Piero G Delprete
email: pierog.delprete@ird.fr

Sushil K Paudyal

Solenandra Adolphoduckea

& Greuter

Rankin-Rodríguez

Rebuttal to Greuter & Rankin-Rodríguez's (2831) proposal to conserve Exostema against Coutarea (Rubiaceae) and to their expanded circumscription of Exostema

Keywords: Chiococceae, Coutarea, Exostema, genus-level classification, Rubiaceae, phylogeny

Paudyal & al. performed the most comprehensive phylogenetic study of the tribe Chiococceae (Rubiaceae) using two nuclear and two plastid datasets, and resolved four well-supported main clades. Within Clade B, Paudyal & al. recognized the genera Exostema, Hintonia, Coutareopsis, Motleyothamnus, Coutarea,

■ INTRODUCTION

The tribe Chiococceae Hook.f. sensu [START_REF] Paudyal | Using molecular, morphological, and palynological evidence to transfer Strumpfia maritima to the monotypic tribe Strumpfieae (Cinchonoideae, Rubiaceae), and a re-delimitation of the tribe Chiococceae[END_REF][START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] is a group of c. 210 species occurring mostly in the Neotropics, with c. 160 species in the Greater Antilles, 25 species in Central and South America, as well as c. 26 species in the South Pacific (Philippines, Marianas, New Caledonia, Melanesia, Tonga Islands). Numerous molecular phylogenies have long established that the tribe is a strongly supported monophyletic group (e.g., [START_REF] Motley | Molecular systematics of the Catesbaeeae-Chiococceae complex (Rubiaceae): Flower and fruit evolution and biogeographic implications[END_REF][START_REF] Bremer | Time tree of Rubiaceae phylogeny and dating the family, subfamilies, and tribes[END_REF][START_REF] Manns | Towards a better understanding of intertribal relationships and stable tribal delimitations within Cinchonoideae s.s. (Rubiaceae)[END_REF][START_REF] Paudyal | Using molecular, morphological, and palynological evidence to transfer Strumpfia maritima to the monotypic tribe Strumpfieae (Cinchonoideae, Rubiaceae), and a re-delimitation of the tribe Chiococceae[END_REF][START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. For more information regarding the taxonomic history and delimitation of the tribe, see [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. The tribe Chiococceae is a morphologically diverse group, with habits ranging from subshrubs, shrubs, vines, treelets to tall trees, with axillary or terminal inflorescences, corollas widely ranging in size and shape, from c. 3 mm long (e.g., Erithalis P.Browne) to 27 cm long (e.g., Osa Aiello), ovaries with several kinds of placentation, and fruits either capsular, with various modes of dehiscence, or baccate. [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] performed the most comprehensive phylogenetic study of the Chiococceae using two nuclear (ETS, ITS) and two plastid (petD, trnL-F) datasets analyzed with Bayesian and maximum likelihood methods. The phylogenetic trees generated from the analyses of the combined dataset (all markers) resulted in the most fully resolved phylogeny, and the majority consensus tree was used by Paudyal & al. (2018: fig. 3) for taxonomic decisions. This tree has four well-supported main clades, designated A to D. Clade B (Fig. 1) comprises the group of c. 40 species previously included in Exostema (Pers.) Bonpl., Solenandra Hook.f., Hintonia Bullock, and Coutarea Aubl. In recognition of the monophyletic groups within Clade B, Paudyal & al. recircumscribed three of these four genera (except Hintonia, which was maintained as traditionally delimited) and named three additional small genera, Coutareopsis Paudyal & Delprete, Motleyothamnus Paudyal & Delprete, and Adolphoduckea Paudyal & Delprete. The assessment and recognition of monospecific genera was in agreement with Razafimandimbison & al. (2011: 945-946), who presented the following explanation "[monospecific genera] are discussed on the basis of a combination of the following criteria [START_REF] Backlund | To be or not to be -Principles of classification and monotypic plant families[END_REF][START_REF] Razafimandimbison | Phylogeny and classification of Naucleeae s.l. (Rubiaceae) inferred from molecular (ITS, rbcL, and trnT-F) and morphological data[END_REF]: (1) If they are not nested within other well-defined genera; (2) If they have at least one autapomorphic character or a combination of plesiomorphic characters, allowing them to be recognized easily."

The changes in generic delimitations made by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] were discussed within their article, and are succinctly addressed below. The relevant clades are referenced in Fig. 1, which corresponds to Clade B of figure 3 in [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. In addition, the geographical distribution, ecology, and main morphological characters of the genera of Clade B recognized by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], treated as sections of Exostema by Greuter & Rankin-Rodríguez, are summarized in Table 1.

In clade B of the phylogenies obtained by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], Exostema, as traditionally delimited, was confirmed to be paraphyletic, and required most taxonomic changes. In the well-supported subclade B1 (BPP 0.96; BPP = Bayesian posterior probability) were retrieved the species of Exostema with axillary inflorescences. This result agrees with the morphology-based delimitation of E. sect. Exostema of Mc-Dowell (1996). Because E. caribaeum (Jacq.) Roem. & Schult. is the type of the genus, [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] treated the species of this clade as Exostema s.str., a genus of 8 species that occur in Cuba and Hispaniola, with the exception of E. caribaeum, which is also present in other Antillean islands, southern Florida, Mexico and Central America. The presence of axillary vs. terminal inflorescences is a significant character that has been used to define many genera in the Rubiaceae (see below for some examples), and axillary inflorescence represents a strong morphological synapomorphy for Exostema s.str.

