Evolution,phylogeography and taxonomy of allopolyploid Dactylorhiza (Orchidaceae) and its implications for conservation

This review is based on recent molecular studies of Dactylorhiza (Orchidaceae). Most of the studies have focused on the allotetraploid members of the genus in general and on D. majalis ssp. lapponica in particular. It was concluded that most of the allotetraploid taxa have derived from hybridizations between the parental lineages D. maculata s.l. and D. incarnata s.l., with D. maculata s.l. serving as the seed parent. Evidence of multiple origins was found both among northern European allotetraploids as well as among Greek allotetraploids. Introgression from both parental lineages and hybridizations between independently derived polyploid lineages was also detected. The three morphologically similar taxa D. majalis ssp. traunsteineri, ssp. lapponica and ssp. russowii should be treated as one and most of the Greek allotetraploids should be regarded as regional variants of the southeastern European D. majalis ssp. cordigera. The Balkans and the Alps most probably served as refugia for the genus during the last glaciations and at least two waves of immigration reached Scandinavia. Finally, we suggest that the conservation of allotetraploid Dactylorhiza should emphasize important geographic areas and habitats and that the allopolyploids should have the same conservation status as the diploids.     

Plastid DNA variation in the Dactylorhiza incarnata/maculata polyploid complex and the origin of allotetraploid D. sphagnicola (Orchidaceae)

To obtain further information on the polyploid dynamics of the the Dactylorhiza incarnata/maculata polyploid complex and the origin of the allotetraploid D. sphagnicola (Orchidaceae), plastid DNA variation was studied in 400 plants from from Sweden and elsewhere in Europe and Asia Minor by means of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and sequencing. Allotetraploid taxa in Europe are known have evolved by multiple independent polyploidization events following hybridization between the same set of two distinct ancestral lineages. Most allotetraploids have inherited the plastid genome from parents similar to D. maculata sensu lato, which includes, e.g. the diploid D. fuchsii and the autotetraploid D. maculata sensu stricto. D. sphagnicola carries a separate plastid haplotype different from the one found in other allotetraploid taxa, which is in agreement with an independent origin from the parental lineages. Some of the remaining allotetraploids have local distributions and appear to be of postglacial origin, whereas still other allotetraploids may be of higher age, carrying plastid haplotypes that have not been encountered in present day representatives of the parental lineages. Introgression and hybridization between diploids and allotetraploids, and between different independently derived allotetraploids may further have contributed to genetic diversity at the tetraploid level. Overall, the Dactylorhiza polyploid complex illustrates how taxon diversity and genetic diversity may be replenished rapidly in a recently glaciated area.     

Patterns of polyploid evolution in Greek marsh orchids (Dactylorhiza; Orchidaceae) as revealed by allozymes, AFLPs, and plastid DNA data

Polyploidy is common in higher plants, and speciation in polyploid complexes is usually the result of reticulate evolution. We examined variation in nuclear AFLP fingerprints, nuclear isozymes, and hypervariable plastid DNA loci to describe speciation patterns and species relationships in the Dactylorhiza incarnata/maculata polyploid complex (marsh orchids; Orchidaceae) in Greece. Several endemic taxa with restricted distribution have been described from this area, and to propose meaningful conservation priorities, detailed relationships need to be known. We identified four independently derived allopolyploid lineages, which is a pattern poorly correlated with prevailing taxonomy. Three lineages were composed of populations restricted to small areas and may be of recent origins from extant parental lineages. One lineage with wide distribution in northern Greece was characterized by several unique plastid haplotypes that were phylogenetically related and evidently older. The D. incarnata/maculata polyploid complex in Greece has high levels of genetic diversity at the polyploid level. This diversity has accumulated over a long time and may include genetic variants originating from now extinct parental populations. Our data also indicate that the Balkans may have constituted an important refuge from which northern European Dactylorhiza were recruited after the Weichselian ice age.     

