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.     

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.     

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.     

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

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

Genetic differentiation and postglacial migration of the Dactylorhiza majalis ssp. traunsteineri/lapponica complex into Fennoscandia

Eight variable regions (microsatellites, insertion/deletion and duplication regions) from the plastid DNA genome were analyzed for 91 populations belonging to Dactylorhiza majalis ssp. traunsteineri and closely related taxa. A total of 36 composite plastid haplotypes were found. The two dominating haplotypes had a clear geographic distribution suggesting at least two separate immigration routes into Scandinavia after the last glaciation: one southwestern route and one or two southeastern routes. D. majalis ssp. traunsteineri could not be clearly separated from any of the other taxa included in the study except for D. majalis ssp. sphagnicola. The morphologically similar taxa D. majalis ssp. traunsteineri, D. majalis ssp. lapponica and D. majalis ssp. russowii showed no genetic differentiation, and therefore we suggest an amalgamation of the three taxa into one broadly circumscribed subspecies; D. majalis ssp. lapponica. The plastid data also revealed incidents of hybridization and possible introgression between D. majalis ssp. lapponica and other members of the genus, e.g., D. incarnata.     

Comparison of reproductive success in two orchids: the nectarless Dactylorhiza majalis s.s. and the nectar-producing Gymnadenia conopsea s.l.

Differences in reproductive biology between two orchid species were examined: Eight Danish populations of the nectarless Dactylorhiza majalis ssp. majalis and six Swedish populations of the nectar-producing Gymnadenia conopsea ssp. densiflora . Population size varied from 24 to 1700 flowering individuals in D. majalis and from 100 to 1200 in G. conopsea. Dactylorhiza majalis had only half as many flowers per spike as G. conopsea . Fruit-set of D. majalis ranged from 16 to 39%, much lower than the fruit-set of G. conopsea (78–91%). Thus in the species with a few flowers and no nectar a lower fruit-set was observed than in the species with many flowers and presence of nectar. The low fruit-set of D. majalis was pollen-limited and fruit-set was positively correlated with pollinia removal and the latter was negatively correlated with population size. In both species, fruit-set and population size were uncorrelated.     

High levels of genetic diversity in marginal populations of the marsh orchid Dactylorhiza majalis subsp. majalis

Because of harsh conditions, suboptimal habitat quality and poor connectivity to other populations, plant populations at the margin of a distribution are expected to be less genetically diverse, but to be more divergent from each other than populations in the centre of a distribution. In northern Europe, northern marginal populations may also be younger than populations further to the south, and may have had less time to accumulate genetic diversity by mutation and gene flow. However, orchids have very small seeds, which are easily dispersed long distances by wind, and orchids are therefore expected to show less differentiation between marginal and central populations than other groups of seed plants. Here, we analysed whether Scandinavian populations of the tetraploid marsh orchid Dactylorhiza majalis subsp. majalis differ from central European populations in genetic diversity patterns. A total of 220 plants from eight central European and ten Scandinavian populations was examined for variation at five nuclear microsatellite loci, nuclear ITS and 13 polymorphic sites in noncoding regions of the plastid genome. The total genetic diversity was slightly lower in Scandinavia than in central Europe, both in plastid and nuclear markers, but the differences were small. Also, the Scandinavian populations were less diverse and somewhat more strongly differentiated from each other than the central European ones. Dactylorhiza majalis subsp. majalis has apparently colonized Scandinavia on multiple independent occasions and from different source areas in the south. Seed flow between Scandinavian populations has still not fully erased the patterns imprinted by early colonization. Our results suggest that marginal populations of orchids may be as important as central ones in preserving genetic diversity through Pleistocene glacial cycles. We also predict that orchids with their light seeds are better adapted than many other plants to respond to future climate changes by dispersing into new suitable areas.     

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.     

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

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

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.     

