Mosaic variation in allozyme and plastid DNA markers in the European ranges of Silene vulgaris and its partially sympatric relative S. uniflora (Caryophyllaceae)

The perennial herbs Silene vulgaris and S. uniflora are closely related, partially sympatric and interfertile, yet morphologically distinct. We used nuclear (allozyme) and plastid (polymerase chain reaction–restriction fragment length polymorphism) DNA markers to investigate whether these species have a shared history of postglacial colonization and hybridization in Europe, as inferred from large‐scale patterns of geographic variation. The majority of plastid haplotypes and allozyme alleles were widespread and patchily distributed within both species and there was no geographic structure in the distributions of shared allozymes or haplotypes. The mosaic variation is consistent with a scenario in which repeated episodes of interspecific hybridization pre‐dated the largely allopatric range expansion of the two species during the postglacial period. Our overall results are not consistent with a scenario of extensive hybridization and introgression during the postglacial range expansion of the species or within their current areas of sympatry, but we found some evidence for local, postglacial evolution and hybridization in the Baltic region. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 166 , 127–148.     

Complex fine‐scale phylogeographical patterns in a putative refugial region for Fagus sylvatica (Fagaceae)

Broad‐scale plastid (chloroplast) DNA studies of beech (Fagus sylvatica) populations suggest the existence of glacial refugia and introgression zones in south‐eastern Europe. We choose a possible refugium of beech in northern Greece, Mt. Paggeo, which hosts a private plastid haplotype for beech, to conduct a fine‐scale genetic study. We attempt to confirm or reject the hypothesis of the existence of a small‐scale refugium and to gain an understanding of the ecological and topographical factors affecting the spatial distribution of plastid haplotypes in the area. Our results reveal a high haplotype diversity on Mt. Paggeo, but the overall distribution of haplotypes shows no significant correlation with the ecological characteristics of the beech forests. However, the private haplotype is found at high frequencies in beech forests located in or near ravines, having a high spatial overlap with a relict vegetation type occurring in ecological conditions found mainly in ravines. This result emphasizes the importance of topography in the existence of glacial refugia in the wider area. Furthermore, haplotypes originating from two more widespread beech lineages in Greece are found on Mt. Paggeo, indicating a possible mixing of populations originating from a local refugium with populations from remote refugia that possibly migrated into the area after the last glaciation. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 516–528.     

Evaluating the role of ecological isolation in maintaining the species boundary between <Emphasis Type="Italic">Silene dioica</Emphasis> and <Emphasis Type="Italic">S. latifolia</Emphasis>

The relative importance of floral versus ecological isolation in preventing introgression remains unclear. This study examines whether ecological isolation can explain the continuing integrity of Silene dioica and S. latifolia where floral isolation is weak and hybrids are fully viable. Eighteen small replicate founder populations of 6 individuals (3 males and 3 females) of either S. latifolia, S. dioica or hybrids were created in woodland and in open sites in southern UK. Survival, reproduction and introgression of these populations were examined over 9 years. S. latifolia and hybrid plants suffered higher mortality than S. dioica in woodland. In open sites, there was extensive introgression, with few or no pure S. latifolia or S. dioica surviving by the end of the experiment. The experiment suggests that the integrity of S. dioica is maintained by its ability to survive in shaded habitats where S. latifolia and hybrids cannot persist. However, how S. latifolia survives as a distinct species in the study area remains a puzzle. Immigration from regions where S. latifolia occurs in isolation (i.e. large-scale ecological isolation) may balance introgression in the study area.     

Pollen competition as an asymmetric reproductive barrier between two closely related Silene species

Reproductive barriers are important determinants of gene flow between divergent populations or species. We studied pollen competition as a post‐mating reproductive barrier between Silene dioica and S. latifolia. Gene flow between these species is extensive, but early‐generation hybrids are rare. In an experiment with conspecific, heterospecific and 50 : 50 mixed pollinations in the two species, pollination treatments did not significantly affect seed set and seed weight. However, molecular determination of siring success after mixed pollinations showed that fewer than expected hybrids were produced in S. latifolia (18% hybrids) but not in S. dioica (51% hybrids). This constitutes an asymmetric post‐mating reproductive barrier and likely contributes to the rarity of early‐generation hybrids. Our study shows that pollen competition can be an effective barrier to hybridization between closely related species that likely acts in concert with other reproductive barriers.     

