Jacques Hausser

MICROSATELLITES CAN BE MISLEADING: AN EMPIRICAL AND SIMULATION STUDY

Abstract. 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‐statististics 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.

Is the Gibraltar Strait a barrier to gene flow for the bat Myotis myotis (Chiroptera: Vespertilionidae)?

Because of their role in limiting gene flow, geographical barriers like mountains or seas often coincide with intraspecific genetic discontinuities. Although the Strait of Gibraltar represents such a potential barrier for both plants and animals, few studies have been conducted on its impact on gene flow. Here we test this effect on a bat species (Myotis myotis) which is apparently distributed on both sides of the strait. Six colonies of 20 Myotis myotis each were sampled in southern Spain and northern Morocco along a linear transect of 1350 km. Results based on six nuclear microsatellite loci reveal no significant population structure within regions, but a complete isolation between bats sampled on each side of the strait. Variability at 600 bp of a mitochondrial gene (cytochrome b) confirms the existence of two genetically distinct and perfectly segregating clades, which diverged several million years ago. Despite the narrowness of the Gibraltar Strait (14 km), these molecular data suggest that neither males, nor females from either region have ever reproduced on the opposite side of the strait. Comparisons of molecular divergence with bats from a closely related species (M. blythii) suggest that the North African clade is possibly a distinct taxon warranting full species rank. We provisionally refer to it as Myotis cf punicus Felten 1977, but a definitive systematic understanding of the whole Mouse‐eared bat species complex awaits further genetic sampling, especially in the Eastern Mediterranean areas.

Trophic Resource Partitioning and Competition between the Two Sibling Bat Species Myotis myotis and Myotis blythii

1. Niche theory predicts that the stable coexistence of species within a guild should be associated, if resources are limited, with a mechanism of resource partitioning. Using extensive data on diets, the present study attempts: (i) to test the hypothesis that, in sympatry. the interspecific overlap between the trophic niches of the sibling bat species Myotis myotis and M. biythii-which coexist intimately in their roosts-is effectively lower than the two intraspecific overlaps; (ii) to assess the role played by interspecific competition in resource partitioning through the study of trophic niche displacement between several sympatric and allopatric populations. 2. Diets were determined by the analysis of faecal samples collected in the field from individual bats captured in various geographical areas. Trophic niche overlaps were calculated monthly for all possible intraspecific and interspecific pairs of individuals from sympatric populations. Niche breadth was estimated from: (i) every faecal sample; (ii) all the faecal samples collected per month in a given population (geographical area). 3. In every population, the bulk of the diets of M. myotis and M. blythii consisted of, respectively, terrestrial (e.g. carabid beetles) and grass-dwelling (mostly bush crickets) prey. All intraspecific trophic niche overlaps were significantly greater than the interspecific one, except in Switzerland in May when both species exploited mass concentrations of cockchafers, a non-limiting food source. This clearcut partitioning of resources may allow the stable, intimate coexistence observed under sympatric conditions. 4. Relative proportions of ground- and grass-dwelling prey, as well as niche breadths (either individual or population), did not differ significantly between sympatry and allopatry, showing that, under allopatric conditions, niche expansion does not take place. This suggests that active interspecific competition is not the underlying mechanism responsible for the niche partitioning which is currently observed between M. myotis and M. blythii.

Chromosomal versus mitochondrial DNA evolution: tracking the evolutionary history of the southwestern european populations of the Sorex araneus group (Mammalia Insectivora)

The shrews of the Sorex araneus group have undergone a spectacular chromosome evolution. The karyotype of Sorex granarius is generally considered ancestral to those of Sorex coronatus and S. araneus. However, a sequence of 777 base pairs of the cytochrome b gene of the mitochondrial DNA (mtDNA) produces a quite different picture: S. granarius is closely related to the populations of S. araneus from the Pyrenees and from the northwestern Alps, whereas S. coronatus and S. araneus from Italy and the southern Alps represent two well‐separated lineages. It is suggested that mtDNA and chromosomal evolution are in this case largely independant processes. Whereas mtDNA haplotypes are closely linked to the geographical history of the populations, chromosomal mutations were probably transmitted from one population to another. Available data suggest that the impressive chromosome polymorphism of this group is quite a recent phenomenon.

