Structure and evolution of the sexually dimorphic integumentary swelling on the hands of dendrobatid poison frogs and their relatives (Amphibia: Anura: Dendrobatoidea).

The hands of adult males of many dendrobatid poison frogs and their relatives possess swelling formed by glandular tissue hypothesized to secrete courtship pheromones delivered to the female during cephalic amplexus. Variation in the occurrence and external morphology of the swelling of finger IV has provided important evidence for dendrobatoid systematics for decades, but its underlying structure has not been investigated. We undertook a detailed comparative analysis of the integument of the hand, including both external morphology and histology, of 36 species representing the diversity of dendrobatoid frogs and several close relatives. The swelling is caused by four densely packed, hypertrophic, morpho-histochemical types of specialized mucous glands (SMGs). We observed type I SMGs on fingers II-V and the wrist, including areas that are not swollen, types II and IV exclusively on finger IV, and type III on finger IV and the wrist. Type I SMGs occur either in isolation or together with types II, III, or IV; types II, III, and IV never occur together or without type I. We delimited 15 characters to account for the variation in external morphology and the occurrence of SMGs. Our data suggest that type I SMGs are a new synapomorphy for Dendrobatoidea and that type II SMGs originated in either the most recent common ancestor (MRCA) of Dendrobatidae or independently in the MRCAs of Aromobatidae and Colostethinae, respectively, while types III and IV are restricted to Anomaloglossus. The discovery of these SMGs adds a new dimension to studies of poison frog reproductive biology, which have investigated acoustic, visual, and tactile cues in courtship, mating, and parental care across the diversity of Dendrobatoidea for decades but have almost entirely overlooked the possible role of chemical cues.

An empirical test of the relationship between the bootstrap and likelihood ratio support in maximum likelihood phylogenetic analysis.

In maximum likelihood (ML), the support for a clade can be calculated directly as the likelihood ratio (LR) or log-likelihood difference (S, LLD) of the best trees with and without the clade of interest. However, bootstrap (BS) clade frequencies are more pervasive in ML phylogenetics and are almost universally interpreted as measuring support. In addition to theoretical arguments against that interpretation, BS has several undesirable attributes for a support measure. For example, it does not vary in proportion to optimality or identify clades that are rejected by the evidence and can be overestimated due to missing data. Nevertheless, if BS is a reliable predictor of S, then it might be an efficient indirect method of measuring support-an attractive possibility, given the speed of many BS implementations. To assess the relationship between S and BS, we analyzed 106 empirical datasets retrieved from TreeBASE. Also, to evaluate the degree to which S and BS are affected by the number of replicates during suboptimal tree searches for S and pseudoreplicates during BS estimation, we randomly selected 5 of the 106 datasets and analyzed them using variable numbers of replicates and pseudoreplicates, respectively. The correlation between S and BS was extremely weak in the datasets we analyzed. Increasing the number of replicates during tree search decreased the estimated values of S for most clades, but the magnitude of change was small. In contrast, although increasing pseudoreplicates affected BS values for only approximately 40% of clades, values both increased and decreased, and they did so at much greater magnitudes. Increasing replicates/pseudoreplicates affected the rank order of clades in each tree for both S and BS. Our findings show decisively that BS is not an efficient indirect method of measuring support and suggest that even quite superficial searches to calculate S provide better estimates of support.

Evidence of absence treated as absence of evidence: The effects of variation in the number and distribution of gaps treated as missing data on the results of standard maximum likelihood analysis.

