2011 NORTHEASTERN NATURALIST 18(4):497–508 Cytochrome-b Sequence Variation in Water Shrews (Sorex palustris) from Eastern and Western North America Erin E. Mycroft1, Aaron B.A. Shafer2, and Donald T. Stewart3,* Abstract - Mitochondrial DNA sequence data for the cytochrome-b gene was used to assess genetic divergence among samples of Sorex palustris (Water Shrew) from eastern and western North America. Phylogenetic analysis revealed the presence of three previously observed clades—Cordilleran (S. p. navigator and S. p. brooksi), Coastal (S. bendirii), Boreal (S. p. palustris)—along with a novel Eastern clade (S. p. gloveralleni and/or S. p. albibarbis). Intraspecific divergence between the Boreal and Eastern clades was 3.42%, exceeding interspecific divergence between the Cordilleran and Coastal clades (3.35%). Application of the genetic species concept to the results of this study suggests that the Boreal and Eastern clades of Water Shrews warrant further investigation for recognition as two distinct sister species (i.e., S. palustris and S. albibarbis, respectively). Additional samples and analysis of nuclear markers will be required to substantiate this proposed taxonomic revision. Introduction Sorex palustris Richardson (American Water Shrew) is the world’s smallest diving mammal. It has evolved a number of morphological (e.g., stiff hairs on the feet to increase surface area for propulsion underwater) and physiological (e.g., a high basal metabolic rate to fight heat loss in water) adaptations to its semiaquatic lifestyle (Beneski and Stinson 1987, Gusztak et al. 2005, van Zyll de Jong 1983). The American Water Shrew has an extensive distribution; records have been documented as far west as Vancouver Island, across the boreal region of Canada to Labrador, and throughout the Maritime provinces. The species’ range extends up the Rocky Mountains from California to Alaska, and along the Appalachians in the northeastern United States (Banfield 1974, van Zyll de Jong 1983, Whitaker and Hamilton 1998). The American Water Shrew shares many of the aforementioned features with the closely related Sorex bendirii Merriam (Marsh Shrew), which has a more limited distribution along the Pacific coastline from southern British Columbia to California (Pattie 1973). Morphological characteristics such as skull and dental traits have been used to distinguish the American Water Shrew from the Marsh Shrew (Findley 1955). The American Water Shrew has a straighter rostrum than the Marsh Shrew, a distinctly bicolour tail, and lighter underparts (van Zyll de Jong 1983). Allozyme electrophoresis analysis by George (1988) confirmed the two as separate sister species, and mitochondrial 1Faculty of Forestry, 33 Willcocks Street, University of Toronto, Toronto, ON M5S 3B3, Canada. 2Department of Biological Sciences, CW 405, Biological Sciences Building, University of Alberta, Edmonton, AB T6G 2E9, Canada. 3Department of Biology, Acadia University, Wolfville, NS B4P 2R6, Canada. *Corresponding author - don.stewart@ acadiau.ca. 498 Northeastern Naturalist Vol. 18, No. 4 DNA cytochrome-b (mtDNA Cytb) sequence data also supported this relationship (Demboski and Cook 2001, O’Neill et al. 2005). At least nine subspecies of the American Water Shrew have been described (van Zyll de Jong 1983). These subspecies do not differ significantly in size or morphology, and were described mostly on the basis of color and tail characteristics. Sorex palustris navigator is found in the southern Yukon Territory, British Columbia, and in western Alberta. It has a distinct change in color between the dorsal and ventral pelage and a bicolor tail. Sorex palustris brooksi is restricted to Vancouver Island, and is also characterized by a black back and white hair on the underside of the tail. These western subspecies appear to be derived from a single refugium in the Pacific Northwest (Himes and Kenagy 2010), where