Comparative genomics uncovers the evolutionary history, demography, and molecular adaptations of South American canids.

The remarkable radiation of South American (SA) canids produced 10 extant species distributed across diverse habitats, including disparate forms such as the short-legged, hypercarnivorous bush dog and the long-legged, largely frugivorous maned wolf. Despite considerable research spanning nearly two centuries, many aspects of their evolutionary history remain unknown. Here, we analyzed 31 whole genomes encompassing all extant SA canid species to assess phylogenetic relationships, interspecific hybridization, historical demography, current genetic diversity, and the molecular bases of adaptations in the bush dog and maned wolf. We found that SA canids originated from a single ancestor that colonized South America 3.9 to 3.5 Mya, followed by diversification east of the Andes and then a single colonization event and radiation of Lycalopex species west of the Andes. We detected extensive historical gene flow between recently diverged lineages and observed distinct patterns of genomic diversity and demographic history in SA canids, likely induced by past climatic cycles compounded by human-induced population declines. Genome-wide scans of selection showed that disparate limb proportions in the bush dog and maned wolf may derive from mutations in genes regulating chondrocyte proliferation and enlargement. Further, frugivory in the maned wolf may have been enabled by variants in genes associated with energy intake from short-chain fatty acids. In contrast, unique genetic variants detected in the bush dog may underlie interdigital webbing and dental adaptations for hypercarnivory. Our analyses shed light on the evolution of a unique carnivoran radiation and how it was shaped by South American topography and climate change.

A comparative test of inequity aversion in domestic dogs (Canis familiaris) and dingoes (Canis dingo).

Despite much recent empirical work on inequity aversion in nonhuman species, many questions remain about its distribution across taxa and the factors that shape its evolution and expression. Past work suggests that domestic dogs (Canis familiaris) and wolves (Canis lupus) are averse to inequitable resource distributions in contexts that call upon some degree of training such as 'give paw' and 'buzzer press' tasks. However, it is unclear whether inequity aversion appears in other canid species and in other experimental contexts. Using a novel inequity aversion task that does not require specific training, this study helps address these gaps by investigating inequity aversion in domestic dogs and a closely related but non-domesticated canid, the dingo (Canis dingo). Subjects were presented with equal and unequal reward distributions and given the opportunity to approach or refuse to approach allocations. Measures of interest were (1) subjects' refusal to approach when getting no food; (2) approach latency; and (3) social referencing. None of these measures differed systematically across the inequity condition and control conditions in either dogs or dingoes. These findings add to the growing literature on inequity aversion in canids, providing data from a new species and a new experimental context. Additionally, they raise questions about the experimental features that must be in place for inequity aversion to appear in canids.

Ancient DNA from the Asiatic Wild Dog (Cuon alpinus) from Europe.

The Asiatic wild dog (Cuon alpinus), restricted today largely to South and Southeast Asia, was widespread throughout Eurasia and even reached North America during the Pleistocene. Like many other species, it suffered from a huge range loss towards the end of the Pleistocene and went extinct in most of its former distribution. The fossil record of the dhole is scattered and the identification of fossils can be complicated by an overlap in size and a high morphological similarity between dholes and other canid species. We generated almost complete mitochondrial genomes for six putative dhole fossils from Europe. By using three lines of evidence, i.e., the number of reads mapping to various canid mitochondrial genomes, the evaluation and quantification of the mapping evenness along the reference genomes and phylogenetic analysis, we were able to identify two out of six samples as dhole, whereas four samples represent wolf fossils. This highlights the contribution genetic data can make when trying to identify the species affiliation of fossil specimens. The ancient dhole sequences are highly divergent when compared to modern dhole sequences, but the scarcity of dhole data for comparison impedes a more extensive analysis.

Anatomical variations in cervical vertebrae in two species of neotropical canids: What is the meaning?

