Colonization history of Scots pine in Eastern Europe and North Asia based on mitochondrial DNA variation

During Quaternary glaciations, the ranges of Northern Eurasia forest species periodically experienced contraction followed by subsequent re-colonizations in the interglacial intervals. However, unlike the broadleaf trees of temperate forests, taiga species seem not to have retreated fully to southern regions in unfavorable periods and possibly survived at mid-latitudes in multiple refugia. Here, we report a study of genetic variation of three mitochondrial DNA markers in 90 populations of Scots pine (Pinus sylvestris) located from Eastern Europe to Eastern Siberia. The geographic distribution of seven mitotypes demonstrated the split between western and eastern populations approximately along the 38th meridian. Genetic diversity in the western part was significantly higher than in the eastern one. Five mitotypes were western- and one eastern-specific. One mitotype was common in both regions, but in the eastern part it occurred only in the South Urals and adjacent areas. The geographic structure in the mitotype distribution supports a hypothesis of post-glacial re-colonization of the studied territory from the European and Ural refugia.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

Phylogeography of Pinus tabulaeformis Carr. (Pinaceae), a dominant species of coniferous forest in northern China

How coniferous trees in northern China changed their distribution ranges in response to Quaternary climatic oscillations remains largely unknown. Here we report a study of the phylogeography of Pinus tabulaeformis, an endemic and dominant species of coniferous forest in northern China. We examined sequence variation of maternally inherited, seed‐dispersed mitochondrial DNA (mtDNA) (nad5 intron 1 and nad4/3–4) and paternally inherited, pollen‐ and seed‐dispersed chloroplast DNA (cpDNA) (rpl16 and trnS‐trnG) within and among 30 natural populations across the entire range of the species. Six mitotypes and five chlorotypes were recovered among 291 trees surveyed. Population divergence was high for mtDNA variation (G_ST = 0.738, N_ST = 0.771) indicating low levels of seed‐based gene flow and significant phylogeographical structure (N_ST > G_ST, P < 0.05). The spatial distribution of mitotypes suggests that five distinct population groups exist in the species: one in the west comprising seven populations, a second with a north–central distribution comprising 15 populations, a third with a southern and easterly distribution comprising five populations, a fourth comprising one central and one western population, and a fifth comprising a single population located in the north‐central part of the species’ range. Each group apart from the fourth group is characterized by a distinct mitotype, with other mitotypes, if present, occurring at low frequency. It is suggested, therefore, that most members of each group apart from Group 4 are derived from ancestors that occupied different isolated refugia in a previous period of range fragmentation of the species, possibly at the time of the Last Glacial Maximum. Possible locations for these refugia are suggested. A comparison of mitotype diversity between northern and southern subgroups within the north‐central group of populations (Group 2) showed much greater uniformity in the northern part of the range both within and between populations. This could indicate a northward migration of the species from a southern refugium in this region during the postglacial period, although alternative explanations cannot be ruled out. Two chlorotypes were distributed across the geographical range of the species, resulting in lower levels of among‐population chlorotype variation. The geographical pattern of variation for all five chlorotypes provided some indication of the species surviving past glaciations in more than one refugium, although differentiation was much less marked, presumably due to the greater dispersal of cpDNA via pollen.     

Comparative Restriction Enzyme Analysis of Mitochondrial DNA in Several Populations of Lake Resident Chars of the Genus Salvelinus

Restriction enzyme analysis was employed in studying the mitochondrial DNA (mtDNA) ATPase6/ND4L region in several Northeast Asian populations of resident lake char of the genus Salvelinus. On evidence of mitotypes, genetic similarity was assumed for populations of neiva (Ueginskoe Lake), lake resident char from the northern coast of the Sea of Okhotsk (Mak-Mak Lake and Elekchanskie lakes), and dolly varden. Mitotype AAAA proved to be common for these populations. Lake char populations of the Juliet and Maxi lakes (the basin of the Kolyma River) had mitotype DBAA, which is similar to mitotype DBAB observed earlier in Taranetz char from Chukotka. The divergence between mitotypes AAAA and DBAA was estimated at 0.3%. Different origins were assumed for the lake resident char populations from the basins of the Sea of Okhotsk and of the Kolyma River, the former originating in the Pacific and the latter, in the Arctic basins.     

