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© 2014 University of Washington. In North America, terrestrial records of biodiversity and climate change that span Marine Oxygen Isotope Stage (MIS) 5 are rare. Where found, they provide insight into how the coupling of the ocean-atmosphere system is manifested in biotic and environmental records and how the biosphere responds to climate change. In 2010-2011, construction at Ziegler Reservoir near Snowmass Village, Colorado (USA) revealed a nearly continuous, lacustrine/wetland sedimentary sequence that preserved evidence of past plant communities between ~. 140 and 55. ka, including all of MIS 5. At an elevation of 2705. m, the Ziegler Reservoir fossil site also contained thousands of well-preserved bones of late Pleistocene megafauna, including mastodons, mammoths, ground sloths, horses, camels, deer, bison, black bear, coyotes, and bighorn sheep. In addition, the site contained more than 26,000 bones from at least 30 species of small animals including salamanders, otters, muskrats, minks, rabbits, beavers, frogs, lizards, snakes, fish, and birds. The combination of macro- and micro-vertebrates, invertebrates, terrestrial and aquatic plant macrofossils, a detailed pollen record, and a robust, directly dated stratigraphic framework shows that high-elevation ecosystems in the Rocky Mountains of Colorado are climatically sensitive and varied dramatically throughout MIS 5.

More than tools for managing physical and digital objects, museum collection management systems (CMS) serve as platforms for structuring, integrating, and making accessible the rich data embodied by natural history collections. Here we describe Arctos, a scalable community solution for managing and publishing global biological, geological, and cultural collections data for research and education. Specific goals are to: (1) Describe the core features and implementation of Arctos for a broad audience with respect to the biodiversity informatics principles that enable high quality research; (2) Highlight the unique aspects of Arctos; (3) Illustrate Arctos as a model for supporting and enhancing the Digital Extended Specimen concept; and (4) Emphasize the role of the Arctos community for improving data discovery and enabling cross-disciplinary, integrative studies within a sustainable governance model. In addition to detailing Arctos as both a community of museum professionals and a collection database platform, we discuss how Arctos achieves its richly annotated data by creating a web of knowledge with deep connections between catalog records and derived or associated data. We also highlight the value of Arctos as an educational resource. Finally, we present the financial model of fiscal sponsorship by a nonprofit organization, implemented in 2022, to ensure the long-term success and sustainability of Arctos., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cicero et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The genetic basis of adaptive traits has rarely been used to predict future vulnerability of populations to climate change. We show that light versus dark seasonal pelage in white-tailed jackrabbits ( Lepus townsendii ) tracks snow cover and is primarily determined by genetic variation at endothelin receptor type B ( EDNRB ), corin serine peptidase ( CORIN ), and agouti signaling protein ( ASIP ). Winter color variation was associated with deeply divergent alleles at these genes, reflecting selection on both ancestral and introgressed variation. Forecasted reductions in snow cover are likely to induce widespread camouflage mismatch. However, simulated populations with variation for darker winter pelage are predicted to adapt rapidly, providing a trait-based genetic framework to facilitate evolutionary rescue. These discoveries demonstrate how the genetic basis of climate change adaptation can inform conservation.

Discovery of cryptic species is essential to understand the process of speciation and assessing the impacts of anthropogenic stressors. Here, we used genomic data to test for cryptic species diversity within an ecologically well-known radiation of North American rodents, western chipmunks (Tamias). We assembled a de novo reference genome for a single species (Tamias minimus) combined with new and published targeted sequence-capture data for 21,551 autosomal and 493 X-linked loci sampled from 121 individuals spanning 22 species. We identified at least two cryptic lineages corresponding with an isolated subspecies of least chipmunk (T. minimus grisescens) and with a restricted subspecies of the yellow-pine chipmunk (Tamias amoenus cratericus) known only from around the extensive Craters of the Moon lava flow. Additional population-level sequence data revealed that the so-called Crater chipmunk is a distinct species that is abundant throughout the coniferous forests of southern Idaho. This cryptic lineage does not appear to be most closely related to the ecologically and phenotypically similar yellow-pine chipmunk but does show evidence for recurrent hybridization with this and other species., (© 2022 The Authors. Evolution © 2022 The Society for the Study of Evolution.)

