White-footed Mice Peromyscus Leucopus Research Articles

Signatures of positive selection and local adaptation to urbanization in white-footed mice (Peromyscus leucopus).

Urbanization significantly alters natural ecosystems and has accelerated globally. Urban wildlife populations are often highly fragmented by human infrastructure, and isolated populations may adapt in response to local urban pressures. However, relatively few studies have identified genomic signatures of adaptation in urban animals. We used a landscape genomic approach to examine signatures of selection in urban populations of white-footed mice (Peromyscus leucopus) in New York City. We analysed 154,770 SNPs identified from transcriptome data from 48 P.leucopus individuals from three urban and three rural populations and used outlier tests to identify evidence of urban adaptation. We accounted for demography by simulating a neutral SNP data set under an inferred demographic history as a null model for outlier analysis. We also tested whether candidate genes were associated with environmental variables related to urbanization. In total, we detected 381 outlier loci and after stringent filtering, identified and annotated 19 candidate loci. Many of the candidate genes were involved in metabolic processes and have well-established roles in metabolizing lipids and carbohydrates. Our results indicate that white-footed mice in New York City are adapting at the biomolecular level to local selective pressures in urban habitats. Annotation of outlier loci suggests selection is acting on metabolic pathways in urban populations, likely related to novel diets in cities that differ from diets in less disturbed areas.

INTERSPECIFIC COMPARISON OF HANTAVIRUS PREVALENCE IN PEROMYSCUS POPULATIONS FROM A FRAGMENTED AGRO-ECOSYSTEM IN INDIANA, USA.

Comparatively little is known about hantavirus prevalence within rodent populations from the Midwestern US, where two species of native mice, the prairie deer mouse ( Peromyscus maniculatus bairdii) and the white-footed mouse ( Peromyscus leucopus noveboracensis), are dominant members of rodent communities. We sampled both species in central Indiana and tested individuals for presence of hantavirus antibodies to determine whether seroprevalence (percent of individuals with antibodies reactive to Sin Nombre virus antigen) differed between species, or among different habitat types within fragmented agro-ecosystems. Prevalence of hantavirus antibodies varied significantly between species, with seroprevalence in prairie deer mice (21.0%) being nearly four times higher than in white-footed mice (5.5%). Seroprevalence was almost eight times higher within the interior of row-crop fields (37.7%) occupied solely by prairie deer mouse populations, relative to field edges (5.2%) or adjacent forest habitat (6.1%). In the fragmented Midwestern agro-ecosystem of this study, prairie deer mice appear to be the dominant hantavirus reservoir, with particularly high seroprevalence in populations within the interior of row-crop fields.

New York's mice are evolving to eat a city diet

There are always better restaurants in the city, and that could be making the town mice of New York genetically distinct from their country cousins. Stephen Harris at the State University of New York and Jason Munshi-South of Fordham University in New York City caught 48 white-footed mice (Peromyscus leucopus) from three New York parks and three nearby rural areas. The mice are native to this part of North America, so the pair wanted to find out whether some had begun to evolve for city living. They examined the mice's RNA to see if the rural and urban populations expressed different genes

Admixture on the northern front: population genomics of range expansion in the white-footed mouse (Peromyscus leucopus) and secondary contact with the deer mouse (Peromyscus maniculatus).

Range expansion has genetic consequences expected to result in differentiated wave-front populations with low genetic variation and potentially introgression from a local species. The northern expansion of Peromyscus leucopus in southern Quebec provides an opportunity to test these predictions using population genomic tools. Our results show evidence of recent and post-glacial expansion. Genome-wide variation in P. leucopus indicates two post-glacial lineages are separated by the St. Lawrence River, with a more recent divergence of populations isolated by the Richelieu River. In two of three transects we documented northern populations with low diversity in at least one genetic measure, although most relationships were not significant. Consistent with bottlenecks and allele surfing during northward expansion, we document a northern-most population with low nucleotide diversity, divergent allele frequencies and the most private alleles, and observed heterozygosity indicates outcrossing. Ancestry proportions revealed putative hybrids of P. leucopus and P. maniculatus. A formal test for gene flow confirmed secondary contact, showing that a reticulate population phylogeny between P. maniculatus and P. leucopus was a better fit to the data than a bifurcating model without gene flow. Thus, we provide the first genomic evidence of gene flow between this pair of species in natural populations. Understanding the evolutionary consequences of secondary contact is an important conservation concern as climate-induced range expansions are expected to result in new hybrid zones between closely related species.

