Fine-scale population genetic structure and sex-biased dispersal in the smooth snake (Coronella austriaca) in southern England.

Human-induced alteration of natural habitats has the potential to impact on the genetic structuring of remnant populations at multiple spatial scales. Species from higher trophic levels, such as snakes, are expected to be particularly susceptible to land-use changes. We examined fine-scale population structure and looked for evidence of sex-biased dispersal in smooth snakes (Coronella austriaca), sampled from 10 heathland localities situated within a managed coniferous forest in Dorset, United Kingdom. Despite the limited distances between heathland areas (maximum <6 km), there was a small but significant structuring of populations based on eight microsatellite loci. This followed an isolation-by-distance model using both straight line and 'biological' distances between sampling sites, suggesting C. austriaca's low vagility as the causal factor, rather than closed canopy conifer forest exerting an effect as a barrier to dispersal. Within population comparisons of male and female snakes showed evidence for sex-biased dispersal, with three of four analyses finding significantly higher dispersal in males than in females. We suggest that the fine-scale spatial genetic structuring and sex-biased dispersal have important implications for the conservation of C. austriaca, and highlight the value of heathland areas within commercial conifer plantations with regards to their future management.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 April 2010 - 31 May 2010.

This article documents the addition of 396 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anthocidaris crassispina, Aphis glycines, Argyrosomus regius, Astrocaryum sciophilum, Dasypus novemcinctus, Delomys sublineatus, Dermatemys mawii, Fundulus heteroclitus, Homalaspis plana, Jumellea rossii, Khaya senegalensis, Mugil cephalus, Neoceratitis cyanescens, Phalacrocorax aristotelis, Phytophthora infestans, Piper cordulatum, Pterocarpus indicus, Rana dalmatina, Rosa pulverulenta, Saxifraga oppositifolia, Scomber colias, Semecarpus kathalekanensis, Stichopus monotuberculatus, Striga hermonthica, Tarentola boettgeri and Thermophis baileyi. These loci were cross-tested on the following species: Aphis gossypii, Sooretamys angouya, Euryoryzomys russatus, Fundulus notatus, Fundulus olivaceus, Fundulus catenatus, Fundulus majalis, Jumellea fragrans, Jumellea triquetra Jumellea recta, Jumellea stenophylla, Liza richardsonii, Piper marginatum, Piper aequale, Piper darienensis, Piper dilatatum, Rana temporaria, Rana iberica, Rana pyrenaica, Semecarpus anacardium, Semecarpus auriculata, Semecarpus travancorica, Spondias acuminata, Holigarna grahamii, Holigarna beddomii, Mangifera indica, Anacardium occidentale, Tarentola delalandii, Tarentola caboverdianus and Thermophis zhaoermii.

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 F(ST) estimates based on the two types of loci were correlated. MHC F(ST) 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.

A comparison of single-sample effective size estimators using empirical toad (Bufo calamita) population data: genetic compensation and population size-genetic diversity correlations.

The accuracy and precision of four single-sample estimators of effective population size, N(e) (heterozygote excess, linkage disequilibrium, Bayesian partial likelihood and sibship analysis) were compared using empirical data (microsatellite genotypes) from multiple natterjack toad (Bufo calamita) populations in Britain (n = 16) and elsewhere in Europe (n = 10). Census size data were available for the British populations. Because toads have overlapping generations, all of these methods estimated the number of effective breeders N(b) rather than N(e). The heterozygote excess method only provided results, without confidence limits, for nine of the British populations. Linkage disequilibrium gave estimates for 10 British populations, but only six had finite confidence limits. The Bayesian and sibship methods both produced estimates with finite confidence limits for all the populations. Although the Bayesian method was the most precise, on most criteria (insensitivity to locus number, correlation with other effective and census size estimates and correlation with genetic diversity) the sibship method performed best. The results also provided evidence of genetic compensation in natterjack toads, and highlighted how the relationship between effective size and genetic diversity can vary as a function of geographical scale.

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.

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 world's 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.

Defining population boundaries: use of three Bayesian approaches with microsatellite data from British natterjack toads (Bufo calamita).