Evidence from natural systems suggests that hybridization between animal species is more common than traditionally thought, but the overall contribution of introgression to standing genetic variation within species remains unclear for most animal systems. Here, we use targeted exon capture to sequence thousands of nuclear loci and complete mitochondrial genomes from closely related chipmunk species in the Tamias quadrivittatus group that are distributed across the Great Basin and the central and southern Rocky Mountains of North America. This recent radiation includes six overlapping, ecologically distinct species (Tamias canipes, Tamias cinereicollis, Tamias dorsalis, T. quadrivittatus, Tamias rufus, and Tamias umbrinus) that show evidence for widespread introgression across species boundaries. Such evidence has historically been derived from a handful of markers, typically focused on mitochondrial loci, to describe patterns of introgression; consequently, the extent of introgression of nuclear genes is less well characterized. We conducted a series of phylogenomic and species-tree analyses to resolve the phylogeny of six species in this group. In addition, we performed several population-genomic analyses to characterize nuclear genomes and infer coancestry among individuals. Furthermore, we used emerging quartets-based approaches to simultaneously infer the species tree (SVDquartets) and identify introgression (HyDe). We found that, in spite of rampant introgression of mitochondrial genomes between some species pairs (and sometimes involving up to three species), there appears to be little to no evidence for nuclear introgression. These findings mirror other genomic results where complete mitochondrial capture has occurred between chipmunk species in the absence of appreciable nuclear gene flow. The underlying causes of recurrent massive cytonuclear discordance remain unresolved in this group but mitochondrial DNA appears highly misleading of population histories as a whole. Collectively, it appears that chipmunk species boundaries are largely impermeable to nuclear gene flow and that hybridization, while pervasive with respect to mtDNA, has likely played a relatively minor role in the evolutionary history of this group. [Cytonuclear discordance; hyridization; introgression, phylogenomics; SVDquartets; Tamias.]., (© The Author(s) 2021. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The evolution of obligate parasites is often interpreted in light of their hosts' evolutionary history. An expanded approach is to examine the histories of multiple lineages of parasites that inhabit similar environments on a particular host lineage. Western North American chipmunks (genus Tamias) have a broad distribution, a history of divergence with gene flow, and host two species of sucking lice (Anoplura), Hoplopleura arboricola and Neohaematopinus pacificus. From total genomic sequencing, we obtained sequences of over 1100 loci sampled across the genomes of these lice to compare their evolutionary histories and examine the roles of host association in structuring louse relationships. Within each louse species, clades are largely associated with closely related chipmunk host species. Exceptions to this pattern appear to have a biogeographic component, but differ between the two louse species. Phylogenetic relationships among these major louse clades, in both species, are not congruent with chipmunk relationships. In the context of host associations, each louse lineage has a different evolutionary history, supporting the hypothesis that host-parasite assemblages vary both across the landscape and with the taxa under investigation. In addition, the louse Hoplopleura erratica (parasitizing the eastern Tamias striatus) is embedded within H. arboricola, rendering it paraphyletic. This phylogenetic result, together with comparable divergences within H. arboricola, indicate a need for taxonomic revision. Both host divergence and biogeographic components shape parasite diversification as demonstrated by the distinctive diversification patterns of these two independently evolving lineages that parasitize the same hosts., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Phylosymbiosis refers to a congruent pattern between the similarity of microbiomes of different species and the branching pattern of the host phylogeny. Phylosymbiosis has been detected in a variety of vertebrate and invertebrate hosts, but has only been assessed in geographically isolated populations. We tested for phylosymbiosis in eight (sub)species of western chipmunks with overlapping ranges and ecological niches; we used a nuclear (Acrosin) and a mitochondrial (CYTB) phylogenetic marker because there are many instances of mitochondrial introgression in chipmunks. We predicted that similarity among microbiomes increases with: (1) increasing host mitochondrial relatedness, (2) increasing host nuclear genome relatedness and (3) decreasing geographic distance among hosts. We did not find statistical evidence supporting phylosymbiosis in western chipmunks. Furthermore, in contrast to studies of other mammalian microbiomes, similarity of chipmunk microbiomes is not predominantly determined by host species. Sampling site explained most variation in microbiome composition, indicating an important role of local environment in shaping microbiomes. Fecal microbiomes of chipmunks were dominated by Bacteroidetes (72.2%), followed by Firmicutes (24.5%), which is one of the highest abundances of Bacteroidetes detected in wild mammals. Future work will need to elucidate the effects of habitat, ecology and host genomics on chipmunk microbiomes., (© FEMS 2019.)

