Lucie Bauret
email: lu.bauret@gmail.com

Myriam Gaudeul

Michael A Sundue

Barbara S Parris

Tom A Ranker

France Rakotondrainibe

Sabine Hennequin

Jaona Ranaivo

Marc-André Selosse

Germinal Rouhan

Madagascar sheds new light on the molecular systematics and biogeography of grammitid ferns: new unexpected lineages and numerous long-distance dispersal events

Keywords: Biogeography, Ferns, Long-distance dispersal, Madagascar, Phylogeny, Western Indian Ocean

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Madagascar is a continental island of the Western Indian Ocean (WIO) known to host an extraordinary biodiversity, with about 12000 vascular plant species [START_REF] Callmander | The endemic and non-endemic vascular flora of Madagascar updated[END_REF], and listed as one of the world's hotspots of biodiversity [START_REF] Myers | Biodiversity hotspsots for conservation priorities[END_REF]. Eighty-three percent of vascular plants, 52% of birds and 86% of invertebrates are endemics, and this proportion reaches 100% for terrestrial Gastropoda and non-volant Mammalia [START_REF] Goodman | Updated estimates of biotic diversity and endemism for Madagascar[END_REF]. However, this biological diversity is still under-documented and the evolutionary processes explaining its emergence are poorly known, since only a few phylogenetic studies are available. In particular, assessing the relative contributions of vicariance due to plate tectonics, trans-oceanic long-distance dispersal (LDD) and in situ species diversification remains a central question.

Investigating the origin and evolutionary history of Malagasy lineages is challenging because Madagascar is not strongly isolated from the East African coast, and is also surrounded by several archipelagos. Thus, Madagascar must be studied in the broader context of the WIO, involving a complex geologic history of continental and volcanic islands [START_REF] Agnarsson | The Generation of a Biodiversity Hotspot: Biogeography and Phylogeography of the Western Indian Ocean Islands[END_REF]. Most importantly, Madagascar was once part of the supercontinent Gondwana that began to breakup 155-118 Ma, with the separation of the America/Africa block from the Madagascar/India/Australia/Antarctica block. America and Africa split shortly after (110 Ma). Later on (118-84 Ma), India/Seychelles broke away from Madagascar before colliding with Asia 50 Ma, followed by the separation of the Seychelles Plateau (100-95 Ma; [START_REF] Seward | An insight into the breakup of Gondwana: identifying events through low-temperature thermochronology from the basement rocks of Madagascar[END_REF]. In addition, the WIO is characterized by two volcanic archipelagos, the Comoros and Mascarenes, which respectively emerged within the last ca. 15 Ma [START_REF] Emerick | Age progressive volcanism in the Comores Archipelago, western Indian Ocean and implications for Somali plate tectonics[END_REF][START_REF] Nougier | The Comores archipelago in the western Indian Ocean: volcanology, geochronology and geodynamic setting[END_REF] and 8 Ma [START_REF] Mcdougall | Isotopic Dating and Geomagnetic Polarity Studies on Volcanic Rocks from Mauritius, Indian Ocean[END_REF]. Whereas some studies underlined the impact of continental drift on the distribution of species occurring in Madagascar, others have more recently emphasized a greater role of LDD during the Cenozoic period [START_REF] Agnarsson | The Generation of a Biodiversity Hotspot: Biogeography and Phylogeography of the Western Indian Ocean Islands[END_REF][START_REF] Bacon | Biogeography of the Malagasy Celastraceae: Multiple independent origins followed by widespread dispersal of genera from Madagascar[END_REF][START_REF] Buerki | Spatio-temporal history of the endemic genera of Madagascar[END_REF][START_REF] Renner | Biogeography of the Monimiaceae (Laurales): a role for East Gondwana and long-distance dispersal, but not West Gondwana[END_REF][START_REF] Warren | Why does the biota of the Madagascar region have such a strong Asiatic flavour?[END_REF][START_REF] Yoder | Has vicariance or dispersal been the predominant biogeographic force in Madagascar? Only time will tell[END_REF]. At least, the role of dispersal is undisputable in the origin of lineages on the recent volcanic islands surrounding Madagascar.

In this context, ferns are a very interesting biological model to study the biogeographic origin of the Malagasy and WIO diversity. As many as 600 fern species are listed in Madagascar, among which ca. 45% are endemic [START_REF] Goodman | Updated estimates of biotic diversity and endemism for Madagascar[END_REF][START_REF] Rakotondrainibe | Checklist of the pteridophytes of Madagascar[END_REF][START_REF] Roux | Synopsis of the Lycopodiophyta and Pteridophyta of Africa, Madagascar and neighbouring islands[END_REF]. This proportion is high, although not as high as for flowering plants and some other groups cited above, probably because ferns dust-like spores have stronger dispersal abilities that facilitate species arrival and ultimately slow down the speciation process [START_REF] Smith | Comparison of Fern and Flowering Plant Distributions with Some Evolutionary Interpretations for Ferns[END_REF]. Spore dispersal is assumed to occur mostly through air currents, although dispersal mediated by bats [START_REF] Sugita | Possible spore dispersal of a birdnest fern Asplenium setoi by bonin flying foxes Pteropus pselaphon[END_REF] or birds [START_REF] Lewis | First evidence of bryophyte diaspores in the plumage of transequatorial migrant birds[END_REF] has been documented. This high capacity of spore movement, combined to the relatively recent origin of extant fern lineages (especially in the species-rich Polypodiales) since the Cretaceous (<150 Ma; [START_REF] Rothfels | The evolutionary history of ferns inferred from 25 lowcopy nuclear genes[END_REF][START_REF] Schuettpelz | Evidence for a Cenozoic radiation of ferns in an angiospermdominated canopy[END_REF], suggests a major influence of LDD compared to Gondwanan vicariance in the origin of the African-WIO groups. In particular, connections between the Neotropics and Madagascar were previously highlighted based either on floristic affinities [START_REF] Moran | Phytogeographic relationships between neotropical and African-Madagascan pteridophytes[END_REF] or on molecular phylogenetic analyses.

Among the latter studies, LDD between the two areas were hypothesized for numerous Elaphoglossum lineages [START_REF] Rouhan | Molecular phylogeny of the fern genus Elaphoglossum (Elaphoglossaceae) based on chloroplast non-coding DNA sequences: Contributions of species from the Indian Ocean area[END_REF][START_REF] Vasco | Circumscription and phylogeny of the Elaphoglossum ciliatum group (E. sect. Lepidoglossa, Dryopteridaceae) based on cpDNA sequences[END_REF], and in the genera Lomariopsis [START_REF] Rouhan | Preliminary phylogenetic analysis of the fern genus Lomariopsis (Lomariopsidaceae)[END_REF], Stenogrammitis (as Lellingeria in [START_REF] Labiak | Molecular phylogeny, character evolution, and biogeography of the grammitid fern genus Lellingeria (Polypodiaceae)[END_REF], Leucotrichum [START_REF] Rouhan | Not so neotropical after all: the Grammitid fern genus Leucotrichum (Polypodiaceae) is also paleotropical, as revealed by a new species from Madagascar[END_REF] and other Polypodiaceae [START_REF] Janssen | Origin and diversification of African ferns with special emphasis on Polypodiaceae[END_REF]. Transatlantic LDD was also inferred based on dated phylogenies in Alsophila [START_REF] Janssen | Neoendemism in Madagascan scaly tree ferns results from recent, coincident diversification bursts[END_REF] and Parapolystichum [START_REF] Labiak | Phylogeny and historical biogeography of the lastreopsid ferns (Dryopteridaceae)[END_REF]. Claims for Asiatic origins have also been documented, highlighting dispersal routes from Asia e.g. in the genera Pteris [START_REF] Chao | Molecular phylogeny and biogeography of the fern genus Pteris (Pteridaceae)[END_REF], Nephrolepis [START_REF] Hennequin | Phylogenetics and biogeography of Nephrolepisa tale of old settlers and young tramps[END_REF], and Deparia [START_REF] Kuo | Historical biogeography of the fern genus Deparia (Athyriaceae) and its relation with polyploidy[END_REF]. At a more restricted scale, data on ferns are almost completely lacking within the African-WIO region, although a few insights from Platycerium [START_REF] Kreier | Phylogeny and biogeography of the staghorn fern genus Platycerium (Polypodiaceae, Polypodiidae)[END_REF] and Alsophila [START_REF] Janssen | Neoendemism in Madagascan scaly tree ferns results from recent, coincident diversification bursts[END_REF] might support continental Africa as a source of dispersal to Madagascar. This trend, implying migration across the Mozambique Channel, is strongly supported in other plant groups [START_REF] Agnarsson | The Generation of a Biodiversity Hotspot: Biogeography and Phylogeography of the Western Indian Ocean Islands[END_REF].

Our study focused on grammitid ferns that had long been considered as a family, 'Grammitidaceae', until they were shown to form a clade nested within the larger Polypodiaceae family using molecular data [START_REF] Ranker | Phylogeny and evolution of Grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy[END_REF][START_REF] Schneider | Unraveling the phylogeny of polygrammoid ferns (Polypodiaceae and Grammitidaceae): exploring aspects of the diversification of epiphytic plants[END_REF][START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]. Comprising around 1500 species, Polypodiaceae is the second largest family of ferns. The estimated 900 species of grammitid ferns [START_REF] Perrie | Chloroplast DNA sequences indicate the grammitid ferns (Polypodiaceae) in New Zealand belong to a single clade, Notogrammitis gen. nov. New Zeal[END_REF] belong to 33 genera, many of which were described in the last decade based on molecular phylogenetic studies that revealed extensive polyphyly as a recurrent issue resulting from morphological homoplasy [START_REF] Ranker | Phylogeny and evolution of Grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy[END_REF]Sundue et al., 2010a).

