Characterization of seven (CTT)(n) microsatellite loci in the Pyrenean endemic Borderea pyrenaica (Dioscoreaceae): remarks on ploidy level and hybrid origin assessed through allozymes and microsatellite analyses

We present the identification and characterization of microsatellite loci in the Pyrenean endemic Borderea pyrenaica Miégeville (Dioscoreaceae). Seven microsatellite loci were isolated from a (CTT)(n)-enriched partial genomic library. Electropherograms patterns suggest that B. pyrenaica is a tetraploid species, as is its congener B. chouardii. One microsatellite locus was monomorphic, whereas the remaining ones presented from 2 to 10 alleles when analyzed in a sample of 60 individuals. Microsatellites have revealed higher levels of genetic variability than those in previous studies based on allozymes. Levels of genetic diversity are discussed in terms of tetrasomic (autotetraploidy) or duplicated disomic (allotetraploidy) modes of allele segregation. According to the first hypothesis, mean levels of genetic variability (H(min)-H(max)) range between 0.36 and 0.41, whereas, according to the second hypothesis, the 7 primer pairs amplified 11 chromosomal loci, and mean levels of observed and expected heterozygosities were 0.217 and 0.229, respectively, and did not differ significantly from HW expectations. These results suggest a hybrid allopolyploid origin for the Borderea taxa.     

Evidence for a Polymorphism in Gametic Segregation Using a Malate Dehydrogenase Locus in the Tetraploid Treefrog HYLA VERSICOLOR

Artificial cross combinations of tetraploid Hyla versicolor were analyzed electrophoretically using a polymorphic malate dehydrogenase locus (MDH-1) to determine the mechanism of chromosome segregation. Models for differentiating between disomic and tetrasomic inheritance are presented and tested. In some crosses progeny genotypes fit a disomic mode of segregation. In other crosses there is only evidence for a tetrasomic mode of segregation. Additional crosses produced genotypic ratios which conformed to either a disomic or tetrasomic mode of segregation. The same type of inheritance was demonstrated for any individual when used in multiple cross combinations. These results suggest that there exists in H. versicolor a polymorphism with respect to segregation of gametes, resulting from differences in chromosome pairings during meiosis I.     

Single-Locus Inheritance in the Tetraploid Treefrog Hyla versicolor with an Analysis of Expected Progeny Ratios in Tetraploid Organisms

The recently evolved autotetraploid frog, Hyla versicolor , was examined electrophoretically for evidence of genomic restructuring leading to diploidization. Loci were tested against the progeny ratios expected if inheritance was disomic vs. tetrasomic. Two loci (Mpi and Sod-2) appeared to be inherited tetrasomically, one (Mdh-2) appeared to be inherited disomically, and one (Tpi) appeared to be inherited disomically in one family and tetrasomically in another family, when tested conventionally against 1:2:1 and 1:4:1 segregation ratios. The minimum number of progeny required for this type of analysis for codominant alleles is shown to be 92. Progeny resulting from double reduction were observed, and the occurrence of a null allele class at Mpi was noted. A reexamination of expected progeny ratios in tetraploid organisms reveals that tetrasomic inheritance patterns cannot be predicted without adequate knowledge of the amount of crossing-over, the proportion of tetravalents vs. random bivalents that are formed, and the ratio and types of centromere segregation (alternate and adjacent) that occur from tetravalents in the species being studied. However, disomic inheritance can be unambiguously confirmed only by the production of all heteroallelic gametes from homobivalent, symmetrically heterozygous individuals. In addition, a method is described for estimating genecentromere distances using the ratio of progeny genotypes in certain crosses in tetraploid species.     

