Splitting one species into 22: an unusual tripling of molecular, morphological, and geographical differentiation in the fern family Didymochlaenaceae (Polypodiales).

The pantropical fern genus Didymochlaena (Didymochlaenaceae) has long been considered to contain one species only. Recent studies have resolved this genus/family as either sister to the rest of eupolypods I or as the second branching lineage of eupolypods I, and have shown that this genus is not monospecific, but the exact species diversity is unknown. In this study, a new phylogeny is reconstructed based on an expanded taxon sampling and six molecular markers. Our major results include: (i) Didymochlaena is moderately or weakly supported as sister to the rest of eupolypods I, highlighting the difficulty in resolving the relationships of this important fern lineage in the polypods; (ii) species in Didymochlaena are resolved into a New World clade and an Old World clade, and the latter further into an African clade and an Asian-Pacific clade; (iii) an unusual tripling of molecular, morphological and geographical differentiation in Didymochlaena is detected, suggesting single vicariance or dispersal events in individual regions and no evidence for reversals at all, followed by allopatric speciation at more or less homogeneous rates; (iv) evolution of 18 morphological characters is inferred and two morphological synapomorphies defining the family are recognized-the elliptical sori and fewer than 10 sori per pinnule, the latter never having been suggested before; (v) based on morphological and molecular variation, 22 species in the genus are recognized contrasting with earlier estimates of between one and a few; and (vi) our biogeographical analysis suggests an origin for Didymochlaena in the latest Jurassic-earliest Cretaceous and the initial diversification of the extant lineages in the Miocene-all but one species diverged from their sisters within the last 27 Myr, in most cases associated with allopatric speciation owing to geologic and climatic events, or dispersal.

A global plastid phylogeny of the fern genus Asplenium (Aspleniaceae).

The infrageneric relationships and taxonomy of the largest fern genus, Asplenium (Aspleniaceae), have remained poorly understood. Previous studies have focused mainly on specific species complexes involving a few or dozens of species only, or have achieved a large taxon sampling but only one plastid marker was used. In the present study, DNA sequences from six plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL and trnL-F) of 1030 accessions (616 of them newly sequenced here) representing c. 420 species of Asplenium (60% of estimated species diversity), 16 species of Hymenasplenium, three Diplaziopsidaceae, and four Rhachidosoraceae were used to produce the largest genus-level phylogeny yet for ferns. Our major results include: (i) Asplenium as broadly circumscribed is monophyletic based on our inclusion of representatives of 32 of 38 named segregate genera; (ii) 11 major clades in Asplenium are identified, and their relationships are mostly well-resolved and strongly supported; (iii) numerous species, unsampled in previous studies, suggest new relationships and numerous cryptic species and species complexes in Asplenium; and (iv) the accrued molecular evidence provides an essential foundation for further investigations of complex patterns of geographical diversification, speciation and reticulate evolution in this family.

A plastid phylogeny of the Old World fern genus Leptochilus (Polypodiaceae): Implications for cryptic speciation and progressive colonization from lower to higher latitudes.

The newly defined fern genus Leptochilus contains about 50 species occurring in subtropical to tropical Asia and adjacent Pacific islands. The circumscription and phylogeny of the genus have been ambiguous and its species had been included in various genera such as Colysis, Dendroglossa, Kontumia, Microsorum, and Paraleptochilus. Previous molecular studies sampled only 2-4 molecular markers and 2-16 accessions of Leptochilus. In the present study, DNA sequences of six plastid markers of 105 accessions representing ca. 40 species of Leptochilus, including types of Colysis, Kontumia, Leptochilus, and Paraleptochilus, 39 species of six non-Leptochilus genera of Microsoroideae, and one species of Pyrrosia, are used to infer a phylogeny. Our major results include: (1) Leptochilus is monophyletic and resolved as nested within the microsoroid ferns, but its relationships with other members of Microsoroideae are not well resolved; (2) Six well-supported major clades in Leptochilus are recognized, differing from one another in molecular, morphological, and geographical features; (3) Species related to L. macrophyllus representing earliest split in Leptochilus are identified; (4) The inclusion of Microsorum pteropus in Leptochilus is confirmed, whereas M. insigne is closely related to Leptochilus but not resolved as a member of the genus; (5) The species number of the genus is likely to double the most recent estimate following our study, and quite a few cryptic species should be recognized; and (6) A basal grade formed by three major clades is recovered and they are composed of species almost exclusively distributed at lower latitudes (the Malay Archipelago), whereas the shallow-level clades contain species distributed at mainly higher latitudes, suggesting that Leptochilus might have evolved at lower latitudes and progressively dispersed to and colonized higher latitudes.

A plastid phylogeny of the fern genus Arachniodes (Dryopteridaceae).