Evolutionary history of the Dactylorhiza maculata polyploid complex (Orchidaceae)

Taxonomic complexity may be associated with migration history and polyploidy. We used plastid and nuclear DNA markers to investigate the evolutionary history of the systematically challenging Dactylorhiza maculata polyploid complex. A total of 1833 individuals from 298 populations from throughout Europe were analysed. We found that gene flow was limited between the two major taxa, diploid ssp. fuchsii (including ssp. saccifera) and tetraploid ssp. maculata. A minimum of three autotetraploid lineages were discerned: (1) southern/western ssp. maculata; (2) northern/eastern ssp. maculata; and (3) Central European ssp. fuchsii. The two ssp. maculata lineages, which probably pre‐date the last glaciation, form a contact zone with high genetic diversity in central Scandinavia. Intermediate plastid haplotypes in the contact zone hint at recombination. Central Europe may have been a source area for the postglacial migration for the southern/western lineage of ssp. maculata, as well as for ssp. fuchsii. The northern/eastern lineage of ssp. maculata may have survived the LGM in central Russia west of the Urals. The tetraploid lineage of ssp. fuchsii is indistinguishable from diploid ssp. fuchsii, and is probably of postglacial origin. The Mediterranean region and the Caucasus have not contributed to the northward migration of either ssp. fuchsii or ssp. maculata. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 503–525.     

Systematics and phylogeography of the Dactylorhiza maculata complex (Orchidaceae) in Scandinavia: insights from cytological, morphological and molecular data

Flow cytometry, morphometry and molecular markers [plastid DNA and internal transcribed spacers (ITS) of nuclear ribosomal DNA] were used to determine taxonomic and phylogeographic patterns in Dactylorhiza maculata s.l. from Scandinavia. A total of 238 individuals from 27 populations from throughout all of Scandinavia, including the adjacent Kola Peninsula of Russia, were analyzed. Diploid D. maculata ssp. fuchsii and autotetraploid D. maculata ssp. maculata are morphologically differentiated. Fragment size variants from 10 plastid DNA loci (seven microsatellite loci and three loci with indel variation) were combined to give 43 haplotypes. Three major groups of haplotypes were found. Group I haplotypes were prevalent in the north and the northeast, whereas Group II haplotypes were prevalent in the south and the southwest. Group III was represented by only a single haplotype and appeared to be the result of introgression from D. incarnata s.l. Group I and Group II haplotypes did not correspond with cytologically and morphologically defined D. maculata ssp. fuchsii or D. maculata ssp. maculata. Past introgressive gene flow rather than recent hybridization is envisaged. Intermediate Group I haplotypes between Group II and the rest of Group I were detected in a zone of contact in central Sweden, which may suggest plastid DNA recombination. The six ITS alleles scored showed strong positive correlation with taxonomy. All data sets obtained for ssp. maculata were significantly correlated with geography. Three different autotetraploid lineages are hypothesized. One lineage may represent postglacial immigration from the south and the other two lineages may represent eastern immigration routes. Morphology and ITS data suggested that subarctic populations of ssp. maculata should be recognized as var. kolaënsis.     

Patterns of chloroplast diversity among western European Dactylorhiza species (Orchidaceae)

In Europe, the genus Dactylorhiza comprises a bewildering variety of forms that are difficult to sort into discrete species. Most Dactylorhiza species are diploid or tetraploid and contrasting hypotheses have been proposed to explain the complex variation within this group. Using PCR-RFLP analysis in eight putative species, we could identify four highly differentiated chloroplast DNA lineages. The first lineage (clade A) included the unique haplotype found in D. sambucina. Clade B grouped four haplotypes belonging mostly to D. incarnata. Clades C and D included 27 haplotypes detected in diploid D. fuchsii and in all tetraploid species investigated. Eighty percent of the chloroplast variation were consistent with currently accepted species boundaries. The imperfect agreement between chloroplast variation and species boundaries may be ascribed to incomplete lineage sorting and/or reticulation. Our cpDNA results provide strong evidence that the allotetrapolyploids have been formed through asymmetric hybridization with a member of the D. fuchsii / maculata group as the maternal parent.     