Still within clade B, a subset of the species traditionally positioned in Exostema were retrieved on the wellsupported (BPP 1) subclade B4, comprising the terminalflowered sister groups of Solenandra (clade B4b, BPP 1) with many short flowers per inflorescence, and (excepting one species) the "E. sect. Pitonia" group (clade B4a, BPP 1), with many long flowers per inflorescence. [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] merged all the species present on subclades B4a and B4b into the expanded genus Solenandra s.l., and published the necessary new combinations. The monophyly of Solenandra sensu Borhidi [START_REF] Borhidi | Revalidación del genero Solenandra Hook.f. (Rubiaceae)[END_REF] was retrieved in previous molecular studies [START_REF] Mcdowell | Phylogeny, diversity, and distribution in Exostema (Rubiaceae): Implications of morphological and molecular analyses[END_REF][START_REF] Mcdowell | Biogeography of Exostema (Rubiaceae) in the Caribbean region in light of molecular phylogenetic analyses[END_REF][START_REF] Manns | Towards a better understanding of intertribal relationships and stable tribal delimitations within Cinchonoideae s.s. (Rubiaceae)[END_REF][START_REF] Manns | Biogeography of the predominantly Neotropical subfamily Cinchonoideae (Rubiaceae): Into or out of America?[END_REF]. Solenandra, as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], is a genus of 22 species, characterized by terminal inflorescences, infundibular, white corollas, capsular fruits Exostema maynense Poepp. & Endl., within Clade B3, a tall canopy tree from the lowlands of the western Amazon Basin, was retrieved a strongly supported clade (BPP 1) as a sister taxon of Coutarea hexandra (Jacq.) K.Schum. These two taxa are similar in having terminal inflorescences, 6-merous flowers, laterally compressed, septicidal capsules, and winged seeds. Coutarea hexandra differs from Exostema maynense in having zygomorphic flowers (vs. actinomorphic in E. maynense), campanulate, gibbous corollas (vs. straight, narrowly infundibular), corolla tube campanulate (vs. narrowly cylindrical), corolla lobes deltoid to ovate, 0.6-2.3 cm long (vs. linear, 4.5-5.5 cm long), unequal, sigmoidal stamens bending towards the gibbous side of the corolla (vs. equal, rectilinear stamens), and two stigmatic lines along the style (vs. stigmatic portion clavate to subcapitate). Because of the numerous and conspicuous morphological differences between these two taxa, [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] transferred E. maynense to the monospecific genus Adolphoduckea Paudyal & Delprete. An added impetus for naming the monotypic Adolphoduckea was to maintain the traditionally recognized genus Coutarea, whose type C. hexandra is the most geographically widespread species within the entire Exostema-Coutarea-Hintonia Clade B assemblage. Coutarea hexandra is unique within the Chiococceae in having zygomorphic flowers, and is additionally characterized by campanulate, gibbous corollas, well-exserted, unequal stamens, bending towards the gibbous side of the corolla, stigma in two lines along the style, capsules that are elliptic, oblong, obovate to round in outline, laterally compressed, and broadly winged seeds vertically arranged, laterally inserted. In addition, C. hexandra is distributed in 22 countries, from southern Mexico to Argentina, and is the most widespread species in Clade B of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. Whereas, Adolphoduckea is a rainforest tree of western Amazon and eastern Andean slopes from Ecuador, Peru, Bolivia and Brazil, with actinomorphic flowers, with a narrowly cylindrical corolla tube and linear lobes, style clavate or capitate, weakly laterally compressed capsules, and vertically imbricate, acrobasipetally aligned seeds. Aside from the numerous morphological differences between two taxa, which are summarized in Table 1, Adolphoduckea was established to maintain the traditional usage of the name Coutarea hexandra, which applies to the most widespread species in Clade B of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF].

Exostema corymbosum (Ruiz. & Pav.) Spreng., within clade B, was resolved on a strongly supported clade (BPP 1) as sister taxon of Coutarea fuchsioides C.M.Taylor and C. andrei Standl. These taxa are ecologically similar in occurring in dry vegetation of high altitudes on the Andes of Ecuador and Peru. Exostema corymbosum differs from C. fuchsioides and C. andrei by its salverform, white corollas (vs. tubular to funnelform or slightly flared, pink to red in C. fuchsioides and C. andrei), linear anthers (vs. narrowly oblong), linear style branches (vs. ovate to oblong), capsules obpyriform to round in outline, strongly laterally compressed (vs. ellipsoid to obovate in outline, faintly laterally compressed). Because of the significant sets of morphological characters that distinguish the two clades, [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] Greuter & Rankin-Rodríguez (2021, 2022a) interpreted the clades within Clade B (Fig. 1) of Paudyal & al. (2018: fig. 3) differently than [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], and instead of recognizing the phylogenetically and morphologically wellsupported genera, as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], opted to treat the entire Clade B as the broadly expanded, morphologically diverse genus Exostema. Their wide circumscription submerges the long-established genera Coutarea and Hintonia, as well as the more recently recognized segregates genera into Exostema. Because the genus Coutarea is positioned within Clade B, Greuter & Rankin-Rodríguez (2021) published a formal proposal (2831) to conserve the name Exostema against Coutarea, as the latter has nomenclatural priority.

■ DISCUSSION

We strongly disagree with [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) naming the entire Clade B of Paudyal & al. (2018: fig. 3; i.e., Fig. 1 included here) as a widely expanded, highly heteromorphic Exostema, and we prefer to maintain recognition of the phylogenetically and morphologically well-supported genera as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF].

Greuter & Rankin-Rodríguez (2021: 906) argued that the use of the name Exostema for all the species of clade B (Fig. 1) would require fewer new combinations than using the name Coutarea for the whole clade. Several additional arguments presented by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) to recognize their expanded delimitation of Exostema are supported by erroneous information, and are discussed below.

Greuter & Rankin-Rodríguez (2021: 906) supported their proposal by stating, "For reasons of priority the name of that genus would be Coutarea, unless the present proposal is accepted, in which case Exostema could be retained for it. Currently, for the 47 taxa of sectional, specific and subspecific rank recognized by us, only 9 names are available under Coutarea, as contrasted with 37 already available under Exostema. If our proposal were to be rejected, 37 new combinations would have to be published, as contrasted with 9 in case of acceptance (we refrain from proposing these combinations as long as the decision on the proposal is pending). It is Version of Record 5 of note that acceptance of the present proposal would not compromise the use of the generic name Coutarea for the traditionally defined, small genus (47 species) that excludes the type of Exostema." However, according to contemporary Rubiaceae specialists, Coutarea is a genus of 2 or 3 species (Ochoterena, 2012;[START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], and not "47" as stated by Greuter & Rankin-Rodríguez (2021: 906).

Greuter & Rankin-Rodríguez (2021: 906) defended their proposal to conserve the name Exostema against Coutarea because within Clade B (Fig. 1) is positioned the Coutarea, which has nomenclatural priority, by adding that "expanding the use of Coutarea to include Exostema, resulting from rejection of the proposal, would have the unwelcome consequence of increasing the risk of confusion between Coutarea and the similarly spelled name Coussarea Aubl., in general use for a large genus of woody Neotropical Rubiaceae". As far as we know, in all specialized literature examined on Neotropical Rubiaceae (e.g., Standley, Steyermark, C.M. Taylor, Delprete) the names Coutarea and Coussarea have never been confused with each other, as the two genera are morphologically very different, and their names are sufficiently distinct.

