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.     

Habitat differentiation, hybridization and gene flow patterns in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. (Orchidaceae)

Detailed ecological, morphological and molecular analyses were performed in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. in Scandinavia. Comparisons were made with pure populations of either diploid ssp. fuchsii or tetraploid ssp. maculata. It was shown that mixed populations are the result of secondary contact between ssp. fuchsii and ssp. maculata. No patterns of recent and local autopolyploidization were found. Morphology and nuclear DNA markers (internal transcribed spacers of nuclear ribosomal DNA) showed that diploids and tetraploids from mixed populations have similar levels of differentiation to diploids and tetraploids from pure populations. Vegetation analyses, as well as analyses of environmental variables, revealed that diploid and tetraploid individuals in mixed populations are ecologically well differentiated on a microhabitat level. Diploids and tetraploids in pure populations have wider ecological amplitudes than they do in mixed populations. Triploid hybrids grew in intermediate microhabitats between diploids and tetraploids in the mixed populations. Plastid DNA markers indicated that both diploids and tetraploids may act as the maternal parent. Based on morphology and nuclear markers triploids are more similar to tetraploids than to diploids. There were indications of introgressive gene flow between ploidy levels. Plastid markers indicated that gene flow from diploid to tetraploid level is most common, but nuclear markers suggested that gene flow in opposite direction also may occur. Similar patterns of differentiation and gene flow appeared in localities that represented contrasting biogeographic regions. Disturbance and topography may explain why hybridization was slightly more common and the differentiation patterns somewhat less clear in the Scandinavian mountains than in the coastal lowland. An erratum to this article can be found at     

A new cytochrome b phylogroup of the common vole (Microtus arvalis) endemic to the Balkans and its implications for the evolutionary history of the species

The phylogeographic architecture of the common vole, Microtus arvalis, has been well‐studied using mitochondrial DNA and used to test hypotheses relating to glacial refugia. The distribution of the five described cytochrome b (cyt b) lineages in Europe west of Russia has been interpreted as a consequence of postglacial expansion from both southern and central European refugia. A recently proposed competing model suggests that the ‘cradle’ of the M. arvalis lineages is in western central Europe from where they dispersed in different directions after the Last Glacial Maximum. In the present study, we report a new cyt b lineage of the common vole from the Balkans that is not closely related to any other lineage and whose presence might help resolve these issues of glacial refugia. The Balkan phylogroup occurs along the southern distributional border of M. arvalis in central and eastern Bosnia and Herzegovina, Montenegro, and eastern Serbia. Further north and west in Slovenia, Bosnia and Herzegovina, and Serbia, common voles belong to the previously‐described Eastern lineage, whereas both lineages are sympatric in one site in Bosnia and Herzegovina. The Balkan phylogroup most reasonably occupied a glacial refugium already known for various Balkan endemic species, in contrast to the recently proposed model. South‐east Europe is an absolutely crucial area for understanding the postglacial colonization history of small mammals in Europe and the present study adds to the very few previous detailed phylogeographic studies of this region. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 788–796.     

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 diversity and phylogeography of the European roe deer: the refuge area theory revisited

The extant taxa of central and northern Europe are commonly believed to derive from Pleistocene ancestors, who moved to the north from three separate glacial refugia: the Iberian and Italian peninsulae, as well as the southern Balkans. The issue of postglacial dispersal patterns was addressed through the investigation of population structure and phylogeography of the European roe deer, Capreolus capreolus . The genetic diversity in 376 individuals representing 14 allegedly native populations across their European range was assessed, using ten autosomal microsatellite loci and restriction fragment length polymorphisms of the mitochondrial D-loop and NADH dehydrogenase 1 gene segments. Our results suggest the existence of three major genetic lineages of roe deer in Europe. One comprises populations in the south-western limit of the species' distribution (i.e. Iberia), where an internal substructure splits a northern from a southern sublineage. A second lineage includes populations of southern and eastern Europe, as well as a separate sublineage sampled in central-southern Italy, where the existence of the subspecies Capreolus c. italicus was supported. In central-northern Europe, a third lineage is present, which appeared genetically rather homogeneous, although admixed, and equally divergent from both the eastern and western lineages. Current patterns of intraspecific genetic variation suggest that postglacial recolonization routes of this cervid to northern Europe could be due to range expansion from one or more refugia in central-eastern Europe, rather than proceeding from the Mediterranean areas.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 85–100.     