Plastid DNA haplotype diversity and morphological variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in northern Poland

To gain an overview of the variation in the Dactylorhiza incarnata/maculata complex in northern Poland, ten plastid DNA regions (seven microsatellite and three indel loci) and 23 morphometric characters were used. In total, 972 and 480 samples from 64 and 31 populations were utilized for the genetic and morphometric analyses, respectively. One hundred and forty‐one haplotypes that have not been reported previously were recognized. The continuity of morphological characters between the studied species and the impact of post‐glacial colonization on the observed complexity in the Dactylorhiza incarnata/maculata complex were concluded. It was confirmed that the allotetraploid group of D. majalis s.s. has inherited its plastid genome from D. maculata s.l., specifically from D. maculata ssp. fuchsii. In addition, some of the haplotypes found in D. majalis s.s. were distinct and evidently not present in the preserved D. maculata s.l. Although possible gene flow and introgression between two subspecies of the D. maculata s.l. group were indicated, we suggest that they should be treated as separate evolutionary units. Both the common and rare haplotypes show a similar pattern of geographical distribution for all four taxa analysed, which suggests that hybridization took place relatively recently, shortly after the retreat of the ice sheet. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 121–137.     

Genetic differentiation and admixture between sibling allopolyploids in the Dactylorhiza majalis complex

Allopolyploidization often happens recurrently, but the evolutionary significance of its iterative nature is not yet fully understood. Of particular interest are the gene flow dynamics and the mechanisms that allow young sibling polyploids to remain distinct while sharing the same ploidy, heritage and overlapping distribution areas. By using eight highly variable nuclear microsatellites, newly reported here, we investigate the patterns of divergence and gene flow between 386 polyploid and 42 diploid individuals, representing the sibling allopolyploids Dactylorhiza majalis s.s. and D. traunsteineri s.l. and their parents at localities across Europe. We make use in our inference of the distinct distribution ranges of the polyploids, including areas in which they are sympatric (that is, the Alps) or allopatric (for example, Pyrenees with D. majalis only and Britain with D. traunsteineri only). Our results show a phylogeographic signal, but no clear genetic differentiation between the allopolyploids, despite the visible phenotypic divergence between them. The results indicate that gene flow between sibling Dactylorhiza allopolyploids is frequent in sympatry, with potential implications for the genetic patterns across their entire distribution range. Limited interploidal introgression is also evidenced, in particular between D. incarnata and D. traunsteineri. Altogether the allopolyploid genomes appear to be porous for introgression from related diploids and polyploids. We conclude that the observed phenotypic divergence between D. majalis and D. traunsteineri is maintained by strong divergent selection on specific genomic areas with strong penetrance, but which are short enough to remain undetected by genotyping dispersed neutral markers.     

Systematics and conservation genetics of Dactylorhiza majalis ssp. elatior (Orchidaceae) on Gotland

A tall allotetraploid member of the Dactylorhiza incarnata/maculata complex with unspotted leaves and large pinkish flowers from the island of Gotland in the Baltic was examined for molecular variation patterns at five nuclear microsatellite loci, nuclear ITS and in plastid haplotypes. The allotetraploid was well separated from allopatric allotetraploids of similar appearance, including the western European D. majalis ssp. integrata (syn. D. praetermissa) and forms of D. majalis ssp. lapponica from mainland Sweden. It also differed from other allotetraploids distributed in the Baltic Sea region, including D. majalis ssp. baltica and D. majalis ssp. lapponica. It is here recognized as D. majalis ssp. elatior (Fr.) Hedrén & H. A. Pedersen. Dactylorhiza osiliensis Pikner, described from Saaremaa (Estonia) is regarded as a synonym. The distribution covers Gotland, Saaremaa and possibly Hiiumaa. Dactylorhiza majalis ssp. elatior may have one or several recent origins within its present distribution area, and it contains no other molecular markers than those found in the parental D. incarnata var. incarnata and D. maculata ssp. fuchsii in the same area. It appears to have weak barriers towards secondary hybridization with its parental lineages. The situation is reminiscent to that of other young allotetraploids in the D. majalis s.l. complex, suggesting that introgression may be an underestimated process explaining the accumulation of genetic diversity in evolving allopolyploid plants.     

