By how much do we underestimate species diversity of liverworts using morphological evidence? An example from Australasian Plagiochila (Plagiochilaceae: Jungermanniopsida).

As a framework for revisionary study of the leafy liverwort Plagiochila in Australia, two methods for species delimitation on molecular sequence data, General Mixed Yule Coalescence model (GMYC) and Automatic Barcode Gap Discovery (ABGD) were applied to a dataset including 265 individuals from Australia, New Zealand, and the Pacific. Groups returned by GMYC and ABGD were incongruent in some lineages, and ABGD tended to lump groups. This may reflect underlying heterogeneity in the history of diversification within different lineages of Plagiochila. GMYC from trees calculated using three different molecular clocks were compared, in some lineages different primary species hypotheses were returned by analyses of trees estimated under different clock models, suggesting clock model selection should be a routine component of phylogeny reconstruction for tree-based species delimitation methods, such as GMYC. Our results suggest that a minimum of 71 Plagiochilaceae species occur in Australasia, 16 more than currently accepted for the region, comprising 8 undetermined species and 8 synonyms requiring reinstatement. Despite modern taxonomic investigation over a four decade period, (1) real diversity is 29% higher than currently recognized; and (2) 12 of 33, or 36%, of currently accepted and previously untested Australasian species have circumscription issues, including polyphyly, paraphyly, internal phylogenetic structure, or combinations of two or more of these issues. These both reflect the many challenges associated with grouping decisions based solely on morphological data in morphologically simple yet polymorphic plant lineages. Our results highlight again the critical need for combined molecular-morphological datasets as a basis for resolving robust species hypotheses in species-rich bryophyte lineages.

Species delimitation and biogeography of a southern hemisphere liverwort clade, Frullania subgenus Microfrullania (Frullaniaceae, Marchantiophyta).

Frullania subgenus Microfrullania is a clade of ca. 15 liverwort species occurring in Australasia, Malesia, and southern South America. We used combined nuclear and chloroplast sequence data from 265 ingroup accessions to test species circumscriptions and estimate the biogeographic history of the subgenus. With dense infra-specific sampling, we document an important role of long-distance dispersal in establishing phylogeographic patterns of extant species. At deeper time scales, a combination of phylogenetic analyses, divergence time estimation and ancestral range estimation were used to reject vicariance and to document the role of long-distance dispersal in explaining the evolution and biogeography of the clade across the southern Hemisphere. A backbone phylogeny for the subgenus is proposed, providing insight into evolution of morphological patterns and establishing the basis for an improved sectional classification of species within Microfrullania. Several species complexes are identified, the presence of two undescribed but genetically and morphologically distinct species is noted, and previously neglected names are discussed.

The first fossil of a bolbitidoid fern belongs to the early-divergent lineages of Elaphoglossum (Dryopteridaceae).

UNLABELLED: • PREMISE OF THE STUDY: Closing gaps in the fossil record and elucidating phylogenetic relationships of mostly incomplete fossils are major challenges in the reconstruction of the diversification of fern lineages through time. The cosmopolitan family Dryopteridaceae represents one of the most species-rich families of leptosporangiate ferns, yet its fossil record is sparse and poorly understood. Here, we describe a fern inclusion in Miocene Dominican amber and investigate its relationships to extant Dryopteridaceae.• METHODS: The morphology of the fossil was compared with descriptions of extant ferns, resulting in it being tentatively assigned to the bolbitidoid fern genus Elaphoglossum. This assignment was confirmed by reconstructing the evolution of the morphological characters preserved in the inclusion on a molecular phylogeny of 158 extant bolbitidoid ferns. To assess the morphology-based assignment of the fossil to Elaphoglossum, we examined DNA-calibrated divergence time estimates against the age of the amber deposits from which it came.• KEY RESULTS: The fossil belongs to Elaphoglossum and is the first of a bolbitidoid fern. Its assignment to a particular section of Elaphoglossum could not be determined; however, sects. Lepidoglossa, Polytrichia, and Setosa can be discounted because the fossil lacks subulate scales or scales with acicular marginal hairs. Thus, the fossil might belong to either sects. Amygdalifolia, Wrightiana, Elaphoglossum, or Squamipedia or to an extinct lineage.• CONCLUSIONS: The discovery of a Miocene Elaphoglossum fossil provides remarkable support to current molecular clock-based estimates of the diversification of these ferns.