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 R‐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. FST 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.

A taxonomical re-evaluation of the Valais chromosome race of the common shrew Sorex araneus [Insectivora: Soricidae]

The common shrewSorex araneus Linnaeus, 1758 is subject to intense chromosomal polymorphism. About 65 chromosome races are presently known. One of these chromosome races (the Valais race) is karyologically, morphologically, biochemically, and genetically clearly distinct from all other chromosome races of the species. Recent studies of hybrid zones between the Valais race and other chromosome races in the Swiss and French Alps add further strong evidence for the specific taxonomic status of the Valais race. Chromosomes and diagnostic protein markers reveal sharp frequency clines and strong heterozygote deficits. In one hybrid zone, the maintenance of the strong genetic differentiation of the hybridizing taxa was confirmed by a study with autosomal microsatellites indicating minimal gene flow. A microsatellite marker on the Y-chromosome showed complete absence of male mediated gene flow suggesting hybrid male sterility. To clarify the taxonomic status of this taxon, additional analyses were conducted. A morphometric analysis of the mandible indicated the Valais race is morphologically as distinct from neighbouring chromosome races ofS. araneus as from other relatedSorex species. In a phylogeny based on complete mitochondrial DNA cytochromeb gene sequences, the Valais race clearly appears as the sister taxon to all other races ofS. araneus. Therefore, the chromosome race Valais ofS. araneus herein is elevated to specific status and the nameSorex antinorii Bonaparte, 1840 is applied.

Phylogeographical structure, postglacial recolonization and barriers to gene flow in the distinctive Valais chromosome race of the common shrew (Sorex araneus)

Using one male‐inherited and eight biparentally inherited microsatellite markers, we investigate the population genetic structure of the Valais chromosome race of the common shrew (Sorex araneus) in the Central Alps of Europe. Unexpectedly, the Y‐chromosome microsatellite suggests nearly complete absence of male gene flow among populations from the St‐Bernard and Simplon regions (Switzerland). Autosomal markers also show significant genetic structuring among these two geographical areas. Isolation by distance is significant and possible barriers to gene flow exist in the study area. Two different approaches are used to better understand the geographical patterns and the causes of this structuring. Using a principal component analysis for which testing procedure exists, and partial Mantel tests, we show that the St‐Bernard pass does not represent a significant barrier to gene flow although it culminates at 2469 m, close to the highest altitudinal record for this species. Similar results are found for the Simplon pass, indicating that both passes represented potential postglacial recolonization routes into Switzerland from Italian refugia after the last Pleistocene glaciations. In contrast with the weak effect of these mountain passes, the Rhône valley lowlands significantly reduce gene flow in this species. Natural obstacles (the large Rhône river) and unsuitable habitats (dry slopes) are both present in the valley. Moreover, anthropogenic changes to landscape structures are likely to have strongly reduced available habitats for this shrew in the lowlands, thereby promoting genetic differentiation of populations found on opposite sides of the Rhône valley.

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.

Isolation and characterization of simple sequence repeats in the genome of the common shrew.