Southern isolation and northern long‐distance dispersal shaped the phylogeography of the widespread,but highly disjunct,European high mountain plant Artemisia eriantha (Asteraceae)

We investigated the range dynamics of Artemisia eriantha, a widespread, but rare, mountain plant with a highly disjunct distribution in the European Alpine System. We focused on testing the roles of vicariance and long‐distance dispersal in shaping the current distribution of the species. To this end, we collected AFLP and plastid DNA sequence data for 17 populations covering the entire distributional range of the species. Strong phylogeographical structure was found in both datasets. AFLP data suggested that almost all populations were genetically strongly differentiated, with 58% of the overall genetic variation partitioned among populations. Bayesian clustering identified five groups of populations: Balkans, Pyrenees, Central Apennines, one southwestern Alpine population and a Widespread cluster (eastern Pyrenees, Alps, Carpathians). Major groups were supported by neighbor‐joining and NeighbourNet analyses. Fourteen plastid haplotypes were found constituting five strongly distinct lineages: Alps plus Pyrenees, Apennines, Balkans, southern Carpathians, and a Widespread group (eastern Pyrenees, northern Carpathians, Mt. Olympus). Plastid DNA data suggested that A. eriantha colonized the European Alpine System in a westward direction. Although, in southern Europe, vicariant differentiation among the Iberian, Italian and Balkan Peninsulas predominated, thus highlighting their importance as glacial refugia for alpine species, in temperate mountain ranges, long‐distance dispersal prevailed. This study emphasizes that currently highly disjunct distributions can be shaped by both vicariance and long‐distance dispersal, although their relative importance may be geographically structured along, for instance, latitude, as in A. eriantha. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 214–226.     

Diversity and biogeography of Ni‐hyperaccumulators of Alyssum section Odontarrhena (Brassicaceae) in the central western Mediterranean: evidence from karyology,morphology and DNA sequence data

The diversity of Alyssum section Odontarrhena in the central–western Mediterranean region was investigated to elucidate relationships and biogeography of Ni‐hyperaccumulators in the group. Karyological, morphometric and molecular phylogenetic analyses were performed on accessions of Ni‐hyperaccumulators from serpentine outcrops and non‐hyperaccumulators from calcareous–dolomitic soils in the region. Alpine and Apennine populations of A. argenteum, Sardinian A. tavolarae and some Tuscan A. bertolonii had a tetraploid chromosome complement and larger silicles, seeds and seed wings than diploid accessions. DNA sequences from the plastid rpoC1 locus corroborated the monophyly of section Odontarrhena but species relationships were poorly resolved. Bayesian analysis of combined ITS‐rpoC1 sequences retrieved three main lineages including hyperaccumulators and non‐hyperaccumulators of contrasting geographical origin. One lineage was mainly continental and included alpine and northern Apennine populations of A. argenteum, the Balkan complex of A. murale and the Iberian group of A. serpyllifolium, sister to Corsican A. robertianum as suggested by their similar diploid karyotype. In this clade no divergence was found between typical A. serpyllifolium and related Ni‐hyperaccumulator races from the Iberian peninsula, supporting their conspecific status. A second lineage was prevalently Mediterranean and included the sister species A. bertolonii and A. tavolarae, and other endemics from Sicily, the southern Balkans and Turkey from dolomite and serpentine habitats. The present data suggest new model systems consisting of hyperaccumulators and non‐hyperaccumulators of proven phylogenetic affinity for further research on the molecular mechanisms of Ni‐hyperaccumulation and serpentine tolerance at the diploid and tetraploid level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 269–289.     

