Chromosomal evolution of the Canidae. I. Species with high diploid numbers

The Giemsa banding patterns of seven canid species, including the grey wolf (Canis lupus), the maned wolf (Chrysocyon brachyurus), the bush dog (Speothos venaticus), the crab-eating fox (Cerdocyon thous), the grey fox (Urocyon cinereoargenteus), the bat-eared fox (Otocyon megalotis), and the fennec (Fennecus zerda), are presented and compared. Relative to other members of Canidae, these species have high diploid complements (2n greater than 64) consisting of largely acrocentric chromosomes. They show a considerable degree of chromosome homoeology, but relative to the grey wolf, each species is either missing chromosomes or has unique chromosomal additions and rearrangements. Differences in chromosome morphology among the seven species were used to reconstruct their phylogenetic history. The results suggest that the South American canids are closely related to each other and are derived from a wolf-like progenitor. The fennec and the bat-eared fox seem to be recent derivatives of a lineage that branched early from the wolf-like canids and which also includes the grey fox.     

External brain anatomy in relation to the phylogeny of Caninae (Carnivora: Canidae)

Caninae is one of the most studied mammalian groups, nevertheless there are relatively few comparative studies on their neuroanatomy. This work contributes to a better knowledge of this subfamily, since it describes the external cerebrum anatomy of 29 out of the 35 living Caninae species, 11 of which are described for the first time. Information about their frontal region appears to be a welcome supplement to the study of the phylogeny. Two distinctive features are recognized, that can be traced back in the fossil record: the sulcal pattern medial to the coronal sulci, and the shape and relative size of the proreal gyrus. Four types are described for the first feature: (1) orthogonal: Canis, Lycaon, Cuon, Atelocynus, Speothos , (2) pentagonal: Vulpes, Alopex, Otocyon, † Eucyon , (3) parenthesis-like: † Dusicyon, Pseudalopex, Chrysocyon , (4) heart-shaped: Urocyon, Cerdocyon, Pseudalopex culpaeus, Nyctereutes . Three types are described for the second feature: (1) elongated and bilaterally compressed: Canis, Cuon, Lycaon, Atelocynus, Speothos, Cerdocyon, † Dusicyon, Chrysocyon, Pseudalopex , † Nyctereutes sinensis , † N. tingi , (2) small: Vulpes, Otocyon, Urocyon, Alopex , (3) wide and low: Nyctereutes procyonoides. On the basis of these features some phylogenetic interpretations are presented: the fossil Asian Nyctereutes is close to Cerdocyon, Speothos is close to Atelocynus , Chrysocyon is not related to Canis , Urocyon differs from Vulpes and Pseudalopex culpaeus differs from the rest of the Pseudalopex species.  © 2003 The Linnean Society of London. Zoological Journal of the Linnean Society , 2003, 138 , 505–522.     

Survey of antibodies to Trypanosoma cruzi and Leishmania spp. in gray and red fox populations from North Carolina and Virginia

American trypanosomiasis and leishmaniasis are caused by related hemoflagellate parasites, Trypanosoma cruzi and Leishmania spp., which share several common host species. Both zoonotic protozoans are endemic in the United States. Canines, including domestic and wild canids, are reservoir hosts for human infections with T. cruzi and Leishmania spp. The present study examined the seroprevalence of T. cruzi and Leishmania spp. in wild canids from North Carolina and Virginia. Wild canine species tested in this work included 49 gray foxes (Urocyon cinereoargenteus) and 5 red foxes (Vulpes vulpes). Overall, sera samples from 54 foxes (North Carolina = 43; Virginia = 11) were tested by immunochromatographic strip assays (ICT). Antibodies to T. cruzi were found in 4 (9%) gray foxes from North Carolina and 2 (18%) gray foxes from Virginia. Antibodies to Leishmania spp. were detected in 1 (2%) gray fox from North Carolina. Our results indicate that wild canids are exposed more frequently to T. cruzi in North Carolina than Leishmania spp. and only T. cruzi in Virginia.     

