Extralimital terrestrials: A reassessment of range limits in Alaska's land mammals.

Understanding and mitigating the effects of anthropogenic climate change on species distributions requires the ability to track range shifts over time. This is particularly true for species occupying high-latitude regions, which are experiencing more extreme climate change than the rest of the world. In North America, the geographic ranges of many mammals reach their northernmost extent in Alaska, positioning this region at the leading edge of climate-induced distribution change. Over a decade has elapsed since the publication of the last spatial assessments of terrestrial mammals in the state. We compared public occurrence records against commonly referenced range maps to evaluate potential extralimital records and develop repeatable baseline range maps. We compared occurrence records from the Global Biodiversity Information Facility for 61 terrestrial mammal species native to mainland Alaska against a variety of range estimates (International Union for Conservation of Nature, Alaska Gap Analysis Project, and the published literature). We mapped extralimital records and calculated proportions of occurrences encompassed by range extents, measured mean direction and distance to prior range margins, evaluated predictive accuracy of published species models, and highlighted observations on federal lands in Alaska. Range comparisons identified 6,848 extralimital records for 39 of 61 (63.9%) terrestrial mainland Alaskan species. On average, 95.5% of Alaska Gap Analysis Project occurrence records and ranges were deemed accurate (i.e., > 90.0% correct) for 31 of 37 species, but overestimated extents for 13 species. The International Union for Conservation of Nature range maps encompassed 68.1% of occurrence records and were > 90% accurate for 17 of 39 species. Extralimital records represent either improved sampling and digitization or actual geographic range expansions. Here we provide new data-driven range maps, update standards for the archiving of museum-quality locational records and offer recommendations for mapping range changes for monitoring and conservation.

Ancient DNA indicates a century of overhunting did not reduce genetic diversity in Pacific Walruses (Odobenus rosmarus divergens).

Pacific Walruses (Odobenus rosmarus divergens [Illiger 1815]) are gregarious marine mammals considered to be sentinels of the Arctic because of their dependence on sea ice for feeding, molting, and parturition. Like many other marine mammal species, their population sizes were decimated by historical overhunting in the nineteenth and twentieth centuries. Although they have since been protected from nearly all commercial hunting pressure, they now face rapidly accelerating habitat loss as global warming reduces the extent of summer sea ice in the Arctic. To investigate how genetic variation was impacted by overhunting, we obtained mitochondrial DNA sequences from historic Pacific Walrus samples in Alaska that predate the period of overhunting, as well as from extant populations. We found that genetic variation was unchanged over this period, suggesting Pacific Walruses are resilient to genetic attrition in response to reduced population size, and that this may be related to their high vagility and lack of population structure. Although Pacific Walruses will almost certainly continue to decline in number as the planet warms and summer sea ice is further reduced, they may be less susceptible to the ratcheting effects of inbreeding that typically accompany shrinking populations.

A re-assessment of diversity among Philippine gymnures (Mammalia: Erinaceidae: Podogymnura), with a new species from eastern Mindanao.

Podogymnura, a Philippine endemic genus of gymnures ("soft-furred hedgehogs"), has been known previously only from the highlands of central Mindanao (P. truei truei and P. t. minima) and from two small islands off the northeast tip of Mindanao (P. aureospinula). Based on a combination of mitochondrial genetic and qualitative and quantitative morphological data, we recognize P. minima, formerly considered a subspecies of P. truei, as a distinct species. We also recognize specimens from two little-studied montane regions of southeastern Mindanao, Mt. Hamiguitan and Mt. Kampalili, as a distinctive new species, P. intermedia n. sp. We estimate that dispersal of a common ancestor from Borneo to Mindanao ca. 1.9 to 3.5 Ma gave rise to Podogymnura, followed by speciation within Mindanao beginning 0.53 to 0.97 Ma. These results further increase the rich diversity of mammals on Mindanao, and underscore the importance of the eastern Mindanao highlands as a likely important but little-studied center of Philippine mammalian diversity.

