DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora.     

EARLIEST KNOWN CARNIVORAN AUDITORY BULLA AND SUPPORT FOR A RECENT ORIGIN OF CROWN-GROUP CARNIVORA (EUTHERIA, MAMMALIA)

Abstract:  An ossified auditory bulla of a basal carnivoran is described for the first time. Although broken, the bulla of Viverravus acutus (Viverravidae, Carnivora) appears to have enclosed the middle ear and to have been composed exclusively of an ectotympanic bone. The structure of the bulla and other basicranial features support the hypothesis that viverravids lie phylogenetically outside crown-group Carnivora and that the last common ancestor of living carnivorans may have existed as recently as 42 million years ago, not 60–70 million years ago as suggested by some authors.     

Phylogenetic relationships among eutherian orders estimated from inferred sequences of mitochondrial proteins: Instability of a tree based on a single gene

The phylogenetic relationships among Primates (human), Artiodactyla (cow), Cetacea (whale), Carnivora (seal), and Rodentia (mouse and rat) were estimated from the inferred amino acid sequences of the mitochondrial genomes using Marsupialia (opossum), Aves (chicken), and Amphibia (Xenopus) as an outgroup. The overall evidence of the maximum likelihood analysis suggests that Rodentia is an outgroup to the other four eutherian orders and that Cetacea and Artiodactyla form a clade with Carnivora as a sister taxon irrespective of the assumed model for amino acid substitutions. Although there remains an uncertainty concerning the relation among Artiodactyla, Cetacea, and Carnivora, the existence of a clade formed by these three orders and the outgroup status of Rodentia to the other eutherian orders seems to be firmly established. However, analyses of individual genes do not necessarily conform to this conclusion, and some of the genes reject the putatively correct tree with nearly 5% significance. Although this discrepancy can be due to convergent or parallel evolution in the specific genes, it was pointed out that, even without a particular reason, such a discrepancy can occur in 5% of the cases if the branching among the orders in question occurred within a short period. Due to uncertainty about the assumed model underlying the phylogenetic inference, this can occur even more frequently. This demonstrates the importance of analyzing enough sequences to avoid the danger of concluding an erroneous tree.     

FGF gene family characterization provides insights into its adaptive evolution in Carnivora

Fibroblast growth factors (FGFs) encoded by the FGF gene family can regulate development and physiology in animals. However, their evolutionary characteristics in Carnivora are largely unknown. In this study, we identified 660 sequences of three types of FGF genes from 30 unannotated genomes of Carnivora animals (before 7th May 2020), and the FGF genes from 52 Carnivora species were analyzed through the method of comparative genomics. Phylogenetic and selective pressure analyses were carried out based on the FGF genes of these 52 Carnivora species. The phylogenetic analysis results demonstrated that the FGF gene family was divided into 10 subfamilies and that FGF5 formed one clade rather than belonging to the subfamilies of FGF4 and FGF6. The evolutionary analysis results showed that the FGF genes were prominently subjected to purifying selection and were highly conserved in the process of Carnivora evolution. We also carried out phylogenetic comparative analyses, which indicated that the habitat was one of the factors that shaped the evolution of Carnivora FGF genes. The FGF1 and FGF6 genes were positively selected in the Carnivora animals, and positive selection signals were detected for the FGF19 gene in semiaquatic Carnivora animals. In summary, we clarified the phylogenetic and evolutionary characteristics of Carnivora FGF genes and provided valuable data for future studies on evolutionary characterization of Carnivora animals.     

Evolution and systematics of the feliform Carnivora

Recent studies have improved our knowledge about the evolution and phylogeny of feliform taxa. Detailed study on new fossil remains of extinct feliform nimravides allows a new hypothesis concerning interrelationships within this family. Many factors indicate lack of sister relationships of Nimravinae and Barbourofelinae. However, only further investigations may bring full acceptance of this hypothesis. The paraphyly of Viverridae has been resolved by excluding the taxa Nandinia, Prionodon and Cryptoprocta and Fossa, which today are placed basally to all remaining Feliformia (family Nandiniidae), as sister taxon of Felidae (family Prionodontidae) and as Malagasy Carnivora lineage basal to hyaenid–herpestid clade, respectively. Still, incongruence among results concerning the systematic position of these taxa exhibits the necessity of further investigation. Detailed study revealed inconsistencies within genet and genet-like taxa phylogeny, which have still to be resolved. Malagasy Carnivora belong to a separate lineage, which originated from herpestid–hyaenid ancestors and colonised Madagascar during a single colonisation event. However, interrelationships among Malagasy Carnivora are poorly resolved. The situation of the social mongooses clade was resolved by including ethologic data to phylogenetic analyses; however, there is little information on solitary mongooses, which have a paraphyletic status today. Felid morphology and taxonomic revision attained during recent years show greater evolutionary differentiation. Nevertheless, no clear taxonomy has been achieved. New investigating methods are required. In the hyaenid family, which includes only four living species, some investigations related to the ecomorphological evolutionary path have been performed. The comparisons of fossil and subfossil remains with modern feliforms, combined with recent molecular methods, may improve our knowledge.     