Identification of mitochondrial genome rearrangements unique to novel cytoplasmic male sterility in radish (Raphanus sativus L.)

A novel cytoplasmic male-sterility (CMS) radish (Raphanus sativus L.) and its associated mitotype (DCGMS) were previously identified; however, no mtDNA fragments flanking the atp6 gene were found in the DCGMS mitotype. Unlike three other mitotypes in this study, a unique mtDNA organization, atp6–nad3–rps12, was found to be the major mtDNA structure associated with this mitotype. This organization may have arisen from short repeat sequence-mediated recombination events. The short repeat clusters involved in the mtDNA rearrangement around the atp6 gene also exist as repetitive sequences in the complete mitochondrial genomes of other members of the Brassicaceae family, including rapeseed and Arabidopsis. These sequences do not exist as repetitive elements in the mtDNA of tobacco, sugar beet, or rice. While studying the regions flanking atp6, we identified a truncated atp6 mtDNA fragment which consists of the 5′ part of the atp6 gene linked to an unidentified sequence. This mtDNA structure was present in all mitotypes; however, a single nucleotide insertion mutation leading to a frame-shift was identified only in the DCGMS mitotype. Although this truncated atp6 organization was transcribed, there was no significantly different expression between male-sterile and fertile segregating individuals from the BC₁F₁ population originating from a cross between male-sterile and restorer parents. Comprehensive survey of the single base-pair insertion showed that it was maternally inherited and unique to the DCGMS mitotype. Therefore, this single nucleotide polymorphism (SNP) in the coding sequence of the mtDNA will be a useful molecular marker for the detection of the DCGMS mitotype. Young-Pyo Lee and Sunggil Kim equally contributed to this work.     

Mitochondrial and chloroplast phylogeography of Picea crassifolia Kom. (Pinaceae) in the Qinghai-Tibetan Plateau and adjacent highlands

The disjunct distribution of forests in the Qinghai-Tibetan Plateau (QTP) and adjacent Helan Shan and Daqing Shan highlands provides an excellent model to examine vegetation shifts, glacial refugia and gene flow of key species in this complex landscape region in response to past climatic oscillations and human disturbance. In this study, we examined maternally inherited mitochondrial DNA (nad1 intron b/c and nad5 intron 1) and paternally inherited chloroplast DNA (trnC-trnD) sequence variation within a dominant forest species, Picea crassifolia Kom. We recovered nine mitotypes and two chlorotypes in a survey of 442 individuals from 32 populations sampled throughout the species' range. Significant mitochondrial DNA population subdivision was detected (G(ST) = 0.512; N(ST) = 0.679), suggesting low levels of recurrent gene flow through seeds among populations and significant phylogeographical structure (N(ST) > GST, P < 0.05). Plateau haplotypes differed in sequence from those in the adjacent highlands, suggesting a long period of allopatric fragmentation between the species in the two regions and the presence of independent refugia in each region during Quaternary glaciations. On the QTP platform, all but one of the disjunct populations surveyed were fixed for the same mitotype, while most populations at the plateau edge contained more than one haplotype with the mitotype that was fixed in plateau platform populations always present at high frequency. This distribution pattern suggests that present-day disjunct populations on the QTP platform experienced a common recolonization history. The same phylogeographical pattern, however, was not detected for paternally inherited chloroplast DNA haplotypes. Two chlorotypes were distributed throughout the range of the species with little geographical population differentiation (G(ST) = N(ST) = 0.093). This provides evidence for highly efficient pollen-mediated gene flow among isolated forest patches, both within and between the QTP and adjacent highland populations. A lack of isolation to pollen-mediated gene flow between forests on the QTP and adjacent highlands is surprising given that the Tengger Desert has been a geographical barrier between these two regions for approximately the last 1.8 million years.     