Ultraconserved elements improve resolution of marmot phylogeny and offer insights into biogeographic history.

Marmots (Marmota spp.) comprise a lineage of large-bodied ground squirrels that diversified rapidly in the Pleistocene, when the planet quickly transitioned to a drier, colder, and highly seasonal climate-particularly at high latitudes. Fossil evidence indicates the genus spread from North America, across Beringia, and into the European Alps over the course of only a few million years, beginning in the late Pliocene. Marmots are highly adapted to survive long and severely cold winters, and this likely favored their expansion and diversification over this time period. Previous phylogenetic studies have identified two major subgenera of marmots, but the timing of important speciation events and some species relationships have been difficult to resolve. Here we use ultraconserved elements and mitogenomes, with samples from all 15 extant species, to more precisely retrace how and when marmots came to inhabit a vast Holarctic range. Our results indicate marmots arose in North America in the Early Miocene (∼16.3 Ma) and dispersed across the Bering Land Bridge in the Pliocene (∼3-4 Ma); in addition, our fossil-calibrated timeline is suggestive of the rise and spread of open grasslands as being particularly important to marmot diversification. The woodchuck (M. monax) and the Alaska marmot (M. broweri) are found to be more closely related to the Eurasian species than to the other North American species. Paraphyly is evident in the bobak marmot (M. bobak) and the hoary marmot (M. caligata), and in the case of the latter the data are highly suggestive of a second, cryptic species in the Cascade Mountains of Washington.

Recent and rapid ecogeographical rule reversals in Northern Treeshrews.

Two of the most-studied ecogeographical rules describe patterns of body size variation within species. Bergmann's rule predicts that individuals have larger body sizes in colder climates (typically at higher latitudes), and the island rule predicts that island populations of small-bodied species average larger in size than their mainland counterparts (insular gigantism). These rules are rarely tested in conjunction or assessed across space and time simultaneously. We investigated these patterns in the Northern Treeshrew (Tupaia belangeri) using museum specimens collected across a wide spatial and temporal range. Contrary to Bergmann's rule, size increases with temperature in T. belangeri, a signal that is highly consistent across space and time. We also show that these rules are intertwined: Bergmann's rule is reversed on the mainland but holds on islands, and therefore the island rule is upheld at higher, but not lower, latitudes. Moreover, we demonstrate a rapid reversal of both rules over time. The mechanism behind these inversions remains unclear, though temperature and precipitation are significant predictors of body size. Ecogeographical rules rely on the assumption of a constant relationship between size and the factors driving its variation. Our results highlight the need to question this assumption and reevaluate these rules in the context of accelerating and uneven climate change.

Speciation and gene flow in two sympatric small mammals from Madagascar, Microgale fotsifotsy and M. soricoides (Mammalia: Tenrecidae).

Madagascar's shrew tenrecs (Mammalia: Tenrecidae; Microgale, Nesogale) represent an excellent system for studying speciation. Most species are endemic to the island's eastern humid forests, a region renowned for high levels of biodiversity and a high rate of in situ diversification. We set out to understand the speciation dynamics in a clade of recently described taxa: Microgale fotsifotsy and M. soricoides, which have nearly identical distributions in the moist evergreen forest, and M. nasoloi, which occurs in the western dry deciduous forest. A phylogenetic analysis using mitochondrial DNA data recovered two distinct clades of M. fotsifotsy: a south clade that is sister to, and broadly sympatric with, M. soricoides, and a north clade that is sister to the dry-forest and distantly allopatric species M. nasoloi. To better understand this result, we analysed cranioskeletal measurements and performed demographic analyses using nuclear sequence data from ultraconserved elements. Nuclear data did not support a sister relationship between M. soricoides and the south clade of M. fotsifotsy but did demonstrate introgression between these clades, which probably explains the discordance between nuclear and mitochondrial phylogenies. Demographic analyses also revealed the absence of gene flow between the north and south clades of M. fotsifotsy. Morphometric data revealed several major differences between M. soricoides and M. fotsifotsy, as well as more subtle differences between the two clades of M. fotsifotsy. In light of these results, we treat the south clade of M. fotsifotsy as a new candidate species. Our findings demonstrate the utility of integrating multiple data types to understand complex speciation histories, and contribute to a growing body of evidence that species diversity on Madagascar is underestimated.