Grammitid ferns are mostly found as epiphytes in tropical montane forests, and distributed in two main phytogeographic zones [START_REF] Parris | The distribution of Grammitidaceae (Filicales) inside and outside Malesia[END_REF]): i) around 400 species in the Neotropics, tropical Africa, and the WIO (including Madagascar, Mascarenes, Comoros and Seychelles); and ii) around 500 species in tropical Asia s.l., including Malesia and the Pacific Ocean islands. The Neotropics and the WIO were grouped in the same phytogeographic zone because they share several taxa: the genera Alansmia, Ceradenia, Cochlidium, Enterosora, Grammitis, Leucotrichum, Melpomene, Stenogrammitis, and Zygophlebia, and even some species (Alansmia elastica, Cochlidium serrulatum and Melpomene flabelliformis). In contrast, only a few taxa are known to be shared by the two phytogeographic zones: i) Ctenopterella is mainly distributed in Malesia and the South-Pacific Ocean islands, but also in the WIO although its monophyly has never been tested; ii)

Stenogrammitis is mainly Neotropical but also distributed in Africa, the WIO, Hawaii and the South-Pacific Ocean islands. iii) Notogrammitis has a circumaustral distribution, from Australia and New Zealand to South Africa and South America.

A recent phylogenetic and biogeographic study showed that grammitids evolved from a Neotropical ancestor between the end of the Eocene and beginning of the Paleocene (45-37 Ma; [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]. In agreement with the distribution patterns and delimitation of the two phytogeographic zones, the inferred phylogeny exhibited a clear geographic pattern: a single clade from tropical Asia s.l. (comprising Australia and Melanesia) was nested in a Neotropical grade, which also included the WIO species. This result was explained by one LDD from the Neotropics to tropical Asia s.l. at the end of the Paleocene, and repeated LDD from the Neotropics to the WIO since molecular dating suggested relatively recent diversification and allowed excluding vicariance hypotheses linked to Gondwana breakup. However, the sampling of [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF] included only eight species occurring in the WIO, so that many questions remain about the systematics and biogeography of grammitid species in this area.

For the WIO and Africa, Roux (2009) listed 51 species of grammitid ferns belonging to 9 genera, and in particular 31 species in Madagascar including 13 endemics (Table 1; Fig. 1). However, taking into account several taxa awaiting description based on morphological evidence, the actual diversity could reach >40 species in Madagascar alone. In addition, generic delimitations have to be revised for some genera already identified as non-monophyletic at the worldwide scale (especially Grammitis and Zygophlebia), and may also have to be for some others (in particular Ctenopterella, which is shared between Tropical Asia s.l. and the WIO; Table 1).

Based on an extended sampling and using phylogenetic inference, molecular dating and ancestral area estimation, we aim at improving the systematics of the Malagasy and other WIO grammitids, better understanding their biogeographic history, and shedding light on the evolutionary mechanisms that led to their emergence. More specifically, our goals are: i) to further test the monophyly of grammitid genera, particularly those diversified in the WIO, namely Grammitis, Zygophlebia and for the first time Ctenopterella; ii) more broadly, to improve the systematics of grammitid ferns by delimiting species as monophyletic groups; iii) to test the global biogeographic scenario proposed by [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]; iv) to infer the biogeographic origin of the Malagasy and other WIO species (i.e., did they emerge after dispersal events from the Neotropics only, as suggested by [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]? How many such events explain the extant diversity in the area?); iv) to assess to what extent dispersal occurred and the predominant migration routes at the regional scale, in the WIO; and v) to check whether diversification occurred within the WIO.

Materials and methods

Taxonomic sampling

The sampling was composed of two parts: the first one, based on the worldwide grammitid phylogeny of [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF], included 199 samples representing 180 grammitid species and provided a global framework. The second one was focused primarily on the WIO and Africa, and even more specifically on Madagascar: 193 new samples represented 61 species (Appendix 1). In total, the ingroup comprised 396 samples representing 224 species, including 36 species from Africa and WIO islands, of which 31 occur in Madagascar (taking into account a few undescribed species), and 12 of which are endemic (representing 87% of the described species, and ca. 77% of the described endemics). A total of 31 out of 33 recognized grammitid genera were represented (Archigrammitis Parris and Luisma M.T.Murillo & A.R.Sm. were lacking) and one type species (Tomophyllum subsecundodissectum) was newly sampled, for a total of 21 type species (Appendix 1). Five outgroups were added, belonging to four other Polypodiaceae genera that belong to the sister clade of grammitids [START_REF] Sundue | Morphological innovation, ecological opportunity, and the radiation of a major vascular epiphyte lineage[END_REF]: Microgramma C.Presl, Niphidium J.Sm., Polypodium L. and Serpocaulon A.R.Sm. Voucher information is listed in Appendix 1.

DNA sequencing

Total genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit (Valencia, California, USA) either from silica-dried leaves whenever available, or from herbarium specimens. The manufacturer's protocol was followed but slightly modified for herbarium samples, adding 30 µL proteinase K (20 mg/mL) and 30 µL beta-mercaptoethanol for the initial lysis step, which was carried out at 42 °C during 24 hrs on a tipping plate.

The polymerase chain reaction (PCR) was used to amplify five plastid DNA regions: the two coding genes atpB and rbcL, and the three non-coding intergenic spacers: rps4-trnS, trnG-trnR and trnL-trnF. PCR were carried out in a 25 μL volume containing 1x PCR buffer, 2.5 mM MgCl 2 , 250 μM of each dNTP, 1M betaine, 0.4 μM of each primer, 0.75 U Taq polymerase (Taq CORE kit; MP Biomedicals, Illkirch, France), and 0.75 μL of template DNA. Primer sequences and thermal cycling conditions are reported in Table 2. The resulting PCR products were checked on a 1% agarose gel and sequenced in both directions at the Centre National de Séquençage (Evry, France), using the amplification primers and additional, internal primers for atpB, rbcL and trnG-trnR (Table 2). Forward and reverse DNA strands were assembled in Sequencher 4.9 (GeneCodes Corporation, Ann Arbor, Michigan, USA). The newly obtained consensus sequences were submitted to GenBank (accession numbers listed in Appendix 1).

Phylogenetic analyses

The sequences were aligned with the MAFFT plugin [START_REF] Katoh | Parallelization of the MAFFT multiple sequence alignment program[END_REF] in Jalview 2.8.2 [START_REF] Waterhouse | Jalview Version 2--a multiple sequence alignment editor and analysis workbench[END_REF] using the default parameters. The alignments were then checked and revised manually when necessary. For each DNA region, the most suitable model of nucleotide substitution was determined with MrModelTest 2.3 [START_REF] Nylander | MrModeltest ver. 2.3. Program distributed by author[END_REF], following the Akaike Information Criterion. A data matrix was then built by concatenation of all five regions using Sequence Matrix 1.7.8 [START_REF] Vaidya | SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information[END_REF], each DNA region constituting a distinct partition in the final dataset.

Phylogenetic trees were inferred based on two probabilistic methods: Maximum Likelihood (ML) and Bayesian Inference (BI), as respectively implemented in RAxML-HPC2 8.2.6 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] and MrBayes 3.2.3 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF]. Gaps were treated as missing data and analyses were performed on the CIPRES science gateway [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF]. Parameters for the RAxML analysis were determined on the CIPRES interface. We used the GTRGAMMA+I model of nucleotide substitution for each partition, performed 1000 rapid bootstrap (BS) replicates and searched for the best-scoring ML tree. In MrBayes, we conducted two independent but parallel analyses with four chains each (one cold and three incrementally heated at a temperature of 0.1).

The BI analyses were run for 15 million generations each, sampled every 1500 generations, and performed with the GTR+I+Γ model of nucleotide substitution for each partition and parameters unlinked. Tracer 1.6.0 [START_REF] Rambaut | Tracer 1.6[END_REF] was used to check that effective sample sizes were higher than recommended (>200) and that the two runs had converged to the stationary distribution. The 25% initial trees were discarded as burn-in, the post-burn-in trees (15000 total trees) were pooled and a 50% majority-rule consensus was computed with average branch lengths and posterior probability (PP) estimates for all nodes. This tree and the best ML tree were visualised with FigTree 1.4.2 [START_REF] Rambaut | FigTree 1.4. 2 software[END_REF].

Divergence time estimation

Divergence time estimation was performed on the CIPRES science gateway using BEAST 1.8.0 [START_REF] Drummond | Bayesian Phylogenetics with BEAUti and the BEAST 1.7[END_REF]. Our biogeographic analyses were performed at the species level and focused on Madagascar. Therefore, given that the BEAST outputs were used as inputs for the estimation of ancestral areas, we reduced our ingroup sampling to 149 samples representing 139 species for the dating analysis. For genera that included Malagasy species, we kept one sample per species that was retrieved as monophyletic and as many samples as retrieved lineages when species were poly-or paraphyletic. For genera without any Malagasy species, we retained at least two samples of two distinct species including the type species of the genus whenever available, and the species that was recovered as the most early-diverging lineage (in order not to underestimate the diversification date of the genus). For genera with no resolution on the crown node, we kept all the sampled species branched on the polytomous node.