Tramps, narrow endemics and morphologically cryptic species in the epiphyllous liverwort Diplasiolejeunea.

Diplasiolejeunea is a pantropical, epiphytic genus of leafy liverworts that occurs from the lowlands to more than 4000m altitude. Phylogenetic analyses of a molecular dataset consisting of three markers (nuclear ribosomal ITS region, plastidic trnL-F region and rbcL gene) and 122 accessions (plus two outgroups, Colura and Cololejeunea) indicate that the evolutionary diversity of Diplasiolejeunea is underestimated by current morphology-based classification. Four morphologically semi-cryptic species have been recovered. The molecular phylogenies support a deep split into a Neotropical and a Paleotropical clade, the latter structured into Australasian, Asian and Afromadacascan lineages. Presented results confirm the ranges of two pantropical species (D. cavifolia, D. rudolphiana), provide evidence for dispersal from the Neotropics into the Paleotropics, indicate speciation along altitudinal gradients and demonstrate extensive morphological homoplasy. We propose a revised supraspecific classification of Diplasiolejeunea into a predominantly Paleotropical subgenus Physolejeunea and predominantly Neotropical subgenera Austrolejeuneopsis and Diplasiolejeunea, the former containing mainly epiphytic species, the latter mainly epiphylls. Several clades are supported by combinations of morphological character states, and could be assigned to sections at some later point. This is the first comprehensive phylogeny of a largely epiphyllous genus of liverworts.

A phylogeny of the northern temperate leafy liverwort genus Scapania (Scapaniaceae, Jungermanniales).

Scapania is a northern temperate genus with a few disjunctions in the south. Despite receiving considerable attention, the supraspecific classification of this genus remains unsatisfactorily solved. We use three molecular markers (nrITS, cpDNA trnL-F region, atpB-rbcL spacer) and 175 accessions belonging to 50 species (plus eight outgroup taxa) to estimate the phylogeny and to test current classification systems. Our data support the classification of Scapania into six rather than three subgenera, rearrangements within numerous sections, and inclusion of Macrodiplophyllum microdontum. Scapania species with a plicate perianth form three early diverging lineages; the most speciose subgenus, Scapania s.str., represents a derived clade. Most morphological species concepts are supported by the molecular topologies but classification of sect. Curtae requires further study. Southern lineages are nested in northern hemispheric clades. Palearctic-Nearctic distribution ranges are supported for several species.

Diversification of land plants: insights from a family-level phylogenetic analysis.

BACKGROUND: Some of the evolutionary history of land plants has been documented based on the fossil record and a few broad-scale phylogenetic analyses, especially focusing on angiosperms and ferns. Here, we reconstructed phylogenetic relationships among all 706 families of land plants using molecular data. We dated the phylogeny using multiple fossils and a molecular clock technique. Applying various tests of diversification that take into account topology, branch length, numbers of extant species as well as extinction, we evaluated diversification rates through time. We also compared these diversification profiles against the distribution of the climate modes of the Phanerozoic. RESULTS: We found evidence for the radiations of ferns and mosses in the shadow of angiosperms coinciding with the rather warm Cretaceous global climate. In contrast, gymnosperms and liverworts show a signature of declining diversification rates during geological time periods of cool global climate. CONCLUSIONS: This broad-scale phylogenetic analysis helps to reveal the successive waves of diversification that made up the diversity of land plants we see today. Both warm temperatures and wet climate may have been necessary for the rise of the diversity under a successive lineage replacement scenario.

Ectomycorrhizas from a Lower Eocene angiosperm forest.

