Discussion
The present study is among the first to compare the gut microbiome of coexisting insular mesocarnivores within the context of fine-scale niche differentiation. More specifically, we examined gut microbial diversity and composition between island foxes and island spotted skunks inhabiting Santa Cruz Island and Santa Rosa Island off the coast of southern California. We found that both island foxes and island spotted skunks harbor the core mammalian microbiome, while also exhibiting species-specific microbial communities. Within species, island of origin significantly influenced gut microbial communities, with more pronounced differences observed in island spotted skunks compared to island foxes. This distinction may reflect the more specialized nature of island spotted skunk ecology, in contrast to the more generalist nature of island fox ecology. It may further reflect evolutionary processes operating during a longer period of separation between island spotted skunk populations, which diverged roughly 10,000 years ago, compared to island foxes, which colonized the islands roughly 7,000 years ago .
Regarding their similarities, the shared phylogenetic history of island foxes and island spotted skunks within the class Mammalia, order Carnivora, and suborder Caniformia contributes to both host species harboring phyla that comprise the core mammalian microbiome. These phyla include Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria, with the additional phylum Fusobacteria often reported in mammalian carnivores . Interestingly, we observed higher levels of Fusobacteria in island foxes, which is consistent with other studies characterizing the microbiome of species within Canidae . These results reflect the importance of evolutionary history in shaping the core features of mammalian microbiomes at higher taxonomic levels .
Yet amid these high-level similarities, we also observed significant differentiation between island foxes and island spotted skunks. In fact, species identity explained the largest percentage of variation in our dataset by a significant margin, with 24% of microbial abundance and 19.1% of microbial presence attributed to host species. This result may reflect the evolutionary divergence between ancestral lineages of the Canidae and Mephitidae families roughly 49 million years ago, as well as ecological differences between these species that likely recur throughout their range. We posit that the reported patterns of diversity likely reflect both of these eco-evolutionary processes, and in particular, the breadth of each species’ ecological niche.
For example, examination of alpha diversity revealed a wider range of bacterial ASVs detected in island foxes compared to island spotted skunks, likely resulting from their more generalist resource utilization . Island foxes encounter a wider variety of food sources and environments that may contribute to their higher bacterial richness, as microbiomes are known to reflect an organism’s diet and environmental setting . Conversely, as island spotted skunks are more specialized in resource use, they likely experience reduced exposure to these external sources of microbial variation, thus contributing to their lower bacterial richness. Their diet provides a likely candidate underlying this result. Compared to more omnivorous island foxes, island spotted skunk diets are mostly carnivorous and may therefore require fewer microbes to digest their smaller range of food sources . Previous research further showed that carnivore microbiomes tend to exhibit lower richness than omnivore microbiomes, as meat proteins are easier to digest than plant compounds . We therefore concluded that species identity exerted a strong influence on gut microbial communities, likely due to their dietary differences.
These interspecific differences were further reflected in the relative abundance of bacterial taxa, as numerous classes significantly differed between island foxes and island spotted skunks. On both Santa Cruz and Santa Rosa Islands, island foxes harbored higher relative abundances of Gammaproteobacteria and Deltaproteobacteria than their island spotted skunks counterparts. Gammaproteobacteria have been linked to coastal amphipod species similar to those preyed upon by island foxes . They have also been associated with soil invertebrates such as earthworms , which were recently introduced to the Channel Islands . These results potentially reflect a dietary preference towards these food sources among island foxes. It may also reflect evolutionary history, as Gammaproteobacteria has been isolated from other species within Canidae . The other class consistently more abundant in island foxes, Deltaproteobacteria, has been linked to marine environments , and may further reflect island fox foraging behavior on beaches .
