Search

In order to improve the molecular resources available for conservation management of Madagascar's threatened ichthyofauna, we elaborated a curated database of 2860 mitochondrial sequences of the mitochondrial COI, 16S and ND2 genes of Malagasy fishes, of which 1141 sequences of freshwater fishes were newly sequenced for this data set. The data set is mostly composed of COI (2015 sequences) while 16S and ND2 sequences from partly the same samples were used to match the COI sequences to reliably identified reference sequences of these genes. We observed COI uncorrected pairwise genetic distances of 5.2‒31.0% (mean 20.6%) among species belonging to different genera, and 0.0‒22.4% (mean 6.4%) for species belonging to the same genus. Deeply divergent mitochondrial lineages of uncertain attribution were found among Malagasy freshwater eleotrids and gobiids, confirming these groups are in need of taxonomic revision. DNA barcodes assigned to introduced cichlids (tilapias) included Coptodon rendallii, C. zillii, Oreochromis aureus (apparently a new country record), O. cf. mossambicus, O. niloticus, and one undetermined species of Oreochromis, with sequences of up to three species found per location. In aplocheiloid killifishes of the genus Pachypanchax, most species from northern Madagascar had only low mitochondrial divergences, three of these species (P. omalonotus, P. patriciae, and P. varatraza) were not reciprocally monophyletic, and one genetically deviant lineage was discovered in a northern locality, suggesting a need for partial taxonomic revision of this genus. While the lack of voucher specimens for most of the samples sequenced herein precludes final conclusions, our first step towards a DNA barcoding reference library of Madagascar's fishes already demonstrates the value of such a data set for improved taxonomic inventory and conservation management. We strongly suggest further exploration of Madagascar's aquatic environments, which should include detailed photographic documentation and tissue sampling of large numbers of specimens, and collection of preserved voucher specimens as well as of living fish for the buildup of ex situ assurance populations of threatened species complying with the One Plan Approach proposed by the IUCN SSC Conservation Breeding Specialist Group (CBSG). [ABSTRACT FROM AUTHOR]

In the processes that give rise to new species, changes first occur at the population level. But with the continuous nature of the divergence process, change in biological properties delimiting the shift from "individuals of divergent populations" towards "individuals of distinct species", as well as abiotic factors driving the change, remain largely ambivalent. Here we study diversification processes at the population level in a semi-aquatic frog, Mantidactylus (Brygoomantis) bellyi, across the diverse vegetation types of Montagne d'Ambre National Park (MANP), Madagascar. Genetic diversity was assessed with seven newly developed microsatellite markers as well as mitochondrial DNA sequences and concordance with patterns of ecological, morphological, and bioacoustic divergence evaluated. We found M. bellyi lacking mitochondrial differentiation within MANP, while microsatellite datasets partitioned them into three highly differentiated, geographically separated subpopulations (with indications for up to five subpopulations). The molecular grouping–primarily clustering individuals by geographic proximity–was coincident with differences in mean depth and width of waters, suggesting a possible role of fluvial characteristics in genetic exchange in this stream-breeding species. Genetic clustering not consistent with differences in call properties, except for dominant call frequencies under the two-subpopulations model. Morphological divergence was mostly consistent with the genetic clustering; subpopulations strongly differed by their snout-vent length, with individuals from high-elevation subpopulations smaller than those from populations below 1000 m above sea level. These results exemplify how mountains and environmental conditions might primarily shape genetic and morphological divergence in frog populations, without strongly affecting their calls. [ABSTRACT FROM AUTHOR]

