Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa.

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

Species limits and diversification of the Dendropsophus rubicundulus subgroup (Anura, Hylidae) in Neotropical savannas.

Understanding the processes that generate and maintain biodiversity at and below the species level is a central goal of evolutionary biology. Here we explore the spatial and temporal drivers of diversification of the treefrog subgroup Dendropsophus rubicundulus, a subgroup of the D. microcephalus species group, over periods of pronounced geological and climatic changes in the Neotropical savannas that they inhabit. This subgroup currently comprises 11 recognized species distributed across the Brazilian and Bolivian savannas, but the taxonomy has been in a state of flux, necessitating reexamination. Using newly generated single nucleotide polymorphism (SNP) data from restriction-site associated DNA sequencing (RADseq) and mitochondrial 16S sequence data for ∼150 specimens, we inferred phylogenetic relationships, tested species limits using a model-based approach, and estimated divergence times to gain insights into the geographic and climatic events that affected the diversification of this subgroup. Our results recognized at least nine species: D. anataliasiasi, D. araguaya, D. cerradensis, D. elianeae, D. jimi, D. rubicundulus, D. tritaeniatus, D. rozenmani, and D. sanborni. Although we did not collect SNP data for the latter two species, they are likely distinct based on mitochondrial data. In addition, we found genetic structure within the widespread species D. rubicundulus, which comprises three allopatric lineages connected by gene flow upon secondary contact. We also found evidence of population structure and perhaps undescribed diversity in D. elianeae, which warrants further study. The D. rubicundulus subgroup is estimated to have originated in the Late Miocene (∼5.45 million years ago), with diversification continuing through the Pliocene and Early Pleistocene, followed by the most recent divergence of D. rubicundulus lineages in the Middle Pleistocene. The epeirogenic uplift followed by erosion and denudation of the central Brazilian plateau throughout the Pliocene and Pleistocene, in combination with the increasing frequency and amplitude of climatic fluctuations during the Pleistocene, was important for generating and structuring diversity at or below the species level in the D. rubicundulus subgroup.

Linking Predator Responses to Alkaloid Variability in Poison Frogs.

Many chemically-defended/aposematic species rely on diet for sequestering the toxins with which they defend themselves. This dietary acquisition can lead to variable chemical defenses across space, as the community composition of chemical sources is likely to vary across the range of (an aposematic) species. We characterized the alkaloid content of two populations of the Dyeing Poison Frog (Dendrobates tinctorius) in northeastern French Guiana. Additionally, we conducted unpalatability experiments with naive predators, Blue Tits (Cyanistes caeruleus), using whole-skin secretion cocktails to assess how a model predator would respond to the defense of individuals from each population. While there was some overlap between the two D. tinctorius populations in terms of alkaloid content, our analysis revealed that these two populations are markedly distinct in terms of overall alkaloid profiles. Predator responses to skin secretions differed between the populations. We identified 15 candidate alkaloids (including three previously undescribed) in seven classes that are correlated with predator response in one frog population. We describe alkaloid profile differences between populations for D. tinctorius and provide a novel method for assessing unpalatability of skin secretions and identifying which toxins may contribute to the predator response. In one population, our results suggest 15 alkaloids that are implicated in predator aversive response. This method is the first step in identifying the causal link between alkaloids and behavioral responses of predators, and thus makes sense of how varying alkaloid combinations are capable of eliciting consistent behavioral responses, and eventually driving evolutionary change in aposematic characters (or characteristics).

Phylogenomics Reveals Ancient Gene Tree Discordance in the Amphibian Tree of Life.

Molecular phylogenies have yielded strong support for many parts of the amphibian Tree of Life, but poor support for the resolution of deeper nodes, including relationships among families and orders. To clarify these relationships, we provide a phylogenomic perspective on amphibian relationships by developing a taxon-specific Anchored Hybrid Enrichment protocol targeting hundreds of conserved exons which are effective across the class. After obtaining data from 220 loci for 286 species (representing 94% of the families and 44% of the genera), we estimate a phylogeny for extant amphibians and identify gene tree-species tree conflict across the deepest branches of the amphibian phylogeny. We perform locus-by-locus genealogical interrogation of alternative topological hypotheses for amphibian monophyly, focusing on interordinal relationships. We find that phylogenetic signal deep in the amphibian phylogeny varies greatly across loci in a manner that is consistent with incomplete lineage sorting in the ancestral lineage of extant amphibians. Our results overwhelmingly support amphibian monophyly and a sister relationship between frogs and salamanders, consistent with the Batrachia hypothesis. Species tree analyses converge on a small set of topological hypotheses for the relationships among extant amphibian families. These results clarify several contentious portions of the amphibian Tree of Life, which in conjunction with a set of vetted fossil calibrations, support a surprisingly younger timescale for crown and ordinal amphibian diversification than previously reported. More broadly, our study provides insight into the sources, magnitudes, and heterogeneity of support across loci in phylogenomic data sets.[AIC; Amphibia; Batrachia; Phylogeny; gene tree-species tree discordance; genomics; information theory.].