Potential Sources of High Frequency and Biphonic Vocalization in the Dhole (Cuon alpinus)

Biphonation, i.e. two independent fundamental frequencies in a call spectrum, is a prominent feature of vocal activity in dog-like canids. Dog-like canids can produce a low (f0) and a high (g0) fundamental frequency simultaneously. In contrast, fox-like canids are only capable of producing the low fundamental frequency (f0). Using a comparative anatomical approach for revealing macroscopic structures potentially responsible for canid biphonation, we investigated the vocal anatomy for 4 (1 male, 3 female) captive dholes (Cuon alpinus) and for 2 (1 male, 1 female) wild red fox (Vulpes vulpes). In addition, we analyzed the acoustic structure of vocalizations in the same dholes that served postmortem as specimens for the anatomical investigation. All study dholes produced both high-frequency and biphonic calls. The anatomical reconstructions revealed that the vocal morphologies of the dhole are very similar to those of the red fox. These results suggest that the high-frequency and biphonic calls in dog-like canids can be produced without specific anatomical adaptations of the sound-producing structures. We discuss possible production modes for the high-frequency and biphonic calls involving laryngeal and nasal structures.     

A noninvasive method for distinguishing among canid species: amplification and enzyme restriction of DNA from dung

Endangered San Joaquin kit foxes Vulpes macrotis mutica can be sympatrically distributed with as many as four other canids: red fox, gray fox, coyote and domestic dog. Canid scats are often found during routine fieldwork, but cannot be reliably identified to species. To detect and study the endangered kit fox, we developed mitochondrial DNA markers that can be amplified from small amounts of DNA extracted from scats. We amplified a 412-bp fragment of the mitochondrial cytochrome- b gene from scat samples and digested it with three restriction enzymes. The resulting restriction profiles discriminated among all five canid species and correctly identified 10 'unknown' fox scats to species in blind tests. We have applied our technique to identify canids species for an environmental management study and a conservation study. We envision that our protocol, and similar ones developed for other endangered species will be greatly used for conservation management in the future.     

GENETIC SUBDIVISIONS AMONG SMALL CANIDS: MITOCHONDRIAL DNA DIFFERENTIATION OF SWIFT,KIT, AND ARCTIC FOXES

Gene flow can effectively suppress genetic divergence among widely separated populations in highly mobile species. However, the same may not be true of species that typically disperse over shorter distances. Using mtDNA restriction-site and sequence analyses, we evaluate the extent of divergence among populations of two small relatively sedentary North American canids, the kit and swift foxes (genus Vulpes). We determine the significance of genetic differentiation among populations separated by distance and those separated by discrete topographic barriers. Our results show the among-population component of genetic variation in kit and swift foxes is large and similar to that of small rodents with limited dispersal ability. In addition, we found two distinct groupings of genotypes, separated by the Rocky Mountains, corresponding to the traditional division between kit and swift fox populations. Previous workers have characterized these morphologically similar populations either as separate species or subspecies. Our mtDNA data also suggest that kit and swift fox populations hybridize over a limited geographic area. However, the sequence divergence between kit and swift foxes is similar to that between these taxa and the arctic fox (Alopex lagopus), a morphologically distinct species commonly placed in a separate genus. This result presents a dilemma for species concepts, and we conclude that kit and swift foxes should be recognized as separate 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.     

Chromosomal evolution of the Canidae. II. Divergence from the primitive carnivore karyotype

The Giemsa-banding patterns of chromosomes from the arctic fox (Alopex lagopus), the red fox (Vulpes vulpes), the kit fox (Vulpes macrotis), and the raccoon dog (Nyctereutes procyonoides) are compared. Despite their traditional placement in different genera, the arctic fox and the kit fox have an identical chromosome morphology and G-banding pattern. The red fox has extensive chromosome arm homoeology with these two species, but has only two entire chromosomes in common. All three species share some chromosomes with the raccoon dog, as does the high diploid-numbered grey wolf (Canis lupus, 2n = 78). Moreover, some chromosomes of the raccoon dog show partial or complete homoeology with metacentric feline chromosomes which suggests that these are primitive canid chromosomes. We present the history of chromosomal rearrangements within the Canidae family based on the assumption that a metacentric-dominated karyotype is primitive for the group.     

