Inga Zeisset

Amphibian phylogeography: a model for understanding historical aspects of species distributions

Phylogeographic analysis has become a major tool for investigating historical aspects of biogeography and population genetic structure. Anuran amphibians are particularly informative subjects for phylogeographic research on account of their global distribution, high degree of population genetic structure and ease of sampling. Studies on all the worlds inhabited continents have demonstrated the nature and locations of refugia, including the Gulf Coast in North America and the Mediterranean peninsulas in Europe during the Pleistocene glaciations; the importance of vicariance events such as the uplift of the Andes in shaping modern distributions; and colonization routes in temperate zones during postglacial climatic amelioration. Features identified as important to amphibian biogeography, notably mountain ranges, large rivers such as the Amazon and climatic fluctuations, are common to many other taxa. New analytical methods based on coalescent, Bayesian and likelihood approaches permit more rigorous hypothesis testing than has hitherto been possible and offer the prospect of even more detailed insights into species and population history in future work.

Population genetics of a successful invader: the marsh frog Rana ridibunda in Britain.

We investigated the genetic outcome of successful invasion by an alien species, the marsh frog Rana ridibunda, in Britain. Twelve adults translocated from Hungary into Kent (Romney) in 1935 resulted rapidly in a large localized population. A further successful translocation in 1973 from Romney to Sussex (Lewes), together with other range extensions, provided an opportunity to test bottleneck effects during colonization events. Romney and Lewes frogs had similar genetic diversities to those in Hungary at 14 random amplified polymorphic DNA marker (RAPD) and five microsatellite loci. The introduced populations were, however, differentiated genetically from each other and from a reference population in Hungary. Fitness assessments (larval growth and survival) revealed no differences between the Lewes and Romney populations. Despite starting with few founders, significant bottleneck effects on R. ridibunda in Britain were therefore undetectable, presumably because population expansions were rapid immediately after the translocations.

Determination of biogeographical range: an application of molecular phylogeography to the European pool frog Rana lessonae

Understanding how species are constrained within their biogeographical ranges is a central problem in evolutionary ecology. Essential prerequisites for addressing this question include accurate determinations of range borders and of the genetic structures of component populations. Human translocation of organisms to sites outside their natural range is one factor that increasingly complicates this issue. In areas not far beyond presumed natural range margins it can be particularly difficult to determine whether a species is native or has been introduced. The pool frog (Rana lessonae) in Britain is a specific example of this dilemma.Weused variation at six polymorphic microsatellite loci for investigating the phylogeography of R. lessonae and establishing the affinities of specimens from British populations. The existence and distribution of a distinct northern clade of this species in Norway, Sweden and England infer that it is probably a long-standing native of Britain, which should therefore be included within its natural range. This conclusion was further supported by posterior probability estimates using Bayesian clustering.The phylogeographical analysis revealed unexpected patterns of genetic differentiation across the range of R. lessonae that highlighted the importance of historical colonization events in range structuring.

Population structure of a large blue butterfly and its specialist parasitoid in a fragmented landscape

Habitat fragmentation may interrupt trophic interactions if herbivores and their specific parasitoids respond differently to decreasing connectivity of populations. Theoretical models predict that species at higher trophic levels are more negatively affected by isolation than lower trophic level species. By combining ecological data with genetic information from microsatellite markers we tested this hypothesis on the butterfly Maculinea nausithous and its specialist hymenopteran parasitoid Neotypus melanocephalus. We assessed the susceptibility of both species to habitat fragmentation by measuring population density, rate of parasitism, overall genetic differentiation (θST) and allelic richness in a large metapopulation. We also simulated the dynamics of genetic differentiation among local populations to asses the relative effects of migration rate, population size, and haplodiploid (parasitoid) and diploid (host) inheritance on metapopulation persistence. We show that parasitism by N. melanocephalus is less frequent at larger distances to the nearest neighbouring population of M. nausithous hosts, but that host density itself is not affected by isolation. Allelic richness was independent of isolation, but the mean genetic differentiation among local parasitoid populations increased with the distance between these populations. Overall, genetic differentiation in the parasitoid wasp was much greater than in the butterfly host and our simulations indicate that this difference is due to a combination of haplodiploidy and small local population sizes. Our results thus support the hypothesis that Neotypus parasitoid wasps are more sensitive to habitat fragmentation than their Maculinea butterfly hosts.

Molecular characterization of major histocompatibility complex class II alleles in the common frog, Rana temporaria

Major histocompatibility complex (MHC) class II genes, which play a major role in the immune system response, are some of the most polymorphic genes in vertebrates. We developed polymerase chain reaction primers for part of the second exon of an expressed MHC class II gene in the common frog, Rana temporaria. We genotyped this locus in five frog populations in southeast England and detected eight alleles in 215 individuals. Five or six alleles were detected in each population with a maximum of two alleles per individual, indicating that only a single locus was amplified. We also inferred the possible existence of a null allele. There were 23 variable nucleotide sites (out of 136) and 13 variable amino acid sites (out of 44), many of which corresponded to amino acids involved in antigen recognition. We detected a significant excess of nonsynonymous substitutions at antigen binding sites, indicating that this gene is under positive selection. The level of variation we found was similar to that in other amphibian MHC class II loci, such as those in Bombina bombina, Xenopus laevis and Ambystoma tigrinum.