Availability of Recently Described Ptychadena (Anura: Ptychadenidae) Nomina from Ethiopia.

Frogs of the genus Ptychadena (Boulenger, 1917) have long been identified as harboring cryptic diversity and comprising numerous species-complexes (Largen 1997; Zimkus et al. 2017), and many authors have recognized the particularly high hidden richness in the Ethiopian highlands (Largen 1997; see Largen Spawls 2010 and refs. within). This cryptic diversity was confirmed by recent molecular studies (Freilich et al. 2014; Smith et al. 2017a, Reyes-Velasco et al. 2018). Those authors identified a congruent set of evolutionarily distinct candidate species using both mitochondrial and nuclear DNA, and described the geographic and ecological isolation of these species in detail (Freilich et al. 2014; Smith et al. 2017a).

Cryptic diversity among Yazoo Darters (Percidae: Etheostoma raneyi) in disjunct watersheds of northern Mississippi.

The Yazoo Darter, Etheostoma raneyi (Percidae), is an imperiled freshwater fish species endemic to tributaries of the Yocona and Little Tallahatchie rivers of the upper Yazoo River basin, in northern Mississippi, USA. The two populations are allopatric, isolated by unsuitable lowland habitat between the two river drainages. Relevant literature suggests that populations in the Yocona River represent an undescribed species, but a lack of data prevents a thorough evaluation of possible diversity throughout the range of the species. Our goals were to estimate phylogenetic relationships of the Yazoo Darter across its distribution and identify cryptic diversity for conservation management purposes. Maximum likelihood (ML) phylogenetic analyses of the mitochondrial cytochrome b (cytb) gene returned two reciprocally monophyletic clades representing the two river drainages with high support. Bayesian analysis of cytb was consistent with the ML analysis but with low support for the Yocona River clade. Analyses of the nuclear S7 gene yielded unresolved relationships among individuals in the Little Tallahatchie River drainage with mostly low support, but returned a monophyletic clade for individuals from the Yocona River drainage with high support. No haplotypes were shared between the drainages for either gene. Additional cryptic diversity within the two drainages was not indicated. Estimated divergence between Yazoo Darters in the two drainages occurred during the Pleistocene (<1 million years ago) and was likely linked to repeated spatial shifts in suitable habitat and changes in watershed configurations during glacial cycles. Individuals from the Yocona River drainage had lower genetic diversity consistent with the literature. Our results indicate that Yazoo Darters in the Yocona River drainage are genetically distinct and that there is support for recognizing Yazoo Darter populations in the Yocona River drainage as a new species under the unified species concept.

Is Niagara Falls a barrier to gene flow in riverine fishes? A test using genome-wide SNP data from seven native species.

Since the early Holocene, fish population genetics in the Laurentian Great Lakes have been shaped by the dual influences of habitat structure and post-glacial dispersal. Riverscape genetics theory predicts that longitudinal habitat corridors and unidirectional downstream water-flow drive the downstream accumulation of genetic diversity, whereas post-glacial dispersal theory predicts that fish genetic diversity should decrease with increasing distance from glacial refugia. This study examines populations of seven native fish species codistributed above and below the 58 m high Niagara Falls - a hypothesized barrier to gene flow in aquatic species. A better understanding of Niagara Falls' role as a barrier to gene flow and dispersal is needed to identify drivers of Great Lakes genetic diversity and guide strategies to limit exotic species invasions. We used genome-wide SNPs and coalescent models to test whether populations are: (a) genetically distinct, consistent with the Niagara Falls barrier hypothesis; (b) more genetically diverse upstream, consistent with post-glacial expansion theory, or downstream, consistent with the riverscape habitat theory; and (c) have migrated either upstream or downstream past Niagara Falls. We found that genetic diversity is consistently greater below Niagara Falls and the falls are an effective barrier to migration, but two species have probably dispersed upstream past the falls after glacial retreat yet before opening of the Welland Canal. Models restricting migration to after opening of the Welland Canal were generally rejected. These results help explain how river habitat features affect aquatic species' genetic diversity and highlight the need to better understand post-glacial dispersal pathways.

