Laura Lagourgue
email: laura.lagourgue@ird.fr

Nicolas Puillandre

Claude Payri

Exploring the Udoteaceae diversity (Bryopsidales, Chlorophyta) in the Caribbean region based on molecular and morphological data

Keywords: Bryopsidales, Udoteaceae, Udotea, species delimitation, Multilocus phylogeny, Morphological clustering Declarations of interests: none

The Udoteaceae family (Bryopsidales, Chlorophyta) is known to be highly diverse morphologically in the Caribbean region, but only few studies have studied its genetic diversity. Using an integrative taxonomic approach, this study aimed at (1) exploring the Udoteaceae species diversity using a combination of five DNA-based species delimitation methods and morpho-anatomical data for confirmation; (2) estimating the discriminatory power of traditional diagnostic characters using a morphology-based clustering method and statistical analyses focused on the genus Udotea; and (3) reconstructing the phylogeny of the family based on a multilocus analysis (tufA, rbcL, 18S rDNA). Our results revealed strong congruence between species hypotheses across delimitation methods and markers. Morpho-anatomical characters proved essential to validate these hypotheses, to assign species names and to unveil new species. Morphological analyses led to relevant results for accurately discriminating Udotea morphospecies. Siphon features and cortication were key characters to define supra-specific groups and to revise the taxonomy of the genus Udotea. Phylogenetic analyses confirmed the polyphyly of Udotea, Rhipocephalus and Penicillus, which led us to propose a revised definition of Udotea sensu stricto based on both genetic and morphological data. Finally, our study emphasizes the importance of combining genetic and morphological data for the taxonomic revision of the Udoteaceae, but stresses the need of including more taxa from other geographical regions to better resolve taxonomic issues. Highlights:

 A species delimitation approach combining distance and tree-based methods is proposed  High congruence is reached between methods and chloroplast markers  A morphology-based clustering method proved relevant for discriminating the morphospecies in the genus Udotea  Udotea, Penicillus and Rhipocephalus revealed polyphyletic in our multilocus phylogeny  The genus Udotea sensu stricto is redefined based on congruent genetic and morphological data

INTRODUCTION

Udoteaceae is a family of green macroalgae belonging to the order Bryopsidales (Chlorophyta). It comprises both calcified and non-calcified species, and has a significant ecological function through its contribution to primary production and carbonate fluxes. The Udoteaceae are particularly diverse morphologically, the most among the bryopsidalean families. Their internal structure is siphonous and characterized by a unique giant multinucleate cell that ranges from simple siphonous filaments to complex multiaxial structures. They include tufts of uncalcified and free filaments (e.g., Chlorodesmis), brush-like calcified thalli (e.g., Penicillus) or calcareous compact fan-shaped blades (e.g., Udotea). Although they occur in the Mediterranean Sea, Udoteacean species are mostly tropical and particularly abundant and diverse in the Caribbean zone. Several authors have investigated the North Atlantic marine flora over the last decades (see [START_REF] Wynne | A checklist of benthic marine algae of the tropical and subtropical Western Atlantic : fourth revision[END_REF] for a review), and several species of Udotea were morphologically described by [START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF] and recorded by [START_REF] Collado-Vides | Una revisión taxonómica del género Udotea en el Caribe mexicano y cubano[END_REF] in their revision of the genus diversity from Cuba and Mexico. According to these works, only four out of the fourteen currently accepted Udoteaceae genera are present in the tropical western Atlantic, with one of them endemic to this region (Rhipocephalus). All genera included, twenty-four species have been listed, of which seventeen are only found in the tropical western Atlantic and ten are restricted to the Caribbean region. However, most of these species are based on morphological descriptions and genetic data is still unavailable or fragmentary, a common trend in the Udoteaceae. Indeed, since J. Agardh (1887) described the family, the taxonomy of the Udoteaceae has been established predominantly on morphological and anatomical traits [START_REF] Ernst | Beiträge zur Kenntris der Codiaceen[END_REF][START_REF] Farghaly | Algues Benthiques de la Mer Rouge et du bassin occidental de l'océan Indien[END_REF][START_REF] Gepp | The Codiaceae of the Siboga Expedition[END_REF][START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF]. The most significant taxonomical work on Udoteaceae is that of [START_REF] Gepp | The Codiaceae of the Siboga Expedition[END_REF] based on material sampled during the Siboga expedition to the East Indian archipelago. Life cycles and sexual reproduction patterns were also the subject of a few studies but have not been reviewed since the work of [START_REF] Meinesz | Connaissances actuelles et contribution à l'étude de la reproduction et du cycle des Udotéacées (Caulerpales, Chlorophytes)[END_REF] and [START_REF] Vroom | Cladistics of the Bryopsidales: A preliminary analysis[END_REF], the latter performed cladistics analyses on morphological, anatomical and reproductive characters. The first phylogenetic analysis has been realized using nuclear-encoded ribosomal DNA (nrDNA) [START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF]. This study emphasized the polyphyly of several genera among the Udoteaceae (Udotea, Penicillus, Chlorodesmis), which was later confirmed by [START_REF] Lam | Phylogenetic analyses of the Bryopsidales (Ulvophyceae, Chlorophyta) based on Rubisco large subunit gene sequences[END_REF], [START_REF] Curtis | Phylogenetic analysis of the large subunit rubisco gene supports the exclusion of Avrainvillea and Cladocephalus from the Udoteaceae (Bryopsidales, Chlorophyta)[END_REF] and [START_REF] Coppejans | Rhipidosiphon lewmanomontiae sp. nov. (Bryopsidales, Chlorophyta), a calcified udoteacean alga from the central Indo-Pacific based on morphological and molecular investigations[END_REF] based on the chloroplast rbcL marker, and by Verbruggen et al. (2009aVerbruggen et al. ( , 2009b) ) based on multilocus analyses. All the results, together with the presence of cryptic diversity and/or morphological variability, illustrate how challenging the study of Udoteaceae can be and emphasize the need for a taxonomic revision. Sequence-based species delimitation approaches are efficient tools for discriminating species and could help to better describe the Udoteaceae diversity and to resolve taxonomic ambiguities. A number of methods have been developed to detect discontinuities in DNA sequence variation associated with species boundaries. They have been applied at various taxonomic levels for the Phaeophyceae [START_REF] Montecinos | Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae)[END_REF][START_REF] Silberfeld | Species Diversity, Phylogeny and Large Scale Biogeographic Patterns of the Genus Padina (Phaeophyceae, Dictyotales)[END_REF][START_REF] Vieira | Toward an inordinate fondness for stars, beetles and Lobophora ? Species diversity of the genus Lobophora (Dictyotales, Phaeophyceae) in New Caledonia[END_REF], the Rhodophyta (e. g., [START_REF] Jesus | Species-delimitation and phylogenetic analyses of some cosmopolitan species of Hypnea (Rhodophyta) reveal synonyms and misapplied names to H.?cervicornis , including a new species from Brazil[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF][START_REF] Pardo | A Multilocus Species Delimitation Reveals a Striking Number of Species of Coralline Algae Forming Maerl in the OSPAR Maritime Area[END_REF] and the Chlorophyta [START_REF] Leliaert | DNA taxonomy in morphologically plastic taxa: Algorithmic species delimitation in the Boodlea complex (Chlorophyta: Cladophorales)[END_REF][START_REF] Sauvage | A metabarcoding framework for facilitated survey of endolithic phototrophs with tufA[END_REF][START_REF] Zou | Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta)[END_REF]. In this study, we have chosen five exploratory species delimitation methods: the Automatic Barcode Gap Discovery (ABGD, Puillandre et al., 2012), the General Mixed Yule Coalescent (GMYC) [START_REF] Pons | Sequence-based species delimitation for the DNA taxonomy of undescribed insects[END_REF] and its Bayesian implementation, bGMYC [START_REF] Reid | Phylogenetic estimation error can decrease the accuracy of species delimitation : a Bayesian implementation of the general mixed Yule-coalescent model[END_REF], the Poisson tree process model (PTP, [START_REF] Zhang | A general species delimitation method with applications to phylogenetic placements[END_REF], and the Multirate PTP (mPTP, [START_REF] Kapli | Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo[END_REF]. The association of these methods is interesting for the different assumptions they rely on. On the one hand, ABGD is a distance-based method which aims at identifying a barcode gap that delimits the intraspecific from the interspecific distances in the distribution of genetic distances. On the other hand, the (b)GMYC and (m)PTP methods use information from a phylogenetic tree and focus on branching and mutation rates to identify a shift between speciation (between species) and coalescence events (within species). While (b)GMYC rely on branching rates through time and thus require an ultrametric time-calibrated tree, (m)PTP relies on the number of substitutions along branches, and, in the case of mPTP, accounts for divergent intraspecific variation. The second group of methods (tree-based methods) assumes the monophyly of the delimited species, which is not the case of the distance-based methods such as ABGD. To converge towards robust species hypotheses, several authors strongly recommend using multilocus datasets and assessing the congruence between several methods [START_REF] Carstens | How to fail at species delimitation[END_REF][START_REF] Carstens | Estimating Species Phylogeny from Gene-Tree Probabilities Despite Incomplete Lineage Sorting : An Example from Melanoplus Grasshoppers[END_REF][START_REF] Dupuis | Multi-locus species delimitation in closely related animals and fungi: one marker is not enough[END_REF][START_REF] Leliaert | DNA-based species delimitation in algae[END_REF]Puillandre et al., 2012b;[START_REF] Rannala | The art and science of species delimitation[END_REF]. The analysis of non-genetic data is also recommended by many authors to discuss DNA-based species hypotheses [START_REF] Carstens | How to fail at species delimitation[END_REF][START_REF] Carstens | Estimating Species Phylogeny from Gene-Tree Probabilities Despite Incomplete Lineage Sorting : An Example from Melanoplus Grasshoppers[END_REF][START_REF] Fujita | Coalescent-based species delimitation in an integrative taxonomy[END_REF][START_REF] Talavera | Factors affecting species delimitations with the GMYC model : insights from a butterfly survey[END_REF][START_REF] Wiens | Species delimitation: new approaches for discovering diversity[END_REF]. Following these recommendations, we based our study on molecular data, using several genetic markers to propose Primary Species Hypotheses (PSHs) and infer their phylogenetic relationship, combined with morpho-anatomical data to propose Secondary Species Hypothesis (SSHs), following an integrative taxonomy strategy. Our objectives were to: (i) explore the diversity of the Udoteaceae in the Caribbean region, (ii) clarify species boundaries and (iii) produce a more accurate phylogeny of the family. To reach these objectives, we (i) applied the five species delimitation methods described above using two chloroplast markers; (ii) validated the resulting DNA-based species hypotheses using cladistic analyses of morpho-anatomical traits, and (iii) reconstructed the phylogeny of the family based on the analysis of the chloroplast tufA and rbcL, and the nuclear 18S rDNA markers.

