Evolution of a novel adrenal cell type that promotes parental care.

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

Differential Expression in Testis and Liver Transcriptomes from Four Species of Peromyscus (Rodentia: Cricetidae).

The genus Peromyscus represents a rapidly diverged clade of Cricetid rodents that contains multiple cryptic species and has a propensity for morphologic conservation across its members. The unresolved relationships in previously proposed phylogenies reflect a suspected rapid adaptive radiation. To identify functional groups of genes that may be important in reproductive isolation in a reoccurring fashion across the Peromyscus phylogeny, liver and testis transcriptomes from four species (P. attwateri, P. boylii, P. leucopus, and P. maniculatus) were generated and differential expression (DE) tests were conducted. Taxa were selected to represent members diverged from a common ancestor: P. attwateri + P. boylii (clade A), and P. leucopus + P. maniculatus (clade B). Comparison of clades (A vs. B) suggested that 252 transcripts had significant DE in the liver data set, whereas significant DE was identified for 657 transcripts in the testis data set. Further, 45 genes had DE isoforms in the 657 testis transcripts and most of these functioned in major reproductive roles such as acrosome assembly, spermatogenesis, and cell cycle processes (meiosis). DE transcripts in the liver mapped to more broad gene ontology terms (metabolic processes, catabolic processes, response to chemical, and regulatory processes), and DE transcripts in the testis mapped to gene ontology terms associated with reproductive processes, such as meiosis, sperm motility, acrosome assembly, and sperm-egg fusion. These results suggest that a suite of genes that conduct similar functions in the testes may be responsible for the adaptive radiation events and potential reoccurring speciation of Peromyscus in terms of reproduction through varying expression levels.

Genomes, expression profiles, and diversity of mitochondria of the White-footed Deermouse Peromyscus leucopus, reservoir of Lyme disease and other zoonoses.

The cricetine rodents Peromyscus leucopus and P. maniculatus are key reservoirs for several zoonotic diseases in North America. We determined the complete circular mitochondrial genome sequences of representatives of 3 different stock colonies of P. leucopus, one stock colony of P. maniculatus and two wild populations of P. leucopus. The genomes were syntenic with that of the murids Mus musculus and Rattus norvegicus. Phylogenetic analysis confirmed that these two Peromyscus species are sister taxa in a clade with P. polionotus and also uncovered a distinction between P. leucopus populations in the eastern and the central United States. In one P. leucopus lineage four extended regions of mitochondrial pseudogenes were identified in the nuclear genome. RNA-seq analysis revealed transcription of the entire genome and differences from controls in the expression profiles of mitochondrial genes in the blood, but not in liver or brain, of animals infected with the zoonotic pathogen Borrelia hermsii. PCR and sequencing of the D-loop of the mitochondrion identified 32 different haplotypes among 118 wild P. leucopus at a Connecticut field site. These findings help to further establish P. leucopus as a model organism for studies of emerging infectious diseases, ecology, and in other disciplines.

Characterizing the influence of chronic hypobaric hypoxia on diaphragmatic myofilament contractile function and phosphorylation in high-altitude deer mice and low-altitude white-footed mice.

Deer mice, Peromyscusmaniculatus, live at high altitudes where limited O2 represents a challenge to maintaining oxygen delivery to tissues. Previous work has demonstrated that hypoxia acclimation of deer mice and low altitude white-footed mice (P. leucopus) increases the force generating capacity of the diaphragm. The mechanism behind this improved contractile function is not known. Within myocytes, the myofilament plays a critical role in setting the rate and level of force production, and its ability to generate force can change in response to changes in physiological conditions. In the current study, we examined how chronic hypobaric hypoxia exposure of deer mice and white-footed mice influences the Ca2+ activation of force generation by skinned diaphragmatic myofilaments, and the phosphorylation of myofilament proteins. Results demonstrate that myofilament force production, and the Ca2+ sensitivity of force generation, were not impacted by acclimation to hypobaric hypoxia, and did not differ between preparations from the two species. The cooperativity of the force-pCa relationship, and the maximal rate of force generation were also the same in the preparations from both species, and not impacted by acclimation. Finally, the relative phosphorylation of TnT, and MLC was lower in deer mice than white-footed mice, but was not affected by acclimation. These results indicate that species differences in diaphragm function, and the increase in force production with hypoxia acclimation, are not due to differences, or changes, in myofilament function. However, it appears that diaphragmatic myofilament function in these species is not affected by chronic hypobaric hypoxia exposure.

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.

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.