Weak warning signals can persist in the absence of gene flow.

Aposematic organisms couple conspicuous warning signals with a secondary defense to deter predators from attacking. Novel signals of aposematic prey are expected to be selected against due to positive frequency-dependent selection. How, then, can novel phenotypes persist after they arise, and why do so many aposematic species exhibit intrapopulation signal variability? Using a polytypic poison frog (Dendrobates tinctorius), we explored the forces of selection on variable aposematic signals using 2 phenotypically distinct (white, yellow) populations. Contrary to expectations, local phenotype was not always better protected compared to novel phenotypes in either population; in the white population, the novel phenotype evoked greater avoidance in natural predators. Despite having a lower quantity of alkaloids, the skin extracts from yellow frogs provoked higher aversive reactions by birds than white frogs in the laboratory, although both populations differed from controls. Similarly, predators learned to avoid the yellow signal faster than the white signal, and generalized their learned avoidance of yellow but not white. We propose that signals that are easily learned and broadly generalized can protect rare, novel signals, and weak warning signals (i.e., signals with poor efficacy and/or poor defense) can persist when gene flow among populations, as in this case, is limited. This provides a mechanism for the persistence of intrapopulation aposematic variation, a likely precursor to polytypism and driver of speciation.

Differential responses of avian and mammalian predators to phenotypic variation in Australian Brood Frogs.

Anti-predator signaling is highly variable with numerous examples of species employing cryptic coloration to avoid detection or conspicuous coloration (often coupled with a secondary defense) to ensure detection and recollection. While the ends of this spectrum are clear in their function, how species use intermediate signals is less clear. Australian Brood Frogs (Pseudophryne) display conspicuous coloration on both their dorsum and venter. Coupled with the alkaloid toxins these frogs possess, this coloration may be aposematic, providing a protective warning signal to predators. We assessed predation rates of known and novel color patterns and found no difference for avian or mammalian predators. However, when Pseudophryne dorsal phenotypes were collectively compared to the high-contrast ventral phenotype of this genus, we found birds, but not mammals, attacked dorsal phenotypes significantly less frequently than the ventral phenotype. This study, importantly, shows a differential predator response to ventral coloration in this genus which has implications for the evolution of conspicuous signaling in Pseudophryne.

Avian learning favors colorful, not bright, signals.

A few colors, such as red and yellow, are commonly found in aposematic (warning) signaling across taxa, independent of evolutionary relationships. These colors have unique traits (i.e., hue, brightness) that aid in their differentiation, and perhaps, their effectiveness in promoting avoidance learning. This repeated use calls into question the influence of selection on specific warning colors adopted by aposematic prey-predator systems. To disentangle the influence of color characteristics on this process, we trained week-old chickens (Gallus gallus domesticus) to learn to avoid distasteful food that was associated with one of three color signals (yellow, white, red) that varied in both hue and in brightness in order to assess which of these traits most influenced their ability to learn avoidance. Our results show that while chicks learned to avoid all three colors, avoidance was based on the hue, not brightness of the different signals. We found that yellow was the most effective for avoidance learning, followed by red, and finally white. Our results suggest that while these three colors are commonly used in aposematic signaling, predators' ability to learn avoidance differs among them. These results may explain why yellow is among the most common signals across aposematic taxa.

A cytochrome P450 CYP71 enzyme expressed in Sorghum bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone.

Sorgoleone, a major component of the hydrophobic root exudates of Sorghum spp., is probably responsible for many of the allelopathic properties attributed to members of this genus. Much of the biosynthetic pathway for this compound has been elucidated, with the exception of the enzyme responsible for the catalysis of the addition of two hydroxyl groups to the resorcinol ring. A library prepared from isolated Sorghum bicolor root hair cells was first mined for P450-like sequences, which were then analyzed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to identify those preferentially expressed in root hairs. Full-length open reading frames for each candidate were generated, and then analyzed biochemically using both a yeast expression system and transient expression in Nicotiana benthamiana leaves. RNA interference (RNAi)-mediated repression in transgenic S. bicolor was used to confirm the roles of these candidates in the biosynthesis of sorgoleone in planta. A P450 enzyme, designated CYP71AM1, was found to be capable of catalyzing the formation of dihydrosorgoleone using 5-pentadecatrienyl resorcinol-3-methyl ether as substrate, as determined by gas chromatography-mass spectroscopy (GC-MS). RNAi-mediated repression of CYP71AM1 in S. bicolor resulted in decreased sorgoleone contents in multiple independent transformant events. Our results strongly suggest that CYP71AM1 participates in the biosynthetic pathway of the allelochemical sorgoleone.

