Systematics and biogeography of the Old World fern genus Antrophyum.

Antrophyum is one of the largest genera of vittarioid ferns (Pteridaceae) and is most diverse in tropical Asia and the Pacific Islands, but also occurs in temperate Asia, Australia, tropical Africa and the Malagasy region. The only monographic study of Antrophyum was published more than a century ago and a modern assessment of its diversity is lacking. Here, we reconstructed a comprehensively sampled and robustly supported phylogeny for the genus based on four chloroplast markers using Bayesian inference, maximum likelihood and maximum parsimony analyses. We then explored the evolution of the genus from the perspectives of morphology, systematics and historical biogeography. We investigated nine critical morphological characters using a morphometric approach and reconstructed their evolution on the phylogeny. We describe four new species and provide new insight into species delimitation. We currently recognize 34 species for the genus and provide a key to identify them. The results of biogeographical analysis suggest that the distribution of extant species is largely shaped by both ancient and recent dispersal events.

Reticulate evolution in the Pteris fauriei group (Pteridaceae).

The Pteris fauriei group (Pteridaceae) has a wide distribution in Eastern Asia and includes 18 species with similar but varied morphology. We collected more than 300 specimens of the P. fauriei group and determined ploidy by flow cytometry and inferred phylogenies by molecular analyses of chloroplast and nuclear DNA markers. Our results reveal a complicated reticulate evolution, consisting of seven parental taxa and 58 hybrids. The large number of hybrid taxa have added significant morphological complexity to the group leading to difficult taxonomic issues. The hybrids generally had broader ranges and more populations than their parental taxa. Genetic combination of different pairs of parental species created divergent phenotypes of hybrids, exhibited by both morphological characteristics and ecological fidelities. Niche novelty could facilitate hybrid speciation. Apogamy is common in this group and potentially contributes to the sustainability of the whole group. We propose that frequent hybridizations among members of the P. fauriei group generate and maintain genetic diversity, via novel genetic combinations, niche differentiation, and apogamy.

Providing the missing links in fern life history: Insights from a phenological survey of the gametophyte stage.

Premise: The entire life cycle of ferns has been documented, yet their life histories are still poorly understood. In particular, the phenology of fern gametophytes remains largely unknown. To address this issue, we demonstrated a new ecological approach to explore the phenological link between spore release and gametophyte maturation within the life history of a tree fern species. Methods: We conducted a serial survey of Alsophila podophylla gametophyte abundance in the field, and recorded the time of its spore release. Every two months for one year, all terrestrial fern gametophytes in an unsampled subplot were collected and identified using tissue-direct PCR. Results: We found temporal differences in gametophyte abundances, with a sevenfold difference between the highest and lowest months. The number of spores released was linked to the gametophyte abundance two months later. The switch from gametophyte to juvenile sporophyte was found to be most correlated with precipitation. Discussion: The observed fluctuation in gametophyte abundance and population structure was likely associated with the phenology of spore release and environmental factors. Importantly, these findings provide the first evidence of phenological links between different developmental stages in a fern's life history.

Organelle Genome Inheritance in Deparia Ferns (Athyriaceae, Aspleniineae, Polypodiales).

Organelle genomes of land plants are predominately inherited maternally but in some cases can also be transmitted paternally or biparentally. Compared to seed plants (>83% genera of angiosperms and >12% genera of gymnosperms), plastid genome (plastome) inheritance has only been investigated in fewer than 2% of fern genera, and mitochondrial genome (mitogenome) from only one fern genus. We developed a new and efficient method to examine plastome and mitogenome inheritance in a fern species-Deparia lancea (Athyriaceae, Aspleniineae, Polypodiales), and found that plastid and mitochondrial DNAs were transmitted from only the maternal parentage to a next generation. To further examine whether both organelle genomes have the same manner of inheritance in other Deparia ferns, we sequenced both plastid and mitochondrial DNA regions of inter-species hybrids, and performed phylogenetic analyses to identify the origins of organellar DNA. Evidence from our experiments and phylogenetic analyses support that both organelle genomes in Deparia are uniparentally and maternally inherited. Most importantly, our study provides the first report of mitogenome inheritance in eupolypod ferns, and the second one among all ferns.