Acclimation to hypoxia increases carbohydrate use during exercise in high-altitude deer mice.

The low O2 experienced at high altitude is a significant challenge to effective aerobic locomotion, as it requires sustained tissue O2 delivery in addition to the appropriate allocation of metabolic substrates. Here, we tested whether high- and low-altitude deer mice (Peromyscus maniculatus) have evolved different acclimation responses to hypoxia with respect to muscle metabolism and fuel use during submaximal exercise. Using F1 generation high- and low-altitude deer mice that were born and raised in common conditions, we assessed 1) fuel use during exercise, 2) metabolic enzyme activities, and 3) gene expression for key transporters and enzymes in the gastrocnemius. After hypoxia acclimation, highland mice showed a significant increase in carbohydrate oxidation and higher relative reliance on this fuel during exercise at 75% maximal O2 consumption. Compared with lowland mice, highland mice had consistently higher activities of oxidative and fatty acid oxidation enzymes in the gastrocnemius. In contrast, only after hypoxia acclimation did activities of hexokinase increase significantly in the muscle of highland mice to levels greater than lowland mice. Highland mice also responded to acclimation with increases in muscle gene expression for hexokinase 1 and 2 genes, whereas both populations increased mRNA expression for glucose transporters. Changes in skeletal muscle with acclimation suggest that highland mice had an increased capacity for the uptake and oxidation of circulatory glucose. Our results demonstrate that highland mice have evolved a distinct mode of hypoxia acclimation that involves an increase in carbohydrate use during exercise.

A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species.

Peromyscus mice as a model for studying natural variation.

The deer mouse (genus Peromyscus) is the most abundant mammal in North America, and it occupies almost every type of terrestrial habitat. It is not surprising therefore that the natural history of Peromyscus is among the best studied of any small mammal. For decades, the deer mouse has contributed to our understanding of population genetics, disease ecology, longevity, endocrinology and behavior. Over a century's worth of detailed descriptive studies of Peromyscus in the wild, coupled with emerging genetic and genomic techniques, have now positioned these mice as model organisms for the study of natural variation and adaptation. Recent work, combining field observations and laboratory experiments, has lead to exciting advances in a number of fields-from evolution and genetics, to physiology and neurobiology.

Transcriptome markers of viral persistence in naturally-infected andes virus (bunyaviridae) seropositive long-tailed pygmy rice rats.

Long-tailed pygmy rice rats (Oligoryzomys longicaudatus) are principal reservoir hosts of Andes virus (ANDV) (Bunyaviridae), which causes most hantavirus cardiopulmonary syndrome cases in the Americas. To develop tools for the study of the ANDV-host interactions, we used RNA-Seq to generate a de novo transcriptome assembly. Splenic RNA from five rice rats captured in Chile, three of which were ANDV-infected, was used to generate an assembly of 66,173 annotated transcripts, including noncoding RNAs. Phylogenetic analysis of selected predicted proteins showed similarities to those of the North American deer mouse (Peromyscus maniculatus), the principal reservoir of Sin Nombre virus (SNV). One of the infected rice rats had about 50-fold more viral burden than the others, suggesting acute infection, whereas the remaining two had levels consistent with persistence. Differential expression analysis revealed distinct signatures among the infected rodents. The differences could be due to 1) variations in viral load, 2) dimorphic or reproductive differences in splenic homing of immune cells, or 3) factors of unknown etiology. In the two persistently infected rice rats, suppression of the JAK-STAT pathway at Stat5b and Ccnot1, elevation of Casp1, RIG-I pathway factors Ppp1cc and Mff, and increased FC receptor-like transcripts occurred. Caspase-1 and Stat5b activation pathways have been shown to stimulate T helper follicular cell (TFH) development in other species. These data are also consistent with reports suggestive of TFH stimulation in deer mice experimentally infected with hantaviruses. In the remaining acutely infected rice rat, the apoptotic pathway marker Cox6a1 was elevated, and putative anti-viral factors Abcb1a, Fam46c, Spp1, Rxra, Rxrb, Trmp2 and Trim58 were modulated. Transcripts for preproenkephalin (Prenk) were reduced, which may be predictive of an increased T cell activation threshold. Taken together, this transcriptome dataset will permit rigorous examination of rice rat-ANDV interactions and may lead to better understanding of virus ecology.

Hematologic and serum biochemical values of 4 species of Peromyscus mice and their hybrids.