MATERIAL AND METHODS

Sampling and study region

A total of 205 specimens of Udoteaceae from the Caribbean region were included in the study. Most of the samples (127) were collected in the Lesser Antilles from 10 islands, including 102 specimens from the scientific campaign PACOTILLES in 2015 (DOI, 10.17600/15005200) onboard the vessel Antea, and 25 specimens collected in 2014 in Guadeloupe (Supplementary Figure A.1). All our samples were collected by SCUBA from a total of 20 sites between the surface and 40m deep. Sites were georeferenced and habitats described using bathymetric and substrata descriptors. When possible, specimens were photographed on site prior to collection in order to document their in-situ habits. Samples were sorted, labeled and photographed while fresh. Subsamples were preserved in both 95 % ethanol and silica gel for later DNA analyses. Specimens were then pressed-dried as herbarium vouchers and are currently housed at NOU in New Caledonia. The other 78 specimens were collected and sequenced by collaborators of the National Museum of Natural History and the School of Biosciences of the University of Melbourne, which enabled us to extend our geographical coverage to most subregions of the Caribbean zone. The latter specimens are housed at PC in Paris and GENT respectively (herbarium abbreviations follow [START_REF] Thiers | Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff[END_REF], continuously updated).

DNA extraction, amplification and sequencing

From the 127 Udoteaceae specimens collected in the Lesser Antilles, 114 samples were successfully extracted using the DNeasy Plant Mini Kit (Qiagen Inc, Valencia, CA, USA) following a modified and optimized protocol based on the manufacturer's instructions (available on request). Sequences for two chloroplast and one nuclear genes were produced: tufA (approximately 800 base pairs -bp), rbcL (approx. 1400 bp) and 18S rDNA (approx. 1300 bp) using previously published primers [START_REF] Händeler | Slugs' last meals: molecular identification of sequestered chloroplasts from different algal origins in Sacoglossa (Opisthobranchia, Gastropoda)[END_REF][START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF][START_REF] Lam | Phylogenetic analyses of the Bryopsidales (Ulvophyceae, Chlorophyta) based on Rubisco large subunit gene sequences[END_REF]Verbruggen et al., 2009a) (Supplementary material, Table A .1). The rbcL and 18S rDNA markers were sequenced in two fragments. PCR reactions were conducted in a final volume of 25 µL including of 12.5 µL of AmpliTaq Gold 360 Master Mix (Applied Biosystems), 0.75 µL of dimethylsulfoxyde (DMSO), 1 µL of bovine serum albumin (BSA), 1 µL of each primer (10 µM), 2.5 µL of DNA, and 6.25 µL of ultra-pure water (PCR programs available on request). The Sanger sequencing reaction was carried out using 20 µL of PCR product by Genoscreen (Lille, FRANCE). Sequences were edited in Geneious version 7.1.9 (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] and aligned for each marker separately using the MUSCLE algorithm available in the software. In addition to the sequences produced during this study, Udoteaceae sequences obtained from collaborators (National Museum of Natural History: 42 tufA sequences, and the School of Biosciences, University of Melbourne: 33 tufA and 6 rbcL sequences) and GenBank (16 rbcL and 15 18S rDNA sequences) were added to the datasets. The detailed list of specimens used and the accession numbers are recorded in Supplementary material (Table A.2).

