Genetic differentiation of common shrew Sorex araneus populations among different alpine valleys revealed by microsatellites

Geographical barriers may affect the genetic structure of populations by reducing gene exchanges among them. In Switzerland, the common shrew Sorer araneus Linnaeus, 1758 is mostly confined to mountainous areas because of a competing sister species, Millet's shrew S. coronatus Millet, 1828, which occupies most of the Swiss lowlands. The structure of common shrew populations found in different alpine valleys may therefore be affected by the topography. Using microsatellites, genetic structuring of seven shrew populations is investigated among four different valleys of, the Swiss Alps. Using the exact G-test, significant genetic structuring is detected between several valleys. Isolation by distance does not fully explain our results. It appears that high mountain ridges (> 2400 m) can significantly reduce gene flow. F- and R-statistics are estimated and compared to the exact G-tests results. Mantel tests show that F-ST, unlike R-ST, is significantly correlated with differentiation. F-ST remains however low even at high differentiation levels, while R-ST has a high variance. We discuss how these results may have wider implications with regards the interpretation of microsatellite data. Finally, a new microsatellite locus, L99, appears to discriminate S. araneus of the Vaud and Cordon races from both S. araneus Valais and S. coronatus.

Microsatellites can be misleading: an empirical and simulation study.

It has been long recognized that highly polymorphic genetic markers can lead to underestimation of divergence between populations when migration is low. Microsatellite loci, which are characterized by extremely high mutation rates, are particularly likely to be affected. Here, we report genetic differentiation estimates in a contact zone between two chromosome races of the common shrew (Sorex araneus), based on 10 autosomal microsatellites, a newly developed Y-chromosome microsatellite, and mitochondrial DNA. These results are compared to previous data on proteins and karyotypes. Estimates of genetic differentiation based on F- and R-statistics are much lower for autosomal microsatellites than for all other genetic markers. We show by simulations that this discrepancy stems mainly from the high mutation rate of microsatellite markers for F-statistics and from deviations from a single-step mutation model for R-statistics. The sex-linked genetic markers show that all gene exchange between races is mediated by females. The absence of male-mediated gene flow most likely results from male hybrid sterility.

DNA degradation in avian faecal samples and feasibility of non-invasive genetic studies applied to threatened capercaillie populations

We evaluated the feasibility of using faeces as a non-invasively collected DNA source for the genetic study of an endangered bird population (capercaillie; Tetrao urogallus). We used a multitube approach, and for our panel of 11 microsatellites genotyping reliability was estimated at 98% with five repetitions. Experiments showed that free DNases in faecal material were the major cause of DNA degradation. Our results demonstrate that using avian faeces as a source of DNA, reliable microsatellite genotyping can be obtained with a reasonable number of PCR replicates.

Hierarchical analyses of genetic differentiation in a hybrid zone of Sorex araneus (Insectivora : Soricidae)

Microsatellites are used to unravel the fine-scale genetic structure of a hybrid zone between chromosome races Valais and Cordon of the common shrew (Sorex araneus) located in the French Alps. A total of 269 individuals collected between 1992 and 1995 was typed for seven microsatellite loci. A modified version of the classical multiple correspondence analysis is carried out. This analysis clearly shows the dichotomy between the two races. Several approaches are used to study genetic structuring. Gene flow is clearly reduced between these chromosome races and is estimated at one migrant every two generations using X-statistics and one migrant per generation using F-statistics. Hierarchical F- and R-statistics are compared and their efficiency to detect inter- and intraracial patterns of divergence is discussed. Within-race genetic structuring is significant, but remains weak. F-ST displays similar values on both sides of the hybrid zone, although no environmental barriers are found on the Cordon side, whereas the Valais side is divided by several mountain rivers. We introduce the exact G-test to microsatellite data which proved to be a powerful test to detect genetic differentiation within as well as among races. The genetic background of karyotypic hybrids was compared with the genetic background of pure parental forms using a CRT-MCA. Our results indicate that, without knowledge of the karyotypes, we would not have been able to distinguish these hybrids from karyotypically pure samples.

Microsatellites reveal high population viscosity and limited dispersal in the ant Formica paralugubris

We used microsatellites to study the fine-scale genetic structure of a highly polygynous and largely uni-colonial population of the ant Formica paralugubris. Genetic data indicate that long-distance gene flow between established nests is limited and new queens are primarily recruited from within their natal nest. Most matings occur between nestmates and are random at this level. In the center of the study area, budding and permanent connections between nests result in strong population viscosity, with close nests being more similar generically than distant nests. In contrast, nests located outside of this supercolony show no isolation by distance, suggesting that they have been initiated by queens that participated in mating flights rather than by budding from nearby nests in our sample population. Recruitment of nestmates as new reproductive individuals and population viscosity in the supercolony increase genetic differentiation between nests. This in turn inflates relatedness estimates among worker nestmates (r = 0.17) above what is due to close pedigree links. Local spatial genetic differentiation may favor the maintenance of altruism when workers raise queens that will disperse on foot and compete with less related queens from neighboring nests or disperse on the wing and compete with unrelated queens.