Correction to 'The role of climatic and geological events in generating diversity in Ethiopian grass frogs (genus Ptychadena)'.

[This corrects the article DOI: 10.1098/rsos.170021.].

The role of climatic and geological events in generating diversity in Ethiopian grass frogs (genus Ptychadena).

Ethiopia is a world biodiversity hotspot and harbours levels of biotic endemism unmatched in the Horn of Africa, largely due to topographic-and thus habitat-complexity, which results from a very active geological and climatic history. Among Ethiopian vertebrate fauna, amphibians harbour the highest levels of endemism, making amphibians a compelling system for the exploration of the impacts of Ethiopia's complex abiotic history on biotic diversification. Grass frogs of the genus Ptychadena are notably diverse in Ethiopia, where they have undergone an evolutionary radiation. We used molecular data and expanded taxon sampling to test for cryptic diversity and to explore diversification patterns in both the highland radiation and two widespread lowland Ptychadena. Species delimitation results support the presence of nine highland species and four lowland species in our dataset, and divergence dating suggests that both geologic events and climatic fluctuations played a complex and confounded role in the diversification of Ptychadena in Ethiopia. We rectify the taxonomy of the endemic P. neumanni species complex, elevating one formally synonymized name and describing three novel taxa. Finally, we describe two novel lowland Ptychadena species that occur in Ethiopia and may be more broadly distributed.

Phylogenetic Analysis of Bacterial Communities in Different Regions of the Gastrointestinal Tract of Agkistrodon piscivorus, the Cottonmouth Snake.

Vertebrates are metagenomic organisms in that they are composed not only of their own genes but also those of their associated microbial cells. The majority of these associated microorganisms are found in the gastrointestinal tract (GIT) and presumably assist in processes such as energy and nutrient acquisition. Few studies have investigated the associated gut bacterial communities of non-mammalian vertebrates, and most rely on captive animals and/or fecal samples only. Here we investigate the gut bacterial community composition of a squamate reptile, the cottonmouth snake, Agkistrodon piscivorus through pyrosequencing of the bacterial 16S rRNA gene. We characterize the bacterial communities present in the small intestine, large intestine and cloaca. Many bacterial lineages present have been reported by other vertebrate gut community studies, but we also recovered unexpected bacteria that may be unique to squamate gut communities. Bacterial communities were not phylogenetically clustered according to GIT region, but there were statistically significant differences in community composition between regions. Additionally we demonstrate the utility of using cloacal swabs as a method for sampling snake gut bacterial communities.

Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.

Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.

Multi-gene phylogeny of Madagascar's plated lizards, Zonosaurus and Tracheloptychus (Squamata: Gerrhosauridae).

We analyzed the phylogenetic relationships of the Malagasy plated lizards in the family Gerrhosauridae based on DNA sequence fragments of four mitochondrial and five nuclear genes. Various clades were strongly supported by the concatenated data set and also recovered by separate analyses of mtDNA and nucDNA. In particular, two clades here named the Z. rufipes group (containing Z. bemaraha , Z. brygooi , Z. rufipes , Z. subunicolor , Z. tsingy and an undescribed candidate species from northern Madagascar) and the Z. ornatus group (containing Z. anelanelany , Z. laticaudatus , Z. karsteni , Z. ornatus , Z. quadrilineaus, and Z. trilineatus) were resolved with strong support. A third clade named the Z. madagascariensis group contains Z. madagascariensis with a nested Z. haraldmeieri; the status of that species requires further investigation. Tentatively we also include Z. aeneus in this species group although its phylogenetic relationships were poorly resolved. A fourth clade with less support included Z. boettgeri and Z. maximus. The phylogenetic position of the genus Tracheloptychus remains uncertain: whereas in the species tree it was recovered as the sister group to Zonosaurus, other methods indicated that it was nested within Zonosaurus, albeit alternative topologies were rejected with only marginal statistical support.

