Identification of Fragmentary Late Pleistocene Musteloids through Morphometric Analyses

1. Identification of Fragmentary Late Pleistocene Musteloids through Morphometric Analyses Previous studies of Guy Wilson Cave (GWC) in Sullivan County, Tennessee revealed an abundance of late Pleistocene large mammal fossils, mostly herbivores such as deer (Odocoileus sp.) and flat-headed peccary (Platygonuscompressus). In addition were some bones of dire wolf (Canusdirus) and bear (Ursus sp.). Based on analysis of wear patterns on peccary long bones, it has been suggested that GWC was a dire wolf den. Other fossils found in GWC were mostly fragmentary remains of small carnivorans. In particular, several partial mandibles with lower first molars (or lower “carnassials”) were recovered that appeared to be from musteloids. Geometric morphometrics has been successfully used to identify fragmentary specimens of other fossil taxa, and so a project was initiated to look at several sets of closely related taxa, some of which appeared to be in the GWC sample. Specifically, this thesis developed a landmark-based, 2-dimensional approach for identifying musteloids, focusing on the lower first molar. Digital images of the lower carnassials of several GWC fossils and of extant reference musteloids were combined using the morphometrics programs tpsDIG1, tpsUtil and tpsSuper. Statistical analysis of the landmark data was performed in the program PASW Statistics (also known as SPSS). Results have been successful not only in separating the ‘sister’ taxa Mephitis mephitis (Striped skunk) and M. macroura (Hooded skunk), but also with genetically and ecologically similar taxa as Martesamericana (American marten) and M.pennanti (fisher). A by-product of this study was the opportunity to search for both gender-based and geographical patterns within the statistical data. Sex appears to separate out somewhat via the lower carnassial, but more work is needed to be certain. Regional variances discovered in that same tooth suggest an interesting biogeographical history for each taxon. Abstract Joel A. Christine1 and Steven C. Wallace2 Department of Geosciences, Don Sundquist Center of Excellence in Paleontology, East Tennessee State University, Johnson City, TN, USA.1joelalvinchristine@yahoo.com 2wallaces@mail.etsu.edu Introduction Materials & Methods(con’d) Discussion Conclusions References Anderson, E. 1970. Quaternary evolution of the genus Martes (Carnivora, Mustelidae). ActaZoologicaFennica 130:1–130. Daitch, D. J. and R. P. Guralnick. 2007. Geographic variation in tooth morphology of the arctic fox, Vulpes (Alopex) lagopus. Journal of Mammalogy 88(2):384–393. Dayan, T., D. Wool and D. Simberloff. 2002. Variation and covariation of skulls and teeth: modern carnivores and the interpretation of fossil mammals. Paleobiology 28(4):508–526. Dragoo, J. W. and R. L. Honeycutt. 1997. Systematics of mustelid-like carnivores. Journal of Mammalogy 78(2):426–443. Flynn, J. J., J. A. Finarelli, S. Zehr, J. Hsu and M. A. Nedbal. 2005. Molecular phylogeny of the Carnivora (Mammalia): assessing the impact of increased sampling on resolving enigmatic relationships. Systematic Biology 54(2):317–337. Fulton, T. L. and C. Strobeck. 2006. Molecular phylogeny of the Arctoidea (Carnivora): effect of missing data on supertree and supermatrix analyses of multiple gene data sets. Molecular Phylogenetics and Evolution 41:165–181. Fulton, T. L. and C. Strobeck. 2006. Molecular phylogeny of the Arctoidea (Carnivora): effect of missing data on supertree and supermatrix analyses of multiple gene data sets. Molecular Phylogenetics and Evolution 41:165–181. Giuliano, W. M., J. A. Litvaitis and C. L. Stevens. 1989. Prey selection in relation to sexual dimorphism of fishers (Martespennanti) in New Hampshire. Journal of Mammalogy70(3):639–641. Hughes, S. S. 2009. Noble marten (Martesamericananobilis) revisited: its adaptation and extinction. Journal of Mammalogy 90(1):74–92. Loy, A., O. Spinosi and R. Carlini. 2004. Cranial morphology of Martesfoina and M. martes (Mammalia, Carnivora, Mustelidae): the role of size and shape in sexual dimorphism and interspecific diiferentiation. Italian Journal of Zoology 71:27–35. Lyman, R. L. 2010. Paleoecological and biogeographical implications of late Pleistocene noble marten (Martesamericananobilis) in eastern Washington State, USA. Quaternary Research 75:176–182. Meiri, S., T. Dayan, and D. Simberloff. 