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).     

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.     

Matrilinear phylogeography and demographical patterns of Rutilus rutilus: implications for taxonomy and conservation

A phylogeographic analysis of mitochondrial DNA sequence variation was carried out to infer the geographical distribution of the genealogical lineages and the historical demography of roach Rutilus rutilus (L.). A total of 265 individuals from 52 sites covering most of the Eurasian distribution range were sequenced for a 475 bp fragment of the mitochondrial cytochrome b gene. The monophyletic roach contained two deep clades that dated back to the Pliocene. The Ponto-Caspian clade comprised populations from Greece to Siberia with a likely palaeorefugium at the west coast of the Caspian Sea. This clade largely corresponds to individuals with morphological features described as Rutilus heckelii . The west European clade included individuals from central and western Europe with the Danube and Dniester basins as possible palaeorefugia. This clade largely corresponds to individuals with morphological features described as R. rutilus . A suture-zone of the two main lineages was observed along the coastal region of the Black Sea. The neutrality tests and the mismatch distributions indicated a demographic expansion during the Middle-Pleistocene for both clades.     

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.     

Evolution, phylogeography, and taxonomy within the Viola alba complex (Violaceae)

 Taxa of the Viola alba complex were investigated using allozymes and morphometry. A taxonomic revision is presented. A wide delimitation of V. alba with only three morphological and geographical subspecies is suggested: (1) ssp. dehnhardtii distributed in the Mediterranean eastwards to Turkey; (2) ssp. alba flanking ssp. dehnhardtii in the north and east; and (3) ssp. cretica endemic to Crete. Ssp. cretica, up to now treated as a separate species, is particularly close to ssp. dehnhardtii. Viola cadevallii (NW Mediterranean) is included in the synonymy of ssp. dehnhardtii. Ssp. scotophylla (S Europe), ssp. thessala (Balkan), V. armena (Turkey), and V. besseri (Caucasus) are reduced to synonyms of V. alba ssp. alba. Viola pentelica (Greece) might represent transitional forms between ssp. alba and ssp. dehnhardtii. Glacial refugia for ssp. alba are suggested from the eastern Mediterranean via Turkey to the Caucasus, for ssp. dehnhardtii in the Mediterranean area in general, and for ssp. cretica in Crete. A key to the subspecies is provided. Taxonomic recombination: Viola alba Bess. ssp. cretica (Boiss. & Heldr.) Marcussen, comb. nov. Received June 17, 2002; accepted November 27, 2002 Published online: March 20, 2003     

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.     

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.     

The allopolyploid origin of Spiranthes hongkongensis (Orchidaceae)

Morphological and ecological similarities between Spiranthes hongkongensis Hu et Barretto and S. sinensis (Pers.) Ames suggest a close evolutionary relationship. The hypothesis that S. hongkongensis evolved through natural hybridization between S. sinensis and S. spiralis was tested using isozyme electrophoresis. A total of 28 isozyme loci were interpreted in the diploid species S. sinensis and S. spiralis, and “fixed heterozygosity” owing to gene duplication was observed at ten of these loci in S. hongkongensis. Isozyme phenotypes provided strong evidence for an allotetraploid origin of S. hongkongensis, with S. sinensis and most likely S. spiralis as its diploid progenitors. Chromosomal counts of S. hongkongensis gave a tetraploid number of ≈60. The allotetraploid species is self-fertile, and completely isolated by sterility barriers from S. sinensis. Premating isolation also exists between S. hongkongensis and S. sinensis due to earlier flowering and autogamy in S. hongkongensis, which may account for the rarity of natural hybrids in the sites of sympatry. The role of the autogamous breeding system in the establishment and spread of S. hongkongensis is discussed.     

Rangewide phylogeography in the greater horseshoe bat inferred from microsatellites: implications for population history, taxonomy and conservation

The distribution of genetic variability across a species' range can provide valuable insights into colonization history. To assess the relative importance of European and Asian refugia in shaping current levels of genetic variation in the greater horseshoe bats, we applied a microsatellite-based approach to data collected from 56 localities ranging from the UK to Japan. A decline in allelic richness from west Asia to the UK and analyses of F(ST) both imply a northwestward colonization across Europe. However, sharp discontinuities in gene frequencies within Europe and between the Balkans and west Asia (Syria/Russia) are consistent with suture zones following expansion from multiple refugia, and a lack of recent gene flow from Asia Minor. Together, these results suggest European populations originated from west Asia in the ancient past, and experienced a more recent range expansion since the Last Glacial Maximum. Current populations in central Europe appear to originate from the Balkans and those from west Europe from either Iberia and/or Italy. Comparisons of R(ST )and F(ST) suggest that stepwise mutation has contributed to differentiation between island and continental populations (France/UK and China/Japan) and also among distant samples. However, pairwise R(ST) values between distant populations appear to be unreliable, probably due to size homoplasy. Our findings also highlight two priorities for conservation. First, stronger genetic subdivision within the UK than across 4000 km of continental Eurasia is most likely the result of population fragmentation and highlights the need to maintain gene flow in this species. Second, deep splits within China and between Europe and China are indicative of cryptic taxonomic divisions which need further investigation.     