In Greuter & Rankin-Rodríguez (2021: 906), it is unclear if they refer to Exostema including or excluding Solenandra or other genera. As they wrote "Currently, for the 47 taxa of sectional, specific and subspecific rank recognized by us [in Exostema]" it seems that they refer to their broadly delimited Exostema. [START_REF] Mcdowell | Phylogeny, diversity, and distribution in Exostema (Rubiaceae): Implications of morphological and molecular analyses[END_REF] recognized Exostema (which was shown to be paraphyletic, e.g., [START_REF] Manns | Towards a better understanding of intertribal relationships and stable tribal delimitations within Cinchonoideae s.s. (Rubiaceae)[END_REF][START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], as a genus of 25 species, with E. sect. Exostema (8 spp.), sect. Brachyantha (6 spp.), and sect. Pitonia (11 spp.). Hence, Greuter & Rankin-Rodríguez's (2021: 906) summary refers to the genus Exostema as expanded by their proposal, which would combine the entire Clade B of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] into a genus with different morphology, distribution, and evolutionary context.

Greuter & Rankin-Rodríguez (2021) proposed a widely delimited, highly heteromorphic Exostema with several sections that generally correspond with the main clades of Clade B (Fig. 1) retrieved by Paudyal & al. (2018: fig. 3). In the introduction of their article, Greuter & Rankin-Rodríguez (2022a: 210) wrote that " [START_REF] Mcdowell | Exostema (Rubiaceae): Taxonomic history, nomenclature, position and subgeneric classification[END_REF], in a cladistic analysis based on both morphological and molecular (ITS) data, recognized three morphologically defined sections in the genus: E. sect. Exostema, characterized by long flowers in axillary, 1-3-flowered inflorescences; E. sect. Brachyanthum DC. (as 'Brachyantha'), with numerous shorter flowers in terminal inflorescences; and E. sect. Pitonia DC., again with terminal inflorescences with fewer and still longer flowers. [START_REF] Mcdowell | Phylogeny, diversity, and distribution in Exostema (Rubiaceae): Implications of morphological and molecular analyses[END_REF], combining morphological and molecular data, confirmed these sections as monophyletic groups as did further, more broadly based molecular analyses, in particular that of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. [START_REF] Mcdowell | Phylogeny, diversity, and distribution in Exostema (Rubiaceae): Implications of morphological and molecular analyses[END_REF] removed the two South American species from within their section (E. maynense from E. sect. Pitonia, E. corymbosum from E. sect. Brachyanthum), moving them to a basal position within the genus." Greuter & Rankin-Rodríguez failed to explain that, as previously stated by [START_REF] Motley | Molecular systematics of the Catesbaeeae-Chiococceae complex (Rubiaceae): Flower and fruit evolution and biogeographic implications[END_REF] and [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], the phylogenies produced by [START_REF] Mcdowell | Exostema (Rubiaceae): Taxonomic history, nomenclature, position and subgeneric classification[END_REF] and [START_REF] Mcdowell | Phylogeny, diversity, and distribution in Exostema (Rubiaceae): Implications of morphological and molecular analyses[END_REF] did not truly test the monophyly of Exostema, because in the ingroup of the phylogenetic study were present only Exostema species, as no other genera of basal Chiococceae were included in the ingroup. Because of the reduced sampling of taxa, and the absence of additional genera in the ingroup, the phylogenies obtained by [START_REF] Mcdowell | Exostema (Rubiaceae): Taxonomic history, nomenclature, position and subgeneric classification[END_REF] and [START_REF] Mcdowell | Phylogeny, diversity, and distribution in Exostema (Rubiaceae): Implications of morphological and molecular analyses[END_REF] were not appropriate to show the paraphyletic condition of Exostema as delimited by [START_REF] Mcdowell | Exostema (Rubiaceae): Taxonomic history, nomenclature, position and subgeneric classification[END_REF].

Greuter & Rankin-Rodríguez (2022a: 211) regarding the generic re-delimitations within Clade B of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] wrote: "We remain unconvinced of the soundness of that approach because, based on the same data and analysis, there is at least one morphologically and biogeographically more convincing while less disruptive alternative solution that fulfils the monophyly criterion. We opt for widening the circumscription of Exostema, adding to it two clades (each as an independent section), one corresponding to the genus Hintonia Bullock, the second to Coutarea Aubl. plus its recent segregate Coutareopsis Paudyal & Delprete plus the two S American Exostema species that correspond to the newly described genera Motleyothamnus Paudyal & Delprete and Adolphoduckea Paudyal & Delprete." Their argument for increased stability by proposing to name all the taxa in Clade B as Exostema ignores the option of simply maintaining the generic boundaries of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], which are all strongly supported by molecular phylogenies and sets of morphological data.

Greuter & Rankin-Rodríguez (2022a: 213), under Exostema sect. Brachyanthum, wrote "All nine species of our E. sect. Brachyanthum inhabit mainland South America, but one of them, Coutarea hexandra, extends northward through Central America to S Mexico and to Venezuela's offshore islands." However, C. hexandra is widespread throughout the whole Neotropical region and outside of it, occurring in at least 22 countries, ranging from Mexico throughout the whole of Brazil and to northern Argentina, and is the species with the widest distribution in Exostema sensu Greuter & Rankin-Rodríguez.

Greuter & Rankin-Rodríguez (2022a) explained the extreme morphological variation encountered in their widely delimited Exostema by simply describing the variation in characters and characters states present in their broadly delimited Exostema. However, many morphological characters discussed in Greuter & Rankin-Rodríguez's Exostema provide numerous diagnostic/synapomorphic character states that have traditionally been used to distinguish genera in numerous Rubiaceae tribes, as, e.g., inflorescence position (terminal vs. axillary), flower merosity, and stamen presentation (well exserted vs. included or partially exserted), and style morphology. Inflorescence position is an important synapomorphic/diagnostic character for the recognition of genera in the same tribe. For example, in the Condamineeae 6 Version of Record [START_REF] Delprete | Rondeletieae (Rubiaceae), part I ([END_REF][START_REF] Kainulainen | Molecular systematics and morphological character evolution of the Condamineeae[END_REF] the inflorescences are axillary in, e.g., Chimarrhis Jacq. and Macrocnemum P.Browne, or terminal in, e.g., Condaminea DC., Ferdinandusa Pohl, and Pogonopus Klotzsch. In the tribe Sipaneeae [START_REF] Delprete | A phylogenetic study of the tribe Sipaneeae (Rubiaceae, Ixoroideae), using trnL-F and ITS sequence data[END_REF][START_REF] Delprete | Monograph of tribe Sipaneeae (Rubiaceae, Ixoroideae): A Neotropical group with its center of diversity on the Guiana Shield[END_REF] inflorescences are axillary in Chalepophyllum Hook.f., Maguireothamnus Steyerm., Neblinathamnus Steyerm. and Steyermarkia Standl., or terminal in Dendrosipanea Ducke, Limnosipanea Hook.f., Neobertiera Wernham, Pteridocalyx Wernham, Sipanea Aubl. and Sipaneopsis Steyerm., without exceptions within each genus. In the Sipaneeae [START_REF] Delprete | A phylogenetic study of the tribe Sipaneeae (Rubiaceae, Ixoroideae), using trnL-F and ITS sequence data[END_REF][START_REF] Delprete | Monograph of tribe Sipaneeae (Rubiaceae, Ixoroideae): A Neotropical group with its center of diversity on the Guiana Shield[END_REF], stamens presentation is an important diagnostic-synapomorphic character, as they are well exserted beyond the corolla only in Limnosipanea, or included or partially exserted in the other genera of the tribe. The list could go on, but we refrain from citing more examples, as we wish to cite only a few cases where certain morphological characters have traditionally been used and continue to be used by Rubiaceae specialists to distinguish genera demonstrated to be monophyletic by molecular phylogenies within the same tribe.