Parasitism is a common symbiotic interaction across diverse natural systems. Using a comparative evolutionary approach, we investigated the contributions of both host phylogeny and abiotic factors toward diversification of phylogenetically independent endoparasites that inhabit essentially the same physical space. We tested for host-parasite and parasite-parasite phylogenetic concordance in western North American chipmunks (Rodentia: Sciuridae) and two distantly related species of pinworms (Nematoda: Oxyurida). Deep structure in molecular phylogenies revealed signals of host-associated divergence in both parasite species, while shallower phylogeographic structure varied between the two parasites. This suggests that although these parasites experienced similar landscapes and cyclic climate processes, temporally distinctive diversification events were associated with differences in the initiation of their association with host lineages. When climate cycles initiate diversification, partially congruent, but asynchronous, host-associated parasite phylogenies may emerge.

The extinct passenger pigeon was once the most abundant bird in North America, and possibly the world. Although theory predicts that large populations will be more genetically diverse, passenger pigeon genetic diversity was surprisingly low. To investigate this disconnect, we analyzed 41 mitochondrial and 4 nuclear genomes from passenger pigeons and 2 genomes from band-tailed pigeons, which are passenger pigeons' closest living relatives. Passenger pigeons' large population size appears to have allowed for faster adaptive evolution and removal of harmful mutations, driving a huge loss in their neutral genetic diversity. These results demonstrate the effect that selection can have on a vertebrate genome and contradict results that suggested that population instability contributed to this species's surprisingly rapid extinction., (Copyright © 2017, American Association for the Advancement of Science.)

Delimiting species can be challenging, but is a key step for the critical examination of evolutionary history and for prioritizing conservation efforts. Because systematic relationships are often determined iteratively using tests based on taxonomy, such methods can fail to detect cryptic variation and result in biased conclusions. Conversely, discovery-based approaches provide a powerful way to define operational taxonomic units and test species boundaries. We compare both approaches (taxonomy-based delimitation - TBD and discovery-based delimitation - DBD) within North American jumping mice (Zapodinae) using broad sampling, multilocus analyses, and ecological tests. This group diversified through the dynamic glacial-interglacial periods of the Quaternary and phylogeographic tests reveal 28 lineages that correspond poorly with current taxonomy (4 species, 32 nominal subspecies). However, neither the 4-species or 28-lineage hypotheses are optimal for species-level classification. Rather, information theoretic approaches (Bayes Factors) indicate a 15-species hypothesis is best for characterizing genetic variation in this group, with subsequent iterative pairwise ecological tests failing to confirm four species pairs. Taken together, evolutionary and ecological tests capture divergence among 11 putative species that, if upheld by additional tests, will lead to taxonomic revision and reevaluation of conservation plans., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Many species are not completely reproductively isolated, resulting in hybridization and genetic introgression. Organellar genomes, such as those derived from mitochondria (mtDNA) and chloroplasts, introgress frequently in natural systems; however, the forces shaping patterns of introgression are not always clear. Here, we investigate extensive mtDNA introgression in western chipmunks, focusing on species in the Tamias quadrivittatus group from the central and southern Rocky Mountains. Specifically, we investigate the role of selection in driving patterns of introgression. We sequenced 51 mtDNA genomes from six species and combine these sequences with other published genomic data to yield annotated mitochondrial reference genomes for nine species of chipmunks. Genomic characterization was performed using a series of molecular evolutionary and phylogenetic analyses to test protein-coding genes for positive selection. We fit a series of maximum likelihood models using a model-averaging approach, assessed deviations from neutral expectations, and performed additional tests to search for codons under the influence of selection. We found no evidence for positive selection among these genomes, suggesting that selection has not been the driving force of introgression in these species. Thus, extensive mtDNA introgression among several species of chipmunks likely reflects genetic drift of introgressed alleles in historically fluctuating populations.