New evidence from mitochondrial DNA of a progenitor-derivative species relationship between black spruce and red spruce (Pinaceae)

Mitochondrial DNA (mtDNA) markers were used to assess the genetic diversity in allopatric populations of black spruce (Picea mariana [Mill.] BSP) and red spruce (P. rubens Sarg.). Patterns of mitochondrial haplotypes (mitotypes) were strikingly different between the two species. All mtDNA markers surveyed were polymorphic in black spruce, revealing four different mitotypes and high levels of mtDNA diversity (P(p) = 100%, A = 2.0, H = 0.496). In contrast, populations of red spruce had only two mitotypes and harbored low levels of ggenetic diversity (P(p) = 13.2%, A = 1.1, H = 0.120). When the southernmost allopatric populations of red spruce were considered, only one mitotype was detected. As previously reported for nuclear gene loci, the diversity observed for mtDNA in red spruce was a subset of that found in black spruce. Comparison of present and previously published data supports the hypothesis of a recent progenitor-derivative relationship between these species, red spruce presumably being derived by allopatric speciation of an isolated population of black spruce during the Pleistocene.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in mosquitoes of the <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera,Culicidae) complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquitoes C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Shaikevich, Vinogradova, Platonov, Karan, Zakharov.     

Diverging patterns of mitochondrial and nuclear DNA diversity in subarctic black spruce: imprint of a founder effect associated with postglacial colonization

High-latitude ecotonal populations at the species margins may exhibit altered patterns of genetic diversity, resulting from more or less recent founder events and from bottleneck effects in response to climate oscillations. Patterns of genetic diversity were investigated in nine populations of the conifer black spruce (Picea mariana [Mill.] BSP.) in northwestern Québec, Canada, using seed-dispersed mitochondrial (mt) DNA and nuclear (nc) DNA. mtDNA diversity (mitotypes) was assessed at three loci, and ncDNA diversity was estimated for nine expressed sequence tag polymorphism (ESTP) loci. Sampling included populations from the boreal forest and the southern and northern subzones of the subarctic forest-tundra, a fire-born ecotone. For ncDNA, populations from all three vegetation zones were highly diverse with little population differentiation (thetaN = 0.014); even the northernmost populations showed no loss of rare alleles. Patterns of mitotype diversity were strikingly different: within-population diversity and population differentiation were high for boreal forest populations [expected heterozygosity per locus (HE) = 0.58 and thetaM = 0.529], but all subarctic populations were fixed for a single mitotype (HE = 0). This lack of variation suggests a founder event caused by long-distance seed establishment during postglacial colonization, consistent with palaeoecological data. The estimated movement of seeds alone (effective number of migrants per generation, NmM < 2) was much restricted compared to that estimated from nuclear variants, which including pollen movement (NmN > 17). This could account for the conservation of a founder imprint in the mtDNA of subarctic black spruce. After reduction, presumably in the early Holocene, the diversity in ncDNA would have been replenished rapidly by pollen-mediated gene flow, and maintained subsequently through vegetative layering during the current cooler period covering the last 3000 years.     

Migration barriers protect indigenous brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) populations from introgression with stocked hatchery fish

Brown trout populations in the Belgian rivers Scheldt and Meuse have been intensively stocked in the past decades, often with material of uncertain origin. Moreover, the species habitat has become increasingly fragmented, preventing gene flow between neighboring populations. We assessed how this impacted genetic diversity and population structure by analyzing 12 wild populations (total n=309) and seven hatchery stocks (n=200) at the mitochondrial control region with SSCP and at 27 RAPD loci. Historical records indicate that brown trout from distant locations have been used to supplement hatchery stocks; nevertheless we detected non-Atlantic mitochondrial genomes in only one population of the Scheldt basin and in one hatchery. In general, the hatchery samples displayed a higher genetic diversity and differentiated less among each other (global F_ST(mtDNA)=0.311/F_ST(RAPD)=0.029) compared to the wild populations (global F_ST(mtDNA)=0.477/F_ST(RAPD)=0.204). This is due to frequent exchanges between hatcheries and regular supplementation from several indigenous populations. Gene pools present in most downstream sections from tributaries of the Meuse were similar to each other and to the hatchery samples, despite the presence of migration barriers. Assignment analyses indicated that the contribution of hatchery material to the upstream parts was limited or even completely absent in populations separated by a physical barrier. Intensive stocking and exchange between hatcheries has homogenized the downstream sections of the Meuse River, whereas the migration barriers preserved the indigenous upstream populations. As such, uncontrolled removal of barriers might result in an irreversible loss of the remnant indigenous gene pools.     

