Evolution of Dactylorhiza baltica (Orchidaceae) in European Russia: evidence from molecular markers and morphology

Four plastid markers, four nuclear markers and 14 morphometric characters were used in this study to investigate the evolution of Dactylorhiza baltica (Orchidaceae) in European Russia. In total, 98, 214 and 775 samples from 85, 112 and 121 populations were involved in the combined and separate molecular and morphometric analyses, respectively. In most cases, morphometric measures were done on exactly the same plants that were used for DNA studies. Dactylorhiza baltica plants from European Russia are most probably the products of several recent and mostly local hybridization events between the diploids D. fuchsii and D. incarnata , which have each been the maternal parent on different occasions. Considerable introgression into the parental diploids via the allopolyploid D. baltica is also hypothesized. Several morphological characters, such as length of the lip lateral lobe and the length of longest leaf, were found to be robust and could be useful in identification of D. baltica . This study demonstrates the advantage of 'combined' techniques, especially in the case of taxonomically complex taxa.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 257–274.     

Genetic diversity in Dactylorhiza majalis subsp. majalis populations (Orchidaceae) of northern Poland

We analysed 16 populations of Dactylorhiza majalis subsp. majalis from northern Poland, simultaneously utilizing both morphological and molecular data. Genetic differentiation was examined using five microsatellite loci, and morphological variation was assessed for 23 characters. At the species level, our results showed a moderate level of genetic diversity (A = 6.00; A_e = 1.86; H_o = 0.387; F_IS = 0.139) which varied between the studied populations (A = 2.60–4.20; A_e = 1.68–2.39; H_o = 0.270–0.523; F_IS = ?0.064–0.355). A significant excess of homozygotes was detected in five population, while excess of heterozygotes was observed in four populations, but the latter values were statistically insignificant. Moderate, but clear between population genetic differentiation was found (F_ST = 0.101; p < 0.001). Considering pairwise‐F_ST and number of migrants among populations, we recognized three population groups (I, II, III), where the first could be further divided into two subgroups (Ia, Ib). These three groups differed with respect to gene flow values (N_m = 0.39–1.12). The highest number of migrants per generation was noticed among populations of subgroup Ia (8.58), indicative of a central panmictic population with free gene flow surrounded by peripatric local populations (Ib) with more limited gene flow. Geographic isolation, habitat fragmentation and limited seed dispersal are inferred to have caused limitations to gene flow among the three indicated population groups. There was a significant correlation between the morphological and genetic distance matrices. A weak but significant pattern of isolation by distance was also observed (r = 0.351; p < 0.05).     

Apochromic populations of Dactylorhiza incarnata s.l. (Orchidaceae): diversity and systematic significance as revealed by allozyme markers and morphology

Apochromic forms of the Eurasian Dactylorhiza incarnata s.l. were studied in northern Europe to reveal their genetic (allozyme) and morphological diversity and to assess their systematic significance. The study included eight localities with sympatric populations of plants with anthocyanin‐pigmented and apochromic flowers. Parallel samples of the two morphs were taken from each locality. Genetic variation was only found at the allozyme loci pgd, pgi and ugpp. Statistically significant differences in allele frequencies between the two colour morphs were found in two localities and demonstrate that the occurrence of apochromic individuals in D. incarnata s.l. is not always because of spontaneous mutation. At least in some localities the apochromic plants form distinct breeding groups (but local populations of different colour morphs may also be composed of several more or less distinct breeding groups). Based on molecular and morphometric data, it is proposed that the apochromic study populations from calcareous fens should be referred to D. incarnata var. ochroleuca, whereas the apochromic study populations from non‐calcareous fens are better treated as aberrant local populations of var. incarnata s.l. Possible evolutionary patterns and processes are discussed and guidelines for identification of var. ochroleuca from morphological features are given. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 396–407.     

Age-related morphological differentiation among populations of Dactylorhiza traunsteineri (Orchidaceae) in eastern Sweden

Andersson, E. 1995. Age-related morphological differentiation among populations of Dactylorhiza traunsteineri (Orchidaceae) in eastern Sweden. - Nord. J. Bot. 15: 127–137. Copenhagen. ISSN 0107–055X.
Evolution takes time. It is generally believed that for a given species older populations have diversified more than younger ones. To test this hypothesis, two regions in southern Sweden of different age, determined based on land uplift data, were chosen for studying morphological differentiation within the orchid species Dactylorhiza traunsteineri . Individuals from 10 populations in an older region, Gotland, and 11 populations in a younger region, Uppland, were scored for 54 morphological characters. Patterns of morphological variation and the structure of diversity were analyzed using multivariate and simple statistics; the degrees of correspondence among matrices of morphological and environmental factors were calculated. Morphological variation among populations was greater in the older region. This pattern was consistent in all analyses, although more pronounced in the analyses of floral morphology than in analyses of vegetative morphology. Orchid populations on Gotland appear to be relatively old, small and isolated, but genetic drift does not seem to have been important for the differentiation among populations. Other factors, such as habitat heterogeneity, pollinator-mediated selection and accumulation of neutral mutations, were more important. Although populations from Uppland are young, the founder effect does not seem to have been responsible for the low level of diversity among populations. Differentiation appeared to have been limited by low habitat diversity, and a lack of time for genetic drift and for the accumulation of mutations. This study shows that patterns of differentiation within this species are indeed time-dependent.     