Isolation, characterization, inheritance and linkage of microsatellite DNA markers in white spruce (Picea glauca) and their usefulness in other spruce species

Microsatellite DNA/simple-sequence-repeat (SSR) loci were identified, isolated and characterized in white spruce (Picea glauca) by screening both a non-enriched partial genomic library and a partial genomic library enriched for (AG/TC)n-containing clones. Inheritance and linkage of polymorphic SSR loci were determined in F1 progeny of four controlled crosses. We also assessed the compatibility and usefulness of the P. glauca microsatellite DNA markers in five other Picea species. Twenty-four microsatellites were identified by sequencing 32 clones selected from screens of 5,400 clones from the two libraries. The (AG/TC)n microsatellites were the most abundant in the non-enriched library. Eight microsatellite DNA loci were of the single-copy type, and six of these were polymorphic. A total of 87 alleles were detected at the six polymorphic SSR loci in 32 P. glauca individuals drawn from several populations. The number of alleles found at these six SSR loci ranged from 2 to 22, with an average of 14.5 alleles per locus, and the observed heterozygosity ranged from 0.48 to 0.91, with a mean of 0.66 per locus. Parents of the controlled crosses were polymorphic for five of the six polymorphic SSR loci. Microsatellite DNA variants at each of these five SSR loci followed a single-locus, codominant, Mendelian inheritance pattern. Joint two-locus segregation tests indicated complete linkage between PGL13 and PGL14, and no linkage between any of the remaining SSR loci. Each of the 32 P. glauca individuals examined had unique single or two-locus genotypes. With the exception of non-amplification of PGL12 in P. sitchensis, P. mariana, and P. abies and the monomorphic nature of PGL7 in P. mariana, primer pairs for all six polymorphic SSR loci successfully amplified specific fragments from genomic DNA and resolved polymorphic microsatellites of comparable sizes in P. engelmanni, P. sitchensis, P. mariana, P. rubens, and P. abies. The closely related species P. mariana and P. rubens, and P. glauca and P. sitchensiss could be distinguished by the PGL12 SSR marker. The microsatellite DNA markers developed and reported here could be used for assisting various genetics, breeding, biotechnology, tree forensics, genome mapping, conservation, restoration, and sustainable forest management programs in spruce species.     

Non-Mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)(n) and (AG)(n) microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable.     

Development and standardization of disomic microsatellite markers for lake sturgeon genetic studies

Lake sturgeon (Acipenser fulvescens) are of conservation concern in North America. To facilitate the recovery of this fish species, an understanding of their population genetic structure is necessary to develop and implement spatially and temporally appropriate management actions. Until recently, few genetic data using nuclear loci have been collected, primarily due to the paucity of suitable genetic markers because most microsatellite loci in lake sturgeon appeared to be tetrasomic. The authors identified nine microsatellite loci (from 254 examined) that were putative polymorphic disomic loci and tested their conformance to a disomic mode of inheritance using three lake sturgeon families. The objectives of the study were to: (i) confirm the disomic status of the nine loci through inheritance testing, and (ii) standardize the genetic markers among participating laboratories. At all nine loci, disomic inheritance were confirmed, and all nine loci segregated independently in the 26 of 36 loci pairs possible to test. One of the nine loci showed non‐Mendelian segregation, possibly due to meiotic drive and/or selection. Three progeny had peak patterns inconsistent with disomy at one or more loci. The nine loci when combined with four microsatellite loci previously confirmed in other studies as disomic in lake sturgeon now yield a suite of 13 microsatellite markers. These 13 markers have been standardized among four other laboratories to facilitate building an inter‐laboratory genetic database for lake sturgeon.     

Bayesian Procedures for Discriminating among Hypotheses with Discrete Distributions: Inheritance in the Tetraploid Astilbe Biternata

Discrimination between disomic and tetrasomic inheritance aids in determining whether tetraploids originated by allotetraploidy or autotetraploidy, respectively. Past assessments of inheritance in tetraploids have used analyses whereby each inheritance hypothesis is tested independently. I present a Bayesian analysis that is appropriate for discriminating among several inheritance hypotheses and can be used in any case where hypotheses are defined by discrete distributions. The Bayesian approach incorporates prior knowledge of the probability of occurrence of disomic and tetrasomic hypotheses so that the results of the analysis are not biased by the fact that there is a single tetrasomic hypothesis and multiple disomic hypotheses. This analysis is used to interpret data from crosses in the tetraploid Astilbe biternata, a herbaceous plant native to the southern Appalachians. The progeny ratios from all crosses favored the hypothesis of disomic inheritance at both the PGM and slow-PGI loci. These results support earlier cytogenetic evidence for the allotetraploid origin of Astilbe biternata.     

Inheritance of microsatellite loci in the polyploid lake sturgeon (Acipenser fulvescens).