Greuter & Rankin-Rodríguez (2022a: 211) stated that "As traditionally circumscribed, Exostema is characterized by a capsular fruit and flattened, winged seeds; and by a salvershaped (hypocrateriform), 4-5-merous corolla with a narrow tubular basis, long, linear, ± recurved lobes, and widely exserted stamens with linear, basifix anthers." In their broadly delimited Exostema, they included Hintonia, which has 6-8-merous flowers with campanulate corollas, Coutareopsis, which has 5-7-merous flowers, Adolphoduckea, which has 6-merous flowers, and Coutarea, which has 6-merous, zygomorphic flowers with campanulate, gibbous corollas and unequal stamens. They proceeded by explaining further significant morphological traits peculiar to certain clades as shifts in "reproductive strategies (to pollination by hummingbirds or bats rather than moths; or to water rather than wind dispersal)" and made wild unsupported assumptions in attributing the sets of morphological features that characterize each clade to shifts in pollination strategy. As a result of the "logic" presented in their article, Greuter & Rankin-Rodríguez divided their widely delimited Exostema into five sections. The genera of Clade B recognized by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], treated as sections of Exostema by Greuter & Rankin-Rodríguez, with their geographical distribution, ecology, and main morphological characters, are summarized in Table 1.

Greuter & Rankin-Rodríguez (2022a: 213) explained the wide morphological variation within their broad delimitation of Exostema sect. Brachyanthum by saying "All [the taxa] share the flattened, winged seeds characteristic of our redefined Exostema, but they show pronounced diversity in flower characters, presumably linked with pollinator shifts. In terms of reproductive biology, this indicates adaptation to bee or moth pollination, and wind dispersal. It is to be expected that a shift in reproductive strategies (to pollination by hummingbirds or bats rather than moths; or to water rather than wind dispersal) may entail quantum changes in correlated morphological characters; such shifts are bound to make the genus less uniform and more difficult to define. This is what we assume has happened in Exostema: A pollinator shift has likely occurred at the basis of E. sect. Hintonia, and two independent such shifts appear to have happened within E. sect. Brachyanthum." This assertion of a broad suite of character changes consequent of a pollination syndrome shift is stated with no reference to scientific evidence. Hence, they supposed that the wide morphological variation present in their broadly delimited Exostema is mainly due to shift in pollination syndromes, without taking into account the strong set of additional morphological features that characterize each clade within Clade B [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], as inflorescence position, flower merosity, corolla symmetry, ovary placentation, style morphology, among many others.

If we follow the same line of thinking that Greuter & Rankin-Rodríguez (2022a) applied in defining genera within Clade B, to be consistent with their interpretation in all the clades of the tribe Chiococceae, within clade C of the phylogeny presented in figure 3 of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], we will have to synonymize Cubanola Aiello, Osa Aiello, Nernstia Urb., Catesbaea L., Portlandia P.Browne, and Isidorea A.Rich. ex DC. under a sole genus. [START_REF] Aiello | A reexamination of Portlandia (Rubiaceae) and associated taxa[END_REF] segregated Osa from Hintonia, and Cubanola and Nernstia (as Cigarrilla Aiello) from Portlandia based mainly on placentation and seed morphology, and the monophyly of all those genera has been shown to be strongly supported in all subclades of Clade C of Paudyal & al. (2018: fig. 3), confirming the taxonomic value of those morphological characters. For example, Portlandia and Isidorea are positioned on a strongly supported clade (BPP 1) as sister taxa, and their morphological differences are only vegetative characters, which are synapomorphic and diagnostic for each genus. Portlandia (subclade C4, BPP 1) is a genus of six species endemic to Jamaica [START_REF] Aiello | A reexamination of Portlandia (Rubiaceae) and associated taxa[END_REF][START_REF] Delprete | Portlandia proctorii (Rubiaceae, Catesbaeeae), a new combination for a narrow endemic Jamaican species[END_REF] with broadly triangular stipules and nonpungent leaves. Isidorea (subclade C5, BPP 89) is a genus with about 15 species endemic to Cuba and Hispaniola, and differs from Portlandia in having stiff, pungent leaves, and stipules divided at the base into two parts, looking like four apically pungent stipules per node [START_REF] Aiello | A reexamination of Portlandia (Rubiaceae) and associated taxa[END_REF]. The two genera are nearly identical in capsule shape, dehiscence and placentation, and seed morphology. The corolla shape and size vary within each genus and do not present any particular feature to separate the two genera, with the exception that they are generally larger, 5-22 cm long in Portlandia, and smaller, 1.2-4 cm long in Isidorea. However, Portlandia proctorii (Aiello) Delprete has corollas 2.5-5.4 cm long, which are the smallest in the genus, and have intermediary dimensions between the two genera [START_REF] Liogier | Flora de Cuba, vol. 5, Rubiales -Valerianales -Cucurbitales -Campanulales -Asterales[END_REF][START_REF] Liogier | La flora de la Española[END_REF][START_REF] Aiello | A reexamination of Portlandia (Rubiaceae) and associated taxa[END_REF][START_REF] Delprete | Portlandia proctorii (Rubiaceae, Catesbaeeae), a new combination for a narrow endemic Jamaican species[END_REF]. Apparently, Greuter & Rankin-Rodríguez treated those vegetative features as sufficient for generic delimitations, as they recognized Portlandia and Isidorea as two distinct genera in their checklist of Cuban plants (Greuter & Rankin-Rodríguez, 2022b). Eriksson & al. (2022: 494) in an article discussing several scenarios in delimiting genera within the tribe Potentillinae (Rosaceae) wrote: "The International Code of Nomenclature for algae, fungi, and plants governs current plant nomenclature and describes how to name groups of plants assigned to ranked categories [START_REF] Turland | International Code of nomenclature for algae, fungi, and plants (Shenzhen Code): Adopted by the Nineteenth International Botanical Congress[END_REF]" and cited the standards detailed by [START_REF] Backlund | To be or not to be -Principles of classification and monotypic plant families[END_REF] as follows: "maximising stability, phylogenetic information, support for monophyly, and ease of identification. These criteria have been followed in the Angiosperm Phylogeny Group classifications (Angiosperm Phylogeny Group, 1998), with group size as an additional consideration, and we think it is fair to state that the monophyly of taxa above the species level as primary classification principle is now well established, although not universally accepted (e.g., [START_REF] Hörandl | Paraphyletic versus monophyletic taxa -Evolutionary versus cladistic classifications[END_REF][START_REF] Ertter | Potentilla Linnaeus[END_REF]." Here, we wish to address [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) wide delimitation of Exostema and their proposal to reject Coutarea vs. Exostema, in vision of the four secondary principles proposed by [START_REF] Backlund | To be or not to be -Principles of classification and monotypic plant families[END_REF] and presented by Eriksson & al. (2022: 494).