genetic clades do not appear to correspond to subspecies. Sorex palustris palustris occurs in the boreal region of Canada from northeastern British Columbia through to northern Ontario and Québec; it is characterized by a distinct change in color between the dorsal and ventral area, and a bicolor tail. Sorex palustris albibarbis occupies a range from southern Ontario and southern Québec through to western New Brunswick; it does not possess as distinct a colour demarcation or a bicolour tail. Sorex palustris gloveralleni is found in Prince Edward Island, central and eastern New Brunswick, and Nova Scotia including Cape Breton. It is described as having a pale grey pelage with a bicolor tail (van Zyll de Jong 1983). Subspecies S. p. hydrobadistes, S. p. turneri, S. p. labradorensis, and S. p. punctulatus have been proposed to occur in Michigan and Wisconsin, Northern Québec, Labrador, and the southern Appalachians, respectively (for detailed accounts of these ranges see Banfield [1974] and Hall [1981]). Variation in Cytb gene sequences of the American Water Shrew and the Marsh Shrew from western North America was assessed by O’Neill et al. (2005). Inter- and intra-specific divergence values and phylogenetic analyses showed S. p. brooksi and S. p. navigator to be more closely related to S. b. palmeri and S. b. bendirii than to another putative member of their own species, S. p. palustris. The authors suggested that Water Shrew taxonomy be revised to recognize the western Cordilleran subspecies as a distinct species, Sorex navigator (Navigator Shrew; O’Neill et al. 2005). In the present study, geographic variation of additional Water Shrew specimens from across North America was investigated by examining genetic variation in the mtDNA Cytb gene of samples obtained from both eastern and western regions. Based on our phylogenetic and genetic distance analyses, we discuss possible taxonomic revisions to the Water Shrew species complex. Methods Samples Partial mitochondrial Cytb gene sequences (994 base pairs) were generated from 10 specimens of Water Shrews from Alberta, Manitoba, Nova Scotia, and New Brunswick. These samples correspond to the putative subspecies S. p. palustris, S. p. albibarbis, and S. p. gloveralleni. Thirty-four additional Cytb sequences, twenty-five of which are from Water or Marsh Shrews, were obtained 2011 E.E. Mycroft, A.B.A. Shafer, and D.T. Stewart 499 from Fumagalli et al. (1999), Demboski and Cook (2001), and O’Neill et al. (2005) through the GenBank database (Table 1). Mitochondrial DNA sequencing Total DNA was extracted from frozen or ethanol-preserved tissue. Approximately 0.2 g of tissue per sample was used for DNA extraction (DNeasy Table 1. GenBank accession numbers, location data, specimen/tissue numbers, articles referenced from, and length of sequences for samples used in the analysis. Additional names not defined in the text include Sorex vagrans Baird (Vagrant Shrew) and S. fumeus Miller (Smoky Shrew). Specimen/ GenBank Phylogenetic accession No. Location Article reference reference S. p. navigator AY954934 Yoho National Park, BC O’Neill et al. 2005 (BC1) AY954936 Yoho National Park, BC O’Neill et al. 2005 (BC2) AY954937 Cayoosh Creek, BC O’Neill et al. 2005 (BC3) AY954933 Yoho National Park, BC O’Neill et al. 2005 (BC4) AY954935 Yoho National Park, BC O’Neill et al. 2005 (BC5) AY954932 Yoho National Park, BC O’Neill et al. 2005 (BC6) AJ000448 Salt Lake County, UT Fumagalli et al.1999 (UT1) AJ000449 Watson Lake, YT Fumagalli et al.1999 (YK1) AF238033 Petersburg Quad, AK Demboski and Cook 2001 (AK1) S. p. brooksii AY954926 Hamilton Creek, Vancouver O’Neill et al. 2005 (BC1) Island, BC AY954927 Hamilton Creek, Vancouver O’Neill et al. 2005 (BC2) Island, BC AY954931 Lower Lost Shoe Creek, O’Neill et al. 2005 (BC3) Vancouver Island, BC AY954928 Lowry Lake, Vancouver Island, BC O’Neill et al. 2005 (BC4) AY954929 Lowry Lake, Vancouver