Origins and introgression of polyploid species in Mentzelia section Trachyphytum (Loasaceae)

? Premise of the study: Polyploid speciation has been important in plant evolution. However, the conditions that favor the origination and persistence of polyploids are still not well understood. Here, we examine origins of 16 polyploid species in Mentzelia section Trachyphytum. ? Methods: We used phylogeny reconstructions based on DNA sequences from plastid regions and the nuclear gene isocitrate dehydrogenase (idh) to construct hypotheses of introgression and polyploidization. ? Key results: Molecular data suggest that homoploid hybridization has been surprisingly common in Trachyphytum. Diploid species had unequal involvement in polyploid origins, but most polyploid taxa had allopolyploid origins from extant progenitors. A few polyploids with extreme phenotypes did not appear to have extant progenitors. We infer that the progenitors of these species were derived from extinct diploid lineages or ancestral lineages of multiple extant diploids. In agreement with other recent studies, we recovered molecular evidence of multiple phylogenetically distinct origins for several polyploid taxa, including the widespread octoploid M. albicaulis. ? Conclusions: Evidence of high levels of introgression and allopolyploidy suggests that hybridization has played an important role in the evolution of Trachyphytum. Although idh sequences exhibited complicated evolution, including gene duplication, deletion, and recombination, they provided a higher percentage of informative characters for phylogeny reconstruction than the most variable plastid regions, allowing tests of hypotheses regarding polyploid origins. Given the necessity for rapidly evolving low-copy nuclear genes, researchers studying hybridization and polyploidy may increasingly turn to complex sequence data.     

Polyploid formation in cotton is not accompanied by rapid genomic changes.

Recent work has demonstrated that allopolyploid speciation in plants may be associated with non-Mendelian genomic changes in the early generations following polyploid synthesis. To address the question of whether rapid genomic changes also occur in allopolyploid cotton (Gossypium) species, amplified fragment length polymorphism (AFLP) analysis was performed to evaluate nine sets of newly synthesized allotetraploid and allohexaploid plants, their parents, and the selfed progeny from colchicine-doubled synthetics. Using both methylation-sensitive and methylation-insensitive enzymes, the extent of fragment additivity in newly combined genomes was ascertained for a total of approximately 22,000 genomic loci. Fragment additivity was observed in nearly all cases, with the few exceptions most likely reflecting parental heterozygosity or experimental error. In addition, genomic Southern analysis on six sets of synthetic allopolyploids probed with five retrotransposons also revealed complete additivity. Because no alterations were observed using methylation-sensitive isoschizomers, epigenetic changes following polyploid synthesis were also minimal. These indications of genomic additivity and epigenetic stasis during allopolyploid formation provide a contrast to recent evidence from several model plant allopolyploids, most notably wheat and Brassica, where rapid and unexplained genomic changes have been reported. In addition, the data contrast with evidence from repetitive DNAs in Gossypium, some of which are subject to non-Mendelian molecular evolutionary phenomena in extant polyploids. These contrasts indicate polyploid speciation in plants is accompanied by a diverse array of molecular evolutionary phenomena, which will vary among both genomic constituents and taxa.     