The development of mycorrhizal associations is considered a key innovation that enabled vascular plants to extensively colonize terrestrial habitats. Here, we present the first known fossil ectomycorrhizas from an angiosperm forest. Our fossils are preserved in a 52 million-yr-old piece of amber from the Tadkeshwar Lignite Mine of Gujarat State, western India. The amber was produced by representatives of Dipterocarpaceae in an early tropical broadleaf forest. The ectomycorrhizas were investigated using light microscopy and field emission scanning electron microscopy. Dissolving the amber surrounding one of the fossils allowed ultrastructural analyses and Raman spectroscopy. Approx. 20 unramified, cruciform and monopodial-pinnate ectomycorrhizas are fossilized adjacent to rootlets, and different developmental stages of the fossil mycorrhizas are delicately preserved in the ancient resin. Compounds of melanins were detectable in the dark hyphae. The mycobiont, Eomelanomyces cenococcoides gen. et spec. nov., is considered to be an ascomycete; the host is most likely a dipterocarp representative. An early ectomycorrhizal association may have conferred an evolutionary advantage on dipterocarps. Our find indicates that ectomycorrhizas occurred contemporaneously within both gymnosperms (Pinaceae) and angiosperms (Dipterocarpaceae) by the Lower Eocene.

Formalizing morphologically cryptic biological entities: new insights from DNA taxonomy, hybridization, and biogeography in the leafy liverwort Porella platyphylla (Jungermanniopsida, Porellales).

PREMISE OF THE STUDY: Recognition and formalization of morphologically cryptic species is a major challenge to modern taxonomy. An extreme example in this regard is the Holarctic Porella platyphylla s.l. (P. platyphylla plus P. platyphylloidea). Earlier studies demonstrated the presence of three isozyme groups and two molecular lineages. The present investigation was carried out to elucidate the molecular diversity of P. platyphylla s.l. and the distribution of its main clades, and to evaluate evidence for the presence of one vs. several species. METHODS: We obtained chloroplast (atpB-rbcL, trnL-trnF) and nuclear ribosomal (ITS) DNA sequences from 101 Porella accessions (P. platyphylla s.l., P. × baueri, P. cordaeana, P. bolanderi, plus outgroup species) to estimate the phylogeny using parsimony and likelihood analyses. To facilitate the adoption of Linnean nomenclature for molecular lineages, we chose a DNA voucher as epitype. KEY RESULTS: Phylogenies derived from chloroplast vs. nuclear data were congruent except for P. platyphylla s.l., including a North American lineage that was placed sister to P. cordaeana in the chloroplast DNA phylogeny but sister to the Holarctic P. platyphylla s.str. in the nuclear DNA phylogeny. European and North American accessions of P. cordaeana and P. platyphylla form sister clades. CONCLUSIONS: The genetic structure of P. platyphylla s.l. reflects morphologically cryptic or near cryptic speciation into Holarctic P. platyphylla s.str. and North American P. platyphylloidea. The latter species is possibly an ancient hybrid resulting from crossings of P. cordaeana and P. platyphylla s.str. and comprises several distinct molecular entities.

The mid-Miocene Zhangpu biota reveals an outstandingly rich rainforest biome in East Asia.

During the Mid-Miocene Climatic Optimum [MMCO, ~14 to 17 million years (Ma) ago], global temperatures were similar to predicted temperatures for the coming century. Limited megathermal paleoclimatic and fossil data are known from this period, despite its potential as an analog for future climate conditions. Here, we report a rich middle Miocene rainforest biome, the Zhangpu biota (~14.7 Ma ago), based on material preserved in amber and associated sedimentary rocks from southeastern China. The record shows that the mid-Miocene rainforest reached at least 24.2°N and was more widespread than previously estimated. Our results not only highlight the role of tropical rainforests acting as evolutionary museums for biodiversity at the generic level but also suggest that the MMCO probably strongly shaped the East Asian biota via the northern expansion of the megathermal rainforest biome. The Zhangpu biota provides an ideal snapshot for biodiversity redistribution during global warming.

A phylogeny of Adelanthaceae (Jungermanniales, Marchantiophyta) based on nuclear and chloroplast DNA markers, with comments on classification, cryptic speciation and biogeography.