Breeding system and genetic variance in the monogamous, semi-social shrew, Crocidura russula

The population-genetic consequences of monogamy and male philopatry (a rare breeding system in mammals) were investigated using microsatellite markers in the semisocial and anthropophilic shrew Crocidura russula. A hierarchical sampling design over a 16-km geographical transect revealed a large genetic diversity (h = 0.813) with significant differentiation among subpopulations (F-ST = 5-6%), which suggests an exchange of 4.4 migrants per generation. Demic effective-size estimates were very high, due both to this limited gene inflow and to the inner structure of subpopulations. These were made of 13-20 smaller units (breeding groups), comprising an estimate of four breeding pairs each. Members of the same breeding groups displayed significant coancestries (F-LS = 9-10%), which was essentially due to strong male kinship: syntopic males were on average related at the half-sib level. Female dispersal among breeding groups was not complete (similar to 39%), and insufficient to prevent inbreeding. From our results, the breeding strategy of C. russula seems less efficient than classical mammalian systems (polygyny and male dispersal) in disentangling coancestry from inbreeding, but more so in retaining genetic variance.

A genetic isolation gradient of populations of the Balearic green toad (Bufo balearicus) follows rising eastward fragmentation of the rural landscapes on the island of Menorca.

We present the first approach to the genetic diversity and structure of the Balearic toad (Bufo balearicus Boettger, 1880) for the island of Menorca. Forty-one individ- uals from 21 localities were analyzed for ten microsatellite loci. We used geo-refer- enced individual multilocus genotypes and a model-based clustering method for the inference of the number of populations and of the spatial location of genetic dis- continuities between those populations.¦Only six of the microsatellites analyzed were polymorphic. We revealed a northwest- ern area inhabited by a single population with several well-connected localities and another set of populations in the southeast that includes a few unconnected small units with genetically significant differences among them as well as with the individ- uals from the northwest of the island. The observed fragmentation may be explained by shifts from agricultural to tourism practices that have been taking place on the island of Menorca since the 1960s. The abandonment of rural activities in favor of urbanization and concomitant service areas has mostly affected the southeast of the island and is currently threatening the overall geographic connectivity between the different farming areas of the island that are inhabited by the Balearic toad.

Chromosomal rearrangement and genetic structure in the shrews of the Sorex araneus group