2005. Variation and correlations in carnivore and crania and dentition. Functional Ecology 19:337–343. Moors, P. J. 1980. Sexual dimorphism in the body size of mustelids (Carnivora): the roles of food habits and breeding systems. Oikos34(2):147-158. Nye, A. S. 2007. Pleistocene Peccaries from Guy Wilson Cave, Sullivan County, Tennessee. Unpublished M.S. thesis, East Tennessee State University, Johnson City, TN. Nye, A. S., B. W. Schubert and S. C. Wallace. 2007. Late Pleistocene Peccaries from Guy Wilson Cave, Sullivan County, Tennessee. Journal of Vertebrate Paleontology 27:w125A. Powell, R. A. and R. D. Leonard. 1983. Sexual dimorphism and energy expenditure for reproduction in female fisher Martespennanti. Oikos 34(2):147-158. Reig, S. 1992. Geographic variation in pine marten (Martesmartes) and beech marten (M. foina) in Europe. Journal of Mammalogy73(4):744–769. Rohlf, F. J. (2004a) tpsDig, version 1.40. Department of Ecology and Evolution, State University of New York, Stony Brook. Available at http://life.bio.sunysb.edu/morph/. Rohlf, F. J. (2004b) tpsSuper, version 1.14. Department of Ecology and Evolution, State University of New York, Stony Brook. Available at http://life.bio.sunysb.edu/morph/. Rohlf, F. J. (2009) tpsUtil, version 1.44. Department of Ecology and Evolution, State University of New York, Stony Brook. Available at http://life.bio.sunysb.edu/morph/. Sealfon, R. A. 2007. Dental divergence supports the species status of the extinct sea mink (Carnivora: Mustelidae: Neovisonmacrodon). Journal of Mammalogy88(2):371–383. Stone, K. D., R. W. Flynn and J. A. Cook. 2002. Post-glacial colonization of northwestern North America by the forest-associated American marten (Martes Americana, Mammalia: Carnivora: Mustelidae). Molecular Ecology 11:2049-2063. Szuma, E. 2004. Evolutionary implications of morphological variation in the lower carnassial of red fox Vulpesvulpes. ActaTheriologica49(4):433–447. Thom, M. D., L. A. Harrington and D. W. Macdonald. 2004. Why are American mink sexually dimorphic? A role for niche separation. Oikos 105(3):525-535. Wang, X., D. P. Whistler and G. T. Takeuchi. 2005. A new basal skunk Martinogale (Carnivora, Mephitinae) from late Miocene Dove Spring Formation, California, and origin of New World mephitines. Journal of Vertebrate Paleontology 25(4):936-949. Wang, X. and Ó. Carranza-Castañeda. 2008. Earliest hog-nosed skunk, Conepatus (Mephitidae, Carnivora), from the early Pliocene of Guanajuato, Mexico and origin of South American skunks. Zoological Journal of the Linnean Society 154:386-407. Wolsan, M., A. L. Ruprecht and T. Buchalczyk. 1985. Variation and asymmetry in the dentition of the Pine and Stone martens (Martesmartes and M. foina) from Poland. ActaTheriologica 30(3):79-114. Zakrzewski, R. J. 1967. The systematic position of Canimartes? from the Upper Pliocene of Idaho. Journal of Mammalogy 48(2):293–297. Zalewski, A. 2007. Does sexual dimorphism reduce competition between sexes? The diet of male and female pine martens at local and wider geographical scales. ActaTheriologica 52(3):237–250. Acknowledgements Vertebrate teeth are not only extremely durable but are very useful in identification, particularly in the case of mammals (e.g., Dayan and Simberloff, 2002). Members of Carnivora, for instance, generally share dental features such as extended-length canines for stabbing prey and blade-like carnassials(Figure 1) for slicing meat (Meiriet al, 2005). Variation in the genetics and in the bones and teeth of carnivores has been used to search for and study geographic patterns (Daitch and Guralnick, 2007; Hughes, 2009; Lyman, 2010; Reig, 1992; Stone et al, 2002; Szuma, 2004). Within the Carnivora are two divisions: the Feliformia– “cat-shaped” – and the Caniformia – “dog-shaped” (Dragoo and Honeycutt, 1997; Flynn et al, 2005; Fulton and Strobeck, 2006). Based on recent genetic analyses, skunks and stink badgers belong in the Mephitidaeas a sister clade to the Mustelidae, which includes otters, badgers and weasels (Dragoo and Honeycutt, 1997; Flynn et al, 2005; Fulton and Strobeck, 2006). The Mephitidaeand Mustelidae themselves fall within the larger superfamily of Musteloidea, which also includes the red panda, raccoons and ringtails; this superfamily is itself part of a larger division of the Caniformia(Dragoo and Honeycutt, 1997; Flynn et al, 2005; Fulton and Strobeck, 2006). For a number of years researchers at East Tennessee State University have been studying fossils from Guy Wilson Cave (GWC) in Sullivan County, Tennessee (Figure 2). They have found that GWC holds a variety of late Pleistocene mammal bones, most likely as a result of it being used as a den for dire wolves, Canusdirus (Nye, 2007; Nye