Although numerous studies have demonstrated the theoretical and empirical importance of treating gaps as insertion/deletion (indel) events in phylogenetic analyses, the standard approach to maximum likelihood (ML) analysis employed in the vast majority of empirical studies codes gaps as nucleotides of unknown identity ("missing data"). Therefore, it is imperative to understand the empirical consequences of different numbers and distributions of gaps treated as missing data. We evaluated the effects of variation in the number and distribution of gaps (i.e., no base, coded as IUPAC "." or "-") treated as missing data (i.e., any base, coded as "?" or IUPAC "N") in standard ML analysis. We obtained alignments with variable numbers and arrangements of gaps by aligning seven diverse empirical datasets under different gap opening costs using MAFFT. We selected the optimal substitution model for each alignment using the corrected Akaike Information Criterion in jModelTest2 and searched for optimal trees using GARLI. We also employed a Monte Carlo approach to randomly replace nucleotides with gaps (treated as missing data) in an empirical dataset to understand more precisely the effects of varying their number and distribution. To compare alignments, we developed four new indices and used several existing measures to quantify the number and distribution of gaps in all alignments. Our most important finding is that ML scores correlate negatively with gap opening costs and the amount of missing data. However, this negative relationship is not due to the increase in missing data per se-which increases ML scores-but instead to the effect of gaps on nucleotide homology. These variables also cause significant but largely unpredictable effects on tree topology.

The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae).

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.

The phylogeny of the Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini).

The South American and West Indian Casque-headed Treefrogs (Hylidae: Hylinae: Lophyohylini) include 85 species. These are notably diverse in morphology (e.g. disparate levels of cranial hyperossification) and life history (e.g. different reproductive modes, chemical defences), have a wide distribution, and occupy habitats from the tropical rainforests to semiarid scrubland. In this paper, we present a phylogenetic analysis of this hylid tribe based on sequence fragments of up to five mitochondrial (12S, 16S, ND1, COI, Cytb) and six nuclear genes (POMC, RAG-1, RHOD, SIAH, TNS3, TYR). We included most of its species (> 96%), in addition to a number of new species. Our results indicate: (i) the paraphyly of Trachycephalus with respect to Aparasphenodon venezolanus; (ii) the nonmonophyly of Aparasphenodon, with Argenteohyla siemersi, Corythomantis galeata and Nyctimantis rugiceps nested within it, and Ap. venezolanus nested within Trachycephalus; (iii) the polyphyly of Corythomantis; (iv) the nonmonophyly of the recognized species groups of Phyllodytes; and (v) a pervasive low support for the deep relationships among the major clades of Lophyohylini, including C. greeningi and the monotypic genera Itapotihyla and Phytotriades. To remedy the nonmonophyly of Aparasphenodon, Corythomantis, and Trachycephalus, we redefined Nyctimantis to include Aparasphenodon (with the exception of Ap. venezolanus, which we transferred to Trachycephalus), Argenteohyla, and C. galeata. Additionally, our results indicate the need for taxonomic work in the following clades: (i) Trachycephalus dibernardoi and Tr. imitatrix; (ii) Tr. atlas, Tr. mambaiensis and Tr. nigromaculatus; and (iii) Phyllodytes. On the basis of our phylogenetic results, we analyzed the evolution of skull hyperossification and reproductive biology, with emphasis on the multiple independent origins of phytotelm breeding, in the context of Anura. We also analyzed the inter-related aspects of chemical defences, venom delivery, phragmotic behaviour, co-ossification, and prevention of evaporative water loss.

Lens transmittance shapes ultraviolet sensitivity in the eyes of frogs from diverse ecological and phylogenetic backgrounds.

The amount of short wavelength (ultraviolet (UV), violet and blue) light that reaches the retina depends on the transmittance properties of the ocular media, especially the lens, and varies greatly across species in all vertebrate groups studied previously. We measured the lens transmittance in 32 anuran amphibians with different habits, geographical distributions and phylogenetic positions and used them together with eye size and pupil shape to evaluate the relationship with diel activity pattern, elevation and latitude. We found an unusually high lens UV transmittance in the most basal species, and a cut-off range that extends into the visible spectrum for the rest of the sample, with lenses even absorbing violet light in some diurnal species. However, other diurnal frogs had lenses that transmit UV light like the nocturnal species. This unclear pattern in the segregation of ocular media transmittance and diel activity is shared with other vertebrates and is consistent with the absence of significant correlations in our statistical analyses. Although we did not detect a significant phylogenetic effect, closely related species tend to have similar transmittances, irrespective of whether they share the same diel pattern or not, suggesting that anuran ocular media transmittance properties might be related to phylogeny.

