Ronald M. Bonett

New Amphibians and Global Conservation: A Boost in Species Discoveries in a Highly Endangered Vertebrate Group

Abstract 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.

Phylogenetic analysis of ecomorphological divergence, community structure, and diversification rates in dusky salamanders (Plethodontidae: Desmognathus)

Abstract 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 lifehistory 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.

Evolution of leptin structure and function.

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.

Discovery of the first Asian plethodontid salamander

Nearly 70% of the 535 species of salamanders in the world are members of a single family, the Plethodontidae, or lungless salamanders. The centre of diversity for this clade is North and Middle America, where the vast majority (99%) of species are found. We report the discovery of the first Asian plethodontid salamander, from montane woodlands in southwestern Korea. The new species superficially resembles members of North American genera, in particular the morphologically conservative genus Plethodon. However, phylogenetic analysis of the nuclear encoded gene Rag-1 shows the new taxon to be widely divergent from Plethodon. The new salamander differs osteologically from putative relatives, especially with respect to the tongue (attached protrusible) and the derived tarsus. We place the species in a new genus on the basis of the morphological and molecular data. The distribution of the new salamander adds to the enigma of Old World plethodontids, which are otherwise restricted to the western Mediterranean region, suggesting a more extensive past distribution of the family.

Evolution of paedomorphosis in plethodontid salamanders: ecological correlates and re-evolution of metamorphosis.

Life‐history modes can profoundly impact the biology of a species, and a classic example is the dichotomy between metamorphic (biphasic) and paedomorphic (permanently aquatic) life‐history strategies in salamanders. However, despite centuries of research on this system, several basic questions about the evolution of paedomorphosis in salamanders have not been addressed. Here, we use a nearly comprehensive, time‐calibrated phylogeny of spelerpine plethodontids to reconstruct the evolution of paedomorphosis and to test if paedomorphosis is (1) reversible; (2) associated with living in caves; (3) associated with relatively dry climatic conditions on the surface; and (4) correlated with limited range size and geographic dispersal. We find that paedomorphosis arose multiple times in spelerpines. We also find evidence for re‐evolution of metamorphosis after several million years of paedomorphosis in a lineage of Eurycea from the Edwards Plateau region of Texas. We also show for the first time using phylogenetic comparative methods that paedomorphosis is highly correlated with cave‐dwelling, arid surface environments, and small geographic range sizes, providing insights into both the causes and consequences of this major life history transition.

Early Miocene origin and cryptic diversification of South American salamanders

BackgroundThe currently recognized species richness of South American salamanders is surprisingly low compared to North and Central America. In part, this low richness may be due to the salamanders being a recent arrival to South America. Additionally, the number of South American salamander species may be underestimated because of cryptic diversity. The aims of our present study were to infer evolutionary relationships, lineage diversity, and timing of divergence of the South American Bolitoglossa using mitochondrial and nuclear sequence data from specimens primarily from localities in the Andes and upper Amazon Basin. We also estimated time of colonization of South America to test whether it is consistent with arrival via the Panamanian Isthmus, or land bridge connection, at its traditionally assumed age of 3 million years.ResultsDivergence time estimates suggest that Bolitoglossa arrived in South America from Central America by at least the Early Miocene, ca. 23.6 MYA (95% HPD 15.9-30.3 MYA), and subsequently diversified. South American salamanders of the genus Bolitoglossa show strong phylogeographic structure at fine geographic scales and deep divergences at the mitochondrial gene cytochrome b (Cytb) and high diversity at the nuclear recombination activating gene-1 (Rag1). Species often contain multiple genetically divergent lineages that are occasionally geographically overlapping. Single specimens from two southeastern localities in Ecuador are sister to the equatoriana-peruviana clade and genetically distinct from all other species investigated to date. Another single exemplar from the Andes of northwestern Ecuador is highly divergent from all other specimens and is sister to all newly studied samples. Nevertheless, all sampled species of South American Bolitoglossa are members of a single clade that is one of several constituting the subgenus Eladinea, one of seven subgenera in this large genus.ConclusionsThe ancestors of South American salamanders likely arrived at least by the Early Miocene, well before the completion of the Late Pliocene Panamanian land bridge (widely accepted as ca. 3 MYA). This date is in agreement with recent, controversial, arguments that an older, perhaps short-lived, land connection may have existed between South America and present-day Panama 23–25 MYA. Since its arrival in South America, Bolitoglossa has diversified more extensively than previously presumed and currently includes several cryptic species within a relatively small geographic area. Rather than two upper Amazonian species currently recorded for this region, we propose that at least eight should be recognized, although these additional lineages remain to be formally described.

Deciphering the Regulatory Logic of an Ancient, Ultraconserved Nuclear Receptor Enhancer Module

Cooperative, synergistic gene regulation by nuclear hormone receptors can increase sensitivity and amplify cellular responses to hormones. We investigated thyroid hormone (TH) and glucocorticoid (GC) synergy on the Krüppel-like factor 9 (Klf9) gene, which codes for a zinc finger transcription factor involved in development and homeostasis of diverse tissues. We identified regions of the Xenopus and mouse Klf9 genes 5-6 kb upstream of the transcription start sites that supported synergistic transactivation by TH plus GC. Within these regions, we found an orthologous sequence of approximately 180 bp that is highly conserved among tetrapods, but absent in other chordates, and possesses chromatin marks characteristic of an enhancer element. The Xenopus and mouse approximately 180-bp DNA element conferred synergistic transactivation by hormones in transient transfection assays, so we designate this the Klf9 synergy module (KSM). We identified binding sites within the mouse KSM for TH receptor, GC receptor, and nuclear factor κB. TH strongly increased recruitment of liganded GC receptor and serine 5 phosphorylated (initiating) RNA polymerase II to chromatin at the KSM, suggesting a mechanism for transcriptional synergy. The KSM is transcribed to generate long noncoding RNAs, which are also synergistically induced by combined hormone treatment, and the KSM interacts with the Klf9 promoter and a far upstream region through chromosomal looping. Our findings support that the KSM plays a central role in hormone regulation of vertebrate Klf9 genes, it evolved in the tetrapod lineage, and has been maintained by strong stabilizing selection.