Trees inference for species delimitation

The species delimitation methods were used for the analysis of the two chloroplast markers, tufA and rbcL, known for their discriminatory power at the species level in green macroalgae [START_REF] Leliaert | DNA-based species delimitation in algae[END_REF][START_REF] Saunders | An evaluation of rbcL, tufA, UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae[END_REF]Verbruggen et al., 2009a). The 18s rDNA was not used for species delimitation due to the low intra and interspecific variability observed in preliminary analyses (results available upon request). Both Bayesian ultrametric and Maximum Likelihood (ML) trees were constructed to produce the two types of input required for tree-based delimitation methods. Identical sequences (numerous for the tufA dataset) were removed using the Collapsetypes v4.6 perl script [START_REF] Chesters | Collapsetypes[END_REF] to keep only one sequence for each haplotype, in order to prevent potential analysis bias (e.g., calculation of an infinite coalescent λ and over-partition of the dataset; see [START_REF] Pons | Sequence-based species delimitation for the DNA taxonomy of undescribed insects[END_REF] and [START_REF] Reid | Phylogenetic estimation error can decrease the accuracy of species delimitation : a Bayesian implementation of the general mixed Yule-coalescent model[END_REF] and to decrease computation time [START_REF] Kapli | Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo[END_REF][START_REF] Talavera | Factors affecting species delimitations with the GMYC model : insights from a butterfly survey[END_REF]. Codium taylorii P.C. Silva (Codiaceae, Bryopsidales), Caulerpa sertularioides (S.G. Gmelin) M. Howe (Caulerpaceae, Bryopsidales) and Avrainvillea lacerata J Agardh (Dichotomosiphonaceae, Bryopsidales) were used as outgroup taxa for tree reconstruction but removed before running the species delimitation analyses. Best substitution models were identified under the Akaike information Criterion (AIC) implemented in jModelTest 2 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF][START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] as follows: GTR+I+G for tufA and HKY+I+G for rbcL. Ultrametric trees were obtained using BEAST v. 1.8.3 (Drummond et al., 2012 ) through the CIPRES web portal [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF] from two independent runs of 50 and 120 million generations, starting from a random tree and sampled every 5,000 and 12,000 generations for tufA and rbcL, respectively. Because the null clock hypothesis was rejected for both datasets (Likelihood ratio test in MEGA 6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF]), the BEAST analyses were run under a relaxed log-normal molecular clock [START_REF] Drummond | Relaxed Phylogenetics and Dating with Confidence[END_REF] with a coalescent constant size tree prior as recommended by [START_REF] Michael | Accelerated Species Inventory on Madagascar Using Coalescent-Based Models of Species Delineation[END_REF]. The output of each run was checked in Tracer 1.5 [START_REF] Rambaut | Tracer version 1.5[END_REF] to confirm the convergence of the Markov Chains Monte Carlo (MCMC), with effective sample sizes all above 200. Outputs were then combined using Log Combiner (included in the Beast package), removing the first 10 % generations as burn-in. The Maximum Clade Credibility Tree (MCCT) was calculated using Tree Annotator (included in the Beast package). The maximum likelihood analyses were run using RAxML [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] through the CIPRES web portal [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF], using the rapid bootstrapping and search for best-scoring ML tree algorithm. A multi-parametric bootstrap resampling with 1,000 iterations provided bootstrap supports [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF]. The Bayesian MCCTs were used as the input tree for the GMYC method. The bGMYC (multitree) analysis was run using a subsample of 100 trees from the Beast analyses for each marker. The PTP method was applied to ML rooted trees and mPTP was applied on both ML trees and MCCTs. 2.4 Exploratory species delimitation and validation strategy ABGD was performed through the website (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html) and applied to both tufA and rbcL alignments. The rbcL marker was analyzed as two distinct datasets (rbcL5' and rbcL3') following the two-fragment scheme of sequencing, in order to avoid potential bias in the calculation of genetic distance due to different dataset sizes and/or lack of species representatives. Default values for all the parameters were used, except for the X value, for which the default value returned partitions with only one species. As advised on the ABGD website, we thus decreased the X value until we obtained partitions with more than one species: the X-value was set to 1 for rbcL5' and rbcL3', and 0.8 for tufA. The GMYC method was applied using the "splits" package under the R environment (R Development Core Team, 2017). For the likelihood ratio test, a P-value < 0.112 was seen as significant following the revised model of [START_REF] Fujisawa | Delimiting species using single-locus data and the Generalized Mixed Yule Coalescent approach: a revised method and evaluation on simulated data sets[END_REF]. The bGMYC method was also conducted in the R environment using the "bGMYC" package [START_REF] Reid | Phylogenetic estimation error can decrease the accuracy of species delimitation : a Bayesian implementation of the general mixed Yule-coalescent model[END_REF]. After exploratory tests, the method was run for 30,000 generations with the 15,000 first generations removed as burn-in for tufA, and 60,000 generations with the 20,000 first generations removed as burn-in for rbcL. Sampling was set every 100 generations for both analyses. The PTP method was carried out on the Exelixis Lab web server (http://sco.hits.org/exelixis/web/software/PTP/index.html). It was run for 500,000 generations with a thinning value of 100 and a burn-in of 10%. MCMC convergence was visually checked before the interpretation of the results as recommended by [START_REF] Zhang | A general species delimitation method with applications to phylogenetic placements[END_REF]. The mPTP analysis was run through the mPTP web server (http://mPTP.h-its.org) with the default settings. The assessment of the species hypotheses provided by ABGD, GMYC and PTP consisted in a search for common PSHs among the different methods. In case of conflict, the most prevalent partition (or PSHs) found among the methods was preferred (i.e., the partitions had to be shared at least by three out of the five methods) in order to define a SSHs. We then used morpho-anatomical traits to confirm the SSHs and assign species names.