MULTIPLE ORIGINS OF GENDER-ASSOCIATED MITOCHONDRIAL DNA LINEAGES IN BIVALVES (MOLLUSCA: BIVALVIA)

Previous studies have shown that marine mussels (genus Mytilus) and a freshwater mussel (Pyganodon grandis) contain two distinct gender-associated mitotypes, which is a characteristic feature of the phenomenon of doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA). Here we present evidence for the presence of distinct male (M) and female (F) mitotypes in three other bivalve species, the mytilid Geukensia demissa, and the unionid species P. fragilis and Fusconaia flava. Nucleotide sequences of a segment of the COI gene from the M and F mitotypes from each of the three mytilid species (M. edulis, M. trossulus, G. demissa) and three unionid species (P. grandis, P. fragilis, F. flava) were used for phylogenetic analysis. The analysis suggests three independent origins of M and F mitotypes for the six species examined; one for the three unionid species, one for the two Mytilus species, and one for Geukensia. The first of these F/M divergence events, while of uncertain age, predates the divergence of the two unionid genera and is likely older than either of the two F/M divergence events in the mytilid taxa. The most parsimonious explanation of multiple F/M divergence events is that they represent independent origins of DUI. Another possibility is that, in a given taxon, an F or M mitotype assumes the role of the opposite mitotype (by virtue of a mechanism that remains to be clarified) and subsequently was fixed within its new gender. The fixation of a mtDNA lineage derived from a mitotype of switched function would reset the divergence of the gender-associated lineages to zero, thereby mimicking a de novo split of F and M lineages from a preexisting mtDNA genome that was not gender specific. Further broad-scale taxonomic studies of the occurrence of distinct M and F mitotypes may allow for the evaluation of the latter hypothesis.     

Decoupled mitochondrial and chloroplast DNA population structure reveals Holocene collapse and population isolation in a threatened Mexican-endemic conifer

Chihuahua spruce (Picea chihuahuana Martínez) is a montane subtropical conifer endemic to the Sierra Madre Occidental in northwestern México. Range-wide variation was investigated using maternally inherited mitochondrial (mtDNA) and paternally inherited chloroplast (cpDNA) DNA markers. Among the 16 mtDNA regions analysed, only two mitotypes were detected, while the study of six cpDNA microsatellite markers revealed eight different chlorotypes. The average cpDNA diversity (H = 0.415) was low but much higher than that for mtDNA (H = 0). The distribution of mitotypes revealed two clear nonoverlapping areas (G(ST) = N(ST) = 1), one including northern populations and the second one including the southern and central stands, suggesting that these two regions may represent different ancestral populations. The cpDNA markers showed lower population differentiation (G(ST) = 0.362; R(ST) = 0.230), implying that the two ancestral populations continued to exchange pollen after their initial geographic separation. A lack of a phylogeographic structure was revealed by different spatial analyses of cpDNA (G(ST) > R(ST); and samova), and reduced cpDNA gene flow was noted among populations (Nm = 0.873). Some stands deviated significantly from the mutation-drift equilibrium, suggesting recent bottlenecks. Altogether, these various trends are consistent with the hypothesis of a population collapse during the Holocene warming and suggest that most of the modern P. chihuahuana populations are now effectively isolated with their genetic diversity essentially modelled by genetic drift. The conservation efforts should focus on most southern populations and on the northern and central stands exhibiting high levels of genetic diversity. Additional mtDNA sequence analysis confirmed that P. martinezii (Patterson) is not conspecific with P. chihuahuana, and thus deserves separate conservation efforts.     