ABSTRACT The role of chromosomal rearrangements in the speciation process is much debated and many theoretical models have been developed. The shrews of the Sorex araneus group offer extraordinary opportunities to study the relationship between chromosomal variation and speciation. Indeed, this group of morphologically very similar species received a great deal of attention due to its karyotypic variability, which is mainly attributed to Robertsonian fusions. To explore the impact of karyotypic changes on genetic differentiation, we first studied the relationship between genetic and karyotypic structure among Alpine species and among chromosome races of the S. araneus group using Bayesian admixture analyses. The results of these analyses confirmed the taxonomic status of the studied species even though introgression can still be detected between species. Moreover, the strong spatial sub-structure highlighted the role of historical factors (e.g. geographical isolation) on genetic structure. Next, we studied gene flow at the chromosome level to address the question of the impact of chromosomal rearrangements on genetic differentiation. We used flow sorted chromosomes from three different karyotypic taxa of the S. araneus group to map microsatellite markers at the chromosóme arm level. We have been able to map 24 markers and to show that the karyotypic organisation of these taxa is well conserved, which suggests that these markers can be used for further inter-taxa studies. A general prediction of chromosomal speciation models is that genetic differentiation between two taxa should be larger across rearranged chromosomes than across chromosomes common to both taxa. We combined two approaches using mapped microsatellites to test this prediction. First, we studied the genetic differentiation among five shrew taxa placed at different evolutionary levels (i.e. within and among species). In this large scale study, we detected an overall significant difference in genetic structure between rearranged vs. common chromosomes. Moreover, this effect varied among pairwise comparisons, which allowed us to differentiate the role of the karyotypic complexity of hybrids and of the evolutionary divergence between taxa. Secondly, we compared the levels of gene flow measured across common vs. rearranged chromosomes in two karyotypically different hybrid zones (strong vs. low complexity of hybrids), which show similar levels of genetic structure. We detected a significantly stronger genetic structure across rearranged chromosomes in the hybrid zone showing the highest level of hybrid complexity. The large variance observed among loci suggested that other factors, such as the position of markers within the chromosome, also certainly affects genetic structure. In conclusion, our results strongly support the role of chromosomal rearrangements in the reproductive barrier and suggest their importance in speciation process of the S. araneus group. RESUME Le rôle des réarrangements chromosomiques dans les processus de spéciation est fortement débattu et de nombreux modèles théoriques ont été développés sur le sujet. Les musaraignes du groupe Sorex araneus présentent de nombreuses opportunités pour étudier les relations entre les variations chromosomiques et la spéciation. En effet, ce groupe d'espèces morphologiquement très proches a attiré l'attention des chercheurs en raison de sa variabilité caryotypique principalement attribuée à des fusions Robertsoniennes. Pour explorer l'impact des changements caryotypiques sur la différenciation génétique, nous avons tout d'abord étudié les relations entre la structure génétique et caryotypique de races chromosomiques et d'espèces alpine du groupe S. araneus en utilisant des analyses Bayesiennes d' « admixture ». Les résultats de ces analyses ont confirmé le statut taxonomique des espèces étudiées bien que nous ayons détecté de l'introgression entre espèces. L'observation d'une sous structure spatiale relativement forte souligne l'importance des facteurs historiques (telle que l'isolation géographique) sur la structure génétique de ce groupe. Ensuite, nous avons étudié le flux de gène au niveau des chromosomes pour aborder de manière directe la question de l'impact des réarrangements chromosomiques sur la différenciation génétique. En conséquence, nous avons utilisé des tris de chromosomes de trois taxons du groupe S. araneus pour localiser des marqueurs microsatellites au niveau du bras chromosomique. Au cours de cette étude, nous avons pu localiser 24 marqueurs et montrer une forte conservation dans l'organisation du caryotype de ces taxa. Ce résultat suggère que leur utilisation est appropriée pour des études entre taxa. Une prédiction générale à tous les modèles de spéciation chromosomique correspond à la plus grande différenciation génétique des chromosomes réarrangés que des chromosomes communs. Nous avons combiné deux approches utilisant des microsatellites localisés au niveau du bras chromosomique pour tester cette prédiction. Premièrement, nous avons étudié la différenciation génétique entre cinq taxa du groupe S. araneus se trouvant à des niveaux évolutifs différents (i.e. à l'intérieur et entre espèce). Au cours de cette étude, nous avons détecté une différenciation globale significativement plus élevée sur les chromosomes réarrangés. Cet effet varie entre les comparaisons, ce qui nous a permis de souligner le rôle de la complexité caryotypique des hybrides et du niveau de divergence évolutive entre taxa. Deuxièmement, nous avons comparé le flux de gènes des chromosomes communs et réarrangés dans deux zones d'hybridation caryotypiquement différentes (forte vs. Faible complexité des hybrides) mais présentant un niveau de différenciation génétique similaire. Ceci nous a permis de détecter une structure génétique significativement plus élevée sur les chromosomes réarrangés au centre de la zone d'hybridation présentant la plus grande complexité caryotypic. La forte variance observée entre loci souligne en outre le fait que d'autres facteurs, tel que la position du marqueur sur le chromosome, affectent probablement aussi la structure génétique mesurée. En conclusion, nos résultats supportent fortement le rôle des réarrangements chromosomiques dans la barrière reproductive entre espèces ainsi que leur importance dans les processus de spéciation des musaraignes du groupe S. araneus.

Are invasive marsh frogs (Pelophylax ridibundus) replacing the native P. lessonae /P. esculentus hybridogenetic complex in Western Europe? Genetic evidence from a field study.

The water-frog L-E system, widespread in Western Europe, comprises the pool frog Pelophylax lessonae and the hybridogenetic edible frog P. esculentus, which originated from hybridization between pool frogs and marsh frogs (P. ridibundus). In P. esculentus, the lessonae (L) genome is eliminated during meiosis and has to be gained anew each generation from a P. lessonae partner, while the ridibundus (R') genome is transmitted clonally. It therefore accumulates deleterious mutations, so that R'R' offspring from P. esculentus x P. esculentus crosses are normally unviable. This system is now threatened by invasive P. ridibundus (RR) imported from Eastern Europe and the Balkans. We investigated the genetic interactions between invasive marsh frogs and native water frogs in a Swiss wetland area, and used genetic data collected in the field to validate several components of a recently postulated mechanism of species replacement. We identified neo-ridibundus individuals derived from crosses between invasive ridibundus and native esculentus, as well as newly arisen hybridogenetic esculentus lineages stemming from crosses between invasive ridibundus (RR) and native lessonae (LL). As their ridibundus genomes are likely to carry less deleterious mutations, such lineages are expected to produce viable ridibundus offspring, contributing to species replacement. However, such crosses with invasive ridibundus only occurred at a limited scale; moreover, RR x LL crosses did not induce any introgression from the ridibundus to the lessonae genome. We did not find any ridibundus stemming from crosses between ancient esculentus lineages. Despite several decades of presence on the site, introduced ridibundus individuals only represent 15% of sampled frogs, and their spatial distribution seems shaped by specific ecological requirements rather than history of colonization. We therefore expect the three taxa to coexist stably in this area.