On the distinction of Dactylorhiza baltica and D. pardalina (Orchidaceae) and the systematic affinities of geographically intermediate populations

The eastern European Dactylorhiza baltica (Klinge) N. I. Orlova and the western European D. pardalina (Pugsl.) Aver. (= D. praetermissa var. junialis (Verm.) Sengh) are usually considered to have non-overlapping geographic distributions, for which reason it has rarely been realized that they are morphologically similar. They have not previously been thoroughly compared by molecular methods, and no existing flora or revision has convincingly demonstrated that they can be distinguished by morphological characters. In reality, they might be 'political' rather than natural taxa. Prompted by the recent discovery of geographically intermediate populations (in eastern Denmark), originally identified as D. baltica , we have addressed this problem by analysis of morphometric data as well as molecular data from allozyme markers, plastid haplotypes, nuclear ITS alleles and nuclear microsatellites. Dactylorhiza baltica and D. pardalina turned out to be clearly distinguished genetically, and although they are morphologically similar, a few characters were identified that distinguish with 81–85% certainty between the two taxa. Molecular and morphometric data place the geographically intermediate populations in D. pardalina . Both taxa were confirmed to be allotetraploids combining diploid genomes from the D. incarnata s.l. and D. maculata s.l. lineages, and they should therefore be recognized as infraspecic taxa under D. majalis s.l. Thus, D. baltica should be called D. majalis subsp. baltica ; D. pardalina is identical with D. praetermissa var. junialis , but the nomenclatural consequences for D. praetermissa , if treated as subspecies under D. majalis , are still unresolved.     

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.     

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     

Flowering regimes of terrestrial orchids: unpredictability or regularity?

Abstract. Empirical data on many species of terrestrial orchids suggest that their flowering pattern over the years is extremely irregular and unpredictable. A long search for the reason has hitherto proved inconclusive. Irregular flowering was attributed to costs associated with sexual reproduction, to herbivory, or to the chaotic behaviour of the system represented by difference equations describing growth of the vegetative and reproductive organs. Data on the seasonal growth of leaves and inflorescence of Dactylorhiza majalis are used here to test alternative explanations of the irregular flowering patterns of orchids. These patterns are found to be extremely rare in our data set. Neither costs of reproduction nor grazing seem to explain the rare events of a transition from flowering one year to sterility or absence the next year. These transitions are almost exclusively characteristic of one of four experimental sites, the only unmown site, where the mean leaf area and incidence of flowering in the whole population is also in decline. It is therefore hypothesized that irregular flowering regimes may be characteristic of sites with temporarily or steadily declining populations and that they are usually not present in prosperous ones - at least for D. majalis.     

Early Responses of Fishes and Crustaceans to Restoration of a Tidally Restricted New England Salt Marsh

Nekton (fishes and decapod crustaceans) is an abundant and productive faunal component of salt marshes, yet nekton responses to tidal manipulations of New England salt marshes remain unclear. This study examined nekton use of a tidally restricted salt marsh in Narragansett, Rhode Island relative to an unrestricted marsh during summer. In addition, a before‐after‐control‐impact design was used to examine early responses of nekton to the reintroduction of natural tidal flushing. Species richness and densities of Cyprinodon variegatus, Lucania parva, Menidia beryllina, and Palaemonetes pugio were higher in the restricted marsh compared with the unrestricted marsh. The unrestricted marsh supported higher densities of Menidia menidia and Fundulus majalis. Mean lengths of Carcinus maenas and P. pugio were greater in the restricted marsh. Tidal restoration resulted in increased tidal flushing, salinity, and water depth in the restricted marsh. Densities of Fundulus heteroclitus, F. majalis, and Callinectes sapidus were higher after 2 years of restoration. Density of L. parva decreased after restoration, probably in response to a loss of macroalgal habitat. Species richness also decreased after 2 years, from 20.9 species when the marsh was restricted to 13.0 species. Total nekton density did not change with restoration, but shifts in community composition were evident. In this study restoration induced rapid changes in the composition, density, size, and distribution of nekton species, but additional monitoring is necessary to quantify longer‐term effects of salt marsh restoration on nekton.     

Genetic differentiation of metallicolous and non-metallicolous Armeria maritima (Mill.) Willd. taxa (Plumbaginaceae) in Central Europe

This study investigates the genetic differentiation within the Central European Armeria maritima (Mill.) Willd. complex with special reference to the metallicolous populations using AFLP markers. Our sampling comprised all metallicolous (ssp. halleri, hornburgensis, bottendorfensis, eifeliaca, calaminaria), and non-metallicolous taxa (ssp. maritima, elongata, alpina). Geographical and genetic distances between populations were moderately positively correlated. Genetic variability of metallicolous and non-metallicolous populations was not significantly different. Lowland populations were clearly differentiated from the alpine populations. Within the lowland group metallicolous and non-metallicolous populations were not genetically differentiated. All lowland populations show a regional differentiation and close relationships to ssp. elongata. Thus, the metallicolous taxa should not be maintained as subspecies. Likewise, their treatment as varieties of a ssp. halleri s.l. is critical because this taxon cannot be consistently characterized throughout its geographical range and may be an artefact itself. If a taxonomical recognition should be considered necessary it is advisable to treat the microendemics as varieties of ssp. elongata.     

