Recent studies found major conflicts between traditional taxonomy and genetic differentiation of grass snakes and identified previously unknown secondary contact zones. Until now, little is known about gene flow across these contact zones. Using two mitochondrial markers and 13 microsatellite loci, we examined two contact zones. One, largely corresponding to the Rhine region, involves the western subspecies Natrix natrix helvetica and the eastern subspecies N. n. natrix, whereas in the other, more easterly, contact zone two lineages meet that are currently identified with N. n. natrix and N. n. persa. This second contact zone runs across Central Europe to the southern Balkans. Our analyses reveal that the western contact zone is narrow, with parapatrically distributed mitochondrial lineages and limited, largely unidirectional nuclear gene flow. In contrast, the eastern contact zone is very wide, with massive nuclear admixture and broadly overlapping mitochondrial lineages. In combination with additional lines of evidence (morphology, phylogeny, divergence times), we conclude that these differences reflect different stages in the speciation process and that Natrix helvetica should be regarded as a distinct species. We suggest a nomenclatural framework for presently recognized grass snake taxa and highlight the need for reconciling the conflicts between genetics and taxonomy.

In this study we use sensitivity analysis sensu Wheeler (1995) for a matrix entirely composed of DNA sequences. We propose that not only congruence but also phylogenetic structure, as measured by character resampling, should be used to choose among competing weighting regimes. An extensive analysis of a five-gene data set for Themira (Sepsidae: Diptera) reveals that even with different ways of partitioning the data, measures of topological congruence, character incongruence, and phylogenetic structure favor similar weighting regimes involving the down-weighting of transitions. We furthermore use sensitivity analysis for obtaining empirical evidence that allows us to select weights for third positions, deciding between treating indels as fifth character states or missing values, and choosing between manual and computational alignments. For our data, sensitivity analysis favors manual alignment over a Clustal-generated numerical alignment, the treatment of indels as fifth character states over considering them missing values, and equal weights for all positions in protein-encoding genes over the down-weighting of third positions. Among the topological congruence measures compared, symmetric tree distance performed best. Partitioned Bremer Support analysis reveals that COI contributes the largest amount of support for our phylogenetic tree for Themira.Ó The Willi Hennig Society 2005.Reconstructing phylogenetic relationships based on DNA sequence data is sometimes naı¨vely considered more objective than reconstructing trees based on morphological characters. This belief is rooted in the objectivity of raw nucleotide sequence data, but overlooks that there are many different analysis strategies, weighting schemes, and ⁄ or evolutionary models that can be used to derive trees from sequences (Wheeler, 1995;Wheeler and Hayashi, 1998). It is thus not surprising that much of the recent literature in systematic biology treats the advantages and disadvantages of different analysis philosophies (e.g., Maximum Likelihood, Bayesian Likelihood, Neighbor Joining, Parsimony) and discusses the best techniques for choosing appropriate evolutionary models and ⁄ or weighting schemes. In maximum likelihood and Bayesian likelihood estimation, different methods for model choice have been established (e.g., Aris-Brosou, 2003;Posada and Crandall, 1998). In parsimony, some proponents strongly favor equal weighting under all circumstances (e.g., Kluge and Wolf, 1993), while others prefer to choose analysis conditions for their data sets based on a variety of more or less explicit criteria.In parsimony, one of the most popular methods for choosing among weighting regimes (Giribet, 2003) is Wheeler's (1995) sensitivity analysis, which uses the congruence between different data partitions for this selection. Traditionally, such sensitivity analyses have

R. 2002. Morphometric, biochemical and molecular traits in Caucasian wood mice (Apodemus/Sylvaemus), with remarks on species divergence. Acta Theriologica 47: 389-416.We analysed Caucasian wood mice from Georgia (n = 60) and supplementary reference material of the Apodemus/Sylvaemus species group to evaluate the reliability of taxon identification. Traditional "expert knowledge" plus three different methodological approaches were employed and combined to perceive their discriminatory power for a reliable taxon assignment. Graphs of principal component scores derived from the analysis of 14 skull metrics displayed taxon membership of individuals. Individual multi--locus (L = 18) electrophoretic profiles were used to re-assess specimens to a specific genepool by an assignment test based on allele frequencies indicative of populational taxon samples of the respective sampling locations. Genotyped individuals were re-allocated to those taxa, for which they yielded the highest probability score. Genetic distances among the taxa were computed and clustered in a neighbour-joining tree. PCR-fragments of 1074bp amplified from the mitochondrial cytochrome b gene were cut with 2 six-and 4 four-cutter restriction enzymes, and resulting RFLP patterns were analysed phenetically to classify the specimens according to their molecular similarity. Partial cytochrome b sequences were used to construct a phylogenetic tree by computing neighbour-joining clusters from a matrix of percent nucleotide differences. The power of the combined classification approaches and their congruence is discussed. It is concluded that the joint application of traditional, morphometric and biochemical or genetic techniques for taxon allocation of specimens of wood mice encountered problems in species delimitation. The mtDNA topology obtained was not congruent with protein polymorphism that indicated differential historical and/or recent introgression and incomplete lineage sorting in substructured populations. Cytochrome b sequence DNA data analysed were not as adequate as expected to resolve phylogenetic relationships among Caucasian and European members of the Apodemus-Sylvaemus complex. Altogether, morphometric, biochemical and sequence data sets did not support the hypothesis of the evolutionary independence of European and Caucasian lineages of wood mice. Nonetheless, extended combined morphological and genetic analyses are considered necessary prerequisites to an in-depth study of the evolutionary lineages of the Apodemus/Sylvaemus group. More sequence data of a variety of genes (and plenty of nuclear markers) are needed to resolve the various levels of differentiation of the extant lineages.

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Eighteen microsatellite loci developed for a range of snake species (New World natricines, elapids, crotalids) were tested against European natricines (Natrix natrix, N. maura, and N. tessellata) in cross-species amplification experiments. Five loci were polymorphic (average expected heterozygosity 0.749 for a population of N. natrix in Amsterdam, mean sample size 47.8) and three loci were monomorphic. The remainder could not be consistently scored or failed to amplify. Further tests on single individuals of a diverse set of eight species of colubroid snakes showed that 15 of the 18 loci could be cross-amplified in at least one of these species. We conclude that our results show promise for the utilization of these markers for experimental assessments of genetic variation in the phylogenetically closely related group of European natricine snakes with emphasis on N. natrix. The full suite of microsatellite markers now available for snakes may show additional potential for subsequent investigation across a broader range of colubroid snakes.