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.     

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 .     

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.     

Phylogeography of the threatened crayfish (genus Austropotamobius) in Italy: implications for its taxonomy and conservation

A nucleotide sequence analysis of a portion of the mitochondrial large ribosomal subunit was performed to define the phylogeography of the threatened crayfish Austropotamobius (Decapoda; Astacidae) in Italy. We collected 61 specimens from 31 localities across the Italian peninsula. For the phylogenetic inference, we combined the 61 Austropotamobius spp sequences obtained from this study with 18 sequences deposited in GenBank and corresponding to Italian, French, Irish, Swiss, and Slovenian locations. Among the analysed sequences, 34 distinct haplotypes were detected. Our results confirmed the presence of both A. pallipes and A. italicus in the Italian peninsula and the existence within the latter species of a strong intraspecific genetic variation, due to the occurrence of four subspecies with a well-defined geographic distribution. From a conservation viewpoint, Italy, with its high haplotype variability, may be considered a 'hot spot' for the genetic diversity of the European native crayfish Austropotamobius. We suggest that re-introduction programs should be conducted with extreme caution in Italy, since not only the two Austropotamobius species but also the four A. italicus subspecies are genetically and taxonomically separate units and require independent conservation plans.     

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.     

Mitochondrial phylogeography, subspecific taxonomy, and conservation genetics of sandhill cranes (Grus canadensis; Aves: Gruidae)

Six subspecies of sandhill cranes (Gruscanadensis) have been denoted based onperceived morphological and/or breedinglocality differences among them. Threesubspecies are migratory, breeding from thehigh arctic in North America and Siberia(lesser sandhill, G. c. canadensis),south through central Canada (Canadiansandhill, G. c. rowani) and into thenorthern United States (greater sandhill, G. c. tabida). A review of sandhill cranetaxonomy indicates that the size variation, onthe basis of which these subspecies were named,may be clinal and not diagnostic. The otherthree subspecies, all listed as endangered orthreatened, are non-migratory, resident inFlorida (G. c. pratensis), Mississippi(G. c. pulla), and Cuba (G. c.nesiotes). We used analysis of mitochondrialDNA control region (CR) sequences to determinewhether haplotypes representing currentsubspecies show any genetic cohesion or aremore consistent with a pattern of clinalvariation in morphology. CR sequences indicatethat only two highly divergent (5.3%) lineagesof sandhill cranes occur in North America: onelineage composed only of arctic-nesting G.c. canadensis, the other of the remainingNorth American subspecies (we lack data on theCuban population). The deep split betweenlineages is consistent with an estimatedisolation of approximately 1.5 Mya(mid-Pleistocene), while the distribution ofmutational changes within lineages isconsistent with an hypothesis of rapid,post-Pleistocene population expansions. Noother phylogeographic structuring is concordantwith subspecific boundaries, however, analysisof molecular variance indicates that there issignificant population genetic differentiationamong all subspecies except G. c. tabidaand G. c. rowani, which areindistinguishable. We suggest thatrecognition of the recently named G. c.rowani be abandoned.     

Mitochondrial phylogeography and genetic diversity of Tibetan gazelle (Procapra picticaudata): implications for conservation

The Tibetan gazelle (Procapra picticaudata) is a threatened species and distributed on the Qinghai-Tibet Plateau of China (Qinghai Province, Tibet Autonomous Region and the adjacent Gansu Province, Sichuan Province, and Xinjiang Uigur Autonomous Region). Small peripheral populations of Tibetan gazelle were once found in northern Sikkim and Ladakh, but now these are close to extinction. To describe the evolutionary history and to assess the genetic diversity within this monotypic species and population structure among different geographic locations in China, we sequenced mitochondrial DNA from the control region (CR) and cytochrome (cyt) b gene for 46 individuals from 12 geographic localities in Qinghai, Tibet, Xinjiang, Gansu, and Sichuan. A total of 25 CR haplotypes and 16 cyt b haplotypes were identified from these gazelle samples. CR haplotype diversity (0.98+/-0.01) and nucleotide diversity (0.08+/-0.009) were both high. Phylogenetic trees indicate that the Tibetan gazelle in China can be divided into three main clades: Tibet, Sichuan (SCH) and Qinghai-Arjin Shan-Kekexili (QH-ARJ-KKXL). Analysis of molecular variance (AMOVA) and network analysis consistently support this geographic structure in both datasets. Significant differentiation between populations argues for the presence of management units (MUs). Such differentiation may reflect a geographic separation resulting from the uplift of the Qinghai-Tibet Plateau during the Late Pliocene and Pleistocene. Mismatch distribution analysis implies that Tibetan gazelle has undergone complex population changes. We suggest that the present population structure has resulted from habitat fragmentation during the recent glacial period on the Qinghai-Tibet Plateau and population expansion from glacial refugia after the glacial period. It is likely that the present populations of Tibetan gazelle exhibit a pattern reminiscent of several bottlenecks and expansions in the recent past.     

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.