Cross-amplification of polymorphic microsatellites reveals extra-pair paternity and brood parasitism in Sturnus vulgaris

We tested the cross-amplification of 37 microsatellites in a population of starlings (Stumus vulgaris). Twenty-three of them amplified and five exhibited a large number of alleles per locus and high heterozygosity (on average: 14.6 alleles/locus and H. = 0.704). We assessed the occurrence of extra-pair paternity (EPP) and intraspecific brood parasitism GBP) in this population. The EPP rate was 16% to 18% offspring from 43% to 45% of nests. IBP was very variable between two successive years (14% to 27% chicks from 25% to 64% of clutches). These five polymorphic markers will be of potential use in studies of genetic diversity, population structure and reproductive strategy of this species.

Do riverine barriers, history or introgression shape the genetic structuring of a common shrew (Sorex araneus) population?

The common shrew (Sorex araneus) is subdivided into numerous chromosome races. The Valais and Cordon chromosome races meet and hybridize at a mountain river in Les Houches (French Alps). Significant genetic structuring was recently reported among populations found on the Valais side of this hybrid zone. In this paper, a phylogenetic analysis and partial Mantel tests are used to investigate the patterns and causes of this structuring. A total of 185 shrews were trapped at 12 localities. All individuals were typed for nine microsatellite loci. Although several mountain rivers are found in the study area, riverine barriers do not have a significant influence on gene flow. Partial Mantel tests show that our result is caused by the influence of the hybrid zone with the Cordon race. The geographical patterns of this structuring are discussed in the context of the contact zone, which appears to extend up to a group of two rivers. The glacier they originate from is known to have cut the Arve valley as recently as 1818. The recent history of this glacier, its moraine and possibly rivers, may therefore be linked to the history of this hybrid zone.

Clinal variation in a brown lemur (Eulemur spp.) hybrid zone: Combining morphological, genetic and climatic data to examine stability.

Studies of hybrid zones can inform our understanding of reproductive isolation and speciation. Two species of brown lemur (Eulemur rufifrons and E. cinereiceps) form an apparently stable hybrid zone in the Andringitra region of south-eastern Madagascar. The aim of this study was to identify factors that contribute to this stability. We sampled animals at 11 sites along a 90-km transect through the hybrid zone and examined variation in 26 microsatellites, the D-loop region of mitochondrial DNA, six pelage and nine morphological traits; we also included samples collected in more distant allopatric sites. Clines in these traits were noncoincident, and there was no increase in either inbreeding coefficients or linkage disequilibrium at the centre of the zone. These results could suggest that the hybrid zone is maintained by weak selection against hybrids, conforming to either the tension zone or geographical selection-gradient model. However, a closer examination of clines in pelage and microsatellites indicates that these clines are not sigmoid or stepped in shape but instead plateau at their centre. Sites within the hybrid zone also occur in a distinct habitat, characterized by greater seasonality in precipitation and lower seasonality in temperature. Together, these findings suggest that the hybrid zone may follow the bounded superiority model, with exogenous selection favouring hybrids within the transitional zone. These findings are noteworthy, as examples supporting the bounded superiority model are rare and may indicate a process of ecologically driven speciation without geographical isolation.

Scale-specific sex-biased dispersal in the Valais shrew unveiled by genetic variation on the Y chromosome, autosomes, and mitochondrial DNA.

We investigated sex specificities in the evolutionary processes shaping Y chromosome, autosomes, and mitochondrial DNA patterns of genetic structure in the Valais shrew (Sorex antinorii), a mountain dwelling species with a hierarchical distribution. Both hierarchical analyses of variance and isolation-by-distance analyses revealed patterns of population structure that were not consistent across maternal, paternal, and biparentally inherited markers. Differentiation on a Y microsatellite was lower than expected from the comparison with autosomal microsatellites and mtDNA, and it was mostly due to genetic variance among populations within valleys, whereas the opposite was observed on other markers. In addition, there was no pattern of isolation by distance for the Y, whereas there was strong isolation by distance on mtDNA and autosomes. We use a hierarchical island model of coancestry dynamics to discuss the relative roles of the microevolutionary forces that may induce such patterns. We conclude that sex-biased dispersal is the most important driver of the observed genetic structure, but with an intriguing twist: it seems that dispersal is strongly male biased at large spatial scale, whereas it is mildly biased in favor of females at local scale. These results add to recent reports of scale-specific sex-biased dispersal patterns, and emphasize the usefulness of the Y chromosome in conjunction with mtDNA and autosomes to infer sex specificities.