Arachniodes (Dryopteridaceae) is one of the most confusing and controversial fern genera in terms of its circumscription, nomenclature, and taxonomy. Estimates of species number range from 40 to 200. Previous molecular works included only 2-17 accessions representing 2-12 species of Arachniodes and allied genera, leaving most of the Asian species remain unsampled and the infragneric relationships unclear. In this study DNA sequences of seven plastid markers of 343 accessions representing ca. 68 species of Arachniodes (275 accessions), and 64 outgroup accessions from subfam. Dryopteridoideae and subfam. Polybotryoideae were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Two species currently assigned in Arachniodes (A. macrostegia and A. ochropteroides are resolved outside of the core Arachniodes making the currently defined Arachniodes polyphyletic, confirming earlier findings; (2) Lithostegia, Leptorumohra, and Phanerophlebiopsis are indeed synonyms of Arachniodes; (3) Leptorumohra is confirmed to be monophyletic, but Phanerophlebiopsis is polyphyletic; (4) The New World species of Arachniodes are confirmed to be not monophyletic with A. denticulata being nested within the Old World species, suggesting that this species is dispersed from the Old World; (5) Arachniodes s.s is resolved into 12 major clades, some of which are further divisable into recognizable subclades and groups, with A. mutica from Japan being resolved as the sister to the rest of the genus; (6) A number of systematic implications of the phylogeny have been suggested; and (7) the genus is estimated to contain ca. 83 species.

A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae).

The fern genus Hymenasplenium (Aspleniaceae) is one of the two genera in the family. It is generally recognized among modern pteridologists. However, its infrageneric relationships and species diversity have been unclear and controversial. The molecular studies so far have had small taxon and character sampling. In the present study, DNA sequences of six plastid markers of 158 accessions representing ca. 40 out of ca. 50 known species of Hymenasplenium, and 16 species of Asplenium were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Hymenasplenium as currently defined is strongly supported as monophyletic; (2) three major clades representing early splits in Hymenasplenium are identified, with the Old World species being strongly supported as monophyletic; it is ambiguous if the New World species are monophyletic; (3) extensive cryptic speciation in the Old World is discovered demonstrating the complexity of evolution of the genus; and (4) six strongly or moderately supported subclades in the Old World clade are revealed, differing from one another in molecular, morphological, and geographical features.

Phylogeny of the fern subfamily Pteridoideae (Pteridaceae; Pteridophyta), with the description of a new genus: Gastoniella.

As the second most genera-rich fern family, Pteridaceae contain more than 1000 species contributing to ca. 10% of extant leptosporangiate fern diversity. The subfamily Pteridoideae is one of the five subfamilies often recognized. The circumscription of Pteridoideae has not been clear. A large number of species have not yet been included in any molecular analyses before. In this study, DNA sequences of six plastid loci of 154 accessions representing ca. 87 species in 14 genera of Pteridaceae subfam. Pteridoideae and four accessions representing two species in subfam. Parkerioideae and one species of subfam. Adiantoideae as outgroups were used to infer a phylogeny using maximum likelihood and maximum parsimony. Our analyses show that (1) Pteridoideae is monophyletic and the newly defined subfamily is composed of 14 genera including a newly described genus; (2) Pteridoideae is resolved into four strongly supported monophyletic clades: the Pteris clade, the Actiniopteris+Onychium clade, the JAPSTT clade, and the GAPCC clade, these being supported by not only molecular data but also morphological features and distribution information; (3) Onychium is confirmed as monophyletic and accessions of Onychium are resolved into two strongly supported clades, the O. cryptogrammoides clade and the O. siliculosum clade; and (4) Accessions of the traditionally defined Anogramma are resolved as paraphyletic in relation to Cerosora, Cosentinica, and Pityrogramma. Three species traditionally treated in Anogramma are in fact more closely related to Cerosora and Pityrogramma than they are to Anogramma. Gastoniella Li Bing Zhang & Liang Zhang, gen. nov. is described to accommodate these species and three new combinations are provided. Three currently known species of Gastoniella are distributed in the Ascension Island in South Atlantic Ocean, central Mexico, and tropical America, respectively. The new genus is distinct from Anogramma s.s. in having ultimate segments linear not obviously broadening toward the upper portion.

A global phylogeny of the fern genus Tectaria (Tectariaceae: Polypodiales) based on plastid and nuclear markers identifies major evolutionary lineages and suggests repeated evolution of free venation from anastomosing venation.