the most remarkable degrees of chromosomal variability in mammals (Zima et al. in press). The geographical distribution of these races, their complex pattern of genetic and karyotypic differentiation and the existence of several hybrid zones has attracted much attention over the past 10 years (review: Searle 1993). Consequently, the common shrew has become a model species to study the role of chromosomes in the process of population differentiation and speciation. More recently it has become the subject of gene mapping projects (Pack et al. 1995; Matyakhina 1996). At present, using a set of shrew–rodent somatic cell hybrid clones, 17 genes have been mapped (Pack et al. 1995; Matyakhina 1996). However, a major obstacle of using the common shrew in genetic studies is the current lack of sufficient polymorphic markers in the nuclear genome. An attractive source of markers which show abundant polymorphism is provided by simple sequence repeats (SSRs), also called microsatellites. SSRs are tandemly repeated DNA sequences which have proved to be both abundant and highly polymorphic in mammalian genomes (Hamada et al. 1982; Weber 1990; Stallings et al. 1991). Besides their importance in human genetic diseases, such as neurological disorders and cancer (Hamada et al. 1982; Weber 1990; Stallings et al. 1991; Wooster et al. 1994; Sutherland & Richards 1995), and their usefulness in genome mapping (Hamada et al. 1982; Weber 1990; Stallings et al. 1991; Weissenbach et al. 1992; Wooster et al. 1994), there is a great potential for the use of SSRs in population genetic studies (Bruford & Wayne 1993). In this paper we show that the genome of the common shrew contains abundant SSRs. We characterize eight polymorphic loci for use in population genetic studies in Sorex and investigate the level of conservation and polymorphism of these loci in other members of the order Insectivora. A partial genomic library was constructed with genomic DNA purified from common shrew spleen. The protocol as described by Rassmann et al. (1991) was followed with the following modifications: DNA fragments between 300 and 600 bp were cloned in a pBluescript II KS+ vector (Stratagene). The library contained 2000 recombinant clones. Seventy (3.5%) gave a positive hybridization signal after screening with a mixture of (TC)10–(TG)10 oligonucleotide probe labelled with the DIG system (Boehringer) and in subsequent Southern blotting assays. Inserts of 25 clones were sequenced from which 20 clones contained a perfect (AC)n repeat (two compound with a trinucleotide), two clones an imperfect (AC)n repeat and three clones a perfect (AG)n repeat following the nomenclature of Weber (1990). The same procedure was applied to screen 1500 clones with a (CCT)8oligonucleotide probe. Eight positive clones were found, from which three were sequenced and confirmed to be (GGA)n repeats. Primers flanking each repeat were designed for 15 loci using the computer program PRIMER 1.0 (Lincoln et al. 1993). Seven SSR-loci were monomorphic or showed unclear banding patterns, whereas eight SSR-loci gave satisfactory amplification results and, for the most part, showed extensive polymorphism (3–37 different alleles) (Table 1). These data are based on samples from four chromosomal races of Switzerland and France. Within chromosomal races, loci exhibited between three and 15 alleles and only locus 9 had 28 alleles (data not shown). When pooling all data, the allele frequency distributions at the eight loci were irregular showing bimodal or multimodal distributions. In order to test whether the loci were in Hardy– Weinberg equilibrium and if they were independent, we performed exact tests using the G E N E P O P package version 1.2 (Raymond & Rousset 1995). The samples used for this analysis were collected in 1994 in one valley (5 km stretch) of the French Alps and were partitioned into 15 subsets (n = 5–15). There was no departure from panmixia locally, but a classical F-statistic analysis using F S T A T 1.2 (Goudet 1995) indicated some degree of population subdivision (FST = 0.02, P < 0.001) and thus overall a slight departure from Hardy–Weinberg equilibrium. Exact tests for P R I M E R N O T E

Interbreedings between karyotypic Alpine races of the common shrew Sorex araneus (Insectivora, Mammalia)

SUMMARYControlled interbreedings were performed between distinct chromosomal races or forms of Sorex araneus coming from populations of Swiss and French Alps. Four mating pairs including homozygous individuals of the Vaud race, the Valais race, the Intermediate Vaud-Acrocentric form and the Acrocentric form have led to several heterozygous hybrid litters. The karyotypes of the parents were determined from cultures of cartilaginous cells. The karyotypes of the offsprings were determined with a classical method. The production of hybrids between different races suggests the absence of postmating barrier between the Vaud race and the Intermediate form, the Acrocentric form and the Intermediate form, the Acrocentric form and the Valais race.