Island, BC O’Neill et al. 2005 (BC5) AY954930 Lowry Lake, Vancouver Island, BC O’Neill et al. 2005 (BC6) S. b. bendirii AY954944 Sumas Mountain, BC O’Neill et al. 2005 (BC1) AY954945 Sumas Mountain, BC O’Neill et al. 2005 (BC2) AY954946 Seymour River, BC O’Neill et al. 2005 (BC3) AY954947 Aldergrove, BC O’Neill et al. 2005 (BC4) S. p. palustris AY954938 Calling Lake, AB O’Neill et al. 2005 (AB1) AY954940 Calling Lake, AB O’Neill et al. 2005 (AB2) AY954941 Calling Lake, AB O’Neill et al. 2005 (AB3) AY954942 Calling Lake, AB O’Neill et al. 2005 (AB4) AY954939 Calling Lake, AB O’Neill et al. 2005 (AB5) AY954943 Lac La Biche, AB O’Neill et al. 2005 (AB6) DQ788800 Ototoks, AB This study (AB7) DQ995274 Caddy Lake, MB This study (MB1) DQ995275 Piney, MB This study (MB2) S. p. albibarbis DQ995279 Edmundston, NB This study (NB1) DQ995280 Edmundston, NB This study (NB2) 500 Northeastern Naturalist Vol. 18, No. 4 Tissue Kit, QIAGEN). Samples were diluted to 25 ug DNA/ml in preparation for polymerase chain reaction (PCR). Amplification of the Cytb gene was accomplished with a PCR reaction using L14841 (Kocher et al. 1989) or L14724, and H15915 primers (Irwin et al. 1991). PCR reactions were 25 ul each, consisting of 12.45 ul ultra-pure water, 2.5 ul 10x Buffer, 2.5 ul L14724 primer [10 pmol], 2.5 ul H15915 primer [10 pmol], 2.5 ul dNTPs [8mM], 0.3 ul Taq polymerase [5 U/ul] (Promega), 1.25 ul MgCl2 [25 mM], and 1 ul of [25 ug/ ml] DNA template. The PCR program was an initial 94 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 50 °C for 45 s, and extension at 72 °C for 1 min, concluding with 72 °C for 5 min. PCR products were purified using the QIAquick Gel Purification Kit (QIAGEN). Double-stranded sequencing was performed at the Florida State University DNA Sequencing Facility (Tallahassee, FL) using an Applied Biosystems 3100 Genetic Analyzer. Mitochondrial Cytb sequences were aligned with the ClustalW algorithm (Thompson et al. 1994) and edited using BioEdit v.7.0.5.3 (Hall 1999). Data analysis For comparison with previous studies, the traditional neighbor-joining (NJ) method of analysis was performed using the Kimura 2-parameter model of substitution in MEGA (Kumar et al. 2004). Maximum parsimony (MP) analysis was conducted using PAUP v.4.0b10 (Swofford 2002). A heuristic search was conducted on all data sets, with 10 replicates of the addition sequence, and equal character weighting. A tree bisection-reconnection (TBR) branch-swapping Table 1, continued. Specimen/ GenBank Phylogenetic accession No. Location Article reference reference S. p. gloveralleni DQ995276 Mount Martock, NS This study (NS1) DQ995277 Waverley, NS This study (NS2) DQ788802 Cole Harbour, NS This study (NS3) DQ788801 Cape Breton, NS This study (NS4) DQ995278 Cape Breton, NS This study (NS5) S. monticolus AJ000451 Tillamook County, OR Fumagalli et al.1999 (OR1) AJ000450 Tillamook County, OR Fumagalli et al.1999 (OR2) S. fumeus AJ000462 Edmundston, NB Fumagalli et al.1999 (NB1) AJ000461 Edmundston, NB Fumagalli et al.1999 (NB2) S. hoyi AJ000460 Ogilvie Mountains, YT Fumagalli et al.1999 (YK1) AF238040 Hughes Quad, AK Demboski and Cook 2001 (AK1) S. vagrans AJ000455 Sagehen Creek, CA Fumagalli et al.1999 (CA1) AJ000454 Sagehen Creek, CA Fumagalli et al. 1999 (CA2) S. araneus AY936837 Sweden Andersson et al. 2005 2011 E.E. Mycroft, A.B.A. Shafer, and D.T. Stewart 501 algorithm was also implemented. For both NJ and MP analyses, the robustness of the resulting tree was evaluated by performing 1000 bootstrap replicates. The appropriate model for the maximum likelihood (ML) analysis was determined with MODELTEST (Posada and Crandall 1998), with the model TrN+G chosen as the best-fit model of nucleotide substitution. Taxa were added to the tree using a random addition sequence (x10), and the TBR branchswapping algorithm was employed. Due to the computational time required, the robustness of the