New insights into the evolution of the fern family Dennstaedtiaceae from an expanded molecular phylogeny and morphological analysis.

Dennstaedtiaceae has 270 species, a worldwide distribution, and an edge-colonizing habit that is unusual among ferns. Aneuploidy, polyploidy, and hybrids are common in the family. Combining morphology, anatomy, chromosome number, and geographical distributions with our newly generated molecular phylogeny, we provide new insights into the evolution of the family. We paid special attention to Hypolepis. Our molecular dataset of five cpDNA markers is the most comprehensive to date, comprising 72 species (and a total of 98 taxa), of which 33 are Hypolepis (45 taxa). We also generated divergence-time estimates through BEAST with four fossil calibrations. We recovered three sub-families in Dennstaedtiaceae: Monachosoroideae (monogeneric), Dennstaedtioideae, and Hypolepidoideae. Monachosoroideae has a chromosome base number of x = 28; Hypolepidoideae of x = 26; while in Dennstaedtioideae this is still obscure, with different numbers ranging from 30 to 47. Dennstaedtioideae genera require re-circumscriptions because Dennstaedtia is polyphyletic. In Hypolepidoideae, the six genera are monophyletic. Within Hypolepis, seven geographically distinct clades were recovered; but we found no strong morphological characters to define them. Within the family, the long-creeping rhizome evolved with a change in habit: from shade-tolerant to edge-colonizers, to thicket-formers. Short or extremely large leaves are derived conditions. Sorus shape and position, glandular hairs, and prickles are homoplastic. Hybridization/allotetraploidy in Hypolepis can be suggested by the combined data. In our phylogenetic hypothesis, Dennstaedtiaceae originated around 135 Ma, with the split of Monachosoroideae around 94 Ma, and the split between Dennstaedtioideae/Hypolepidoideae around 78 Ma. All extant genera are inferred to be relatively young. Hypolepis started to diversify around 10 Ma, and it probably originated in east Asia and/or Oceania. Hypolepis reached the Neotropics twice: through elements of the Hypolepis rugosula clade (which originated at 7 Ma), and through the ancestor of the Neotropical clade, which originated at 3.1 Ma and was prickly.

Inferring the impacts of evolutionary history and ecological constraints on spore size and shape in the ferns.

PREMISE: In the ferns, cell size has been explored with spores, which are largely uniform within species, produced in abundance, and durable. However, spore size and shape have been variously defined, and the relationship of these traits to genome size has not been well established. Here, we explore the variation in fern spore size and shape by ploidy level and genome size. METHODS: Measurements of spore dimensions for two study sets of ferns, Polystichum and Adiantum, both including diploid and tetraploid taxa, provided the basis for computing estimates of shape and size as defined here. These traits were compared between and within ploidy levels and regressed with genome size estimates from flow cytometry analysis. RESULTS: All size traits were strongly correlated with genome size; the shape trait was weakly correlated. Tetraploids were larger than diploids as expected; the spores of some closely related diploid species were also significantly different in size. DISCUSSION: Researchers with access to a student-grade compound microscope can develop a valid estimate of relative genome size for ferns. These estimates provide enough resolution to infer ploidy level and explore the relationship between genome size, habitat, and physiological constraints for species within ploidy level.

Historical biogeography of the fern genus Polystichum (Dryopteridaceae) in Austral South America.

A group of seven endemic Polystichum species inhabit Patagonia, the southern region of South America. To date, evolutionary relationships of these Austral South American Polystichum remain unknown. The biota of the Southern Andes appears to be more closely related to the temperate Australasian species than to northern South American ones. Differences in morphological characters suggested that Austral South American Polystichum follows that biogeographical pattern, not being closely related to their congeners in the Northern and Central Andes. We sought to reveal the evolutionary relationships, estimate the divergence times and reconstruct both ancestral areas and ancestral ploidy levels of Austral South America Polystichum. Phylogenetic relationships were estimated using maximum likelihood and Bayesian approaches. The seven Austral South American species plus 31 Polystichum species spanning all other major biogeographic regions were sampled for three DNA markers. Divergence times were estimated in BEAST and Bayesian binary Markov chain Monte Carlo reconstruction was applied in order to infer ancestral areas. The evolution of ploidy was reconstructed on the maximum clade credibility tree, using stochastic character mapping. Austral South American Polystichum was recovered as monophyletic. The earliest divergence reconstructed within the Austral South American Clade was that of Polystichum andinum; subsequently two other lineages diverged comprising the remaining Austral South American species. The Austral South American lineage is not closely allied to North and Central Andes congeners. Long-distance dispersal of an ancestral tetraploid from Australasia during the late Miocene is the most likely explanation for the origin of Patagonian Polystichum. Then, Pliocene and Pleistocene orogenic and climatic changes may have shaped its diversification in Patagonia.