Partitions and models of nucleotide substitution were the same as for the BI. An uncorrelated lognormal relaxed clock was used, with a Yule tree prior [START_REF] Gernhard | The conditioned reconstructed process[END_REF] and a random starting tree. Based on a large-scale, fossil-based study on leptosporangiate ferns [START_REF] Schuettpelz | Evidence for a Cenozoic radiation of ferns in an angiospermdominated canopy[END_REF], we used four secondary calibration points corresponding to four well-supported clades within the Polypodiaceae (nodes 367, 368, 378, 382 in Schuettpelz and Pryer 2009; figured as black squares on Fig. 4) that were also used by [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]. These points were modelled by normal distribution priors on crown nodes, with 10% standard deviation (S.D.) in all cases: (1) the age of the grammitid clade was set to 31.2 Ma (S.D.= 3.1); ( 2 3); and (4) the diversification of the genus Serpocaulon, in the outgroup, was dated to 15.5 Ma (S.D. = 1.5). The prior for ucld.mean was defined by continuous-time Markov chain (CTMC) Rate Reference, as advised when no prior knowledge is available [START_REF] Ferreira | Bayesian analysis of elapsed times in continuous-time Markov chains[END_REF]. Two analyses of 20 million generations each were performed with tree and parameters sampling every 2000 generations. Tracer 1.6.0 was used to define burn-in length and the first 1000 trees (10%) were discarded for each run. We used LogCombiner v1.8.0 to combine results from the two runs and TreeAnnotator v1.8.0 [START_REF] Drummond | Bayesian Phylogenetics with BEAUti and the BEAST 1.7[END_REF] to compute the maximum clade credibility tree. FigTree 1.4.2 [START_REF] Rambaut | FigTree 1.4. 2 software[END_REF] was used to visualize the maximum clade credibility tree and the associated chronogram.

Ancestral areas estimation

We used both the statistical Dispersal-Vicariance Analysis S-DIVA [START_REF] Yu | S-DIVA (Statistical Dispersal-Vicariance Analysis): a tool for inferring biogeographic histories[END_REF] and the Bayes-Lagrange S-DEC [START_REF] Beaulieu | A Southern Hemisphere origin for campanulid angiosperms, with traces of the break-up of Gondwana[END_REF] models implemented in the RASP software [START_REF] Yu | RASP (Reconstruct Ancestral State in Phylogenies): a tool for historical biogeography[END_REF] to estimate ancestral ranges at all nodes. Nine biogeographic regions were defined (see Fig. Geographic distributions were coded for all species, based on [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF], and on herbarium specimens (mostly from the Paris Herbarium) and personal observations for all other samples. The taxonomic identification of all herbarium specimens was checked. We considered polyphyletic species as distinct lineages and coded as many distinct distributions as retrieved lineages. The maximal number of ancestral areas was set to two and, owing to the high winddispersal ability of fern spores over long distances, we considered that dispersal probability was not correlated to geographic distance. The frequencies of an ancestral range at a node were averaged over 100 random, post burn-in trees taken from the BEAST output. The condensed tree was the maximum clade credibility tree estimated in BEAST.

Results

Phylogenetic analyses

Our combined DNA sequence matrix of the five plastid regions was composed of 401 specimens and 5593 base pairs. Table 3 reports the percentage of samples successfully sequenced and other statistics, for each DNA region and the combined alignment. The phylogenetic hypotheses produced by the ML analysis and BI resulted in very similar topologies, with a few minor, not statistically supported differences. Consequently, only the BI topology is summarized in Fig. 2 (see Appendix 2 for the full tree with all terminals).

Both the ML analysis and BI supported grammitid ferns as a strongly-supported clade (BS=100; PP=1; Fig. 2). The resulting phylogeny was almost fully resolved at the species level. Most nodes were strongly supported, especially those supporting genera (BS≥95 and/or PP≥0.95; Fig. 2), although more moderate support values were retrieved for the backbone nodes and some nodes within clade-1 (Fig. 2). The inferred topology almost fully agreed with the most recent phylogenetic study on the group [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF], with two notable exceptions: we recovered Grammitis s.s. as monophyletic (BS=100; PP=1), and three new clades were supported (highlighted as grey-filled triangles, Fig. 2) involving species currently classified in Grammitis, Ctenopterella and Enterosora (as detailed later).

The phylogeny was geographically structured, with a Paleotropical clade (BS=78; PP=0.86) nested within a large grade (Fig. 2). The Paleotropical clade included 15 genera mostly distributed in tropical Asia s.l. and the Pacific Ocean islands. Notably, the Paleotropical clade also comprised eight species from the WIO and Africa, belonging to Ctenopterella and Grammitis and all together forming clade-3 (BS=100; PP=1; Fig. 2). This clade is a newly recovered one for grammitids. The relationships between clade-3, Chrysogrammitis, and the rest of the Paleotropical clade were not resolved.

The rest of the tree consisted of a large grade, hereafter called amphi-Atlantic grade. It included 17 genera showing either an exclusively Neotropical distribution for six genera, or a distribution on both sides of the Atlantic Ocean (including Africa and the WIO) for 10 genera (with the exceptions of Stenogrammitis and Grammitis s.s., which include a few additional species from the Pacific Ocean islands, tropical Asia s.l. or the circumaustral region; and Adenophorus restricted to Hawaii; Fig. 2).

Out of the 31 genera that we sampled, monophyly could not be assessed for Acrosorus and Lomaphlebia (represented by only one species each). Twenty-two genera were recovered as monophyletic, including Moranopteris that was exclusively described from the Neotropics to date and that was also represented in this study by an undescribed, endemic species to Madagascar (Moranopteris sp. 1). By contrast, seven genera were not recovered as monophyletic: Ceradenia, Ctenopterella, Enterosora, Grammitis, Oreogrammitis, Radiogrammitis and Zygophlebia (indicated in bold, Fig. 2). In particular, Grammitis, Ctenopterella and Enterosora were retrieved as highly polyphyletic: Grammitis was retrieved in the clade-1 of the amphi-Atlantic grade (as Grammitis s.s.), but also as four distinct lineages within the Paleotropical clade (two lineages in clade-3, one clade sister-group to Notogrammitis, and G. stenophylla as sister lineage to the Acrosorus-Ctenopterella denticulata clade; Figs. 2 &3). Ctenopterella species formed three distinct lineages within the Paleotropical clade: C. denticulata was included in the clade Acrosorus-Grammitis stenophylla clade, C. lasiostipes and two other undescribed species were sister-group to Grammitis-Notogrammitis, and two species were included in clade-3; Figs. 2 &3). Enterosora was retrieved as four lineages belonging to the amphi-Atlantic grade: two species were sister-group to Adenophorus in clade-1 and, in clade-2, two distinct lineages were nested within Zygophlebia (counting four and one species, respectively) and one species within Ceradenia (Fig. 3).

Our molecular sampling included multiple individuals per species, especially for those from the WIO, and also allowed the identification of several cases of polyphyly at the species level. Twentyeight species were thus recovered as non-monophyletic (Appendix 2). They belonged to 15 genera and included 12 species from the WIO and Africa (Table 4). These 12 non-monophyletic species were mostly found in Grammitis, with four non-monophyletic species in Grammitis s.s.

(highlighted in distinct colors; Fig. 3, clade-1; Table 4) and six non-monophyletic species which could be classified in one to several new genera in order to accommodate their placement in clade-3 (Fig. 3; Table 4).

Divergence time and ancestral areas estimations

Divergence time estimation

Molecular dating showed that grammitid ferns diverged from other Polypodiaceae about 39.3 Ma (stem age; 95% Highest Posterior Density (HPD) interval: 31.2-50.6) and started to diversify at least 34.1 Ma (crown age; 95% HPD: 28.3-40.2) at the end of the Eocene (Fig. 4A). The Paleotropical clade was dated to 24.9 Ma for the stem age (95% HPD: 19.6-28.1; Fig. 4B).

Divergence dates (stem ages) of generic lineages ranged between 13.1 Ma (divergence between Acrosorus and Ctenopterella denticulata; 95% HPD: 9.0-18.1; Fig. 4B) and 32.1 Ma (divergence between Terpsichore and its sister-clade; 95% HPD: 26.5-38.7; Fig. 4A), while their diversification (crown) ages ranged between 2.0 and 14.6 Ma for Xiphopterella and Ascogrammitis respectively (Fig. 4B). In fact, diversification could also have occurred earlier: several new genera will have to be newly described based on this study (see Table 3 and Discussion) and as a result, the potential genus corresponding to clade-V would have started to diversify 21.1 Ma.

Ancestral areas estimation

S-DIVA and S-DEC analyses produced very similar results. When they differed, S-DEC inferred slightly broader ancestral areas (i.e., including an additional region, but with a low probability), but the areas inferred by S-DIVA were also always the most probable ones inferred by S-DEC (results not shown). The only notable difference was an ancestral area covering both Madagascar and the Neotropics for the crown node of Stenogrammitis and its next outer node by S-DEC, vs.

Madagascar only by S-DIVA (Fig. 4B).