The bacterial communities colonizing amphibian skin have been intensively studied due to their interactions with pathogenic chytrid fungi that are causing drastic amphibian population declines. Bacteria of the family Alcaligenaceae, and more specifically of the genus Pigmentiphaga, have been found to be associated specifically to arboreal frogs. Here we analyze their occurrence in a previously assembled global skin microbiome dataset from 205 amphibian species. Pigmentiphaga made up about 5% of the total number of reads in this global dataset. They were mostly found in unrelated arboreal frogs from Madagascar (Mantellidae and Hyperoliidae), but also occurred at low abundances on Neotropical frogs. Based on their 16S sequences, most of the sequences belong to a clade within Pigmentiphaga not assignable to any type strains of the five described species of the genus. One isolate from Madagascar clustered with Pigmentiphaga aceris (>99% sequence similarity on 16S rRNA gene level). Here, we report the full genome sequence of this bacterium which, based on 16S sequences of >97% similarity, has previously been found on human skin, floral nectar, tree sap, stream sediment and soil. Its genome consists of a single circular chromosome with 6,165,255 bp, 5,300 predicted coding sequences, 57 tRNA genes, and three rRNA operons. In comparison with other known Pigmentiphaga genomes it encodes a higher number of genes associated with environmental information processing and cellular processes. Furthermore, it has a biosynthetic gene cluster for a nonribosomal peptide syntethase, and bacteriocin biosynthetic genes can be found, but clusters for β-lactones present in other comparative Pigmentiphaga genomes are lacking. [ABSTRACT FROM AUTHOR]

Immediate and reliable pathogen detection in large numbers of samples is essential in wildlife disease monitoring and is often realized by DNA-based techniques. Pooling samples increases processing efficiency and reduces processing costs, and has been suggested as a viable technique for quantitative PCR detection of fungal amphibian pathogens of the genus Batrachochytrium. For these fungi, this diagnostic method has been validated by in vitro set ups that provided controlled test conditions but did not take into account potential effects from amphibian skin compounds (e.g. skin secretions and Microbiota) on the approach. Some of these skin compounds are known to cause PCR inhibition in single sample applications and could lead to false negative reactions and thereby hamper pathogen detection. In this study we examined the effect of skin compounds on the pooled extraction method by swabbing individuals of seven amphibian species (one Anura and six Caudata) prior to the inoculation of the swabs with chytrid zoospores. For each species, swabs were extracted in pools of different sizes (from one to four swabs) with only one swab per pool being inoculated with zoospores. There were no significant differences regarding the ability to detect zoospores when comparing pool sizes for any species, with a tendency for more false negatives when the inoculated swab had been inoculated with a single zoospore. This study provides further in vivo evidence for the viability of the pooled extraction method for DNA-based detection of pathogens. [ABSTRACT FROM AUTHOR]

Miniaturised frogs form a fascinating but poorly understood amphibian ecomorph and have been exceptionally prone to taxonomic underestimation. The subfamily Cophylinae (family Microhylidae), endemic to Madagascar, has a particularly large diversity of miniaturised species which have historically been attributed to the single genus Stumpffia largely based on their small size. Recent phylogenetic work has revealed that several independent lineages of cophyline microhylids evolved towards highly miniaturised body sizes, achieving adult snout–vent lengths under 16 mm. Here, we describe five new species belonging to three clades that independently miniaturised and that are all genetically highly divergent from their relatives: (i) a new genus (Mini gen. nov.) with three new species from southern Madagascar, (ii) one species of Rhombophryne, and (iii) one species of Anodonthyla. Mini mum sp. nov. from Manombo in eastern Madagascar is one of the smallest frogs in the world, reaching an adult body size of 9.7 mm in males and 11.3 mm in females. Mini scule sp. nov. from Sainte Luce in southeastern Madagascar is slightly larger and has maxillary teeth. Mini ature sp. nov. from Andohahela in southeast Madagascar is larger than its congeners but is similar in build. Rhombophryne proportionalis sp. nov. from Tsaratanana in northern Madagascar is unique among Madagascar’s miniaturised frogs in being a proportional dwarf, exhibiting far less advanced signs of paedomorphism than other species of similar size. Anodonthyla eximia sp. nov. from Ranomafana in eastern Madagascar is distinctly smaller than any of its congeners and is secondarily terrestrial, providing evidence that miniaturisation and terrestriality may be evolutionarily linked. The evolution of body size in Madagascar’s microhylids has been more dynamic than previously understood, and future studies will hopefully shed light on the interplay between ecology and evolution of these remarkably diverse frogs. [ABSTRACT FROM AUTHOR]