The rise of the Andes promoted rapid diversification in Neotropical Phlegmariurus (Lycopodiaceae).

Tropical mountains are disproportionately biodiverse relative to their surface area, but the processes underlying their exceptional diversity require further study. Here, we use comparative phylogenetic methods to examine the impact of the Andean orogeny on the diversification of Neotropical Phlegmariurus, a species-rich lycophyte clade. We generated a time-calibrated phylogeny of 105 species of Neotropical Phlegmariurus and estimated lineage diversification rates. We tested for correlations between lineage diversification rates and species range size, niche breadth, elevational range amplitude, and mean elevation of occurrence. A recently developed macroevolutionary model was used to incorporate geological data and test for an association between diversification rates and the Andean uplift. Diversification rates of Neotropical Phlegmariurus are negatively correlated with species range size and positively correlated with mean elevation of species occurrence. The rise of the Andes is strongly associated with increased rates of diversification in Neotropical Phlegmariurus during the last 10 Myr. Our study demonstrates the importance of mountain-building events and geographical isolation of alpine populations as drivers of rapid diversification, even in spore-dispersed plants. This work also highlights the usefulness of combined phylogenetic, geological and ecological datasets, and the promise of comparative environment-dependent diversification models in better understanding the evolutionary origins of biodiversity.

Vacuolar Protein Sorting 26C encodes an evolutionarily conserved large retromer subunit in eukaryotes that is important for root hair growth in Arabidopsis thaliana.

The large retromer complex participates in diverse endosomal trafficking pathways and is essential for plant developmental programs, including cell polarity, programmed cell death and shoot gravitropism in Arabidopsis. Here we demonstrate that an evolutionarily conserved VPS26 protein (VPS26C; At1G48550) functions in a complex with VPS35A and VPS29 necessary for root hair growth in Arabidopsis. Bimolecular fluorescence complementation showed that VPS26C forms a complex with VPS35A in the presence of VPS29, and this is supported by genetic studies showing that vps29 and vps35a mutants exhibit altered root hair growth. Genetic analysis also demonstrated an interaction between a VPS26C trafficking pathway and one involving the SNARE VTI13. Phylogenetic analysis indicates that VPS26C, with the notable exception of grasses, has been maintained in the genomes of most major plant clades since its evolution at the base of eukaryotes. To test the model that VPS26C orthologs in animal and plant species share a conserved function, we generated transgenic lines expressing GFP fused with the VPS26C human ortholog (HsDSCR3) in a vps26c background. These studies illustrate that GFP-HsDSCR3 is able to complement the vps26c root hair phenotype in Arabidopsis, indicating a deep conservation of cellular function for this large retromer subunit across plant and animal kingdoms.

Widespread sampling biases in herbaria revealed from large-scale digitization.

Nonrandom collecting practices may bias conclusions drawn from analyses of herbarium records. Recent efforts to fully digitize and mobilize regional floras online offer a timely opportunity to assess commonalities and differences in herbarium sampling biases. We determined spatial, temporal, trait, phylogenetic, and collector biases in c. 5 million herbarium records, representing three of the most complete digitized floras of the world: Australia (AU), South Africa (SA), and New England, USA (NE). We identified numerous shared and unique biases among these regions. Shared biases included specimens collected close to roads and herbaria; specimens collected more frequently during biological spring and summer; specimens of threatened species collected less frequently; and specimens of close relatives collected in similar numbers. Regional differences included overrepresentation of graminoids in SA and AU and of annuals in AU; and peak collection during the 1910s in NE, 1980s in SA, and 1990s in AU. Finally, in all regions, a disproportionately large percentage of specimens were collected by very few individuals. We hypothesize that these mega-collectors, with their associated preferences and idiosyncrasies, shaped patterns of collection bias via 'founder effects'. Studies using herbarium collections should account for sampling biases, and future collecting efforts should avoid compounding these biases to the extent possible.

