John R. Demboski

INVESTIGATING THE EVOLUTIONARY HISTORY OF THE PACIFIC NORTHWEST MESIC FOREST ECOSYSTEM: HYPOTHESIS TESTING WITHIN A COMPARATIVE PHYLOGEOGRAPHIC FRAMEWORK

Abstract We examine the evolution of mesic forest ecosystems in the Pacific Northwest of North America using a statistical phylogeography approach in four animal and two plant lineages. Three a priori hypotheses, which explain the disjunction in the mesic forest ecosystem with either recent dispersal or ancient vicariance, are tested with phylogenetic and coalescent methods. We find strong support in three amphibian lineages (Ascaphus spp., and Dicampton spp., and Plethodon vandykei and P. idahoensis) for deep divergence between coastal and inland populations, as predicted by the ancient vicariance hypothesis. Unlike the amphibians, the disjunction in other Pacific Northwest lineages is likely due to recent dispersal along a northern route. Topological and population divergence tests support the northern dispersal hypothesis in the water vole (Microtus richardsoni) and northern dispersal has some support in both the dusky willow (Salix melanopsis) and whitebark pine (Pinus albicaulis). These analyses demonstrate that genetic data sampled from across an ecosystem can provide insight into the evolution of ecological communities and suggest that the advantages of a statistical phylogeographic approach are most pronounced in comparisons across multiple taxa in a particular ecosystem. Genetic patterns in organisms as diverse as willows and salamanders can be used to test general regional hypotheses, providing a consistent metric for comparison among members of an ecosystem with disparate life‐history traits.

Ancient hybridization and mitochondrial capture between two species of chipmunks

Models that posit speciation in the face of gene flow are replacing classical views that hybridization is rare between animal species. We use a multilocus approach to examine the history of hybridization and gene flow between two species of chipmunks (Tamias ruficaudus and T. amoenus). Previous studies have shown that these species occupy different ecological niches and have distinct genital bone morphologies, yet appear to be incompletely isolated reproductively in multiple areas of sympatry. We compared data from four sequenced nuclear loci and from seven microsatellite loci to published cytochrome b sequences. Interspecific gene flow was primarily restricted to introgression of the T. ruficaudus mitochondrial genome into a sympatric subspecies of T. amoenus, T. a. canicaudus, with the four sequenced nuclear loci showing little to no interspecific allele sharing. Microsatellite data were consistent with high levels of differentiation between the species and also showed no current gene flow between broadly sympatric populations of T. a. canicaudus and T. ruficaudus. Coalescent analyses date the mtDNA introgression event from the mid‐Pleistocene to late Pliocene. Overall, these data indicate that introgression has had a minimal impact on the nuclear genomes of T. amoenus and T. ruficaudus despite multiple independent hybridization events. Our findings challenge long‐standing assumptions on patterns of reproductive isolation in chipmunks and suggest that there may be other examples of hybridization among the 23 species of Tamias that occur in western North America.

Phylogeography of the dusky shrew, Sorex monticolus (Insectivora, Soricidae): insight into deep and shallow history in northwestern North America

Phylogenetic relationships among the dusky shrew (Sorexmonticolus) and eight related species (S. bairdi, S. bendirii, S. neomexicanus, S.ornatus, S. pacificus, S. palustris, S. sonomae and S.vagrans) were assessed using sequences from the mitochondrial cytochrome b gene (801 bp). Analyses using parsimony and maximum likelihood revealed significant molecular variation not reflected in previous morphological studies of these species. Conversely, three morphologically defined species (S.bairdi, S.neomexicanusandS.pacificus) were poorly differentiated. SorexornatusandS.vagrans represented basal taxa for a more inclusive group that included: (i) a widespread Continental clade containing S.monticolus (Arizona to Alaska, including S. neomexicanus); (ii) a Coastal clade containing S.monticolus (Oregon to south‐east Alaska, including S. bairdiandS. pacificus); (iii) the semiaquatic species (S. bendiriiandS. palustris); and (iv) S.sonomae. Additional subdivision was observed within the Continental clade corresponding to populations from the northern and southern Rocky Mountains. Average uncorrected sequence divergence between the Coastal and Continental clades was 5.3% (range 4.5–6.2%), which exceeds many interspecific comparisons within this species complex and within the genus Sorex. Lack of resolution of internal nodes within topologies suggests a deep history of rapid diversification within this group. Late Pleistocene/Holocene glacial perturbations are reflected in the shallow phylogeographic structure within these clades in western North America. Our results suggest also that S. monticolus is not monophyletic under current taxonomic nomenclature. This perspective on phylogeographic history was developed within a growing comparative framework for other organisms in western North America.