The number of species of frogs in the South American genus Adelophryne has increased in recent years, and it has become apparent that this group contains a substantial amount of undescribed diversity. Currently the genus contains nine described species and five candidate species. Here we describe the tenth species of the genus Adelophryne from the municipality of Igrapiúna, southern Bahia state, Brazil. The new species is characterized by its small body size, indistinct tympanum, and two phalanges in the finger IV. The species of the genus are distributed in three groups, Northern Amazonia Clade, Northern Atlantic Forest Clade and Southern Atlantic Forest Clade. The new species is phylogenetically related to species of the Northern Atlantic Forest Clade of Adelophryne and restricted to forested habitat, as typical for other Adelophryne. The species is restricted to the pristine forests in the type locality, and we consider its conservation status as Near Threatened. New morphological and molecular data of other Adelophryne species are presented, extending the distribution of Adelophryne sp. 2, Adelophryne sp. 4, Adelophryne mucronata and Adelophryne glandulata. However, a more comprehensive revision of the diversity and phylogenetic position of most Adelophryne species is needed, and the evolutionary relationships of A. meridionalis and A. pachydactyla remain unknown. [ABSTRACT FROM AUTHOR]

The fungal pathogen Batrachochytrium dendrobatidis (Bd) drives declines and extinctions in amphibian communities. However, not all regions and species are equally affected. Here, we show that association with amphibian aquatic habitat types (bromeliad phytotelmata versus stream) across Central America results in the odds of being threatened by Bd being five times higher in stream microhabitats. This differential threat of Bd was supported in our study by a significantly lower prevalence of Bd in bromeliad-associated amphibian species compared to riparian species in Honduran cloud forests. Evidence that the bromeliad environment is less favorable for Bd transmission is exemplified by significantly less suitable physicochemical conditions and higher abundance of Bd-ingesting micro-eukaryotes present in bromeliad water. These factors may inhibit aquatic Bd zoospore survival and the development of an environmental reservoir of the pathogen. Bromeliad phytotelmata thus may act as environmental refuges from Bd, which contribute to protecting associated amphibian communities against chytridiomycosis-driven amphibian declines that threaten the nearby riparian communities. [ABSTRACT FROM AUTHOR]

Assemblages that are exposed to recurring temporal environmental changes can show changes in their ecological properties. These can be expressed by differences in diversity and assembly rules. Both can be identified using two measures of diversity: functional (FD) and phylogenetic diversity (PD). Frog communities are understudied in this regard, especially during the tadpole life stage. We utilised tadpole assemblages from Madagascan rainforest streams to test predictions of seasonal changes on diversity and assemblage composition and on diversity measures. From the warm-wet to the cool-dry season, species richness (SR) of tadpole assemblages decreased. Also FD and PD decreased, but FD less and PD more than expected by chance. During the dry season, tadpole assemblages were characterised by functional redundancy (among assemblages—with increasing SR), high FD (compared to a null model), and low PD (phylogenetic clustering; compared to a null model). Although mutually contradictory at first glance, these results indicate competition as tadpole community assembly driving force. This is true during the limiting cool-dry season but not during the more suitable warm-wet season. We thereby show that assembly rules can strongly depend on season, that comparing FD and PD can reveal such forces, that FD and PD are not interchangeable, and that conclusions on assembly rules based on FD alone are critical. [ABSTRACT FROM AUTHOR]