Morpho-anatomical analyses

The morphological and anatomical observations were made on fragments of herbarium specimens decalcified in HCL solution (5%) and observed under a microscope (Olympus BH-2) equipped with an Olympus Camedia C-5050 digital camera (Tokyo, Japan). In order to validate the SSHs resulting from the species delimitation analysis, and to assign them species names, up to five specimens per SSHs were studied (depending on availability). Species identification followed the morphological keys of [START_REF] Gepp | The Codiaceae of the Siboga Expedition[END_REF] and [START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF]. The morpho-anatomical characters assessed in this study are summarized in Table 2. A morphology-based delimitation analysis was also performed. The aim was to evaluate the efficiency and robustness of the morpho-anatomical characters in discriminating morphospecies compared to DNA-based species delimitation methods. Here, we consider morphospecies as entities that can be identified based on a combination of both morphological and anatomical diagnostic criteria. The analysis was focused on Udotea species, because the genus is predominant in the Caribbean. The analysis was based on 57 individuals for which 22 morpho-anatomical variables (two quantitative and 20 qualitative) were compiled in an encoded matrix (see Supplementary data A.1). Information for type species and species not sampled in this study were extracted from [START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF] and added to the matrix. Based on this dataset, we performed a cluster analysis using the packages "cluster" and "ade4" in R. After exploratory tests, we applied a hierarchical clustering (hclust) analysis with the "average" cluster method and an Euclidean calculation of distances to discriminate the specimens. With the aim to obtain delimited clades of morphospecies, we applied a 95 % confidence clustering with an a priori number of species. Simultaneously, using the "pvclust" package in R [START_REF] Suzuki | Pvclust: an R package for assessing the uncertainty in hierarchical clustering[END_REF] and under the same parameters (dendrogram topologies were congruent between both methods), we estimated approximately unbiased p-values of the nodes as computed by multiscale bootstraps resampling with 1,000 bootstrap replications. To further observe the discriminating power of the diagnostic morpho-anatomical characters, a Hill-Smith analysis was carried out. This multivariate analysis performs with continuous and multi-state discrete characters [START_REF] Hill | Principal Component Analysis of Taxonomic Data with Multi-State Discrete Characters[END_REF]. All the variables were thus integrated in a single analysis, whether they are qualitative or quantitative. 2.6 Phylogenetic analysis of DNA sequences Phylogenetic trees were produced using maximum likelihood and Bayesian inference implemented on individual alignments as well as a concatenated multilocus matrix including tufA, rbcL and 18S rDNA sequences. The resulting concatenated dataset included only one representative per SSHs. Outgroup taxa were the same as those used in species delimitation analyses (Supplementary material, Table A.2). The best partition scheme for the dataset was identified using Partition Finder v1.1.0 under the Akaike Information Criterion (AIC). Results suggested a partition by genes and codon positions for the chloroplast protein-coding genes tufA (GTR+I, GTR+I, GTR+G) and rbcL (5' fragment: GTR+G, GTR+I, GTR+I+G; 3' fragment: JC, GTR+I, GTR+G), and a unique partition for the 18S rDNA (GTR+I+G). The Bayesian phylogenetic analysis was performed in MrBayes v.3.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF] through the CIPRES portal. Two independent runs of four incrementally heated chains were run for 50 million generations. The convergence of the Markov chain Monte Carlo (MCMC) and the ESS values were checked in TRACER v. 1.5 (Rambaut and Drummond, 2007). The 10 % first generations were discarded as "burn-in" prior to computing a consensus topology and posterior probabilities. The ML analysis was conducted in RAxML [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] on the partitioned dataset under a GTR + GAMMA +I nucleotide model. Parameters included a rapid bootstrapping and search for best-scoring ML tree algorithm, as well as 1,000 bootstrap replicates [START_REF] Stamatakis | A Rapid Bootstrap Algorithm for the RAxML Web Servers[END_REF]. 3. RESULTS 3.1 Sequence variability, species delimitation analysis, definition of the SSHs and assignment A total of 162 tufA sequences (834 bp, 46 unique haplotypes, 272 variable sites), 91 rbcL sequences (1322 bp, 40 unique haplotypes, 258 variable sites) and 62 rDNA 18S sequences (1248 bp, 47 unique haplotypes, 197 variables sites) (from targeted specimens for the latter) were obtained. The rbcL sequences were sequenced in two parts, the rbcL 5' part was 718 bp long with 155 variable sites, and the rbcL 3' part was 604 bp long with 103 variable sites. tufA dataset -Results obtained with the five species delimitation methods for the tufA dataset are summarized in Fig. 1. With the single threshold model, the GMYC analysis resulted in 16 PSHs (confidence interval of 12-19; Likelihood ratio-test (LRT) = 0.033), the bGMYC and ABGD analyses suggested 15 PSHs, while PTP and mPTP delimited 20 (and 12 PSHs , respectively. The bootstraps values supporting the delimited PSHs with the PTP are given in Supplementary material (Table A.3). Focusing on differences in the partition results, GMYC separated LL057 from the rest of the clade 4, while PTP split the same clade in four PSHs. PTP also split in two PSHs the clades 1, 11 and 12, and grouped the clades 2 and 3 in a single PSH. The mPTP method grouped the clades 6, 7, 8 and 9 in a single PSH. rbcL dataset -Results obtained with the five species delimitation methods for the rbcL dataset are summarized in Supplementary material (Figure A.2). The GMYC and ABGD methods both resulted in 15 PSHs (confidence interval of 3-42, LRT= 0.086, threshold time= -0.0070 for GMYC). The bGMYC and mPTP methods both produced 16 PSHs, whereas PTP produced 23 PSHs ) (see Supplementary Table A.4 for the PSHs bs values for PTP and bGMYC methods). The clade 7 was split in two PSHs by bGMYC and PTP. The clade 10 was recovered as a single PSHs for all methods except PTP for which the clade was split into five PSHs. In addition, PTP separated NOU087132 from the rest of the clade 6 and split the clade 3 in three PSHs. mPTP singled out HV405 from the rest of clade 9, DQ469318 from the rest of clade 6, and HV02674 from the rest of clade 13. mPTP also grouped in a single PSHs the clades 2+3 and 16+17. SSHs definition -Overall, our analyses produced relatively congruent PSHs between the different delimitation methods and both marker datasets (Table 2). However, the GMYC multiple-threshold method was not considered because it provided much higher number of PSHs, compared to the other methods. Besides, we only presented the results obtained using the MCCTs for the mPTP method (but see Supplementary data A.2 and A.3 for the results of mPTP applied on ML rooted trees). Despites several tests with ML rooted trees, the number of PSHs obtained were surprisingly low (respectively 9 and 5 for tufA and rbcL dataset), we decided to not include these results in the PSHs/SSHs discussion. ABGD, GMYC, bGMYC and mPTP provided similar partitions and are more conservative than PTP, generally defined more PSHs for both datasets. Seven and five of the 15 PSHs were recovered by all five methods for rbcL and tufA, respectively. All PSHs were supported by at least three of the five delimitation methods, which led to the definition of 18 SSHS (i.e., previously identified as clades). Among these, 12 SSHs were represented in both datasets. Some SSHs could not be compared between the two datasets as some sequences were missing for one or the other marker. Morpho-anatomical assignment of the SSHs -Based on morpho-anatomical observations, 15 out of the 18 SSHs were assigned a known species name (Figures 1 andA.2). The three remaining SSHs could not be identified either because of insufficient material for morpho-anatomical observations (SSHs 1 and 4) or because the observed characters did not match any known species (SSH 5). 3.2 Independent morpho-anatomical analysis of the Udotea morphospecies This part of the study was focused on morphology-based delimitation of species in the genus Udotea, investigated through two different analyses: morphological-based clustering and multivariate analysis. The discrimination of the specimens and the morphospecies clusters obtained under an a priori number of species (18 or 22 depending on the consideration of species forms and varieties or not) are illustrated on Figure 2. Most of the specimens were grouped in mostly well-supported clusters containing one of the reference species described by [START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF]; those that did not, appeared to be juvenile forms (e.g., NOU087081, NOU087164). Noticeable incongruences included Udotea cyathiformis var. flabellifolia which clustered separately from Udotea cyathiformis var. cyathiformis and its forms, and instead grouped with Udotea conglutinata. Udotea cyathiformis var. flabellifolia is indeed markedly different from other forms and varieties of the species (fanshaped blade for the variety "flabellifolia" versus the cup-shaped blade of the variety "cyathiformis"). The two species Udotea luna and U. looensis also grouped together in a single cluster, as well as Udotea spinulosa and U. spinulosa f. palmettoidea. Beyond morphospecies level, the multivariate analysis considered all morpho-anatomical variables (Supplementary Figure A.3) and clearly recovered specimens in three groups (component axes 1 and 2 explained 58.23% of total variability). This subdivision in three groups was also very clear in the independent analysis of two variables: the cortication and siphon features (Figure3a, b). In the cortication analysis, all the specimens were perfectly grouped according to the three states that were considered: absence of cortex (1), complete cortex (2) and incomplete cortex (3). For siphon features, the five states that were considered clustered in three groups as follow: the first group matched the "absence of cortex" group and included species with smooth siphons (state 1) or with a few basal scattered protuberances only on siphons from the blade/stipe basal transition zone (state 2); the second group matched the "complete cortex" group and included species with appendages (state 5); the third group matched the "incomplete cortex" group and included species with a few scattered protuberances on blade siphons, most of the time only on exposed areas (state 3) and species with numerous protuberances all along blade siphons (state 4). The groups highlighted in the multivariate analysis were also distinct on the dendrogram resulting from the morphology-based clustering analysis (Figure 2 and Table 3). Non-corticated species (C) were distinctively separated from completely corticated species (B) and incompletely corticated species (A). Only one species did not follow this classification and was recovered in the corticated group on the dendrogram reconstruction but not in the multivariate analysis (U. wilsonii, with protuberances).