Insights into evolution in Andean Polystichum (Dryopteridaceae) from expanded understanding of the cytosolic phosphoglucose isomerase gene.

Cytosolic phosphoglucose isomerase (pgiC) is an enzyme essential to glycolysis found universally in eukaryotes, but broad understanding of variation in the gene coding for pgiC is lacking for ferns. We used a substantially expanded representation of the gene for Andean species of the fern genus Polystichum to characterize pgiC in ferns relative to angiosperms, insects, and an amoebozoan; assess the impact of selection versus neutral evolutionary processes on pgiC; and explore evolutionary relationships of selected Andean species. The dataset of complete sequences comprised nine accessions representing seven species and one hybrid from the Andes and Serra do Mar. The aligned sequences of the full data set comprised 3376 base pairs (70% of the entire gene) including 17 exons and 15 introns from two central areas of the gene. The exons are highly conserved relative to angiosperms and retain substantial homology to insect pgiC, but intron length and structure are unique to the ferns. Average intron size is similar to angiosperms; intron number and location in insects are unlike those of the plants we considered. The introns included an array of indels and, in intron 7, an extensive microsatellite array with potential utility in analyzing population-level histories. Bayesian and maximum-parsimony analysis of 129 variable nucleotides in the Andean polystichums revealed that 59 (1.7% of the 3376 total) were phylogenetically informative; most of these united sister accessions. The phylogenetic trees for the Andean polystichums were incongruent with previously published cpDNA trees for the same taxa, likely the result of rapid evolutionary change in the introns and contrasting stability in the exons. The exons code a total of seven amino-acid substitutions. Comparison of non-synonymous to synonymous substitutions did not suggest that the pgiC gene is under selection in the Andes. Variation in pgiC including two additional accessions represented by incomplete sequences provided new insights into reticulate relationships among Andean taxa.

Typifications and synonymy in Polystichum (Dryopteridaceae) from Chile and Argentina.

Polystichum Roth is one of the largest and most taxonomically challenging fern genera. South American species have a rich and complex nomenclatural history; many of the early names are inadequately typified. Based on extensive examination of original type material, we designate eleven lectotypes (including Aspidium mohrioides, Aspidium montevidense f. imbricata, Aspidium montevidense f. squamulosa, Aspidium plicatum, Aspidium pycnolepis, Dicksonia andina, Polystichum elegans, Polystichum mohrioides f. latifolia, Polystichum multifidum var. autranii, Polystichum platyphyllum var. kurtziana, and Polypodium polystichoides), and one neotype (Polystichum brongniartianum) for Polystichum taxa. Furthermore, three new synonyms are proposed.

Resumen Polystichum Roth es uno de los géneros de helechos más grandes y de mayor complejidad taxonómica. Las especies sudamericanas tienen una rica y compleja historia nomenclatural, con muchos de los nombres inadecuadamente tipificados. En base a un minucioso examen del material tipo original, designamos lectotipos para once taxones de Polystichum (que incluyen Aspidium mohrioides, Aspidium montevidense f. imbricata, Aspidium montevidense f. squamulosa, Aspidium plicatum, Aspidium pycnolepis, Dicksonia andina, Polystichum elegans, Polystichum mohrioides f. latifolia, Polystichum multifidum var. autranii, Polystichum platyphyllum var. kurtziana y Polypodium polystichoides) y un neotipo (Polystichum brongniartianum). Además, se proponen 3 nuevos sinónimos.

Dynamics of asymmetrical hybridization in North American wood ferns: reconciling patterns of inheritance with gametophyte reproductive biology.

Hybridization is an important evolutionary force in plants, but the mechanisms underlying it have not been well studied for many groups. In particular, the drivers of non-random patterns of interspecific gene flow (asymmetrical hybridization) remain poorly understood, especially in the seed-free vascular plants. Here, we examine patterns of asymmetrical hybridization in two widespread fern hybrids from eastern North America and study the role of gametophyte ecology in the determination of hybridization bias. We characterized the maternal parentage of > 140 hybrid sporophytes by sequencing a c. 350-bp region of chloroplast DNA (cpDNA). To identify factors contributing to patterns of asymmetrical hybridization, we cultured gametophytes of the parental species and evaluated critical aspects of their reproductive biology. We found that asymmetrical hybridization was prevalent across the populations of both hybrids. Reproductive traits varied across species and suggest that selfing potential, antheridiogen responsiveness, sperm dispersal capacity and gamete size all contribute to the mediation of the direction of hybridization in this group. Our findings suggest that asymmetrical hybridization in ferns is driven by an array of reproductive traits. This study helps to sharpen and define a mechanistic understanding of patterns of hybridization in this group and demonstrates the importance of considering gametophyte biology when studying evolutionary processes in ferns.

