Molecules and morphology in concert. II. The Festuca brachyphylla complex (Poaceae) in Svalbard

We used a combined molecular and morphological approach to unravel variation in the autogamous Festuca brachyphylla polyploid complex in the arctic archipelago of Svalbard. Forty populations were analyzed for random amplified polymorphic DNAs (RAPDs) and 46 morphological characters. Eighteen RAPD multilocus phenotypes were observed in the 86 plants analyzed, based on 30 polymorphic markers. Multivariate analyses of the RAPD data revealed four distinct groups of multilocus phenotypes; in contrast, the variation was more or less continuous in multivariate analyses of the morphological data. However, we identified several individual morphological characters that unambiguously discriminated among the four groups of RAPD multilocus phenotypes. Analysis of type material suggests that the four groups in Svalbard can be referred to Festuca baffinensis, F. brachyphylla, F. hyperborea, and F. edlundiae. This study shows that concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.     

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Salpha rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa-IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Salpha rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Salpha rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Salpha rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Salpha rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.     

Incongruent phylogeographies in spite of similar morphology, ecology, and distribution:Phippsia algida andP. concinna (Poaceae) in the North Atlantic region

Phylogeographic and taxonomic relationships among 54 North Atlantic populations of the snowbed grass genusPhippsia were investigated based on isozymes and genetically based morphological variation. The results support recognition of two distinct species,P. algida andP. concinna, the latter with at least two subspecies. Both of these self-fertilizing arctic pioneer species were genetic allotetraploids almost without intrapopulational variation. The two species showed strikingly different phylogeographies in the North Atlantic region in spite of their similarity in morphology, habitat ecology, mating system, and dispersal ecology, and in spite of their present cooccurrence in many geographic areas, sometimes even in the same snowbeds. The same electrophoretic multilocus phenotype was observed in all populations ofP. algida, and although this species showed considerable morphological variation, the variation was unstructured geographically. Thus,P. algida showed a pattern similar to other arctic species investigated in the North Atlantic region; it has probably dispersed postglacially across the sea barriers among Greenland, Svalbard, Iceland, and Scandinavia. In contrast,P. concinna was virtually fixed for different multilocus phenotypes in the three main geographic areas analyzed (S Norway, Svalbard, Greenland), corresponding to fairly distinct divergence in morphology. This pattern suggests absence of postglacial among-area dispersal ofP. concinna in spite of all of its similarities with its congener; it may have immigrated to the North Atlantic from different source areas and/or survived the last glaciationin situ.     

EVOLUTIONARY AND ECOLOGICAL DIFFERENTIATION IN THE PANTROPICAL TO WARM-TEMPERATE SEAWEED DIGENEA SIMPLEX (RHODOPHYTA)1

Genetic differentiation among geographic isolates of the pantropical to warm-temperate red alga Digenea simplex (Wulfen) C. Agardh was investigated using random amplified polymorphic DNA (RAPD) markers, crossing studies, and temperature tolerances experiments. Eleven isolates representing populations from the Caribbean, eastern Atlantic, and Indo-West Pacific were compared. RAPD analysis clearly revealed an Indo-West Pacific group, a Caribbean/Cape Verde Islands group, and a Canary Islands group. Crossing studies showed different levels of inter fertility. In most crosses between Western Australian and Atlantic isolates, no hybrid tetrasporophytes were formed. In crosses between Caribbean and Cape Verde Islands isolates, tetrasporophytes developed, but the viability of tetraspores was reduced. Full sexual compatibility was observed among Cape Verde Islands isolates and among isolates from Bonaire. Temperature tolerance studies indicate that Pacific isolates have a broader temperature survival range than Atlantic isolates, which may be correlated to local temperature extremes. Despite the reduced level of sexual compatibility between Caribbean and Cape Verde Islands isolates, their shared position in the RAPD analysis and similar temperature responses suggest trans-Atlantic dispersal in the near geological past. In addition to their discrete position in the RAPD distance analysis, the Canary Islands isolates were significantly more cold-tolerant than the other Atlantic isolates. This finding is consistent with the hypothesis that the Canary Islands were recolonized from cold-adapted eastern Mediterranean populations after the last Pleistocene glaciation.     