Deer mice (Peromyscus maniculatus) and congeneric species are used in a wide variety of research applications, particularly studies of developmental, physiologic, and behavioral characteristics associated with habitat adaptation and speciation. Because peromyscine mice readily adapt to colony conditions, animals with traits of interest in the field are moved easily into the laboratory where they can be studied under controlled conditions. The purpose of this study was to determine the serum chemistry and hematologic parameters of 4 frequently used species from the Peromyscus Genetic Stock Center species (P. californicus, P. leucopus, P. maniculatus, and P. polionotus) and to determine quantitative differences in these parameters among species and between sexes. Triglyceride values were substantially higher in female compared with male mice in all 4 species. Similar cross-species differences in MCH were present. Overall there was considerable interspecific variation for most blood parameters, with little evidence for covariation of any 2 or more parameters. Because crosses of P. maniculatus and P. polionotus produce fertile offspring, segregation analyses can be applied to determine the genetic basis of any traits that differ between them, such as their 3.8- and 2.1-fold interspecific differences in cholesterol and triglyceride levels, respectively. The current data provide a set of baseline values useful for subsequent comparative studies of species experiencing different circumstances, whether due to natural variation or anthropogenic environmental degradation. To enable such comparisons, the raw data are downloadable from a site maintained by the Stock Center (http://ww2.biol.sc.edu/∼peromyscus).

Effects of inbreeding on skeletal size and fluctuating asymmetry of Peromyscus polionotus mice.

Measurements of size and asymmetry in morphology might provide early indications of damaging effects of inbreeding or other genetic changes in conservation breeding programs. We examined the effects of inbreeding on size and fluctuating asymmetry (FA) in skull and limb bone measurements in experimental populations of three subspecies of Peromyscus polionotus mice that had previously been shown to suffer significant reductions in reproductive success when inbred. Inbreeding caused significant depression in mean size in two of the subspecies (P. p. rhoadsi and P. p. subgriseus), but the effects were smaller in the third (P. p. leucocephalus). Inbreeding caused an increase in FA of just one of eight bilateral traits in one subspecies (P. p. rhoadsi). Inbreeding depression in size was more easily detected than the effects of inbreeding on FA. FA may be much less sensitive to inbreeding and other stresses than are more direct measures of fitness such as reproductive output and body mass growth rate. Given the large sample sizes and statistical complexity required to assess changes to typically very small levels of FA in captive populations, FA will not likely provide a useful measure of inbreeding depression in captive populations.

Regulatory changes contribute to the adaptive enhancement of thermogenic capacity in high-altitude deer mice.

In response to hypoxic stress, many animals compensate for a reduced cellular O(2) supply by suppressing total metabolism, thereby reducing O(2) demand. For small endotherms that are native to high-altitude environments, this is not always a viable strategy, as the capacity for sustained aerobic thermogenesis is critical for survival during periods of prolonged cold stress. For example, survivorship studies of deer mice (Peromyscus maniculatus) have demonstrated that thermogenic capacity is under strong directional selection at high altitude. Here, we integrate measures of whole-organism thermogenic performance with measures of metabolic enzyme activities and genomic transcriptional profiles to examine the mechanistic underpinnings of adaptive variation in this complex trait in deer mice that are native to different elevations. We demonstrate that highland deer mice have an enhanced thermogenic capacity under hypoxia compared with lowland conspecifics and a closely related lowland species, Peromyscus leucopus. Our findings suggest that the enhanced thermogenic performance of highland deer mice is largely attributable to an increased capacity to oxidize lipids as a primary metabolic fuel source. This enhanced capacity for aerobic thermogenesis is associated with elevated activities of muscle metabolic enzymes that influence flux through fatty-acid oxidation and oxidative phosphorylation pathways in high-altitude deer mice and by concomitant changes in the expression of genes in these same pathways. Contrary to predictions derived from studies of humans at high altitude, our results suggest that selection to sustain prolonged thermogenesis under hypoxia promotes a shift in metabolic fuel use in favor of lipids over carbohydrates.

Genetic impacts of Anacapa deer mice reintroductions following rat eradication.