Species phylogeny

Phylogenetic analyses of individual markers produced highly congruent topologies for chloroplast genes. The topology of the 18s rDNA gene tree showed polyphyly at the species level, although not supported. However, the three gene trees all confirmed the polyphyly of the udoteacean genera (individual gene trees are available on request to the first author). ML and Bayesian analyses, applied to concatenated alignment (tufA, rbcL and 18S rDNA), produced similar topologies, except for one node in clade D (Figure 4) that was not supported by the ML analysis (bs: 26) and absent in the BI analysis. The BI tree was generally better supported than the ML tree. We considered nodes with bs values > 95 and Posterior Probability (PP) > 0.98 as highly supported, and nodes with bs values comprised between 95 and 60 and PP between 0.98 and 0.70 as moderately supported. Below these values, nodes are considered not supported. In both trees, the representative of Udoteaceae included in this study formed a fully supported clade (clade A, Figure 4), but all genera represented by at least two species appeared polyphyletic. The Udoteaceae was divided in two main subclades: clade B (moderately supported, bs: 79; PP: 0.70) with four species of Udotea including the type species of the genus (U. flabellum), and clade C (strongly supported, bs: 100, PP: 1) with the rest of the species. Remaining Udotea species appeared in both clades D (strongly supported, bs: 99; PP: 1) and G (moderately supported bs: 65; PP: 0.85). Three species of Penicillus clustered in Clade F (moderately to strongly supported, bs: 88; PP: 1) while the fourth Penicillus species clustered in Clade D with Udotea and Rhipocephalus species. 4. DISCUSSION 4.1 Diversity of the Udoteaceae in the Caribbean region The DNA-based species delimitation results highlighted 18 different SSHs in our Caribbean dataset. We assigned species names to 15 SSHs using morpho-anatomical observations. These 15 taxa were previously recorded by [START_REF] Gepp | The Codiaceae of the Siboga Expedition[END_REF] and [START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF] and belong to Udotea (8), Penicillus (4), Rhipocephalus (2), and Rhipidosiphon (1). One species may be new to science (clade 5 in Figures 21 andA.2) as its morpho-anatomical characters did not match any of the known species, but we recommend the prior revision and redefinition of the genus Udotea before this species can be described with confidence. Two other species (clades 1 and 4, Figure 1) could not be identified because no specimen was available for morpho-anatomical analyses. Eight of the Udotea species described by [START_REF] Gepp | The Codiaceae of the Siboga Expedition[END_REF] and [START_REF] Littler | Systematics of Udotea species (Bryopsidales, Chlorophyta) in the tropical western Atlantic[END_REF] were not found during our survey and no sequence was available in public DNA databases. It cannot be excluded that some of these species could correspond to clades 1 and 4. Additional effort is required to sample these species and check their taxonomic status. Among all species, 16 were present in the Lesser Antilles, thus highlighting the diversity of this sub-region.

Variability and relevance of the genetic markers used

The results of the species delimitation analyses strongly rely on the selection of the genetic markers. In this study, we selected markers based on their known variability and their different evolutionary rates [START_REF] Leliaert | DNA-based species delimitation in algae[END_REF][START_REF] Saunders | An evaluation of rbcL, tufA, UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae[END_REF]Verbruggen et al., 2009a). Our results demonstrated that the tufA and rbcL chloroplast markers are well suited to study the Udoteaceae at species level. The tufA sequences contained more variable sites than rbcL ones (32.61% versus 19.51 %). The first fragment of the rbcL gene (rbcL 5') is slightly more variable (21.59% of variable sites) than the entire rbcL marker (19.51%) or the rbcL3' fragment (17.05%), contrary to what has been found in other Bryopsidales families [START_REF] Saunders | An evaluation of rbcL, tufA, UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae[END_REF]: this pattern needs to be confirmed in other lineages of Udoteaceae. Chloroplast genes have been widely used for barcoding purposes in the study of green macroalgae, i.e., to assign unknown specimens to known species ("specimen identification", sensu Collins and Cruickshank, 2012). Here, we show that they are also suitable markers to propose species hypotheses ("species delimitation", sensu Collins et al., 2012). In contrast, the nuclear 18S rDNA gene appeared less variable than the chloroplast markers and was not a relevant choice for species delimitation methods. The insufficient variability of the 18S rDNA at the species level has already been shown for another bryopsidalean family, the Caulerpaceae [START_REF] Kazi | Molecular Phylogeny and Barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA Genes[END_REF]. Nevertheless, the nuclear 18S rDNA proved useful for phylogenetic analyses, as already suggested by [START_REF] Kooistra | Molecular systematics, historical ecology, and phylogeography of Halimeda (Bryopsidales)[END_REF] for Halimeda and [START_REF] Kazi | Molecular Phylogeny and Barcoding of Caulerpa (Bryopsidales) based on the tufA, rbcL, 18S rDNA and ITS rDNA Genes[END_REF] for Caulerpa, and applied for multilocus analyses of Bryopsidales lineages (Verbruggen et al., 2009a(Verbruggen et al., , 2009b)). We believe that our results emphasize the need of markers from different genomic compartments to better understand species diversity and their phylogenetic relationships. Indeed, combining markers from different cellular compartments or from unlinked nuclear markers allows the identification of any lack of genetic divergence or potential incomplete lineage sorting or introgression events that would otherwise not be detected with a single locus [START_REF] Carstens | How to fail at species delimitation[END_REF][START_REF] Carstens | Estimating Species Phylogeny from Gene-Tree Probabilities Despite Incomplete Lineage Sorting : An Example from Melanoplus Grasshoppers[END_REF][START_REF] Leliaert | DNA-based species delimitation in algae[END_REF][START_REF] Rannala | The art and science of species delimitation[END_REF].

