Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.

Evolutionary developmental biology ("evo-devo") has revolutionized evolutionary biology but has had relatively little impact on systematics. We show that similar large-scale developmental changes in distantly related lineages can dramatically mislead phylogenetic analyses based on morphological data. Salamanders are important model systems in many fields of biology and are of special interest in that many species are paedomorphic and thus never complete metamorphosis. A recent study of higher-level salamander phylogeny placed most paedomorphic families in a single clade based on morphological data. Here, we use new molecular and morphological data to show that this result most likely was caused by the misleading effects of paedomorphosis. We also provide a well-supported estimate of higher-level salamander relationships based on combined molecular and morphological data. Many authors have suggested that paedomorphosis may be problematic in studies of salamander phylogeny, but this hypothesis has never been tested with a rigorous phylogenetic analysis. We find that the misleading effects of paedomorphosis on phylogenetic analysis go beyond the sharing of homoplastic larval traits by paedomorphic adults, and the problem therefore is not solved by simply excluding suspected paedomorphic characters. Instead, two additional factors are critically important in causing paedomorphic species to be phylogenetically "misplaced": (1) the absence of clade-specific synapomorphies that develop during metamorphosis in nonpaedomorphic taxa and allow their "correct" placement and (2) parallel adaptive changes associated with the aquatic habitat of the larval stage. Our results suggest that the effects of paedomorphosis on phylogenetic analyses may be complex, difficult to detect, and can lead to results that are both wrong and statistically well supported by parsimony and Bayesian analyses.

Abstract. The transition from aquatic to terrestrial eggs is a key evolutionary change that has allowed vertebrates to successfully colonize and exploit the land. Although most amphibians retain the primitive biphasic life cycle (eggs deposited in water that hatch into free-living aquatic larvae), direct development of terrestrial eggs has evolved repeatedly and may have been critical to the evolutionary success of several amphibian groups. We provide the first conclusive evidence for evolutionary reversal of direct development in vertebrates. The family Plethodontidae (lungless salamanders) contains the majority of salamander species, including major radiations of direct developers. We reconstruct the higher level phylogenetic relationships of plethodontid salamanders using molecular and morphological data and use this phylogeny to examine the evolution of direct development. We show that the predominantly biphasic desmognathines, previously considered the sister group of other plethodontids, are nested inside a group of directdeveloping species (Plethodontini) and have re-evolved the aquatic larval stage. Rather than being an evolutionary dead end, the reversal from direct developing to biphasic life history may have helped communities in eastern North America to achieve the highest local diversity of salamander species in the world.

An important dimension of adaptive radiation is the degree to which diversification rates fluctuate or remain constant through time. Focusing on plethodontid salamanders of the genus Desmognathus, we present a novel synthetic analysis of phylogeographic history, rates of ecomorphological evolution and species accumulation, and community assembly in an adaptive radiation. Dusky salamanders are highly variable in life history, body size, and ecology, with many endemic lineages in the southern Appalachian Highlands of eastern North America. Our results show that life-history evolution had important consequences for the buildup of plethodontid-salamander species richness and phenotypic disparity in eastern North America, a global hot spot of salamander biodiversity. The origin of Desmognathus species with aquatic larvae was followed by a high rate of lineage accumulation, which then gradually decreased toward the present time. The peak period of lineage accumulation in the group coincides with evolutionary partitioning of lineages with aquatic larvae into seepage, stream-edge, and stream microhabitats. Phylogenetic simulations demonstrate a strong correlation between morphology and microhabitat ecology independent of phylogenetic effects and suggest that ecomorphological changes are concentrated early in the radiation of Desmognathus. Deep phylogeographic fragmentation within many codistributed ecomorph clades suggests long-term persistence of ecomorphological features and stability of endemic lineages and communities through multiple climatic cycles. Phylogenetic analyses of community structure show that ecomorphological divergence promotes the coexistence of lineages and that repeated, independent evolution of microhabitat-associated ecomorphs has a limited role in the evolutionary assembly of Desmognathus communities. Comparing and contrasting our results to other adaptive radiations having different biogeographic histories, our results suggest that rates of diversification during adaptive radiation are intimately linked to the degree to which community structure persists over evolutionary time.