From middens to molecules: phylogeography of the piñon pine,Pinus edulis

Aim Data from packrat middens have established a hypothesized historical biogeography of piñon pine, Pinus edulis, including locations of glacial refugia in the south‐western USA and subsequent migration out of the refugia. In this study, we used molecular techniques to test the glacial refugial hypotheses inferred from packrat (Neotoma) midden data for P. edulis. Location South‐western USA. Methods Two fragments of chloroplast DNA (a portion of the matK gene and a portion of the rbcL gene) for a total of 1045 base pairs were amplified and sequenced for 100 individuals. Thirty‐one populations were sampled throughout the range of P. edulis. Phylogenetic analyses included maximum parsimony and maximum likelihood. Results Very little variation existed among the individuals sampled. Four haplotypes were identified. The inferred ancestral haplotype was the most widespread; it was most common in Texas and New Mexico where, with the exception of one individual, it was the only haplotype found. Arizona and Utah populations were more diverse, with almost half of the populations containing two or more haplotypes. The most derived haplotype was most abundant in Arizona. Main conclusions The distribution of haplotypes is geographically informative. Only one haplotype exists in the south‐eastern portion of the range of P. edulis whereas up to four haplotypes are found in other populations, suggesting one of two hypotheses: either all modern populations are descended from a refugial population in central Arizona, or modern populations are descended from two refugial populations, one in central Arizona and another in Texas–southern New Mexico. Interpreting these data in the light of packrat midden data gives more support for the latter hypothesis.     

Global phylogeography in Sanionia uncinata (Amblystegiaceae: Bryophyta)

Global phylogeographic patterns in Sanionia uncinata are addressed based on information in internal transcribed spacer (ITS) (214 specimens) and the plastid markers trnL‐trnF (221) and rpl16 (217). ITS suggests a monophyletic Sanionia and a paraphyletic S. uncinata; this was neither supported nor rejected by plastid data. Northern or Eastern Eurasia and Alaska appear important in the early evolution of Sanionia and some populations dispersed into the Southern Hemisphere relatively early. Some haplotypes or groups of haplotypes are morphologically and ecologically distinct, biologically meaningful units that correspond with S. orthothecioides, S. symmetrica and S. georgicouncinata s.l. The latter includes two species that are indistinguishable by morphology, S. georgicouncinata s.s. (Southern Hemisphere) and S. nivalis (Northern Hemisphere). Tropical African and South American S. uncinata populations have separate origins and the Southern Hemisphere was colonized at least twice. In the northern circum‐Arctic region, the haplotype composition differs between the North Atlantic and Beringian areas. Eastern Eurasia has a higher S. uncinata haplotype diversity than other Holarctic regions, implying less devastating effects of recurrent glacial periods. For Eastern and Western Eurasia, North America and the Southern Hemisphere, most of the haplotype variation was found within the regions, but 14–18% can be referred to among region variation. Plastid haplotype diversity was lower in the Southern Hemisphere than in the Arctic to subarctic, possibly attributable to founder effects. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 19–42.     

Ecological divergence plays an important role in strong but complex reproductive isolation in campions (Silene)*

New species arise through the evolution of reproductive barriers between formerly interbreeding lineages. Yet, comprehensive assessments of potential reproductive barriers, which are needed to make inferences on processes driving speciation, are only available for a limited number of systems. In this study, we estimated individual and cumulative strengths of seven prezygotic and six postzygotic reproductive barriers between the recently diverged taxa Silene dioica (L.) Clairv. and S. latifolia Poiret using both published and new data. A combination of multiple partial reproductive barriers resulted in near‐complete reproductive isolation between S. dioica and S. latifolia, consistent with earlier estimates of gene flow between the taxa. Extrinsic barriers associated with adaptive ecological divergence were most important, while intrinsic postzygotic barriers had moderate individual strength but contributed only little to total reproductive isolation. These findings are in line with ecological divergence as driver of speciation. We further found extensive variation in extrinsic reproductive isolation, ranging from sites with very strong selection against migrants and hybrids to intermediate sites where substantial hybridization is possible. This situation may allow for, or even promote, heterogeneous genetic divergence.     