Grammitid ferns originated and diversified in the Neotropics (Fig. 4A) and a Neotropical origin was also inferred for all the generic lineages of the amphi-Atlantic grade, with two exceptions: the ancestral area of Adenophorus was inferred in the Neotropics and the Pacific Ocean islands, while Stenogrammitis would have originated in the Neotropics and Madagascar.

The Paleotropical clade originated in tropical Asia s.l. and the Neotropics (Fig. 4B). Within this clade, all genera have an exclusively tropical Asian origin, except Notogrammitis (for which the circumaustral area was also inferred) and clade-V if considered as a new genus (for which Madagascar was also inferred).

Focus on Madagascar, surrounding islands and Africa

The lineages occurring in this region were dispersed throughout the phylogeny. They originated by 13 LDD events dated from the end of the Oligocene or more recently (indicated by black arrows on branches; Fig. 4): 12 from the Neotropics and one from tropical Asia s.l., leading to clade-V.

However, this latter event was not statistically supported since the node grouping the African-WIO clade-V with the rest of the Paleotropical clade (excluding the Asian genus Chrysogrammitis) had PP=0.45. Five LDD were inferred to Madagascar (black arrows for LDD-3, LDD-5, LDD-10, LDD-12 and LDD-13; Fig. 4). These events resulted in three species endemic to Madagascar:

Grammitis ebenina-Rouhan1180 (LDD-3), Leucotrichum madagascariense (LDD-5) and Moranopteris sp. 1 (LDD-12). The two other dispersal events to Madagascar were at the origin of the genus Stenogrammitis (LDD-10) and of clade-V (LDD-13 from Asia). One LDD only reached Africa (LDD-8). Seven other LDD gave rise to lineages distributed in several areas, with no possibility to infer whether one or several dispersal events occurred, and which area(s) was(were) colonized at first (LDD-1, 2, 4, 6, 7, 9 and 11). Only one transatlantic LDD from Madagascar back to the Neotropics was suggested, within the genus Stenogrammitis. At a regional scale, several dispersal events were also inferred in all directions between Madagascar, Africa and the neighbouring islands, but most of them originated from Madagascar to give rise to Grammitis melanoloma endemic to Réunion, and for others to expand their original ranges (Grammitis cryptophlebia, Zygophlebia devoluta-Rouhan1379, Zygophlebia torulosa, Enterosora barbatula-Rouhan1186, Stenogrammitis oosora, Grammitis synsora, Grammitis pygmaea, Ctenopterella zenkeri and Ctenopterella parvula). In two cases, dispersal originated in other WIO islands: Ceradenia sechellarum dispersed to Africa, and Ceradenia comorensis to Madagascar. Last, only one dispersal was inferred from Africa towards Madagascar and the other WIO islands, which gave rise to clade-III (Zygophlebia-Enterosora; Fig. 4).

The analysis suggested that out of the 13 LDD that gave rise to lineages of the WIO and Africa, five were followed by species diversifications in the region (clades I to V, Fig. 4).

Discussion

Our phylogenetic analyses included 25% of all grammitid species and especially filled major gaps in the Malagasy and other WIO taxa (e.g., 87% of the described Malagasy diversity and 77% of the known endemics are now included vs. 22% and 0%, respectively in [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]). This allowed improving our understanding of both the worldwide systematics and the biogeographic history of the group, as hereafter discussed.

Systematic implications

Our results generally supported those previously obtained with a more limited sampling (Sundue et al., 2014): grammitid ferns formed a strongly supported clade, and the same global topology was recovered although support values of deeper nodes were more moderate. In addition, 22 genera were confirmed as monophyletic and four as polyphyletic (Grammitis, Oreogrammitis, Radiogrammitis and Zygophlebia; the assessment was not possible for Lomaphlebia and Acrosorus, represented by only one species each, and for Luisma and Archigrammitis still to be sampled; Fig.

2).

Besides these, our results revealed six new major findings: i) Grammitis s.s. was resolved as monophyletic; ii) all other species of Grammitis s.l. were retrieved across four lineages, reinforcing the polyphyly of this genus; iii) for the first time, Ctenopterella (newly tested here) and Enterosora were recovered as polyphyletic; iv) three new major clades were supported (grey-filled triangles 1, 2, and 3, in Fig. 2), involving species currently classified in Grammitis s.l., Ctenopterella and Enterosora; v) three new species, suspected based on morphological evidence, were confirmed as distinct molecular lineages and will be described elsewhere (Moranopteris sp. 1 as endemic to Madagascar, whereas the genus was known only from the Neotropics to date; Stenogrammitis sp. 1 as endemic to Amsterdam Island, which is the new southernmost occurrence for the genus; and Zygophlebia sp. 1 as endemic to the Comoros); and vi) a high level of cryptic species was suggested. Some of these findings being related to each other, they are hereafter discussed by genus or new major clade.

4.1.1. Grammitis s.s. is monophyletic Grammitis s.s. was newly resolved as monophyletic and sister-clade to Cochlidium (Fig. 2), whereas it was previously interpreted as paraphyletic with respect to Cochlidium [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]. Our result is in agreement with morphological observations, which suggested that all the ca. 20 species showing distinct, blackish sclerotic lamina margins (e.g. G. copelandii; Fig. 1E) were close relatives [START_REF] Parris | Five new genera and three new species of Grammitidaceae (Filicales) and the reestablishment of Oreogrammitis[END_REF]. All such species are indeed placed within Grammitis s.s. in our molecular phylogeny, and we also anticipate that the type species of the genus (G. marginella, not included here) belongs to this group. However, G. cryptophlebia and G. coriaceifolia ined. are also both nested within Grammitis s.s. based on molecular data, although their laminae do not exhibit blackish margins (Fig. 3, clade-1). It suggests that this character was lost at least once during the evolution of the clade. Consequently, the blackish sclerotic margin adds to the long list of homoplastic morphological characters in the evolution of grammitids [START_REF] Ranker | Phylogeny and evolution of Grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy[END_REF], which resulted in the recent description of many genera. 4.1.2. Grammitis s.l. and Ctenopterella are highly polyphyletic, and lead to the identification of two new major clades.

Apart from the Grammitis s.s. clade, other Grammitis species were retrieved in four lineages (Fig. 2) that are consequently in need of new generic concepts. These phylogenetic placements were described previously for G. stenophylla (sister lineage to Acrosorus and Ctenopterella denticulata), and for the three-species clade sister to Notogrammitis [START_REF] Perrie | Chloroplast DNA sequences indicate the grammitid ferns (Polypodiaceae) in New Zealand belong to a single clade, Notogrammitis gen. nov. New Zeal[END_REF][START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]. In addition, we identified two new lineages nested within clade-3 (Fig. 2).

The latter clade-3 is new for the grammitid phylogeny. Exclusively composed of African and WIO species, it was unexpected due to its singular placement within the large Paleotropical clade otherwise only composed of Asian and Oceanian taxa, and due to its grouping of six species of Grammitis with two species of Ctenopterella (Figs. 2 & 3). Laminar dissection was commonly used to distinguish Grammitis (simple, undivided laminae; Fig. 1E) from Ctenopterella (pinnatisect laminae; Fig. 1C) but the grouping of species from the two genera, in clade-3, supports leaf dissection as homoplastic as evidenced within other grammitid genera like Ceradenia (Ranker et al., 2004). This explains why the modern generic classification of grammitids is rather based on characters of the rhizomes, rhizome scales and frond hairs [START_REF] Parris | New genera of Malesian Grammitidacae (Monilophyta)[END_REF][START_REF] Sundue | A morphological cladistic analysis of Terpsichore (Polypodiaceae)[END_REF]Sundue et al., 2010a). However, many of these morphological characters are variable among the species clustered in clade-3 (e.g., rhizome symmetry is either dorsiventral or radial, rhizomes scales are concolorous or bicolor, and clathrate or not). Clade-3 could nevertheless be supported by the presence of hydathodes, hypothesizing one reversion in the anhydathodous G. obtusa (Fig. 3). This clade could be treated as one or several new genera distinct from Grammitis, which should ultimately be restricted to Grammitis s.s. Another possibility would be to accommodate the six species of Grammitis of clade-3 in an expanded concept of Ctenopterella. However, the placement of Ctenopterella in the whole tree is uncertain because the type species of the genus (C. blechnoides) has never been sampled and moreover, we recovered the three other sampled species of Ctenopterella as another distinct, new clade of the grammitid phylogeny (Fig. 2).

Of this new Ctenopterella lineage, retrieved as sister to the clade formed by Notogrammitis and three species of Grammitis (Fig. 2), two species are endemic to New Caledonia (C. lasiostipes and Ctenopterella sp. 1) and the third, Ctenopterella sp. 2, occurs in eastern Papua New Guinea, Solomon Islands and Vanuatu (Parris unpubl.; Appendix 2). We speculate that most, if not all other species from Oceania and South-eastern Asia (including the type species C. blechnoides) could belong here, i.e., 16 out of the ca. 21 species in the genus [START_REF] Parris | Ctenopterella gabonensis, a new species of grammitid fern (Polypodiaceae) from Gabon, Africa[END_REF][START_REF] Parris | New genera of Malesian Grammitidacae (Monilophyta)[END_REF]. In contrast, the four Ctenopterella species from Africa and the WIO [START_REF] Parris | Ctenopterella gabonensis, a new species of grammitid fern (Polypodiaceae) from Gabon, Africa[END_REF] likely all cluster in clade-3 (as observed for the two species included here, C. zenkeri and C. parvula).