A phylogeographic perspective on endemism in the Alexander Archipelago of southeast Alaska

Conservation plans for the North Pacific Coast are beginning to acknowledge that the old-growth forests remaining on the extensive archipelagos of the region may require a new management paradigm. Extensive timber harvesting activities on these islands may impact wildlife populations and be particularly detrimental to the large number of endemics. Because insular biotas have suffered a disproportionate number of documented extinctions worldwide, the documentation of taxonomic validity and spatial distribution of endemics should be a priority for management efforts. To date, few North Pacific coastal endemics have been reevaluated since their original descriptions. This study provides an independent view of endemism in the Alexander Archipelago of southeast Alaska through a phylogeographic assessment of eight species of mammals (Sorex monticolus, Glaucomys sabrinus, Microtus longicaudus, Clethrionomys rutilus, Clethrionomys gapperi, Martes americana, Mustela erminea, Ursus americanus). The molecular data (mitochondrial sequence variation of the cytochrome b gene) suggest a dynamic history of faunal interchange in the region. While some purported endemics show minimal levels of genetic divergence from other conspecific populations, other taxa appear to be more divergent than recognized by current taxonomy. Some species in the region are comprised of multiple clades or evolutionarily significant units. These reciprocally monophyletic lineages may be the result of multiple Holocene invasions (neoendemics) or they may have persisted in refugia (paleoendemics) in the region during Pleistocene glacial advances. This emerging historical perspective should have direct implications for the management of these endemic taxa.

PHYLOGEOGRAPHY AND INTROGRESSIVE HYBRIDIZATION: CHIPMUNKS (GENUS TAMIAS) IN THE NORTHERN ROCKY MOUNTAINS

Abstract. If phylogeographic studies are to be broadly used for assessing population‐level processes relevant to speciation and systematics, the ability to identify and incorporate instances of hybridization into the analytical framework is essential. Here, we examine the evolutionary history of two chipmunk species, Tamias ruficaudus and Tamias amoenus, in the northern Rocky Mountains by integrating multivariate morphometrics of bacular (os penis) variation, phylogenetic estimation, and nested clade analysis with regional biogeography. Our results indicate multiple examples of mitochondrial DNA introgression layered within the evolutionary history of these nonsister species. Three of these events are most consistent with recent and/or ongoing asymmetric introgression of mitochondrial DNA across morphologically defined secondary contact zones. In addition, we find preliminary evidence where a fourth instance of nonconcordant characters may represent complete fixation of introgressed mitochondrial DNA via a more ancient hybridization event, although alternative explanations of convergence or incomplete sorting of ancestral polymorphisms cannot be dismissed with these data. The demonstration of hybridization among chipmunks with strongly differentiated bacular morphology contradicts long‐standing assumptions that variation within this character is diagnostic of complete reproductive isolation within Tamias. Our results illustrate the utility of phylogeographic analyses for detecting instances of reticulate evolution and for incorporating this and other information in the inference of the evolutionary history of species.

Extensive mtDNA variation within the yellow-pine chipmunk, Tamias amoenus (Rodentia: Sciuridae), and phylogeographic inferences for northwest North America

The yellow-pine chipmunk, Tamias amoenus, is common in xerophytic forests throughout much of northwest North America. We analyzed cytochrome b sequence variation from 155 individuals representing 57 localities across the distribution of T. amoenus including 10 additional species of Tamias. Maximum likelihood and parsimony tree estimation methods were used in conjunction with nested clade analysis to infer both deep and population-level processes. Our results indicate that two currently recognized subspecies of T. amoenus (T. a. canicaudus and T. a. cratericus) are not nested within other samples of T. amoenus. Maximum uncorrected levels of intraspecific sequence divergence within remaining samples of T. amoenus are >7%. Substantial geographic variation is characterized by 12 well-supported clades that correspond to distinct mountain ranges, but do not necessarily follow existing subspecific taxonomy. Significant association between geography and genealogy was detected within many of these clades and can be attributed to different population-level processes including past fragmentation, recent range expansion, and isolation by distance.

Phylogenetic Diversification within the Sorex cinereus Group (Soricidae)

Abstract Phylogenetic relationships among 8 members of the Sorex cinereus group (S. camtschatica, S. cinereus, S. haydeni, S. jacksoni, S. portenkoi, S. preblei, S. pribilofensis, and S. ugyunak) and S. longirostris were estimated using DNA sequence data from 2 mitochondrial genes, cytochrome b (1,140 base pairs) and nicotinamide adenine dinucleotide dehydrogenase 4 (582 base pairs). S. hoyi, S. monticolus, S. palustris, S. tenellus, S. trowbridgii, and S. vagrans also were included in our analyses. Phylogenetic analyses of the combined data recovered 2 major clades within the species group: a northern clade that includes the Beringian species (S. camtschatica, S. jacksoni, S. portenkoi, S. pribilofensis, and S. ugyunak), S. haydeni, and S. preblei and a southern clade that includes S. cinereus and S. longirostris. Mitochondrial DNA clades generally corresponded to previously identified morphological groups with 2 exceptions: inclusion of S. longirostris with S. cinereus in the southern clade and inclusion of S. preblei within the northern clade. With the exception of the 5 Beringian species, taxa were readily differentiated with strong bootstrap support in our topologies. We also noted phylogenetic concordance with the general ecological affiliations of each species; the northern clade generally includes xeric-affiliated species, whereas the southern clade includes species with mesic habitat affinities.