Phylogeography of the large Myotis bats (Chiroptera: Vespertilionidae) in Europe,Asia Minor,and Transcaucasia

The large Myotis complex in continental Europe, Asia Minor, and Transcaucasia comprises two sibling bat species, the greater mouse‐eared bat, Myotis myotis, and the lesser mouse‐eared bat, Myotis blythii, also referred to as Myotis oxygnathus. Here, we investigate the phylogeography of these bats using two mitochondrial markers: the second hypervariable domain of the control region (HVII) and a fragment of the cytochrome b gene (cyt b). The HVII haplotypes formed six distinct haplogroups associated with different geographical regions. Most of the European HVII haplotypes were exclusive to M. myotis, whereas the majority of HVII haplotypes found in Asia Minor were exclusive to M. blythii/M. oxygnathus. The phylogenetic reconstruction based on the concatenated cyt b and HVII fragments recovered two major lineages. The first lineage comprised samples from Europe (western lineage), and the second lineage included samples from Asia Minor, Transcaucasia, Crimea, Western Ukraine, Thrace, the Balkans, and Eastern Europe (eastern lineage). The mitochondrial lineage of M. blythii, reported from Kyrgyzstan, was not present in Asia Minor and Transcaucasia. Therefore, we consider the possibility that the M. blythii/M. oxygnathus found in Europe, Asia Minor, and Transcaucasia are not recent descendants of the Central Asian M. blythii. Instead, we suggest that M. blythii/M. oxygnathus and M. myotis diverged through allopatric speciation in Asia Minor and Europe, and that they are represented by the eastern and western mitochondrial lineages. We also examine an alternative hypothesis: that the large Myotis complex consists of more than two species that diverged independently in Asia Minor and Europe through ecological speciation. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ??, ??–??.     

A morphologically intergrading population facilitates plastid introgression from diploid to tetraploid Dodecatheon (Primulaceae)

Polyploidy may promote diversification by generating reproductive isolation between ploidy levels, but this reproductive barrier may not be absolute. Several recent analyses of diploid–tetraploid contact zones have found evidence for hybridization. In these cases, inter‐cytotype gene flow is often associated with morphologically intergrading populations. In this study, we combine cytological, fitness and population genetic data to examine the evolutionary role of a morphologically intergrading population at a contact zone between species with different ploidy levels in Dodecatheon. Diploid D. frenchii and tetraploid D. meadia are usually distinguished by leaf‐shape characters. In southern Illinois, where these taxa occur in parapatry, a morphologically intergrading population includes the first documented tetraploid with D. frenchii morphology. Most plants in this intergrading population are fertile, and a nearby typical population of D. meadia has plastid DNA haplotypes that only occur in D. frenchii elsewhere in southern Illinois. These results suggest that fit neo‐tetraploids in this intergrading population have facilitated local introgression between ploidy levels. Similar patterns in other regions where these taxa co‐occur may explain weak range‐wide genetic differentiation between these species. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 91–100.     

Global phylogeography of the genus Capreolus (Artiodactyla: Cervidae), a Palaearctic meso‐mammal

Areas of sympatry and hybridization of closely related species can be difficult to assess through morphological differences alone. Species which coexist and are similar morphologically may be distinguished only with molecular techniques. The roe deer (Capreolus spp.) is a meso‐mammal having a Palaearctic distribution, with two closely related species: the European C. capreolus and the Siberian C. pygargus. We analysed mtDNA sequences from 245 individuals, sampled through all the entire range of the genus, to investigate the distribution of genetic lineages and outline phylogeographical patterns. We found that: (1) a C. pygargus lineage occurs in Poland and Lithuania, much farther west than the area which so far was believed its westernmost limit; (2) no haplotype of this C. pygargus lineage matches any found in East Europe and Asia – this should rule out human introductions and may indicate Pleistocene–Holocene migrations from the east; (3) no geographical structuring of C. pygargus lineages occurs, questioning the existence of putative subspecies; (4) several genetic lineages of C. capreolus can be recognized, consistent with the existence of two subspecies, respectively in central–southern Italy and southern Spain. Coalescence times suggest that intraspecific variation in C. capreolus and C. pygargus developed approximately 100–10 kya. The extant mitochondrial lineages pre‐dated the Last Glacial Maximum. Capreolus pygargus must have moved westward to Central Europe, where at least one genetic lineage still survives, coexisting with C. capreolus. © 2013 The Linnean Society of London     