Caught in the act: Incipient speciation across a latitudinal gradient in a semifossorial mammal from Madagascar, the mole tenrec Oryzorictes hova (Tenrecidae).

Madagascar is one of the world's foremost biodiversity hotspots, yet a large portion of its flora and fauna remains undescribed and the driving forces of in situ diversification are not well understood. Recent studies have identified a widespread, latitudinally structured phylogeographic pattern in Madagascar's humid-forest mammals, amphibians, reptiles, and insects. Several factors may be driving this pattern, namely biogeographic barriers (i.e., rivers or valleys) or past episodes of forest contraction and expansion. In this study, we describe the phylogeographic structure of the small, semifossorial mammal Oryzorictes hova, one of Madagascar's two species of mole tenrec, found throughout Madagascar's eastern humid forest belt, from high-elevation montane forest to low-elevation forests, as well as disturbed habitat such as rice fields. Using one mitochondrial locus, four nuclear loci, and 31 craniomandibular measurements, we identified three distinct populations of O. hova associated with the northern, central, and southern regions of the island. We found little evidence of gene flow among these populations, so we treated each population as a potential species. We validated species limits using two Bayesian methods: BP&P, employing only DNA sequence data, and iBPP using both DNA and morphological data, and we assessed whether these methods are susceptible to producing false positive errors. Molecular and morphological data support the recognition of each of the three populations of O. hova as distinct species, but formal species descriptions will require additional data from type specimens. This study illustrates the importance of using integrative datasets, multiple methodological approaches, and extensive geographic sampling for species delimitation and adds evidence for a widespread phylogeographic pattern in Madagascar's humid forest taxa.

Rule reversal: Ecogeographical patterns of body size variation in the common treeshrew (Mammalia, Scandentia).

There are a number of ecogeographical "rules" that describe patterns of geographical variation among organisms. The island rule predicts that populations of larger mammals on islands evolve smaller mean body size than their mainland counterparts, whereas smaller-bodied mammals evolve larger size. Bergmann's rule predicts that populations of a species in colder climates (generally at higher latitudes) have larger mean body sizes than conspecifics in warmer climates (at lower latitudes). These two rules are rarely tested together and neither has been rigorously tested in treeshrews, a clade of small-bodied mammals in their own order (Scandentia) broadly distributed in mainland Southeast Asia and on islands throughout much of the Sunda Shelf. The common treeshrew, Tupaia glis, is an excellent candidate for study and was used to test these two rules simultaneously for the first time in treeshrews. This species is distributed on the Malay Peninsula and several offshore islands east, west, and south of the mainland. Using craniodental dimensions as a proxy for body size, we investigated how island size, distance from the mainland, and maximum sea depth between the mainland and the islands relate to body size of 13 insular T. glis populations while also controlling for latitude and correlation among variables. We found a strong negative effect of latitude on body size in the common treeshrew, indicating the inverse of Bergmann's rule. We did not detect any overall difference in body size between the island and mainland populations. However, there was an effect of island area and maximum sea depth on body size among island populations. Although there is a strong latitudinal effect on body size, neither Bergmann's rule nor the island rule applies to the common treeshrew. The results of our analyses demonstrate the necessity of assessing multiple variables simultaneously in studies of ecogeographical rules.

Reconstructing the molecular phylogeny of giant sengis (Macroscelidea; Macroscelididae; Rhynchocyon).