The cranium of Proviverra typica (Mammalia,Hyaenodonta) and its impact on hyaenodont phylogeny and endocranial evolution

We describe the first endocast reconstruction of a hyaenodont mammal based on X‐ray microtomography. The endocast belongs to the type material of the European hyaenodont Proviverra typica. We performed phylogenetic analysis to contextualize the evolution of endocranial size and complexity in Hyaenodonta. We added several European hyaenodonts and modified several codings of the most recent character–taxon matrix established to question the relationships within Hyaenodonta. Including these new species in a phylogenetic analysis reveals a new clade: Hyaenodontoidea. Comparisons with several previously described endocasts show that there was an increase in complexity in the convolutions of the encephalon within Hyaenodontidae history. Moreover, the analysis of the encephalization quotient reveals that the endocranium of the Hyaenodonta is not smaller than those of fossil Carnivora or some extant Carnivora. Therefore, the extinction of Hyaenodonta may not be linked to the relative size of hyaenodont brains.     

Carnivora Population Dynamics Are as Slow and as Fast as Those of Other Mammals: Implications for Their Conservation

Of the 285 species of Carnivora 71 are threatened, while many of these species fulfill important ecological roles in their ecosystems as top or meso-predators. Population transition matrices make it possible to study how age-specific survival and fecundity affect population growth, extinction risks, and responses to management strategies. Here we review 38 matrix models from 35 studies on 27 Carnivora taxa, covering 11% of the threatened Carnivora species. We show that the elasticity patterns (i.e. distribution over fecundity, juvenile survival and adult survival) in Carnivora cover the same range in triangular elasticity plots as those of other mammal species, despite the specific place of Carnivora in the food chain. Furthermore, reproductive loop elasticity analysis shows that the studied species spread out evenly over a slow-fast continuum, but also quantifies the large variation in the duration of important life cycles and their contributions to population growth rate.These general elasticity patterns among species, and their correlation with simple life history characteristics like body mass, age of first reproduction and life span, enables the extrapolation of population dynamical properties to unstudied species. With several examples we discuss how this slow-fast continuum, and related patterns of variation in reproductive loop elasticity, can be used in the formulation of tentative management plans for threatened species that cannot wait for the results of thorough demographic studies. We argue, however, that such management programs should explicitly include a plan for learning about the key demographic rates and how these are affected by environmental drivers and threats.     

Phylogenetic relationships within mammalian order Carnivora indicated by sequences of two nuclear DNA genes

Phylogenetic relationships among 37 living species of order Carnivora spanning a relatively broad range of divergence times and taxonomic levels were examined using nuclear sequence data from exon 1 of the IRBP gene (approximately 1.3 kb) and first intron of the TTR gene (approximately 1 kb). These data were used to analyze carnivoran phylogeny at the family and generic level as well as the interspecific relationships within recently derived Felidae. Phylogenetic results using a combined IRBP+TTR dataset strongly supported within the superfamily Califormia, the red panda as the closest lineage to procyonid-mustelid (i.e., Musteloidea) clade followed by pinnipeds (Otariidae and Phocidae), Ursidae (including the giant panda), and Canidae. Four feliform families, namely the monophyletic Herpestidae, Hyaenidae, and Felidae, as well as the paraphyletic Viverridae were consistently recovered convincingly. The utilities of these two gene segments for the phylogenetic analyses were extensively explored and both were found to be fairly informative for higher-group associations within the order Carnivora, but not for those of low level divergence at the species level. Therefore, there is a need to find additional genetic markers with more rapid mutation rates that would be diagnostic at deciphering relatively recent relationships within the Carnivora.     

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.     

Mandible shape in marsupial and placental carnivorous mammals: a morphological comparative study using geometric morphometrics

We analysed mandible shape of the orders Dasyuromorpha, Didelphimorphia, and Carnivora using two‐dimensional geometric morphometrics, in order to explore the relationship between shape, size, and phylogeny. We studied 541 specimens, covering most of the genera of the terrestrial Carnivora (115 species) and a wide sample of marsupials (36 species). The observed shape variation had an ecological component. As an example, omnivorous carnivores have thick mandibles and large talonids in the carnassials, while hypercarnivores possess short mandibles and reduced talonids. There is also a discrimination between different taxonomic groups (i.e. marsupials and Carnivora), indicating some kind of constraint. Size explains a large percentage of total variance (large species had shorter and stronger mandibles, with anteriorly displaced carnassials), was significant when phylogeny was taken into account with a comparative method, but not when size and shape were optimized on the phylogeny. Carnivora presents a larger disparity and variation in body size, which could be related to the difference in teeth replacement. The optimization of mandible shape on the phylogenetic tree indicates that functional aspects, such as diet, are a key factor in the evolution of the carnivore mandible, but also that there is a phylogenetic pattern that cannot be explained by differences in diet alone. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 836–855.     