et al., 2007). Among the many bones found in the cave were a skull and several lower jaw fragments of smaller carnivorans. Soon after their recovery, some of these bones were identified as belonging to different musteloids: an American marten, Martesamericana(Figure 3); a fisher, Martespennanti(Figure 4); and a striped skunk, Mephitis mephitis (Figure 5). Overlaps in tooth size and morphology warranted a more robust method of identification; more specifically, identification through the use of landmark analysis. Initially, the goal was alpha taxonomy. However, since a significant number of reference specimens were obtained, analyses focused on spatial and morphometric patterns that might be present. The authors would like to thank Drs. Blaine Schubert and Jim Mead of East Tennessee State University for their advice and encouragement throughout the course of this project. Sincere thanks is also owed to Ms. Amy Nye and Mr. Brett Woodward, collections managers at the East Tennessee State University and General Shale Brick Natural History Museum and Visitor Center, for their courtesy and professionalism in allowing access to the Guy Wilson Cave fossils. A special mention goes to Ms. Sandra Swift for her advice and assistance in getting high-quality photographic images of the various specimens involved. Last but by no means least, a debt of thanks is owed to Ms. Suzanne C. Peurach, Collections Manager for North American mammals at the Smithsonian Institution’s National Museum of Natural History, for allowing access to an extensive number of musteloid specimens being kept at the Smithsonian’s Museum Support Center (MSC) in Suitland, Maryland. Figure 2: Tennessee county map; Guy Wilson Cave indicated by star in upper right corner. Adapted from Nye 2007. Figure 8: tpsDIG1 screen-shot of the lower right carnassial for skunk reference specimen NMNH 120100, showing the locations of all 23 landmarks. The first eleven lay out the apparent edge of the tooth crown, while the other 12 mark specific features across the occlusal surface. Figure 7: tpsDIG1 screen-shot of the lower left carnassial for fisher reference specimen ETMNH 598, showing all 23 landmarks. The first eleven landmarks describe the tooth crown’s apparent edge as seen from above, while the other twelve mark specific features across the occlusal surface. Figure 9: Discriminant Analysis histogram plot of all M. americana and M. pennanti specimens, Guy Wilson Cave included. The M. pennanti specimens form a roughly ‘bell-curve’ shape while the M. americana show a more skewed distribution, which could simply reflect the chosen bin size for this plot. The GWC specimens fall where their original identifications placed them. Figure 10: Discriminant Analysis plot of all skunk specimens, including the Guy Wilson Cave fossil. All three genera cleanly separate out from each other, but the Mephitis species remain intermingled. The Guy Wilson Cave fossil appears to clearly be a Mephitis in this plot, but its exact species is not obvious here. Figure 21: Current range for the striped skunk (Mephitis mephitis). Note how much more of the continent is inhabited by M. mephitis than by either of the Martes spp. Source is http://en.wikipedia.org/wiki/File:Mephitis_mephitis_range_map.png; accessed on 10/28/2011. Figure 6: Guy Wilson Cave fossil teeth studied for this thesis: a) ETMNH 6242, Martesamericana; b) ETMNH 6243, Martespennanti; c) ETMNH 6244, Mephitis mephitis. Images not to same scale. Figure 11: Discriminant Analysis histogram of all Mephitis specimens, Guy Wilson Cave included. A tall, narrow roughly bell-like shape denotes each of the sister taxa. Here the separation between M. mephitis and M. macroura is clear and the GWC fossil is distinctly identified as belonging with M. mephitis. Figure 15: Discriminant Analysis plot for all of the M. americana specimens, by geographic region. Interestingly, the data separates out not north-to-south but also east-to-west. This would be very unlikely without true regional differences in the lower carnassial, in this case consistent with four distinct sub-populations. It is no surprise that the two Alaskan specimens (green diamonds) fall closest to the West Canadian group. Figure 12: Discriminant Analysis histogram for all of the Martesamericana specimens, by sex. While this appears to distinguish males from females, without the unknowns’ sexes it is difficult to make a conclusion. The lack of a true ‘bell-curve’ shape for each sex could indicate the m1’s unsuitability in separating the sexes. Figure 13: Discriminant Analysis histogram