Leptin, the protein product of the obese(ob or Lep) gene, is a hormone synthesized by adipocytes that signals available energy reserves to the brain, and thereby influences development, growth, metabolism and reproduction. In mammals, leptin functions as an adiposity signal: circulating leptin fluctuates in proportion to fat mass, and it acts on the hypothalamus to suppress food intake. Orthologs of mammalian Lep genes were recently isolated from several fish and two amphibian species, and here we report the identification of two Lep genes in a reptile, the lizard Anolis carolinensis. While vertebrate leptins show large divergence in their primary amino acid sequence, they form similar tertiary structures, and may have similar potencies when tested in vitro on heterologous leptin receptors (LepRs). Leptin binds to LepRs on the plasma membrane, activating several intracellular signaling pathways. Vertebrate LepRs signal via the Janus kinase (Jak) and signal transducer and activator of transcription (STAT) pathway. Three tyrosine residues located within the LepR cytoplasmic domain are phosphorylated by Jak2 and are required for activation of SH2-containing tyrosine phosphatase-2, STAT5 and STAT3 signaling. These tyrosines are conserved from fishes to mammals, demonstrating their critical role in signaling by the LepR. Leptin is anorexigenic in representatives of all vertebrate classes, suggesting that its role in energy balance is ancient and has been evolutionarily conserved. In addition to its integral role as a regulator of appetite and energy balance, leptin exerts pleiotropic actions in development, physiology and behavior.

Metazoans display a tremendous diversity of developmental patterns, including complex life cycles composed of morphologically disparate stages. In this regard, the evolution of life cycle complexity promotes phenotypic diversity. However, correlations between life cycle stages can constrain the evolution of some structures and functions. Despite the potential macroevolutionary consequences, few studies have tested the impacts of life cycle evolution on broad-scale patterns of trait diversification. Here we show that larval and adult salamanders with a simple, aquatic-only (paedomorphic) life cycle had an increased rate of vertebral column and body form diversification compared to lineages with a complex, aquatic-terrestrial (biphasic) life cycle. These differences in life cycle complexity explain the variations in vertebral number and adult body form better than larval ecology. In addition, we found that lineages with a simple terrestrial-only (direct developing) life cycle also had a higher rate of adult body form evolution than biphasic lineages, but still 10-fold lower than aquatic-only lineages. Our analyses demonstrate that prominent shifts in phenotypic evolution can follow long-term transitions in life cycle complexity, which may reflect underlying stage-dependent constraints.

Summary Several models explain how a complex integrated system like the rodent mandible can arise from multiple developmental modules. The models propose various integrating mechanisms, including epigenetic effects of muscles on bones. We test five for their ability to predict correlations found in the individual (symmetric) and fluctuating asymmetric (FA) components of shape variation. We also use exploratory methods to discern patterns unanticipated by any model. Two models fit observed correlation matrices from both components: (1) parts originating in same mesenchymal condensation are integrated, (2) parts developmentally dependent on the same muscle form an integrated complex as do those dependent on teeth. Another fits the correlations observed in FA: each muscle insertion site is an integrated unit. However, no model fits well, and none predicts the complex structure found in the exploratory analyses, best described as a reticulated network. Furthermore, no model predicts the correlation between proximal parts of the condyloid and coronoid, which can exceed the correlations between proximal and distal parts of the same process. Additionally, no model predicts the correlation between molar alveolus and ramus and/or angular process, one of the highest correlations found in the FA component. That correlation contradicts the basic premise of all five developmental models, yet it should be anticipated from the epigenetic effects of mastication, possibly the primary morphogenetic process integrating the jaw coupling forces generated by muscle contraction with those experienced at teeth.

Amphibians are characterized both by a strongly increasing number of newly discovered species and by a high degree of decline. The observed increase in species numbers, over 25 percent in 11 years, is largely due to the intensified exploration of tropical areas and the application of more efficient techniques such as bioacoustics and molecular genetics, rather than to the elevation of subspecies to species rank or the distinction of species that were formerly considered synonymous. In the mantellid frogs of Madagascar, the many species newly described between 1992 and 2004 were as genetically divergent as those described in previous research periods, and most had not been collected previously, corroborating the lack of "taxonomic inflation" in this vertebrate class. Taxonomic exploration is still desperately needed to avoid misinterpretations in global conservation policy.