Morphological characterization of infra-generic lineages in Deparia (Athyriaceae: Polypodiales).

Deparia, including the previously recognized genera Lunathyrium, Dryoathyrium (=Parathyrium), Athyriopsis, Triblemma, and Dictyodroma, is a fern genus comprising about 70 species in Athyriaceae. In this study, we inferred a robust Deparia phylogeny based on a comprehensive taxon sampling (~81% of species) that captures the morphological diversity displayed in the genus. All Deparia species formed a highly supported monophyletic group. Within Deparia, seven major clades were identified, and most of them were characterized by inferring synapomorphies using 14 morphological characters including leaf architecture, petiole base, rhizome type, soral characters, spore perine, and leaf indument. These results provided the morphological basis for an infra-generic taxonomic revision of Deparia.

Pteris latipinna sp. nov. (Pteridaceae), a new species segregated from Pteris fauriei.

Pteris fauriei is widely distributed in Eastern Asia and has high morphological variation. Some morphologically similar plants related to this species are difficult to distinguish. We showed that the new Pteris species from Taiwan, previously identified as P. fauriei, can be morphologically distinguished by its wide pinnae, larger terminal pinnae than the lateral pinnae in sterile fronds, and triangular basal segments of the lateral pinnae. It was confirmed that this species is phylogenetically separated from the other East Asian Pteris species, except for a morphologically distinct species P. arisanensis, by means of chloroplast genes, rbcL and matK. The new species is named as Pteris latipinnasp. nov., referring to its wide pinnae. Here, we provide a key to facilitate the identification of the morphologically similar Pteris species in Asia. The morphological descriptions, images, ecology, and distribution are also presented.

Evaluating the spore genome sizes of ferns and lycophytes: a flow cytometry approach.

Ferns and lycophytes produce spores to initiate the gametophyte stage for sexual reproduction. Approximately 10% of these seedless vascular plants are apomictic, and produce genomic unreduced spores. Genome size comparisons between spores and leaves are a reliable, and potentially easier way to determine their reproductive mode compared to traditional approaches. However, estimation of the spore genome sizes of these plants has not been attempted. We attempted to evaluate the spore genome sizes of ferns and lycophytes using flow cytometry, collected spores from selected species representing different spore physical properties and taxonomic groups, and sought to optimize bead-vortexing conditions. By evaluating the spore and sporophyte genome sizes, we examined whether reproductive modes could be ascertained from these flow cytometry results. We proposed two separate sets of optimized bead-vortexing conditions for the nuclear extraction of green and nongreen spores. We further successfully extracted spore nuclei of 19 families covering most orders, and the qualities and quantities of these extractions satisfied the C-value criteria. These evaluated genome sizes further supported the reproductive modes reported previously. In the current study, flow cytometry was used for the first time to evaluate the spore genome sizes of ferns and lycophytes. This use of spore flow cytometry provides a new, efficient approach to ascertaining the reproductive modes of these plants.

Not only in the temperate zone: independent gametophytes of two vittarioid ferns (Pteridaceae, Polypodiales) in East Asian subtropics.