Exploration of stable isotope analysis for tick host identification

Due to the problem of tick-borne diseases, there is a need to better understand the importance of different host species in maintaining enzootic disease cycles. We explored the utility of stable isotope analysis to identify the larval hosts of questing ixodid ticks. In laboratory experiments, we used Ixodes scapularis and two host species that are important in the Lyme disease system in eastern North America. First, we tested how effectively a short-term dietary tracer (13C in corn) was reflected in molted ticks. Second, we attempted to identify the host species (either white-footed mouse (Peromyscus leucopus) or eastern chipmunk (Tamias striatus)), based on the isotopic signature of the ticks that had fed on them.The corn isotopic signal was easily detectable in the ticks that fed on corn-diet hosts despite the brief feeding period (96h). However, we were not able to differentiate between flat Ixodes scapularis nymphs that had fed as larvae on mice vs. those fed on chipmunks. Isotopic signatures of fur from mice and chipmunks were also indistinguishable, probably due to the similar diets of these two species in the wild. We conclude that, while stable isotope analysis of ticks may not be able to distinguish between ecologically similar host species, it may be useful in sorting ticks to the level of feeding guild of the host.

Secondary dispersal of forest herb seeds from raccoon dung: contrasting service by multiple vectors

Secondary movement of seeds deposited in dung potentially improves their chances of seedling establishment but has rarely been documented in temperate-zone deciduous forest. We followed mayapple (Podophyllum peltatum) seeds after deposition in raccoon (Procyon lotor) dung to test for secondary dispersal in a long-established deciduous forest in southeastern Ohio, USA. Marked seeds were incorporated into raccoon dung, placed in natural forest sites, and observed at regular intervals until the subsequent Spring. Abiotic seed movement was monitored during natural and simulated rain events. To identify animal vectors and describe their interaction with seeds, motion-triggered wildlife cameras were trained on dung with embedded mayapple seeds. Seed movement was initially limited by adhesion in the viscid dung matrix, but 15% of seeds eventually moved >20 cm from the point of placement, a minimal estimate of a shoot’s competitive neighborhood (maximum movement distance = 130 cm). Significantly greater movement occurred on slopes and in the absence of leaf litter. Rainfall moved seeds as far as 51 cm. White-footed mice (Peromyscus leucopus) and chipmunks (Tamias striatus) began removing seeds within 24 h of dung placement, presumably caching a portion of them outside the 1.5-m radius of observation. At least four other animal species interacted with dung in ways unrelated to seed presence, which nevertheless resulted in seed movement on a scale of centimeters. Thus, multiple secondary processes caused seed movement, each resulting in a distinctive seed distribution. The forest herb potentially benefits through reduction in seed density and distribution to a variety of microsites.

Functional connectivity of the white-footed mouse in Southern Quebec, Canada

The white-footed mouse (Peromyscus leucopus) is an important reservoir host for the pathogen responsible for Lyme disease in eastern North-America. Indigenous cases of Lyme disease in southern Quebec have increased from two cases in 2004 to 160 cases in 2015. Because of the strong relationship between the white-footed mouse occurrence and the Lyme disease pathogen prevalence, we can estimate the risk of Lyme disease by finding areas of high contact between mice. In this study, we inferred the movement patterns and contact rate of the white-footed mouse in southern Quebec. We used pattern-orientated modelling to estimate a directional measure of functional connectivity from an Individual-Based Model. We replicated the spatial pattern observed in previously published molecular analysis of a white-footed mouse population. A perceptual range of 80 m best explained the genetic structure of the white-footed mouse in the region. The paths of individuals generally overlapped the edges of urban centers and the boundary of linear obstacles such as highways and water bodies. We show that the contact probability of mice was a good predictor of the number of mice caught in the field. Our findings highlight the usefulness of individual-based models to potentially predict high contact areas and disease hotspots across landscapes.

Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses.

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1–30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host) with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN) response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1) or the type I IFN receptor (IFNAR1) by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic insight into efficient control of virus replication and may inform the development of antiviral therapeutics.

Low-basal area treatment and prescribed fire to restore oak-pine savannas alter small mammal communities

Following decades of fire suppression many oak-pine savanna ecosystems have shifted to closed-canopied forests. Restoration of these ecosystems to their savanna condition is seen as a way to reduce woody species encroachment and dangerous fuel loads, and restore community species composition. The management practices to achieve these goals typically involve thinning and prescribed fire. We assessed how thinning to reduce basal area combined with frequent prescribed fire influenced small mammal communities and their habitat. We focused on six habitat variables that can influence small mammal abundance and species composition: stand basal area of live trees and snags, volume of coarse woody debris, percent ground cover, forest floor depth, and distance of vulnerability, a measure of understory vegetation structure and animal vulnerability to predators. Although savanna restoration reduced basal area by 80%, there was no change in snag density or coarse woody debris volume. Savanna restoration significantly increased the ground cover of graminoids, forbs, bare ground, and down woody debris and reduced forest floor depth and distance of vulnerability. These habitat changes likely contributed to the significant differences between small mammal communities in restored and non-restored stands. Restoration treatments caused a large increase in abundance of White-footed Mouse (Peromyscus leucopus) and important changes in community assemblages. Least Shrew (Cryptotis parva), Fulvous Harvest Mouse (Reithrodontomys fulvescens), Eastern Harvest Mouse (R. humulis), and Hispid Cotton Rat (Sigmodon hispidus) were caught only in restored stands, while House Mouse (Mus musculus), Plains Harvest Mouse (R. montanus), and Texas Mouse (P. attwateri) were caught only in non-restored stands.

Effects of parasite and historic driven selection on the diversity and structure of a MHC-II gene in a small mammal species (Peromyscus leucopus) undergoing range expansion

Genetic diversity may decrease from the centre to the margin of a species distribution range due to neutral stochastic processes. Selection may also alter genetic diversity in non-neutral markers, such as genes associated with the immune system. Both neutral processes and selection on the immune system are thus expected to affect the spatial distribution of such markers, but the relative strength of each has been scarcely studied. Here, we compared the diversity of a neutral marker (mitochondrial cytochrome b) and a selected marker (DRB gene from the MHC-II), in eastern-North American populations of white-footed mice (Peromyscus leucopus), a species known for its role of main reservoir of the Lyme disease. We observed distinct phylogeographic patterns with these two markers, which may be the result of selection pressure acting upon the DRB gene. As predicted by the central marginal hypothesis, we observed a loss of neutral genetic diversity toward the margin of the species distribution. A decrease in diversity was also observed for the DRB gene, likely due to genetic drift and positive selection operated by helminth parasites. Such a loss in genetic diversity at the range margin may slow down the ongoing expansion of P. leucopus, by counterbalancing the effect of global warming on the mouse survival at higher latitude.

Isolation of the Lyme Disease Spirochete Borrelia mayonii From Naturally Infected Rodents in Minnesota.

Borrelia mayonii is a newly described member of the Borrelia burgdorferi sensu lato complex that is vectored by the black-legged tick (Ixodes scapularis Say) and a cause of Lyme disease in Minnesota and Wisconsin. Vertebrate reservoir hosts involved in the enzootic maintenance of B. mayonii have not yet been identified. Here, we describe the first isolation of B. mayonii from naturally infected white-footed mice (Peromyscus leucopus Rafinesque) and an American red squirrel (Tamiasciurus hudsonicus Erxleben) from Minnesota, thus implicating these species as potential reservoir hosts for this newly described spirochete.

