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For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.

Batesian mimicry, in which harmless species (mimics) deter predators by deceitfully imitating the warning signals of noxious species (models), generates striking cases of phenotypic convergence that are classic examples of evolution by natural selection. However, mimicry of venomous coral snakes has remained controversial because of unresolved conflict between the predictions of mimicry theory and empirical patterns in the distribution and abundance of snakes. Here we integrate distributional, phenotypic and phylogenetic data across all New World snake species to demonstrate that shifts to mimetic coloration in nonvenomous snakes are highly correlated with coral snakes in both space and time, providing overwhelming support for Batesian mimicry. We also find that bidirectional transitions between mimetic and cryptic coloration are unexpectedly frequent over both long- and short-time scales, challenging traditional views of mimicry as a stable evolutionary ‘end point’ and suggesting that insect and snake mimicry may have different evolutionary dynamics. Toxic and venomous species often have conspicuous warning colouration that is mimicked by harmless species. Here, Davis Rabosky et al . combine phylogenetic and biogeographic analyses to reveal that mimicry of venomous coral snakes has been a major driver of snake colour evolution in the New World.

How biotic interactions, current and historical environment, and biogeographic barriers determine community structure is a fundamental question in ecology and evolution, especially in diverse tropical regions. To evaluate patterns of local and regional diversity, we quantified the phylogenetic composition of 189 hummingbird communities in Ecuador. We assessed how species and phylogenetic composition changed along environmental gradients and across biogeographic barriers. We show that humid, low-elevation communities are phylogenetically overdispersed (coexistence of distant relatives), a pattern that is consistent with the idea that competition influences the local composition of hummingbirds. At higher elevations communities are phylogenetically clustered (coexistence of close relatives), consistent with the expectation of environmental filtering, which may result from the challenge of sustaining an expensive means of locomotion at high elevations. We found that communities in the lowlands on opposite sides of the Andes tend to be phylogenetically similar despite their large differences in species composition, a pattern implicating the Andes as an important dispersal barrier. In contrast, along the steep environmental gradient between the lowlands and the Andes we found evidence that species turnover is comprised of relatively distantly related species. The integration of local and regional patterns of diversity across environmental gradients and biogeographic barriers provides insight into the potential underlying mechanisms that have shaped community composition and phylogenetic diversity in one of the most species-rich, complex regions of the world.

Background Mitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods. Results With the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80–90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes. Conclusions This method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O ₂ affinity for O ₂ loading in the pulmonary circulation and O ₂ unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O ₂ affinity and native elevation. Protein engineering experiments and ancestral-state reconstructions revealed that this correlation is attributable to derived increases in Hb-O ₂ affinity in highland lineages, as well as derived reductions in Hb-O ₂ affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O ₂ affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation.

Herein, we describe a new species of terrestrially-nesting fanged frog from Sulawesi Island, Indonesia. Though male nest attendance and terrestrial egg deposition is known in one other Sulawesi fanged frog ( Limnonectes arathooni ), the new species exhibits a derived reproductive mode unique to the Sulawesi assemblage; male frogs guard one or more clutches of eggs festooned to leaves or mossy boulders one to two meters above small slow-moving streams, trickles, or seeps. This island endemic has thus far been collected at three sites on Sulawesi: one in the Central Core of the island, and two on the Southwest Peninsula—south of the Tempe Depression (a major biogeographical boundary). The new Limnonectes has the smallest adult body size among its Sulawesi congeners—with a maximum snout-vent length of about 30 millimeters. Beyond its unique reproductive behavior and body size, the species is further diagnosed on the basis of advertisement call and genetic distance from sympatric fanged frogs. The discovery and description of the new species highlights the remarkable reproductive trait diversity that characterizes the Sulawesi fanged frog assemblage despite that most species in this radiation have yet to be formally described.