Using the dog genome to find single nucleotide polymorphisms in red foxes and other distantly related members of the Canidae

Single nucleotide polymorphisms (SNP) are the ideal marker for characterizing genomic variation but can be difficult to find in nonmodel species. We explored the usefulness of the dog genome for finding SNPs in distantly related nonmodel canids and evaluated so-ascertained SNPs. Using 40 primer pairs designed from randomly selected bacterial artificial chromosome clones from the dog genome, we successfully sequenced 80-88% of loci in a coyote (Canis latrans), grey fox (Urocyon cinereoargenteus), and red fox (Vulpes vulpes), which compared favourably to a 60% success rate for each species using 10 primer pairs conserved across mammals. Loci were minimally heterogeneous with respect to SNP density, which was similar, overall, in a discovery panel of nine red foxes to that previously reported for a panel of eight wolves (Canis lupus). Additionally, individual heterozygosity was similar across the three canids in this study. However, the proportion of SNP sites shared with the dog decreased with phylogenetic divergence, with no SNPs shared between red foxes and dogs. Density of interspecific SNPs increased approximately linearly with divergence time between species. Using red foxes from three populations, we estimated F(ST) based on each of 42 SNPs and 14 microsatellites and simulated null distributions conditioned on each marker type. Relative to SNPs, microsatellites systematically underestimated F(ST) and produced biased null distributions, indicating that SNPs are superior markers for these functions. By reconstituting the frequency spectrum of SNPs discovered in nine red foxes, we discovered an estimated 77-89% of all SNPs (within the region screened) present in North American red foxes. In sum, these findings indicate that information from the dog genome enables easy ascertainment of random and gene-linked SNPs throughout the Canidae and illustrate the value of SNPs in ecological and evolutionary genetics.     

Phylogenetic implications of the 38 putative ancestral chromosome segments for four canid species.

Chromosome homologies between the Japanese raccoon dog (Nectereutes procyonoides viverrinus, 2n = 39 + 2-4 B chromosomes) and domestic dog (Canis familiaris, 2n = 78) have been established by hybridizing a complete set of canine paint probes onto high-resolution G-banded chromosomes of the raccoon dog. Dog chromosomes 1, 13, and 19 each correspond to two raccoon dog chromosome segments, while the remaining 35 dog autosomes each correspond to a single segment. In total, 38 dog autosome paints revealed 41 conserved segments in the raccoon dog. The use of dog painting probes has enabled integration of the raccoon dog chromosomes into the previously established comparative map for the domestic dog, Arctic fox (Alopex lagopus), and red fox (Vulpes vulpes). Extensive chromosome arm homologies were found among chromosomes of the red fox, Arctic fox, and raccoon dog. Contradicting previous findings, our results show that the raccoon dog does not share a single biarmed autosome in common with the Arctic fox, red fox, or domestic cat. Comparative analysis of the distribution patterns of conserved chromosome segments revealed by dog paints in the genomes of the canids, cats, and human reveals 38 ancestral autosome segments. These segments could represent the ancestral chromosome arms in the karyotype of the most recent ancestor of the Canidae family, which we suggest could have had a low diploid number, based on comparisons with outgroup species.     