A dune with a view: the eyes of a neotropical fossorial lizard.

BACKGROUND: Lizards are excellent models to study the adaptations of the visual system to different scenarios, and surface-dwelling representatives have been relatively well studied. In contrast, very little is known about the functional anatomy of the eyes of fossorial lineages, and properties such as the light transmission by the ocular media have never been characterised in any fossorial species. Some lizards in the family Gymnophthalmidae endemic to the sand dunes of North Eastern Brazil have evolved sand-burrowing habits and nocturnal activity. Lizards in the sister group to Gymnophthalmidae, the family Teiidae, have decidedly diurnal and epigeal lifestyles, yet they are equally poorly known in terms of visual systems. We focussed on the eye anatomy, photoreceptor morphology and light transmittance properties of the ocular media and oil droplets in the gymnophthalmid Calyptommatus nicterus and the teiid Ameivula ocellifera. RESULTS: The general organisation of the eyes of the fossorial nocturnal C. nicterus and the epigeal diurnal A. ocellifera is remarkably similar. The lenses are highly transmissive to light well into the ultraviolet part of the spectrum. The photoreceptors have the typical cone morphology, with narrow short outer segments and oil droplets. The main difference between the two species is that C. nicterus has only colourless oil droplets, whereas A. ocellifera has colourless as well as green-yellow and pale-orange droplets. CONCLUSIONS: Our results challenge the assumption that fossorial lizards undergo loss of visual function, a claim that is usually guided by the reduced size and external morphology of their eyes. In the case of C. nicterus, the visual system is well suited for vision in bright light and shows specialisations that improve sensitivity in dim light, suggesting that they might perform some visually-guided behaviour above the surface at the beginning or the end of their daily activity period, when light levels are relatively high in their open dunes habitat. This work highlights how studies on the functional anatomy of sensory systems can provide insights into the habits of secretive species.

Differences in ocular media transmittance in classical frog and toad model species and its impact on visual sensitivity.

The transmittance properties of the cornea, lens and humours of vertebrates determine how much light across the visible spectrum reaches the retina, influencing sensitivity to visual stimuli. Amphibians are the only vertebrate class in which the light transmittance of these ocular media has not been thoroughly characterised, preventing large-scale comparative studies and precise quantification of visual stimuli in physiological and behavioural experiments. We measured the ocular media transmittance in some commonly used species of amphibians (the bufonids Bufo bufo and Rhinella ornate, and the ranids Lithobates catesbeianus and Rana temporaria) and found low transmittance of short wavelength light, with ranids having less transmissive ocular media than bufonids. Our analyses also show that these transmittance properties have a considerable impact on spectral sensitivity, highlighting the need to incorporate this type of measurement into the design of stimuli for experiments on visual function.

A total evidence analysis of the phylogeny of hatchet-faced treefrogs (Anura: Hylidae: Sphaenorhynchus).

The Neotropical hylid genus Sphaenorhynchus includes 15 species of small, greenish treefrogs widespread in the Amazon and Orinoco basins, and in the Atlantic Forest of Brazil. Although some studies have addressed the phylogenetic relationships of the genus with other hylids using a few exemplar species, its internal relationships remain poorly understood. In order to test its monophyly and the relationships among its species, we performed a total evidence phylogenetic analysis of sequences of three mitochondrial and three nuclear genes, and 193 phenotypic characters from all species of Sphaenorhynchus. Our results support the monophyly of Sphaenorhynchus with molecular and phenotypic evidence, with S. pauloalvini as the earliest diverging taxon, followed by S. carneus, as the sister taxon of all remaining species of the genus. We recognize three species groups in Sphaenorhynchus (the S. lacteus, S. planicola and S. platycephalus groups), to facilitate its taxonomic study; only three species (S. carneus, S. pauloalvini and S. prasinus) remain unassigned to any group. Sequence data were not available for only two species (S. bromelicola and S. palustris) for which we scored phenotypic data; wildcard behaviour was detected only in S. bromelicola nested inside the S. platycephalus group. On the basis of the resulting phylogenetic hypothesis, we discuss the evolution of oviposition site and a number of phenotypic characters that could be associated with heterochronic events in the evolutionary history of this group.