Maximizing stability. -Both the wide delimitation of Exostema and the proposal to reject Coutarea vs. Exostema published by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) are already causing and will continue to produce disruption in nomenclatural stability of traditional and current usage of generic names within the Chiococceae. With their wide delimitation of Exostema, they disregarded significant morphological features that are commonly used as diagnostic characters by Rubiaceae specialists for delimitation of rubiaceous genera, as inflorescence position, flower merosity, ovary placentation, corolla symmetry, style morphology, seed morphology, among many others. There are several recent examples of acceptance of the genera of Clade B as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. [START_REF] Borhidi | Addiciones y correciones a la monografía Rubiáceas de Cuba[END_REF] in an article dealing with additions and corrections to their floristic treatment of Cuban Rubiaceae [START_REF] Borhidi | Rubiáceas de Cuba[END_REF], followed all generic delimitations proposed by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] 2023) recognized the South-American genera Adolphoduckea, Coutareopsis, and Motleyothamnus as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. The multi-authored internet site Flora e Funga do Brasil (2023) accepted Adolphoduckea and Coutarea occurring in Brazil as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]. The website Plants of the World Online (POWO, 2023) accepted all the genera of the Chiococceae as delimited by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF].

Phylogenetic information. -The reduction of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] entire clade B to the single genus Exostema, as proposed by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a), entails the lumping of six genera (Coutarea, Adolphoduckea, Coutareopsis, Motleyothamnus, Hintonia, Solenandra). This extreme synonymisation decreases the value of the diagnostic information of each monophyletic genus present in the clade.

Greuter & Rankin-Rodríguez's reduction of the genera of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] to sections of Exostema is also not an optimal solution, as it reduces the systematic and morphological information of the individual species as members of monophyletic genera. For example, in most floristic treatments, sections of genera are usually not contemplated, and the usage of a broadly polymorphic Exostema will diminish the diagnostic value of the morphological characters that distinguish internal monophyletic groups.

As Eriksson & al. (2022: 499) emphasized, "Genus-level classification is important in this context, because it reflects on the species names, the general expectation being that the species with the same genus name are closest relatives." Those authors, after considering several scenarios about recognizing genera within the Potentillinae (Rosaceae) phylogenies obtained, opted to name Potentilla clade C because it provides "the most stable and the least disruptive Potentilla in a monophyletic classification of Potentillinae, and it is our view that clade C is the best candidate for the Potentilla genus name. […]. The species of clade C are easily distinguished from the species of its sister clade, the Anserina clade (the genera Argentina and Tylosperma), which have 'ventral stipular auricles' of basal leaves […]. This means that the Anserina clade needs to be classified separately, and we favor the recognition of a well-diagnosed Argentina (herbaceous perennials with interruptedly pinnate leaves and yellow flowers) and its sister Tylosperma (whiteflowered low shrubs with pinnate leaves)" (p. 502).

Numerous other authors followed the basic principles enunciated by [START_REF] Backlund | To be or not to be -Principles of classification and monotypic plant families[END_REF] in delimiting genera according to the molecular phylogenies obtained. [START_REF] Razafimandimbison | Molecular phylogenetics and generic assessment in the tribe Morindeae (Rubiaceae-Rubioideae): How to circumscribe Morinda L. to be monophyletic?[END_REF], assessing the molecular phylogenies obtained and generic assessment of the tribe Morindeae (Rubiaceae), after considering several scenarios stated "We favor […] the recognition of the four major lineages (A-D in Fig. 1) as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in Morindeae and the phylogenetic and taxonomic distinctness of its newly delimited genera" (Razafimandimbison & al., 2009: 884). [START_REF] Ji | Phylogeny and classification of Paris (Melanthiaceae) inferred from DNA sequence data[END_REF] evaluating genus delimitations within the family Melianthaceae also followed the general principles of classification outlined by [START_REF] Backlund | To be or not to be -Principles of classification and monotypic plant families[END_REF], and stated that "a genus should be not only monophyletic with strong statistical support, but should also be recognizable from morphological characters" (p. 254), and they delimited three genera according to the strongly supported clades, recognizable by significant sets of morphological characters.

Another example is that of [START_REF] Mello-Silva | Five vicarious genera from Gondwana: The Velloziaceae as shown by molecules and morphology[END_REF], who, in assessing the delimitation of genera within the family Velloziaceae to interpret molecular phylogenies and morphological datasets, stated that "Taxonomic decisions about lumping versus splitting were based on the priorities as discussed in Backlund & Bremer (1998)" (p. 88). Consequently, [START_REF] Mello-Silva | Five vicarious genera from Gondwana: The Velloziaceae as shown by molecules and morphology[END_REF] delimited genera within the family according to the basic principles enunciated by Backlund & Bremer "for reasons of maximizing phylogenetic information and ease of identification" (p. 97).

Support for monophyly. -All the internal clades of clade B (Fig. 1) retrieved by Paudyal & al.'s (2018: fig. 3) 8 Version of Record are well-to strongly supported, and each one is characterized by a unique set of morphological characters. In Table 1 are summarized the genera recognized by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] in Clade B, compared with the Exostema sections proposed by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a), with corresponding number of species, geographic distribution, ecology, and main morphological characters.