Late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae): variation patterns and taxonomic inferences

Principal components analysis (PCA) was applied to summarize variation in 23 morphological characters scored from nine late-flowering dune populations of Dactylorhiza incarnata (Orchidaceae) in Wales, The Netherlands, and Denmark. The Dutch and Danish groups of populations were vaguely separated in a PCA conducted on populations, and a very broad overlap between them was found in a PCA conducted on individual specimens. On the other hand, a clear morphological distinction between the Welsh and continental groups of populations appeared from both PCAs. The probability of each character to distinguish correctly between the main groups of populations was estimated. Three characters were found to distinguish reliably (success ≥ 90 %) between the Welsh and continental groups of populations, while no character was found to distinguish reliably between the Dutch and Danish groups of populations. It is concluded that the Welsh and Dutch/Danish populations represent two distinct taxa distributed in the British Isles and in continental Northwestern Europe, respectively. The British taxon is recognized as D. incarnata subsp. coccinea and the continental one as D. incarnata subsp. lobelii (stat. nov.). Brief taxonomic accounts are provided.     

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.     

A secondary hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta and allotetraploid D. lapponica (Orchidaceae)

Secondary hybrid zones are not uncommon in Dactylorhiza, but knowledge of ecological and evolutionary consequences of hybridization are scarce. Here, we assess interploidal gene flow and introgression in a hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta (2n = 2x = 40) and its putative allotetraploid derivative D. lapponica (2n = 4x = 80). Photometric quantification of DNA content and morphology confirmed that triploids are abundant in sympatric populations in our study area. Allozyme segregation patterns in D. lapponica supported an allopolyploid origin, although unbalanced genotypes suggested rare pairings between homoeologous chromosomes. Photometric data and chromosome counts suggest backcrossing between the triploid hybrid and D. lapponica, and hence some hybrid fertility. Triploids are morphologically more similar to the tetraploids than the diploids, maybe owing to the hybrid origin of both triploids and tetraploids. The diploids and tetraploids were not more similar in the parapatric populations compared to when they occur in allopatry. This indicates that backcrossing rarely leads to introgression, or alternatively that allopatric populations are not isolated enough to prevent influx of pollen from the other species. Despite some evidence of backcrossing, our study gives few indications that widespread hybridization entails local breakdown of species boundaries. Rather, the hybrid zone may be a transient phenomenon due to intensive mowing, resulting in the opening of habitats and hence bringing the parental species into close contact.     

Reproductive isolation and hybridization in sympatric populations of three Dactylorhiza species (Orchidaceae) with different ploidy levels

Background and Aims

The potential for gene exchange between species with different ploidy levels has long been recognized, but only a few studies have tested this hypothesis in situ and most of them focused on not more than two co-occurring species. In this study, we examined hybridization patterns in two sites containing three species of the genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative D. praetermissa).

Methods

To compare the strength of reproductive barriers between diploid species, and between diploid and tetraploid species, crossing experiments were combined with morphometric and molecular analyses using amplified fragment length polymorphism markers, whereas flow cytometric analyses were used to verify the hybrid origin of putative hybrids.

Key Results

In both sites, extensive hybridization was observed, indicating that gene flow between species is possible within the investigated populations. Bayesian assignment analyses indicated that the majority of hybrids were F₁ hybrids, but in some cases triple hybrids (hybrids with three species as parents) were observed, suggesting secondary gene flow. Crossing experiments showed that only crosses between pure species yielded a high percentage of viable seeds. When hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating strong post-zygotic reproductive isolation and high sterility.

Conclusions

Strong post-mating reproductive barriers prevent local breakdown of species boundaries in Dactylorhiza despite frequent hybridization between parental species. However, the presence of triple hybrids indicates that in some cases hybridization may extend the F₁ generation.     

Allozyme variation and genetic integrity of Dactylorhiza incarnata (Orchidaceae)

Eleven Danish populations of the diploid Dactylorhiza incarnata s.lat. have been examined for allozyme variation in DIA, PGD, PGI, PGM, SKD, TP1, and UGPP. The results reveal a deficiency of heterozygotes in several populations. Possible reasons for this deficiency are considered. The results also indicate that no genetic barrier seems to prevent gene flow between D. incarnata var. incarnata , var. dunensis , and var. ochrantha — and some indication exists that a considerable amount of gene exchange takes place between sympatric populations of var. incarnata and var. ochrantha . On the other hand, there is no sign of recent introgression between D. incarnata s.lat. and any of the sympatric tetraploid species ( D. maculata, D. majalis, D. purpurella s.lat.). It is suggested that knowledge on the various degrees of genetic integrity of morphologically recognizable entities should be elaborated and subsequently utilized for a biosystematic approach to Dactylorhiza .     