Efficiency and limits of the species delimitation methods

In our study, the GMYC single-threshold method produced results congruent with other methods -if not similar-and with the morpho-anatomical observations. The high similarity between GMYC and ABGD in our study is worth pointing out as these two methods are usually seen as complementary for the contrasting results they produce [START_REF] Montecinos | Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae)[END_REF]Puillandre et al., 2012b). However, the multiple-threshold method produced partitions with much more species hypotheses compared to other methods, which is a tendency emphasized in previous studies [START_REF] Esselstyn | Single-locus species delimitation : a test of the mixed Yule -coalescent model , with an empirical application to Philippine round-leaf bats[END_REF][START_REF] Guillemin | The bladed Bangiales (Rhodophyta) of the South Eastern Pacific: Molecular species delimitation reveals extensive diversity[END_REF][START_REF] Modica | A good compromise: Rapid and robust species proxies for inventorying biodiversity hotspots using the Terebridae (Gastropoda: Conoidea)[END_REF][START_REF] Montecinos | Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae)[END_REF][START_REF] Tang | Effects of phylogenetic reconstruction method on the robustness of species delimitation using single-locus data[END_REF]. The results obtained with bGMYC did not differ from the other methods and were consistent between both the tufA and the rbcL datasets. However, the computation of the bGMYC rates unveiled more Yule events (speciation) than coalescent events, which could have had an impact on the analysis [START_REF] Reid | Phylogenetic estimation error can decrease the accuracy of species delimitation : a Bayesian implementation of the general mixed Yule-coalescent model[END_REF]. In the present study, because our dataset included several genera and numerous species with low intraspecific variation, it is possible that coalescent events have been under-represented. The bGMYC results need to be interpreted carefully and in conjunction with other methods. The results of the distance-based method, ABGD, were not very different from the other methods with partition schemes similar to at least one of the tree-based method for each marker. However, the method produced contrasted genetic distance distributions for the two markers. There was a clear barcode gap for tufA, which was not the case for rbcL. For rbcL, one of the distributions displayed two clear gaps but the other didn't show any gap. There are several possible explanations for the absence of a unique and clear barcode gap, which include limited species sampling or variable rates of evolution among species or genera. Incomplete lineage sorting or introgression could also explain an absence of barcode gap [START_REF] Talavera | Factors affecting species delimitations with the GMYC model : insights from a butterfly survey[END_REF]. For both markers, the PTP method tended to overestimate the number of species while the mPTP method appeared more congruent with the other methods (similar PSHs) and morphology. The differences between the two methods can be explained by the ability of mPTP to consider variability in exponential distributions while PTP cannot accommodate intraspecific diversity. However, the mPTP method provided partitions congruent with the other methods only when using MCCTs: partitions obtained with ML trees largely underestimated the number of PSHs, compared to the number of SSHs finally retained. Both PTP and mPTP methods were primarily designed for nonultrametric trees, so it remains unclear why PTP accommodated both ultrametric (MCCTs) and nonultrametric (ML) trees while the mPTP methods failed with non-ultrametric trees. Combining different delimitation methods was thus necessary to balance their respective assumptions, bias or limitations, and to search for congruence among theoretically different models.

In the present study, tree-based methods were relatively congruent (whether they rely on ultrametric tree or not), and also concordant with the distance-based method, ABGD. This congruence reinforces the reliability of the resulting SSHs [START_REF] Fujita | Coalescent-based species delimitation in an integrative taxonomy[END_REF][START_REF] Ross | Species delimitation: A case study in a problematic ant taxon[END_REF]. 4.4 Leverages and limits of involving morphological data to assign species names Morpho-anatomical observations were used to assess SSHs, to assign species names, and to eventually determine if some of the genetically defined PSHs had never been described before and may correspond to new species. However, not all the PSHs have been assigned a species name. This can be explained by the absence, fragmentary state or inaccuracy of the available information for the family (morphological or genetic). Hence our study was hindered by ( 1) insufficient or unavailable voucher material for morpho-anatomical observations; (2) the limited number of reference sequences, particularly lacking for holotypes, to properly allow molecular identification; and (3) unreliable species identification for available sequences, often due to insufficient morpho-anatomical analysis (relying exclusively on gross morphology). Difficulties are also encountered from the morpho-anatomical characters themselves. Indeed, the morphological identification of Udoteaceae species can easily be misled by different factors such as phenotypic plasticity, cryptic diversity, life stages, observation interpretability, etc. Besides, it has been shown that a large set of specimens is required to cover the range of species polymorphism (see [START_REF] Wiens | Species delimitation in systematics: inferring diagnostic differences between species[END_REF] and therefore to accurately describe species, which might not always have been the case for the description of some Udoteaceae species. When morphological variability is large, species identification is more difficult because only a few anatomical characters remain relevant to discriminate and diagnose the species. For instance, we observed that the quantitative variables (e.g., size of filaments) tend to follow a continuum, meaning that size ranges overlap between species rather than being clearly distinct (see also [START_REF] Collado-Vides | Una revisión taxonómica del género Udotea en el Caribe mexicano y cubano[END_REF]. A morphometry study, such as those realized on the genus Halimeda (Verbruggen et al., 2005a(Verbruggen et al., , 2005b)), referencing the values of the continuous traits of Udoteaceae species could be relevant to further analyze these characters beyond their overlapping measure ranges. Finally, some morphological discrete traits can also be interpreted differently according to the observer [START_REF] Fujita | A coalescent perspective on delimiting and naming species: a reply to Bauer et al[END_REF] and the assessment of a form (e.g., the shape of the blade appendages apices) can be very subjective. That leads to even more confusion when species identification relies on a single character or when this latter may vary among or within specimens.