4.1.3. Enterosora is highly polyphyletic, notably revealing a new major grammitid clade Enterosora was here recovered as polyphyletic for the first time (Fig. 2). Although the genus was previously hypothesized as paraphyletic with respect to Zygophlebia (Sundue et al., 2010a), it was recently supported as monophyletic [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF]. Our extended sampling included nine out of ca. 17 Zygophlebia species plus seven out of 11 Enterosora species, and led to two findings.

First, the close relationship between these two genera was confirmed, two lineages of Enterosora being nested within Zygophlebia (Fig. 3, clade-2). The lack of morphological apomorphy supporting Enterosora [START_REF] Ranker | Phylogeny and evolution of Grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy[END_REF][START_REF] Sundue | A morphological cladistic analysis of Terpsichore (Polypodiaceae)[END_REF] argues for including the five concerned species (E. enterosoides, E. trifurcata, E. trichosora, E. percrassa and E. barbatula) into Zygophlebia (the additional segregate lineage of E. barbatula from Réunion Island, retrieved within Ceradenia, is discussed in the next section, 4.1.4).

Second, the two species E. parietina and E. sprucei formed a new clade, placed as sister-group to Adenophorus (Figs. 2 &3, clade-1). This grouping is meaningful since E. parietina and E. sprucei are morphologically most closely related to each other within the genus. It is also in agreement with [START_REF] Bishop | Revision of the Fern Genus Enterosora (Grammitidaceae) in the New World[END_REF], who underlined that the affinity of E. sprucei (as E. gilpinae) and E. parietina to other species of Enterosora was not obvious, especially considering unique characters like medial soral placement and the number of annulus cells in the sporangia. All these characters should help to define a new genus accommodating this new clade. The type species of the genus, E. campbellii, has not been sampled yet, however we expect it to reside within clade-2 based on its assemblage of morphological characters. Within the new clade, E. parietina and E. sprucei were not monophyletic with respect to each other, in agreement with [START_REF] Bishop | Revision of the Fern Genus Enterosora (Grammitidaceae) in the New World[END_REF] who regarded them as conspecific in the Neotropics (as E. parietina). In fact, our results even support recognition of only one species on both sides of the Atlantic Ocean, as suggested by [START_REF] Parris | New Species and New Combinations in African Grammitidaceae (Filicales)[END_REF][START_REF] Parris | Grammitidaceae[END_REF] and contrary to [START_REF] Bishop | Revision of the Fern Genus Enterosora (Grammitidaceae) in the New World[END_REF] who distinguished E. parietina in the Neotropics from E. gilpinae in Africa and Madagascar. 4.1.4. High level of cryptic species as revealed by a dense sampling on Madagascar Our sampling, especially focused on Madagascar and other WIO islands and Africa, included at least two individuals from each of 30 species from this geographical region. This allowed assessment of monophyly at the species level. Based on our molecular data, a total of 12 species were revealed as non-monophyletic, although we could not identify morphological characters to differentiate the distinct lineages recovered for each nominal species (Table 4, Appendix 2).

Therefore, nine of these species are best interpreted as cryptic species [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF]. The three remaining ones (Stenogrammitis oosora, Stenogrammitis sp. 1, and Enterosora sprucei) deserve further investigation since they could actually turn out to represent a single species each (see Table 4). In three cases of polyphyly (Grammitis ebenina (Fig. 3, clade-1), Grammitis kyimbilensis (Fig. 3, clade-1) and Enterosora barbatula (Fig. 3, clade-2), the segregate lineages occurred in disjunct geographic areas. This provides an additional line of evidence to describe new species (respectively, two new species for Madagascar and one for Madagascar/Africa) that would accommodate the segregate, allopatric species with monophyly (Table 4). By contrast, for the six other cryptic species, only an in-depth study of integrative taxonomy combining molecular data, morphology, anatomy, cytometry and cytology might help to identify discriminating characters.

Given that most grammitid ferns are epiphytes, a possibility of differential host-plant specificity should also be explored.

We do not know the evolutionary processes underlying cryptic speciation, but the apparent morphological similarity must be either a plesiomorphy due to shared ancestry (morphological stasis), or a homoplastic result of parallel or convergent evolution. Morphological stasis is rather expected to result from non-ecological vicariant speciation, e.g., when the topography is complex and populations are isolated [START_REF] Britton | Topography as a driver of cryptic speciation in the high-elevation cape sedge Tetraria triangularis (Boeck.) C. B. Clarke (Cyperaceae: Schoeneae)[END_REF]. In contrast, parallel or convergent evolution usually suggests the influence of selection, e.g., to adapt to extreme environmental conditions [START_REF] Bickford | Cryptic species as a window on diversity and conservation[END_REF] such as high elevations for many grammitid ferns [START_REF] Parris | New genera of Malesian Grammitidacae (Monilophyta)[END_REF][START_REF] Sylvester | The world's highest vascular epiphytes found in the Peruvian Andes[END_REF]. We tend to favour the latter mechanism given the high dispersal capacities of ferns and the well-known homoplastic nature of several morphological traits in this group [START_REF] Ranker | Phylogeny and evolution of Grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy[END_REF]. Hybridization might also be at the origin of some cryptic species [START_REF] Mallet | Hybrid speciation[END_REF] and is thought to be especially frequent in ferns compared to angiosperms [START_REF] Barrington | Hybridization, reticulation, and species concepts in the ferns[END_REF][START_REF] Haufler | Species and Speciation[END_REF]. Hybrids are often postulated based on morphological intermediacy between the two putative parents, but they sometimes retain the overall morphology of only one parent. In such a case, the comparison of plastid-and nuclear-based phylogenetic analyses may eventually provide evidence of such reticulate evolution, and explain the incongruence between morphological and molecular plastid data.

Our results showed that the identification of morphologically cryptic lineages increased the number of sampled species from 36 to >50 in the African-WIO region (some cryptic species have to be confirmed, and their exact number cannot be firmly established). Whatever the evolutionary origins of the cryptic species, failure to distinguish them greatly underestimates species richness [START_REF] Gill | Cryptic species diversity reveals biogeographic support for the "mountain passes are higher in the tropics" hypothesis[END_REF], and consequently the impact of habitat destruction that is already a very serious threat in the Malagasy region [START_REF] Harper | Fifty years of deforestation and forest fragmentation in Madagascar[END_REF]. The inclusion of several samples per a priori delimited species is therefore crucial in a conservation perspective, to uncover the possible hidden but threatened Malagasy diversity.

Finally, taking into account all our phylogenetic findings, we suggest that the African-WIO region harbours >68 grammitid species (>50 analyzed here, plus 18 non-sampled species) instead of the 51 previously listed [START_REF] Roux | Synopsis of the Lycopodiophyta and Pteridophyta of Africa, Madagascar and neighbouring islands[END_REF]; that is, we document a >30% increase in the species richness of the region. Although some taxa are still missing and the extinction of some lineages may bias the inferences as in all biogeographic studies, our extended sampling provides valuable insights into the biogeographic events and evolutionary processes underlying the emergence of the species in the region.

Biogeographic history of grammitid ferns, and origins of the African-WIO species

4.2.1. At the worldwide scale: a Neotropical origin, followed by numerous LDD events At the global scale, as in [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF], our results supported the Neotropical origin of grammitids and the divergence and start of diversification of the group were dated to the Eocene and early Oligocene (39 and 34 Ma, respectively). This relatively recent timing confirms that dispersal, rather than vicariance linked to continental fragmentation, explains species distribution patterns. The high dispersal ability of dust-like spores has been previously invoked to explain LDD in fern lineages of diverse ages and in different areas (e.g., [START_REF] Chao | Molecular phylogeny and biogeography of the fern genus Pteris (Pteridaceae)[END_REF][START_REF] Chen | A phylogeny and biogeographic analysis for the Cape-Pondweed family Aponogetonaceae (Alismatales)[END_REF][START_REF] Hennequin | Phylogenetics and biogeography of Nephrolepisa tale of old settlers and young tramps[END_REF][START_REF] Labiak | Phylogeny and historical biogeography of the lastreopsid ferns (Dryopteridaceae)[END_REF][START_REF] Labiak | Molecular phylogeny, character evolution, and biogeography of the grammitid fern genus Lellingeria (Polypodiaceae)[END_REF][START_REF] Le Péchon | A wellsampled phylogenetic analysis of the polystichoid ferns (Dryopteridaceae) suggests a complex biogeographical history involving both boreotropical migrations and recent transoceanic dispersals[END_REF][START_REF] Lu | Phylogenetic relationships of Chinese Adiantum based on five plastid markers[END_REF]Wang et al., 2012;[START_REF] Wei | Eurasian origin, boreotropical migration and transoceanic dispersal in the pantropical fern genus Diplazium (Athyriaceae)[END_REF]. In addition, epiphytism with occurrence of long-lived and slow growing gametophytes, as observed in grammitids [START_REF] Parris | Grammitidaceae[END_REF][START_REF] Stokey | The gametophyte of the Grammitidaceae[END_REF], may allow to delay sexual reproduction until a second gametophyte germinates nearby and confer an additional advantage for successful colonization [START_REF] Dassler | Significance of gametophyte form in long-distance colonization by tropical, epiphytic ferns[END_REF][START_REF] Moran | Phytogeographic relationships between neotropical and African-Madagascan pteridophytes[END_REF][START_REF] Sessa | On the widespread capacity for, and functional significance of, extreme inbreeding in ferns[END_REF]. In contrast to those previous findings, we recovered the existence of an African-WIO clade nested within the Paleotropical clade, and composed of species sampled for the first time (clade-V, Fig. 4).