ML tree topology was tested in PAUP v.4.0b10 with 100 bootstrap replicates, and the number of trees saved per replicate was reduced to one (MAXTREE = 1). The Bayesian analysis was conducted using MrBayes v.3.1 (Huelsenbeck and Ronquist 2001). Codon positions were treated separately with models determined from MRMODELTEST (Nylander 2004), as the frequency of substitution varies greatly among codon positions in Cytb (Irwin et al. 1991). Each position (1st, 2nd, and 3rd, which had the models HKY+G, HKY+I, and HKY+I+G, respectively) were unlinked, and were allowed to evolve separately. The Markov chain Monte Carlo algorithm was run with four chains and sampled every 1000 generations until the average standard deviation of split frequencies stabilized at less than 0.01. All chains prior to the stabilization of likelihood values were removed as a burn-in, and the remainder were used to construct a 50% consensus tree. Pairwise genetic distances were determined with MEGA using Kimura’s two-parameter model with complete deletion (Table 2). This model was chosen to enable comparison of results with those of other studies (e.g., Demboski and Cook 2001, 2003; Fumagalli et al. 1999; O’Neill et al. 2005). Table 2. Percent divergence in Cytochrome-b sequences between the four major clades (consisting of S. palustris and S. bendirii) and outgroups using the Kimura 2-parameter model and complete deletion. S. h. = Sorex hoyi, S. f. = S. fumeus, S. m. = S. monticolus, and S. v. = S. vagrans. Cordilleran Coastal Boreal Eastern S. h. S. f. S. m. S. v. Group 1 “Cordilleran” (S. p. navigator/ - - - - - - - - S. p. brooksi) Group 2 “Coastal” (S. bendirii) 3.35 - - - - - - - Group 3 “Boreal” (S. p. palustris) 7.21 6.61 - - - - - - Group 4 “Eastern” (S. p. albibarbis/ 6.12 6.48 3.42 - - - - - S. p. gloveralleni) S. hoyi 13.35 11.96 12.91 12.18 - - - - S. fumeus 13.36 12.84 13.09 13.16 14.15 - - - S. monticolus 5.09 4.41 7.01 5.82 12.47 13.16 - - S. vagrans 8.25 7.13 8.61 7.90 12.18 13.24 7.90 - S. araneus 15.96 16.33 17.00 16.41 17.39 18.59 15.52 17.50 502 Northeastern Naturalist Vol. 18, No. 4 Figure 1 (opposite page). Phylogenetic relationships for members of the genus Sorex, inferred from 994bp of mtDNA Cytb sequence data, rooted with Sorex araneus L. (European Common Shrews) as an outgroup. Bootstrap support values are for the neighbor-joining maximum-parsimony (above the branch) and maximum-likelihood (below the branch), respectively. For the first two methods, 1000 bootstrap replicates were performed; for maximum-likelihood analysis, 100 bootstrap replicates were used. For clarity, only bootstrap values of major clades are displayed on branches. Identifiers in brackets correspond to Genbank accession numbers and can be found in Table 1. Results Phylogenetic analysis Neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian methods of analysis produced near-identical trees with similar support values for most clades (see Figs. 1 and 2). Four distinct clades were evident: a “Cordilleran” clade (S. p. navigator and S. p. brooksi), a “Coastal” clade (S. b. bendirii), a “Boreal” clade (S. p. palustris), and an “Eastern” clade (S. p. gloveralleni, S. p. albibarbis). The subspecies contained within the “Eastern” clade have been inferred using putative geographical ranges (Banfield 1974) and are not definitive. The “Cordilleran” and “Coastal” clades form one group, and the “Boreal” and “Eastern” clades form another. Sorex monticolus Merriam (Montane Shrew) Cytb sequences formed a sister-group to the combined “Cordilleran” and “Coastal” clades of Water Shrews. The neighbor-joining tree had high bootstrap support values, ranging from 99–100% for each major clade, as did the maximumparsimony tree (95–100%; Fig. 1). For the Bayesian analysis, we ran 500,000 generations with the first 10% removed as a burn-in (based on plot of likelihood values). The average standard deviation of split