New available SCAR markers: potentially useful in distinguishing a commercial strain of the superior type from other strains of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in China

At present, more than 100 strains of Lentinula edodes are cultivated on a commercial scale in China. A simple, reliable, and effective method to distinguish some commercial strains of the superior type from other commercial strains is very important for the Lentinula industry. In this study, 23 commercial strains of L. edodes cultivated widely in China at present were collected and analyzed with randomly amplified polymorphic DNA (RAPD) technique. Three informative dominant sequence characterized amplified region (SCAR) markers were developed by designing three pairs of specific SCAR primers from three sequenced differential RAPD bands, respectively. Based on the three SCAR markers, three different multiplex polymerase chain reaction (PCR) phenotypes were detected among the 23 studied commercial strains and in which a multilocus phenotype characterizing a commercial strain Cr02 of the superior type could potentially be used to distinguish this strain from the other 22 studied commercial strains. To our knowledge, this study is the first to describe the development of a multiplex PCR technique based on SCAR markers for detecting the molecular phenotypes among commercial strains of L. edodes in China.     

Glacial survival does not matter – II: RAPD phylogeography of Nordic Saxifraga cespitosa

It has been suggested that many arctic-alpine plant species have limited dispersal ability and cannot have arrived in Scandinavia and the arctic archipelago of Svalbard by long-distance dispersal after a total glaciation. It has therefore been proposed that such species must have survived the entire glaciation(s) in ice-free refugia in southern Norway, northern Norway and Svalbard. We investigated random amplified polymorphic DNA (RAPD) variation among 28 populations from Norway and Svalbard of one of these arctic-alpine 'short-distance dispersers', the selfing polyploid Saxifraga cespitosa . In an analysis of molecular variance ( AMOVA ), more variation was found among populations within the three postulated refugia regions (45%) than among these regions (25%). Spatial autocorrelation (Mantel) analyses showed that the genetic distance monotonously increased with increasing geographical distance. In UPGMA and PCO analyses, the populations from Norway and Svalbard formed a south–north cline that continued across the Barents Sea barrier. The results suggest that there has been recent dispersal among the three postulated refugia regions and thus that postglacial dispersal into these refugia regions from other distant areas also must represent a possibility. The observed geographical pattern of the genetic variation may have been established after expansion from different source areas outside the North European ice sheet and/or from different refugia areas 'within' the ice sheet, but it is probably not possible to distinguish among these alternatives. The results for S. cespitosa are consistent with a dynamic late- and postglacial scenario with extensive plant dispersal, and support the conclusion from our previous study of the outbreeding Saxifraga oppositifolia ; the hypothesis of glacial survival in Norway and/or Svalbard is superfluous.     

The extreme disjunction between Beringia and Europe in Ranunculus glacialis s. l. (Ranunculaceae) does not coincide with the deepest genetic split – a story of the importance of temperate mountain ranges in arctic–alpine phylogeography

The arctic–alpine Ranunculus glacialis s. l. is distributed in high‐mountain ranges of temperate Europe and in the North, where it displays an extreme disjunction between the North Atlantic Arctic and Beringia. Based on comprehensive sampling and employing plastid and nuclear marker systems, we (i) test whether the European/Beringian disjunction correlates with the main evolutionary diversification, (ii) reconstruct the phylogeographic history in the Arctic and in temperate mountains and (iii) assess the susceptibility of arctic and mountain populations to climate change. Both data sets revealed several well‐defined lineages, mostly with a coherent geographic distribution. The deepest evolutionary split did not coincide with the European/Beringian disjunction but occurred within the Alps. The Beringian lineage and North Atlantic Arctic populations, which reached their current distribution via rapid postglacial colonization, show connections to two divergent pools of Central European populations. Thus, immigration into the Arctic probably occurred at least twice. The presence of a rare cpDNA lineage related to Beringia in the Carpathians supports the role of these mountains as a stepping stone between temperate Europe and the non‐European Arctic, and as an important area of high‐mountain biodiversity. The temperate and arctic ranges presented contrasting phylogeographic histories: a largely static distribution in the former and rapid latitudinal spread in the latter. The persistence of ancient lineages with a strictly regional distribution suggests that the ability of R. glacialis to survive repeated climatic changes within southern mountain ranges is greater than what recently was predicted for alpine plants from climatic envelope modelling.     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.     