Defining boundaries between populations is often difficult in the absence of information about current levels of gene flow. Such definitions can be important, however, both for the understanding of population dynamics and for conservation planning. Recently developed Bayesian methods for analysing genetic data now provide a powerful approach to this problem. Natterjack toads Bufo calamita are endangered in Britain, where their distribution is restricted to four geographically discrete regions. In three of these regions the boundaries between populations are often uncertain. We therefore used Bayesian approaches with microsatellite data to try and define British natterjack population structure, and thus inform conservation management. A large sample of natterjack toads from all 38 locations in Britain where the species is native was genotyped at eight microsatellite loci. The genetic diversity of natterjack populations declined as a function of increasing latitude, echoing postglacial colonization dynamics. Comparisons of three assignment methods (structure, baps and geneland) generated some broad similarities but also some inconsistencies in the definitions of population structure, especially in the most complex region (south Cumbria). Implications of the analyses for the future conservation of Bufo calamita in Britain are discussed.

Low gene flow but high genetic diversity in the threatened Mallorcan midwife toad Alytes muletensis.

We investigated fine-scale genetic structuring in the rare and vulnerable Mallorcan midwife toad Alytes muletensis using eight polymorphic microsatellite markers. The current range of this amphibian is restricted to some 19 sites of which six are derived from reintroductions, all located in the mountain ranges of Mallorca. We sampled tadpoles from 14 pools covering 10 natural sites and two reintroduction sites for microsatellite DNA analyses. Relatively high levels of genetic variation were found in most pools (H(E) = 0.38-0.71, allelic richness = 2.6-6.2). Only at one pool has the population recently gone through a bottleneck. Dispersal between pools in different torrents does not occur whereas downstream dispersal between pools within the same torrent does happen at low frequencies. This occasional exchange of individuals does not lead to neighbouring pools in the same torrent being panmictic. This can be concluded because all F(ST) values (0.12-0.53) differ significantly from zero and STRUCTURE analyses identified neighbouring pools as separate populations. Furthermore, assignment and migration tests showed little exchange between neighbouring pools. If upstream locations or complete torrents go extinct, they are unlikely to be recolonized naturally. For conservation purposes, reintroductions of tadpoles to sites where local extinctions have occurred may therefore be advisable.

Conservation genetics of amphibians.

Amphibians are good models for investigating the genetics of wild animal populations because they are: (1) widely distributed in most ecosystems; (2) easy to sample in breeding assemblages; (3) often philopatric to breeding sites, generating high levels of population genetic structure; (4) amenable to controlled crossings in the laboratory; and (5) of major conservation concern. Neutral genetic markers, mostly microsatellites, have been used successfully in studies of amphibian effective population sizes and structures, and in assessing the consequences of hybridisation. Phylogeography has provided important insights into population histories and the fates of introductions. Quantitative genetic methods have demonstrated adaptive variation in life history traits of importance to fitness and therefore to population viability.

Contrasting population structures in two sympatric anurans: implications for species conservation.

A general prediction of the neutral theory of evolution is that genetic diversity should correlate positively with effective population size. We show here that diversity across eight microsatellite loci was consistently and substantially lower in one common amphibian (Bufo bufo) than in another with similar life history traits (Rana temporaria) despite B. bufo having the larger breeding assemblage sizes. However, B. bufo breeding assemblages were much more highly differentiated than those of R. temporaria according to both Fst and Rst estimators. These differences occurred in shared habitats across identical geographical distances. The patterns of genetic diversity and differentiation detected in these two species were probably a consequence of high gene flow in R. temporaria but much lower gene flow among the larger but more dispersed B. bufo assemblages. These observations highlight the difficulty of defining the boundaries of wild populations, and show how two broadly similar species can exhibit very different population dynamics.

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 . We used 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.

Fitness and microsatellite diversity estimates were not correlated in two outbred anuran populations.

Larval fitness traits were investigated in two anuran species (Bufo calamita and Rana temporaria) under controlled laboratory conditions, and allelic diversity measured in the same individuals at five and seven microsatellite loci, respectively. For both species there were significant differences among sibships in larval growth and development rates, and in some cases also in microsatellite heterozygosity and mean d2 (a measure of diversity based on differences in allele sizes). However, there were no significant correlations between any of the fitness and genetic measures either among all individuals, among sibships or among individuals within sibships. Under the conditions and within the statistical power of the study there was no relationship between fitness-related quantitative trait variation and that seen at presumed-neutral microsatellite loci in these outbred populations.

Competition between Bufo larvae in a eutrophic pond.