Madagascar has become a model region for testing hypotheses of species diversification and biogeography, and many studies have focused on its diverse and highly endemic herpetofauna. Here we combine species distribution models of a near-complete set of species of reptiles and amphibians known from the island with body size data and a tabulation of herpetofaunal communities from field surveys, compiled up to 2008. Though taxonomic revisions and novel distributional records arose since compilation, we are confident that the data are appropriate for inferring and comparing biogeographic patterns among these groups of organisms. We observed species richness of both amphibians and reptiles was highest in the humid rainforest biome of eastern Madagascar, but reptiles also show areas of high richness in the dry and subarid western biomes. In several amphibian subclades, especially within the Mantellidae, species richness peaks in the central eastern geographic regions while in reptiles different subclades differ distinctly in their richness centers. A high proportion of clades and subclades of both amphibians and reptiles have a peak of local endemism in the topographically and bioclimatically diverse northern geographic regions. This northern area is roughly delimited by a diagonal spanning from 15.5°S on the east coast to ca. 15.0°S on the west coast. Amphibian diversity is highest at altitudes between 800–1200 m above sea-level whereas reptiles have their highest richness at low elevations, probably reflecting the comparatively large number of species specialized to the extended low-elevation areas in the dry and subarid biomes. We found that the range sizes of both amphibians and reptiles strongly correlated with body size, and differences between the two groups are explained by the larger body sizes of reptiles. However, snakes have larger range sizes than lizards which cannot be readily explained by their larger body sizes alone. Range filling, i.e., the amount of suitable habitat occupied by a species, is less expressed in amphibians than in reptiles, possibly reflecting their lower dispersal capacity. Taxonomic composition of communities assessed by field surveys is largely explained by bioclimatic regions, with communities from the dry and especially subarid biomes distinctly differing from humid and subhumid biomes. [ABSTRACT FROM AUTHOR]

Scincine lizards in Madagascar form an endemic clade of about 60 species exhibiting a variety of ecomorphological adaptations. Several subclades have adapted to burrowing and convergently regressed their limbs and eyes, resulting in a variety of partial and completely limbless morphologies among extant taxa. However, patterns of limb regression in these taxa have not been studied in detail. Here we fill this gap in knowledge by providing a phylogenetic analysis of DNA sequences of three mitochondrial and four nuclear gene fragments in an extended sampling of Malagasy skinks, and microtomographic analyses of osteology of various burrowing taxa adapted to sand substrate. Based on our data we propose to (i) consider Sirenoscincus Sakata & Hikida, 2003, as junior synonym of Voeltzkowia Boettger, 1893; (ii) resurrect the genus name Grandidierina Mocquard, 1894, for four species previously included in Voeltzkowia; and (iii) consider Androngo Brygoo, 1982, as junior synonym of Pygomeles Grandidier, 1867. By supporting the clade consisting of the limbless Voeltzkowia mira and the forelimb-only taxa V. mobydick and V. yamagishii, our data indicate that full regression of limbs and eyes occurred in parallel twice in the genus Voeltzkowia (as hitherto defined) that we consider as a sand-swimming ecomorph: in the Voeltzkowia clade sensu stricto the regression first affected the hindlimbs and subsequently the forelimbs, whereas the Grandidierina clade first regressed the forelimbs and subsequently the hindlimbs following the pattern prevalent in squamates. Timetree reconstructions for the Malagasy Scincidae contain a substantial amount of uncertainty due to the absence of suitable primary fossil calibrations. However, our preliminary reconstructions suggest rapid limb regression in Malagasy scincids with an estimated maximal duration of 6 MYr for a complete regression in Paracontias, and 4 and 8 MYr respectively for complete regression of forelimbs in Grandidierina and hindlimbs in Voeltzkowia. [ABSTRACT FROM AUTHOR]

Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence Ctax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index Rtax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies. [ABSTRACT FROM AUTHOR]