Anatomical variation in wild canids is rarely described. Altered shapes, uni- or bilaterally situated, were observed in ventral laminae of C6 and/or in C7 vertebral body of two fox species: Lycalopex gymnocercus and Cerdocyon thous. The specimens were young adults, collected dead on highways in southern Brazil. Deformities were considered anatomical variations because apparently there was no interference in function. The occurrence of such variations in two related species from the same area can suggest an environmental common cause rather than genetic issues. Future studies may deepen the understanding between cause and effect of these vertebral deformities in wild canids.

Rapid evolution of the primate larynx?

Tissue vibrations in the larynx produce most sounds that comprise vocal communication in mammals. Larynx morphology is thus predicted to be a key target for selection, particularly in species with highly developed vocal communication systems. Here, we present a novel database of digitally modeled scanned larynges from 55 different mammalian species, representing a wide range of body sizes in the primate and carnivoran orders. Using phylogenetic comparative methods, we demonstrate that the primate larynx has evolved more rapidly than the carnivoran larynx, resulting in a pattern of larger size and increased deviation from expected allometry with body size. These results imply fundamental differences between primates and carnivorans in the balance of selective forces that constrain larynx size and highlight an evolutionary flexibility in primates that may help explain why we have developed complex and diverse uses of the vocal organ for communication.

Genomic regions under selection in the feralization of the dingoes.

Dingoes are wild canids living in Australia, originating from domestic dogs. They have lived isolated from both the wild and the domestic ancestor, making them a unique model for studying feralization. Here, we sequence the genomes of 10 dingoes and 2 New Guinea Singing Dogs. Phylogenetic and demographic analyses show that dingoes originate from dogs in southern East Asia, which migrated via Island Southeast Asia to reach Australia around 8300 years ago, and subsequently diverged into a genetically distinct population. Selection analysis identifies 50 positively selected genes enriched in digestion and metabolism, indicating a diet change during feralization of dingoes. Thirteen of these genes have shifted allele frequencies compared to dogs but not compared to wolves. Functional assays show that an A-to-G mutation in ARHGEF7 decreases the endogenous expression, suggesting behavioral adaptations related to the transitions in environment. Our results indicate that the feralization of the dingo induced positive selection on genomic regions correlated to neurodevelopment, metabolism and reproduction, in adaptation to a wild environment.

Ancient RNA from Late Pleistocene permafrost and historical canids shows tissue-specific transcriptome survival.

While sequencing ancient DNA (aDNA) from archaeological material is now commonplace, very few attempts to sequence ancient transcriptomes have been made, even from typically stable deposition environments such as permafrost. This is presumably due to assumptions that RNA completely degrades relatively quickly, particularly when dealing with autolytic, nuclease-rich mammalian tissues. However, given the recent successes in sequencing ancient RNA (aRNA) from various sources including plants and animals, we suspect that these assumptions may be incorrect or exaggerated. To challenge the underlying dogma, we generated shotgun RNA data from sources that might normally be dismissed for such study. Here, we present aRNA data generated from two historical wolf skins, and permafrost-preserved liver tissue of a 14,300-year-old Pleistocene canid. Not only is the latter the oldest RNA ever to be sequenced, but it also shows evidence of biologically relevant tissue specificity and close similarity to equivalent data derived from modern-day control tissue. Other hallmarks of RNA sequencing (RNA-seq) data such as exon-exon junction presence and high endogenous ribosomal RNA (rRNA) content confirms our data's authenticity. By performing independent technical library replicates using two high-throughput sequencing platforms, we show not only that aRNA can survive for extended periods in mammalian tissues but also that it has potential for tissue identification. aRNA also has possible further potential, such as identifying in vivo genome activity and adaptation, when sequenced using this technology.

Noninvasive DNA-based species and sex identification of Asiatic wild dog (Cuon alpinus).