Phylogeographical patterns and genetic diversity in three species of Eurasian boreal forest beetles

Many species that occur in formerly glaciated areas of Fennoscandia have reached their current ranges from glacial refugial areas in Eurasia. Little is known of the refugia and postglacial colonization routes of insect species that are confined to boreal forests. Here, we investigate the phylogeography of three species of saproxylic beetles distributed across Eurasia: two rare boreal forest specialists, Pytho kolwensis and Pytho abieticola , and a common, less specialized species, Pytho depressus . In all species, there were two well-defined haplotype clades based on 645 bp of cytochrome oxidase subunit I gene sequence. In each species one clade was found only in China. The other clade occurred from China to north-western Europe in both P. kolwensis and P. depressus , but was apparently absent from China in P. abieticola. In spite of common phylogeographical patterns, the distribution of genetic variation differed markedly between the three species. In P. kolwensis , a highly-threatened species in old-growth forests in Fennoscandia, there was an extremely low level of genetic variation throughout Eurasia. One common haplotype, represented by 86% of the samples, dominated in all sampling localities. Levels of genetic variation were higher in both P. abieticola and P. depressus , with 31% and 58%, respectively, of the samples representing a unique haplotype. In each species, relationships between haplotypes were not well resolved, and haplotypes from one sampling locality were generally not clustered in either Neighbour-joining trees or statistical parsimony networks. These patterns in the distribution of genetic variation can be attributed to differences in the species' population sizes, ecologies, glacial refugial areas, and postglacial colonization dynamics.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 267–279.     

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica

Background and Aims

Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible.

Methods

Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers.

Key Results

The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers.

Conclusions

The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.     

The colonization history of the Mediterranean dwarf palm (<Emphasis Type="Italic">Chamaerops humilis</Emphasis> L., Palmae)

Chamaerops humilis L. (Mediterranean dwarf palm) is an important floristic element of the western Mediterranean region because it is the only palm species naturally distributed in both Europe and Africa. The combination of a time-calibrated phylogeny, a haplotype network and genetic diversity analyses based on plastid sequences, together with previous nuclear DNA fingerprint results, helped reconstruct the colonization history of the dwarf palm. Based on a sample of 218 individuals taken from 29 geographical areas that cover the current distribution of Chamaerops, we detected four plastid DNA (petA-psbJ) haplotypes distributed in two haplotype groups (lineage 1: haplotypes A/B; lineage 2: haplotypes C/D). Haplotypes A, B, and C showed a widespread geographical distribution in both Africa and Europe, whereas haplotype D was restricted to two African localities. Paleobotanical data, species distribution modeling and divergence time estimates suggest that Chamaerops diverged from Trachycarpus in the Miocene (27.05–6.05 Ma), followed by a split of the two C. humilis lineages that remained isolated during the Miocene-Pliocene. Divergence estimates also support a derived split into two haplotypes (A/B) in the Pleistocene, when the Mediterranean Sea barrier was in existence. This, together with geographical distribution of haplotypes A and B, strongly suggests that the disjunct distribution of C. humilis haplotypes in Europe and Africa is the result of long-distance dispersal (LDD) events rather than vicariance. In agreement with recurrent gene flow (events of LDD colonization), AMOVA revealed that most of the genetic variance was found among populations (61.52%). Irrespective of predominant plant translocations by humans or seed dispersal by natural means, our results support that current populations are the result of relatively recent contacts between Africa and Europe in the Quaternary.     

Understanding the spectacular failure of DNA barcoding in willows (Salix): Does this result from a trans‐specific selective sweep?