Unravelling the evolutionary history and future prospects of endemic species restricted to former glacial refugia

The contemporary distribution and genetic composition of biodiversity bear a signature of species’ evolutionary histories and the effects of past climatic oscillations. For many European species, the Mediterranean peninsulas of Iberia, Italy and the Balkans acted as glacial refugia and the source of range recolonization, and as a result, they contain disproportionately high levels of diversity. As these areas are particularly threatened by future climate change, it is important to understand how past climatic changes affected their biodiversity. We use an integrated approach, combining markers with different evolutionary rates and combining phylogenetic analysis with approximate Bayesian computation and species distribution modelling across temporal scales. We relate phylogeographic processes to patterns of genetic variation in Myotis escalerai, a bat species endemic to the Iberian Peninsula. We found a distinct population structure at the mitochondrial level with a strong geographic signature, indicating lineage divergence into separate glacial refugia within the Iberian refugium. However, microsatellite markers suggest higher levels of gene flow resulting in more limited structure at recent time frames. The evolutionary history of M. escalerai was shaped by the effects of climatic oscillations and changes in forest cover and composition, while its future is threatened by climatically induced range contractions and the role of ecological barriers due to competition interactions in restricting its distribution. This study warns that Mediterranean peninsulas, which provided refuge for European biodiversity during past glaciation events, may become a trap for limited dispersal and ecologically limited endemic species under future climate change, resulting in loss of entire lineages.     

Evolution and polyploid origins in North American Arctic Puccinellia (Poaceae) based on nuclear ribosomal spacer and chloroplast DNA sequences

The proportion of polyploid plant species increases at higher latitudes, and it has been suggested that original postglacial Arctic immigrants of some large groups, including grasses, were polyploid. We analyzed noncoding nuclear and chloroplast DNA of all North American diploid Puccinellia (Poaceae) and a subset of arctic polyploids to hypothesize evolutionary relationships among diploids and to evaluate the parentage of polyploids. Diploids formed three lineages: one uniting arctic species P. arctica and P. banksiensis; a second comprising arctic species P. tenella, P. alaskana, P. vahliana, and P. wrightii; and a third uniting the two temperate species P. lemmonii and P. parishii. The arctic species P. angustata (hexaploid) and P. andersonii (primarily octoploid) apparently derive from the P. arctica-P. banksiensis lineage based on ITS and chloroplast sequences, and share an ancestor with arctic triploid/tetraploid P. phryganodes based on nrDNA sequences. Sequence comparisons also suggest tetraploid P. bruggemannii evolved from two arctic lineages: P. vahliana-P. wrightii and P. arctica-P. banksiensis. These patterns and the predominance of arctic rather than temperate diploid species support the idea that diploid Puccinellia recolonized the Arctic from northern glacial refugia like Beringia, and also formed stabilized polyploid hybrids during these refugial events or subsequently during postglacial colonization.     

Invasion history of <Emphasis Type="Italic">Cardamine hirsuta</Emphasis> in Japan inferred from genetic analyses of herbarium specimens and current populations