Analysis of the Contact Zone between the Dusky Salamanders Desmognathus fuscus fuscus and Desmognathus fuscus conanti (Caudata: Plethodontidae)

Abstract Two most widely distributed “subspecies” of the Desmognathus fuscus complex are the northern dusky salamander (Desmognathus fuscus fuscus) and the spotted dusky salamander (Desmognathus fuscus conanti). Previous mitochondrial DNA and allozyme studies of this complex have suggested that these two forms should be granted species-level recognition. However, detailed examination of populations inhabiting the region where the two taxa come into contact have not been performed to corroborate this taxonomic change. In this study, specimens were collected from four transects that traverse the putative contact zone. Allozyme and color pattern analysis were employed to determine the nature of variation across the area and evaluate the taxonomic status of D. f. conanti. Phylogenetic analyses of allozyme data divide the populations sampled into at least four major groups, two of which are referable to Desmognathus fuscus (groups A and C) and two referable to D. conanti (groups B and D). This study supports the distinctiveness of D. conanti by revealing parapatry between populations of groups A and B in western Kentucky with only a minor amount of hybridization between them and identifies color pattern differences that are effective for characterizing most group B individuals. The data presented here suggest that a longitudinal division may exist within D. conanti in central Alabama. Group C occurs primarily on the Piedmont of North Carolina and Virginia and appears to hybridize with group A in central Virginia. Groups C and D are treated, conservatively, as D. fuscus and D. conanti, respectively; but further study may reveal that each is a distinct species.

Considering alternative life history modes and genetic divergence in conservation: a case study of the Oklahoma salamander

Alternative life history strategies can provide important variation for the long-term persistence of a lineage. However, conservation of such lineages can be complicated because each life history mode may have different habitat requirements and may be vulnerable to different environmental perturbations. The Oklahoma salamander (Eurycea tynerensis) is endemic to the Ozark Plateau of North America, and has two discrete life history modes, biphasic (metamorphic) and aquatic (paedomorphic). Until recently, these modes were considered separate species and conservation attention focused only on paedomorphic populations. We perform phylogenetic analyses of the mitochondrial gene cytochrome b (Cytb) and nuclear gene proopiomelanocortin (POMC) to assess patterns of historical isolation in E. tynerensis, and test whether life history mode is randomly distributed with respect to the phylogeny and geography. We find three divergent Cytb lineages and significant shifts in POMC allele frequencies between the eastern, western, and southwestern portions of the distribution. Life history mode varies extensively, but paedomorphosis is largely restricted to the widespread western clade. Therefore, the two most divergent and narrowly distributed clades (southwestern and eastern) were previously overlooked due to their metamorphic life history. Paedomorphosis has allowed E. tynerensis to drastically increase its niche breadth and distribution size. Nevertheless, metamorphosis is also an important attribute, and metamorphic populations are the ultimate source for paedomorphic evolution. Preservation of divergent genetic lineages, and regions that include adjacent habitat for both life history modes, may be the most effective way to maintain historical and adaptive variation and provide gateways for ongoing life history evolution.

Distribution, abundance, and genetic diversity of Clinostomum spp. metacercariae (Trematoda:Digenea) in a modified Ozark stream system.

Abstract Land-use alterations can have profound influences on faunal distributions, including host-parasite relationships. Yellow grub trematodes (Clinostomum spp.) have complex life cycles involving 3 hosts: a snail, a fish or amphibian, and a bird. Here, we analyze the distribution, prevalence, intensity, abundance, and genetic diversity of encysting metacercariae of Clinostomum spp. in salamanders and fishes throughout an aquatic system that includes a natural Ozark stream and man-made ponds. We found Clinostomum sp. infecting permanently aquatic Oklahoma salamanders (Eurycea tynerensis; 56% prevalence) and larval grotto salamanders (Eurycea spelaea) immediately downstream from a man-made pond. However, Clinostomum sp. did not infect any salamanders in the spring that supplies this pond, or in sections farther downstream (∼0.5 and 2 km). Metacercariae of Clinostomum sp. were present in ∼90% of introduced largemouth bass (Micropterus salmoides) in the man-made pond adjunct to the stream. Morphological examination and phylogenetic analyses based on the mitochondrial gene cytochrome oxidase 1 (Co1) and the nuclear ribosomal gene 18S show that fishes and salamanders at this site are primarily infected with Clinostomum marginatum. There is a relatively high degree of mitochondrial haplotype diversity in C. marginatum at this site but no consistent genetic difference between parasites in largemouth bass from the man-made pond and those in salamanders from the stream. Based on the microgeographic distribution and relationships of metacercariae of C. marginatum at this site, we hypothesize that the adjunct man-made pond has created an ecological situation that brings the cercariae of this parasite into contact with novel stream salamander hosts.