Giant sengis (Macroscelidea; Macroscelididae; Rhynchocyon), also known as giant elephant-shrews, are small-bodied mammals that range from central through eastern Africa. Previous research on giant sengi systematics has relied primarily on pelage color and geographic distribution. Because some species have complex phenotypic variation and large geographic ranges, we used molecular markers to evaluate the phylogeny and taxonomy of the genus, which currently includes four species: R. chrysopygus, R. cirnei (six subspecies), R. petersi (two subspecies), and R. udzungwensis. We extracted DNA from fresh and historical museum samples from all taxa except one R. cirnei subspecies, and we generated and analyzed approximately 4700 aligned nucleotides (2685 bases of mitochondrial DNA and 2019 bases of nuclear DNA) to reconstruct a molecular phylogeny. We genetically evaluate Rhynchocyon spp. sequences previously published on GenBank, propose that the captive R. petersi population in North American zoos is likely R. p. adersi, and suggest that hybridization among taxa is not widespread in Rhynchocyon. The DNA sample we have from the distinctive but undescribed giant sengi from the Boni forest of northern coastal Kenya is unexpectedly nearly identical to R. chrysopygus, which will require further study. Our analyses support the current morphology-based taxonomy, with each recognized species forming a monophyletic clade, but we propose elevating R. c. stuhlmanni to a full species.

Novel Orthopoxvirus Infection in an Alaska Resident.

BACKGROUND.: Human infection by orthopoxviruses is being reported with increasing frequency, attributed in part to the cessation of smallpox vaccination and concomitant waning of population-level immunity. In July 2015, a female resident of interior Alaska presented to an urgent care clinic with a dermal lesion consistent with poxvirus infection. Laboratory testing of a virus isolated from the lesion confirmed infection by an Orthopoxvirus. METHODS.: The virus isolate was characterized by using electron microscopy and nucleic acid sequencing. An epidemiologic investigation that included patient interviews, contact tracing, and serum testing, as well as environmental and small-mammal sampling, was conducted to identify the infection source and possible additional cases. RESULTS.: Neither signs of active infection nor evidence of recent prior infection were observed in any of the 4 patient contacts identified. The patient's infection source was not definitively identified. Potential routes of exposure included imported fomites from Azerbaijan via the patient's cohabiting partner or wild small mammals in or around the patient's residence. Phylogenetic analyses demonstrated that the virus represents a distinct and previously undescribed genetic lineage of Orthopoxvirus, which is most closely related to the Old World orthopoxviruses. CONCLUSIONS.: Investigation findings point to infection of the patient after exposure in or near Fairbanks. This conclusion raises questions about the geographic origins (Old World vs North American) of the genus Orthopoxvirus. Clinicians should remain vigilant for signs of poxvirus infection and alert public health officials when cases are suspected.

Multiple Loci and Complete Taxonomic Sampling Resolve the Phylogeny and Biogeographic History of Tenrecs (Mammalia: Tenrecidae) and Reveal Higher Speciation Rates in Madagascar's Humid Forests.