Phylogeny and biogeography of exindusiate Andean Polystichum (Dryopteridaceae).

PREMISE OF THE STUDY: Uplift of the tropical Andes had a significant impact on the diversification of South American flora and fauna. Recent biogeographic inquiries have established patterns of Andean divergence, but investigations on ferns are scant. The fern genus Polystichum Roth (Dryopteridaceae) combines widespread geographic and elevational distribution with a large number of species to form an ideal system for investigation of the origin and diversification patterns of a fern lineage in the tropical Andes. METHODS: The relationships among 42 Polystichum species, including taxa from all major biogeographic regions, were analyzed with 2591 aligned nucleotides from four plastid markers using maximum parsimony and Bayesian inference. The resulting phylogeny was then used to estimate divergence times and reconstruct both ancestral areas and ancestral elevations. KEY RESULTS: Tropical Andean South American polystichums that lack an indusium (sori exindusiate) were confirmed to form a monophyletic group. This exindusiate Andean Polystichum clade diverged from a middle-elevation forest lineage now rich in species endemic to Mexico during the middle Miocene (13.12 million years ago). The majority of diversification that followed took place in the montane regions of the central Andes with radiations to the northern Andes, southeastern Brazil, and alpine regions. CONCLUSIONS: The monophyletic exindusiate Andean Polystichum lineage diverged from a Mexican lineage in the middle Miocene and diversified in the central Andes before dispersing northward. This south-to-north dispersal pattern, documented for many other Andean lineages, corresponds with episodes of uplift in the tropical Andes.

Phylogenetic analyses place the Australian monotypic Revwattsia in Dryopteris (Dryopteridaceae).

Revwattsia fragilis (Watts) D.L. Jones (Dryopteridaceae), originally described as a Polystichum Roth by the pioneer Australian botanist Reverend W.W. Watts in 1914, is a rare epiphytic fern endemic to northeastern Queensland, Australia. Known from only a few populations, it is restricted to tropical rainforests in the Atherton Tablelands. We used the cpDNA markers psbA-trnH, rbcL, rbcL-accD, rps4-trnS, trnG-trnR, trnL-trnF, and trnP-petG to infer the relationships of Revwattsia fragilis within Dryopteridaceae. Based on our molecular analysis, we were able to reject Watts's 1914 hypothesis of a close relationship to Polystichum. Its closest allies are a suite of Asian Dryopteris Adans. species including Dryopteris labordei, Dryopteris gymnosora, Dryopteris erythrosora and Dryopteris cystolepidota; maintaining Revwattsia renders Dryopteris paraphyletic. The epiphytic habit and distinctive long-creeping rhizome of Revwattsia appear to be autapomorphies and do not warrant its generic status. In the course of our investigation we confirmed that polyphyly of Dryopteris is also sustained by the inclusion of Acrorumohra (H.Itô) H.Itô, Acrophorus C.Presl, Arachniodes Blume, Diacalpe Blume, Dryopsis Holttum & P.J.Edwards, and Peranema D.Don. The epithet fragilis is occupied in Dryopteris, therefore we provide the name Dryopteris wattsiinom. nov. to accommodate Revwattsia fragilis in Dryopteris.

Systematics of the Carex aquatilis and C. lenticularis lineages: Geographically and ecologically divergent sister clades of Carex section Phacocystis (Cyperaceae).

Carex aquatilis is a highly diverse and geographically widespread member of one of the largest genera of flowering plants, Carex, and is ideally suited for the study of the role of hybridization and niche partitioning in ecological speciation. Phylogenetic analyses of nuclear ITS and ETS 1f and chloroplast psbA-trnH DNA sequences support the monophyly of a broadly defined Carex aquatilis-Carex lenticularis lineage, which includes C. aquatilis and C. lenticularis and their allies within section Phacocystis. However, neither taxon is monophyletic as currently circumscribed. The C. aquatilis lineage includes C. aquatilis and four morphologically and molecularly distinct salt-tolerant maritime taxa with which C. aquatilis s.s. is reported to form stabilized homoploid hybrids. The C. lenticularis lineage includes a paraphyletic C. lenticularis and seven allied species from both the New and Old World. The data provided here allow recognition of four species within the North American endemic C. lenticularis and suggest a neotropical origin for the C. lenticularis lineage with subsequent radiation and divergence through northwestern North America to Asia and via northeastern North America to Europe and southern South America. Evolutionary rate analyses indicate an origin for the C. aquatilis-C. lenticularis group around 1.89 million years ago during the early Pleistocene.

Phylogeny of Chinese Polystichum (Dryopteridaceae) based on chloroplast DNA sequence data (trnL-F and rps4-trnS).

Polystichum is one of the largest and most taxonomically complex fern genera in China. The evolutionary relationships of Chinese Polystichum and related genera, and the relationship between our Polystichum phylogeny and ecogeographic distribution, were tested by the use of DNA sequence data. Fifty-one species of Polystichum and 21 species in allied genera were sequenced for the plastid intergenic spacers rps4-trnS and trnL-F. Maximum parsimony and Bayesian phylogenetic analyses of both individual and combined data sets showed that Chinese Polystichum as commonly recognized was paraphyletic: one clade (the CCPC clade) included Cyrtomidictyum lepidocaulon, two Cyrtogonellum species, three Cyrtomium species, and a small number of Polystichum species usually occurring on limestone. A second clade, Polystichum sensu stricto, included the remainder of the Polystichum species; these often occur on non-limestone substrates. The remaining Cyrtomium species formed the third clade. Three subclades resolved within Polystichum sensu stricto (s.s.) clade do not correspond with recent sectional classifications, and we outline the issues relevant to a new classification for the genus.

Origin of Hawaiian Polystichum (Dryopteridaceae) in the context of a world phylogeny.

A genus-wide molecular phylogeny for Polystichum and allied genera (Dryopteridaceae) was reconstructed to address the biogeographic origin and evolution of the three Hawaiian Polystichum species, all endemic there. The analysis was based on the cpDNA sequences rbcL and the trnL-F spacer from a taxonomically and geographically diverse sample. Parsimony and Bayesian phylogenetic analyses of the combined data support a monophyletic Polystichum and corroborate recent hypotheses as to membership and sequence of origin of the major groups within the genus. The Hawaiian Polystichum species are polyphyletic; two separate lineages appear to have arrived independently from the Old World. The provenance of the diploid Polystichum hillebrandii is continental eastern Asia, while the source of the polyploid lineage comprising tetraploid P. haleakalense and octoploid P. bonseyi is likely continental Asia. From our results, the origin of the Hawaiian species of Polystichum, like many Hawaiian fern genera with several species, is the result of multiple migrations to the islands, rather than single migrations yielding nearly all the local diversity as in the angiosperms. This emerging pattern provides a modern test of the premise that propagule vagility has a central role in determining pattern of evolution.

Major evolutionary events in the origin and diversification of the fern genus Polystichum (Dryopteridaceae).

Recent advances in molecular systematics of the ferns make it possible to address long-standing questions about classification of the major fern genera, such as the worldwide genus Polystichum (Dryopteridaceae), comprising at least 200 species. In this study we examined rbcL sequences and morphological characters from 55 fern taxa: 34 were from Polystichum and 21 were from other genera in the Dryopteridaceae. We found that Phanerophlebia, possibly including Polystichopsis, is the sister group to Polystichum sensu lato (s.l.), including Cyrtomium. Polystichum as commonly recognized is paraphyletic. Our results lead us to suggest recognizing the clade of earliest diverging Polystichum species as a distinct genus (Cyrtomidictyum) and to continue to recognize Cyrtomium as a separate genus, leaving a monophyletic Polystichum sensu stricto (s.s.). We resolved a tropical American clade and an African clade within Polystichum s.s. However, the resemblance between the once-pinnate, bulb-bearing calciphilic species found in Asia and the West Indies appears to be the result of convergent evolution. Optimizing our morphological character transformations onto the combined phylogeny suggests that the common ancestor of Polystichum s.l. and Phanerophlebia had evolved the common features of the alliance, including ciliate petiole-base scales, once-pinnate fronds, ultimate segments with scarious tips, peltate indusia, and microscales.