The Anacapa deer mouse is an endemic subspecies that inhabits Anacapa Island, part of Channel Islands National Park, California. We used mitochondrial DNA cytochrome c oxidase subunit II gene (COII) and 10 microsatellite loci to evaluate the levels of genetic differentiation and variation in ~1400 Anacapa deer mice sampled before and for 4 years after a black rat (Rattus rattus) eradication campaign that included trapping, captive holding and reintroduction of deer mice. Both mitochondrial and microsatellite analyses indicated significant differentiation between Anacapa deer mice and mainland mice, and genetic variability of mainland mice was significantly higher than Anacapa mice even prior to reintroduction. Bayesian cluster analysis and Principal Coordinates Analysis indicated that East, Middle and West Anacapa mice were genetically differentiated from each other, but translocation of mice among islands resulted in the East population becoming less distinct as a result of management. Levels of heterozygosity were similar before and after management. However, numerous private alleles in the founder populations were not observed after reintroduction and shifts in allele frequencies occurred, indicating that the reintroduced populations experienced substantial genetic drift. Surprisingly, two mitochondrial haplotypes observed in an earlier study of Anacapa deer mice were lost in the 20 years prior to the rat eradication program, leaving only a single haplotype in Anacapa deer mice. This study demonstrates how genetic monitoring can help to understand the re-establishment of endemic species after the eradication of invasive species and to evaluate the effectiveness of the management strategies employed.

Competition drives cooperation among closely related sperm of deer mice.

Among the extraordinary adaptations driven by sperm competition is the cooperative behaviour of spermatozoa. By forming cooperative groups, sperm can increase their swimming velocity and thereby gain an advantage in intermale sperm competition. Accordingly, selection should favour cooperation of the most closely related sperm to maximize fitness. Here we show that sperm of deer mice (genus Peromyscus) form motile aggregations, then we use this system to test predictions of sperm cooperation. We find that sperm aggregate more often with conspecific than heterospecific sperm, suggesting that individual sperm can discriminate on the basis of genetic relatedness. Next, we provide evidence that the cooperative behaviour of closely related sperm is driven by sperm competition. In a monogamous species lacking sperm competition, Peromyscus polionotus, sperm indiscriminately group with unrelated conspecific sperm. In contrast, in the highly promiscuous deer mouse, Peromyscus maniculatus, sperm are significantly more likely to aggregate with those obtained from the same male than with sperm from an unrelated conspecific donor. Even when we test sperm from sibling males, we continue to see preferential aggregations of related sperm in P. maniculatus. These results suggest that sperm from promiscuous deer mice discriminate among relatives and thereby cooperate with the most closely related sperm, an adaptation likely to have been driven by sperm competition.

Evolutionary and functional insights into the mechanism underlying high-altitude adaptation of deer mouse hemoglobin.

Adaptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. Here we investigate these possibilities by conducting a combined evolutionary and functional analysis of duplicated globin genes in natural populations of deer mice (Peromyscus maniculatus) that are adapted to different elevational zones. A multilocus analysis of nucleotide polymorphism and linkage disequilibrium revealed that high-altitude adaptation of deer mouse hemoglobin involves parallel functional differentiation at multiple unlinked gene duplicates: two alpha-globin paralogs on chromosome 8 and two beta-globin paralogs on chromosome 1. Differences in O(2)-binding affinity of the alternative beta-chain hemoglobin isoforms were entirely attributable to allelic differences in sensitivity to 2,3-diphosphoglycerate (DPG), an allosteric cofactor that stabilizes the low-affinity, deoxygenated conformation of the hemoglobin tetramer. The two-locus beta-globin haplotype that predominates at high altitude is associated with suppressed DPG-sensitivity (and hence, increased hemoglobin-O(2) affinity), which enhances pulmonary O(2) loading under hypoxia. The discovery that allelic differences in DPG-sensitivity contribute to adaptive variation in hemoglobin-O(2) affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. Investigation into the functional significance of the deer mouse beta-globin polymorphism was motivated by the results of population genetic analyses which revealed evidence for a history of divergent selection between elevational zones. The experimental measures of O(2)-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.

Factors associated with the risk of Giardia species infection in two sympatric species of Peromyscus mice.

An epidemiologic case-control study was conducted to identify factors that predispose Peromyscus spp. to the risk of infection with Giardia sp. in watersheds. A total of 200 Giardia sp.-positive mice (cases) and a similar number of Giardia sp.-negative mice (controls) were selected from a population 2,528 mice captured in a watershed in southeastern New York State. The Giardia sp. infection status of the mice was determined by centrifugation concentration flotation and an enzyme-linked immunosorbent assay. The mice were later classified into 2 species, Peromyscus maniculatus and P. leucopus, using cellulose acetate electrophoresis of individual saliva samples. The risk of infection was evaluated while controlling for indigenous factors (age and sex) and exogenous factors (habitat and land use) that were hypothesized to influence the likelihood of infection with Giardia sp. The study population consisted of 257 (59%) P. leucopus and 165 (41%) P. maniculatus. Peromyscus leucopus mice were at a higher risk of becoming infected with Giardia in comparison to P. maniculatus (adjusted odds ratio = 40). The risk of Giardia sp. infection varied with the age of the animals, since adult animals were at higher risk than juvenile animals. This study suggested a difference in susceptibility to Giardia sp. between different species of mice.