Maximum likelihood analysis of gene-based and structure-based process partitions,using mammalian mitochondrial genomes

Aligned protein-coding genes from 19 completely sequenced mammalian mitochondrial genomes were examined by parsimony and maximum likelihood analyses. Particular attention is given to a comparison between gene-based and structure-based data partitions. Because actual structures are not known for most of the mitochondrially encoded proteins, three different surrogate partitioning schemes were examined, each based on the identity of the consensus amino acid at a specific homologous position. One of the amino-acid-based partitioning schemes gave the highest likelihood, but that scheme was based on concordance with a well-corroborated phylogeny from an earlier parsimony analysis. The gene-based partitioning scheme gave a significantly higher likelihood compared to the only structure-based scheme examined that could be generated without prior assumptions about the phylogeny. Two contrasting phylogenetic inferences were supported by the analyses. Both unpartitioned analyses and analyses in which all partitions were constrained to have identical patterns of branch lengths supported ((Artiodactyla, Cetacea) (Perissodactyla, Carnivora)), whereas all analyses with that constraint relaxed supported (((Artiodactyla, Cetacea) Carnivora) Perissodactyla).     

Supernumerary deciduous incisors and the order of eruption of the incisor teeth in the albino ferret

A radiological study of the developing dentition of the domestic albino ferret has revealed the presence of four upper and three lower deciduous functionless incisor teeth in each half of the jaw. The presence of the additional upper incisor tooth can only be accounted for in two ways:
(1) It arose as the result of a gene mutation within the closed colony.
(2) It is indicative of the fact that at some early date in the evolution of the Carnivora, four deciduous incisors were functional in each half of the jaw.     

A phylogeny of the Caniformia (order Carnivora) based on 12 complete protein-coding mitochondrial genes

Evolutionary relationships of the order Carnivora have been extensively studied. However, phylogenetic studies based on different types of data, species samples, and methods of analysis provide contradictory results. Consequently, phylogenetic relationships of Carnivora remain contentious. Here, the sequence of 12 mitochondrial genes (10,842 nucleotides) from a total of 38 carnivore species was used to investigate the phylogeny of the caniform (dog-like) carnivores. An analysis using maximum parsimony, maximum likelihood, and Bayesian approaches provided a unique and well-supported solution to most contentious relationships within Caniformia. The clade Arctoidea was shown to consist of three major monophyletic groups: Pinnipedia, Ursidae, and Musteloidea. Within Pinnipedia, the families Otariidae and Odobenidae formed a clade, sister to Phocidae. Within Musteloidea, there was a sister relationship between true mustelids (i.e., excluding the skunks) and procyonids, and between ailurids and mephitids (skunks). Despite a high level of confidence obtained at most nodes, uncertainty remained about the relative position of the three major arctoid clades.     

Differential enzyme targeting as an evolutionary adaptation to herbivory in carnivora

Not all members of the order Carnivora are carnivorous. Some are omnivorous, and a few, such as the giant panda, Ailuropoda melanoleuca, are almost exclusively herbivorous. Although a number of adaptations to increased plant-eating are recognized within Carnivora, few have been studied at the molecular level. One molecular adaptation to diet that is spread widely across Mammalia is the differential intracellular targeting of the intermediary metabolic enzyme alanine:glyoxylate aminotransferase (AGT), which tends to be mitochondrial in carnivores, peroxisomal in herbivores, and both mitochondrial and peroxisomal in omnivores. In the present study, we have analyzed the targeting of AGT in Carnivora in relation to species' natural diets. We show not only that there has been an adaptive shift in AGT targeting from the mitochondrion toward the peroxisome as diets have shifted from being mainly carnivorous to ones that are more omnivorous and herbivorous but also that in one lineage, namely that of the giant panda, there is evidence for positive selection pressure at the molecular level on the AGT mitochondrial targeting sequence to decrease its efficiency, thereby allowing more AGT to be targeted to the peroxisomes.     