This suggests the occurrence of a newly inferred dispersal from Tropical Asia s.l. to the African-WIO region (see map, Fig. 4), which would be the oldest LDD event to this region observed in grammitids, at the end of the Oligocene. However, this inference remains to be confirmed since the branching pattern of the African-WIO clade with the rest of the Paleotropical clade was not wellsupported statistically. Therefore, we cannot fully exclude that dispersal actually occurred from the Neotropics to Africa-WIO, and either later on from Africa-WIO to Tropical Asia s.l., or independently from the Neotropics to Tropical Asia s.l.

In spite of this new finding, our study strengthened the scenario of [START_REF] Sundue | Global phylogeny and biogeography of grammitid ferns (Polypodiaceae)[END_REF] of repeated dispersals from the Neotropics to the African-WIO region (Fig. 4): while they inferred at least six such events, our data showed that at least 12 (occurring within the last 20 Ma and including species of 10 distinct genera) must actually be hypothesized to explain the current species richness of the region. This was in line with the observed distribution patterns: indeed, no genus is shared between the African-WIO region and Tropical Asia s.l. (only Ctenopterella and Grammitis were thought to be shared but turned out to be polyphyletic, with distinct lineages in both regions), whereas 11 genera are shared between the African-WIO region and the Neotropics. We do not exclude that the generic affinities between the Neotropics and the African-WIO region could be even greater since two genera were discovered recently in Madagascar, namely Leucotrichum [START_REF] Rouhan | Not so neotropical after all: the Grammitid fern genus Leucotrichum (Polypodiaceae) is also paleotropical, as revealed by a new species from Madagascar[END_REF] and Moranopteris (this study), which were previously considered endemic to the Neotropics. At the species level, even four species (A. elastica, C. serrulatum, E. sprucei and M. flabelliformis) are shared: this co-occurrence in the Neotropics and WIO may be explained by recent dispersal events (<4 Ma; and, therefore, insufficient time for geographically disjunct populations to diverge genetically) or by repeated dispersals, causing recurrent gene flow and preventing genetic differentiation.

Another eastward transatlantic migration of grammitid ferns is exemplified by the sister relationships between the Neotropical G. paramicola and G. ebenina, which is endemic to the mid-South Atlantic Ocean island of St. Helena, although this island is geographically closer to Africa (Fig. 4). A similar closer evolutionary relationship between species from St. Helena and the Neotropics has been reported in the genus Elaphoglossum [START_REF] Vasco | Circumscription and phylogeny of the Elaphoglossum ciliatum group (E. sect. Lepidoglossa, Dryopteridaceae) based on cpDNA sequences[END_REF].

In contrast to these 12 eastward dispersal events, only one back-dispersal from the African-WIO region to the Neotropics was inferred, in Stenogrammitis (clade-IV; Fig. 4). An additional such westward back-dispersal may have occurred in the Zygophlebia-Enterosora clade, where the African Zygophlebia cf. devoluta was retrieved as basal to a dichotomy of a Neotropical and an African-WIO clades: this pattern suggested either two independent dispersal events from the Neotropics to Africa-WIO, or one migration followed by a back-dispersal. In any case, transatlantic migrations appear highly skewed in their directions, with a predominant eastward direction.

The much higher affinity and closer phylogenetic relationships of grammitid ferns of the WIO with those of the Neotropics, compared to those of Tropical Asia s.l., may be due neither to ecological specificities -in all regions, most species occur in mountain forests-nor to lower dispersal abilities of taxa occurring in Tropical Asia s.l. -most of which have large distribution areas. Therefore, we rather hypothesize a higher potential for dispersal from the Neotropics than from Tropical Asia s.l. due to environmental factors, even if the Neotropics do not seem to be a major area in the biogeographic history of other Malagasy vascular plants: in their broad-scale survey, [START_REF] Buerki | Spatio-temporal history of the endemic genera of Madagascar[END_REF] identified only two undisputable cases of American ancestry for Malagasy angiosperms (in Haematodendron, Myristicaceae; and Tsoala, Solanaceae). More recently, [START_REF] Smedmark | Inferring geographic range evolution of a pantropical tribe in the coffee family (Lasiantheae, Rubiaceae) in the face of topological uncertainty[END_REF] and [START_REF] Janssens | Dispersing towards Madagascar: Biogeography and evolution of the Madagascan endemics of the Spermacoceae tribe (Rubiaceae)[END_REF] provided two additional examples of such dispersal from the Neotropics to Africa-WIO in Saldinia and Astiella and closely-related genera, respectively (both Rubiaceae).

However, this pattern remains scarce in flowering plants. In contrast, it has been inferred in several fern groups (see Introduction). In some of them (e.g., Parapolystichum and Lastreopsis; [START_REF] Labiak | Phylogeny and historical biogeography of the lastreopsid ferns (Dryopteridaceae)[END_REF] connexions between the Neotropics and the WIO could be facilitated by overland dispersal events via Antarctica, which were possible until the Late Cretaceous [START_REF] Buerki | Spatio-temporal history of the endemic genera of Madagascar[END_REF][START_REF] Yoder | Has vicariance or dispersal been the predominant biogeographic force in Madagascar? Only time will tell[END_REF]. However, it cannot be invoked in grammitids, whose stem lineage is younger (here dated to the Eocene at the earliest). Therefore, the most likely hypothesis for the strong link between the Neotropical and the WIO grammitids involves transoceanic LDD. Their highly predominant occurrence in the eastward direction could be due to transport of spores through winds blowing from the west, either surface winds over the Central Atlantic Ocean (10°S-5°N; even if these are subject to significant year-to-year changes), or the jet streams always blowing from the west and that may extend across the entire Atlantic basin [START_REF] Renner | Bayesian analysis of combined chloroplast loci, using multiple calibrations, supports the recent arrival of Melastomataceae in Africa and Madagascar[END_REF]. In fact, prevailing westerly trade winds existing at even higher latitudes (30-60° S latitude) could also have contributed to skewed dispersals [START_REF] Munoz | Wind as a Long-Distance Dispersal Vehicle in the Southern Hemisphere[END_REF], as suggested for many other fern genera with a circumaustral distribution including Madagascar [START_REF] Labiak | Phylogeny and historical biogeography of the lastreopsid ferns (Dryopteridaceae)[END_REF][START_REF] Parris | Circum-Antarctic continental distribution patterns in pteridophyte species[END_REF]Sundue et al., 2010b). Such a very southern trajectory is likely to reach Madagascar and the other WIO islands through the counter clockwise gyre over the South Atlantic Ocean, moving over the ocean south of Africa and arriving in Madagascar, as modelled for spores of Ophioglossum reticulatum [START_REF] Meza Torres | Distribution of Ophioglossum reticulatum L. in South America. A case of long-distance jump dispersal?[END_REF]. 4.2.3. At the regional African-WIO scale: Madagascar, rather than Africa, acts as a major source of dispersal Many species co-occur in Africa, Madagascar or the other WIO islands: 20 species are shared by at least two of these areas, out of the 51 species recorded in the overall region [START_REF] Roux | Synopsis of the Lycopodiophyta and Pteridophyta of Africa, Madagascar and neighbouring islands[END_REF]. In agreement with these shared distribution patterns, species that are endemic to one territory were most often retrieved as sister-species to others distributed in nearby areas, showing a high level of dispersal at this regional scale.

Strikingly, we inferred only one possible dispersal event from Africa towards Madagascar and the other WIO islands, which gave rise to clade-III (Fig. 4). In all other cases, the origin of dispersal within the region appeared to be either Madagascar or the other WIO islands. Therefore, Africa does not seem to be a source region for grammitid species in the WIO, contrary to what could be expected based on the Theory of Island Biogeography [START_REF] Macarthur | The theory of island biogeography[END_REF] and empirical evidence for most of the angiosperms studied [START_REF] Bacon | Biogeography of the Malagasy Celastraceae: Multiple independent origins followed by widespread dispersal of genera from Madagascar[END_REF][START_REF] Buerki | Spatio-temporal history of the endemic genera of Madagascar[END_REF][START_REF] Janssens | Dispersing towards Madagascar: Biogeography and evolution of the Madagascan endemics of the Spermacoceae tribe (Rubiaceae)[END_REF][START_REF] Yoder | Has vicariance or dispersal been the predominant biogeographic force in Madagascar? Only time will tell[END_REF]. Actually, there is a growing body of evidence against the paradigm postulating that continents and islands respectively act as sources and sinks of species diversity [START_REF] Heaney | Is a new paradigm emerging for oceanic island biogeography?[END_REF], and an increasing awareness of the importance of islands as cradles of diversity themselves: it has been documented for the WIO islands [START_REF] Agnarsson | The Generation of a Biodiversity Hotspot: Biogeography and Phylogeography of the Western Indian Ocean Islands[END_REF][START_REF] Krüger | Molecular phylogeny of the tribe Danaideae (Rubiaceae: Rubioideae): Another example of out-of-Madagascar dispersal[END_REF] and for the Macaronesian islands [START_REF] Patiño | Approximate Bayesian Computation Reveals the Crucial Role of Oceanic Islands for the Assembly of Continental Biodiversity[END_REF]. Although the observed patterns may be overestimated due to missing African species in our study (we included 14 out of 26 African species), grammitid ferns corroborate the latter scenario, and provide another example contradicting the traditional view. This was also suggested by the higher species diversity described from Madagascar compared to continental Africa, which harbour ca. 31 and 26 species, respectively, although Africa is 50 times larger in area. Such a lower taxonomic diversity in Africa compared to Madagascar was also observed in many other fern genera [START_REF] Kornas | The Significance of Historical Factors and Ecological Preference in the Distribution of African Pteridophytes[END_REF][START_REF] Roux | Synopsis of the Lycopodiophyta and Pteridophyta of Africa, Madagascar and neighbouring islands[END_REF].