Geographically congruent large‐scale patterns of plastid haplotype variation in the European herbs Silene dioica and S. latifolia (Caryophyllaceae)

The closely related dioecious herbs Silene latifolia and Silene dioica are widespread and predominantly sympatric in Europe. The species are interfertile, but morphologically and ecologically distinct. A study of large‐scale patterns of plastid DNA (polymerase chain reaction–restriction fragment length polymorphism) haplotypes in a sample of 198 populations from most of the European ranges of both species revealed extensive interspecific haplotype sharing. Four of the 28 detected haplotypes were frequent (found in > 40 populations) and widespread. Three of these frequent haplotypes occurred in both species and the geographic distribution of each haplotype was broadly congruent in both species. Each of these three, shared and widespread haplotypes is likely to have colonized central and/or northern Europe after the last glaciation from one or more of refugial areas in southern Europe. Interspecific hybridization and plastid introgression within refugial regions and/or during the early stages of postglacial expansion is the most plausible explanation for the broadly similar distribution patterns of the shared, frequent chloroplast haplotypes in the two species. The fourth frequent, widespread haplotype was absent from S. latifolia and almost entirely restricted to Nordic S. dioica. It is most likely that this haplotype spread into the Nordic countries from a central or northern European source or from a refugial area in Russia. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 153–170.     

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.     

Parnassia palustris: a genetically diverse species in Scandinavia

Patterns of variation at nine enzyme loci were examined in 528 plants representing diploid and tetraploid populations of Parnassia palustris s. l. in Europe to assess genetic variation patterns and migration history. Half of the plants showed a unique multilocus phenotype and 75% of all phenotypes occurred only in Scandinavia. Diploid populations showed similar levels of genetic diversity as other widespread outbreeding species with animal-mediated pollination and F -statistics indicated excessive heterozygosity and low rates of gene flow among them. In spite of dramatic population histories caused by the ice ages, diploid populations have maintained the same genetic diversity in Scandinavia as in central and southern Europe. Northern populations have apparently been established through the gradual advance of genetically variable populations and patterns of variation at individual loci indicate different migration routes, from the south-south-west and the east-north-east, respectively. The data strongly support a repeated autoploid origin of the tetraploid cytotype which has been much more successful than the diploid progenitors in colonizing new land since the last ice age. High genetic diversity in Scandinavia has apparently been obtained by a combination of immigration of plants from different source areas and recurrent formation of autotetraploids from diploid progenitors.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 347−372.     

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.     

Evidence of a complex phylogeographic structure in the common dormouse,Muscardinus avellanarius (Rodentia: Gliridae)

This is the first mitochondrial phylogeography of the common dormouse, Muscardinus avellanarius (Linnaeus, 1758), a hibernating rodent strictly protected in Europe (Habitat Directive, annex IV; Bern Convention, annex III). The 84 individuals of M. avellanarius, sampled throughout the distributional range of the species, have been sequenced at the mitochondrial DNA gene (cytochrome b, 704 base pairs). The results revealed two highly divergent lineages, with an ancient separation around 7.7 Mya and a genetic divergence of 7.7%. Lineage 1 occurs in Western Europe (France, Belgium, and Switzerland) and Italy, and lineage 2 occurs in Central–Northern Europe (Poland, Germany, Latvia, and Lithuania), on the Balkan Peninsula, and in Turkey. Furthermore, these two lineages are subdivided into five sublineages genetically isolated with a strong geographical association. Therefore, lineage 1 branches into two further sublineages (Western European and Italian), whereas lineage 2 contained three sublineages (Central–Northern European, Turkish, and Balkan). We observed low genetic diversity within the sublineages, in contrast to the significant level of genetic differentiation between them. The understanding of genetic population structure is essential for identifying units to be conserved. Therefore, these results may have important implications for M. avellanarius conservation. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 648–664.     

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.