The family Tenrecidae (tenrecs) is one of only four extant terrestrial mammal lineages to have colonized and diversified on Madagascar. Over the last 15 years, several studies have disagreed on relationships among major tenrec lineages, resulting in multiple reinterpretations of the number and timing of historical transoceanic dispersal events between Africa and Madagascar. We reconstructed the phylogeny of Tenrecidae using multiple loci from all recognized extant species and estimated divergence timing using six fossil calibrations within Afrotheria. All phylogenetic analyses strongly support monophyly of the Malagasy tenrecs, and our divergence timing analysis places their colonization of the island at 30-56 Ma. Our comprehensive phylogeny supports three important taxonomic revisions that reflect the evolutionary history of tenrecs: (1) we formally elevate the African otter shrews to their own family Potamogalidae, thereby rendering extant Tenrecidae entirely endemic to Madagascar; (2) we subsume the semiaquatic genus Limnogale within the shrew tenrec genus Microgale; and (3) we re-elevate the two largest-bodied shrew tenrecs, Microgale dobsoni and Microgale talazaci, to the genus Nesogale Thomas (1918) Finally, we use recently summarized habitat data to test the hypothesis that diversification rates differ between humid and arid habitats on Madagascar, and we compare three common methods for ancestral biogeographic reconstruction. These analyses suggest higher speciation rates in humid habitats and reveal a minimum of three and more likely five independent transitions to arid habitats. Our results resolve the relationships among previously recalcitrant taxa, illuminate the timing and mechanisms of major biogeographic patterns in an extraordinary example of an island radiation, and permit the first comprehensive, phylogenetically consistent taxonomy of Madagascar's tenrecs.

Colonization from divergent ancestors: glaciation signatures on contemporary patterns of genomic variation in Collared Pikas (Ochotona collaris).

Identifying the genetic structure of a species and the factors that drive it is an important first step in modern population management, in part because populations evolving from separate ancestral sources may possess potentially different characteristics. This is especially true for climate-sensitive species such as pikas, where the delimitation of distinct genetic units and the characterization of population responses to contemporary and historical environmental pressures are of particular interest. We combined a restriction site-associated DNA sequencing (RADSeq) data set containing 4156 single nucleotide polymorphisms with ecological niche models (ENMs) of present and past habitat suitability to characterize population composition and evaluate the effects of historical range shifts, contemporary climates and landscape factors on gene flow in Collared Pikas, which are found in Alaska and adjacent regions of northwestern Canada and are the lesser-studied of North America's two pika species. The results suggest that contemporary environmental factors contribute little to current population connectivity. Instead, genetic diversity is strongly shaped by the presence of three ancestral lineages isolated during the Pleistocene (~148 and 52 kya). Based on ENMs and genetic data, populations originating from a northern refugium experienced longer-term stability, whereas both southern lineages underwent population expansion - contradicting the southern stability and northern expansion patterns seen in many other taxa. Current populations are comparable with respect to generally low diversity within populations and little-to-no recent admixture. The predominance of divergent histories structuring populations implies that if we are to understand and manage pika populations, we must specifically assess and accurately account for the forces underlying genetic similarity.

Comparative phylogeography highlights the double-edged sword of climate change faced by arctic- and alpine-adapted mammals.

Recent studies suggest that alpine and arctic organisms may have distinctly different phylogeographic histories from temperate or tropical taxa, with recent range contraction into interglacial refugia as opposed to post-glacial expansion out of refugia. We use a combination of phylogeographic inference, demographic reconstructions, and hierarchical Approximate Bayesian Computation to test for phylodemographic concordance among five species of alpine-adapted small mammals in eastern Beringia. These species (Collared Pikas, Hoary Marmots, Brown Lemmings, Arctic Ground Squirrels, and Singing Voles) vary in specificity to alpine and boreal-tundra habitat but share commonalities (e.g., cold tolerance and nunatak survival) that might result in concordant responses to Pleistocene glaciations. All five species contain a similar phylogeographic disjunction separating eastern and Beringian lineages, which we show to be the result of simultaneous divergence. Genetic diversity is similar within each haplogroup for each species, and there is no support for a post-Pleistocene population expansion in eastern lineages relative to those from Beringia. Bayesian skyline plots for four of the five species do not support Pleistocene population contraction. Brown Lemmings show evidence of late Quaternary demographic expansion without subsequent population decline. The Wrangell-St. Elias region of eastern Alaska appears to be an important zone of recent secondary contact for nearctic alpine mammals. Despite differences in natural history and ecology, similar phylogeographic histories are supported for all species, suggesting that these, and likely other, alpine- and arctic-adapted taxa are already experiencing population and/or range declines that are likely to synergistically accelerate in the face of rapid climate change. Climate change may therefore be acting as a double-edged sword that erodes genetic diversity within populations but promotes divergence and the generation of biodiversity.