Phenotypic characters with a complex physical basis may have a correspondingly complex evolutionary history. Males in the “bee” hummingbird clade court females with sound from tail-feathers, which flutter during display dives. On a phylogeny of 35 species, flutter sound frequency evolves as a gradual, continuous character on most branches. But on at least six internal branches fall two types of major, saltational changes: mode of flutter changes, or the feather that is the sound source changes, causing frequency to jump from one discrete value to another. In addition to their tail “instruments,” males also court females with sound from their syrinx and wing feathers, and may transfer or switch instruments over evolutionary time. In support of this, we found a negative phylogenetic correlation between presence of wing trills and singing. We hypothesize this transference occurs because wing trills and vocal songs serve similar functions and are thus redundant. There are also three independent origins of self-convergence of multiple signals, in which the same species produces both a vocal (sung) frequency sweep, and a highly similar nonvocal sound. Moreover, production of vocal, learned song has been lost repeatedly. Male bee hummingbirds court females with a diverse, coevolving array of acoustic traits.

We describe a new species of fanged frog (Limnonectes larvaepartus) that is unique among anurans in having both internal fertilization and birth of tadpoles. The new species is endemic to Sulawesi Island, Indonesia. This is the fourth valid species of Limnonectes described from Sulawesi despite that the radiation includes at least 15 species and possibly many more. Fewer than a dozen of the 6455 species of frogs in the world are known to have internal fertilization, and of these, all but the new species either deposit fertilized eggs or give birth to froglets.

Comparison of the taxonomic, phylogenetic, and trait dimensions of beta diversity may uncover the mechanisms that generate and maintain biodiversity, such as geographic isolation, environmental filtering, and convergent adaptation. We developed an approach to predict the relationship between environmental and geographic distance and the dimensions of beta diversity. We tested these predictions using hummingbird assemblages in the northern Andes. We expected taxonomic beta diversity to result from recent geographic barriers limiting dispersal, and we found that cost distance, which includes barriers, was a better predictor than Euclidean distance. We expected phylogenetic beta diversity to result from historical connectivity and found that differences in elevation were the best predictors of phylogenetic beta diversity. We expected high trait beta diversity to result from local adaptation to differing environments and found that differences in elevation were correlated with trait beta diversity. When combining beta diversity dimensions, we observe that high beta diversity in all dimensions results from adaption to different environments between isolated assemblages. Comparisons with high taxonomic, low phylogenetic, and low trait beta diversity occurred among lowland assemblages separated by the Andes, suggesting that geographic barriers have recently isolated lineages in similar environments. We provide insight into mechanisms governing hummingbird biodiversity patterns and provide a framework that is broadly applicable to other taxonomic groups.

Lineage separation and divergence form a temporally extended process whereby populations may diverge genetically, morphologically, or ecologically, and these contingent properties of species provide the operational criteria necessary for species delimitation. We inferred the historical process of lineage formation in the coast horned lizard (Phrynosoma coronatum) species complex by evaluating a diversity of operational species criteria, including divergence in mtDNA (98 specimens; 2,781 bp) and nuclear loci (RAG-1, 1,054 bp; BDNF 529 bp), ecological niches (11 bioclimatic variables; 285 unique localities), and cranial horn shapes (493 specimens; 16 landmarks). A phylogenetic analysis of mtDNA recovers 5 phylogeographic groups arranged latitudinally along the Baja California Peninsula and in California. The 2 southern phylogeographic groups exhibit concordance between genetic, morphological, and ecological divergence; however, differentiation is weak or absent at more recent levels defined by phylogeographic breaks in California. Interpreting these operational species criteria together suggests that there are 3 ecologically divergent and morphologically diagnosable species within the P. coronatum complex. Our 3-species taxonomic hypothesis invokes a deep coalescence event when fitting the mtDNA genealogy into the species tree, which is not unexpected for populations that have diverged recently. Although the hypothesis that the 3 phylogeographic groups distributed across California each represent distinctive species is not supported by all of the operational species criteria evaluated in this study, the conservation status of the imperiled populations represented by these genealogical units remains critical.