Analyses of phylogenetic topology and estimates of divergence timing have facilitated a reconstruction of Madagascar’s colonization events by vertebrate animals, but that information alone does not reveal the major factors shaping the island’s biogeographic history. Here, we examine profiles of Malagasy vertebrate clades through time within the context of the island’s paleogeographical evolution to determine how particular events influenced the arrival of the island’s extant groups. First we compare vertebrate profiles on Madagascar before and after selected events; then we compare tetrapod profiles on Madagascar to contemporary tetrapod compositions globally. We show that changes from the Mesozoic to the Cenozoic in the proportions of Madagascar’s tetrapod clades (particularly its increase in the representation of birds and mammals) are tied to changes in their relative proportions elsewhere on the globe. Differences in the representation of vertebrate classes from the Mesozoic to the Cenozoic reflect the effects of extinction (i.e., the non-random susceptibility of the different vertebrate clades to purported catastrophic global events 65 million years ago), and new evolutionary opportunities for a subset of vertebrates with the relatively high potential for transoceanic dispersal potential. In comparison, changes in vertebrate class representation during the Cenozoic are minor. Despite the fact that the island’s isolation has resulted in high vertebrate endemism and a unique and taxonomically imbalanced extant vertebrate assemblage (both hailed as testimony to its long isolation), that isolation was never complete. Indeed, Madagascar’s extant tetrapod fauna owes more to colonization during the Cenozoic than to earlier arrivals. Madagascar’s unusual vertebrate assemblage needs to be understood with reference to the basal character of clades originating prior to the K-T extinction, as well as to the differential transoceanic dispersal advantage of other, more recently arriving clades. Thus, the composition of Madagascar’s endemic vertebrate assemblage itself provides evidence of the island's paleogeographic history. [ABSTRACT FROM AUTHOR]

Background: DNA barcoding of non-avian reptiles based on the cytochrome oxidase subunit I (COI) gene is still in a very early stage, mainly due to technical problems. Using a newly developed set of reptile-specific primers for COI we present the first comprehensive study targeting the entire reptile fauna of the fourth-largest island in the world, the biodiversity hotspot of Madagascar Methodology/Principal Findings: Representatives of the majority of Madagascan non-avian reptile species (including Squamata and Testudines) were sampled and successfully DNA barcoded. The new primer pair achieved a constantly high success rate (72.7-100%) for most squamates. More than 250 species of reptiles (out of the 393 described ones; representing around 64% of the known diversity of species) were barcoded. The average interspecific genetic distance within families ranged from a low of 13.4% in the Boidae to a high of 29.8% in the Gekkonidae. Using the average genetic divergence between sister species as a threshold, 41-48 new candidate (undescribed) species were identified. Simulations were used to evaluate the performance of DNA barcoding as a function of completeness of taxon sampling and fragment length. Compared with available multi-gene phylogenies, DNA barcoding correctly assigned most samples to species, genus and family with high confidence and the analysis of fewer taxa resulted in an increased number of well supported lineages Shorter marker-lengths generally decreased the number of well supported nodes, but even mini-barcodes of 100 bp correctly assigned many samples to genus and family Conclusions/Significance:The new protocols might help to promote DNA barcoding of reptiles and the established library of reference DNA barcodes will facilitate the molecular identification of Madagascan reptiles. Our results might be useful to easily recognize undescribed diversity (i.e. novel taxa), to resolve taxonomic problems, and to monitor the international pet trade without specialized expert knowledge. [ABSTRACT FROM AUTHOR]

Background: One clade of Malagasy leaf chameleons, the Brookesia minima group, is known to contain species that rank among the smallest amniotes in the world. We report on a previously unrecognized radiation of these miniaturized lizards comprising four new species described herein. Methodology/Principal Findings: The newly discovered species appear to be restricted to single, mostly karstic, localities in extreme northern Madagascar: Brookesia confidens sp. n. from Ankarana, B. desperata sp. n. from Forê t d'Ambre, B. micra sp. n. from the islet Nosy Hara, and B. tristis sp. n. from Montagne des Français. Molecular phylogenetic analyses based on one mitochondrial and two nuclear genes of all nominal species in the B. minima group congruently support that the four new species, together with B. tuberculata from Montagne d'Ambre in northern Madagascar, form a strongly supported clade. This suggests that these species have diversified in geographical proximity in this small area. All species of the B. minima group, including the four newly described ones, are characterized by very deep genetic divergences of 18-32% in the ND2 gene and >6% in the 16S rRNA gene. Despite superficial similarities among all species of this group, their status as separate evolutionary lineages is also supported by moderate to strong differences in external morphology, and by clear differences in hemipenis structure. Conclusion/Significance: The newly discovered dwarf chameleon species represent striking cases of miniaturization and microendemism and suggest the possibility of a range size-body size relationship in Malagasy reptiles. The newly described Brookesia micra reaches a maximum snout-vent length in males of 16 mm, and its total length in both sexes is less than 30 mm, ranking it among the smallest amniote vertebrates in the world. With a distribution limited to a very small islet, this species may represent an extreme case of island dwarfism. [ABSTRACT FROM AUTHOR]