Willows (Salix: Salicaceae) form a major ecological component of Holarctic floras and consequently are an obvious target for a DNA‐based identification system. We surveyed two to seven plastid genome regions (~3.8 kb; ~3% of the genome) from 71 Salix species across all five subgenera, to assess their performance as DNA barcode markers. Although Salix has a relatively high level of interspecific hybridization, this may not sufficiently explain the near complete failure of barcoding that we observed: only one species had a unique barcode. We recovered 39 unique haplotypes, from more than 500 specimens, that could be partitioned into six major haplotype groups. A unique variant of group I (haplotype 1*) was shared by 53 species in three of five Salix subgenera. This unusual pattern of haplotype sharing across infrageneric taxa is suggestive of either a massive nonrandom coalescence failure (incomplete lineage sorting), or of repeated plastid capture events, possibly including a historical selective sweep of haplotype 1* across taxonomic sections. The former is unlikely as molecular dating indicates that haplotype 1* originated recently and is nested in the oldest major haplotype group in the genus. Further, we detected significant non‐neutrality in the frequency spectrum of mutations in group I, but not outside group I, and demonstrated a striking absence of geographical (isolation by distance) effects in the haplotype distributions of this group. The most likely explanation for the patterns we observed involves recent repeated plastid capture events, aided by widespread hybridization and long‐range seed dispersal, but primarily propelled by one or more trans‐species selective sweeps.     

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.     

Immigration history and gene dispersal: allozyme variation in Nordic populations of the red campion, Silene dioica (Caryophyllaceae)

Most of the Nordic region was ice-covered during the last (Weichselian) glaciation. During the postglacial period, plant and animal species recolonized the region from several directions and the geographic structuring of genetic variation within Nordic species may still contain a historic component that reflects patterns of postglacial immigration. The present investigation of 69 populations of Silene dioica represents the first large-scale allozyme study of a widespread herbaceous plant in the Nordic region. Although the frequencies of individual alleles showed a range of different geographic patterns, mapping of the axis scores from an ordination of variation at eight polymorphic loci revealed a division into two main geographic groups of populations. The broadly south-western and north-eastern distributions of these two groups of populations suggest that immigration into the region may have involved both eastern and southern geographic sources. However, the geographic boundaries between the two groups of populations are diffuse, and the relatively low between-population component of genetic diversity (G_ST = 16.4%) suggests a history of extensive gene dispersal by pollen.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77 , 23–34.     

Rhytidium rugosum (Bryophyta) colonized Scandinavia from at least two glacial refugial source populations

The Scandinavian post‐glacial history of the moss Rhytidium rugosum is traced on the basis of information from the nuclear markers ITS and gpd for 229 Scandinavian and 81 other specimens. Some haplotypes, groups or lineages identified in a NeighborNet split network are predominantly northern Scandinavian, whereas others are southern. With the distributions of individual haplotypes and the timing of the deglaciation in different parts of Scandinavia, this implies colonization from the south and from the north or north‐east. High haplotype and nucleotide diversity and the occurrence of certain private haplotypes in the north suggest that the species may have survived the Last Glacial Maximum in local refugia. Slightly higher numbers of private haplotypes in Scandinavia than in central or north‐eastern Europe also favour an explanation with at least some local glacial survival. Low diversity in the southernmost contiguous region of the Scandinavian mountain range is probably a result of recent land uplift and late colonization. The Scandinavian lowland regional populations probably represent remains of an earlier widespread population that became increasingly restricted to small and isolated areas when the vegetation closed during the post‐glacial period. Some of the lowland populations require extensive management to survive. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 635–657.     

Chloroplast Genome of Native Silene latifolia subsp. alba from Fennoscandia Shows High Level of Differences from Invasive White Campion

Silene latifolia is an herbaceous plant with great invasive potential. Spread along trade routes from Europe to almost all continents, white campion became particularly widespread in North America. We sequenced the chloroplast genome of S. latifolia subsp. alba from a native range in southeast Fennoscandia. The chloroplast genome of native S. latifolia subsp. alba forms a 151,747-bp circle, has two inverted repeat regions (25,993 bp each), large single copy (82,708 bp), and small single copy (17,106 bp) regions. It contains 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. SSRs and long DNA repeats were identified. Comparison of a newly sequenced plastome of S. latifolia subsp. alba with plastomes of invasive specimens of species from North America and Japan revealed a high level of single nucleotide polymorphisms (SNPs) among them. A total of 214 SNPs were found, among which 110 were identified in intergenic spacers, 74 in exons, and 30 in introns. Intraspecific shifts in inverted repeat boundaries were identified. Our research suggests that high polymorphic regions may be potential molecular markers for population studies and that high intraspecific genetic polymorphism may contribute to a species’ invasive success.