Multiple introductions of a species are thought to enhance its invasion success by increasing genotypic diversity; this involves frequent crossing among different lineages. However, genetic diversity through crossing is less likely in autogamous species. To understand the impact of multiple introductions on the colonization success of autogamous species, we studied hairy bittercress, Cardamine hirsuta, which invaded Japan several decades ago. We detected temporal changes in its population structure using nine microsatellite markers amplified from leaf samples collected from 87 sites between 2009 and 2010, and herbarium specimens collected between 1988 and 2007. To examine whether the phenotypic variation corresponded with the genetic population structure, we also investigated the geographic variation in the lateral stamen number of this species across 49 sites. The present populations can be divided into three genetic groups, which are distributed in northern, eastern, and western Japan. This finding suggests that there are three invasive lineages (North, East, and West) in Japan. The geographic variation in lateral stamen number corresponded to the distributions of these lineages. The former distributions of the North and West lineages mostly corresponded to those found at present, but they were also historically found in eastern Japan. However, the East lineage has apparently expanded into eastern Japan, resulting in a change in dominant lineages over only a few decades. For the autogamous C. hirsuta, multiple introductions contributed toward colonization success over a wider range, which was associated with a local change in the dominant lineages.     

Patterns of genetic variation detected by RAPDs suggest a single origin with subsequent mutations and long-distance dispersal in the apomictic fern Dryopteris remota (Dryopteridaceae)

Debates on speciation processes in pteridophytes have revived. In order to study the evolutionary origin of an apomictic fern species, we investigated the genetic variation in the strictly agamosporous Dryopteris remota. We determined the genotypes of 22 individuals from many different locations within the species' European distribution and of 20 individuals from a Swiss population. A previous study on isozyme variation showed no intraspecific genetic variation in a similar sample set (Schneller and Holderegger, 1994, American Fern Journal 84: 94-98). In contrast to this, four out of 12 random amplified polymorphic DNA (RAPD) primers tested revealed low genetic diversity among individuals of D. remota from different locations. Intrapopulational genetic variation was also very low, but in the single population studied, a unique multiband genotype could be detected. The geographic distribution of genetic variation found in D. remota was best explained by the assumption of a single origin, the accumulation of somatic mutations during spread, and occasional, but effective, events of dispersal over large distances. The present study thus stresses the importance of long-distance dispersal in evolutionary processes and biogeography of ferns.     

Discordant temporal and geographic patterns in maternal lineages of eastern North American frogs, Rana catesbeiana (Ranidae) and Pseudacris crucifer (Hylidae)

Whether the Pleistocene has had a disproportionate impact on the recent diversification of temperate species, or played a lesser role in a more protracted process, has been a prominent evolutionary debate for the past decade. We used cytochrome b sequences to reconstruct the evolutionary histories of two widely co-distributed, and ecologically divergent frogs (Rana catesbeiana and Pseudacris crucifer) to examine the role of the Pleistocene in structuring these species. Results for R. catesbeiana reflect a pattern of allopatric fragmentation, likely in Coastal Plain refugia on either side of the Mississippi River dating to the mid to early Pleistocene. In contrast, P. crucifer contains numerous divergent lineages, including one west of the Mississippi River in the Interior Highlands, and in the east, multiple lineages that likely expanded from a number of southern Appalachian refugia with lineage sundering originating in the late Pliocene. Large-scale phylogeographic comparisons between these and other eastern North American species reflect both congruent and independent patterns of diversification, possibly reflecting the relative importance of dispersal ability and habitat associations. Although intra-lineage diversification has been structured by repeated Pleistocene glaciations, lineage sundering likely dates at least to the Pliocene in most (but not all) northern temperate amphibian and reptile species studied to date.     

Speciation below ground: Tempo and mode of diversification in a radiation of endogean ground beetles

Dispersal is a critical factor determining the spatial scale of speciation, which is constrained by the ecological characteristics and distribution of a species’ habitat and the intrinsic traits of species. Endogean taxa are strongly affected by the unique qualities of the below‐ground environment and its effect on dispersal, and contrasting reports indicate either high dispersal capabilities favoured by small body size and mediated by passive mechanisms, or low dispersal due to restricted movement and confinement inside the soil. We studied a species‐rich endogean ground beetle lineage, Typhlocharina, including three genera and more than 60 species, as a model for the evolutionary biology of dispersal and speciation in the deep soil . A time‐calibrated molecular phylogeny generated from >400 individuals was used to delimit candidate species, to study the accumulation of lineages through space and time by species–area–age relationships and to determine the geographical structure of the diversification using the relationship between phylogenetic and geographic distances across the phylogeny. Our results indicated a small spatial scale of speciation in Typhlocharina and low dispersal capacity combined with sporadic long distance, presumably passive dispersal events that fuelled the speciation process. Analysis of lineage growth within Typhlocharina revealed a richness plateau correlated with the range of distribution of lineages, suggesting a long‐term species richness equilibrium mediated by density dependence through limits of habitat availability. The interplay of area‐ and age‐dependent processes ruling the lineage diversification in Typhlocharina may serve as a general model for the evolution of high species diversity in endogean mesofauna.     