Dactylorhiza majalis s.l. (Orchidaceae) in acid habitats: variation patterns, taxonomy, and evolution

Morphometric and allozyme data were collected from 11 populations of Dactylorhiza majalis s.l. in the Ardennes (Belgium, France) and North Jutland (Denmark). All study populations were growing in obviously acid habitats. Genetic distances were calculated and illustrated by UPGMA dendrograms, while principal components analyses (PCAs) were used to summarize morphological variation patterns. Based on the PCAs, the probability of each character to distinguish correctly between the main groups of populations was estimated. The study populations from the Ardennes and from the Danish island of Læsø seem to agree with populations from Sweden previously studied by Hedrén—and to belong to D. majalis subsp. sphagnicola (comb. nov.). It should be noted that allozyme data suggest a polytopic origin of this taxon. The study populations from the Danish region of Thy differ morphologically from the others and exhibit much less genetic variation. They are described as a new subspecies, D. majalis subsp. calciñigiens. The evolution and biogeography of the two taxa are discussed, and brief taxonomic accounts are provided.     

Growth and viability of mycorrhizal extraradical mycelia associated with three temperate orchid species

Growth and enzymatic activities of extraradical mycelia (ERM) of native mycorrhizal symbionts associated with three orchid species, Dactylorhiza fuchsii, D. majalis and Platanthera bifolia, were studied. ERM extracted from the mycorrhizosphere of these species showed features typical for fungi that form orchid mycorrhiza. In the first pot experiment, three different treatments were applied on tubers of D. fuchsii transplanted from a natural site: control (no specific treatment), reinoculated (surface-sterilized tubers reinoculated with mycorrhizal fungi-colonised roots), and benomyl (nonsterilized tubers treated with fungicide). However, no significant differences in ERM growth and intensity of root mycorrhizal colonisation at harvest were observed among these treatments. ERM associated with reinoculated D. fuchsii plants showed significantly higher alkaline phosphatase (ALP) enzymatic activity at week 36 than at week 24, but no differences were observed for NADH diaphorase activity. Benomyl application significantly reduced ALP activity in comparison with reinoculated plants at week 36. In the second experiment, plants of all three species were either untreated (control), or repeatedly treated with benomyl. Similarly to the results of the first experiment, benomyl application did not reduce the ERM growth of mycorrhizal symbionts associated with D. majalis and D. fuchsii. The low ERM growth associated with benomyl-treated P. bifolia was probably caused by poor root system development in this treatment. Significantly higher mycorrhizal colonisation was found for D. fuchsii compared to P. bifolia in control treatments at the end of cultivation. The ERM of native symbionts of the three orchid species studied seemed to have a different growth pattern over time and responded differently to fungicide application.     

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     

The wheat curl mite Aceria tosichella (Acari: Eriophyoidea) is a complex of cryptic lineages with divergent host ranges: evidence from molecular and plant bioassay data

Aceria tosichella (the wheat curl mite, WCM) is a global pest of wheat and other cereals, causing losses by direct damage, as well as the transmission of plant viruses. The mite is considered to have an unusually wide host range for an eriophyoid species. The present study tested the commonly held assumption that WCM is a single, highly polyphagous species by assessing the host range of genetically distinct lineages of WCM occurring in Poland on different host plants. Genotyping was performed by analyzing nucleotide sequence data from fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear D2 region of 28S rDNA. Mean between‐lineage distance estimated using COI data was found to be one order of magnitude greater than the within‐clade lineage and, in some cases, comparable to distances between WCM lineages and a congeneric outgroup species. Host acceptance was tested by quantifying population growth for different WCM mitochondrial (mt)DNA lineages when transferred from source host plants to test plants. These experiments revealed significant differences in host colonization ability between mtDNA lineages, ranging from highly polyphagous to more host‐specific. The present study reveals that WCM is composed of several discrete genetic lineages with divergent host‐acceptance and specificity traits. Genetic variation for host acceptance within A. tosichella s.l. may act as a reproductive barrier between these lineages, most of which had narrow host ranges. Two lineages appear to have high pest potential on cereals, whereas several others appear to specialize on wild grass species. We conclude that WCM is not a homogeneous species comprising polyphagous panmictic populations rather it is a complex of genetically distinct lineages with variable host ranges and therefore variable pest potential. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 165–180.