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.     

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.     

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 .     

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.     

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.     

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 .     

Nutrient versus pollination limitation in Platanthera bifolia and Dactylorhiza incarnata (Orchidaceae)

Availability of resources and pollination services have been demonstrated to have impact on reproductive success in some orchid species, but to our knowledge no studies have examined the effects of nutrient application and pollination limitation in the same experiment. In this study, factors limiting reproductive success were studied with two terrestrial orchid species in Central Finland during 1996 and 1997. In a field experiment using a factorial design, plants of nectar-producing Platanthera bifolia and nectarless Dactylorhiza incarnata were treated with nutrient application and hand-pollination. Inflorescence size was considered as an indicator of attractiveness to pollinators as well as of the general condition of the plant. In 1996, fertilizer treatment increased relative capsule production in P. bifolia plants with small inflorescences, indicating poorer store of resources in the underground corm and dependence on nutrient availability during capsule maturation, which was not found in large plants . Hand-pollination and large inflorescence size in P. bifolia did not affect capsule initiation, but increased the proportion of mature capsules. P. bifolia may thus be regarded as both resource- and pollination-limited within a year. Pollination success was observed to be the only factor limiting reproduction of the nectarless D. incarnata within a year, because hand-pollination increased capsule production, and there were no significant effects of fertilizer treatment or inflorescence size in 1996. Capsule production in 1996 did not affect the probability of P. bifolia flowering in the following year, while high capsule production decreased the subsequent probability of flowering in D. incarnata . Species with different pollination strategies differed in the use of resources. The nectar-producing P. bifolia had a lower fruit/flower ratio than the nectarless D. incarnata , also after hand-pollination.     

Systematics of the Dactylorhiza euxina/incarnata/maculata polyploid complex (Orchidaceae) in Turkey: evidence from allozyme data

 Material of Dactylorhiza were sampled from 49 localities in Turkey and investigated for allozyme variation at ten loci (nine enzyme systems). Among diploids, the Anatolian D. osmanica and D. umbrosa were allozymically variable, but not distinct from each other or from D. incarnata. Dactylorhiza saccifera contained the same alleles as the European D. fuchsii. Dactylorhiza iberica and D. euxina were distinct from each other and the other diploids. On basis of allozyme patterns three distinct allotetraploid genotypes were distinguished, and each of them could be treated as a separate species. Dactylorhiza nieschalkiorum is similar to European allotetraploids, and may have arisen from hybridization between D. incarnata s.l. and D. saccifera. Dactylorhiza urvilleana may have arisen from parents related to present-day D. saccifera and D. euxina, but it also contains additional alleles that have not been found in any of the diploids investigated. A third allotetraploid known from four populations in the Ardahan and Kars provinces of north-eastern Turkey combines the allozyme patterns found in material of D. incarnata s.l. from the same area with those from D. euxina. It is here described for the first time as D. armeniaca. Received November 14, 2000 Accepted June 20, 2001     

Reproductive success of Dactylorhiza incarnata ssp. incarnata (Orchidaceae): the effects of population size and plant visibility

Reproduction of plants pollinated solely by flower-visiting animals depends on the ability of the population and each of its flowering member to attract pollinators. Factors affecting the pollination of nectarless species differ somewhat from those affecting the pollination of rewarding species due to the avoidance behaviour of pollinators after visiting empty flowers.
We studied a non-mimic food-deceptive orchid, Dactylorhiza incarnata ssp. incarnata, in 16 populations in central Finland to examine if population properties and plant size affected reproductive success of plants.
We found that the number of flowering plants increased total pollinia removal and seed production of the population, but had no effect on reproduction of individual plants. Dactylorhiza incarnata occurred in open mires with virtually no rewarding species in the neighbourhood, but the distance from the forest edge did not have any effect on reproduction. However, increased variation in the number of flowers among within-population plants enhanced plant reproductive success. The increased variation probably hampered the learning process of flower visitors. Plant size affected reproductive success only in populations with a high general reproductive success indicating that in the rarely visited populations, pollination of individual plants is mainly affected by random effects.     

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.     

Pollen flow and post-pollination barriers in two varieties of Dactylorhiza incarnata s.l. (Orchidaceae)

Dactylorhiza orchids are known for their high variation in morphology and distinct varieties have been named in D. incarnata s.l. However, it is not known how these varieties interact in mixed populations and why they remain stable. We conducted three field experiments in West-Estonian populations of D. incarnata to examine if the two most common varieties co-occurring these are separated from each other either by pre-pollination or post-pollination reproductive barriers. We found that pollinia were far more frequently transferred between the purple-flowered var. incarnata and the pale-flowered var. ochroleuca than between plants of the same variety. Furthermore, in hand-pollination and germination experiments concerning the same two varieties, we found that pollen source (self-pollination, within- or between-varieties pollination) did not affect seed production or probability of fungus infection of the germinating seeds. These two varieties of D. incarnata thus had no pre-pollination or early functioning post-pollination reproductive barriers. Post-pollination barriers may, however, act later in seedling or adult stage.     

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.     

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.     

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.     

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.     

Dactylorhiza (Orchidaceae) in European Russia: combined molecular and morphological analysis

Four plastid and two nuclear (internal transcribed spacer [ITS] ribosomal DNA) markers were used in this study of the Dactylorhiza maculata and D. incarnata complexes (Orchidaceae: Orchidinae) to determine diversity and taxonomic distribution of haplotypes, hybridization frequencies, and maternal parentage of hybrids in 125 samples from 78 populations from European Russia and the Caucasus. A morphometric study of all populations revealed significant correspondence between morphological and plastid DNA data. Most D. maculata sensu stricto (s.s.) specimens from Russia have D. fuchsii haplotypes; this could be evidence for introgression or widespread hybridization between these species in northern Russia. Heterogeneity within populations is much higher for ITS data and is strongly correlated with latitude. Both plastid and nuclear data are significantly correlated with distribution along a south-north axis. Several haplotypes and ITS alleles uncommon in western Europe are more widely distributed in Russia, whereas some frequent haplotypes from western Europe are absent.     

Molecular evidence for allopolyploid speciation and a single origin of the western Mediterranean orchid Dactylorhiza insularis (Orchidaceae)

The hybrid origin of the western Mediterranean orchid Dactylorhiza insularis was demonstrated by genetic markers. Allozyme data showed that throughout its range D. insularis has an allotriploid constitution and reproduces apomictically. The parental species of D. insularis were identified as D. romana andD. sambucina; they contributed 2 alleles and 1 allele, respectively, at the allozyme loci studied. The maternal species of D. insularis was D. romana , as inferred from cpDNA ( trn L(UAA) intron). High genetic similarities were found when comparing present populations of D. romana and D. sambucina with their respective genomes 'frozen' in D. insularis. Dactylorhiza insularis showed fixed (or nearly fixed) heterozygosity at 11 out of the 19 loci studied, and poor genetic variation: eight multilocus genotypes were detected at allozyme level. No multilocus genotype differs from the most similar one by more than one allele substitution. All D. insularis individuals showed the same cpDNA haplotype (I) , regardless of their geographic origin and multilocus genotype. The I haplotype is similar, but not identical to that found in D. romana (R). No recurrent formation of D. insularis was observed in hybrid zones between D. romana and D. sambucina , where diploid sexual hybrids (F_1; F_n, backcrosses) were detected. Available data agree with a single origin for D. insularis , which possibly occurred in the present postglacial, when D. romana and D. sambucina , expanding from their glacial refugia, came into contact. The genetic homogeneity found between D. romana and D. markusii , both from their locus classicus , indicates that the latter is a junior synonym of D. romana; on the other hand, D. romana and D. sambucina are well differentiated species ( D_Nei = 0.59).