In the present study, we had no case of erroneous species descriptions due to the presence of cryptic diversity, or morphological variability, as the number of DNA-based species hypothesis was congruent with morphological identification. However, we were not able to confirm all species of Caribbean Udoteaceae listed in the literature because of the lack of genetic information for some morphospecies or the absence of specimens for morpho-anatomical observations of some PSHs. 4.5 Relevance of morphological characters to discriminate Udotea species The Udotea morpho-anatomical cluster analysis (Figure 2) produced well supported results which largely matched with genetically delimited species hypotheses. This demonstrates the ability and potential of the morphological characters for delineating Udotea species. The studies that used morphological-based methods often found discordance with molecular results [START_REF] Barrett | An integrative approach to delimiting species in a rare but widespread mycoheterotrophic orchid[END_REF][START_REF] Doan | Using morphological and molecular evidence to infer species boundaries within Proctoporus bolivianus Werner (Squamata: Gymnophthalmidae)[END_REF][START_REF] Wiens | Delimiting species using DNA and morphological variation and discordant species limits in spiny lizards ( Sceloporus )[END_REF]. This situation may explain why morphological traits analyses are under-used for delimiting species in recent molecular studies [START_REF] Wiens | Species delimitation: new approaches for discovering diversity[END_REF][START_REF] Wiens | Delimiting species using DNA and morphological variation and discordant species limits in spiny lizards ( Sceloporus )[END_REF]. Here, we would like to emphasize the relevant contribution of morphology to integrative taxonomy approaches, and to promote the value of reassessing morphological diagnostic characters as already pointed out [START_REF] Cianciola | Using Molecular-Assisted Alpha Taxonomy to Better Understand Red Algal Biodiversity in Bermuda[END_REF][START_REF] Vieira | Toward an inordinate fondness for stars, beetles and Lobophora ? Species diversity of the genus Lobophora (Dictyotales, Phaeophyceae) in New Caledonia[END_REF]. We observed that the combination of several morpho-anatomical characters was relevant to accurately delimit species. However, each character considered independently was relevant at higher taxonomic level, such as groups of species or genus, but not at the species level. Finally, we demonstrated that the cortication and the blade siphon features were relevant variables to explain morphospecies discrimination. Taken alone, these two variables were sufficiently effective to explain the clustering pattern observed for Udotea in both the multilocus and the multivariate analyses. Nevertheless, from our results, we argue that a nomenclatural clarification of the term "appendages" is needed to better understand the distinctiveness of Udotea species (as currently defined) and acquire more indicators for the necessary redefinition of the genus. We propose to consider under the term "appendages" only the well-developed secondary structures that are dichotomously divided or lobed with numerous tips, and lead to a cortication (different from "protuberances", which only consist of outgrowths). Through the redefinition of these terms, it becomes easier to highlight the discriminatory power of the siphon features and associate this latter to the cortication variable (see Table 3 for the correlation). These both variables stand out from other morpho-anatomical characters for discriminating Udotea sub-lineages and may be relevant indicators for future taxonomic revision.

Phylogenetic relationship and taxonomic revision

The polyphyly of Udotea and Penicillus, that had been shown in previous molecular phylogenies [START_REF] Curtis | Phylogenetic analysis of the large subunit rubisco gene supports the exclusion of Avrainvillea and Cladocephalus from the Udoteaceae (Bryopsidales, Chlorophyta)[END_REF][START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF][START_REF] Lam | Phylogenetic analyses of the Bryopsidales (Ulvophyceae, Chlorophyta) based on Rubisco large subunit gene sequences[END_REF]Verbruggen et al., 2009a) was retrieved in this study with also, for the first time, the polyphyly of the genus Rhipocephalus. Traditional classification at the genus level relies on gross morphological observations which do not agree with molecular phylogenies. For instance, the flabellate fan-shaped blade species have traditionally been assigned to Udotea while DNA analyses showed that this form emerged in genetically distinct clades and sometimes even grouped with other blade-morphologies (e.g., the flabellate fan-shaped Udotea conglutinata closely related to the brush-like Penicillus capitatus in clade D (Figure 4).

In our phylogenetic analyses, the Caribbean representatives of the Udoteaceae were split in two main supported subclades, the first one being composed of Udotea species only, and the second subclade regrouping sequences of different genera. Furthermore, the split of the genus Udotea in several lineages was always supported, regardless of the type of data used (molecular or morpho-anatomical), suggesting that it is more a taxonomical problem rather than a genetic issue. Based on our results, we consider as Udotea sensu stricto (s. s.), the group of species clustered in clade B of our concatenated phylogeny, which also includes the type-species of the genus, Udotea flabellum (J. Ellis & Sollander) M. Howe. Species included in Udotea s.s. appeared to be morphologically distinct and to have exclusive characters compared to the other taxa traditionally assigned to Udotea. Species of Udotea s .s. all share similar habits with a stocky thallus, a thick and strictly fan-shaped blade with a strong calcification, a well-developed rhizoid system and siphons of moderate size (≤ 80 µm for both stipe and blade filaments). The strongest diagnostic character is the presence of appendages (dichotomously divided or lobbed with rounded knobs) on the filaments of the blade which leads to cortication. This newly redefined Udotea s.s. clade then corresponds to the corticated lineage mentioned by [START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF]. In addition to the four species included in clade B of our phylogeny (Udotea occidentalis, U. dixonii, U. dotyi and U. flabellum), we propose to also include in Udotea s. s. the following species: U. norrisii, and the Indo-Pacific Udotea argentea and U. geppiorum. From nuclear-based phylogeny and morphological observations, [START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF] also considered Udotea norrisii among the ancestral species (equal cortication of blade and stipe) along with Flabellia petiolata, U. dotyi and U. flabellum. However, he excluded U. argentea from this group and rather considered its appendages as reduced small stumps. Thus, our assumptions based on morphological agreement only need to be confirmed with multilocus DNA analysis. The remaining taxa included in our phylogeny all clustered in clade C. The morphology of these taxa is variable, with filaments either assembled in single blade (fan-or cup-shaped), in several blades attached to a main axis, or free in brush-like forms. These taxa have in common the absence of appendages on the blade filaments. All the specimens clustered in clade C currently assigned to Udotea are distinguishable from Udotea s.s. for their more delicate and thinner appearance, and their wider filaments (largest siphons size in species with smooth filaments). All taxa of clade C need to be taxonomically revised, particularly at the genus level. [START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF] suggested that all these species could be considered as part of the same genus. Our phylogenetic results and morphoanatomical analyses rather suggest that species in clade C could be re-classified in several distinct genera. For example, although clade D contains variable thallus morphologies, all the taxa have smooth blade siphons. Similarly, taxa in clade F all have the same distinctive thallus morphology (brush-like capitulum) and smooth blade filaments. Similarly, more genetic data is needed to confirm if Udotea species with protuberances cluster with U. spinulosa (only species for which genetic data is available). However, we believe that more species need to be studied before a taxonomic revision of this group is proposed. Our study highlights the need for in-depth taxonomic revision of the family, especially above the species-level. Ideally, this revision should include all the taxonomical representatives of the family across their distribution range, particularly the Indo-Pacific region for which little data is available.

