermality (BIO2/BIO7) (*100)	Bio3	x	-	-

Table 4 .

 4 Sets of environmental variables used for model calibration of Magnolia subsect. Talauma in Cuba. Source of the variables: WorldClim[START_REF] Hijmans | Very high resolution interpolated climate surfaces for global land areas[END_REF]. Sets 2 and 3 did not include variable(s) with the lowest contribution to the model. set of correlated variables (r≥0.85). To improve the model calibration process, we designed three sets of variables (Table4) as[START_REF] Cobos | Kuenm: An R package for detailed development of ecological niche models using Maxent[END_REF] recommended.

  Table 4). Modeling was performed in Maxent v. 3.4.1 with 50 bootstrap replicates, and the best configurations obtained during the evaluation process. The median models of the logistic output were converted into a binary map in ArcGis v. 10.2 (ESRI 2014). The minimum training presence

(MTP) was used as a threshold. It considers as suitable habitats all the pixels with higher values than the smallest value of probability corresponding to a real presence point. The contribution of each variable to the final model was assessed with a jackknife analysis in Maxent v. 3.4.1, and the total suitable area was calculated for each model.

Table 5 .

 5 Multiplex designed with the 21 microsatellites marker used for the genetic characterization of Magnolia subsect. Talauma in Cuba. The following universal tags were used: T3: 5′ AATTAACCCTCACTAAAGGG 3′, M13

Table 6 .

 6 Values of MTP (minimum training presence) and area of the habitat suitability of the groups of Magnolia subsect. Talauma in Cuba.

	2 )

Table 7 .

 7 Niche identity test between the groups of Magnolia subsect. Talauma in Cuba. Numbers below diagonal refer to Schoener's D and numbers above diagonal refer to Hellinger's-based I values.

2 Cl-3 Cl-4 Cl-5 Cl-6 Cl-7 Cl-8 Cl-9

	Minor	98	31	78	38	49	82	0	1	10
	Oblongifolia	13	2	3	23	9	6	0	0	1
	Ophiticola	8	53	44	16	84	0	0	128	25
	Orbiculata	4	0	0	0	0	0	74	0	0

Table 8 .

 8 Classification matrix obtained after the cluster (Cl) analysis using the environmental data of Magnolia subsect. Talauma in Cuba.

Table 9 .

 9 Average values and standard error of the measures of genetic diversity by groups of

	Magnolia He: expected heterozygosity.
		N	Np	Na	A R	Ne	Ho	He
	Orbiculata	36	11	6.81 ±1.18	6.652 ±1.143	3.618 ±0.543	0.584 0.078	0.564 ±0.064
	Minor	218	48	13.619 ±2.043	9.091 ±1.412	5.732 ±1.169	0.518 ±0.072	0.588 ±0.072
	Oblongifolia	35	7	9.810 ±1.360	9.674 ±1.339	5.420 ±0.929	0.579 ±0.066	0.630 ±0.065
	Ophiticola	172	34	12.524 ±1.896	9.163 ±1.306	5.658 ±1.023	0.574 ±0.060	0.650 ±0.057

subsect. Talauma in Cuba. N: sample size, Np: number of private alleles, Na: number of mean alleles, A R : allele richness, Ne: number of effective alleles, Ho: observed heterozygosity,

Table

Table 11 .

 11 Confusion matrix of the individuals of Magnolia subsect. Talauma from northeastern Cuba, based on the NEWHYBRIDS result with a probability threshold of 0.9 and 0.7. Mm: Minor;

	Groups -0.9	N	Mm pure F1	F2	BC-Mm	BC-Oph Oph pure NC
	Minor	218	127	0	2	3	0	0	86
	Oblongifolia	35	10	0	0	0	0	1	24
	Ophiticola	172	10	0	12	1	0	80	69
	Total	425	147	0	14	4	0	81	179
	Groups -0.7	N	Mm pure F1	F2	BC-Mm	BC-Oph Oph pure NC
	Minor	218	155	0	6	15	0	1	41
	Oblongifolia	35	14	0	0	4	0	1	16
	Ophiticola	172	10	0	40	3	0	89	30
	Total	425	179	0	46	22	0	91	87

Oph: Ophiticola; F1: first generation hybrid; F2: second generation hybrid; BC-Mm: Backcross to Minor; BC-Oph: Backcross to Ophiticola; NR: non-classified (individuals with probabilities under the threshold value).