Tectaria (Tectariaceae) is one of the most confusing fern genera in terms of its circumscription and phylogeny. Since its original description, a number of genera had been moved into or related with this genus, while others had been segregated from it. Tectaria is also among the largest fern genera, comprising 150-210 mostly tropical species. Previous molecular studies have been far from comprehensive (sampling no more than 76 accessions from 52 species), limited in geographic scope (mainly restricted to Asia), and based exclusively on plastid markers. In the present study, DNA sequences of eight plastid and one nuclear marker of 360 accessions representing ca. 130 species of Tectaria, ca. 36 species of six non-Tectaria genera in Tectariaceae, 12 species of Davalliaceae, Oleandraceae, and Polypodiaceae, and 13 species of Lomariopsidaceae were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) the most recently proposed circumscription of Tectaria is strongly supported as monophyletic; (2) the genera Lenda, Microbrochis, Phlebiogonium, and Sagenia, sampled here for the first time, are resolved as part of Tectaria; (3) four superclades representing early splits in Tectaria are identified, with the Old World species being sister to the New World species; (4) 12 well-supported major clades in Tectaria are revealed, differing from one another in molecular, morphological, and geographical features; (5) evolution of 13 morphological characters is inferred in a phylogenetic context and morphological synapomorphies of various clades are identified; and in particular (6) free venation in Tectaria is inferred to be repeatedly derived from anastomosing venation, an evolutionary phenomenon not documented previously in vascular plants in a phylogenetic context based on both plastid and nuclear evidence.

A plastid phylogeny and character evolution of the Old World fern genus Pyrrosia (Polypodiaceae) with the description of a new genus: Hovenkampia (Polypodiaceae).

The Old World fern genus Pyrrosia (Polypodiaceae) offers a rare system in ferns to study morphological evolution because almost all species of this genus are well studied for their morphology, anatomy, and spore features, and various hypotheses have been proposed in terms of the phylogeny and evolution in this genus. However, the molecular phylogeny of the genus lags behind. The monophyly of the genus has been uncertain and a modern phylogenetic study of the genus based on molecular data has been lacking. In the present study, DNA sequences of five plastid markers of 220 accessions of Polypodiaceae representing two species of Drymoglossum, 14 species of Platycerium, 50 species of Pyrrosia, and the only species of Saxiglossum (subfamily Platycerioideae), and 12 species of other Polypodiaceae representing the remaining four subfamilies are used to infer a phylogeny of the genus. Major results and conclusions of this study include: (1) Pyrrosia as currently circumscribed is paraphyletic in relation to Platycerium and can be divided into two genera: Pyrrosia s.s. and Hovenkampia (gen. nov.), with Hovenkampia and Platycerium forming a strongly supported clade sister to Pyrrosia s.s.; (2) Subfamily Platycerioideae should contain three genera only, Hovenkampia, Platycerium, and Pyrrosia s.s.; (3) Based on the molecular phylogeny, macromorphology, anatomical features, and spore morphology, four major clades in the genus are identified and three of the four are further resolved into four, four, and six subclades, respectively; (4) Three species, P. angustissima, P. foveolata, and P. mannii, not assigned to any groups by Hovenkamp (1986) because of their unusual morphology, each form monospecific clades; (5) Drymoglossum is not monophyletic and those species previously assigned to this genus are resolved in two different subclades; (6) Saxiglossum is resolved as the first lineage in the Niphopsis clade; and (7) The evolution of ten major morphological characters in the subfamily is inferred based on the phylogeny and various morphological synapomorphies for various clades and subclades are identified.

A large-scale phylogeny of the lycophyte genus Selaginella (Selaginellaceae: Lycopodiopsida) based on plastid and nuclear loci.

The lycophyte genus Selaginella alone constitutes the family Selaginellaceae, the largest of the lycophyte families. The genus is estimated to contain 700-800 species distributed on all continents except Antarctica, with highest species diversity in tropical and subtropical regions. The monophyly of Selaginella in this broad sense has rarely been doubted, whereas its intrageneric classification has been notoriously contentious. Previous molecular studies were based on very sparse sampling of Selaginella (up to 62 species) and often used DNA sequence data from one genome. In the present study, DNA sequences of one plastid (rbcL) and one nuclear (ITS) locus from 394 accessions representing approximately 200 species of Selaginella worldwide were used to infer a phylogeny using maximum likelihood, Bayesian inference and maximum parsimony methods. The study identifies strongly supported major clades and well resolves relationships among them. Major results include: (i) six deep-level clades are discovered representing the deep splits of Selaginella; and (ii) 20 major clades representing 20 major evolutionary lineages are identified, which differ from one another in molecular, macro-morphological, ecological and spore features, and/or geographical distribution.

Two new species in the fern genus Lomariopsis (Lomariopsidaceae) from East Asia.

Two East Asian Lomariopsis (Lomariopsidaceae, Polypodiales) species, Lomariopsismoorei and Lomariopsislongini, which were previously misidentified as L.spectabilis, are here described as new species based on evidence from morphological characters and a molecular phylogeny. The two species differ from the three other described species in East Asia by their venation, pinna shapes, and perine morphology. A phylogeny based on a combined dataset of three chloroplast regions (rbcL+ rps4-trnS + trnL-L-F) showed that L.moorei and L.longini each formed a well-supported monophyletic group which was distantly related to both L.spectabilis and the other morphologically similar East Asian species, L.boninensis.