Adelanthaceae (including Jamesoniellaceae) represent a major lineage of jungermannialean liverworts that is characterized by ventral-intercalary, often flagelliform branches, succubous leaves, ovoid to cylindrical, plicate perianths with a contracted mouth, often connate bracts and bracteoles, and 4-7 stratose capsule walls. Here we present the first comprehensive molecular phylogeny of Adelanthaceae using five markers (rbcL, psbA, trnL-trnF region, atpB-rbcL spacer, nrITS1-5.8S-ITS2) and 108 accessions from throughout the geographic range of the family. The molecular data support the separation of subfamilies Adelanthoideae and Jamesonielloideae. The Adelanthoideae include the genera Adelanthus, Pseudomarsupidium and Wettsteinia. The Jamesonielloideae include representatives of the genera Anomacaulis, Cryptochila, Cuspidatula, Jamesoniella, and Syzygiella in five main clades. The monophyly of taxa in current morphological classification schemes of Jamesonielloideae is not supported by the molecular data. Based on the outcome of the molecular phylogenetic analyses we propose to include Anomacaulis and Jamesoniella kirkii in Cuspidatula, and to place Cryptochila, Roivainenia, and Jamesoniella in the synonymy of Syzygiella. Molecular data support intercontinental ranges for several species and a range formation of Adelanthaceae by frequent short-distance dispersal, rare long-distance dispersal, extinction, and diversification. Disjunct distribution patterns within the Adelanthaceae cannot be explained by Gondwanan vicariance.

Phylogeny of the paleotropical fern genus Lepisorus (Polypodiaceae, Polypodiopsida) inferred from four chloroplast DNA regions.

Phylogenetic relationships within the paleotropical genus Lepisorus (Polypodiaceae) were investigated using plastid DNA sequences from four regions: rbcL, rps4 and rps4-trnS IGS, trnL intron plus trnL-F IGS, rbcL-atpB IGS. Over 4000 nucleotides were sequenced for 77 specimens belonging to 54 species. Each cpDNA region was analyzed separately and combined into a single dataset. All phylogenetic analyses, maximum parsimony, maximum likelihood and Bayesian Inference of phylogeny, revealed the paraphyly of Lepisorus with the monotypic Drymotaenium miyoshianum and of the paleotropical genus Belvisia nested within the Lepisorus clade. Nine well-supported major clades were found. The phylogenetic results provided new evidence for the sectional classification of Lepisorus. The evolution of three morphological characters, clathrateness of rhizome scales, margin of rhizome scales and defoliated leaves, and the evolution of the karyotype, were reconstructed to identify lineage specific phenotypic character states or combination of characters. Unique character combinations, rather than synapomorphies, were found to be of systematic value in sectional delimitation. The variation of chromosome numbers is largely due to a single aneuploidy event instead of a stepwise reduction during the evolutionary history of this genus.

Phylogeny of the leafy liverwort Ptilidium: cryptic speciation and shared haplotypes between the Northern and Southern Hemispheres.

The small, phylogenetically isolated liverwort genus Ptilidium has been regarded as of cool-Gondwanic origin with the bipolar, terrestrial Ptilidium ciliare giving rise to the Northern Hemisphere epiphytes Ptilidium pulcherrimum and Ptilidium californicum. This hypothesis is examined using a dataset including three chloroplast DNA regions from 134 Ptilidium accessions and one accession each of its closest relatives Trichocoleopsis and Neotrichocolea. Maximum likelihood and parsimony analyses point to a close relationship between P. ciliare and P. pulcherrimum, whereas P. californicum is placed sister to the remainder of the genus, separated by a long branch. Haplotype analysis and our phylogeny indicate the presence of Southern Hemisphere haplotypes of P. ciliare in the Northern Hemisphere, and shared haplotypes of P. ciliare and P. pulcherrimum between Europe and North America. Based on our findings, we reject the Gondwana-scenario and propose recent long distance dispersal as an explanation for the bipolar disjunct range. Ptilidium ciliare is resolved as paraphyletic with P. pulcherrimum nested within it. An isolated Ptilidium lineage with the morphology of P. ciliare from the Himalaya region likely represents a hitherto unrecognized cryptic species. Ptilidium pulcherrimum splits into a Japanese clade and a clade with accessions from Europe and North America.

One species or at least eight? Delimitation and distribution of Frullania tamarisci (L.) Dumort. s. l. (Jungermanniopsida, Porellales) inferred from nuclear and chloroplast DNA markers.