Background: DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species. Methodology/Principal Findings: In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob. Conclusions/Significance: DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species. [ABSTRACT FROM AUTHOR]

Poison frogs acquire chemical defenses from the environment for protection against potential predators. These defensive chemicals are lipophilic alkaloids that are sequestered by poison frogs from dietary arthropods and stored in skin glands. Despite decades of research focusing on identifying poison frog alkaloids, we know relatively little about how environmental variation and subsequent arthropod availability impacts alkaloid loads in poison frogs. We investigated how seasonal environmental variation influences poison frog chemical profiles through changes in the diet of the Climbing Mantella (Mantella laevigata). We collected M. laevigata females on the Nosy Mangabe island reserve in Madagascar during the wet and dry seasons and tested the hypothesis that seasonal differences in rainfall is associated with changes in diet composition and skin alkaloid profiles of M. laevigata. The arthropod diet of each frog was characterized into five groups (i.e. ants, termites, mites, insect larvae, or 'other') using visual identification and cytochrome oxidase 1 DNA barcoding. We found that frog diet differed between the wet and dry seasons, where frogs had a more diverse diet in the wet season and consumed a higher percentage of ants in the dry season. To determine if seasonality was associated with variation in frog defensive chemical composition, we used gas chromatography / mass spectrometry to quantify alkaloids from individual skin samples. Although the assortment of identified alkaloids was similar across seasons, we detected significant differences in the abundance of certain alkaloids, which we hypothesize reflects seasonal variation in the diet of M. laevigata. We suggest that these variations could originate from seasonal changes in either arthropod leaf litter composition or changes in frog behavioral patterns. Although additional studies are needed to understand the consequences of long-term environmental shifts, this work suggests that alkaloid profiles are relatively robust against short-term environmental perturbations., Competing Interests: The authors have declared that no competing interests exist.

Species distributed across vast continental areas and across major biomes provide unique model systems for studies of biotic diversification, yet also constitute daunting financial, logistic and political challenges for data collection across such regions. The tree frog Dendropsophus minutus (Anura: Hylidae) is a nominal species, continentally distributed in South America, that may represent a complex of multiple species, each with a more limited distribution. To understand the spatial pattern of molecular diversity throughout the range of this species complex, we obtained DNA sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and the 16S rhibosomal gene (16S) for 407 samples of D. minutus and closely related species distributed across eleven countries, effectively comprising the entire range of the group. We performed phylogenetic and spatially explicit phylogeographic analyses to assess the genetic structure of lineages and infer ancestral areas. We found 43 statistically supported, deep mitochondrial lineages, several of which may represent currently unrecognized distinct species. One major clade, containing 25 divergent lineages, includes samples from the type locality of D. minutus. We defined that clade as the D. minutus complex. The remaining lineages together with the D. minutus complex constitute the D. minutus species group. Historical analyses support an Amazonian origin for the D. minutus species group with a subsequent dispersal to eastern Brazil where the D. minutus complex originated. According to our dataset, a total of eight mtDNA lineages have ranges >100,000 km2. One of them occupies an area of almost one million km2 encompassing multiple biomes. Our results, at a spatial scale and resolution unprecedented for a Neotropical vertebrate, confirm that widespread amphibian species occur in lowland South America, yet at the same time a large proportion of cryptic diversity still remains to be discovered.