Notes on the esterase variation in Swedish Dactylorhiza incarnata s. l. (Orchidaceae)

Swedish material of Dactylorhiza incarnara s. l. shows little variation at commonly investigated allozyme loci. However, interpretable variation was found at one esterase locus. All plants investigated of D. incarnata var. cruenta from southern Sweden (with spotted leaves) were homozygous for allele a at this locus, whereas all plants investigated of D. incarnata var. ochroleuca were fixed for allele b. In D. incarnata var. incarnata , both alleles were found, although the b allele dominated. In northern Swedish material of D. incarnata s. l., only allele b was found, regardless whether the material had spotted leaves (sometimes referred to as var. cruenta ), or had unspotted leaves.
These results indicate that there is restricted gene flow between var. cruenta and the other varieties in southern Sweden, although they often grow in mixed populations. The northern Swedish material with spotted leaves appears not to be related to the southern var. cruenta .     

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.     

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.     

Systematics and evolution of the Dactylorhiza romana/sambucina polyploid complex (Orchidaceae)

The European–Mediterranean–Oriental Dactylorhiza romana/sambucina polyploid complex was studied with regard to genetic and morphological variation patterns. Allozyme and morphometric data were collected from 24 and 19 populations, respectively, initially identified as D. flavescens, D. insularis, D. markusii, D. romana, D. sambucina, and an indeterminate taxon. Genetic distances were calculated and illustrated by an unweighted pair‐group method using arithmetic averages (UPGMA) dendrogram, and principal components analyses (PCAs) were used to summarize morphological variation patterns. Another PCA was performed on combined allozyme and morphometric data. On the basis of the dendrogram and the PCA plots, main groups of populations were delimited, and the probability that each morphological character would distinguish correctly between these groups was estimated. After combining morphometric interpretations with studies of herbarium material and information from the literature, the following taxa were confidently accepted: D. romana ssp. romana, D. romana ssp. guimaraesii (comb. et stat. nov.) , D. romana ssp. georgica, D. sambucina, D. cantabrica (sp. nov.) , and D. insularis. Levels of genetic diversity suggest that D. romana s.s. is the least derived member of the complex. The evolutionary divergence of the diploid species, D. romana and D. sambucina, was probably the outcome of vicariant speciation, whereas D. romana ssp. georgica and D. romana ssp. guimaraesii appear to have evolved from D. romana s.s. through incomplete vicariant and peripheral isolate speciation events, respectively. In some populations of the diploid taxa, a significant deficiency in heterozygotes was found at one to three loci. It is proposed that this pattern may indicate a Wahlund effect, hypothesizing that local populations are subdivided into demes determined by the commonly sympatric occurrence of two distinct colour morphs combined with partial morph constancy of individual pollinators (bumblebees). Several pathways are possible for the origin of the allotriploid D. insularis and the apparently allotetraploid D. cantabrica. A taxonomic revision is provided. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 152 , 405–434.     

Genetic diversity and differentiation of allopolyploid Dactylorhiza (Orchidaceae) with particular focus on the Dactylorhiza majalis ssp. traunsteineri/lapponica complex

Genetic differentiation of Dactylorhiza majalis ssp. traunsteineri from the Alps, Scandinavia, and Britain was studied and compared with other allotetraploid members of the systematically challenging genus Dactylorhiza . One-hundred and eleven populations from altogether 18 taxa were analysed for eight polymorphic plastid markers and two size-variable fragments from the nuclear internal transcribed spacer (ITS) region. In total, 60 plastid haplotypes and six ITS alleles were found among the 737 individuals analysed. No clear differentiation between populations of ssp. traunsteineri from the three regions was revealed. However, ssp. traunsteineri was genetically differentiated from Dactylorhiza baumanniana , Dactylorhiza elata , and D. majalis ssp. sphagnicola , although the majority of allotetraploid taxa remained inseparable. Judging from the degree of concerted evolution in ITS, D. majalis ssp. alpestris may be regarded as a relatively old allotetraploid, whereas ssp. baltica and ssp. purpurella may be considerably younger. Based on plastid data, the Alp region had the highest genetic diversity followed by Scandinavia and Britain. The geographic distribution of haplotypes provided support for possible refugial areas around the Alps and for several independent immigration routes into Scandinavia after the last ice age.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 52–67.     

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     

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.     

Electrophoretic evidence for all otetraploid origin of Dactylorhiza purpurella (Orchidaceae)

Evidence from all ozyme markers suggests that the NW European Dactylorhiza purpurella (Orchidaceae) is an allotetraploid which originated from taxa closely related to the present-day D. incarnata s. I. and D. fuchsii/D. maculata . However, Dactylorhiza purpurella deviates more strongly from the allotetraploid condition than other taxa previously investigated in Dactylorhiza (i.e., D. majalis, D. traunsteineri, D. sphagnicola , and D. lapponica ), in that the characteristic incarnata alleles occur at lower frequencies than expected at two loci. It is suggested that D. purpurella arose from parents slightly different from those giving rise to the other allotetraploids, or that the tetraploid genome in D. purpurella has been modified by rare recombination events between homoeologous chromosomes, replacing segments of the incarnata chromosomes with the fuchsii/maculata genome.