CONCLUSION

Based on an integrative taxonomical approach, using genetic multilocus and morphological data inferred from a taxa rich dataset, (i) we explored the diversity of the Udoteaceae from the Caribbean region using five DNA-based species delimitation methods and morpho-anatomical analyses, (ii) we produced a phylogeny of the family highlighting the polyphyly of most genera, and (iii) we proposed a revised morphological description of the genus Udotea s. s. Our results demonstrate the adequacy of the chloroplast markers (tufA and rbcL) for delimiting species. We emphasize the importance of (i) searching for congruence between several species delimitation methods and markers, and (ii) using independent data (here, morpho-anatomical) to increase the robustness of the species hypotheses, and facilitate species name assignment. Performed independently, morpho-anatomical analyses led to relevant results and emphasized the discriminatory power of morpho-anatomical characters to delineate Udotea morphospecies. We demonstrate that morpho-anatomical characters represent key data and helpful indicators in the process of taxonomical revision (at least at the genus level). The polyphyly of the Udoteaceae genera was confirmed once more and clearly appeared as a taxonomical issue rather than erroneous genetic signal. The redefinition of the genus Udotea sensu stricto was achievable through the combination of both genetic and morphological information. However, to resolve the Udoteaceae taxonomical issues in depth, a global study is necessary, and it should ideally include all the taxonomical representatives of the family across their distribution range, particularly the Indo-Pacific region.

AKNOWLEDGEMENTS

This work was supported by DUNE Labex-CORAIL project and ENTROPIE funds. The authors are grateful to Dr Florence Rousseau, Line Le Gall, Maggy Grun, Heroen Verbrugen, and Chiela Cremen for providing additional sequences. SSM and ATM are acknowledged for their financial support for sequencing. Thank as well as to Dr Helene Magalon and Cecile Fauvelot for their helpful sampling during PACOTILLE campaign. Samples have been collected during several campaigns on board of the R/V ANTEA (IRD), Pacotille (DOI number: http://dx.doi.org/10.17600/15005200); KARUBENTHOS DOI Number (http://dx.doi.org/10.17600/15005400); Fieldtrip in Guadeloupe organized by Onema and MNHN. LL acknowledges Laurent Millet (Plateforme du Vivant), as well as Bryan Vincent and Patrick Houssard for their support in statistics. Additional thanks to Lydiane Mattio for English translation and her valuable advices.

Table 3: Overview of the three morphological main groups of Udotea species (A, B and C) defined through the morphological-based clustering and the multivariate analysis and also retrieved in the phylogeny (for the one for which molecular data were available). For each group, the morphological states of cortication and siphon features are detailed, and the species list, with illustrations of the corresponding siphon features. andC) highlighted from all the morpho-anatomical characters (detailed in Supplementary data A.1) and described in Table 3. Table 3: Overview of the three morphological main groups of Udotea species (A, B and C) defined through the morphological-based clustering and the multivariate analysis and also retrieved in the phylogeny (for the one for which molecular data were available). For each group, the morphological states of cortication and siphon features are detailed, and the species list, with illustrations of the corresponding siphon features.

Groups of

LIST OF FIGURES

Groups of

Figure 1 :

 1 Figure1: Species delimitation results obtained using the five methods (ABGD, GMYC, bGMYC, PTP and mPTP) applied to the tufA dataset. The tree is the maximum clade credibility tree from the BEAST analysis. Black bars indicate partitions retained as SSHs and indicated in right column as well as the species identification from morphoanatomical observations. Grey bars indicate different partition schemes not retained. Numbers in brackets indicate the number of identical haplotypes in the initial dataset.

Figure 2 :

 2 Figure 2: Dendrogram representation of the morpho-anatomical clustering (hclust analysis) applied on Udotea specimens. The delimitated morphospecies clusters are indicated in the timeline with a priori 18 (continuous lines) or 22 (dotted lines) number of species. Node values indicate approximately unbiased p-values. Different coloration of branches transcribes the three main groups of specimens (A, B, and C) highlighted from all the morpho-anatomical characters (detailed in Supplementary data A.1) and described in Table3.

Figure 3 :

 3 Figure 3: Multivariate component analyses representation of the variables "cortication" (a) and "siphon features" (b) in two component axes. Component axes 1 and 2 respectively explain 43.52 % and 14.71 % of the total variability (cumulative total: 58. 23%). In Fig 3.a, 1=absence of cortex, 2= complete cortex and 3= incomplete cortex. In Fig 3.b, 1=smooth siphons, 2 = smooth siphons with few protuberances in the transition zone between blade and stipe, 3= protuberances on blade siphons only in exposed areas, 4= protuberances all along blade siphons, 5= appendages. In Fig 3.a and b, A=incompletely corticated species (cortication= 3; siphon features= either 3 or 4), B= completely corticated species (cortication= 2, siphon features= 5), C= noncorticated species (cortication= 1, siphon feature= 1 or 2).

Figure 4 :

 4 Figure 4: Maximum likelihood phylogenetic tree inferred from the multilocus matrix. At each node, circles represent ML bootstrap values (left) and Bayesian posterior probabilities (right): black circles indicate high support (bs > 95; PP>0.98), dark grey circles represent moderate support (95 > bs >60; 0.98 > PP > 0.70), and white circles indicate absence of support (bs < 60 and PP< 0.70). Type-species are indicated in bold character. SSHs number ID (as per species delimitation analysis), morphological habit and diagnostic characters are indicated in the right columns. The genus Udotea s. s. is surrounded by the rectangle. Capital letters indicated the main nodes of the tree discussed in the text.

  List of the PSHs obtained with the mPTP method applied on ML rooted tree for the tufA dataset List of the PSHs obtained with the mPTP method applied on ML rooted tree for the rbcL dataset

Table 1 :

 1 Anatomical characters assessed for the identification of Penicillus, Udotea and Rhipocephalus species Aspect (moniliform, tortuous or smooth)  Presence of dichotomies  Presence of constriction above dichotomies  Presence of appendages and aspect (stalk, dichotomously division and apices)

	Udotea	A	B	C
	species
	Cortication state	Incomplete (state 3) cortication	Cortication (state 2) (complete)	No cortication (state 1)
				Smooth
	Blade siphons features	Protuberances (state 3+4)	Appendages (state 5)	(state 1) [or a few protuberances at the transition zone blade-stipe] (state 2)
		Udotea goreaui
			Udotea
			occidentalis
				Udotea cyathiformis
				Udotea conglutinata
		Udotea spinulosa		Udotea unistratea
			Udotea	Udotea caribaea
			flabellum	Udotea fibrosa
	Correspondi			Udotea abbottiorum
	ng species	Udotea		Udotea luna
	and siphons illustrations features	verticillosa	Udotea dotyi	Udotea looensis Udotea sp2
			Udotea
		Udotea wilsonii	dixonii
			Udotea norrisii

LIST OF TABLES

 1); even and elongated (2); uneven and elongated (3); even and well-marked (4); uneven and well-marked ( 5 [START_REF] Kooistra | Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis[END_REF]Verbruggen et al., 2009