Asiatic wild dog (Cuon alpinus) or dhole is an endangered canid with fragmented distribution in South, East and Southeast Asia. The remaining populations of this species face severe conservation challenges from anthropogenic interventions, but only limited information is available at population and demography levels. Here, we describe the novel molecular approaches for unambiguous species and sex identification from noninvasively collected dhole samples. We successfully tested these assays on 130 field-collected dhole faecal samples from the Vidarbha part of central Indian tiger landscape that resulted in 97 and 77% successrates in species and sex identification, respectively. These accurate, fast and cheap molecular approaches prove the efficacy of such methods in gathering ecological data from this elusive, endangered canid and show their application in generating population level information from noninvasive samples.

DNA barcoding of three species (Canis aureus, Canis lupus and Vulpes vulpes) of Canidae.

Sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene have been used for DNA barcoding and determining the genetic diversity of mammal species. In the current study, our intention was to test the validity of COI barcodes for detecting genetic divergence and to reveal whether or not there is a genetic variation at this marker within canids. Three species (Canis aureus, Canis lupus and Vulpes vulpes) from the family Canidae were selected for DNA barcoding using samples collected from Iran and Turkey. All three species had unique barcoding sequences and none of the sequences were shared among these species. The mean sequence divergences within and among the species were 0.61% and 12.32%, respectively, which fell into the mean divergence ranges found in some mammal groups. The genetic diversity of these three canid species was relatively higher than that found in previously reported studies.

Bayesian phylogenetic estimation of fossil ages.

Recent advances have allowed for both morphological fossil evidence and molecular sequences to be integrated into a single combined inference of divergence dates under the rule of Bayesian probability. In particular, the fossilized birth-death tree prior and the Lewis-Mk model of discrete morphological evolution allow for the estimation of both divergence times and phylogenetic relationships between fossil and extant taxa. We exploit this statistical framework to investigate the internal consistency of these models by producing phylogenetic estimates of the age of each fossil in turn, within two rich and well-characterized datasets of fossil and extant species (penguins and canids). We find that the estimation accuracy of fossil ages is generally high with credible intervals seldom excluding the true age and median relative error in the two datasets of 5.7% and 13.2%, respectively. The median relative standard error (RSD) was 9.2% and 7.2%, respectively, suggesting good precision, although with some outliers. In fact, in the two datasets we analyse, the phylogenetic estimate of fossil age is on average less than 2 Myr from the mid-point age of the geological strata from which it was excavated. The high level of internal consistency found in our analyses suggests that the Bayesian statistical model employed is an adequate fit for both the geological and morphological data, and provides evidence from real data that the framework used can accurately model the evolution of discrete morphological traits coded from fossil and extant taxa. We anticipate that this approach will have diverse applications beyond divergence time dating, including dating fossils that are temporally unconstrained, testing of the 'morphological clock', and for uncovering potential model misspecification and/or data errors when controversial phylogenetic hypotheses are obtained based on combined divergence dating analyses.This article is part of the themed issue 'Dating species divergences using rocks and clocks'.

The complete mitochondrial genome of the Tibetan fox (Vulpes ferrilata) and implications for the phylogeny of Canidae.

Canidae is a family of carnivores comprises about 36 extant species that have been defined as three distinct monophyletic groups based on multi-gene data sets. The Tibetan fox (Vulpes ferrilata) is a member of the family Canidae that is endemic to the Tibetan Plateau and has seldom been in the focus of phylogenetic analyses. To clarify the phylogenic relationship of V. ferrilata between other canids, we sequenced the mitochondrial genome and firstly attempted to clarify the relative phylogenetic position of V. ferrilata in canids using the complete mitochondrial genome data. The mitochondrial genome of the Tibetan fox was 16,667 bp, including 37 genes (13 protein-coding genes, 2 rRNA, and 22 tRNA) and a control region. A comparison analysis among the sequenced data of canids indicated that they shared a similar arrangement, codon usage, and other aspects. A phylogenetic analysis on the basis of the nearly complete mtDNA genomes of canids agreed with three monophyletic clades, and the Tibetan fox was highly supported as a sister group of the corsac fox within Vulpes. The estimation of the divergence time suggested a recent split between the Tibetan fox and the corsac fox and rapid evolution in canids. There was no genetic evidence for positive selection related to high-altitude adaption for the Tibetan fox in mtDNA and following studies should pay more attention to the detection of positive signals in nuclear genes involved in energy and oxygen metabolisms.