Carnivores, biases and Bergmann's rule

Studies of Bergmann's rule may encompass a non-random subsample of extant homeotherms. We examined patterns of correlation between skull length and geographical latitude in 44 species of carnivores in order to test the validity of Bergmann's rule in the Carnivora. Results were then compared to those of other studies. Significant positive correlation between skull length and latitude was found in 50% of carnivore species, while significant negative correlation was found in only 11% of species. These results indicate that the occurrence of Bergmann's rule in the Carnivora is less frequent than earlier published data suggest. Publication bias is not detected in published data. Therefore, previous studies of geographical size variation might be biased in favour of species known to follow Bergmann's rule.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 81 , 579–588.     

Phylogenetic reconstruction of carnivore social organizations

It is generally assumed that carnivore social organizations evolved directionally from a solitary ancestor into progressively more advanced forms of group living. Although alternative explanations exist, this evolutionary hypothesis has never been tested. Here, I used literature data and maximum likelihood reconstruction on a complete carnivore phylogeny to test this hypothesis against two others: one assuming directional evolution from a non-solitary ancestor, and one assuming parallel evolutions from a socially flexible ancestor, that is, an ancestor with abilities to live in a variety of social organizations. The phylogenetic reconstructions did not support any of the three hypotheses of social evolution at the root of Carnivora. At the family level, however, there was support for a non-solitary and socially flexible ancestor to Canidae, a socially flexible or solitary ancestor to Mustelidae, a solitary or socially flexible ancestor to Mephitidae, a solitary or group living ancestor to Phocidae, a group living ancestor to Otariidae and a solitary ancestor to Ursidae, Felidae, Herpestidae and Viverridae. There was equivocal support for the ancestral state of Procyonidae and Hyaenidae. It is unclear whether the common occurrence of a solitary ancestry at the family level was caused by a solitary ancestor at the root of Carnivora or by multiple transitions into a solitary state. The failure to support a solitary ancestor to Carnivora calls for caution when using this hypothesis in an evolutionary framework, and I suggest continued investigations of the pathways of the evolution of carnivore social organizations.     

Molecular characterization of an ancient Hepatozoon species parasitizing the 'living fossil' marsupial 'Monito del Monte' Dromiciops gliroides from Chile

The Microbiotheriid Dromiciops gliroides , also known as 'Monito del Monte', is considered to be a threatened species and the only living representative of this group of South American marsupials. During the last few years, several blood samples from specimens of 'Monito del Monte' captured at Chiloé island in Chile have been investigated for blood parasites. Inspection of blood smears detected a Hepatozoon species infecting red blood cells. The sequences of DNA fragments corresponding to small subunit ribosomal RNA gene revealed two parasitic lineages belonging to Hepatozoon genus. These parasite lineages showed a basal position with respect to Hepatozoon species infecting rodents, reptiles, and amphibians but are phylogenetically distinct from Hepatozoon species infecting the order Carnivora. In addition, the Hepatozoon lineages infecting D. gliroides are also different from those infecting other micro-mammals living in sympatry, as well as from some that have been described to infect an Australian species of bandicoot. The potential vector of this parasite appears to be the host-specific tick Ixodes neuquenensis because the sequencing of a long amplicon determined the presence of one of the two lineages found in the marsupial.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 568–576.     

大熊猫的脊柱和胸廓

大熊猫Ailuropoda melanoleuca(David)是我国特有的珍稀动物。关于它的骨学研究,前人的报道多限于头骨和四肢骨,对头骨以外的中轴骨--脊柱和胸廓--则很少涉及,仅Davis(1964)的著作中包括这部分骨学内容,但标本数量较少,且无幼体标本。我们用完整骨骼6例(包括1例幼体)进行了较系统的观察,并和黑熊Sele-narctos thibetanus的成体和幼体、小熊猫Ailurus fulgens、犬Canis familiaris、虎Panthera tigris、狮Panthera leo、云豹Neofelis nebulosa等其他食肉目兽类加以对比,以显示出大熊猫的特点。     

Phylogenetic relationships of the endemic malagasy carnivoreCryptoprocta ferox (Aeluroidea): DNA/DNA hybridization experiments

A molecular and morphological study of several living aeluroid Carnivora was completed to evaluate the evolutionary relationships of the endemicCryptoprocta ferox, a carnivore living on the island of Madagascar. The molecular analysis, based on DNA/DNA hybridization experiments, suggests thatCryptoprocta is more closely related to the Herpestidae (as represented byMungos andIchneumia) than it is to the Viverrinae (Genetta), Paradoxurinae (Paguma, Paradoxurus), Felidae (Felis, Panthera), or Hyaenidae (Crocuta). Based on bootstrapping procedures applied to the individual DNA/DNA results, three branching patterns were observed which differ only by the relative position of the Felidae within the Aeluroidea. The amounts of genetic divergence measured between pairs of compared taxa have been transformed into millions years datings by the molecular clock concept, and this was done by establishing a molecular time scale based on the fossil record of the aeluroid Carnivora.