Independent gametophyte ferns are unique among vascular plants because they are sporophyteless and reproduce asexually to maintain their populations in the gametophyte generation. Such ferns had been primarily discovered in temperate zone, and usually hypothesized with (sub)tropical origins and subsequent extinction of sporophyte due to climate change during glaciations. Presumably, independent fern gametophytes are unlikely to be distributed in tropics and subtropics because of relatively stable climates which are less affected by glaciations. Nonetheless, the current study presents cases of two independent gametophyte fern species in subtropic East Asia. In this study, we applied plastid DNA sequences (trnL-L-F and matK + ndhF + chlL datasets) and comprehensive sampling (~80%) of congeneric species for molecular identification and divergence time estimation of these independent fern gametophytes. The two independent gametophyte ferns were found belonging to genus Haplopteris (vittarioids, Pteridaceae) and no genetic identical sporophyte species in East Asia. For one species, divergence times between its populations imply recent oversea dispersal(s) by spores occurred during Pleistocene. By examining their ex situ and in situ fertility, prezygotic sterility was found in these two Haplopteris, in which gametangia were not or very seldom observed, and this prezygotic sterility might attribute to their lacks of functional sporophytes. Our field observation and survey on their habitats suggest microhabitat conditions might attribute to this prezygotic sterility. These findings point to consideration of whether recent climate change during the Pleistocene glaciation resulted in ecophysiological maladaptation of non-temperate independent gametophyte ferns. In addition, we provided a new definition to classify fern gametophyte independences at the population level. We expect that continued investigations into tropical and subtropical fern gametophyte floras will further illustrate the biogeographic significance of non-temperate fern gametophyte independence.

Historical biogeography of the fern genus Deparia (Athyriaceae) and its relation with polyploidy.

The wide geographical distribution of many fern species is related to their high dispersal ability. However, very limited studies surveyed biological traits that could contribute to colonization success after dispersal. In this study, we applied phylogenetic approaches to infer historical biogeography of the fern genus Deparia (Athyriaceae, Eupolypods II). Because polyploids are suggested to have better colonization abilities and are abundant in Deparia, we also examined whether polyploidy could be correlated to long-distance dispersal events and whether polyploidy could play a role in these dispersals/establishment and range expansion. Maximum likelihood and Bayesian phylogenetic reconstructions were based on a four-region combined cpDNA dataset (rps16-matK IGS, trnL-L-F, matK and rbcL; a total of 4252 characters) generated from 50 ingroup (ca. 80% of the species diversity) and 13 outgroup taxa. Using the same sequence alignment and maximum likelihood trees, we carried out molecular dating analyses. The resulting chronogram was used to reconstruct ancestral distribution using the DEC model and ancestral ploidy level using ChromEvol. We found that Deparia originated around 27.7Ma in continental Asia/East Asia. A vicariant speciation might account for the disjunctive distribution of East Asia-northeast North America. There were multiple independent long-distance dispersals to Africa/Madagascar (at least once), Southeast Asia (at least once), south Pacific islands (at least twice), Australia/New Guinea/New Zealand (at least once), and the Hawaiian Islands (at least once). In particular, the long-distance dispersal to the Hawaiian Islands was associated with polyploidization, and the dispersal rate was slightly higher in the polyploids than in diploids. Moreover, we found five species showing recent infraspecific range expansions, all of which took place concurrently with polyploidization. In conclusion, our study provides the first investigation using phylogenetic and biogeographic analyses trying to explore the link between historical biogeography and ploidy evolution in a fern genus and our results imply that polyploids might be better colonizers than diploids.

Global phylogeny and biogeography of grammitid ferns (Polypodiaceae).

We examined the global historical biogeography of grammitid ferns (Polypodiaceae) within a phylogenetic context. We inferred phylogenetic relationships of 190 species representing 31 of the 33 currently recognized genera of grammitid ferns by analyzing DNA sequence variation of five plastid DNA regions. We estimated the ages of cladogenetic events on an inferred phylogeny using secondary fossil calibration points. Historical biogeographical patterns were inferred via ancestral area reconstruction. Our results supported four large-scale phylogenetic and biogeographic patterns: (1) a monophyletic grammitid clade that arose among Neotropical polypod ancestors about 31.4 Ma; (2) a paraphyletic assemblage of clades distributed in the Neotropics and the Afro-Malagasy region; (3) a large clade distributed throughout the Asia-Malesia-Pacific region that originated about 23.4 Ma; and, (4) an Australian or New Zealand origin of the circumaustral genus Notogrammitis. Most genera were supported as monophyletic except for Grammitis, Oreogrammitis, Radiogrammitis, and Zygophlebia. Grammitid ferns are a well-supported monophyletic group with two biogeographically distinct lineages: a primarily Neotropical grade exhibiting several independent successful colonizations to the Afro-Malagasy region and a primarily Paleotropical clade exhibiting multiple independent dispersals to remote Pacific islands and temperate, austral regions.