Magnolia minor (Urb.) Govaerts, Word

  Juss., Gen. Pl.: 280. 1789. Type: Magnolia L. Magnolia sect. Talauma (Juss.) Baill., Adansonia 7: 3, 66. 1866. Type: Magnolia plumieri Sw. ; apex rounded, retuse, truncate; apex rounded and obtuse, retuse, truncate, subtruncated, emarginate and cuneiform; base from truncate to rounded, base rounded, obtuse; rarely subtruncated; surface glabrous and prominently reticulate-veined in both sides, the midrib flat above, prominent beneath; adaxial surface dark green, abaxial surface light green. Checkl. Bibliogr. Magnoliaceae 71. 1996. ≡ Talauma minor Urb.,Symb. Antill. 7: 222. 1912. ≡ Svenhedinia minor Urb., Repert. Spec. Nov. Regni Veg.

	Magnolia L., Sp. Pl. : 1: 535. 1753. Type: Magnolia virginiana L. Magnolia subsect. Talauma (Juss.) Figlar & Noot., Blumea 49(1): 90. 2004. Trees or little trees, evergreen; stipules adnate to the petioles and soon deciduous, leaving a scar on both edges of the adaxial side of petiole and converging apically; leaf blade generally coriaceous to subcoriaceous, entire margin; leaf orbicular, widely elliptic, ovate, reniform, oblong-37: 3. 2016. ≡ Talauma orbiculata Britton & P. Wilson, Bull. Torrey Bot. Club 50: 37. 1923. ≡ T. minor subsp. orbiculata (Britton & P. Wilson) Borhidi, Acta Bot. Acad. Sci. Hung. 17: 7. 1971. Lectotype (designated here): "Cuba. Prov. Granma, "tree, 6-8 m. tall; Loma de Quintín-Nagua", VII-1922, León LS 10955 [NY #73699!]. Isolectotypes: GH [#39075!], HAC ex LS [#SV 10703!]. = Svenhedinia truncata Mold., Phytologia 2: 142. 1946. ≡ Talauma truncata (Mold.) Howard, Bull Torrey Bot. Club 75: 357. 1948. Holotype: Cuba. Prov. Santiago de Cuba, "Alto Babiney, Sur del Turquino" Acuña SV 14069 [HAC ex SV!]. Trees, up to 25 m tall and 62 cm in trunk diameter, leaves with petiole of 2 -7 cm long, leaf blade de 6 -16 × 6 -13 cm, width-length relation between 0.66 to 1.23 (sometime wider than large), orbiculate, widely elliptic, ovate and reniform; apex rounded, retuse, truncate; base from truncate to rounded, adaxial surface dark green and the abaxial light green; glabrous and prominently reticulate-veined in both sides, the midrib flat above, prominent beneath. 24: 3. 1927. Lectotype (designed by Palmarola et al. 2016: 4): Cuba. Prov. Holguín, "Prope villam Monte Verde elliptical, obovateMagnolia orbiculata (Britton & P. Wilson) Palmarola, Revista Jard. Bot. Nac. Univ. Habana dictam Cuba Orientali", I-VII.1859, Wright 1100 B [#100248228!]. Isolectotypes?: BM [#574752!].

  relation between 0.38 to 0.87; apex rounded and obtuse, sometime retuse or truncate; base rounded and obtuse; adaxial surface dark green, abaxial surface light green; glabrous and prominently reticulateveined in both sides, the midrib flat above, prominent beneath.

	Magnolia oblongifolia (León) Palmarola, Revista Jard. Bot. Nac. Univ. Habana 37: 3. 2016. ≡
	T. minor var. oblongifolia León, Contr. Ocas. Mus. Hist. Nat. Colegio "De La Salle" 9: 5. 1950. ≡
	T. minor subsp. oblongifolia (León) Borhidi, Acta Bot. Acad. Sci. Hung. 17: 6. 1971. ≡ Talauma
	oblongifolia (León) Bisse, Feddes Repert. 85: 589. 1974. Holotype: Cuba. Prov. Holguín, "Monte
	de la Breña, Sierra de Moa, Moa, Oriente", 1-VII-1945, León, Alain, Clemente, Crisógono LS
	22588 (HAC ex LS!).
	= Talauma ophiticola Bisse, Feddes Repert. 85: 9-10: 589-590. 1974. Lectotype (designated
	here): Cuba, Prov. Holguín, "Baracoa, Mina Iberia, en las orillas del arroyo Iberia", IV-1968,
	Bisse & Köhler HFC 6519 [HAJB 634!]; isotypes: JE [#1224!, #1225!], HAJB [#635!].