Background: An understanding of the conservation status of Madagascar's endemic reptile species is needed to underpin conservation planning and priority setting in this global biodiversity hotspot, and to complement existing information on the island's mammals, birds and amphibians. We report here on the first systematic assessment of the extinction risk of endemic and native non-marine Malagasy snakes, lizards, turtles and tortoises., Methodology/principal Findings: Species range maps from The IUCN Red List of Threatened Species were analysed to determine patterns in the distribution of threatened reptile species. These data, in addition to information on threats, were used to identify priority areas and actions for conservation. Thirty-nine percent of the data-sufficient Malagasy reptiles in our analyses are threatened with extinction. Areas in the north, west and south-east were identified as having more threatened species than expected and are therefore conservation priorities. Habitat degradation caused by wood harvesting and non-timber crops was the most pervasive threat. The direct removal of reptiles for international trade and human consumption threatened relatively few species, but were the primary threats for tortoises. Nine threatened reptile species are endemic to recently created protected areas., Conclusions/significance: With a few alarming exceptions, the threatened endemic reptiles of Madagascar occur within the national network of protected areas, including some taxa that are only found in new protected areas. Threats to these species, however, operate inside and outside protected area boundaries. This analysis has identified priority sites for reptile conservation and completes the conservation assessment of terrestrial vertebrates in Madagascar which will facilitate conservation planning, monitoring and wise-decision making. In sharp contrast with the amphibians, there is significant reptile diversity and regional endemism in the southern and western regions of Madagascar and this study highlights the importance of these arid regions to conserving the island's biodiversity.

Delimiting and describing species is fundamental to numerous biological disciplines such as evolution, macroecology, and conservation. Delimiting species as independent evolutionary lineages may and often does yield different outcomes depending on the species criteria applied, but methods should be chosen that minimize the inference of objectively erroneous species limits. Several protocols exploit single-gene or multi-gene coalescence statistics, assignment tests or other rationales related to nuclear DNA (nDNA) allele sharing to automatically delimit species. We apply seven different species delimitation protocols to a taxonomically confusing group of Malagasy lizards (Madascincus), and compare the resulting taxonomies with two newly developed metrics: the Taxonomic index of congruence C tax which quantifies the congruence between two taxonomies, and the Relative taxonomic resolving power index R tax which quantifies the potential of an approach to capture a high number of species boundaries. The protocols differed in the total number of species proposed, between 9 and 34, and were also highly incongruent in placing species boundaries. The Generalized Mixed Yule-Coalescent approach captured the highest number of potential species boundaries but many of these were clearly contradicted by extensive nDNA admixture between sympatric mitochondrial DNA (mtDNA) haplotype lineages. Delimiting species as phenotypically diagnosable mtDNA clades failed to detect two cryptic species that are unambiguous due to a lack of nDNA gene flow despite sympatry. We also consider the high number of species boundaries and their placement by multi-gene Bayesian species delimitation as poorly reliable whereas the Bayesian assignment test approach provided a species delimitation highly congruent with integrative taxonomic practice. The present study illustrates the trade-off in taxonomy between reliability (favored by conservative approaches) and resolving power (favored by inflationist approaches). Quantifying excessive splitting is more difficult than quantifying excessive lumping, suggesting a priority for conservative taxonomies in which errors are more liable to be detected and corrected by subsequent studies.

Background: Amphibians are rapidly vanishing. At the same time, it is most likely that the number of amphibian species is highly underestimated. Recent DNA barcoding work has attempted to define a threshold between intra- and inter-specific genetic distances to help identify candidate species. In groups with high extinction rates and poorly known species boundaries, like amphibians, such tools may provide a way to rapidly evaluate species richness., Methodology: Here we analyse published and new 16S rDNA sequences from 60 frog species of Amazonia-Guianas to obtain a minimum estimate of the number of undescribed species in this region. We combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs., Principal Findings: In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contrary to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species., Conclusions: Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas., Significance: As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised.