Rapid allopolyploid radiation of moonwort ferns (Botrychium; Ophioglossaceae) revealed by PacBio sequencing of homologous and homeologous nuclear regions.

Polyploidy is a major speciation process in vascular plants, and is postulated to be particularly important in shaping the diversity of extant ferns. However, limitations in the availability of bi-parental markers for ferns have greatly limited phylogenetic investigation of polyploidy in this group. With a large number of allopolyploid species, the genus Botrychium is a classic example in ferns where recurrent polyploidy is postulated to have driven frequent speciation events. Here, we use PacBio sequencing and the PURC bioinformatics pipeline to capture all homeologous or allelic copies of four long (∼1 kb) low-copy nuclear regions from a sample of 45 specimens (25 diploids and 20 polyploids) representing 37 Botrychium taxa, and three outgroups. This sample includes most currently recognized Botrychium species in Europe and North America, and the majority of our specimens were genotyped with co-dominant nuclear allozymes to ensure species identification. We analyzed the sequence data using maximum likelihood (ML) and Bayesian inference (BI) concatenated-data ("gene tree") approaches to explore the relationships among Botrychium species. Finally, we estimated divergence times among Botrychium lineages and inferred the multi-labeled polyploid species tree showing the origins of the polyploid taxa, and their relationships to each other and to their diploid progenitors. We found strong support for the monophyly of the major lineages within Botrychium and identified most of the parental donors of the polyploids; these results largely corroborate earlier morphological and allozyme-based investigations. Each polyploid had at least two distinct homeologs, indicating that all sampled polyploids are likely allopolyploids (rather than autopolyploids). Our divergence-time analyses revealed that these allopolyploid lineages originated recently-within the last two million years-and thus that the genus has undergone a recent radiation, correlated with multiple independent allopolyploidizations across the phylogeny. Also, we found strong parental biases in the formation of allopolyploids, with individual diploid species participating multiple times as either the maternal or paternal donor (but not both). Finally, we discuss the role of polyploidy in the evolutionary history of Botrychium and the interspecific reproductive barriers possibly involved in these parental biases.

Observations on bipolar disjunctions of moonwort ferns (Botrychium, Ophioglossaceae).

Peter Raven, in 1963, included two fern taxa of the genus Botrychium in his list of plant species exhibiting American amphitropical bipolar disjunctions. He attributed the southern hemisphere occurrences to post-Pleistocene long-distance dispersal from counterparts in the northern hemisphere, probably assisted by annual bird migrations between the disjunct areas. Using genetic evidence gathered through worldwide analyses of phylogenetic relationship in Botrychium, we now review and reconsider Raven's conclusions. Genetic similarities indicate that South American Botrychium dusenii is an allotetraploid taxon closely related to B. spathulatum, a North American endemic, and that B. lunaria in New Zealand possesses a genotype identical to that of a taxon in North America derived through introgressive hybridization between B. lunaria and an endemic North American species, B. neolunaria. Both North American counterparts exhibit Raven's characteristics of bipolar disjuncts in their occurrence in mountain and coastal meadows, copious production of small propagules (spores in Botrychium), occurrence in habitats frequented by transpolar bird migrants, and ability to found new colonies through inbreeding. We discuss these characteristics in Botrychium and relative to other ferns and suggest further studies on Botrychium and related taxa to address questions of time, number, and mode of bipolar dispersals.

Cryptic speciation in allotetraploids: Lessons from the Botrychium matricariifolium complex.

PREMISE OF THE STUDY: Cryptic species are a challenge for botanists and taxonomists. To improve species delineation in the genus Botrychium (Ophioglossaceae), which includes multiple instances of allotetraploid speciation, we examined a cryptic species complex using genetics and morphology. METHODS: We sampled species in the B. matricariifolium complex, concentrating on the Upper Peninsula of Michigan and including multiple proposed morphospecies. We analyzed over 1500 samples using 10 enzyme systems, measured 42 quantitative and qualitative morphological characters for over 650 individuals, and analyzed 145 samples using AFLPs. We tested for diagnostic enzymes in the morphospecies and calculated the correlation between morphological and genetic distances to determine whether putatively distinct morphotypes warrant taxonomic recognition. KEY RESULTS: Allozyme allelic variation corresponded loosely to some morphotypes of B. matricariifolium, but with lower genetic distinction among them than found between B. matricariifolium and B. michiganense. Botrychium michiganense contains unique alleles, indicating a different hybrid origin from that of B. matricariifolium and supporting its status as a genetically distinct species. CONCLUSIONS: We showed that B. acuminatum morphology and genetics are accommodated taxonomically within B. matricariifolium; B. matricariifolium and B. michiganense likely represent hybridization events between related species; and morphotypes within B. matricariifolium likely represent repeated hybridization events between the same two parental species. These hybridizations have resulted in the array of morphotypes observed by field botanists. By helping to identify diagnostic morphological characters, genetic analyses also help us understand and resolve morphological variation observed in the field.