It should be emphasized that Clade B3 of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] is strongly supported (BPP 1) and divided into two subclades; in one subclade, also strongly supported (BPP 1), the authors recognized the genera Coutareopsis and Motleyothamnus. The other subclade of Clade B3 is also strongly supported (BPP 1) and is divided into two smaller clades, one with Coutarea hexandra, and the other with Exostema maynense, which was transferred by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] to the monospecific Adolphoduckea. Coutarea hexandra, a shrub, medium-sized tree, or a tall canopy tree, distributed from Mexico to Argentina, is the only genus with zygomorphic flowers in the Chiococceae, and is additionally characterized by its campanulate, gibbous corollas, well-exserted, unequal stamens, bending towards the gibbous side of the corolla, stigma in two lines along the style, capsules that are elliptic, oblong, obovate to round in outline and laterally compressed, and broadly winged seeds vertically arranged and laterally inserted. Whereas, Adolphoduckea is a rainforest tree of western Amazon and eastern Andean slopes from Ecuador, Peru, Bolivia and Brazil, with actinomorphic flowers, with a narrowly cylindrical corolla tube and linear lobes, style clavate or capitate, weakly laterally compressed capsules, and vertically imbricate, acrobasipetally aligned seeds. Aside from the numerous morphological differences between the two taxa, which are summarized in Table 1, Adolphoduckea was established to maintain the traditional usage of the name Coutarea hexandra, which applies to the most widespread species in Clade B of [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF].

Ease of identification. -The broad delimitation of Exostema proposed by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) does not fulfill the principle of maximizing the ease of identification of taxa within clade B. Indeed, the genus Exostema is no longer diagnosed by the exserted stamens for which it was originally named. The diagnostic power of the morphological characters presented by [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] is masked by the complex and mixed features of the polymorphic, broadly expanded Exostema delimitation by Greuter & Rankin-Rodríguez. For example, within clade B, axillary inflorescences are present only in Hintonia and Exostema sensu [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF]; 6-merous flowers are characteristic of Adolphoduckea, Coutarea and Coutareopsis (5-7-merous); 6-8-merous flowers with campanulate, actinomorphic corolla would immediately identify Hintonia species; and the only zygomorphic flowers in the group are those of Coutarea. There are many additional diagnostic characters that can be used to identify the genera in Clade B, such as those summarized in Table 1.

If the broad delimitation of Exostema proposed by Greuter & Rankin-Rodríguez (2021, 2022a) is followed, it would result in a genus of about 40 species, ranging throughout and slightly outside the Neotropics (e.g., southern Florida, northern Mexico, southern Brazil, northern Argentina), growing in dry, moist or wet vegetation, from sea level to above 1900 m altitude, of shrubs, medium-sized trees, tall canopy trees, or shrubs or treelets with scandent lateral branches, with axillary or terminal, single-, few-or many-flowered, subfasciculate, subumbellate, cymose, or paniculate inflorescences, 4-8-merous, actinomorphic or zygomorphic flowers, with several flowering strategies and pollination syndromes, tubular, funnelform or salverform, white, pinkish-white, pink, red, violet, lavender or purple corollas, with included, partially exserted, exserted or well-exserted stamens that can be equal or unequal in length, narrowly oblong or linear anthers, stigmatic portion present on two ovate, oblong or linear lobes, or on a clavate or capitate head, or as two lines along the style, ellipsoid, subspherical, subcylindrical, obovate or round in outline capsules that can be round, slightly or strongly laterally compressed in cross section, ovaries with a linear, narrowly ellipsoidal, lanceolate or trapezoidal placentas, and seeds that are vertically, acropetally, centripetally or horizontally arranged, and parallel or imbricate with each other. We are left wondering about the practical utility of dealing with such a polymorphic genus by the international community specialized in, for example, ecology, floristics, and conservation.

■ CONCLUSIONS AND RECOMMENDATIONS

Summarizing what has been presented above: (1) The wide expansion of Exostema and the proposal to reject Coutarea vs. Exostema published by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) are already causing and will continue to produce disruption in nomenclatural stability of traditional and current usage of generic and specific names within the Chiococceae; (2) The broad synonymisation under Exostema s.l. proposed by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) entails the lumping of six genera (Adolphoduckea, Coutarea, Coutareopsis, Hintonia, Motleyothamnus, Solenandra) under a broadly distributed, widely polymorphic genus, decreasing the value of the diagnostic information of each monophyletic taxon present in [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] Clade B and reduces the systematic and morphological information of the species, as members of smaller monophyletic genera; (3) All the internal clades of clade B retrieved in [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] phylogenies are well-to strongly supported, and each of them corresponds to a genus characterized by a unique set of morphological characters; (4) [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF] genera have been accepted by numerous Rubiaceae specialists and managers of specialized websites; (5) The broad circumscription of Exostema proposed by [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF], 2022a) does not fulfill the principle of maximizing the ease of identification of the c. 40 species included in such a highly polymorphic genus, and therefore increases the difficulty of species identification by the botanical community.

Taking into account all of the above considerations, which are in agreement with the basic "principles of classification" followed by the Angiosperm Phylogeny Group (Angiosperm Phylogeny Group, 1998), we advise the Nomenclature Committee to reject [START_REF] Greuter | Proposal to conserve the name Exostema against Coutarea (Rubiaceae)[END_REF] proposal to conserve the generic name Exostema against Coutarea.

Greuter & Rankin-Rodríguez should be encouraged to reconsider their wide expansion of Exostema and avoid producing unnecessary new combinations, which most likely will be treated as synonyms by Rubiaceae specialists. Whether the proposal to reject Coutarea vs. Exostema proposed by Greuter & Rankin-Rodríguez ( 2021) is recommended or not, and whether those authors will eventually proceed in publishing new combinations in either Coutarea or Exostema, to our knowledge, most Rubiaceae specialists will continue to recognize the genera of Clade B as delimited [START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], with the obvious disruption in nomenclatural stability and in the traditional usage of generic names in this group.

Fig. 1 .

 1 Fig. 1. Clade B of the majority-rule consensus tree of Chiococceae from the Bayesian inference analyses of the combined dataset (ETS, ITS, petD, trnL-F) obtained by Paudyal & al. (2018). Numbers above branches represent Bayesian posterior probability values (BPP) and numbers below branches represent bootstrap values. Bootstrap values are given only for the deeper nodes and nodes with taxonomic implications that are discussed in this paper. For information regarding material and methods, vouchers, and for consultation of the whole tree, see Paudyal & al. (2018).

  transferred E. corymbosum to the monospecific genus Motleyothamnus Paudyal & Delprete, and Coutarea fuchsioides, C. andrei and C. coutaportloides C.M.Taylor (the latter species was not included in the phylogenetic study) to the genus Coutareopsis Paudyal & Delprete, with the necessary new combinations.

  and recognized the genera Solenandra sensu Paudyal & Delprete, Exostema sensu Paudyal & Delprete, Adolphoduckea, and Motleyothamnus. Torres-Montúfar & al. (2022) in a synthesis of members of the Chiococceae present in Mexico, recognized the genera Coutarea, Exostema, Hintonia, and Solenandra as delimited by Paudyal & al. (2018). Torres-Montúfar & al. (

Table 1 .