Molecular phylogeny and biogeography of the fern genus Pteris (Pteridaceae).

BACKGROUND AND AIMS: Pteris (Pteridaceae), comprising over 250 species, had been thought to be a monophyletic genus until the three monotypic genera Neurocallis, Ochropteris and Platyzoma were included. However, the relationships between the type species of the genus Pteris, P. longifolia, and other species are still unknown. Furthermore, several infrageneric morphological classifications have been proposed, but are debated. To date, no worldwide phylogenetic hypothesis has been proposed for the genus, and no comprehensive biogeographical history of Pteris, crucial to understanding its cosmopolitan distribution, has been presented. METHODS: A molecular phylogeny of Pteris is presented for 135 species, based on cpDNA rbcL and matK and using maximum parsimony, maximum likelihood and Bayesian inference approaches. The inferred phylogeny was used to assess the biogeographical history of Pteris and to reconstruct the evolution of one ecological and four morphological characters commonly used for infrageneric classifications. KEY RESULTS: The monophyly of Pteris remains uncertain, especially regarding the relationship of Pteris with Actiniopteris + Onychium and Platyzoma. Pteris comprises 11 clades supported by combinations of ecological and morphological character states, but none of the characters used in previous classifications were found to be exclusive synapomorphies. The results indicate that Pteris diversified around 47 million years ago, and when species colonized new geographical areas they generated new lineages, which are associated with morphological character transitions. CONCLUSIONS: This first phylogeny of Pteris on a global scale and including more than half of the diversity of the genus should contribute to a new, more reliable infrageneric classification of Pteris, based not only on a few morphological characters but also on ecological traits and geographical distribution. The inferred biogeographical history highlights long-distance dispersal as a major process shaping the worldwide distribution of the species. Colonization of different niches was followed by subsequent morphological diversification. Dispersal events followed by allopatric and parapatric speciation contribute to the species diversity of Pteris.

Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography-electrospray ionization-tandem mass spectrometry.

A wide variety of sulfur metabolites play important roles in plant functions. We have developed a precise and sensitive method for the simultaneous measurement of several sulfur metabolites based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and (34)S metabolic labeling of sulfur-containing metabolites in Arabidopsis thaliana seedlings. However, some sulfur metabolites were unstable during the extraction procedure. Our proposed method does not allow for the detection of the important sulfur metabolite homocysteine because of its instability during sample extraction. Stable isotope-labeled sulfur metabolites of A. thaliana shoot were extracted and utilized as internal standards for quantification of sulfur metabolites with LC-MS/MS using S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), methionine (Met), glutathione (GSH), and glutathione disulfide (GSSG) as example metabolites. These metabolites were detected using electrospray ionization in positive mode. Standard curves were linear (r(2)>0.99) over a range of concentrations (SAM 0.01-2.0µM, SAH 0.002-0.10µM, Met 0.05-4.0µM, GSH 0.17-20.0µM, GSSG 0.07-20.0µM), with limits of detection for SAM, SAH, Met, GSH, and GSSG of 0.83, 0.67, 10, 0.56, and 1.1nM, respectively; and the within-run and between-run coefficients of variation based on quality control samples were less than 8%.

trnL-F is a powerful marker for DNA identification of field vittarioid gametophytes (Pteridaceae).