Disentangling canid howls across multiple species and subspecies: Structure in a complex communication channel.

Wolves, coyotes, and other canids are members of a diverse genus of top predators of considerable conservation and management interest. Canid howls are long-range communication signals, used both for territorial defence and group cohesion. Previous studies have shown that howls can encode individual and group identity. However, no comprehensive study has investigated the nature of variation in canid howls across the wide range of species. We analysed a database of over 2000 howls recorded from 13 different canid species and subspecies. We applied a quantitative similarity measure to compare the modulation pattern in howls from different populations, and then applied an unsupervised clustering algorithm to group the howls into natural units of distinct howl types. We found that different species and subspecies showed markedly different use of howl types, indicating that howl modulation is not arbitrary, but can be used to distinguish one population from another. We give an example of the conservation importance of these findings by comparing the howls of the critically endangered red wolves to those of sympatric coyotes Canis latrans, with whom red wolves may hybridise, potentially compromising reintroduced red wolf populations. We believe that quantitative cross-species comparisons such as these can provide important understanding of the nature and use of communication in socially cooperative species, as well as support conservation and management of wolf populations.

The complete mitochondrial genome of Hequ Tibetan Mastiff Canis lupus familiaris (Carnivora: Canidae).

The Hequ Tibetan Mastiff Canis lupus familiaris (Carnivora: Canidae) is a primitive breed of large dogs native to the northeastern Qinghai-Tibetan Plateau of China. In this study, its complete mitochondrial genome sequence has been assembled and characterized using high-throughput Illumina sequencing technology. The circular genome is 16 730 bp in length, and possesses all genomic components as typically found in most other metazoan mitogenomes. The gene arrangement is identical to those of most other vertebrates. Except for ND4L with GTG as its start codon, all the other PCGs are initiated with an ATR (ATA/ATG) codon. Three distinct stop codons are employed, i.e. AGA for CYTB, TAA for ATP6, ATP8, COX1, COX2, ND1, ND4L, ND5 and ND6, and an incomplete stop codon T for COX3, ND2, ND3 and ND4. The nucleotide composition is asymmetric (31.6% A, 25.5% C, 14.2% G and 28.7% T) with an overall A + T content of 60.3%. These data would contribute to our better understanding its evolutionary history.

The role of clade competition in the diversification of North American canids.

The history of biodiversity is characterized by a continual replacement of branches in the tree of life. The rise and demise of these branches (clades) are ultimately determined by changes in speciation and extinction rates, often interpreted as a response to varying abiotic and biotic factors. However, understanding the relative importance of these factors remains a major challenge in evolutionary biology. Here we analyze the rich North American fossil record of the dog family Canidae and of other carnivores to tease apart the roles of competition, body size evolution, and climate change on the sequential replacement of three canid subfamilies (two of which have gone extinct). We develop a novel Bayesian analytic framework to show that competition from multiple carnivore clades successively drove the demise and replacement of the two extinct canid subfamilies by increasing their extinction rates and suppressing their speciation. Competitive effects have likely come from ecologically similar species from both canid and felid clades. These results imply that competition among entire clades, generally considered a rare process, can play a more substantial role than climate change and body size evolution in determining the sequential rise and decline of clades.

Analysis of structural diversity in wolf-like canids reveals post-domestication variants.