A phylogenetic analysis of basal metabolism,total evaporative water loss,and life-history among foxes from desert and mesic regions

We measured basal metabolic rate (BMR) and total evaporative water loss (TEWL) of species of foxes that exist on the Arabian Peninsula, Blanfords fox (Vulpes cana) and two subspecies of Red fox (Vulpes vulpes). Combining these data with that on other canids from the literature, we searched for specialization of physiological traits among desert foxes using both conventional least squares regression and regressions based on phylogenetic independent contrasts. Further, we explored the consequences of reduced body size of foxes on life history parameters such as litter size and neonate mass. For Blanfords foxes, Red foxes from the central desert of Arabia, and Red foxes from the more mesic Asir mountains, body mass averaged 1,285±52 g, 1,967±289 g, and 3,060±482 g, respectively, whereas mean BMR, during summer, was 304.5±32.3 kJ/day, 418.0±32.4 kJ/day, and 724.1±120.2 kJ/day (±SD). An analysis of covariance with body mass as a covariate showed no statistical differences in BMR among foxes. Analysis of covariance indicated that Red fox from the Asir mountains had a higher TEWL than Red foxes from central Arabia or than Blanfords foxes also from the mountains. Comparisons of all species of desert and mesic foxes showed no significant differences in BMR, nor did desert foxes have a significantly lower BMR than other carnivores. TEWL of desert foxes was lower than other more mesic carnivores; deviations in TEWL ranged from –17.7% for the Fennec fox (Fennecus zerda) to –57.4% for the Kit fox (Vulpes velox). Although desert foxes have a BMR comparable to other more mesic species, it appears that desert foxes do have a smaller body mass, lowering overall energy requirements. We attribute this reduction in body size to the resource limitation hypothesis whereby natural selection favors smaller individuals in a resource-limited environment, especially during periods of severe food shortage. However, until common garden experiments are performed, developmental plasticity and acclimation cannot be ruled out as contributors to this pattern.Abbreviations BMR basal metabolic rate - CLSR conventional least squares regression - MYA million years ago - PIC phylogenetic independent contrasts - T _a ambient temperature - TEWL total evaporative water loss - TNZ thermoneutral zone - O ₂ oxygen consumption Communicated by G. Heldmaier     

Variation of cranial and dental measurements and dental correlations in the pampean fox (Dusicyon gymnocercus)

The knowledge of the craniodental variability of South American canids is mainly anecdotal, and the variability and dental correlation of living canids have only been studied in several species of holarctic foxes ( Vulpes vulpes , Vulpes lagopus and Urocyon cinereoargeteus ) and Canis lupus . These studies have shown that the last molars, canines and incisors are the most variable and the central molars and premolars are the least variable, and that correlations are highest among the posterior premolars. This pattern has been accounted for by developmental factors, functional utility and tooth complexity. In this paper the pattern of dental correlation and variability of craniodental measurements in the South American fox Dusicyon gymnocercus is studied, with the aim of checking the extension of the pattern observed in other species and contrasting previous hypotheses. The most variable in D. gymnocercus measurements are some cranial widths, width of the incisors, lower canine and lower last molar, whereas others such as width of the braincase, length of the upper carnassials, and first and second lower and upper molars have lower variability. The premolar region is the most correlated, in terms of values and number of significant correlation coefficients. The results show that measurement error is negatively correlated with size of the variables, which could explain the bias found in this index as earlier authors have suggested, but the pattern captured by this index cannot be completely considered to be an artefact. The observed results confirm that the patterns of variability and correlation found in holarctic foxes are widespread in the family Canidae, and suggest the presence of an evolutionary constraint.     

Molecular evolution of the leptin exon 3 in some species of the family Canidae

The structure of the leptin gene seems to be well conserved. The polymorphism of this gene in four species belonging to the Canidae family (the dog (Canis familiaris) – 16 different breeds, the Chinese racoon dog (Nyctereutes procyonoides procyonoides), the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus)) were studied with the use of single strand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and DNA sequencing techniques. For exon 2, all species presented the same SSCP pattern, while in exon 3 some differences were found. DNA sequencing of exon 3 revealed the presence of six nucleotide substitutions, differentiating the studied species. Three of them cause amino acid substitutions as well. For all dog breeds studied, SSCP patterns were identical.     

Convergence and divergence in prey of sympatric canids and felids: opportunism or phylogenetic constraint?