Molecules and morphology in concert: tests of some hypotheses in arctic Potentilla (Rosaceae)

We developed a combined molecular and morphological approach to unravel complex variation at low taxonomic levels, exemplified by some arctic members of Potentilla. Twenty-one populations from Svalbard were analyzed for random amplified polymorphic DNAs (RAPDs) and 64 morphological characters to test the hypotheses that (1) the P. nivea complex (section Niveae) consists of three taxa (P. chamissonis, P. insularis, and P. nivea), (2) three "eco-morphotypes" in P. pulchella (section Multifidae) should be considered different taxa, and (3) P. insularis originated as an intersectional hybrid (Niveae × Multifidae). Twenty-two RAPD multilocus phenotypes were observed in the 136 plants analyzed based on 35 markers. Three fairly distinct groups of RAPD phenotypes were identified in the P. nivea complex based on multivariate analyses and an analysis of molecular variance (AMOVA; 77.6% among-group variation). The variation within the P. nivea complex was more or less continuous in multivariate analyses of the morphological data. We identified, however, several individual morphological characters that separated unambiguously among the three groups of RAPD phenotypes, revealing that these groups correspond to the previously hypothesized taxa. Many identical RAPD multilocus phenotypes were observed in the "eco-morphotypes" of P. pulchella, suggesting that its conspicuous morphological variation is caused by plasticity or by genetic variation at a small number of loci. The hypothesis of the hybrid origin of P. insularis was not supported by the RAPD data. Overall, very little RAPD variation was observed within populations of the four taxa (2.1-16.7% in AMOVA analyses; average genotypic diversity, D, was 0.10-0.30). We conclude that detailed, concerted analysis of molecules and morphology is a powerful tool in low-level taxonomy.     

Use of RAPD technique in evolution studies of four species in the family Canidae

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.     

Lineage origin and expansion of a Neotropical migrant songbird after recent glaciation events

Birds of the Northern Hemisphere often harbour the genetic signature of postglaciation expansion but analyses identifying the location of refugia and the directionality of expansions are rare. Here we explore the evolutionary history of yellow warbler lineages, focusing on how these lineages recolonized their current range. We genotyped samples from 696 yellow warblers via direct sequencing of a 333-bp control region I mitochondrial DNA fragment or lineage-specific genotyping. Phylogenetic analysis revealed two monophyletic clades: a highly migratory group including previously identified eastern and western lineages and a less migratory group including a lineage consisting of tropical residents and a new 'southern' lineage localized in southwest United States. We then modelled the expansion of the eastern and western lineages, identified the location of potential refugia and assessed the importance of migration as a historical factor promoting gene flow. The expansion of the eastern lineage proceeded from a main refugia in the eastern United States, with possible contribution of an additional local refugia. In the western lineage, the expansion proceeded from a single refugia possibly located in western United States. Because two lineages overlapped to varying degrees in central North America, we suggest that the Canadian Prairies offered a bridge of riparian habitats where the lineages met after glacier retreat, while the US Central Great Plains acted as a barrier that limited secondary contact. Finally, gene flow was more important along the north-south axis of migration than away from it, suggesting spring migration played a role in the dispersal of lineages.     