Molecular evolution of cytochrome b in high- and low-altitude deer mice (genus Peromyscus).

Patterns of amino-acid polymorphism in human mitochondrial genes have been interpreted as evidence for divergent selection among populations that inhabit climatically distinct environments. If similar patterns are mirrored in other broadly distributed mammalian species, then adaptive modifications of mitochondrial protein function may be detected in comparisons among locally adapted populations of a single wide-ranging species, or among closely related species that have adapted to different environments. Here, we test for evidence of positive selection on cytochrome b variation within and among species of the ecologically diverse rodent genus Peromyscus. We used likelihood-based comparisons of synonymous and nonsynonymous substitution rates to test for evidence of divergent selection between high- and low-altitude haplogroups of the deer mouse, Peromyscus maniculatus. We also tested for evidence of divergent selection among different species of Peromyscus that inhabit different thermal environments. In contrast to the purported evidence for positive selection on mitochondrial proteins in humans and other nonhuman mammals, results of our tests suggest that the evolution of cytochrome b in Peromyscus is chiefly governed by purifying selection.

The genetic basis of phenotypic convergence in beach mice: similar pigment patterns but different genes.

Convergent evolution is a widespread phenomenon seen in diverse organisms inhabiting similar selective environments. However, it is unclear if similar phenotypes are produced by the same or different genes and mutations. Here we analyze the molecular mechanisms underlying convergent pigment pattern among subspecies of the beach mouse (Peromyscus polionotus) inhabiting the Gulf and Atlantic coasts of Florida. In these two geographic regions, separated by more than 300 km, "beach mice" have lighter colored coats than do their mainland counterparts, produced by natural selection for camouflage against the pale coastal sand dunes. We measured color pattern in eight beach mouse subspecies and showed that three of the Gulf Coast subspecies are more phenotypically similar to an Atlantic coast subspecies than to their Gulf Coast neighbors. However, light-colored beach mice do not form a monophyletic group. Previous results implicated a single derived amino acid change in the melanocortin-1 receptor (Mc1r) as a major contributor to pigment pattern in the Gulf Coast beach mice; despite phenotypic similarities, the derived Mc1r allele was not found in the Atlantic coast beach mouse populations. Here we show that Atlantic coast beach mice have high levels of Mc1r polymorphism but they lack unique alleles. Functional assays revealed that single amino acid mutations segregating in Atlantic coast beach mice do not cause any change in Mc1r activity compared with the activity of Mc1r from dark-colored mice. These joint results show that convergent pigment patterns in recently diverged beach mouse subspecies--whose developmental constraints are presumably similar--have evolved through a diversity of genetic mechanisms.

Old mice, young islands and competing biogeographical hypotheses.

Naturally occurring variation within a small rodent species native to the southeastern USA, Peromyscus polionotus, has interested biologists for nearly a century. This species has contributed significantly to our understanding of geographical variation and has often been presented as an example of adaptive evolution. Much of the interest in this organism has been predicated on assumptions that the species is relatively young (<300 000 bp) and that coastal populations have a very recent history (<10 000 bp). To test these assumptions and the prevailing biogeographical hypothesis (Recurrent Invasion), we examined nucleotide sequence data from the cytochrome b and D-loop mitochondrial regions (2449 bp) for 79 samples of P. polionotus collected across the Gulf Coast region of Florida and Alabama. Samples representing Peromyscus maniculatus bairdii, P. m. sonoriensis, P. m. pallescens, and P. keeni were used as outgroups. The degree of cytochrome b divergence (approximately 4.4%) between P. maniculatus and P. polionotus was higher than expected. Analyses consistently indicated that three distinct groups are represented within P. polionotus from the Gulf Coast region. Among these, coastal populations (beach mice) form a monophyletic group and apparently represent a substantially older group (approximately 200 000 year. separation) than previously recognized. Our results were counter to the core assumptions of the existing biogeographical model but were consistent with an alternative hypothesis (Shore-line Tracking) which provides a more parsimonious explanation for the observed patterns. This research provides new insight into the evolutionary history of P. polionotus and highlights the importance of considering biogeographical history when evaluating extant patterns of natural variation.