Frullania tamarisci is usually regarded as a polymorphic, holarctic-Asian liverwort species with four allopatric subspecies [subsp. asagrayana, moniliata, nisquallensis and tamarisci]. This hypothesis is examined using a dataset including sequences of the nuclear internal transcribed spacer region and the plastid trnL-trnF and atpB-rbcL regions of 88 accessions of F. tamarisci and putatively related taxa. Maximum parsimony and maximum likelihood analyses indicate the presence of at least eight main lineages within F. tamarisci s. l. The long branches leading to the tip nodes of the different F. tamarisci s. l. clades and their partly sympatric distribution reinforce species rank. Within F. tamarisci s. l. we recognize the Asian F. moniliata, the western North American F. californica and F. nisquallensis, the eastern North American F. asagrayana, the eastern North American-European F. tamarisci s. str., the Macaronesian F. sergiae, and two newly identified European lineages assigned to as F. calcarifera and F. tamarisci var. azorica. The considerable sequence differences are not reflected in conspicuous morphological disparities, rendering F. tamarisci s. l. the most explicit example of a complex of semi-cryptic and cryptic liverwort species. The temperate Frullania clades of this study likely went through recent extinction and expansion processes as indicated by the bottleneck pattern of genetic diversity. Species from tropical regions or regions with an Atlantic climate usually contain several geographical lineages. Our findings support frequent short-distance migration, rare successful long-distance dispersal events, extinction and recolonization as an explanation for the range formation in these Frullania species.

An ancient tropical origin, dispersals via land bridges and Miocene diversification explain the subcosmopolitan disjunctions of the liverwort genus Lejeunea.

Understanding the biogeographical and diversification processes explaining current diversity patterns of subcosmopolitan-distributed groups is challenging. We aimed at disentangling the historical biogeography of the subcosmopolitan liverwort genus Lejeunea with estimation of ancestral areas of origin and testing if sexual system and palaeotemperature variations can be factors of diversification. We assembled a dense taxon sampling for 120 species sampled throughout the geographical distribution of the genus. Lejeunea diverged from its sister group after the Paleocene-Eocene boundary (52.2 Ma, 95% credibility intervals 50.1-54.2 Ma), and the initial diversification of the crown group occurred in the early to middle Eocene (44.5 Ma, 95% credibility intervals 38.5-50.8 Ma). The DEC model indicated that (1) Lejeunea likely originated in an area composed of the Neotropics and the Nearctic, (2) dispersals through terrestrial land bridges in the late Oligocene and Miocene allowed Lejeunea to colonize the Old World, (3) the Boreotropical forest covering the northern regions until the late Eocene did not facilitate Lejeunea dispersals, and (4) a single long-distance dispersal event was inferred between the Neotropics and Africa. Biogeographical and diversification analyses show the Miocene was an important period when Lejeunea diversified globally. We found slight support for higher diversification rates of species with both male and female reproductive organs on the same individual (monoicy), and a moderate positive influence of palaeotemperatures on diversification. Our study shows that an ancient origin associated with a dispersal history facilitated by terrestrial land bridges and not long-distance dispersals are likely to explain the subcosmopolitan distribution of Lejeunea. By enhancing the diversification rates, monoicy likely favoured the colonisations of new areas, especially in the Miocene that was a key epoch shaping the worldwide distribution.

DNA taxonomy, cryptic speciation and diversification of the Neotropical-African liverwort, Marchesinia brachiata (Lejeuneaceae, Porellales).

The Neotropical-African liverwort Marchesinia brachiata has long been regarded as a polymorphic species. This hypothesis is examined using a dataset including sequences of the nuclear internal transcribed spacer region and the plastidic trnL-trnF region of 39 Marchesinia accessions. Maximum parsimony, maximum likelihood and Bayesian analyses indicate that Marchesinia robusta is nested within M. brachiata s.l. The molecular topologies support at least three partly sympatric biological species within M. brachiata s.l., the Neotropical M. bongardiana and M. languida, and the Neotropical-African M. brachiata s.s. These species are incompletely separated by subtle differences in underleaf shape and leaf dentation. Long branches within M. brachiata s.s. suggest ongoing speciation processes that are not yet reflected in distinguishable morphological variation. Divergence time estimates based on nrITS sequence variation and the liverwort fossil record indicate an establishment of the species M. bongardiana, M. brachiata, M. languida, M. madagassa, and M. robusta in the Late Oligocene and Miocene. The intraspecific diversity shows distinctive patterns with evidence for constant accumulation of genetic diversity in M. robusta and M. brachiata whereas M. bongardiana and M. languida likely went through a recent extinction or expansion process as indicated by the bottleneck pattern of genetic diversity. The tropical American-African disjunction of M. brachiata is the result of dispersal rather than Western Gondwanan vicariance.