Evaluation of the authenticity of a highly novel environmental sequence from boreal forest soil using ribosomal RNA secondary structure modeling.

The number of sequences from both formally described taxa and uncultured environmental DNA deposited in the International Nucleotide Sequence Databases has increased substantially over the last two decades. Although the majority of these sequences represent authentic gene copies, there is evidence of DNA artifacts in these databases as well. These include lab artifacts, such as PCR chimeras, and biological artifacts such as pseudogenes or other paralogous sequences. Sequences that fall in basal positions in phylogenetic trees and appear distant from known sequences are particularly suspect. Phylogenetic analyses suggest that a novel sequence type (NS1) found in two boreal forest soil clone libraries belongs to the fungal kingdom but does not fall unambiguously within any known phylum. We have evaluated this sequence type using an array of secondary-structure analyses. To our knowledge, such analyses have never been used on environmental ribosomal sequences. Ribosomal secondary structure was modeled for four rRNA loci (ITS1, 5.8S, ITS2, 5' LSU). These models were analyzed for the presence of conserved domains, conserved nucleotide motifs, and compensatory base changes. Minimal free energy (MFE) foldings and GC contents of sequences representing the major fungal clades, as well as NS1, were also compared. NS1 displays secondary rRNA structures consistent with other fungi and many, but not all, conserved nucleotide motifs found across eukaryotes. However, our analyses show that many other authentic sequences from basal fungi lack more of these conserved motifs than does NS1. Together our findings suggest that NS1 represents an authentic gene copy. The methods described here can be used on any rRNA-coding sequence, not just environmental fungal sequences. As new-generation sequencing methods that yield shorter sequences become more widely implemented, methods that evaluate sequence authenticity should also be more widely implemented. For fungi, the adjacent 5.8S and ITS2 loci should be prioritized. This region is not only suited to distinguishing between closely related species, but it is also more informative in terms of expected secondary structure.

Molecular phylogeny of treeshrews (Mammalia: Scandentia) and the timescale of diversification in Southeast Asia.

Resolving the phylogeny of treeshrews (Order Scandentia) has historically proven difficult, in large part because of access to specimens and samples from critical taxa. We used "antique" DNA methods with non-destructive sampling of museum specimens to complete taxon sampling for the 20 currently recognized treeshrew species and to estimate their phylogeny and divergence times. Most divergence among extant species is estimated to have taken place within the past 20 million years, with deeper divergences between the two families (Ptilocercidae and Tupaiidae) and between Dendrogale and all other genera within Tupaiidae. All but one of the divergences between currently recognized species had occurred by 4Mya, suggesting that Miocene tectonics, volcanism, and geographic instability drove treeshrew diversification. These geologic processes may be associated with an increase in net diversification rate in the early Miocene. Most evolutionary relationships appear consistent with island-hopping or landbridge colonization between contiguous geographic areas, although there are exceptions in which extinction may play an important part. The single recent divergence is between Tupaia palawanensis and Tupaia moellendorffi, both endemic to the Philippines, and may be due to Pleistocene sea level fluctuations and post-landbridge isolation in allopatry. We provide a time-calibrated phylogenetic framework for answering evolutionary questions about treeshrews and about evolutionary patterns and processes in Euarchonta. We also propose subsuming the monotypic genus Urogale, a Philippine endemic, into Tupaia, thereby reducing the number of extant treeshrew genera from five to four.

Morphological systematics of the kipunji (Rungwecebus kipunji) and the ontogenetic development of phylogenetically informative characters in the Papionini.