Phylogeny of Riama (Squamata: Gymnophthalmidae), impact of phenotypic evidence on molecular datasets, and the origin of the Sierra Nevada de Santa Marta endemic fauna.

Riama is the most speciose genus of the Neotropical lizard family Gymnophthalmidae. Its more than 30 montane species occur throughout the northern Andes, the Cordillera de la Costa (CC) in Venezuela, and Trinidad. We present the most comprehensive phylogenetic analysis of Riama to date based on a total evidence (TE) approach and direct optimization of molecular and morphological evidence. Analyses use DNA sequences from four loci and 35 phenotypic characters. The dataset consists of 55 ingroup terminals representing 25 of the 30 currently recognized species of Riama plus five undescribed taxa, including an endemic species from the Sierra Nevada de Santa Marta (SNSM) in Colombia, and 66 outgroup terminals of 47 species. Analysis results in a well-supported hypothesis in which Riama is polyphyletic, with its species falling into three clades. The Tepuian Anadia mcdiarmidi nests within one clade of Riama, and the recently resurrected Pantodactylus nests within Cercosaura. Accordingly, we propose a monophyletic taxonomy that reflects historical relationships. Analysis of character evolution indicates that the presence/absence of prefrontals-a cornerstone of the early genus-level taxonomy of cercosaurines-is optimally explained as having been plesiomorphically present in the most recent common ancestor of Cercosaurinae and lost in that of the immediately less inclusive clade. Multiple independent reversals to present and subsequent returns to absent occur within this clade. To evaluate the impact of phenotypic evidence on our results, we compare our TE results with results obtained from analyses using only molecular data. Although phenotypic evidence comprises only 1.2% of the TE matrix, its inclusion alters both the topology and support values of the clades that do not differ. Finally, current phylogenetic evidence reveals a SNSM-CC-Trinidad-tepuis biogeographical link. We hypothesize that an ancient connection facilitated the exchange of species between the SNSM and the CC.

The relationship between poison frog chemical defenses and age, body size, and sex.

INTRODUCTION: Amphibians secrete a wide diversity of chemicals from skin glands as defense against predators, parasites, and pathogens. Most defensive chemicals are produced endogenously through biosynthesis, but poison frogs sequester lipophilic alkaloids from dietary arthropods. Alkaloid composition varies greatly, even among conspecific individuals collected at the same time and place, with some individuals having only a few micrograms of one or a few alkaloids and others possessing >1 mg of >30 alkaloids. The paucity of alkaloids in juveniles and their abundance in adults suggests that alkaloids accumulate over time; however, alkaloid diversity is highly variable among adult poison frogs and has never been studied in relation to individual age. Using skeletochronology to infer individual ages and gas chromatography-mass spectrometry and vapor phase Fourier-transform infrared spectral analysis to identify the defensive chemicals of 63 individuals, we tested the relationship between defensive chemicals and age, size, and sex in the Brazilian red-belly toad, Melanophryniscus moreirae, a poison frog that possesses both sequestered alkaloids and the biosynthesized indolealkylamine bufotenine. RESULTS: Adult females were, on average, older and larger than adult males. Juveniles were smaller but not necessarily younger than adults and possessed bufotenine and 18 of the 37 alkaloids found in adults. Alkaloid richness was positively related to age, but not size, whereas the quantities of sequestered alkaloids and bufotenine were positively related to size, but not age. Defensive chemicals were unrelated to sex, independent of size. CONCLUSIONS: The relationship between alkaloid richness and age appears to result from the gradual accumulation of alkaloids over a frog's lifetime, whereas the relationship between the quantity of defensive chemicals and size appears to be due to the greater storage capacity of larger individuals. The decoupling of age and size effects increases the amount of individual variation that can occur within a population, thereby possibly enhancing anti-predator efficacy. Further, given that both richness and quantity contribute to the overall chemical defense of individual frogs, our results suggest that older, larger individuals are better defended than younger, smaller ones. These considerations underscore the importance of including age in studies of the causes and consequences of variation in poison frog chemical defenses.