BACKGROUND AND AIMS: The gametophyte phase of ferns plays an important role in habitat selection, dispersal, adaptation and evolution. However, ecological studies on fern gametophytes have been impeded due to the difficulty of species identification of free-living gametophytes. DNA barcoding provides an alternative approach to identifying fern gametophytes but is rarely applied to field studies. In this study, an example of field vittarioid gametophyte identification using DNA barcoding, which has not been done before, is given. METHODS: A combination of distance-based and tree-based approaches was performed to evaluate the discriminating power of three candidate barcodes (matK, rbcL and trnL-F) on 16 vittarioid sporophytes. Sequences of the trnL-F region were generated from 15 fern gametophyte populations by tissue-direct PCR and were compared against the sporophyte dataset, using BLAST. KEY RESULTS: trnL-F earns highest primer universality and discriminatory ability scores, whereas PCR success rates were very low for matK and rbcL regions (10·8 % and 41·3 %, respectively). BLAST analyses showed that all the sampled field gametophytes could be successfully identified to species level. Three gametophyte populations were also discovered to be living beyond the known occurrence of their sporophyte counterparts. CONCLUSIONS: This study demonstrates that DNA barcoding (i.e. reference databasing, tissue-direct PCR and molecular analysis), especially the trnL-F region, is an efficient tool to identify field gametophytes, and has considerable potential in exploring the ecology of fern gametophytes.

Xiphopterella devolii (Polypodiaceae), a new species and newly recorded genus in Taiwan.

BACKGROUND: Grammitid ferns are a tropical monophyletic clade nested in Polypodiaceae, containing more than 20 genera and more than 750 species. Many of them also grow in Taiwan. During the survey of recent two decades, an unknown grammitid fern was discovered and the taxonomic treatment is given herein. RESULTS: A new species, collected from Taiwan, is recognized and named, i.e., Xiphopterella devolii S. J. Moore, Parris, & W. L. Chiou. The holotype is deposited in TAIF, and isotypes are in HAST, K, L, US, and TNS. It is also distributed on SE & S China. The genus Xiphopterella is also a new record to Taiwan. CONCLUSION: A new species, Xiphopterella devolii S. J. Moore, Parris, & W. L. Chiou is documented herein. The Xiphopterella is a new recorded genus in Taiwan and is first found beyond Malesia regions.

Asarum pubitessellatum, sp. nov. (sect. Heterotropa, Aristolochiaceae) from Taiwan based on morphological and palynological evidence.

BACKGROUND: Recently, we discovered an unknown Asarum from Taiwan which is closely related to A. crassisepalum S.F. Huang, T.H. Hsieh and T.C. Huang and A. taipingshanianum S.F. Huang, T.H. Hsieh and T.C. Huang by sharing a thick leaf blade, spreading perianth-lobes and a conical to cylindrical perianth-tube. We compared it with other related species and identified this plant as a new species. RESULTS: This new species differs distinctly from the above two related species by having larger plant body, shortened rhizomes and an inner surface of the perianth-tube that is covered with numerous simple trichomes (vs. glandular trichomes). The pollen tectum in this new species is perforate, which differs from the incomplete reticulate with small supratectate granules in A. crassisepalum and the compact rugulate with small supratectate granules in A. taipingshanianum. Furthermore, these three species are geographically separated from one another. CONCLUSIONS: Asarum pubitessellatum C.T. Lu & J.C. Wang, a new species is described and illustrated. The trichomes on the inner surface of the perianth-tube and pollen micromorphology were the valuable characters in the low-level classification of Heterotropa species in Taiwan.

Extreme multiple reticulate origins of the Pteris cadieri complex (Pteridaceae).

The Pteris cadieri complex displays extensive morphological variation and seems to have originated through hybridization. However, the members of this complex reproduce by apogamy, which usually limits genetic variation. To evaluate the hypotheses of hybrid origins, the pattern of evolution in this species complex is reconstructed. Multiple methodologies were used. Diploids, triploids, and tetraploids were identified by chromosome counts and flow cytometry. Nuclear DNA markers (cytosolic phosphoglucose isomerase gene, PgiC) were used, together with chloroplast DNA markers (atpB-rbcL spacer and rbcL gene) to infer the biparental and maternal lineages of the Pteris cadieri complex. The three cpDNA haplotype groups and five PgiC alleles found in this study indicate that the evolution of the Pteris cadieri complex has been extremely reticulate. Up to 11 taxa belonging to eight morphs were identified. By comparing genetic variation in the Pteris cadieri in two independent areas, Hainan and Taiwan, we inferred that hybridization has occurred independently in different areas. Furthermore, we found evidence for phenological divergence (evergreen and deciduous) within Taiwan. We propose that the Pteris cadieri complex originated from different genetic lineages through multiple hybridizations in different geographical areas, leading to its present morphological diversity.