Inbreeding and selection shape genomic diversity in captive populations: Implications for the conservation of endangered species

Captive breeding programs are often initiated to prevent species extinction until reintroduction into the wild can occur. However, the evolution of captive populations via inbreeding, drift, and selection can impair fitness, compromising reintroduction programs. To better understand the evolutionary response of species bred in captivity, we used nearly 5500 single nucleotide polymorphisms (SNPs) in populations of white-footed mice (Peromyscus leucopus) to measure the impact of breeding regimes on genomic diversity. We bred mice in captivity for 20 generations using two replicates of three protocols: random mating (RAN), selection for docile behaviors (DOC), and minimizing mean kinship (MK). The MK protocol most effectively retained genomic diversity and reduced the effects of selection. Additionally, genomic diversity was significantly related to fitness, as assessed with pedigrees and SNPs supported with genomic sequence data. Because captive-born individuals are often less fit in wild settings compared to wild-born individuals, captive-estimated fitness correlations likely underestimate the effects in wild populations. Therefore, minimizing inbreeding and selection in captive populations is critical to increasing the probability of releasing fit individuals into the wild.

Morphological Differentiation in White-Footed Mouse (Mammalia: Rodentia: Cricetidae: Peromyscus leucopus) Populations from the New York City Metropolitan Area

Abstract Genetic studies have shown that New York City white-footed mouse (Peromyscus leucopus) populations exhibit substantial genetic structure and high levels of allelic diversity and heterozygosity. These studies have also identified mutations and genes involved in the divergence of urban and rural P. leucopus populations. To investigate whether morphological change mirrors the genetic differentiation observed in New York City P. leucopus populations, we conducted univariate and multivariate analyses on 4 external and 14 skull variables to compare urban, suburban and rural P. leucopus populations from in and around New York City. The only significant morphological differences among the three populations were in upper and lower toothrow lengths, both of which had high loadings in our principal components analyses. In general, rural individuals were found to have longer upper and lower toothrows than urban ones. This difference is likely due to the relationship between food quality and size of dental oc...

Acquisition of Borrelia burgdorferi Infection by Larval Ixodes scapularis (Acari: Ixodidae) Associated With Engorgement Measures.

Measuring rates of acquisition of the Lyme disease pathogen, Borrelia burgdorferi sensu lato Johnson, Schmid, Hyde, Steigerwalt & Brenner, by the larval stage of Ixodes scapularis Say is a useful tool for xenodiagnoses of B. burgdorferi in vertebrate hosts. In the nymphal and adult stages of I. scapularis, the duration of attachment to hosts has been shown to predict both body engorgement during blood feeding and the timing of infection with B. burgdorferi. However, these relationships have not been established for the larval stage of I. scapularis. We sought to establish the relationship between body size during engorgement of larval I. scapularis placed on B. burgdorferi-infected, white-footed mice (Peromyscus leucopus Rafinesque) and the presence or absence of infection in larvae sampled from hosts over time. Body size, time, and their interaction were the best predictors of larval infection with B. burgdorferi. We found that infected larvae showed significantly greater engorgement than uninfected larvae as early as 24 h after placement on a host. These findings may suggest that infection with B. burgdorferi affects the larval feeding process. Alternatively, larvae that engorge more rapidly on hosts may acquire infections faster. Knowledge of these relationships can be applied to improve effective xenodiagnosis of B. burgdorferi in white-footed mice. Further, these findings shed light on vector-pathogen-host interactions during an understudied part of the Lyme disease transmission cycle.

Human activity and habitat type affect perceived predation risk in urban white-footed mice (Peromyscus leucopus)