Climate and host demographic cycling often shape both parasite genetic diversity and host distributions, processes that transcend a history of strict host-parasite association. We explored host associations and histories based on an evaluation of mitochondrial and nuclear sequences to reveal the underlying history and genetic structure of a pinworm, Rauschtineria eutamii , infecting 10 species of western North American chipmunks (Rodentia: Tamias , subgenus Neotamias ). Rauschtineria eutamii contains divergent lineages influenced by the diversity of hosts and variation across the complex topography of western North America. We recovered six reciprocally monophyletic R. eutamii mitochondrial clades, largely supported by nuclear gene trees, exhibiting divergence levels comparable to intraspecific variation reported for other nematodes. Phylogenetic relationships among pinworm clades suggest that R. eutamii colonized an ancestral lineage of western chipmunks and lineages persisted during historical isolation in diverging Neotamias species or species groups. Pinworm diversification, however, is incongruent and asynchronous relative to host diversification. Secondarily, patterns of shallow divergence were shaped by geography through events of episodic colonization reflecting an interaction of taxon pulses and ecological fitting among assemblages in recurrent sympatry. Pinworms occasionally infect geographically proximal host species; however, host switching may be unstable or ephemeral, as there is no signal of host switching in the deeper history of R. eutamii .

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.

The cyclic climate regime of the late Quaternary caused dramatic environmental change at high latitudes. Although these events may have been brief in periodicity from an evolutionary standpoint, multiple episodes of allopatry and divergence have been implicated in rapid radiations of a number of organisms. Shrews of the Sorex cinereus complex have long challenged taxonomists due to similar morphology and parapatric geographic ranges. Here, multi-locus phylogenetic and demographic assessments using a coalescent framework were combined to investigate spatiotemporal evolution of 13 nominal species with a widespread distribution throughout North America and across Beringia into Siberia. For these species, we first test a hypothesis of recent differentiation in response to Pleistocene climate versus more ancient divergence that would coincide with pre-Pleistocene perturbations. We then investigate the processes driving diversification over multiple continents. Our genetic analyses highlight novel diversity within these morphologically conserved mammals and clarify relationships between geographic distribution and evolutionary history. Demography within and among species indicates both regional stability and rapid expansion. Ancestral ecological differentiation coincident with early cladogenesis within the complex enabled alternating and repeated episodes of allopatry and expansion where successive glacial and interglacial phases each promoted divergence. The Sorex cinereus complex constitutes a valuable model for future comparative assessments of evolution in response to cyclic environmental change., (Published by Elsevier Inc.)

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.

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.

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.

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.

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., (Copyright 2002 Elsevier Science (USA))

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.neomexicanus and S.pacificus) were poorly differentiated. Sorexornatus and S.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. bairdi and S. pacificus); (iii) the semiaquatic species (S. bendirii and S. 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.

Jackrabbits and hares, members of the genus Lepus, comprise over half of the species within the family Leporidae (Lagomorpha). Despite their ecological importance, potential economic impact, and worldwide distribution, the evolution of hares and jackrabbits has been poorly studied. We provide an initial phylogenetic framework for jackrabbits and hares so that explicit hypotheses about their evolution can be developed and tested. To this end, we have collected DNA sequence data from a 702-bp region of the mitochondrial cytochrome b gene and reconstructed the evolutionary history (via parsimony, neighbor joining, and maximum likelihood) of 11 species of Lepus, focusing on North American taxa. Due to problems of saturation, induced by multiple substitutions, at synonymous coding positions between the ingroup taxa and the outgroups (Oryctolagus and Sylvilagus), both rooted and unrooted trees were examined. Variation in tree topologies generated by different reconstruction methods was observed in analyses including the outgroups, but not in the analyses of unrooted ingroup networks. Apparently, substitutional saturation hindered the analyses when outgroups were considered. The trees based on the cytochrome b data indicate that the taxonomic status of some species needs to be reassessed and that species of Lepus within North America do not form a monophyletic entity., (Copyright 1999 Academic Press.)