Genetic variability,community structure,and horizontal transfer of endosymbionts among three Asia II‐Bemisia tabaci mitotypes in Pakistan

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro‐environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II‐1, ‐5, and ‐7 mitotypes of the Asia II major clade. The whitefly–endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II‐1 and II‐7 mitotypes (r² = .9, p < .005), while the Asia II‐5 microbiome was less complex. The microbiome OTU groups were mitotype‐specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype‐microbiome co‐adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%–0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II‐5 and ‐7, and one occurred exclusively in Asia II‐1, two only in Asia II‐5, and one in both Asia II‐1 and ‐7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II‐1 and ‐5, and a single Hemipteriphilus OTU occurred in Asia II‐1 and ‐7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.     

Comparative restriction enzyme analysis of mitochondrial DNA in several populations of lake resident chars of the genus Salvelinus

Restriction enzyme analysis was employed in studying the mitochondrial DNA (mtDNA) ATPase6/ND4L region in several Northeast Asian populations of resident lake char of the genus Salvelinus. On evidence of mitotypes, genetic similarity was assumed for populations of neiva (Ueginskoe Lake), lake resident char from the northern coast of the Sea of Okhotsk (Mak-Mak Lake and Elekchanskie lakes), and dolly varden. Mitotype AAAA proved to be common for these populations. Lake char populations of the Juliet and Maxi lakes (the basin of the Kolyma river) had mitotype DBAA, which is similar to mitotype DBAB observed earlier in Taranetz char S. taranetzi from Chukotka. The divergence between mitotypes AAA and DBAA was estimated at 0.3%. Different origins were assumed for the lake resident char populations from the basins of the Sea of Okhotsk and of the Kolyma River, the former originating in the Pacific and the latter, in the Arctic basins.     

Intraspecific genetic polymorphism of Russian sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis>

Three populations (Azov, Caspian, and Black Sea) of Russian sturgeon Acipenser queldenstaedtii were tested for polymorphism at nuclear (RAPD and microsatellites) and mitochondrial (PCR identification of two mitotypes) markers. In addition, morphometric analysis of he representatives of Azov population was carried out. According to the morphological characters, the Black Sea population occupied an intermediate position between the Caspian and Azov populations, reflecting the phylogeography of this species. In agreement with the morphometric data, genetic distances (the data of STR analysis) also placed the Black Sea population between the Caspian and Azov populations (F _ST = 0.058 and 0.043). The genetic distance between the Azov and Caspian population was somewhat higher (F _ST = 0.070). The highest allelic polymorphism at four microsatellite loci was found observed in Caspian population, while the lowest polymorphism was in the Sea of Azov. RAPD analysis distinguished high polymorphism within the populations, although it was not feasible for interpopulation analysis. Using the method differentiating the “baerii-like” and typical “gueldenstaedtii” mitotypes, the absence of the “baerii-like” marker in the Black Sea population was demonstrated. The frequency of this marker in Caspian and Azov populations constituted 31.1 and 1.8%, respectively. Possible evolutionary reasons for the interpopulation differences observed are discussed.     

Origin of honeybees (Apis mellifera L.) from the Yucatan peninsula inferred from mitochondrial DNA analysis

Honeybees (Apis mellifera L.) sampled at sites in Europe, Africa and South America were analysed using a mitochondrial DNA restriction fragment length polymorphism (RFLP) marker. These samples were used to provide baseline information for a detailed analysis of the process of Africanization of bees from the neotropical Yucatan peninsula of Mexico. Radical changes in mitochondrial haplotype (mitotype) frequencies were found to have occurred in the 13-year period studied. Prior to the arrival of Africanized bees (1986) the original inhabitants of the Yucatan peninsula appear to have been essentially of southeastern European origin with a smaller proportion having northwestern European ancestry. Three years after the migration of Africanized bees into the area (1989), only very low levels of maternal gene flow from Africanized populations into the resident European populations had occurred. By 1998, however, there was a sizeable increase in the proportion of African mitotypes in domestic populations (61%) with feral populations having 87% of mitotypes classified as African derived. The results suggest that the early stages of Africanization did not involve a rapid replacement of European with African mitotypes and that earlier studies probably overestimated the prevalence of African mitotypes.     