We also noted interspecific differences unique to each island. On Santa Rosa Island, island spotted skunks exhibit higher abundances of Epsilonproteobacteria. The genus largely driving this result wasCampylobacter , which is often associated with enteritis in humans that come into contact with soil, untreated water sources, or animals . In island spotted skunks, this may derive from their local environment or prey items, as Campylobacter has previously been isolated from wild mice . On Santa Cruz Island, Actinobacteria proved to be more abundant in island spotted skunks. As Actinobacteria is one of the most abundant bacterial taxa in soils , this may reflect habitat use by island spotted skunks, which spend more time in ravines or covered places while foraging for soil-dwelling prey . We therefore posit that the increased presence of certain bacterial classes in island foxes and island spotted skunks may derive from eco-evolutionary differences in their population histories, diets, and preferred habitat types.
While species identity was the strongest predictor of bacterial composition, we also observed intraspecific differences between island populations. This pattern was more pronounced in island spotted skunks, where 15.9% of bacterial abundance and 14.6% of species presence were attributed to island of origin, compared to 6.3% of bacterial abundance and 5.6% of species presence in island foxes. We again attribute this near three-fold difference to two likely factors: the longer separation time and more specialist ecology of island spotted skunks, as subtle differences between island habitats may have a stronger effect on their gut microbial communities. This contrasts with the more recent colonization events and more generalist ecology of island foxes, which harbor a greater diversity of microbes that may reflect their interactions with numerous dietary items and microhabitats across both islands. This likely has a homogenizing effect on their gut microbial communities, although we do still find evidence of intraspecific variation, as previously reported in island fox gut microbial communities .
Differential abundance testing supported these overarching patterns, as intraspecific comparisons only revealed one bacterial class significantly differing between island spotted skunk populations amid no significant differences between island fox populations. Within skunks, the Santa Cruz Island population harbored significantly more Coriobacteriia, which has been linked with the addition of crickets to the diet of domestic dogs as it helps break down chitin in insect exoskeletons . These results suggest that Santa Cruz Island spotted skunks may be eating larger proportions of native arthropod species than their Santa Rosa counterparts. This link between Coriobacteriia and crickets is particularly compelling, as previous research reported Jerusalem crickets (Stenopelmatus fuscus ) as the most frequently consumed arthropod by Santa Cruz Island spotted skunks . Considered alongside interspecific results, this finding further underscores the importance of dietary preference as a driver of gut microbial composition within and between host populations and species.
In summary, the present study characterized the gut microbiomes of island foxes and island spotted skunks across two of California’s Channel Islands: Santa Cruz Island and Santa Rosa Island. We identified the core mammalian microbiome present in both species likely due to their shared evolutionary history, and reported evidence of niche differentiation likely due to differences in diet and habitat use between these coexisting mesocarnivores. We found that host species was the primary driver of microbial differentiation within our dataset, and additionally reported intraspecific variation between island populations. These intraspecific differences were more pronounced in the longer separated and more specialist island spotted skunks compared to the more recently colonized and more generalist island foxes. These results therefore support the claim that host evolutionary history and contemporary ecology (i.e. , diet, habitat use, and local environmental context) all play a role in shaping the gut microbiome. Commensal bacteria likely reflect – and perhaps even contribute to – the fine-scale niche differentiation that allows these insular terrestrial carnivores to coexist. To better understand this ecological rarity, we recommend further study of the diet, behavior, and bacteria colonizing these known competitors. This will shed additional light on the eco-evolutionary processes that have enabled their coexistence since initial colonization millennia ago, and will hopefully facilitate future coexistence for many millennia to come.
Acknowledgments: We would like to thank Bridget Parrino, Heather Alford, Victor Zhang, and Mason London for collecting rectal swabs from island foxes and island spotted skunks. We thank Robert Fleischer and Nancy McInerney from the Center for Conservation Genomics at Smithsonian’s National Zoo and Conservation Biology Institute (NZCBI) for their support of this project. This work was funded by the Smithsonian Institution Fellowship Program, Friends of the Island Fox Research Grant, Georgetown University Annual Research Grant, and Georgetown University Summer Salary Supplement. Further support was provided by the NZCBI Center for Conservation Genomics and Center for Species Survival.