BACKGROUND: Although a variety of genetic changes have been implicated in causing phenotypic differences among dogs, the role of copy number variants (CNVs) and their impact on phenotypic variation is still poorly understood. Further, very limited knowledge exists on structural variation in the gray wolf, the ancestor of the dog, or other closely related wild canids. Documenting CNVs variation in wild canids is essential to identify ancestral states and variation that may have appeared after domestication. RESULTS: In this work, we genotyped 1,611 dog CNVs in 23 wolf-like canids (4 purebred dogs, one dingo, 15 gray wolves, one red wolf, one coyote and one golden jackal) to identify CNVs that may have arisen after domestication. We have found an increase in GC-rich regions close to the breakpoints and around 1 kb away from them suggesting that some common motifs might be associated with the formation of CNVs. Among the CNV regions that showed the largest differentiation between dogs and wild canids we found 12 genes, nine of which are related to two known functions associated with dog domestication; growth (PDE4D, CRTC3 and NEB) and neurological function (PDE4D, EML5, ZNF500, SLC6A11, ELAVL2, RGS7 and CTSB). CONCLUSIONS: Our results provide insight into the evolution of structural variation in canines, where recombination is not regulated by PRDM9 due to the inactivation of this gene. We also identified genes within the most differentiated CNV regions between dogs and wolves, which could reflect selection during the domestication process.

Comparative chromosome painting in Carnivora and Pholidota.

The order of Carnivora has been very well characterized with over 50 species analyzed by chromosome painting and with painting probe sets made for 9 Carnivora species. Representatives of almost all families have been studied with few exceptions (Otariidae, Odobenidae, Nandiniidae, Prionodontidae). The patterns of chromosome evolution in Carnivora are discussed here. Overall, many Carnivora species retained karyotypes that only slightly differ from the ancestral carnivore karyotype. However, there are at least 3 families in which the ancestral carnivore karyotype has been severely rearranged - Canidae, Ursidae and Mephitidae. Here we report chromosome painting of yet another Carnivora species with a highly rearranged karyotype, Genetta pardina. Recurrent rearrangements make it difficult to define the ancestral chromosomal arrangement in several instances. Only 2 species of pangolins (Pholidota), a sister order of Carnivora, have been studied by chromosome painting. Future use of whole-genome sequencing data is discussed in the context of solving the questions that are beyond resolution of conventional banding techniques and chromosome painting.

Dispelling dog dogma: an investigation of heterochrony in dogs using 3D geometric morphometric analysis of skull shape.

Heterochrony is an evolutionary mechanism that generates diversity via perturbations of the rate or timing of development that requires very little genetic innovation. As such, heterochrony is thought to be a common evolutionary mechanism in the generation of diversity. Previous research has suggested that dogs evolved via heterochrony and are paedomorphic wolves. This study uses three-dimensional landmark-based coordinate data to investigate heterochronic patterns within the skull morphology of the domestic dog. A total of 677 adult dogs representing 106 different breeds were measured and compared with an ontogenetic series of 401 wolves. Geometric morphometric analysis reveals that the cranial shape of none of the modern breeds of dogs resembles the cranial shapes of adult or juvenile wolves. In addition, investigations of regional heterochrony in the face and neurocranium also reject the hypothesis of heterochrony. Throughout wolf cranial development the position of the face and the neurocranium remain in the same plane. Dogs, however, have a de novo cranial flexion in which the palate is tilted dorsally in brachycephalic and mesaticephalic breeds or tilted ventrally in dolichocephalic and down-face breeds. Dogs have evolved very rapidly into an incredibly morphologically diverse species with very little genetic variation. However, the genetic alterations to dog cranial development that have produced this vast range of phylogenetically novel skull shapes do not coincide with the expectations of the heterochronic model. Dogs are not paedomorphic wolves.

Cytogenetic studies and karyotype nomenclature of three wild canid species: maned wolf (Chrysocyon brachyurus), bat-eared fox (Otocyon megalotis) and fennec fox (Fennecus zerda).