Predation risk is one of the largest costs associated with foraging in small mammals. Small mammals select microhabitat features such as tree and shrub canopy cover, woody debris and vegetative ground cover that can lower the risk of detection from predators and provide greater protection if discovered. Small mammals also increase foraging activity and decrease selection for cover when cloud cover increases and moon illumination is less. Often researchers assume small mammals in urban areas respond to these cues in the same manner as in natural areas, but these cues themselves are altered in urban zones. In this study, we investigated how Amur honeysuckle (Lonicera maackii) and coarse woody debris (CWD) affected giving-up density (GUD) in white-footed mice (Peromyscus leucopus). Each of three habitat treatments (open flood channel, the edge and interior of the honeysuckle patch) contained cover treatments with coarse woody debris present or absent. The six treatment combinations were compared to environmental variables (temperature, humidity and illumination) and habitat variables to test their effect on GUD. Peromyscus leucopus foraged to lower densities in areas with CWD present and also under the honeysuckle canopy, using this invasive shrub to decrease predation risk, potentially increasing survivability within this urban park. Increased human presence negatively affected foraging behavior across treatments. Human presence and light pollution significantly influenced P. leucopus, modifying their foraging behavior and demonstrating that both fine- and coarse-scale urban factors can affect small mammals. Foraging increased as humidity increased, particularly under the honeysuckle canopy. Changes in illumination due to moonlight and cloud cover did not affect foraging behavior, suggesting urban light pollution may have altered behavioral responses to changes in light levels. Lonicera maackii seemed to facilitate foraging in P. leucopus, even though it adversely affects the plant community, suggesting that its impact may not be entirely negative.

Invasive exotic shrub modifies a classic animal-habitat relationship and alters patterns of vertebrate seed predation.

Recent evidence suggests that invasive exotic plants can provide novel habitats that alter animal behavior. However, it remains unclear whether classic animal-habitat associations that influence the spatial distribution of plant-animal interactions, such as small mammal use of downed woody debris, persist in invaded habitats. We removed an invasive exotic shrub (buckthorn, Rhamnus cathartica) from 7 of 15 plots in Wisconsin. In each plot, we deployed 200 tagged Quercus rubra seeds in November 2014. After five months, tags were recovered to track spatial patterns of small mammal seed predation. Most recovered tags were associated with consumed seeds (95%); live-trapping, ancillary camera-trapping, and previous behavioral studies suggest that white-footed mice (Peromyscus leucopus) were responsible for most seed predation. In habitats without R.cathartica, most seed predation occurred near woody debris. In habitats with R.cathartica, small mammals rarely consumed seeds near woody debris, and seed predation occurred farther from the plot center and was less spatially clustered. Our results illustrate that invasive exotic shrubs can disrupt an otherwise common animal-habitat relationship. Failing to account for changes in habitat use may diminish our ability to predict animal distributions and outcomes of species interactions in novel habitats created by invasive exotic plants.

Experimental evaluation of Peromyscus leucopus as a reservoir host of the Ehrlichia muris-like agent

BackgroundThe Ehrlichia muris-like agent (EMLA) is a newly recognized human pathogen in the North Central United States. Although blacklegged ticks (Ixodes scapularis) have been identified as capable vectors, wild reservoirs have not yet been established for EMLA. As key hosts for I. scapularis, white-footed mice (Peromyscus leucopus) are important reservoirs for various tick-borne pathogens, and potentially, for EMLA. The objective of this study was to evaluate reservoir competence in P. leucopus using a natural vector.ResultsMice acquired EMLA infection from feeding ticks and were able to transmit infection to naïve ticks. Transmission between simultaneously feeding tick life stages was also demonstrated. Infections in mice were acute and severe, with systemic dissemination. Limited host survival and clearance of infection among survivors resulted in a narrow interval where EMLA could be acquired by feeding ticks.ConclusionsPeromyscus leucopus is a competent reservoir of EMLA and likely to play a role in its enzootic transmission cycle. The duration and severity of EMLA infection in these hosts suggests that tick phenology is a critical factor determining the geographic distribution of EMLA in North America.

The Prevalence of Trypanosoma cruzi, the Causal Agent of Chagas Disease, in Texas Rodent Populations.