Sequestered and Synthesized Chemical Defenses in the Poison Frog Melanophryniscus moreirae.

Bufonid poison frogs of the genus Melanophryniscus contain alkaloid-based chemical defenses that are derived from a diet of alkaloid-containing arthropods. In addition to dietary alkaloids, bufadienolide-like compounds and indolealkylamines have been identified in certain species of Melanophryniscus. Our study reports, for the first time, the co-occurrence of large quantities of both alkaloids sequestered from the diet and an endogenously biosynthesized indolalkylamine in skin secretions from individual specimens of Melanophryniscus moreirae from Brazil. GC/MS analysis of 55 individuals of M. moreirae revealed 37 dietary alkaloids and the biosynthesized indolealkylamine bufotenine. On average, pumiliotoxin 267C, bufotenine, and allopumilitoxin 323B collectively represent ca. 90 % of the defensive chemicals present in an individual. The quantity of defensive chemicals differed between sexes, with males possessing significantly less dietary alkaloid and bufotenine than females. Most of the dietary alkaloids have structures with branched-chains, indicating they are likely derived from oribatid mites. The ratio of bufotenine:alkaloid quantity decreased with increasing quantities of dietary alkaloids, suggesting that M. moreirae might regulate bufotenine synthesis in relation to sequestration of dietary alkaloids.

Does counting species count as taxonomy? On misrepresenting systematics, yet again.

Recent commentary by Costello and collaborators on the current state of the global taxonomic enterprise attempts to demonstrate that taxonomy is not in decline as feared by taxonomists, but rather is increasing by virtue of the rate at which new species are formally named. Having supported their views with data that clearly indicate as much, Costello et al. make recommendations to increase the rate of new species descriptions even more. However, their views appear to rely on the perception of species as static and numerically if not historically equivalent entities whose value lie in their roles as "metrics". As such, their one-dimensional portrayal of the discipline, as concerned solely with the creation of new species names, fails to take into account both the conceptual and epistemological foundations of systematics. We refute the end-user view that taxonomy is on the rise simply because more new species are being described compared with earlier decades, and that, by implication, taxonomic practice is a formality whose pace can be streamlined without considerable resources, intellectual or otherwise. Rather, we defend the opposite viewpoint that professional taxonomy is in decline relative to the immediacy of the extinction crisis, and that this decline threatens not just the empirical science of phylogenetic systematics, but also the foundations of comparative biology on which other fields rely. The allocation of space in top-ranked journals to propagate views such as those of Costello et al. lends superficial credence to the unsupportive mindset of many of those in charge of the institutional fate of taxonomy. We emphasize that taxonomy and the description of new species are dependent upon, and only make sense in light of, empirically based classifications that reflect evolutionary history; homology assessments are at the centre of these endeavours, such that the biological sciences cannot afford to have professional taxonomists sacrifice the comparative and historical depth of their hypotheses in order to accelerate new species descriptions.

Dietary alkaloid sequestration in a poison frog: an experimental test of alkaloid uptake in Melanophryniscus stelzneri (Bufonidae).