 1 Comparison of genera of Clade B as recognized by[START_REF] Paudyal | Molecular phylogenetic analysis and generic delimitations in tribe Chiococceae (Cinchonoideae, Rubiaceae)[END_REF], with reference to Greuter & Rankin Rodríguez's (2022a) sectional classification of Exostema, and corresponding number of species, geographic distribution, elevation, ecology, habit, and main morphological characters. Significant diagnostic features are in bold.

	Solenandra sect.	Solenandra	Exostema sect. So-	lenandra			1 2		Mexico and Cen-	tral America (S.	mexicana), Cuba,	Hispaniola,	Jamaica		From near sea	level to 1500 m	Rocky mountain	slopes and moun-	tain forests		Shrubs, or trees to	15 m tall	Terminal	Cymose or	paniculate	5-merous	(Continues)
	Solenandra sect.	Pitonia	Exostema sect.	Pitonia			1 0		Cuba, Hispan-	iola, Jamaica,	and Lesser An-	tilles			From near sea	level to 1000 m	Rocky sites and	moist forests			Subshrubs 0.5-	1 m tall, shrubs,	or trees to 12 m	tall	Terminal	Cymose or pa-	niculate	5-merous
		Hintonia	Exostema sect.	Hintonia			3		From northern	Mexico to	Costa Rica				100-800 m		Deciduous,	semi-deciduous	or mesophyl-	lous forests	Shrubs or trees	to 10 m tall	Axillary	Single-flow-	ered	6-8-merous
		Coutareopsis	Exostema sect.	Brachyanthum			3		Andes of	Ecuador and	Peru				Above 1900 m		Dry vegetation				Shrubs 1.5-5 m	tall	Terminal	Subfasciculate,	subumbellate or	cymose	5-7-merous
		Coutarea	Exostema sect. Bra-	chyanthum			2		Throughout the Neo-	tropics, from Mexico	to Argentina				Lowland to 300 m		Deciduous or ever-	green forests (C. alba	dry shrublands)		Shrubs or trees, or	trees to 30 m tall and	40 cm dbh in Ama-	zon Basin	Terminal	Simple or compound	dichasia	6-merous
		Adolphoduckea	Exostema sect.	Brachyanthum			1		From Ecuador to	Bolivia, low eleva-	tions on eastern	slopes of the Andes	and contiguous Am-	azon Basin	120-500(-1100) m		Rainforest				Trees 7-12 m tall, or	canopy trees to 30 m	tall and 100 cm dbh	in Amazon Basin	Terminal	Cyme	6-merous
		Motleyothamnus	Exostema sect.	Brachyanthum			1		Andes of Peru						1000-2800 m		Open places and	shrublands			Shrubs or small	trees, 1-6 m tall	Terminal	Compound cyme	5-merous
		Exostema	Exostema sect.	Exostema			8		Cuba and Hispaniola	(E. caribaeum also	in other Antilles,	southern Florida,	Mexico, and Central	America)	From near sea level	to 900(-1200) m	Open rocky sites				Shrubs or small	trees, 1-6(-10) m	tall	Axillary	Simple cyme	4-5-merous
			Greuter &	Rankin-	Rodríguez's	classification	Number of	species	Geographic	distribution					Elevation		Ecology				Habit		Inflorescence	position	Inflorescence	architecture	Flower	merosity
													Version of Record					3

Table 1 .

 1

	Solenandra sect.	Solenandra	Fragrant during	the day (corollas	0.6-2.2(-3) cm	long)	Actinomorphic		Exserted			Equal		Clavate or subca-	pitate		Subcylindrical to	cylindrical or	oblanceolate,	round in cross-	section	Basipetally	aligned, vertically	imbricate
	Solenandra sect.	Pitonia	Fragrant at night	(corollas 4-	21 cm long)		Actinomorphic		Exserted			Equal		Clavate or sub-	capitate		Subcylindrical	to cylindrical or	oblanceolate,	round in cross-	section	Acropetally or	centripetally	aligned, verti-	cally imbricate
		Hintonia	Fragrant from	the afternoon to	the evening		Actinomorphic		Included or	partially ex-	serted	Equal		Stigma in two	lines along the	style	Ellipsoid to	subspherical,	terete, round in	cross-section	Horizontally	or basipetally	imbricate
		Coutareopsis	Not reported	(probably not	fragrant)		Actinomorphic		Included or par-	tially exserted		Equal		Style branches	ovate to oblong		Elliptic to ob-	ovate in outline,	weakly laterally	compressed	Vertically	imbricate
		Coutarea	Fragrant from the	afternoon to the	evening		Zygomorphic		Well exserted			Unequal, sigmoidal		Stigma in two lines	along the style		Elliptic, oblong, ob-	ovate to round in	outline, strongly lat-	erally compressed	Vertically imbricate
		Adolphoduckea	Fragrant during the	day			Actinomorphic		Exserted			Equal		Clavate to subcapi-	tate		Obovate in outline,	weakly laterally	compressed	Acrobasipetally	aligned, vertically	imbricate
		Motleyothamnus	Very fragrant	during the day			Actinomorphic		Exserted			Equal		Capitate			Obpyriform to	round in outline,	strongly	laterally	compressed	Acrobasipetally	arranged,	vertically	imbricate
	Continued.	Exostema	Flower Fragrant during the	fragrancy day			Corolla Actinomorphic	symmetry	Stamens Exserted or partially	presentation exserted		Stamens Equal	proportion	Style shape Clavate or	subcapitate		Capsule Obovate in outline,	shape slightly laterally	compressed	Seeds Basipetally,	position acropetally or	centrally inserted
	4									Version of Record

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A Mouttou

F Lemarchand

M Gourdelier

C Koc

A Moreau

D Muriaux

J Lumeau

C Favard
email: cyril.favard@irim.cnrs.fr

A L Lereu
email: aude.lereu@fresnel.fr

Resonant dielectric multilayers improving fluorescence imaging

We present dedicated dielectric multilayers (DM) optimized to be resonant under TIRFmicroscopy constraints. By investigating DM enhancement and transmission, we could predict and measure a fluorescence signal enhanced by a factor of 3.