Inheritance in the expression of amplicons for four microsatellite primer pairs was determined using 10 families created from gametes of wild lake sturgeon (Acipenser fulvescens). Loci Afu34 and Afu68 expressed a maximum of two even-intensity bands per individual and had progeny genotype ratios that fit disomic inheritance (P > 0.05). Some variation exhibited at Afu34 and Afu68 was attributable to a null allele. Genotype expression at both loci also indicated that one female parent had transmitted unreduced gametes. Primer Afu39 amplified products that exhibited four gene doses, where genotype counts fit expected ratios for disomic inheritance (P > 0.05) indicating amplification of products from two disomic loci that share alleles. Meiotic drive was evident at the Afu39 loci based on a test for random segregation (P < 0.05). Only the expression of Afu19 gave evidence of tetrasomic inheritance based on a single progeny potentially produced by a double reduction gamete. No evidence for proposed octoploid inheritance was observed.     

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.     

Sexual reproduction, hybridization, apomixis, and polyploidization in the genus Boechera (Brassicaceae)

Of the 340 genera in the Brassicaceae, apomictic reproduction is found only in the North American genus Boechera. We investigated phylogenetic relationships, ability to hybridize, mating system, and ploidy levels of 92 lines sampled from 85 populations and representing 19 Boechera species. Phylogenetic analyses based on chloroplast DNA sequences identified three lineages in the genus. Reciprocal crosses of each line were made to a common sexual diploid B. stricta tester. The resulting F(1) progeny were analyzed for the inheritance of polymorphic microsatellite loci, genome size, and seed production. Intraspecific B. stricta crosses confirmed that this species is mostly diploid and sexual. Interspecific crosses revealed many other species were diploid and sexual and could be successfully hybridized with the tester. We also found obligate and facultative apomictic diploid and triploid lines. De novo F(1) polyploids (either triploids or tetraploids) were derived from the union of nonreduced (from an apomictic parent) and reduced (from the tester) gametes. However, seed production of these F(1) plants was generally low, suggesting a failure in the transmission of apomixis. The creation of a wide array of segregating genetic populations will facilitate future research on the evolution and inheritance of quantitative variation in Boechera.     

Isolation, characterization, and inheritance of microsatellite loci in alpine larch and western larch.

Microsatellite loci or simple sequence repeat loci (SSRs) were isolated in alpine larch (Larix lyallii Parl.) and western larch (Larix occidentalis Nutt.). In total, 14 SSR loci were characterized; two [(TCT)4, A7] came from published Larix DNA sequence data, one (CA)17 was obtained from a partial non-enriched alpine larch total genomic DNA library, and the remaining 11 loci were obtained from larch genomic DNAs enriched for (CA)n repeats. The SSR regions in these clones could be divided into three categories: perfect repeat sequences without interruption, imperfect repeat sequences with interruption(s), and compound repeat sequences with adjacent tandem simple dinucleotides. Eight of the 14 loci analyzed were found to be polymorphic and useful markers after silver-staining polyacrylamide gel electrophoresis. In addition, several SSR primers developed for alpine larch were able to successfully amplify polymorphic loci in its related species, western larch, and among other closely related taxa within the Larix genus. The inheritance of microsatellite loci was verified by analysis of haploid megagametophyte and diploid embryo tissues of progeny obtained from controlled crosses between western larch and alpine larch. All microsatellite loci analyzed had alleles that segregated according to expected Mendelian frequencies. Two species-specific markers (UAKLly10a and UAKLla1) allow easy and rapid identification of specific genetic entry of alpine larch and western larch at any stage in the sporophyte phase of the life cycle. Therefore, these markers are efficient in identifying the parental species and to validate controlled crosses between these two closely related species. These results are important in tree improvement programs of alpine larch and western larch aimed at producing genetically improved hybrid stock for reforestation in Western Canada and U.S.A.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis

Artificial tetraploid somatic hybrids have been developed for sterile triploid citrus breeding by sexual hybridization between diploid and tetraploid somatic hybrids. The genetic structure of diploid gametes produced by tetraploid genotypes depends on the mode of chromosome association at meiosis. In order to evaluate tetraploid inheritance in a tetraploid interspecific somatic hybrid between mandarin and lemon, we performed segregation studies using cytogenetic and single sequence repeat molecular markers. Cytogenetic analysis of meiosis in the somatic hybrid revealed 11% tetravalents and 76% bivalents. Inheritance of the tetraploid hybrid was analyzed by genotyping the triploid progeny derived from a cross between a diploid pummelo and the tetraploid somatic hybrid, in order to derive genotypes of the meiospores produced by the tetraploid. A likelihood-based approach was used to distinguish between disomic, tetrasomic, and intermediate inheritance models and to estimate the double reduction rate. In agreement with expectations based the cytogenetic data, marker segregation was largely compatible with tetrasomic and inheritance intermediate between disomic and tetrasomic, with some evidence for preferential pairing of homoeologous chromosomes. This has important implications for the design of breeding programs that involve tetraploid hybrids, and underscores the need to consider inheritance models that are intermediate between disomic and tetrasomic.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Enzyme variation and inheritance in Glechoma hederacea (Lamiaceae), a diploidized tetraploid

The chromosome number of the polyploid species Glechoma hederacea was found to be 2n = 36 in a sample of 93 ramets derived from 27 sites in N and C Europe. Variation in 10 enzymes was surveyed in material from S Sweden and S Czech Republic. The genetic control of variation was investigated using segregating progeny from crosses and self-fertilized heterozygous plants. The genetic analysis comprised 30 of 32 putative alleles detected in the geographical survey. Five loci (Aat-2, Tpi-1, Tpi-2, Pgd-2 and Mnr) behaved as isoloci with one copy of a locus being monomorphic for a common allele, the other di-allelic for a common allele and a variant allele. In four isoloci (Pgd-1, Pgi-2, Mdh-2 and Adh), both copies of the duplicated locus were polymorphic, with one allele common to both copies and with another allele unique for each copy except for Pgd-1 where both copies were tri-allelic. Three loci, Pgm-3, Skd-1 and Skd-2 were regarded as being non-duplicated. Segregation ratios for all enzyme loci were in close agreement with expectations based on disomic inheritance. Our data suggest that the tetraploid G. hederaca is a diploidized autotetraploid.     

Microsatellite DNA somaclonal variation in micropropagated trembling aspen (Populus tremuloides)

Microsatellite DNA markers of ten simple sequence repeat (SSR) loci were used to examine somaclonal variation in randomly selected micropropagated plantlets derived from three different Populus tremuloides donor trees (genotypes). The plantlets were obtained from tissue cultures of dormant vegetative buds, and those derived from the same donor tree, grown in the greenhouse, did not exhibit any sign of visible morphological variation. No microsatellite DNA variation was observed among 13 somaclones of one tree and 4 somaclones of another tree at eight of the ten SSR loci. However, despite the small number of micropropagated progeny per tree sampled, microsatellite DNA variation was detected among the plantlets derived from the same donor trees at two SSR loci. The primer pair for the SSR locus PTR5 revealed somaclonal variation in 1 out of the 13 plantlets obtained from one genotype, while the primer pair for the PTR2 SSR locus revealed somaclonal variation in one out of the four plantlets obtained from another genotype. The variation at the PTR2 locus resulted in the appearance of a new allele of increased size, possibly due to an addition of the repeat units, while the variation at the PTR5 locus resulted in the appearance of third allele, presumably due to the presence of a single extra chromosome or duplication of a chromosomal segment. These results demonstrate that the genetic fidelity of micropropagated plants of P. tremuloides cannot always be assured and somaclonal variation can occur even when tissues of well organized vegetative buds are used for tissue cultures; that somaclonal variation cannot always be detected at the gross morphological level; and that microsatellite DNA markers provide useful and sensitive markers for determining the clonal fidelity and somaclonal variation in P. tremuloides.     