It may be due either to the extinction of lineages after much more severe droughts there than in Madagascar during the Late Miocene and Pleistocene [START_REF] Aldasoro | Diversity and distribution of ferns in sub-Saharan Africa, Madagascar and some islands of the South Atlantic[END_REF][START_REF] Morley | Origin and Evolution of Tropical Rain Forests[END_REF], which led to a decline of African rainforests [START_REF] Kornas | The Significance of Historical Factors and Ecological Preference in the Distribution of African Pteridophytes[END_REF][START_REF] Linder | The evolution of African plant diversity[END_REF][START_REF] Morley | Origin and Evolution of Tropical Rain Forests[END_REF], or to the lack of extensive mountain ranges [START_REF] Moran | The importance of mountains to Pteridophytes, with emphasis on Neotropical montane forests[END_REF], or most probably to several intricate, both historical and more recent causes [START_REF] Couvreur | Odd man out: why are there fewer plant species in African rain forests?[END_REF].

From Madagascar and/or the neighbouring islands, dispersal also occurred over larger geographic distances, as evidenced by the colonization of the Subantarctic island of Amsterdam by the genus Stenogrammitis: the phylogeny showed that the Neotropical S. jamesonioides was sister-species to a clade formed by two species, one from Madagascar and Réunion, and the other from Amsterdam.

The most likely biogeographic scenario involves a dispersal event from the Neotropics to Madagascar and/or Réunion, later followed by dispersal to Amsterdam (Fig. 4). Whereas Sundue et al. (2014) suggested that circumaustral taxa only originated in Tropical Asia, our results therefore suggest that the WIO area also played a role in the range expansion of grammitids to this temperate area.

The occurrence of post-dispersal diversification

In total, at least 13 LDD events may explain the origin of the >50 species (including cryptic lineages) sampled in Africa, Madagascar, and other WIO islands. In particular, five LDD gave rise to diversifications counting six to 14 species each. Species diversification probably proceeds at a reduced pace in ferns compared to flowering plants, because of their strong dispersal capacities that slow down genetic differentiation [START_REF] Gillespie | Longdistance dispersal: A framework for hypothesis testing[END_REF]. We actually noticed that diversification occurred in the most ancient lineages, suggesting that the time elapsed since the colonization of a given lineage may be involved in its diversification. Species diversification was probably favoured by the environmental diversity within Madagascar: with its very diverse ecological conditions, the island is known to host spectacular species radiations [START_REF] Vences | Madagascar as a model region of species diversification[END_REF][START_REF] Soulebeau | The hypothesis of adaptive radiation in evolutionary biology: hard facts about a hazy concept[END_REF]. At the regional scale, the insular system and resulting relative isolation of populations probably also facilitated species diversification, in spite of dispersal. The evolutionary processes involved in such diversification will be investigated in further studies. 4. Taxonomic issues at the species level for African and WIO grammitid fern taxa, raised by the molecular phylogenetic results; taxonomic novelties accommodating these results are suggested whenever possible. Non-monophyletic species are indicated in color (same colors as in Fig. 3). For some species, several issues emerged and were distinguished by i/ and ii/, while the distinct lineages of non-monophyletic species were distinguished by a/ and b/.

TABLES

Species

Ceradenia pearcei

Grey ancestral area, p=1

Grey+white ancestral area, p=1

White+grey ancestral area, p=0.5 Black+grey ancestral area, p=0.5

Enterosora parietina

Zygophlebia humbertii

Terpsichore asplenifolia Grammitis cryptophlebia (Kluge 7936) Grammitis kyimbilensis (Janssen 2663) Grammitis copelandii (Rouhan 1241) Grammitis melanoloma

Grammitis ebenina (Rouhan 1180) Grammitis paramicola Grammitis cryptophlebia (Tamon 16) Grammitis bryophila Grammitis kyimbilensis (Rakotondrainibe 6347) Grammitis copelandii (Janssen 2515) Grammitis coriaceifolia ined.

Grammitis ebenina (Eastwood 199) Stenogrammitis oosora (Rakotondrainibe 6929) Calymmodon gracilis

Ctenopterella parvula

Stenogrammitis hartii

Scleroglossum pusillum

Grammitis obtusa Moranopteris sp.1

Stenogrammitis oosora (Rouhan 1188) Melpomene erecta Calymmodon sp. 1

Themelium decrescens Oreogrammitis sp. 1

Grammitis holophlebia

Galactodenia delicatula

Lellingeria hirsuta

Mycopteris taxifolia

Stenogrammitis prionodes

Grammitis diminuta

Grammitis pellucidovenosa Xiphopterella sparsipilosa

Melpomene flabelliformis

Ascogrammitis anfractuosa

Ctenopterella zenkeri (Rouhan 1354bis) Melpomene melanosticta Prosaptia sp. 1

Moranopteris truncicola

Galactodenia subscabra (Rojas 3211)

Radiogrammitis setigera

Scleroglossum sulcatum Stenogrammitis sp.1

Grammitis stenophylla

Stenogrammitis jamesonii

Stenogrammitis saffordii

Ctenopterella zenkeri (Rouhan 1347) Acrosorus friderici-et-pauli (Chen 1630) Mycopteris subtilis

  ) the age of the Neotropical clade including Mycopteris-Stenogrammitis was set to 23.3 Ma (S.D = 2.3); (3) the age of the clade including the Paleotropical clade plus Moranopteris, was set to 23.4 Ma (S.D. = 2.

  2): (A) Madagascar; (B) Mascarenes (including Réunion, Mauritius, and Rodrigues; but no grammitid species is known from Rodrigues), Comoros, and Seychelles (regions A and B together form the WIO); (C) Continental Africa except North Africa; (D) Neotropics; (E) Tropical Asia s.l. (India to South-East Asia and Japan, North Australia and Melanesia except Fiji); (F) Other Pacific Ocean islands (Fiji, Micronesia, French Polynesia, Hawaiian Islands); (G) Temperate circumaustral region, south of 28°S latitude; (H) South Atlantic Islands (Saint Helena); and (I) North America.

4. 2 . 2 .

 22 Origin of the African-WIO grammitids: a clearly predominant role of the Neotropics, with one possible migration from Tropical Asia s.l. Based on the phylogenetic pattern composed of 'a monophyletic tropical Asian clade nested within a primarily Neotropical and African grade', Sundue et al. (2014) inferred the expansion and diversification of grammitids throughout the tropics. From the Neotropics, they inferred at least six dispersal events to Madagascar, two to the Hawaiian Islands in the Pacific, and only one to tropical Asia s.l. where the group underwent a striking species diversification (referred to as the Paleotropical clade, emerging in the late Oligocene 25 Ma). From tropical Asia s.l., they showed that the group further dispersed towards the Hawaiian Islands on the one hand, and to the circumaustral region on the other hand.

Fig. 2 -

 2 Fig. 2 -Majority rule consensus phylogram of grammitids recovered from the BI based on the

Fig. 3 -

 3 Fig. 3 -Detailed phylograms of clades 1 to 3. Unless mentioned next to the nodes, support values

Fig. 4 -

 4 Fig. 4 -Molecular dating estimates (represented on the maximum clade credibility tree) and

Table 1 .

 1 Grammitid genera occurring in the Western Indian Ocean (WIO, incl. Madagascar) and Africa, based on[START_REF] Roux | Synopsis of the Lycopodiophyta and Pteridophyta of Africa, Madagascar and neighbouring islands[END_REF] updated with more recent genera recognized, and corrected for some distributions.

	Genus	Number of	Number of species	Number of	Comments (at the worldwide
		species	recorded in the	species	scale)
		recorded	WIO region	recorded in
		worldwide	[and in Madagascar	Africa
			only]
	Alansmia	26	1 [1]	1
	Ceradenia	73	5 [2]	2
	Cochlidium	18	1 [1]	1
	Ctenopterella 24	3 [3]	2	monophyly never tested
	Enterosora	11	2 [2]	2	monophyletic but nested in
					Zygophlebia
	Grammitis	26	12 [10]	7	polyphyletic, and Grammitis s.s.
					paraphyletic with respect to
					Cochlidium
	Leucotrichum 6	1 [1]	0
	Melpomene	29	1 [1]	1
	*Moranopteris 30	0	0	monophyly to be tested given the
					newly discovered species in
					Madagascar
	Stenogrammitis 31	3 [3]	7
	Zygophlebia	20	7 [7]	3	paraphyletic with respect to
					Enterosora
	*Only known from the Neotropics to date, but a new species was recently discovered in
	Madagascar

Table 2 .