Development of PCR primer sets for intron 1 of the low-copy gene LEAFY in Davalliaceae.

PREMISE OF THE STUDY: Primers were designed for amplifying intron 1 of the single-copy nuclear LEAFY gene for species of Davalliaceae. METHODS AND RESULTS: New primer sets were designed and successfully amplified for intron 1 of the LEAFY gene in 13 species representing the five genera of Davalliaceae. The orthology of these sequences was further confirmed by phylogenetic analyses. Site variation in LEAFY intron 1 sequences across genera of the Davalliaceae and among accessions of the Humata repens complex were 18% and 8%, respectively. Such variation was greater than that for the cpDNA atpB-rbcL intergenic spacer region across the same taxa and accessions. CONCLUSIONS: Using our newly designed primers, intron 1 of the LEAFY gene could be amplified for all species tested. In addition, this single-copy, biparentally inherited, and quickly evolving region showed considerable potential for addressing infraspecific-level questions.

Overcoming deep roots, fast rates, and short internodes to resolve the ancient rapid radiation of eupolypod II ferns.

Backbone relationships within the large eupolypod II clade, which includes nearly a third of extant fern species, have resisted elucidation by both molecular and morphological data. Earlier studies suggest that much of the phylogenetic intractability of this group is due to three factors: (i) a long root that reduces apparent levels of support in the ingroup; (ii) long ingroup branches subtended by a series of very short backbone internodes (the "ancient rapid radiation" model); and (iii) significantly heterogeneous lineage-specific rates of substitution. To resolve the eupolypod II phylogeny, with a particular emphasis on the backbone internodes, we assembled a data set of five plastid loci (atpA, atpB, matK, rbcL, and trnG-R) from a sample of 81 accessions selected to capture the deepest divergences in the clade. We then evaluated our phylogenetic hypothesis against potential confounding factors, including those induced by rooting, ancient rapid radiation, rate heterogeneity, and the Bayesian star-tree paradox artifact. While the strong support we inferred for the backbone relationships proved robust to these potential problems, their investigation revealed unexpected model-mediated impacts of outgroup composition, divergent effects of methods for countering the star-tree paradox artifact, and gave no support to concerns about the applicability of the unrooted model to data sets with heterogeneous lineage-specific rates of substitution. This study is among few to investigate these factors with empirical data, and the first to compare the performance of the two primary methods for overcoming the Bayesian star-tree paradox artifact. Among the significant phylogenetic results is the near-complete support along the eupolypod II backbone, the demonstrated paraphyly of Woodsiaceae as currently circumscribed, and the well-supported placement of the enigmatic genera Homalosorus, Diplaziopsis, and Woodsia.

rbcL and matK earn two thumbs up as the core DNA barcode for ferns.

BACKGROUND: DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history--an endeavor previously impossible--will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade--Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)--to further evaluate the resolving power of these loci. PRINCIPAL FINDINGS: Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate. CONCLUSIONS: Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development.

First insights into fern matK phylogeny.

MatK, the only maturase gene in the land plant plastid genome, is a very popular phylogenetic marker that has been extensively applied in reconstructing angiosperm phylogeny. However, the use of matK in fern phylogeny is largely unknown, due to difficulties with amplification: ferns have lost the flanking trnK exons, typically the region used for designing stable priming sites. We developed primers that are either universal or lineage-specific that successfully amplify matK across all fern families. To evaluate whether matK is as powerful a phylogenetic marker in ferns as in angiosperms, we compared its sequence characteristics and phylogenetic performance to those of rbcL and atpA. Among these three genes, matK has the highest variability and substitution evenness, yet shows the least homoplasy. Most importantly, applying matK in fern phylogenetics better resolved relationships among families, especially within eupolypods I and II. Here we demonstrate the power of matK for fern phylogenetic reconstruction, as well as provide primers and extensive sequence data that will greatly facilitate future evolutionary studies of ferns.