A MOLECULAR PERSPECTIVE ON THE HISTORICAL BIOGEOGRAPHY OF THE NORTHERN HIGH LATITUDES

Abstract Phylogeographic analyses of arctic organisms provide spatial and temporal frameworks for interpreting the role of climate change on biotic diversity in high-latitude ecosystems. Phylogenetic analyses based on 673 base pairs of the mitochondrial control region from 95 arctic hares (Lepus arcticus, L. othus, L. timidus) and 2 other Lepus species identified 6 strongly or moderately supported clades. The 3 arctic hare species are closely related, but phylogenetic discontinuities were found at the eastern and western boundaries of Beringia, the latter not previously identified as a species boundary. The locations of these discontinuities are congruent with previously described genetic breaks in Arctic plants, birds, and small mammals. Similarly, the finding of a Beringian clade corroborates previous studies identifying Beringia as a refugium. A coalescent view of a population on Seward Peninsula, Alaska (eastern Beringia), did not, however, provide a genetic signature of population expansion. In contrast, a Greenland population did show a signal of expansion.

Phylogeny Estimation of the Radiation of Western North American Chipmunks (Tamias) in the Face of Introgression Using Reproductive Protein Genes

The causes and consequences of rapid radiations are major unresolved issues in evolutionary biology. This is in part because phylogeny estimation is confounded by processes such as stochastic lineage sorting and hybridization. Because these processes are expected to be heterogeneous across the genome, comparison among marker classes may provide a means of disentangling these elements. Here we use introns from nuclear-encoded reproductive protein genes expected to be resistant to introgression to estimate the phylogeny of the western chipmunks (Tamias: subgenus: Neotamias), a rapid radiation that has experienced introgressive hybridization of mitochondrial DNA (mtDNA). We analyze the nuclear loci using coalescent-based species-tree estimation methods and concatenation to estimate a species tree and we use parametric bootstraps and coalescent simulations to differentiate between phylogenetic error, coalescent stochasticity and introgressive hybridization. Results indicate that the mtDNA gene tree reflects several introgression events that have occurred between taxa of varying levels of divergence and at different time points in the tree. T. panamintinus and T. speciosus appear to be fixed for ancient mitochondrial introgressions from T. minimus. A southern Rocky Mountains clade appears well sorted (i.e., species are largely monophyletic) at multiple nuclear loci, while five of six taxa are nonmonophyletic based on cytochrome b. Our simulations reject phylogenetic error and coalescent stochasticity as causes. The results represent an advance in our understanding of the processes at work during the radiation of Tamias and suggest that sampling reproductive-protein genes may be a viable strategy for phylogeny estimation of rapid radiations in which reproductive isolation is incomplete. However, a genome-scale survey that can statistically compare heterogeneity of genealogical process at many more loci will be necessary to test this conclusion.

Divergence with gene flow within the recent chipmunk radiation (Tamias)

Increasing data have supported the importance of divergence with gene flow (DGF) in the generation of biological diversity. In such cases, lineage divergence occurs on a shorter timescale than does the completion of reproductive isolation. Although it is critical to explore the mechanisms driving divergence and preventing homogenization by hybridization, it is equally important to document cases of DGF in nature. Here we synthesize data that have accumulated over the last dozen or so years on DGF in the chipmunk (Tamias) radiation with new data that quantify very high rates of mitochondrial DNA (mtDNA) introgression among para- and sympatric species in the T. quadrivittatus group in the central and southern Rocky Mountains. These new data (188 cytochrome b sequences) bring the total number of sequences up to 1871; roughly 16% (298) of the chipmunks we have sequenced exhibit introgressed mtDNA. This includes ongoing introgression between subspecies and between both closely related and distantly related taxa. In addition, we have identified several taxa that are apparently fixed for ancient introgressions and in which there is no evidence of ongoing introgression. A recurrent observation is that these introgressions occur between ecologically and morphologically diverged, sometimes non-sister taxa that engage in well-documented niche partitioning. Thus, the chipmunk radiation in western North America represents an excellent mammalian example of speciation in the face of recurrent gene flow among lineages and where biogeography, habitat differentiation and mating systems suggest important roles for both ecological and sexual selection.