 2 PCR primers and thermal cycling conditions; primers used as internal primers for sequencing only are marked with an asterisk (*)

					Thermal cycling
	DNA region Primer name	Literature reference	Primer sequence (5' to 3')	conditions
			Schuettpelz and Pryer,	AAT GTT ACT TGT GAA
	atpB	ESATPB172F ESATPE45R q OR 1163F*	2007 Schuettpelz and Pryer, 2007 Wolf, 1997	GTW CAA CAA T ATT CCA AAC WAT TCG ATT WGG AG ATG GCA GAA TRT TTC CGA GAT RTY A TTC CTG YAR AGA NCC	5 min 94°C / 40 × (30s 94°C / 1 min 50°C / 2.5 min 72°C) / 10 min 72°C
		ATPB910R*	Pryer et al., 2004	CAT TTC TGT
			Schuettpelz and Pryer,	ATG TCA CCA CAA ACG
	rbcL	ESRBCL1F ESRBCL1361R ESRBCL628F*	2007 Schuettpelz and Pryer, 2007 Schuettpelz and Pryer, 2007 Schuettpelz and Pryer,	GAG ACT AAA GC TCA GGA CTC CAC TTA CTA GCT TCA CG CCA TTY ATG CGT TGG AGA GAT CG GAA RCG ATC TCT CCA	5 min 94°C / 40 × (30s 94°C / 45s 50°C / 2.5 min 72°C) / 10 min 72°C
		ESRBCL654R*	2007	ACG CAT
				GCG GGT ATA GTT TAG
	trnG-trnR	TRNG1F TRNR22R TRNG43F1*	Nagalingum et al., 2007 Nagalingum et al., 2007 Nagalingum et al., 2007	TGG TAA CTA TCC ATT AGA CGA TGG ACG TGA TGC GGG TTC GAT TCC CG GCG GGA ATC GAA CCC	5 min 94°C / 40 × (30s 94°C / 1 min 50°C / 1.5 min 72°C) / 10 min 72°C
		TRNG63R*	Nagalingum et al., 2007	GCA TCA
				ATT TGA ACT GGT GAC	5 min 94°C / 40
	trnL-trnF	f	Taberlet et al., 1991	ACG AG	× (30s 94°C /
					30s 50°C /
				GGC AGC CCC CAR ATT	1.5min 72°C) /
		Fern-1	Taberlet et al., 1991	CAG GGR AAC C	10min 72°C
				AGT TGT TAG TTG TTG AGT	5 min 94°C / 40
	rps4-trnS	rps4-3r.f	Smith and Cranfill, 2002	AT	× (30s 94°C /
					30s 50°C / 45 s
					72°C) / 10 min
		trnS-r	Smith and Cranfill, 2002	TAC CGA GGG TTC GAA TC	72°C

  Morphology and habit of the 11 genera distributed in Madagascar: (A) Alansmia elastica (Rouhan et al. 1138); (B) Ceradenia deltodon (Rouhan et al. 1350); (C) Ctenopterella zenkeri

	Grammitis		yes	belongs to clade-3, a newly recovered	to be placed in a new genus
	holophlebia Grammitis obtusa FIGURES		yes	clade for grammitids belongs to clade-3, a newly recovered	to be placed in a new genus
				clade for grammitids
	Figure Fig. 2 Fig. 2 Fig. 3, clade-3 Fig.1 -(Rouhan et al. 1347); (D) Enterosora sprucei (Rouhan et al. 1227); (E) Grammitis copelandii Monophyletic Taxonomic issues Ctenopterella parvula yes belongs to clade-3, a newly recovered clade for grammitids Grammis pellucidovenosa yes belongs to clade-3, a newly recovered clade for grammitids Grammitis pervillei untested (1 sample included) belongs to clade-3, a newly recovered clade for grammitids Grammitis pygmaea yes belongs to clade-3, a newly recovered	Taxonomic novelties suggested should possibly be placed in a new genus if the type species of the genus (C. blechnoides) belongs to a distinct clade to be placed in a new genus to be placed in a new genus to be placed in a new genus
	Fig. 3, clade-3 (Rouhan et al. 1190); (F) Leucotrichum madagascariense (Rakotondrainibe 6957); (G) Ctenopterella zenkeri no i/ belongs to clade-3, a newly recovered clade for grammitids clade for grammitids Grammitis synsora yes belongs to clade-3, a newly recovered	i/ should possibly be placed in a new genus if the type species of the genus (C. blechnoides) to be placed in a new genus
	ii/ cryptic species (diphyletic) clade for grammitids genus known only from the Neotropics, to Moranopteris sp. 1 (Bauret et al. 107); (H) Stenogrammitis hildebrandtii (Rouhan et al. 1375); (I) Moranopteris sp. 1 untested	belongs to a distinct clade ii/ to be further investigated 1 new species endemic to Madagascar
	Enterosora barbatula Fig. 3, clade-2 Appendix 2 Melpomene flabelliformis (Bauret et al. 105); (J) Zygophlebia goodmanii (Rouhan et al. 1382); (K) no cryptic species, diphyletic: a/ La Réunion (nested within Ceradenia) (1 sample included) date	a/ to be combined with Ceradenia
	b/ Madagascar-Africa (nested within Zygophlebia) paraphyletic Cochilidum serrulatum (Bauret et al. 7). Photos: G. Rouhan; except G, I, K: L. Bauret. Stenogrammitis oosora no	b/ 1 new species for Madagascar and Africa (type of E. barbatula being from Réunion) to to be further investigated (paraphyly being not well supported, this species might eventually
					be described under Zygophlebia be resolved as monophyletic)
	Enterosora sprucei Stenogrammitis sp. 1	Fig. 2	no no	i/ belongs to a newly recovered clade (E. undescribed species, paraphyletic with	i/ E.sprucei/E.parietina should possibly be 1 new species for Amsterdam Island (but
		Fig. 3, clade-1 Appendix 2		sprucei/E.parietina) for grammitids respect to Stenogrammitis sp. 2	placed in a new genus if the type species of the genus (E. campbellii) belongs to a distinct further investigation should assess its conspecificity with Stenogrammitis sp. 2)
	Stenogrammitis sp. 2		untested	undescribed species (analyzed as S.	clade 1 new species for Madagascar and La
			(1 sample	ii/ paraphyletic with respect to E. reunionensis Parris ined. in Ranker et al.,	ii/ to be synonymized with E. parietina Réunion (but further investigation should
			included)	parietina-Sundue 3097 2010)	(earliest species name) assess its conspecificity with Stenogrammitis
	Grammitis copelandii		no	cryptic species, diphyletic	to be further investigated sp. 1)
	Grammitis Zygophlebia devoluta		no no	cryptic species, diphyletic cryptic species, polyphyletic (4 lineages)	to be further investigated to be further investigated
	cryptophlebia Zygophlebia		no	cryptic species, paraphyletic with respect	to be further investigated
	Grammitis ebenina goodmanii Zygophlebia sp. 1 Grammitis kyimbilensis subpinnata Zygophlebia	Fig. 3, clade-Fig. 3, clade-2 1	no yes no no	cryptic species, diphyletic: a/ St-Helena to Z. subpinnata (Rakotomalaza 129) and Z. sp. (Kessler 12778) undescribed species b/ Madagascar a/ Tanzania cryptic species, diphyletic cryptic species, polyphyletic (3 lineages)	a/ remains as G. ebenina (type specimen from St Helena) 1 new species endemic to the Comoros b/ 1 new species, endemic to Madagascar a/ remains as G. kyimbilensis (type specimen to be further investigated
					from Tanzania)
				b/ Madagascar	b/ 1 new species, endemic to Madagascar

ACKNOWLEDGMENTS

We thank Drs. B. Senterre, D. Middleton and S. Lindsay for providing leaf material; T. Cheminel and I. Fabre for their help in the lab; and Dr. M. S. Vorontsova and another, anonymous reviewer for their valuable comments on an earlier version of the manuscript. Collecting permits in Madagascar were granted by Madagascar National Parks and the Ministère de l'Environnement, de l'Ecologie et des Forêts. We are also grateful, for field assistance, to CNRE-Madagascar, MBG-Madagascar and CNDRS-Comoros (especially Y. Ibrahim). We acknowledge funding from the ATM MNHN 'Emergence des clades, des biotes et des cultures', ATM MNHN 'Taxonomie moléculaire, DNA Barcode & gestion durable des collections' and the FRB project 'Connaître pour conserver : le patrimoine naturel caché des Comores'. This project was also supported by the network 'Bibliothèque du Vivant' funded by the CNRS, the MNHN, the INRA and the CEA (Centre National de Séquençage). All molecular work was performed at the BoEM laboratory of the MNHN.

Genus or genera nov.

Appendix 2

Fully detailed majority rule consensus phylogram of grammitids recovered from the BI based on the combined dataset (atpB, rbcL, trnG-trnR, trnL-trnF and rps4-trnS). Unless mentioned next to the nodes, support values were BS≥95 and PP≥0.95. Non-monophyletic species are highlighted in bold and by one color per species. Vouchers are indicated when several samples were included for the same species. Genera names are on the right, followed by (number of species analyzed / number of samples included), as in Fig. 1.