Phylogenetic relationships within the lizard clade Xantusiidae: using trees and divergence times to address evolutionary questions at multiple levels.

Xantusiidae (night lizards) is a clade of small-bodied, cryptic lizards endemic to the New World. The clade is characterized by several features that would benefit from interpretation in a phylogenetic context, including: (1) monophyletic status of extant taxa Cricosaura, Lepidophyma, and Xantusia; (2) a species endemic to Cuba (Cricosaura typica) of disputed age; (3) origins of the parthenogenetic species of Lepidophyma; (4) pronounced micro-habitat differences accompanied by distinct morphologies in both Xantusia and Lepidophyma; and (5) placement of Xantusia riversiana, the only vertebrate species endemic to the California Channel Islands, which is highly divergent from its mainland relatives. This study incorporates extensive new character data from multiple gene regions to investigate the phylogeny of Xantusiidae using the most comprehensive taxonomic sampling available to date. Parsimony and partitioned Bayesian analyses of more than 7 kb of mitochondrial and nuclear sequence data from 11 loci all confirm that Xantusiidae is monophyletic, and comprises three well-supported clades: Cricosaura, Xantusia, and Lepidophyma. The Cuban endemic Cricosaura typica is well supported as the sister to all other xantusiids. Estimates of divergence time indicate that Cricosaura diverged from the (Lepidophyma+Xantusia) clade ≈ 81 million years ago (Ma), a time frame consistent with the separation of the Antilles from North America. Our results also confirm and extend an earlier study suggesting that parthenogenesis has arisen at least twice within Lepidophyma without hybridization, that rock-crevice ecomorphs evolved numerous times (>9) within Xantusia and Lepidophyma, and that the large-bodied Channel Island endemic X. riversiana is a distinct, early lineage that may form the sister group to the small-bodied congeners of the mainland.

Molecular systematics and historical biogeography of tree boas (Corallus spp.).

Inferring the evolutionary and biogeographic history of taxa occurring in a particular region is one way to determine the processes by which the biodiversity of that region originated. Tree boas of the genus Corallus are an ancient clade and occur throughout Central and South America and the Lesser Antilles, making it an excellent group for investigating Neotropical biogeography. Using sequenced portions of two mitochondrial and three nuclear loci for individuals of all recognized species of Corallus, we infer phylogenetic relationships, present the first molecular analysis of the phylogenetic placement of the enigmatic C. cropanii, develop a time-calibrated phylogeny, and explore the biogeographic history of the genus. We found that Corallus diversified within mainland South America, via over-water dispersals to the Lesser Antilles and Central America, and via the traditionally recognized Panamanian land bridge. Divergence time estimates reject the South American Caribbean-Track as a general biogeographic model for Corallus and implicate a role for events during the Oligocene and Miocene in diversification such as marine incursions and the uplift of the Andes. Our findings also suggest that recognition of the island endemic species, C. grenadensis and C. cookii, is questionable as they are nested within the widely distributed species, C. hortulanus. Our results highlight the importance of using widespread taxa when forming and testing biogeographic hypotheses in complex regions and further illustrate the difficulty of forming broadly applicable hypotheses regarding patterns of diversification in the Neotropical region.

Ancient tepui summits harbor young rather than old lineages of endemic frogs.

The flattop mountains (tepuis) of South America are ancient remnants of the Precambrian Guiana Shield plateau. The tepui summits, isolated by their surrounding cliffs that can be up to 1000 m tall, are thought of as "islands in the sky," harboring relict flora and fauna that underwent vicariant speciation due to plateau fragmentation. High endemicity atop tepui summits support the idea of an ancient "Lost World" biota. However, recent work suggests that dispersal between lowlands and summits has occurred long after tepui formation indicating that tepui summits may not be as isolated from the lowlands as researchers have long suggested. Neither view of the origin of the tepui biota (i.e., ancient vicariance vs. recent dispersal) has strong empirical support owing to a lack of studies. We test diversification hypotheses of the Guiana Shield highlands by estimating divergence times of an endemic group of treefrogs, Tepuihyla. We find that diversification of this group does not support an ancient origin for this taxon; instead, divergence times among the highland species are 2-5 Ma. Our data indicate that most highland speciation occurred during the Pliocene. Thus, this unparalleled landscape known as "The Lost World" is inhabited, in part, not by Early Tertiary relicts but neoendemics.

Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species.

Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.