We have analysed the chromosomes of three wild and endangered canid species: the maned wolf (Chrysocyon brachyurus), the bat-eared fox (Otocyon megalotis) and the fennec fox (Fennecuszerda) using classical and molecular cytogenetic methods. For the first time detailed and encompassing descriptions of the chromosomes are presented including the chromosomal assignment of nucleolar organizer regions and the 5S rRNA gene cluster. We propose a karyotype nomenclature with ideograms including more than 300 bands per haploid set for each of these three species which will form the basis for further research. In addition, we propose four basic different patterns of karyotype organization in the family Canidae. A comparison of these patterns with the most recent molecular phylogeny of Canidae revealed that the karyotype evolution of a species is not always strongly connected with its phylogenetic position. Our findings underline the need and justification for basic cytogenetic work in rare and exotic species.

A molecular phylogeny of the Canidae based on six nuclear loci.

We have reconstructed the phylogenetic relationships of 23 species in the dog family, Canidae, using DNA sequence data from six nuclear loci. Individual gene trees were generated with maximum parsimony (MP) and maximum likelihood (ML) analysis. In general, these individual gene trees were not well resolved, but several identical groupings were supported by more than one locus. Phylogenetic analysis with a data set combining the six nuclear loci using MP, ML, and Bayesian approaches produced a more resolved tree that agreed with previously published mitochondrial trees in finding three well-defined clades, including the red fox-like canids, the South American foxes, and the wolf-like canids. In addition, the nuclear data set provides novel indel support for several previously inferred clades. Differences between trees derived from the nuclear data and those from the mitochondrial data include the grouping of the bush dog and maned wolf into a clade with the South American foxes, the grouping of the side-striped jackal (Canis adustus) and black-backed jackal (Canis mesomelas) and the grouping of the bat-eared fox (Otocyon megalotis) with the raccoon dog (Nycteruetes procyonoides). We also analyzed the combined nuclear+mitochondrial tree. Many nodes that were strongly supported in the nuclear tree or the mitochondrial tree remained strongly supported in the nuclear+mitochondrial tree. Relationships within the clades containing the red fox-like canids and South American canids are well resolved, whereas the relationships among the wolf-like canids remain largely undetermined. The lack of resolution within the wolf-like canids may be due to their recent divergence and insufficient time for the accumulation of phylogenetically informative signal.

Isolation and molecular evolution of the selenocysteine tRNA (Cf TRSP) and RNase P RNA (Cf RPPH1) genes in the dog family, Canidae.

In an effort to identify rapidly evolving nuclear sequences useful for phylogenetic analyses of closely related species, we isolated two genes transcribed by RNA polymerase III (pol III), the selenocysteine tRNA gene (TRSP) and an RNase P RNA (RPPH1) gene from the domestic dog (Canis familiaris). We focus on genes transcribed by pol III because their coding regions are small (generally 100-300 base pairs [bp]) and their essential promoter elements are located within a couple of hundred bps upstream of the coding region. Therefore, we predicted that regions flanking the coding region and outside of the promoter elements would be free of constraint and would evolve rapidly. We amplified TRSP from 23 canids and RPPH1 from 12 canids and analyzed the molecular evolution of these genes and their utility as phylogenetic markers for resolving relationships among species in Canidae. We compared the rate of evolution of the gene-flanking regions to other noncoding regions of nuclear DNA (introns) and to the mitochondrial encoded COII gene. Alignment of TRSP from 23 canids revealed that regions directly adjacent to the coding region display high sequence variability. We discuss this pattern in terms of functional mechanisms of transcription. Although the flanking regions evolve no faster than introns, both genes were found to be useful phylogenetic markers, in part, because of the synapomorphic indels found in the flanking regions. Gene trees generated from the TRSP and RPPH1 loci were generally in agreement with the published mtDNA phylogeny and are the first phylogeny of Canidae based on nuclear sequences.