Several lineages of brightly colored anurans independently evolved the ability to secrete alkaloid-containing defensive chemicals from granular glands in the skin. These species, collectively referred to as 'poison frogs,' form a polyphyletic assemblage that includes some species of Dendrobatidae, Mantellidae, Myobatrachidae, Bufonidae, and Eleutherodactylidae. The ability to sequester alkaloids from dietary arthropods has been demonstrated experimentally in most poison frog lineages but not in bufonid or eleutherodactylid poison frogs. As with other poison frogs, species of the genus Melanophryniscus (Bufonidae) consume large numbers of mites and ants, suggesting they might also sequester defensive alkaloids from dietary sources. To test this hypothesis, fruit flies dusted with alkaloid/nutritional supplement powder were fed to individual Melanophryniscus stelzneri in two experiments. In the first experiment, the alkaloids 5,8-disubstituted indolizidine 235B' and decahydroquinoline were administered to three individuals for 104 days. In the second experiment, the alkaloids 3,5-disubstituted indolizidine 239Q and decahydroquinoline were given to three frogs for 153 days. Control frogs were fed fruit flies dusted only with nutritional supplement. Gas chromatography/mass spectrometry analyses revealed that skin secretions of all experimental frogs contained alkaloids, whereas those of all control frogs lacked alkaloids. Uptake of decahydroquinoline was greater than uptake of 5,8-disubstituted indolizidine, and uptake of 3,5-disubstituted indolizidine was greater than uptake of decahydroquinoline, suggesting greater uptake efficiency of certain alkaloids. Frogs in the second experiment accumulated a greater amount of alkaloid, which corresponds to the longer duration and greater number of alkaloid-dusted fruit flies that were consumed. These findings provide the first experimental evidence that bufonid poison frogs sequester alkaloid-based defenses from dietary sources.

Biological invasions and the acoustic niche: the effect of bullfrog calls on the acoustic signals of white-banded tree frogs.

Invasive species are known to affect native species in a variety of ways, but the effect of acoustic invaders has not been examined previously. We simulated an invasion of the acoustic niche by exposing calling native male white-banded tree frogs (Hypsiboas albomarginatus) to recorded invasive American bullfrog (Lithobates catesbeianus) calls. In response, tree frogs immediately shifted calls to significantly higher frequencies. In the post-stimulus period, they continued to use higher frequencies while also decreasing signal duration. Acoustic signals are the primary basis of mate selection in many anurans, suggesting that such changes could negatively affect the reproductive success of native species. The effects of bullfrog vocalizations on acoustic communities are expected to be especially severe due to their broad frequency band, which masks the calls of multiple species simultaneously.

Genetic structure of American bullfrog populations in Brazil.

Non-native species are a major problem affecting numerous biomes around the globe. Information on their population genetics is crucial for understanding their invasion history and dynamics. We evaluated the population structure of the non-native American bullfrog, Aquarana catesbeiana, in Brazil on the basis of 324 samples collected from feral and captive groups at 38 sites in seven of the nine states where feral populations occur. We genotyped all samples using previously developed, highly polymorphic microsatellite loci and performed a discriminant analysis of principal components together with Jost's D index to quantify pairwise differentiation between populations. We then amplified 1,047 base pairs of the mitochondrial cytochrome b (cytb) gene from the most divergent samples from each genetic population and calculated their pairwise differences. Both the microsatellite and cytb data indicated that bullfrogs comprise two populations. Population grouping 1 is widespread and possesses two cytb haplotypes. Population grouping 2 is restricted to only one state and possesses only one of the haplotypes from Population grouping 1. We show that there were two imports of bullfrogs to Brazil and that there is low genetic exchange between population groupings. Also, we find that there is no genetic divergence among feral and captive populations suggesting continuous releases. The limited genetic variability present in the country is associated to the small number of introductions and founders. Feral bullfrogs are highly associated to leaks from farms, and control measures should focus on preventing escapes using other resources than genetics, as feral and captive populations do not differ.

Dose-dependent alkaloid sequestration and N-methylation of decahydroquinoline in poison frogs.