Introduction

In thin films-based nanophotonics, field enhancement and confinement are classically used for sensing or imaging application. In the framework of imaging, surface plasmon resonances have been used, a prism-based configuration [START_REF] Lakowicz | Radiative decay engineering 3. Surface plasmon-coupled directional emission[END_REF], and in an objective-based configuration [START_REF] Le Moal | Enhanced fluorescence cell imaging with metal-coated slides[END_REF]. Large fluorescence signal enhancements were evidenced over model fluorescent molecules and fluorescently marked cells respectively. However, the use of metallic thin films implies limitations in terms of illumination conditions or due to quenching effects by the coupling fluorophores/metals. Capitalizing upon strong and sharp optical resonances supported by Bloch surface waves in resonant dielectric multilayers (DM), [3 -4] have introduced such concept in fluorescence imaging.

In Total Internal Reflection Fluorescence microscopy (TIRF-M), these resonant effects can act on the local evanescent field involved to improve the fluorescence signal and limit the background noise. Indeed, the major drawbacks in TIRF-M are: 1-its angular divergence leading to an illumination mixing propagative and evanescent waves and 2-the strong evanescent intensity decrease as function of the incident angle. All these degrade TIRF-M performances in terms of fluorescence intensity and signal to noise ratio. To overcome these issues, a possible solution is to locally enhance the evanescent field intensity. However, theoretical and experimental disagreements are usually reported with DM. We thus introduced an innovative resonant DM adapted to the Total Internal Reflection Fluorescence microscopy (TIRF-M) constraints [START_REF] Mouttou | Resonant dielectric multilayer with controlled absorption for enhanced total internal reflection fluorescence microscopy[END_REF]. We present here the numerical and experimental results obtained in all aspects from optimisation, fabrication, to TIRF-M implementation.

Material and methods

Experimental set-up

The experimental setup is based on a commercial microscope (Nikon Eclipse Ti) supplied with a 100x 1.49 NA objective and a coupled to 561 nm TE-polarized laser (See Fig. 1). In this inverted configuration, the fluorescence emission is collected through the objective before being directed to an EM-CCD camera and recorded via the implemented MetaMorph software. The laser excitation residual intensity is also filtered thanks to dichroic mirror and a bandpass filter. As schematized in Fig. 1, the excitation beam is focused at the back focal plane of the objective, with an incident angle controlled by tuning the radial position of the focused beam on the back focal plane. With this system, the incident angle at the coverslip free interface can be changed from 0 to 81.

Dielectric multilayers optimization

The DM were optimized using the absorption-based optimization reported in [6 -9]. The main inconvenient here is the resulting strong and sharp resonance that is not adapted to TIRF-M application. Therefore, we developed a dedicated optimization method in order to consider the intrinsic angular divergence and the fluorescence collection through the objective (i.e. DM transmission efficiency). We optimized these dielectric multilayers for a TEpolarized laser of 561 nm at an incident angle of 68°. In our optimization, we specified the spectral (excitation: exc=561 nm and emission: em ranging from 550 to 700 nm) and angular (beam incident angle: inc=68 and photons collection angle: coll ranging from 0 to 81) ranges together with a maximization of the electric field at the free interface of the thin film stack in contact with the biological medium.

Dielectric multilayers deposition

The designed DM substrates were fabricated plasma assisted reactive magnetron sputtering method (Helios -Bühler/Leybold Optics). They are deposited on a 170 µm thick glass coverslip (Menzed-Gräzer, VWR) adapted to objective-based TIRF-M. To obtain a direct comparison of the field enhancement between the DMs and a bare glass coverslip, the coatings were done over half of the glass surface (see figure 1). The resulting DM are made of alternating low (SiO2) and high (Nb2O5) refractive indices materials with a last dissipating layer. Note that the refractive indices of each material are determined by spectrophotometry between 400 to 2400 nm over deposited single layers to be used in all numerical simulations (for structure optimizations to optical response predictions).

Results

Numerical investigations

By numerical analysis, we estimated an electric field enhancement several hundred times higher than the one on glass surface under plane wave conditions. However, when using an objective-based TIRF-M, both the instrumental characteristics such as the beam angular divergence (acting on the excitation field enhancement) and the fluorescence collection scheme (linked to the transmission efficiency of the DM) has to be considered. Under such conditions, the field enhancement at the free interface is highly defaced and the transmission efficiency of the DM adds an additional decrease on the resulting fluorescence enhancement.

We developed numerical studies to predict the enhancement factor of fluorescence by taking into account these two issues. We estimated a loss of more than 95% in the enhancement factor of the fluorescence signal. However, when measuring the fluorescence signal over 200 nm beads, we still observed large disagreements between the predictions and the experiment. Another parameter of importance over such resonance is the extinction coefficient (k~10 -5 ) of the dissipative layer i.e. top layer. We numerically evidenced that a small variation of k will provokes a strong decrease of the field enhancement. Therefore, we tackled the problem differently and conceived DM with a dissipating top layer of measurable and reproducible k to fully control its absorption.

Experiment

With this improved design, a fluorescence enhancement of factor of 4 is predicted by taking account the beam divergence and the transmission efficiency. Experimental measurements, realized on a commercial TIRF-M using fluorescent 200 nm beads were then carried out as shown in Fig. 2. We compared beads fluorescence signals on the glass surface Fig. 2 (a) and the DM surface Fig. 2 (b). We measured a fluorescence signal 3 times higher with the DM together with an improvement of signal to noise ratio by a factor 3.

Conclusion

We designed resonant dielectric multilayers coated coverslip adapted for TIRF imaging application by acting on the local excitation field in order to enhance the fluorescence intensity. These DM can be designed for any excitation wavelengths and incident angles which permit to consider illumination at larger incident angles already improving the axial resolution. To consider the TIRF-M experimental constraints and get a good agreement between the predicted and measured fluorescence signal, we developed dedicated numerical analysis methods. We successfully demonstrated an increase of the fluorescence signal by a factor 4 with respect to classical glass surface, which is in agreement with the observed experimental enhancement factor of 3. We now pursue optimisation steps to achieve high fluorescence enhancement. This study will impact the field of fluorescence-based microscopies by improving the lateral resolution and speed of acquisition, allowing fast super-resolution imaging on simple microscopes.

Fig. 1 :

 1 Fig. 1: Scheme of an Objective-based TIRF microscopy with a resonant dielectric multilayer coated coverslip inducing an enhanced evanescent field.

Fig. 2 :

 2 Fig. 2: TIRF images of fluorescent beads excited with a laser of 561 nm at an incident angle of 68° on glass surface (a) and DM coated surface (b) where the fluorescence signal is enhanced with a factor of 3.

Acknowledgements

The authors acknowledge the CNRS for financial supports through the MITI -Mission for Transversal and interdisciplinary Initiatives 2017 and the 80|PRIME interdisciplinary program 2019. A. Mouttou is a recipient of a CNRS Ph.D. grant.