Since its discovery and description, the systematic position of the kipunji (Rungwecebus kipunji) has been a matter of debate. Although it was first placed in the mangabey genus Lophocebus, subsequent molecular studies indicated that the kipunji is most closely related to baboons (Papio). However, the kipunji does not appear to possess cranial features typical of Papio, thus necessitating the erection of a new genus, Rungwecebus. The recovery of an M2-stage subadult male kipunji voucher specimen, in addition to the original M1-stage subadult male voucher specimen, has since allowed further study. Here, we describe the craniodental morphology of the newly acquired kipunji specimen and present a phylogenetic analysis of Rungwecebus craniodental morphology using quantitative and qualitative characters. We examined the skulls of 76 M1- and M2-stage subadult males representing all extant papionin genera, taking note of character states that are static throughout ontogeny. To control for ontogenetic changes, only those characters expressing unchanged character states between subadult and adult specimens were coded for Rungwecebus and entered into a larger, recently published 151-character matrix of adult male morphology. To account for allometry, the narrow allometric coding method and the general allometric coding method were applied. The resulting most parsimonious trees suggest that Rungwecebus is phylogenetically closest to Lophocebus, a result consistent with initial morphological descriptions. However, due to the large amount of missing data for Rungwecebus, there are low bootstrap support values associated with any relationships within the larger Theropithecus/Papio/Lophocebus/Rungwecebus grouping. Taken in combination with previous molecular, phenetic, and ecological studies, the results of this study suggest that Rungwecebus is best regarded as a distinct genus closely related to Papio, Lophocebus, and Theropithecus. Adult morphological specimens are necessary to fully understand the adult kipunji morphotype, and its phylogenetic position will only be more precisely resolved with additional morphological and molecular data.

The biogeography of introgression in the critically endangered African monkey Rungwecebus kipunji.

In the four years since its original description, the taxonomy of the kipunji (Rungwecebus kipunji), a geographically restricted and critically endangered African monkey, has been the subject of much debate, and recent research suggesting that the first voucher specimen of Rungwecebus has baboon mitochondrial DNA has intensified the controversy. We show that Rungwecebus from a second region of Tanzania has a distinct mitochondrial haplotype that is basal to a clade containing all Papio species and the original Rungwecebus voucher, supporting the placement of Rungwecebus as the sister taxon of Papio and its status as a separate genus. We suggest that the Rungwecebus population in the Southern Highlands has experienced geographically localized mitochondrial DNA introgression from Papio, while the Ndundulu population retains the true Rungwecebus mitochondrial genome.

Inferring divergence times within pikas (Ochotona spp.) using mtDNA and relaxed molecular dating techniques.

Although several studies have recently addressed phylogenetic relationships among Asian pikas (Ochotona spp.), the North American species have been relatively neglected and their monophyly generally unquestioned or assumed. Given the high degree of intraspecific diversity in pelage and call structure, the recent identification of previously unrecognized species of pika in Asia, and the increasing evidence for multiple trans-Beringian dispersals in several small mammal lineages, the monophyly of North American pikas warrants reexamination. In addition, previous studies have applied an externally calibrated rate to examine the timing of diversification within the genus. This method has been increasingly shown to return results that, at the very least, are overly narrow in their confidence intervals, and at the worst can be entirely spurious. For this study we combined GenBank sequences from the mitochondrial genes cyt b and ND4 with newly generated sequence data from O. hyperborea and O. collaris to investigate the origin of the North American lineages and the timing of phylogenetic diversification within the genus Ochotona. Specifically, we address three goals (1) summarize and reanalyze the molecular evidence for relationships within the genus using statistically supported models of evolution; (2) add additional sequences from O. collaris and O. hyperborea to rigorously test the monophyly of North American pikas; (3) examine the timing of the diversification within the genus using relaxed molecular clock methods. We found no evidence of multiple trans-Beringian dispersals into North America, thereby supporting the traditional hypothesis of a single invasion of North America. We also provide evidence that the major splits within the genus occurred in the Miocene, and the Nearctic pikas diverged sometime before the Pleistocene.