Allopolyploid evolution in Geinae (Colurieae: Rosaceae) – building reticulate species trees from bifurcating gene trees

A previous phylogenetic study of paralogous nuclear low-copy granule-bound starch synthase (GBSSI) gene sequences from polyploid and diploid species in Geinae indicated that the clade has experienced two major allopolyploid events in its history. These were estimated to have occurred several million years ago. In this extended study we test if the reticulate phylogenetic hypothesis for Geinae can be maintained when additional sequences are added. The results are compatible with the hypothesis and strengthen it in minor aspects. We also attempt to identify extant members of one of the inferred ancestral lineages of the allopolyploids. On the basis of previous molecular phylogenies, one specific group has been proposed to be the descendants of this taxon. However, none of the additional paralogues belong to this ancestral lineage. A general method is proposed for converting a bifurcating gene tree, with multiple paralogous low-copy gene sequences from allopolyploid taxa, into a reticulate species tree.     

Occurrence and evolutionary origins of polyploids in the clubmoss genus Diphasiastrum (Lycopodiaceae)

Two polyploid taxa are commonly recognized in the genus Diphasiastrum, D. wightianum from Asia and D. zanclophyllum from South Africa and Madagascar. Here we present results from Feulgen DNA image densitometry analyses providing the first evidence for the polyploid origin of D. zanclophyllum. Also reported for the first time are data confirming that D. multispicatum and D. veitchii, representing the putative parent lineages of D. wightianum, are diploids. Phylogenetic analyses of the nuclear regions RPB2, LEAFY and LAMB4 reveal that putative tetraploid accessions are of allopolyploid origin. Diphasiastrum zanclophyllum shows close relationship to the North American taxon D. digitatum on the maternal side, but the paternal relationship is less clear. Two accessions from Asia, both inferred to be polyploid, have D. veitchii as the maternal parent, whereas the paternal paralogs show relationships to D. multispicatum and D. tristachyum, respectively. None of these parental combinations have previously been hypothesized.     

Development of polymorphic nuclear microsatellite markers for polyploid and diploid members of the orchid genus Dactylorhiza

Dactylorhiza traunsteineri is an allotetraploid species that belongs to the large Dactylorhiza incarnata/maculata polyploid complex of the Eurasian genus Dactylorhiza (Orchidaceae). Here, eight polymorphic microsatellite loci were isolated using selective hybridization according to the FIASCO protocol (fast isolation by AFLP of sequences containing repeats) with slight modifications. The number of alleles per locus ranged from two to seven. All loci were possible to amplify in several other species of Dactylorhiza, using the same primers.     

Allopolyploidy-Induced Rapid Genome Evolution in the Wheat (Aegilops–Triticum) Group

To better understand genetic events that accompany allopolyploid formation, we studied the rate and time of elimination of eight DNA sequences in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. In total, 35 interspecific and intergeneric F1 hybrids and 22 derived allopolyploids were analyzed and compared with their direct parental plants. The studied sequences exist in all the diploid species of the Triticeae but occur in only one genome, either in one homologous pair (chromosome-specific sequences [CSSs]) or in several pairs of the same genome (genome-specific sequences [GSSs]), in the polyploid wheats. It was found that rapid elimination of CSSs and GSSs is a general phenomenon in newly synthesized allopolyploids. Elimination of GSSs was already initiated in F1 plants and was completed in the second or third allopolyploid generation, whereas elimination of CSSs started in the first allopolyploid generation and was completed in the second or third generation. Sequence elimination started earlier in allopolyploids whose genome constitution was analogous to natural polyploids compared with allopolyploids that do not occur in nature. Elimination is a nonrandom and reproducible event whose direction was determined by the genomic combination of the hybrid or the allopolyploid. It was not affected by the genotype of the parental plants, by their cytoplasm, or by the ploidy level, and it did not result from intergenomic recombination. Allopolyploidy-induced sequence elimination occurred in a sizable fraction of the genome and in sequences that were apparently noncoding. This finding suggests a role in augmenting the differentiation of homoeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. In our view, this rapid genome adjustment may have contributed to the successful establishment of newly formed allopolyploids as new species.     