Stable maintenance of duplicated chromosomes carrying the mutant pwB gene in Paramecium tetraurelia

An allele of the behavioural mutant pawn-B96 has been reported as a typical recessive gene but was found to show a peculiar inheritance. When the F2 progeny from crosses between the wild-type and pwB96 were obtained by autogamy, the 1:1 phenotypic segregation ratio was observed as expected. However, two-thirds of the wild-type progeny in the F2 were thought to be heterozygotes because they became mixed progeny of wild-type and pawn clones in successive autogamies. Four marker genes showed the expected segregation ratio and stable phenotypes in these crossings. This result and the results of crossings using segregants from the above crosses indicated that parental pwB96 is a tetrasomy of the chromosome carrying the pwB gene. To determine the cause of chromosomal duplication in the mutant, the stability of the chromosome carrying the pwB locus was examined by genetic analyses. The disomy of both pwB and wild-type and the tetrasomy of pwB showed genotypes that were relatively stable during several autogamous generations. However, in clones initially pure for the tetrasomy of wild-type, disomic cells appeared within a few autogamous generations. The difference between the stabilities of the tetrasomy of pwB96 and that of the wild-type might be due partly to differences between the growth rate of tetrasomy and disomy in pwB96 and the wild-type, but mostly the result of an unknown contribution of the chromosome carrying the pwB96 allele to the tetrasomic composition.     

New Variation Identified by SSR in a Wheat Variety Derived from Synthetic Hexaploid Wheat ( Triticum durum× Aegilops tauschii)

Hybridization and polyploidization are important ways for wheat to evolve and to genetically differentiate. Ninety two simple sequence repeat (SSR) molecular markers, which distributed in A, B, and D genomes , were used to perform genetic comparison between Chuan-W5436 (CW5436), a new wheat variety, and its parents, synthetic hexaploid wheat Syn786 (♀ ) and common wheat Mianyang 26 (My26) (♂). The results indicated that alleles were not genetically transmitted from parents (Syn786 (♀) crossed (My26) (♂) ) to the progeny CW5436 as Mendelian proportions . A new variation on a SSR molecular marker loci with novel additive bands was observed in CW5436 but not found in its parents. It suggested that artificial selective stress was an important factor to promote the frequency of significant deviations of the expected allele, resulting in microsatellite sequences of the progeny changed . The affect of the genetic differentiation of SSR molecular marker loci that occurred in wheat crosses and gene transfer on the genetic evolu1tion of wheat was discussed.     

Gene mutations in rye causing embryo lethality in hybrids with wheat: allelism and chromosomal localisation

In crosses between hexaploid wheat and inbred lines of rye, a small number of rye genotypes produce seeds carrying undifferentiated, non-viable embryos. Hybrids between such lines and those not showing this phenotype were used as pollen donors in crosses with bread wheat in order to determine the genetic basis of disturbed embryo development. A single gene, designated Eml-R1b, is causing this character. Molecular markers associated with F₂ genotypes derived from a contrasting rye inbred progeny were used for a linkage study. Recombinant inbred lines of an F₅ population served as testers. Eml-R1b maps to chromosome arm 6RL, along with two co-segregating microsatellite loci, Xgwm1103 and Xgwm732. Complementary interactions of deleterious genes in wheat and rye are discussed.     

Nunatak survival vs. tabula rasa in the Central Pyrenees: a study on the endemic plant species Borderea pyrenaica (Dioscoreaceae)

Aim  Borderea pyrenaica (Dioscoreaceae) is a Tertiary relict plant endemic to the Central Pyrenees. Because of its narrow distribution in a small geographical area and the fact that it is restricted to high alpine habitats, it constitutes an ideal model species for inferring the historical dynamics of population survival and migration during and after Quaternary glaciations in the Pyrenees.
Location  Central Pyrenees and pre-Pyrenees, Spain–France.
Methods  Eleven primer pairs were used to amplify 18 microsatellite loci in this allotetraploid species in a sample of 804 individuals from 15 populations, revealing a total of 77 alleles. Genotypic data of individuals and populations were analysed using clustering and Bayesian methods of analysis of population structure.
Results  A higher number of private alleles and a significantly higher allelic richness ( A *) were found in the southern area (21, A * = 2.295) than in the northern area (5, A * = 1.791). Furthermore, the allelic composition of the northern area represented a subset of that from the southern area.
Main conclusions  The hypothesis of in situ survival in northern Pyrenean nunataks was rejected, while peripheral refugia were considered to be restricted to the southern Pyrenees and pre-Pyrenees, where historical geographical fragmentation probably caused the divergence among southern Pyrenean populations. Molecular evidence indicates that these refugial populations probably colonized the northern area after sheet-ice retreat. Borderea pyrenaica lineages followed two migratory pathways in their northward colonization, suggesting several founder events for the populations that eventually reached the territory of the Gavarnie cirque.