Comparing complete animal mitochondrial genome sequences is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. Not only are they much more informative than shorter sequences of individual genes for inferring evolutionary relatedness, but these data also provide sets of genome-level characters, such as the relative arrangements of genes, that can be especially powerful. We describe here the protocols commonly used for physically isolating mtDNA, for amplifying these by PCR or RCA, for cloning, sequencing, assembly, validation, and gene annotation, and for comparing both sequences and gene arrangements. On several topics, we offer general observations based on our experiences to date with determining and comparing complete mtDNA sequences.

The evolutionary history of the largest salamander family (Plethodontidae) is characterized by extreme morphological homoplasy. Analysis of the mechanisms generating such homoplasy requires an independent, molecular phylogeny. To this end, we sequenced 24 complete mitochondrial genomes (22 plethodontids and two outgroup taxa), added data for three species from GenBank, and performed partitioned and unpartitioned Bayesian, ML, and MP phylogenetic analyses. We explored four dataset partitioning strategies to account for evolutionary process heterogeneity among genes and codon positions, all of which yielded increased model likelihoods and decreased numbers of supported nodes in the topologies (PP > 0.95) relative to the unpartitioned analysis. Our phylogenetic analyses yielded congruent trees that contrast with the traditional morphology-based taxonomy; the monophyly of three out of four major groups is rejected. Reanalysis of current hypotheses in light of these new evolutionary relationships suggests that 1) a larval life history stage re-evolved from a direct-developing ancestor multiple times, 2) there is no phylogenetic support for the "Out of Appalachia" hypothesis of plethodontid origins, and 3) novel scenarios must be reconstructed for the convergent evolution of projectile tongues, reduction in toe number, and specialization for defensive tail loss. Some of these novel scenarios imply morphological transformation series that proceed in the opposite direction than was previously thought. In addition, they suggest surprising evolutionary lability in traits previously interpreted to be conservative.

The analysis of interactions between lineages at varying levels of genetic divergence can provide insights into the process of speciation through the accumulation of incompatible mutations. Ring species, and especially the Ensatina eschscholtzii system exemplify this approach. The plethodontid salamanders Ensatina eschscholtzii xanthoptica and Ensatina eschscholtzii platensis hybridize in the Central Sierran foothills of California. We compared the genetic structure across two transects (southern and northern Calaveras Co.), one of which was re-sampled over 20 years, and examined diagnostic molecular markers (eight allozyme loci and mitochondrial DNA) and a diagnostic quantitative trait (color pattern). Key results across all studies were: i) cline centers for all markers were coincident and the zones were narrow, with width estimates of 730m to 2000m; ii) cline centers at the northern Calaveras transect were coincident between 1981 and 2001, demonstrating repeatability over 5 generations; iii) there are very few if any putative F1's, but a relatively high number of backcrossed individuals (57-86 percent) in the central portion of transects; iv) we found substantial linkage disequilibrium in all three studies and strong heterozygote deficit both in northern Calaveras, in 2001, and southern Calaveras. Both linkage disequilibrium and heterozygote deficit show maximum values near the center of the zones (R and Fis, approx. equal to 0.5). Using estimates of cline width and dispersal, we infer strong selection against hybrids (s* approx. equal to 46-75 percent). This is sufficient to promote accumulation of differences at loci that are neutral or under divergent selection, but would still allow for introgression of a daptive alleles. The evidence for strong, but incomplete isolation across this centrally located contact is consistent with theory suggesting a gradual increase in postzygotic incompatibility between allopatric populations subject to divergent selection and reinforces the value of Ensatina as a system for the study of divergence and speciation at multiple stages.