  , in the phylogeny of the Caribbean magnolias, and supported by our own phylogeny (seeFigures 18, 19), Magnolia subsect. Talauma in Cuba is more closely related to M. dodecapetala than to the other taxa of Magnoliaceae in Cuba.The genetic differentiation and distance measures (F ST and D JOST ) between Orbiculata and the other groups of the subsection were the highest (see Table10). In the case of the groups from northeastern Cuba, both measures showed very low values, less than 0.01. When compared with other studies of Neotropical Magnolia, the F ST values among northeastern groups of Magnolia subsect. Talauma were lower than values usually found at the interspecific level. For example,

	Rico & Gutiérrez-Becerril (2019) consider M. pedrazae A. Vázquez and M. schiedeana Schltdl.
	as being a single species with pairwise F ST values ranging between 0.053 and 0.283; Chávez-
	Cortázar et al. (2021) propose the synonimization of M. nuevoleonensis A. Vázquez & Domínguez-
	Yescas and M. alejandrae García-Mor. & Iamonico with M. dealbata based on F ST values equal
	to 0.21 and 0.43, respectively. Aldaba-Nuñez et al. (2021) recommend a formal taxonomic
	revision of M. decastroi A. Vázquez & Muñiz-Castro and M. mexicana DC. (F ST =0.12, D JOST =0.19)
	and M. lopezobradorii A. Vázquez and M. zoquepopolucae A. Vázquez (F ST =0.11, D JOST =0.28)
	species complexes. The values reported by

  into three separate species (M. minor, M. oblongifolia, and Talauma ophiticola). morphological and genetic data did not support this proposal. Considering M. minor (Minor group) and M. oblongifolia (Oblongifolia group) as separate species is supported neither by ecological (see Figures4, Table 8), morphological (seeFigures 7, 8, 9, 12) nor by genetic data (see Figures15, 16). Indeed, concerning the individual of M. oblongifolia, the foliar phenotype observed in this species appears to be intermediate between M. minor and T. ophiticola (Ophiticola group); but their similarity, based on genetic and morphological data, with M. minor discharged this idea.

	The

  Supplementary material 2. Number of most probable (highest BIC score) groups (Cl) resulting for the ecological niche data of the taxa of Magnolia subsect. Talauma in Cuba. EEI (diagonal, equal volume and shape), EEV (ellipsoidal, equal volume and shape), BIC (Bayesian information criterion), ICL (integrated complete-data likelihood criterion). Size of the alleles identified in the microsatellite loci used in the characterization of the taxa of Magnolia subsect. Talauma in Cuba.

	Supplementary material 6. Loci	Orbiculata
	MA39_120	131, 135
	Cl MA39_182 2 MA39_185	144, 146	Model EEV	Log-likelihood -13 381.82	BIC -27 007.71	ICL -27 008.17
	3		EEV	-13 382.11	-27 116.77	-27 118.55
	4		EEV	-13 359.55	-27 180.13	-27 201.76
	5		EEV	-13 693.38	-27 956.28	-28 169.34
	6		EEI	-13 837.49	-27 946.18	-27 962.64
	7		EEV	-13 291.58	-27 369.64	-27 441.23
	8		EEI	-13 837.49	-27 946.18	-27 962.64
	9		EEV	-11 134.12	-23 271.68	-23 280.05
	10		EEV	-11 384.64	-23 881.19	-23 921.43

  , 145 Supplementary material 7. Average values of the measures of genetic diversity by loci of Magnolia subsect. Talauma in Cuba. Np: number of private alleles, Nr: number of rare alleles Na: number of mean alleles, Ne: number of effective alleles, Ho: observed heterozygosity, He: expected heterozygosity.

	Loci	Np	Nr	Na	Minor Ne	Ho	He
	MA39_120	1	6	9	1.700	0.404 0.412
	MA39_182	0	2	4	1.207	0.170 0.172
	MA39_185	1	8	14	6.034	0.802 0.834
	MA39_191	3	21	27	14.366 0.876 0.930
	MA39_259	2	6	9	2.515	0.573 0.602
	MA39_280	10	24	28	3.773	0.408 0.735
	MA39_342	3	12	19	8.569	0.857 0.883
	MA39_442	1	1	3	1.285	0.198 0.222
	MA40_045	3	15	22	9.983	0.890 0.900
	MA41_120	5	27	34	19.264 0.881 0.948
	MA41_373	3	4	6	1.193	0.171 0.162
	MA42_028	1	4	7	3.134	0.619 0.681
	MA42_072	0	1	3	1.595	0.137 0.373
	MA42_130	2	3	5	1.243	0.106 0.196
	MA42_150	0	2	7	2.933	0.664 0.659
	MA42_231	3	6	7	1.028	0.028 0.027
	MA42_255	3	7	16	8.950	0.862 0.888
	MA42_421	0	10	20	11.996 0.849 0.917
	MA42_441	2	4	5	1.083	0.042 0.077
	MA42_471	2	11	18	5.457	0.440 0.817
	MA42_495	3	14	23	13.067 0.899 0.923

& P. Wilson)Borhidi. Bisse (1974, 1988) split all the morphological variability, previously reflected