Since the canids and felids diverged in the mid‐Eocene or earlier, each family has developed a suite of morphological and behavioural adaptations for obtaining and consuming prey. We here distinguish between prey taxa captured and eaten as a result of these phylogenetic adaptations, and those because they are fortuitously encountered, and argue that such supplementary prey, often opportunistically caught, create a buffer between sympatric, and potentially competitive, canids and felids and thus enhance coexistence. We base our analysis on dietary data derived from the stomach contents of four sympatric canid and felid species in the Free State Province, South Africa (canids: Cape fox Vulpes chama and black‐backed jackal Canis mesomelas; felids: African wild cat Felis silvestris lybica and caracal Caracal caracal), and from results of studies on these species elsewhere in southern Africa. The two canid species preyed heavily on invertebrates, and thus opportunistically, while the felids (especially the caracal) concentrated on mammals, prey they are phylogenetically adapted to capture. Only three species of mammalian prey are shared by the four species. The ratio of opportunistically‐to‐phylogenetically mediated prey taxa used (the O/P ratio) differ between the species, with the black‐backed jackal having the most opportunistically caught taxa in its diet, and the caracal the least. As predicted, a comparison of this data with those from dietary studies of the same species carried out elsewhere indicates that the number of opportunistically obtained prey taxa varies more than those resulting from phylogenetic adaptations. The largest canid had the widest food spectrum (35 prey taxa) while the smallest felid had the most restricted one (11 prey taxa). We argue that using the O/P distinction allows a better understanding of changes in food niche breadth of particular species, especially in xeric areas, and gives a better indication of possible exploitative competition for food by sympatric carnivores than when regarding all prey taxa as actively pursued. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 527–538.     

Use of RAPD technique in evolution studies of four species in the family Canidae

The RAPD-PCR technique was applied to identify genetic markers able to distinguish between four canid species: the arctic fox (Alopex lagopus), red fox (Vulpes vulpes), Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and six breeds of the domestic dog (Canis familiaris). A total of 29 ten-nucleotide arbitrary primers were screened for their potential use in the differentiation of these species. Ten primers amplified RAPD profiles that made it possible to distinguish between the investigated taxa. A number of species-specific bands was scored within RAPD profiles produced by these primers: 35.6% of all the polymorphic bands were unique to the Chinese raccoon dog, 29.6% were unique to the domestic dog, 21.2% were diagnostic for the red fox and 13.6% for the arctic fox. No breed-specific fragments were amplified from canine DNA; however, three primers produced bands characteristic for the dog, but not present in all of the investigated breeds. A Neighbor-Joining tree constructed on the basis of the analysis of RAPD profiles amplified by six primers revealed that the phylogenetic distance between the dog and the arctic fox is larger than the distance between the dog and the red fox. The phylogenetic branch of the Chinese raccoon dog was the most distinct on the dendrogram, suggesting that this species belongs to a different phylogenetic lineage. Obtained results make it possible to conclude that RAPD analysis can be a powerful tool for developing molecular markers useful in distinguishing between species of the family Canidae and for studying their phylogenetic relations.     

Prevalence of Toxoplasma gondii infection diagnosed by PCR in farmed red foxes, arctic foxes and raccoon dogs

The aim of this study was to compare Toxoplasma gondii infection in three canid species: red fox Vulpes vulpes, arctic fox Vulpes lagopus and raccoon dog Nyctereutesprocyonoides kept at the same farm. Anal swabs were taken from 24 adult and 10 juvenile red foxes, 12 adult arctic foxes, three adult and seven juvenile raccoon dogs. Additionally, muscle samples were taken from 10 juvenile red foxes. PCR was used to detect T. gondii DNA. T. gondii infection was not detected in any of the arctic foxes; 60% ofraccoon dogs were infected; the prevalence of the parasite in material from red fox swabs was intermediate between the prevalence observed in arctic foxes and raccoon dogs. It is possible that susceptibility and immune response to the parasite differ between the three investigated canid species. T. gondii DNA was detected in muscle tissue of five young foxes. The results of this study suggest that T. gondii infection is not rare in farmed canids.     