     

Genetic structure of Eurasian badgers Meles meles (Carnivora: Mustelidae) and the colonization history of Ireland

The present study examined the contemporary genetic composition of the Eurasian badger, Meles meles, in Ireland, Britain and Western Europe, using six nuclear microsatellite loci and a 215‐bp fragment of the mitochondrial DNA control region. Significant population structure was evident within Europe (global multilocus microsatellite F_ST = 0.205, P < 0.001; global mitochondrial control region Φ_ST = 0.399, P < 0.001). Microsatellite‐based cluster analyses detected one population in Ireland, whereas badgers from Britain could be subdivided into several populations. Excluding the island populations of Ireland and Britain, badgers from Western Europe showed further structuring, with evidence of discrete Scandinavian, Central European, and Spanish populations. Mitochondrial DNA cluster analysis grouped the Irish population with Scandinavia and Spain, whereas the majority of British haplotypes grouped with those from Central Europe. The findings of the present study suggest that British and Irish badger populations colonized from different refugial areas, or that there were different waves of colonization from the source population. There are indications for the presence of an Atlantic fringe element, which has been seen in other Irish species. We discuss the results in light of the controversy about natural versus human‐mediated introductions. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Sequence diversity patterns suggesting balancing selection in partially sex‐linked genes of the plant Silene latifolia are not generated by demographic history or gene flow

DNA sequence diversity in genes in the partially sex‐linked pseudoautosomal region (PAR) of the sex chromosomes of the plant Silene latifolia is higher than expected from within‐species diversity of other genes. This could be the footprint of sexually antagonistic (SA) alleles that are maintained by balancing selection in a PAR gene (or genes) and affect polymorphism in linked genome regions. SA selection is predicted to occur during sex chromosome evolution, but it is important to test whether the unexpectedly high sequence polymorphism could be explained without it, purely by the combined effects of partial linkage with the sex‐determining region and the population's demographic history, including possible introgression from Silene dioica. To test this, we applied approximate Bayesian computation‐based model choice to autosomal sequence diversity data, to find the most plausible scenario for the recent history of S. latifolia and then to estimate the posterior density of the most relevant parameters. We then used these densities to simulate variation to be expected at PAR genes. We conclude that an excess of variants at high frequencies at PAR genes should arise in S. latifolia populations only for genes with strong associations with fully sex‐linked genes, which requires closer linkage with the fully sex‐linked region than that estimated for the PAR genes where apparent deviations from neutrality were observed. These results support the need to invoke selection to explain the S. latifolia PAR gene diversity, and encourage further work to test the possibility of balancing selection due to sexual antagonism.     

Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium

Background and Aims

In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids.

Methods

Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL–accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network.

Key Results

Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine.

Conclusions

Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.     

Polyploid evolution and plastid DNA variation in the Dactylorhiza incarnata/maculata complex (Orchidaceae) in Scandinavia

The Dactylorhiza incarnata/maculata complex (Orchidaceae) was used as a model system to understand genetic differentiation processes in a naturally occurring polyploid complex with much of ongoing diversification and wide distribution in recently glaciated areas in northern Europe. Data were obtained for 12 hypervariable regions in the plastid DNA genome. A total of 166 haplotypes were found in a sample of 1099 plants. Allopolyploid taxa have inherited their plastid genomes from D. maculata s.l. Overall haplotype diversity of the combined group of allopolyploid taxa was comparable to that of maternal D. maculata s.l., but populations of allopolyploids were also more strongly differentiated from each other and contained lower numbers of haplotypes than populations of D. maculata s.l. In addition to haplotypes found in extant D. maculata s.l., the allopolyploids also contained several distinct and widespread haplotypes that were not found in any of the parental lineages. Some of these haplotypes were shared between widespread allopolyploids. Divergent allopolyploids with small distributions did not seem to originate from local polyploidization events, but rather as segregates of already existing allopolyploids. Genetic diversification of allopolyploid Dactylorhiza is the result of repeated polyploid formation, secondary hybridization and introgression between already existing polyploids and extant representatives of parental lineages, hybridization between independently derived polyploid lineages, and phyletic diversification in the group of allopolyploids. Although some polyploid taxa must have evolved after the last glaciation, genetic material from the parental lineages has been transferred continuously for longer periods of time. This combination of processes may explain the taxonomic complexity encountered in Dactylorhiza and other polyploid complexes distributed in previously glaciated parts of Europe.