Phylogenomic systematics of the spotted skunks (Carnivora, Mephitidae, Spilogale): Additional species diversity and Pleistocene climate change as a major driver of diversification.

Four species of spotted skunks (Carnivora, Mephitidae, Spilogale) are currently recognized: Spilogale angustifrons, S. gracilis, S. putorius, and S. pygmaea. Understanding species boundaries within this group is critical for effective conservation given that regional populations or subspecies (e.g., S. p. interrupta) have experienced significant population declines. Further, there may be currently unrecognized diversity within this genus as some taxa (e.g., S. angustifrons) and geographic regions (e.g., Central America) never have been assessed using DNA sequence data. We analyzed species limits and diversification patterns in spotted skunks using multilocus nuclear (ultraconserved elements) and mitochondrial (whole mitogenomes and single gene analysis) data sets from broad geographic sampling representing all currently recognized species and subspecies. We found a high degree of genetic divergence among Spilogale that reflects seven distinct species and eight unique mitochondrial lineages. Initial divergence between S. pygmaea and all other Spilogale occurred in the Early Pliocene (~ 5.0 million years ago). Subsequent diversification of the remaining Spilogale into an "eastern" and a "western" lineage occurred during the Early Pleistocene (~1.5 million years ago). These two lineages experienced temporally coincident patterns of diversification at ~0.66 and ~0.35 million years ago into two and ultimately three distinct evolutionary units, respectively. Diversification was confined almost entirely within the Pleistocene during a timeframe characterized by alternating glacial-interglacial cycles, with the origin of this diversity occurring in northeastern Mexico and the southwestern United States of America. Mitochondrial-nuclear discordance was recovered across three lineages in geographic regions consistent with secondary contact, including a distinct mitochondrial lineage confined to the Sonoran Desert. Our results have direct consequences for conservation of threatened populations, or species, as well as for our understanding of the evolution of delayed implantation in this enigmatic group of small carnivores.

PREVALENCE AND SEVERITY OF SKUNK CRANIAL WORM (SKRJABINGYLUS CHITWOODORUM) DAMAGE INCREASE WITH PRECIPITATION IN SPOTTED SKUNKS (SPILOGALE SPP.).

Eastern and western spotted skunks (Spilogale putorius and Spilogale gracilis) serve as definitive hosts for skunk cranial worm (Skrjabingylus chitwoodorum), a metastrongylid nematode that spends its adult stage inhabiting the frontal sinuses of the host cranium. Skunk cranial worm can cause severe damage to the skull of spotted skunks, and this damage is identifiable in preserved specimens. We visited six mammal collections between 2016 and 2018 and used 578 spotted skunk skull specimens to identify patterns in prevalence and severity of skunk cranial worm damage in spotted skunks. Specimens were collected between 1895 and 1981 from across the US. We assessed effects of host genetic clade, collection year, precipitation, and sex on prevalence and severity rates. We hypothesized that the midwestern genetic clade (plains spotted skunk, Spilogale putorius interrupta), which experienced a range-wide population decline in the mid-1900s, would experience the highest infection and severity rates. We expected precipitation, but not sex to influence infection and severity. Our top models indicated that host genetic clade, precipitation in the year prior to specimen collection, and the year prior to specimen collection best predicted prevalence and severity of skunk cranial worm. We suggest the positive association with precipitation is a result of local availability of gastropods, the intermediate host for skunk cranial worm. There was a negative association between prior year and prevalence, but the severity of damage increased over time for all clades. Given concerns over population declines of spotted skunks as well as observations of increasing damage over the past century, there is a need for further research on the impacts of cranial worm infection on individual-scale behavior, reproduction and survival, and on population-scale demographics.

Lack of genetic structure and female-specific effect of dispersal barriers in a rabies vector, the striped skunk (Mephitis mephitis).

Evaluating the permeability of potential barriers to movement, dispersal and gene exchanges can help describe spreading patterns of wildlife diseases. Here, we used landscape genetics methods to assess the genetic structure of the striped skunk (Mephitis mephitis), which is a frequent vector of rabies, a lethal zoonosis of great concern for public health. Our main objective was to identify landscape elements shaping the genetic structure of this species in Southern Québec, Canada, in an area where the raccoon rabies variant has been detected. We hypothesised that geographic distance and landscape barriers, such as highways and major rivers, would modulate genetic structure. We genotyped a total of 289 individuals sampled across a large area (22,000 km²) at nice microsatellite loci. Genetic structure analyses identified a single genetic cluster in the study area. Major rivers and highways, however, influenced the genetic relatedness among sampled individuals. Sex-specific analyses revealed that rivers significantly limited dispersal only for females while highways only had marginal effects. Rivers and highways did not significantly affect male dispersal. These results support the contention that female skunks are more philopatric than males. Overall, our results suggest that the effects of major rivers and highways on dispersal are sex-specific and rather weak and are thus unlikely to prevent the spread of rabies within and among striped skunk populations.

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.

Evidence for evolutionary convergence at MHC in two broadly distributed mesocarnivores.