Survival and Cause-Specific Mortality of Corsac and Red Foxes in Mongolia

ABSTRACT The corsac fox (Vulpes corsac) and red fox (Vulpes vulpes) range widely across northern and central Asia and may be declining in many regions due to overhunting and other causes. However, details of the fundamental causes of survival and mortality of both species remain largely unquantified, but may be crucial for understanding interspecific relationships and developing effective conservation actions. We studied a radiomarked population of sympatric corsac and red foxes in central Mongolia to quantify survival and cause-specific mortality rates from April 2005 to April 2007. Survival probability was 0.34 for corsacs (n = 18) and 0.46 for red foxes (n = 17) and did not vary by year within or between each species. Among both foxes, mortality occurred mainly from hunting by humans, but also from predation by larger canids and unknown causes. Our results suggest that illegal human hunting represents the principal source of mortality for both species and that a recently initiated ranger patrol program in the study area did not affect fox survival. As such, more stringent protective measures will likely be necessary to halt declines of both foxes. Our results also suggest that interference competition occurs between species as red foxes killed but did not consume corsacs. Our results will be useful for developing science-based management strategies to protect foxes in Mongolia, and in understanding the competitive relationships between them.     

A Study on the Predilection Sites of Trichinella spiralis Muscle Larvae in Experimentally Infected Foxes (Alopex lagopus,Vulpes vulpes)

Studies were carried out on the predilection sites of Trichinella spiralis muscle larvae in experimentally infected arctic foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) reared in cages. The highest number of larvae per gramme tissue was found in the muscles of the legs, eyes, diaphragm, and tongue. The 2 fox species showed no significant differences with regard to predilection sites.     

The isozyme spectra of lactate dehydrogenase in the tissues of the raccoon dog <Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis> in the autumn

An electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes in the tissue homogenates of cardiac and skeletal muscles, kidney, lungs, spleen, and liver of the raccoon dog Nyctereutes procyonoides from two different geographic zones, viz., northwestern Russia and Poland, as well as the Arctic blue fox Alopex lagopus L. and the red fox Vulpes vulpes L. was performed during the preparatory period to the winter season. Raccoon dogs, which hibernate under natural conditions, differ from other canids (the red fox and Arctic blue fox) to which they are close taxonomically by their body weight and by the higher proportion of aerobic H subunits of lactate dehydrogenase in all organs except for the heart. A higher content of “fast” anode fractions, lactate dehydrogenase-1 and lactate dehydrogenase-2, in the heart, kidney, lungs, liver, and spleen was detected in the raccoon dogs from the northern region compared to those from the southern geographic zone. The shift in the reaction catalyzed by lactate dehydrogenase towards the production of pyruvate indicates that this metabolite is necessary for the synthesis of fatty acids during lipogenesis in the autumn.     

Use of Volatile Organic Components in Scat to Identify Canid Species

Abstract: Identification of wildlife species from indirect evidence can be an important part of wildlife management, and conventional methods can be expensive or have high error rates. We used chemical characterization of the volatile organic constituents (VOCs) in scat as a method to identify 5 species of North American canids from multiple individuals. We sampled vapors of scats in the headspace over a sample using solid-phase microextraction and determined VOC content using gas chromatography with a flame ionization detector. We used linear discriminant analysis to develop models for differentiating species with bootstrapping to estimate accuracy. Our method correctly classified 82.4% (bootstrapped 95% CI = 68.8–93.8%) of scat samples. Red fox (Vulpes vulpes) scat was most frequently misclassified (25.0% of scats misclassified); red fox was also the most common destination for misclassified samples. Our findings are the first reported identification of animal species using VOCs in vapor emissions from scat and suggest that identification of wildlife species may be plausible through chemical characterization of vapor emissions of scat. (JOURNAL OF WILDLIFE MANAGEMENT 72(3):792–797; 2008)