Geographical variation and systematics of the tetraploid marsh orchid Dactylorhiza majalis subsp. sphagnicola (Orchidaceae) and closely related taxa

Dactylorhiza majalis subsp. sphagnicola is an allotetraploid marsh orchid derived from parents closely similar to present‐day D. incarnata and the western European form of D. maculata subsp. maculata, suggesting that it has a postglacial origin. It extends from northwestern continental Europe into areas formerly covered by the Weichselian ice sheet in mid‐Scandinavia. Here, we studied the variation at both the plastid and nuclear marker systems to describe the geographical variation in subsp. sphagnicola and its evolutionary history. We investigated whether subsp. sphagnicola is affected by secondary hybridization and gene flow from its parental lineages or from other allotetraploid marsh orchids, and we also compared subsp. sphagnicola with other allotetraploids of similar origins. We analysed 492 plants from 50 populations. Thirty‐seven populations were collected as potential Dactylorhiza majalis subsp. sphagnicola, five as subsp. sesquipedalis (D. elata), one as D. elata subsp. brennensis, one as subsp. calcifugiens, one as subsp. occidentalis and the remaining five as populations with some affinity to subsp. lapponica (including D. traunsteineri). All populations were analysed for plastid haplotypes and nuclear internal transcribed spacer (ITS) allele frequencies, and a subset of 43 populations was analysed for five nuclear microsatellite loci. Dactylorhiza majalis subsp. sphagnicola was dominated by a single plastid haplotype that was also dominant in western European D. maculata subsp. maculata, and most of the alternative haplotypes differed by only one mutation from the dominant one. There was more variation in nuclear microsatellites and ITS, and the variation was geographically structured in these markers. Subspecies occidentalis and calcifugiens shared haplotypes with subsp. sphagnicola, whereas subsp. sesquipedalis and brennensis had other haplotypes. Dactylorhiza majalis subsp. sphagnicola may have a postglacial origin within its present continental distribution. It has incorporated genetic material from D. maculata subsp. maculata by secondary hybridization and introgression, and some northern populations have assimilated strongly divergent haplotypes from the northeastern form of D. maculata subsp. maculata. Subspecies sphagnicola has also evolved morphologically divergent local populations in the north that do not differ from the typical populations in genetic markers. It may form mixed populations with other allotetraploid subspecies of D. majalis and, at least at one site, it has become integrated with subsp. lapponica, demonstrating that independently derived allotetraploids may contribute to a common gene pool. Subspecies calcifugiens seems to be derived from subsp. sphagnicola, and further studies based on a larger sample may confirm that it is better recognized as a variety. The so‐called D. elata subsp. brennensis is of hybrid origin and combines markers from subsp. sesquipedalis with markers from the D. majalis core complex, possibly subsp. majalis. The new combination Dactylorhiza majalis subsp. sesquipedalis (Willd.) H.A.Pedersen & Hedrén comb. nov. is provided. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 174–193.     

Molecular phylogenetic analyses of nuclear and plastid DNA sequences support dysploid and polyploid chromosome number changes and reticulate evolution in the diversification of Melampodium (Millerieae, Asteraceae)

Chromosome evolution (including polyploidy, dysploidy, and structural changes) as well as hybridization and introgression are recognized as important aspects in plant speciation. A suitable group for investigating the evolutionary role of chromosome number changes and reticulation is the medium-sized genus Melampodium (Millerieae, Asteraceae), which contains several chromosome base numbers (x = 9, 10, 11, 12, 14) and a number of polyploid species, including putative allopolyploids. A molecular phylogenetic analysis employing both nuclear (ITS) and plastid (matK) DNA sequences, and including all species of the genus, suggests that chromosome base numbers are predictive of evolutionary lineages within Melampodium. Dysploidy, therefore, has clearly been important during evolution of the group. Reticulate evolution is evident with allopolyploids, which prevail over autopolyploids and several of which are confirmed here for the first time, and also (but less often) on the diploid level. Within sect. Melampodium, the complex pattern of bifurcating phylogenetic structure among diploid taxa overlain by reticulate relationships from allopolyploids has non-trivial implications for intrasectional classification.     