ACKNOWLEDGEMENTS

First of all, I want to thank Lucy my wife, my Mom and Dad, Kata, Fabrice, and Emanuel, for all your support during this process. This thesis is definitely both yours and mine. I want to thank as well all the institutions that made it possible for me to be here today and finish

clustering analysis was also performed. A DAPC analysis (Discriminant Analysis of Principal Components) was executed in R v. 4.2.0 (R Core Team 2022) using the adegenet R package [START_REF] Jombart | Discriminant Analysis of Principal Components: A New Method for the Analysis of Genetically Structured Populations[END_REF]. Firstly, a PCA was run on the whole dataset for which the first 200 Principal Components (PCs) were retained. Secondly, a discriminant analysis was executed using the number of genetic clusters defined in the previous step. Parallel to the STRUCTURE analysis, a second DAPC analysis was done without Oblongifolia. An individual was considered a member of a genetic cluster when its probability of belonging to that group was higher than or equal to 0.9.

Testing hybrids

The presence or absence of hybrid individuals was estimated with NEWHYBRIDS [START_REF] Anderson | A model-based method for identifying species hybrids using multilocus genetic data[END_REF][START_REF] Anderson | Bayesian inference of species hybrids using multilocus dominant genetic markers[END_REF], using the 21 microsatellite markers. This analysis computes the posterior probability that an individual in the sample belongs to each of the different hybrid categories. The program was run to identify F1 (First Generation), F2 (Second Generation), and backcrossing hybrids from the whole genotypic data. The posterior probability reflects the level of certainty that an individual belongs to a hybrid category. The analysis was performed based on ten iterations carried out using Jeffreys's prior (see [START_REF] Anderson | A model-based method for identifying species hybrids using multilocus genetic data[END_REF][START_REF] Anderson | Bayesian inference of species hybrids using multilocus dominant genetic markers[END_REF]) and setting the burn-in period to 150 000, with a MCMC length of 500 000 replicates. A first performance was made without previous information on populations or taxonomic identity.

A second analysis was made using prior information about the groups identity based on the individuals perfectly classified based on morphological (mclust) and genetic (STRUCTURE) probabilities. The 75 % of the best-classified individuals were used for calibrating the analysis, and the other 25 % for tested (Minor: 38 individuals for calibration and 13 for a test; Ophiticola: 12 individuals for calibration and 5 for a test). A probability threshold of 0.9 and 0.7 was used to classify the individuals in pure, F1, F2, and backcrosses. variability of the groups from northeastern Cuba (Minor, Oblongifolia, and Ophiticola); or 2) two species: M. minor (Urb.) Govaerts (include the individuals of the groups Minor and Oblongifolia) and M. oblongifolia (León) Palmarola (include the individuals considered in the present as Ophiticola) without very strong limits may be recognized. The decision to treat this group as a complex of two different species that can still be hybridized is validated by the consensus protocol for integrative taxonomy proposed by [START_REF] Padial | The integrative future of taxonomy[END_REF]. The start of this protocol is two groups of specimens with differences in a taxonomic character (e.g. morphology), followed by the search for the existence of congruent differences in another taxonomic character (e.g. population genetics). In the present thesis, differences between both species were found based on morphological characters (see Figure 12) and genetic data (see Figures 15,16).

Furthermore, most evolutionary biologists agree that species are separately evolving lineages of populations or meta-populations (Unified SC) [START_REF] De Queiroz | The general lineage concept of species, species criteria, and the process of speciation: A conceptual unification and terminological recommendations[END_REF][START_REF] De Queiroz | Species Concepts and Species Delimitation[END_REF]. According to de Queiroz (2007) and [START_REF] Naciri | Species delimitation and relationships: The dance of the seven veils[END_REF] this implies that the populations share a common history (largely) and have a common future. Contrary to the biological species concept of [START_REF] Mayr | Systematics and the origin of species[END_REF], this concept allows for some gene flow between species. There is no doubt about the common evolutionary history of Magnolia subsect. Talauma from northeastern Cuba, which is supported by the phylogeny (monophyletic group, see Figure 18) and their biogeographic history (see Figure 19). Also, there is strong evidence of a current or recent gene flow between both taxa (see Figures 15,16,17).

Evolution is a continuous process in which different lineages can separate and diverge (de Queiroz 2007). During the speciation process, the daughter lineages become more and more different from one another through time and acquire different properties relative to each other (morphological, ecological, genetic, reproductive incompatibility, etc.). Definitely, it is not a simple matter to propose an unbiased approach for species delimitation given the nature of the speciation process; in which no order is expected in the appearance of divergences among

Orbiculata-Oblongifolia

Orbiculata-Ophiticola