Patterns of genetic variation in Pinus chiapensis,a threatened Mexican pine,detected by RAPD and mitochondrial DNA RFLP markers

Pinus chiapensis (Pinaceae) is a large conifer, endemic to central and southern Mexico and north-western Guatemala. In order to assess the extent of genetic variation within and between populations of this species, samples were obtained from throughout the natural range and analysed using random amplified polymorphic DNA (RAPD) and mtDNA RFLPs markers. Probes for the CoxI mitochondrial gene enabled two mitotypes to be observed. Populations from the eastern and western limit of the range of the species were fixed for one mitotype ('A'), whereas two populations distributed near the centre of the range were fixed for another ('B'). When the samples were screened with eight 10-mer RAPD primers, a total of 12 polymorphic bands were detected. The proportion of polymorphic bands was unusually low (24.5%) compared with other tree species. AMOVA analysis indicated that a significant proportion of the variation (P < 0.002) was distributed between populations; the extent of population differentiation detected (Phi(st) = 0.226; G(ST ) = 0.194) was exceptionally high for a pine species. Pair-wise comparison of Phi(st) values derived from AMOVA indicated that populations were significantly (P < 0.05) different from each other in virtually every case. These results are interpreted in the context of the evolutionary history of the species, and the implications for its in- and ex situ conservation are discussed.     

The fourth paradigm pattern of post-glacial range expansion of European terrestrial species: the phylogeography of the Marbled White butterfly (Satyrinae, Lepidoptera)

Aim Three paradigm patterns of post‐glacial dispersal are known for terrestrial species in Europe. However, the possibility of a fourth arises with the Italian and the Balkan lineages expanding to central Europe and the Iberian one being trapped by the Pyrenees. We test this hypothesis by analysing the molecular biogeography of the Marbled White butterfly. Location Twelve populations distributed over a major part of the European range of Melanargia galathea and M. lachesis. Methods We studied 18 allozyme loci of 403 individuals from 12 populations. Butterflies were sampled in the field, frozen in liquid nitrogen and stored under these conditions until analysis. We used cellulose acetate plates for allozyme electrophoresis. Results We detected three major genetic lineages within the M. galathea/lachesis complex. The M. lachesis sample from the southern Pyrenees was strongly genetically differentiated from M. galathea (F_CT: 0.312). Melanargia galathea splits into two major genetic lineages (F_CT: 0.115), which both were found in post‐glacially invaded regions. The further differentiation within these lineages was comparably low (F_SC: 0.028). The genetic diversity within populations was high compared with other butterfly species. Main conclusions Our findings support the existence of a fourth pattern with only the Iberian lineage not contributing considerably to the post‐glacial colonization of central Europe. Preliminary studies in other butterfly species of dry grasslands support the importance of this pattern possibly representing a fourth paradigm. The high genetic diversity within populations might be one reason for the recently observed expansions at the northern distribution limits.     

Cytoplasmic DNA variation and biogeography of Larix Mill. in Northeast Asia

Range‐wide variation in 54 populations of Dahurian larch (Larix gmelinii) and related taxa in Northeast Asia was assessed with four mitochondrial PCR‐RFLP and five chloroplast SSR markers. Eleven mitotypes and 115 chlorotypes were detected. The highest diversity was observed in the southern Russian Far East where hybrids of L. gmelinii, L. olgensis and L. kamtschatica are distributed. In contrast, only two mitotypes occurred in L. cajanderi and L. gmelinii. The Japanese larch (L. kaempferi) was found to be closely related to populations of L. kamtschatica inhabiting the Kuril Islands and South Sakhalin, populations from the northern part of Sakhalin being more closely related to continental species. In general, both mitochondrial (G_ST = 0.786; N_ST = 0.823) and chloroplast (G_ST = 0.144; R_ST = 0.432) markers showed a strong phylogeographical structure and evidence of isolation‐by‐distance. Yet both markers did not allow a clear delineation of species borders. In particular, and contrary to expectations, cpDNA was not significantly better than mtDNA at delineating species borders. This lack of concordance between morphological species and molecular markers could reflect extensive ancestral haplotype sharing and past and ongoing introgression. Finally the distribution of mtDNA and cpDNA variation suggests the presence of several refugia during Pleistocene glacial intervals. In particular, mtDNA and cpDNA reveal weak but visible differentiation between L. gmelinii and L. cajanderi, suggesting independent glacial histories of these species.