frequencies was below 0.01, and the potential scale reduction factors were ≈1, indicative of convergence. Every major clade was highly supported, with posterior probabilities ranging from 0.97–1.00 (Fig. 2). Sequence divergence values Sequence divergence values between the four major Water Shrew clades (i.e., “Cordilleran”, “Coastal”, “Boreal”, and “Eastern”) and the outgroups are presented in Table 2. The level of divergence between the “Boreal” and “Eastern” clades consisting of S. palustris (3.42%) was greater than that between the “Cordilleran” and “Coastal” clades (3.35%). Distance calculations grouping “Cordilleran” and “Coastal” clades together as one group and “Boreal” and “Eastern” clades as another revealed a divergence value of 6.70%. Discussion In the genus Sorex, interspecific distance estimates for the Cytb gene of selected species range from 0.7% to 9.3% (Stewart et al. 2002, 2003). In this study, intraspecific distance between the “Boreal” and “Eastern” clades of the American Water Shrew was found to be 3.42%. This value falls within the range 2011 E.E. Mycroft, A.B.A. Shafer, and D.T. Stewart 503 504 Northeastern Naturalist Vol. 18, No. 4 Figure 2. Bayesian tree for members of the genus Sorex, inferred from 994bp of mtDNA Cytb sequence data, rooted with European Common as an outgroup. Numbers correspond to posterior probabilities, using the model TrN+G. Identifiers in brackets correspond to Genbank accession numbers and can be found in Table 1. of interspecific divergence in the genus Sorex, and in accordance with the genetic species concept (sensu Baker and Bradley 2006), suggests these two clades may be distinct species. For example, divergence between two putative species of S. hoyi Baird (American Pygmy Shrew) and S. thompsoni Baird (Thompson’s Pygmy Shrew) is 3.3% (Stewart et al. 2003), while the Marsh Shrew and the Navigator Shrew Cytb sequences differ by approximately 3.1% (O’Neill et al. 2005) to 3.35% (this study). Furthermore, Shafer and Stewart (2007) found evidence for a distinct eastern group of Water Shrews. While our data suggest that the “Boreal” and “Eastern” clades of Water shrews may indeed warrant separate 2011 E.E. Mycroft, A.B.A. Shafer, and D.T. Stewart 505 species status, further evidence from a nuclear locus would be a valuable addition to accurately delineate species. A “genetic” species has been defined as “a group of genetically compatible interbreeding natural populations that is genetically isolated from other such groups” (Baker and Bradley 2006). Some mammalian species, such as those belonging to the genus Sorex, are difficult to distinguish based on morphology alone (Baker and Bradley 2006). To develop a genetic distance metric that could be used to identify potentially cryptic species, Bradley and Baker (2001) analyzed levels of Cytb sequence variation required for speciation in 4 genera of rodents and 7 genera of bats; the authors concluded that a genetic distance of >11% indicates definite interspecific variation; values of 2% to 11% warrant additional investigation, and values of <2% likely indicate intraspecific or population-level variation. The precise “threshold” divergence required for speciation may vary depending on the group being studied (Baker and Bradley 2006, Bradley and Baker 2001). Nonetheless, there is increasing evidence that the general utility of mitochondrial “DNA Barcoding” for identifying species divergence thresholds reflects the intense co-evolutionary selective pressures on mitochondrial genes and nuclear genes to produce the functional components for cellular respiration (Lane 2009). If the mitochondrial and nuclear encoded subunits do not work well together (as is increasingly likely with increasing molecular divergence), then respiration will be less efficient or fail altogether. The major inference to be drawn from the phylogenetic and distance analyses presented here is that the “Eastern” clade of Water Shrews may be a distinct species from the “Boreal” clade. Under this division, the “Boreal” clade would represent S. palustris, while the “Eastern” clade, which contains representative samples from two putative subspecies, S. p. albibarbis and S. p. gloveralleni, would be renamed S. albibarbis Cope, which has historical priority (Cope 1862). A tentative common name for this taxon could be the White-bearded Water Shrew. Additional nuclear data and sampling are required prior to a full taxonomic revision. The phylogenetic affinities of the remaining subspecies in eastern North America (namely S. p. hydrobadistes, S. p. labradorensis, S. p. turneri, and S. p. punctulatus) will also need to be directly assessed before they are assigned to either S. palustris or S. albibarbis. Two scenarios are most probable. First, subspecies distributed throughout the boreal forest (i.e., S. p. palustris, S. p. turneri, and S. p. labradorensis), and those present in the Acadian and Great Lakes-St. Lawrence forest zones as well as those in the Appalachian Mountains (i.e., S. p. gloveralleni, S. p. albibarbis, S. p. hydrobadistes, and S. p. punctulatus) will cluster together with either S. palustris or S. albibarbis, respectively. Alternatively, the St. Lawrence River and Great Lakes waterway may constitute a biogeographic barrier that separates two distinctive mitochondrial clades (e.g., S. palustris to the north and S. albibarbis to the south and east), in which case any putative subspecific designations may be need to be revisited. The phylogenetic position of the two Edmundston, NB, samples (see Fig. 1) is of particular interest in this context. One of these samples (NB2) is embedded 506 Northeastern Naturalist Vol. 18, No. 4 within a clade of Nova Scotia sequences and the other (NB1) forms a sister sequence to this clade. This pattern suggests that the area around Edmundston in northwestern New Brunswick is home to Water Shrews with two distinct, albeit very closely related, haplotypes (0.7% sequence divergence). This is the same pattern that is observed within S. cinereus Kerr (Masked Shrews) in eastern Canada. Specifically, Stewart and Baker (1997) found that northwestern New Brunswick was a zone of secondary contact between two mitochondrial clades within Masked Shrews: one primarily a maritime clade (corresponding to the Acadian forest region), and one primarily a Québec and Ontario clade (corresponding more closely to the Great Lakes-St. Lawrence forest region). Given that Water Shrews and Masked Shrews have similar distributions, it would not be surprising that they share similar biogeographic histories. The Wisconsin glacial events that likely lead to the divergence of the distinct mitochondrial clades within Masked Shrews also likely shaped the pattern of molecular divergence within Water Shrews. In summary, further work is needed to clarify the phylogenetic relationships, taxonomic status, historical biogeography, differences in natural history, and conservation status of all American Water and Marsh shrews. The application of novel species concepts, (e.g., the genetic species concept) can be of enormous practical benefit to the study of shrews or other small mammals that are either morphologically non-descript or challenging to maintain in captivity (see Gusztak and Campbell 2004). More voucher specimens of Water Shrews need to be examined both morphologically and genetically from across the full species range. Efforts should also be focused on geographically intermediate areas of genetically distinct clades to identify potential zones of introgression. Acknowledgments Samples from Manitoba were kindly supplied by K.L. Campbell at the University of Manitoba. Funding for this research was provided by a Natural Sciences and Engineering Council of Canada Discovery Grant to D.T. Stewart (217175). 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