Molecular insights into the phylogeny and subgeneric classification of Frullania Raddi (Frullaniaceae, Porellales).

With an estimated 300-375 species, Frullania is the largest genus of Porellales and forms a major clade of leafy liverworts. The cosmopolitan genus includes mostly epiphytes and represents an important component of the cryptogamic vegetation in various, especially tropical, habitats. There have been abundant changes and modifications to the infrageneric classification of Frullania, with up to fifteen subgenera and numerous sections solely based on morphology. Here we present the first molecular phylogeny of Frullania using four markers (rbcL, psbA, trnL-trnF region of cp DNA, nr5.8S-ITS-2 region) and 171 accessions from throughout the range. The molecular data provide evidence for the monophyly of several subgenera and support intercontinental ranges of these clades. Previous subgeneric assignment for a suite of taxa based on morphological evidence is not supported by the molecular data. Representatives of the genera Amphijubula, Neohattoria and Schusterella are nested in robust subclades of Frullania. Basal relationships within Frullania are largely unsupported. Based on the outcome of the phylogenetic analyses we present a revised supraspecific classification and provide evidence for the monophyly of some morphological species. Disjunct distributional patterns within Frullania cannot be explained by Gondwanan vicariance.

Publisher Correction: A fossil species of the enigmatic early polypod fern genus Cystodium (Cystodiaceae) in Cretaceous amber from Myanmar.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Publisher Correction: A fossil species of the enigmatic early polypod fern genus Cystodium (Cystodiaceae) in Cretaceous amber from Myanmar.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

A fossil species of the enigmatic early polypod fern genus Cystodium (Cystodiaceae) in Cretaceous amber from Myanmar.

The monospecific fern genus Cystodium (Cystodiaceae; Polypodiales) occurs exclusively in the tropical forests of the Malay Archipelago, the Admiralty Islands, the Louisiade Archipelago, and the Solomon Islands. Divergence time estimates suggest that the genus originated in the Mesozoic; however, fossil evidence to validate this suggestion has been lacking. Amber from Myanmar (Burmese amber) is an important source of new information on the diversity of vascular cryptogams in the Cretaceous. This paper describes the fossil taxon Cystodium sorbifolioides nov. sp. based on a fragment of a fertile leaf preserved in Burmese amber that represents the first fossil evidence of the family Cystodiaceae. Cystodium sorbifolioides is used to obtain a minimum age estimate for the Cystodiaceae and the closely related, monogeneric Lonchitidaceae and Lindsaeaceae. The fossil strengthens the hypothesis that the forest ecosystems of Malesia and Melanesia represent refugia for many tropical plant lineages that originated in the Cretaceous.

Multiple Geographical Origins of Environmental Sex Determination enhanced the diversification of Darwin's Favourite Orchids.

Environmental sex determination (ESD) - a change in sexual function during an individual life span driven by environmental cues - is an exceedingly rare sexual system among angiosperms. Because ESD can directly affect reproduction success, it could influence diversification rate as compared with lineages that have alternative reproductive systems. Here we test this hypothesis using a solid phylogenetic framework of Neotropical Catasetinae, the angiosperm lineage richest in taxa with ESD. We assess whether gains of ESD are associated with higher diversification rates compared to lineages with alternative systems while considering additional traits known to positively affect diversification rates in orchids. We found that ESD has evolved asynchronously three times during the last ~5 Myr. Lineages with ESD have consistently higher diversification rates than related lineages with other sexual systems. Habitat fragmentation due to mega-wetlands extinction, and climate instability are suggested as the driving forces for ESD evolution.