Unraveling reticulate evolution in North American dryopteris (dryopteridaceae)

ABSTRACT: BACKGROUND: The thirteen species of Dryopteris in North America have long been suspected of having undergone a complicated history of reticulate evolution via allopolyploid hybridization. Various explanations for the origins of the allopolyploid taxa have been suggested, and though most lines of evidence have supported the so-called "semicristata" hypothesis, contention over the group's history has continued in several recent, conflicting studies. RESULTS: Sequence data from nine plastid and two nuclear markers were collected from 73 accessions representing 35 species of Dryopteris. Sequences from each of the allopolyploids are most closely related to their progenitor species as predicted by the "semicristata" hypothesis. Allotetraploid D. campyloptera appears to be derived from a hybrid between diploid D. expansa and D. intermedia; D. celsa, from diploid D. ludoviciana x D. goldiana; and D. carthusiana and D. cristata, from diploid "D. semicristata" x D. intermedia and D. ludoviciana, respectively. Allohexaploid D. clintoniana appears to be derived from D. cristata x D.goldiana. The earliest estimated dates of formation of the allopolyploids, based on divergence time analyses, were within the last 6 Ma. We found no evidence for recurrent formation of any of the allopolyploids. The sexual allopolyploid taxa are derived from crosses between parents that show intermediate levels of genetic divergence relative to all pairs of potential progenitors. In addition, the four allotetraploids are transgressive with respect to geographic range relative to one or both of their parents (their ranges extend beyond those of the parents), suggesting that ecological advantages in novel habitats or regions may promote long-term regional coexistence of the hybrid taxa with their progenitors. CONCLUSIONS: This study provides the first thorough evaluation of the North American complex of woodferns using extensive sampling of taxa and genetic markers. Phylogenies produced from each of three datasets (one plastid and two nuclear) support the "semicristata" hypothesis, including the existence of a missing diploid progenitor, and allowed us to reject all competing hypotheses. This study demonstrates the value of using multiple, biparentally inherited markers to evaluate reticulate complexes, assess the frequency of recurrent polyploidization, and determine the relative importance of introgression vs. hybridization in shaping the histories of such groups.     

Deciphering the origins of apomictic polyploids in the Cheilanthes yavapensis complex (Pteridaceae)

Deciphering species relationships and hybrid origins in polyploid agamic species complexes is notoriously difficult. In this study of cheilanthoid ferns, we demonstrate increased resolving power for clarifying the origins of polyploid lineages by integrating evidence from a diverse selection of biosystematic methods. The prevalence of polyploidy, hybridization, and apomixis in ferns suggests that these processes play a significant role in their evolution and diversification. Using a combination of systematic approaches, we investigated the origins of apomictic polyploids belonging to the Cheilanthes yavapensis complex. Spore studies allowed us to assess ploidy levels; plastid and nuclear DNA sequencing revealed evolutionary relationships and confirmed the putative progenitors (both maternal and paternal) of taxa of hybrid origin; enzyme electrophoretic evidence provided information on genome dosage in allopolyploids. We find here that the widespread apomictic triploid, Cheilanthes lindheimeri, is an autopolyploid derived from a rare, previously undetected sexual diploid. The apomictic triploid Cheilanthes wootonii is shown to be an interspecific hybrid between C. fendleri and C. lindheimeri, whereas the apomictic tetraploid C. yavapensis is comprised of two cryptic and geographically distinct lineages. We show that earlier morphology-based hypotheses of species relationships, while not altogether incorrect, only partially explain the complicated evolutionary history of these ferns.