Sequestration of chemical defenses from dietary sources is dependent on the availability of compounds in the environment and the mechanism of sequestration. Previous experiments have shown that sequestration efficiency varies among alkaloids in poison frogs, but little is known about the underlying mechanism. The aim of this study was to quantify the extent to which alkaloid sequestration and modification are dependent on alkaloid availability and/or sequestration mechanism. To do this, we administered different doses of histrionicotoxin (HTX) 235A and decahydroquinoline (DHQ) to captive-bred Adelphobates galactonotus and measured alkaloid quantity in muscle, kidney, liver, and feces. HTX 235A and DHQ were detected in all organs, whereas only DHQ was present in trace amounts in feces. For both liver and skin, the quantity of alkaloid accumulated increased at higher doses for both alkaloids. Accumulation efficiency in the skin increased at higher doses for HTX 235A but remained constant for DHQ. In contrast, the efficiency of HTX 235A accumulation in the liver was inversely related to dose and a similar, albeit statistically nonsignificant, pattern was observed for DHQ. We identified and quantified the N-methylation of DHQ in A. galactonotus, which represents a previously unknown example of alkaloid modification in poison frogs. Our study suggests that variation in alkaloid composition among individuals and species can result from differences in sequestration efficiency related to the type and amount of alkaloids available in the environment.

A new species of Leucostethus (Anura, Dendrobatidae) from Gorgona Island, Colombia.

We describe a new species of Leucostethus from Gorgona Island, a small (13 km2) island located 35 km from the Pacific coast of southern Colombia. The new species most resembles L.argyrogaster and L.fugax from western Amazonia at 340-870 m elev. in Peru and Ecuador, with which it shares pale ventral coloration and orange suffusion of the axilla, groin and concealed surfaces of the hind limb, but it is most closely related to L.bilsa from ca. 340 km SW in the southern Chocó at 420-515 m elev., northwestern Ecuador. We report miniscule white spots on the posteroventral surface of the thighs of the new species and, on the basis of our preliminary assessment of their taxonomic distribution, hypothesize that their presence is a synapomorphy of Dendrobatoidea with subsequent losses in a few groups. Given the apparent restriction of the new species to Gorgona Island, it is Vulnerable according to IUCN Red List criteria. In addition to naming the new species, we also propose the following new combinations: L.alacris (Rivero and Granados-Díaz, 1990) comb. nov., L.dysprosium (Rivero and Serna, 2000) comb. nov., and L.yaguara (Rivero and Serna, 1991) comb. nov.

REP provides meaningful measurement of support across datasets.

The relative optimality of the best and next-best hypotheses indicates the strength of support for the optimal hypothesis and may be calculated as either the difference or ratio of their optimality scores. The Goodman-Bremer support measure (GB) calculates the support for a given clade in the most parsimonious tree as the difference between the length of the optimal tree and the optimal tree that lacks that clade. The ratio of explanatory power (REP) support measure calculates support as the ratio of optimality scores, which simplifies to the ratio of observed GB and the maximum possible GB, GB/GB(max). In this paper we show that REP support provides a logical basis for comparison of support across datasets and that recent claims to the contrary are incorrect.

Use of whole-body cryosectioning and desorption electrospray ionization mass spectrometry imaging to visualize alkaloid distribution in poison frogs.

Ambient mass spectrometry is useful for analyzing compounds that would be affected by other chemical procedures. Poison frogs are known to sequester alkaloids from their diet, but the sequestration pathway is unknown. Here, we describe methods for whole-body cryosectioning of frogs and use desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to map the orally administered alkaloid histrionicotoxin 235A in a whole-body section of the poison frog Dendrobates tinctorius. Our results show that whole-body cryosectioning coupled with histochemical staining and DESI-MSI is an effective technique to visualize alkaloid distribution and help elucidate the mechanisms involved in alkaloid sequestration in poison frogs.