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.

Methodologies for soil extraction and conservation analysis of ferns and lycophytes with belowground gametophytes.

Premise: Studies of fern and lycophyte gametophyte biology in natural settings can be challenging, but such analyses are critical to understanding the dispersal, ecology, and conservation of these species. It is especially challenging to study species whose gametophytes and early sporophyte stages develop belowground, as is the case for species of the Ophioglossaceae, Psilotaceae, some species of the Schizeaceae (Actinostachys), and some species of the Lycopodiaceae. To study these taxa, gametophytes and young sporophytes must be extracted from the soil. Methods: In 1989, Mason and Farrar described a methodology for accomplishing the collection of belowground gametophytes and sporophytes using soil centrifugation. Here, we refine this procedure based on subsequent years of experience. Results: We found that many more sporophytes exist belowground than are represented by aboveground leaves, and that belowground sporophytes can survive indefinitely without production of aboveground leaves. Belowground gametophytes are common in areas where spore-releasing sporophyte leaves are present. Gametophytes are bisexual with male and female gametangia intermixed. Some species of Botrychium also reproduce asexually belowground through production of gemmae. Discussion: We conclude that in Botrychium, assessments of population health and structure must include analyses of the belowground plants and their habitat. Conservation management strategies must also include potential changes in the belowground habitat.

Photographic analysis of field-monitored fern gametophyte development and response to environmental stress.

Premise: Ferns differ from seed plants in possessing a life cycle that includes a small, free-living, seemingly vulnerable gametophyte stage in which sexual reproduction occurs. Most research on the response of fern gametophytes to environmental stress has been conducted on gametophytes grown in culture or harvested from natural habitats and subsequently manipulated and tested in laboratory experiments. We present a fixed-distance photographic methodology for monitoring longevity of gametophytes and their response to environmental stress in natural, undisturbed habitats over their life spans. Methods: We present methodology for non-invasive monitoring of growth and development in response to environmental factors, using programmed, fixed-distance photography, coupled with computer analyses allowing qualitative and quantitative comparisons. We tracked growth rates and stress responses of individual gametophyte plants to seasonal changes in a temperate climate. Results: Gametophytes and young sporophytes survived freezing and drought in temperate habitats, as we document through photographs and growth measurements. Gametophyte growth was suspended during the cold season and resumed the following spring. Individual gametophytes survived for up to nearly three years with retention of the ability to produce sporophytes. Discussion: Life histories of fern gametophytes in temperate habitats are more similar to those in tropical habitats than previous research has suggested. They survive and maintain reproductive capacity over several growing seasons, allowing extended opportunity for outbreeding. The application of photographic monitoring of additional species and habitats has great potential for a more complete understanding of the ecology of reproduction in homosporous vascular plants.

The sporophyte-less filmy fern of eastern North America Trichomanes intricatum (Hymenophyllaceae) has the chloroplast genome of an Asian species.

Trichomanes intricatum, the sporophyte-less filmy fern of the eastern United States, has been considered to be a species whose sporophyte generation has become extinct or is possibly still present among the many species of Trichomanes s.l. in the new world tropics but unable to grow in a temperate climate. A close relationship to Asian species has heretofore not been considered. Comparison of rbcL and rps4-trnS sequences to species of Trichomanes s.l. reveals that T. intricatum shares its chloroplast genome with Crepidomanes schmidtianum of eastern Asia. Because C. schmidtianum is a sterile triploid and the ploidy level of T. intricatum is unknown, several scenarios leading to their sharing of these maternally inherited genes must be explored.

Genetic variation in populations of the morphologically and ecologically variable fern Stegnogramma pozoi subsp. mollissima (Thelypteridaceae) in Japan.

In Japanese Stegnogramma pozoi subsp. mollissima (Fisher ex Kunze) K. Iwats. there is the intrasubspecific variation among rbcL sequences. Northern and southern plants are genetically differentiated for maternally inherited cpDNA. In the present study we examined allozyme polymorphisms to test the hypothesis that northern and southern plants may be separate species. Based on allozyme data, the degree of gene flow among populations was estimated to be large. The artificial crossing experiments between cpDNA haplotypes also suggested that isolation has not developed among these cpDNA haplotypes. However, interpopulation genetic differentiation in cpDNA was observed even in the small area at the foot of Mt. Hakone, and the cpDNA haplotypes appear to have different habitat preferences.