Phylogeographic structure,demographic history and morph composition in a colour polymorphic lizard

In polymorphic species, population divergence in morph composition and frequency has the potential to promote speciation. We assessed the relationship between geographic variation in male throat colour polymorphism and phylogeographic structure in the tawny dragon lizard, Ctenophorus decresii. We identified four genetically distinct lineages, corresponding to two polymorphic lineages in the Northern Flinders Ranges and Southern Flinders Ranges/Olary Ranges regions respectively, and a monomorphic lineage in the Mt Lofty Ranges/Kangaroo Island region. The degree of divergence between these three lineages was consistent with isolation to multiple refugia during Pleistocene glacial cycles, whereas a fourth, deeply divergent (at the interspecific level) and monomorphic lineage was restricted to western New South Wales. The same four morphs occurred in both polymorphic lineages, although populations exhibited considerable variation in the frequency of morphs. By contrast, male throat coloration in the monomorphic lineages differed from each other and from the polymorphic lineages. Our results suggest that colour polymorphism has evolved once in the C. decresii species complex, with subsequent loss of polymorphism in the Mt Lofty Ranges/Kangaroo Island lineage. However, an equally parsimonious scenario, that polymorphism arose independently twice within C. decresii, could not be ruled out. We also detected evidence of a narrow contact zone with limited genotypic admixture between the polymorphic Olary Ranges and monomorphic Mt Lofty Ranges regions, yet no individuals of intermediate colour phenotype. Such genetic divergence and evidence for barriers to gene flow between lineages suggest incipient speciation between populations that differ in morph composition.     

Mitochondrial phylogeography of the moor frog, Rana arvalis

The moor frog Rana arvalis is a lowland species with a broad Eurasiatic distribution, from arctic tundra through forest to the steppe zone. Its present-day range suggests that glacial refugia of this frog were located outside southern European peninsulas. We studied the species-wide phylogeographical pattern using sequence variation in a 682 base pairs fragment of mtDNA cytochrome b gene; 223 individuals from 73 localities were analysed. Two main clades, A and B, differing by c. 3.6% sequence divergence were detected. The A clade is further subdivided into two subclades, AI and AII differing by 1.0%. All three lineages are present in the Carpathian Basin (CB), whereas the rest of the species range, including huge expanses of Eurasian lowlands, are inhabited solely by the AI lineage. We infer that AII and B lineages survived several glacial cycles in the CB but did not expand, at least in the present interglacial, to the north. The geographical distribution and genealogical relationships between haplotypes from the AI lineage indicate that this group had two glacial refugia, one located in the eastern part of the CB and the other probably in southern Russia. Populations from both refugia contributed to the colonization of the western part of the range, whereas the eastern part was colonized from the eastern refugium only. The effective population size as evidenced by theta(ML) is an order of magnitude higher in the AI lineage than in the AII and B lineages. Demographic expansion was detected in all three lineages.     

Glacial survival may matter after all: nunatak signatures in the rare European populations of two west-arctic species

Biogeographers claimed for more than a century that arctic plants survived glaciations in ice-free refugia within the limits of the North European ice sheets. Molecular studies have, however, provided overwhelming support for postglacial immigration into northern Europe, even from the west across the Atlantic. For the first time we can here present molecular evidence strongly favouring in situ glacial persistence of two species, the rare arctic-alpine pioneer species Sagina caespitosa and Arenaria humifusa. Both belong to the 'west-arctic element' of amphi-Atlantic disjuncts, having their few and only European occurrences well within the limits of the last glaciation. Sequencing of non-coding regions of chloroplast DNA revealed only limited variation. However, two very distinct and partly diverse genetic groups, one East and one West Atlantic, were detected in each species based on amplified fragment length polymorphisms (AFLPs), excluding postglacial dispersal from North America as explanation for their European occurrences. Patterns of genetic diversity and distinctiveness indicate that glacial populations existed in East Greenland and/or Svalbard (A. humifusa) and in southern Scandinavia (S. caespitosa). Despite their presumed lack of long-distance dispersal adaptations, intermixed populations in several regions indicate postglacial contact zones. Both species are declining in Nordic countries, probably due to climate change-induced habitat loss. Little or no current connectivity between their highly fragmented and partly distinct populations call for conservation of several populations in each geographic region.