Competition between larvae of two anuran species (Bufo bufo and B. calamita) was investigated under field conditions likely to disfavour cell-mediated interference mechanisms. The experiment used triplicated cage treatments in an unshaded farm pond, a poor habitat for the unicellular pathogen Anurofeca richardsi implicated in interference competition between these anurans in sand dune ponds. The farm pond experienced lower maximum temperatures than a nearby dune pond but sustained larger numbers of eukaryotic algae and therefore had higher primary productivity. Survival and growth of B. calamita larvae were inversely related to density in all treatments but interspecific effects were much more severe than intraspecific ones. There was no evidence of A. richardsi in any treatment and competition between the Bufo larvae was therefore intense in the absence of Anurofeca-mediated interference effects. Anuran larvae reduced the standing crop and altered the community composition of algae in the treatment cages but larval growth rates were not simply related to food availability. Algal cell numbers in larval guts, a measure of food acquisition, were however inversely related to tadpole density in both species. Feeding niche overlap was high but decreased as larval density increased. Resource competition was implicated as the most probable major mechanism.

Archive contributions to a molecular phylogeography of the toad Bufo calamita in Britain.

A complete phylogeographic analysis of any species requires sampling throughout its biogeographical range. In the case of the natterjack toad Bufo calamita in Britain, recent local extinctions have left substantial areas of its historical range without extant populations. We therefore obtained tissue samples of archived Bufo calamita from four museums in the United Kingdom. A range of tissues (tongue, liver, skin, lung, and larval tail) was sampled from a total of 33 individual animals. DNA was extracted and eight polymorphic microsatellite loci were scored. One or more loci were amplified successfully from 27 individuals, and sufficient data were obtained from regions with few or no surviving populations to supplement a phylogeographic analysis based on extant populations.

Genetic substructuring as a result of barriers to gene flow in urban Rana temporaria (common frog) populations: implications for biodiversity conservation.

The ability to maintain small populations in quasi-natural settings is an issue of considerable importance in biodiversity conservation. The genetic structure of urban common frog (Rana temporaria) populations was determined by allozyme electrophoresis and used to evaluate the effects of restricted intersite migration. Despite the lack of any absolute barrier to movement between ponds, substantial genetic differentiation was found between sites separated by an average of only 2.3 km. Genetic distances between these town ponds correlated positively with geographical distances and were almost twice as great as those found between rural sites separated by an average of 41 km. Measures of genetic diversity and fitness were always lowest in the town, where the degree of subpopulation differentiation (FST = 0.388) was high. Population decline was not evident in the town, but molecular and fitness data indicated the presence of genetic drift and inbreeding depression. The long-term survival of artificially restricted populations, particularly of relatively sedentary species, may require molecular monitoring, if genetic diversity is not to be lost by chance when facets of the species niche prove to be poorly understood.

Preliminary characterisation and partial purification of ribosomal gene promoter-binding proteins from Trypanosoma brucei.

DNA fragments including the promoter region of the major ribosomal RNA gene of Trypanosoma brucei (r-promoter) were identified and subcloned using a synthetic oligonucleotide probe corresponding to the putative core promoter. These fragments were used in mobility shift assays with proteins extracted from T. brucei nuclei, and demonstrated the presence in 0.15 M NaCl extracts of protein(s) with specific binding affinities for the r-promoter region. Binding was stable in the presence of a 100-fold excess of competitor DNA, and occurred at the relatively low salt concentrations (< 50 mM NaCl) characteristic of many enzyme activity optima in this organism. A control DNA fragment not including the r-promoter region was not retarded in the mobility shift assays, and the r-promoter-binding activity had a molecular weight of about 140,000. Nuclear extracts from T. brucei contained large amounts of DNase activity, and the promoter-binding proteins were partially purified from the crude extract using ammonium sulphate precipitation, sephacryl S-200 and Heparin-sepharose chromatography.

Changes in testicular lactate dehydrogenases of the rat (Rattus norvegicus) during growth and development.

1. We have quantified lactate dehydrogenase (LDH) A, B and C enzyme levels during the first twelve weeks of life in rat testes. 2. Proteins were electrophoresed under non-denaturing conditions, isoenzymes identified by activity staining and quantified by densitometry scanning. 3. LDH A declined 2-3-fold during testes growth, changing from the most to the least abundant isoenzyme form. LDH B remained fairly constant, and LDH C increased from undetectable levels at birth to become the most abundant of the three polypeptides from week 3 onwards.

Rat lactate dehydrogenase A and B subunit concentrations are not regulated by mRNA abundance in liver and heart.

RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.