Rodent species were assessed as potential hosts of Trypanosoma cruzi, the etiologic agent of Chagas disease, from five sites throughout Texas in sylvan and disturbed habitats. A total of 592 rodents were captured, resulting in a wide taxonomic representation of 11 genera and 15 species. Heart samples of 543 individuals were successfully analyzed by SybrGreen-based quantitative PCR (qPCR) targeting a 166 bp fragment of satellite DNA of T. cruzi. Eight rodents representing six species from six genera and two families were infected with T. cruzi. This is the first report of T. cruzi in the pygmy mouse (Baiomys taylori) and the white-footed mouse (Peromyscus leucopus) for the USA. All infected rodents were from the southernmost site (Las Palomas Wildlife Management Area). No differences in pathogen prevalence existed between disturbed habitats (5 of 131 tested; 3.8%) and sylvan habitats (3 of 40 tested; 7.5%). Most positives (n=6, 16% prevalence) were detected in late winter with single positives in both spring (3% prevalence) and fall (1% prevalence). Additionally, 30 Triatoma insects were collected opportunistically from sites in central Texas. Fifty percent of these insects, i.e., 13 T. gerstaeckeri (68%), and two T. lecticularia (100%) were positive for T. cruzi. Comparative sequence analyses of 18S rRNA of samples provided identical results with respect to detection of the presence or absence of T. cruzi and assigned T. cruzi from rodents collected in late winter to lineage TcI. T. cruzi from Triatoma sp. and rodents from subsequent collections in spring and fall were different, however, and could not be assigned to other lineages with certainty.

Stable Transmission of Borrelia burgdorferi Sensu Stricto on the Outer Banks of North Carolina.

The spirochaete (Borrelia burgdorferi) associated with Lyme disease was detected in questing ticks and rodents during a period of 18years, 1991-2009, at five locations on the Outer Banks of North Carolina. The black-legged tick (Ixodes scapularis) was collected at varied intervals between 1991 and 2009 and examined for B.burgdorferi. The white-footed mouse (Peromyscus leucopus), house mouse (Mus musculus) marsh rice rat (Oryzomys palustris), marsh rabbit (Sylvilagus palustris), eastern cottontail (Sylvilagus floridanus) and six-lined racerunner (Cnemidophorus sexlineatus) were live-trapped, and their tissues cultured to isolate spirochaetes. Borrelia burgdorferi isolates were obtained from questing adult I.scapularis and engorged I.scapularis removed from P.leucopus, O.palustris and S.floridanus. The prevalence of B.burgdorferi infection was variable at different times and sites ranging from 7 to 14% of examined questing I.scapularis. Mitochondrial (16S) rRNA gene phylogenetic analysis from 65 adult I.scapularis identified 12 haplotypes in two major clades. Nine haplotypes were associated with northern/Midwestern I.scapularis populations and three with southern I.scapularis populations. Sixteen isolates obtained from tick hosts in 2005 were confirmed to be B.burgdorferi by amplifying and sequencing of 16S rRNA and 5S-23S intergenic spacer fragments. The sequences had 98-99% identity to B.burgdorferi sensu stricto strains B31, JD1 and M11p. Taken together, these studies indicate that B.burgdorferi sensu stricto is endemic in questing I.scapularis and mammalian tick hosts on the Outer Banks of North Carolina.

Microsatellite markers reveal low frequency of natural hybridization between the white-footed mouse (Peromyscus leucopus) and deer mouse (Peromyscus maniculatus) in southern Quebec, Canada.

In some parts of southern Quebec, two closely related rodent species - the white-footed mouse (Peromyscus leucopus) and the deer mouse (Peromyscus maniculatus) - have recently come in contact because of climate-driven changes in the distribution of the former. Both species share similar morphology, ecology, and life history traits, which suggests that natural hybridization may be possible. Hybridization among these two species can have important implications on the ecological roles these rodents play in disease transmission, yet few researchers have attempted to examine this phenomenon and results from previous hybridization experiments have remained inconclusive and conflicting. In this study, we attempt to investigate the occurrence of hybridization among white-footed mice and deer mice in southern Quebec by genotyping wild caught specimens with selectively neutral, polymorphic microsatellite markers. Our analyses suggest that hybridization may be occurring at extremely low frequency between both species in our study area. The presence of such hybridization events, even at low frequencies, may have implications on disease transmission risk in the region and further detailed studies are necessary.