Variation within major histocompatibility complex (MHC) genes is important in recognizing pathogens and initiating an immune response. These genes are relevant in enhancing our understanding of how species cope with rapid environmental changes and concomitant fluctuations in selective pressures such as invasive, infectious diseases. Disease-based models suggest that diversity at MHC is maintained through balancing selection arising from the coevolution of hosts and pathogens. Despite intensive balancing selection, sequence motifs or even identical MHC alleles can be shared across multiple species; three potential mechanisms have been put forth to explain this phenomenon: common ancestry, convergent evolution, and random chance. To understand the processes that maintain MHC similarity across divergent species, we examined the variation at two orthologous MHC-DRB genes in widespread North American Musteloid species, striped skunks (Mephitis mephitis), and raccoons (Procyon lotor). These species are often sympatric and exposed to a similar suite of diseases (e.g., rabies, canine distemper, and parvovirus). Given their exposure to similar selective pressures from pathogens, we postulated that similar DRB alleles may be present in both species. Our results indicated that similar motifs are present within both species, at functionally relevant polymorphic sites. However, based on phylogenetic analyses that included previously published MHC sequences of several closely related carnivores, the respective MHC-DRB alleles do not appear to have been maintained through common ancestry and unlikely through random chance. Instead, the similarities observed between the two mesocarnivore species may rather be due to evolutionary convergence.

Chromosome painting shows that skunks (Mephitidae, Carnivora) have highly rearranged karyotypes.

The karyotypic relationships of skunks (Mephitidae) with other major clades of carnivores are not yet established. Here, multi-directional chromosome painting was used to reveal the karyological relationships among skunks and between Mephitidae (skunks) and Procyonidae (raccoons). Representative species from three genera of Mephitidae (Mephitis mephitis, 2n = 50; Mephitis macroura, 2n = 50; Conepatus leuconotus, 2n = 46; Spilogale gracilis, 2n = 60) and one species of Procyonidae (Procyon lotor, 2n = 38) were studied. Chromosomal homology was mapped by hybridization of five sets of whole-chromosome paints derived from stone marten (Martes foina, 2n = 38), cat, skunks (M. mephitis; M. macroura) and human. The karyotype of the raccoon is highly conserved and identical to the hypothetical ancestral musteloid karyotype, suggesting that procyonids have a particular importance for establishing the karyological evolution within the caniforms. Ten fission events and five fusion events are necessary to generate the ancestral skunk karyotype from the ancestral carnivore karyotype. Our results show that Mephitidae joins Canidae and Ursidae as the third family of carnivores that are characterized by a high rate of karyotype evolution. Shared derived chromosomal fusion of stone marten chromosomes 6 and 14 phylogenetically links the American hog-nosed skunk and eastern spotted skunk.

Identification of skunk species submitted for rabies testing in the desert southwest.

Skunks usually are identified by their common name (skunk) when submitted for rabies testing. In the desert southwest (Arizona, New Mexico, Texas, USA; and northern Mexico), there are five species of skunks; four of which can occur in sympatry. To better understand the ecology of skunk rabies in these areas, it is imperative that species be properly identified. We used the displacement loop (d-loop) of the mitochondrial genome to identify to species 24 skunk brain samples submitted for rabies testing in New Mexico from 2001 to 2002. Most were identified as striped skunks (Mephitis mephitis), but hooded (Mephitis macroura) and hog-nosed (Conepatus leuconotus) skunks were also found.

Phylogenetic relationships within caniform carnivores based on analyses of the mitochondrial 12S rRNA gene.

The complete 12S rRNA gene of 32 carnivore species, including four feliforms and 28 caniforms, was sequenced. The sequences were aligned on the basis of their secondary structures and used in phylogenetic analyses that addressed several evolutionary relationships within the Caniformia. The analyses showed an unresolved polytomy of the basic caniform clades; pinnipeds, mustelids, procyonids, skunks, Ailurus (lesser panda), ursids, and canids. The polytomy indicates a major diversification of caniforms during a relatively short period of time. The lesser panda was distinct from other caniforms, suggesting its inclusion in a monotypic family, Ailuridae. The giant panda and the bears were joined on the same branch. The skunks are traditionally included in the family Mustelidae. The present analysis, however, showed a less close molecular relationship between the skunks and the remaining Mustelidae (sensu stricto) than between Mustelidae (sensu stricto) and Procyonidae, making Mustelidae (sensu lato) paraphyletic. The results suggest that the skunks should be included in a separate family, Mephitidae. Within the Pinnipedia, the grouping of walrus, sea lions, and fur seals was strongly supported. Analyses of a combined set of 12S rRNA and cytochrome b data were generally consistent with the findings based on each gene.

Expression of epidermal growth factor receptor in the preimplantation uterus and blastocyst of the western spotted skunk.

The western spotted skunk is unique in that its blastocysts undergo a 180-220-day period of arrested development before implantation. We investigated the potential role of epidermal growth factor (EGF)-related growth factors in regulating uterine and embryonic development in this species by studying the status of EGF receptor (EGF-R) in these tissues during delayed implantation and resumption of embryonic development. The cell-specific distribution of EGF binding sites and the expression of EGF-R mRNA were assayed by autoradiography and Northern blot analysis, respectively. The size of EGF-R was determined by affinity cross-linking studies, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase (PTK) activity. EGF binding sites were localized in the uterine luminal and glandular epithelium, endometrial stroma, myometrium, and blood vessels during both stages of pregnancy. As examined by Northern blot hybridization, a cRNA probe specific to mouse EGF-R hybridized to poly(A)+ RNA of skunk uteri. Transcripts similar to those of mouse uterine EGF-R were identified. [125I]-EGF was cross-linked to a 170-kDa protein both in the uterus and in blastocysts collected during the delayed implantation and periimplantation periods. However, EGF-induced PTK activity was significantly elevated above background levels during the period of renewed embryonic development, but not during arrested embryonic development. The results suggest that EGF-related growth factors may play an important role in regulating embryonic development in this species and that a change in the number and/or functional status of the EGF-R may be a prerequisite for blastocyst activation and implantation in the spotted skunk.