Larval fitness and immunogenetic diversity in chytrid-infected and uninfected natterjack toad (Bufo calamita) populations

Chytrid fungus Batrachochytrium dendrobatidis (Bd), an emerging disease, has been decimating amphibian populations around the world for several decades. We quantified aspects of larval fitness, adaptive (major histocompatibility complex) diversity and neutral (microsatellite) diversity in natterjack toad (Bufo calamita) populations in two regions of north-west England. Toads in region one had no evidence of chytrid infection, whereas in region two there was a substantial prevalence of Bd. Larval fitness (growth rate, time to metamorphosis and survival) of B. calamita did not differ between the regions. Genetic diversity at microsatellite loci was much higher in the infected than in the uninfected region, but the converse was true of MHC diversity indicating that genetic drift was unlikely to explain the differences in MHC between the regions. Furthermore, MHC allele frequencies varied significantly between Bd-infected and uninfected populations. Microsatellite diversity was not a robust indicator of larval fitness in these toad populations while MHC genotype frequencies varied in a way that was consistent with directional selection in response to pathogen prevalence. The acquired immune defences may therefore play an important role in determining the susceptibility of amphibian species to chytridiomycosis.

Drift rather than selection dominates MHC class II allelic diversity patterns at the biogeographical range scale in natterjack toads Bufo calamita

Study of major histocompatibility complex (MHC) loci has gained great popularity in recent years, partly due to their function in protecting vertebrates from infections. This is of particular interest in amphibians on account of major threats many species face from emergent diseases such as chytridiomycosis. In this study we compare levels of diversity in an expressed MHC class II locus with neutral genetic diversity at microsatellite loci in natterjack toad (Bufo (Epidalea) calamita) populations across the whole of the species’ biogeographical range. Variation at both classes of loci was high in the glacial refugium areas (REF) and much lower in postglacial expansion areas (PGE), especially in range edge populations. Although there was clear evidence that the MHC locus was influenced by positive selection in the past, congruence with the neutral markers suggested that historical demographic events were the main force shaping MHC variation in the PGE area. Both neutral and adaptive genetic variation declined with distance from glacial refugia. Nevertheless, there were also some indications from differential isolation by distance and allele abundance patterns that weak effects of selection have been superimposed on the main drift effect in the PGE zone.

Larval fitness, microsatellite diversity and MHC class II diversity in common frog (Rana temporaria) populations

There is increasing interest in using functional rather than neutral loci to assess the genetic health of wild populations. We compared growth and survival of urban and rural frog larvae under three temperature regimes, to vary stress levels, and measured genetic diversities at nine microsatellites and at one major histocompatibility complex (MHC) class II locus. We found no significant differences between urban and rural frogs in larval survivorship, nor in microsatellite and MHC diversities. However, mean larval growth rates were significantly higher (by 4.5–18.9% according to temperature) in urban compared with rural populations. Microsatellite and MHC diversities were uncorrelated, though pairwise inter-site FST estimates based on the two types of loci were correlated. MHC FST estimates averaged about twice as high as those based on microsatellites. There was no evidence of isolation by distance with either marker. There were no correlations at the population level between any larval fitness attribute (growth rate or survival) and any diversity estimate (microsatellite or MHC). There was, however, a weak correlation at the sibship level between mean growth rate and microsatellite expected heterozygosity. MHC alleles varied among dying larvae and survivors, and in one case between urban and rural sites, with one allele being underrepresented in dying larvae and one allele only occurring in the rural sites. Drift was probably the primary cause of genetic population structure at both types of loci. The use of functional loci to assess population genetic health should focus on the roles of specific alleles as well as overall diversity.

RAPD identification of north European water frogs

A technique for the identification of north European water frogs (Rana lessonae, R. ridibunda and R. esculenta) based on PCR (polymerase chain reaction)-amplification of DNA using random primers (RAPD) is presented. The method requires very small amounts (< 2 mg) of tissue and reliably distinguished the three types of frogs using samples taken from two widely separate localities (Britain and Poland). In addition, the primers distinguished the DNA of water frogs from that of a brown frog (R. temporaria) and from that of two toad species (Bufo bufo and B. calamita).

Bufo MHC class II loci with conserved introns flanking exon 2: cross-species amplification with common primers

We characterized the complete second exon with adjacent introns of MHC class II beta genes in the anurans Bufo calamita, B. bufo and B. viridis. Introns were remarkably conserved among species. There were at least two loci (A and B), which we amplified with different forward primers but identical reverse primers. Locus A had low diversity whereas locus B exhibited 42 alleles in B. calamita and 14 in B. bufo. We detected evidence of positive selection at antigen binding sites in both these species and demonstrated expression of locus B in B. calamita.