for all of the Martespennantispecimens by sex. There appears to be separation of males and females, but with unknowns it is not absolutely certain. There are no real ‘bell curves’ for the sexes’ distributions, and gaps in between scattered unknown specimens. Figure 14: Discriminant Analysis histogram for all Mephitis mephitis by sex. This appears to separate males and females into somewhat ‘bell-curve-like’ distributions, but the gaps among the unknowns and lack of data on the sexes of all specimens leave room for doubt. Figure 16: Discriminant Analysis plot for all of the M. pennanti specimens, by geographic region. Here three regions appear to separate out quite clearly, but the East Canada and East US specimens roughly form a vertical ellipse, consistent with a closer relationship between the two regions. The overall result suggests regional differences in the lower carnassials of M. pennanti. Figure 17: Discriminant Analysis plot for all of the M. mephitis specimens, by geographic region. The regions separate out well, suggesting a real distinction in the lower carnassials of animals from different parts of North America. Interestingly, the West US group is alone above the y-axis origin, centered roughly at the x-axis origin, with the others below that line. East US and West Canada sit on opposite sides of the x-axis origin, with West Canada closer to the West US, and the Mexican specimens are on the far right of the x-axis. Lacking specimens from other regions, and an unequal number of specimens from each region, limits the usefulness of this plot. Guy Wilson Cave (GWC) fossils are stored at the East Tennessee State University and General Shale Brick Natural History Museum and Visitor Center. Most of the reference specimens used for comparison with the GWC fossils came from from the Smithsonian Institute’s National Museum of Natural History (NMNH). To provide equal numbers of fishers and martens for the analysis, three fisher specimens from East Tennessee State University’s own vertebrate collection were also used. Taxa included American martens, Martesamericana(N=33); fishers, M. pennanti(N=33); striped skunk, Mephitis mephitis(N=32); hooded skunk, M. macroura(N=33); hog-nosed skunk, Conepatusleuconotus (N=26); and eastern spotted skunk Spilogaleputorius(N=35). Only fully intact lower carnassials that showed a minimum of visible physical wear or damage were selected. Digital images of the GWC fossils (Figure 6) and the Smithsonian’s specimens were taken using an Olympus SP-600UZ digital camera that was set for maximum image quality. During each photo session the camera was mounted on a stand on which both the amount and angle of lighting, as well as the height and angle of the camera, were adjustable. To get proper focus of each specimen the camera’s macro mode was used. Each photograph was of a lower first molar in dorsal view with a six-inch dual metric-and-English ruler (metric side facing the tooth) and the specimen’s data label each placed in very close proximity. Several steps were taken to maintain consistency across the images: the camera was kept at the same height during each photo session, each specimen was photographed against a neutral gray background, and a folded paper wedge was secured beneath the background where each specimen was placed to aim the tooth’s occlusal surface towards the camera. Shortly after being taken, the images were transferred from the camera’s data card to an Apple MacBook Pro. The Macintosh computer program Preview was used to crop each photograph in order to display the carnassial in a close-up at the center of the image. Landmarks were recorded on the m1 molar in each digital photograph using tpsDig(Rohlf, 2004a). A total of 23 landmarks were used for all analyses (Figures 7 and 8). Exact locations of the landmarks differed slightly between the two groups, but in each case the landmarks were divided almost equally between the perimeter of the tooth crown and the occlusal surface. Landmarks were laid out in a predetermined order, first along the crown perimeter from the anterior to the posterior and then at specific points across the occlusal surface. Then tpsUtil(Rohlf, 2004b) was used to combine the digitized landmark files of each study into a single master file, and tpsSuperwas used to superimpose the individual landmark data within the master study file. This was essential to line up the landmarks in each image to a common reference grid. Finally, tpsSuper (Rohlf, 2009) rescaled the image data using a “Procrustes fitting” function to minimize any variations due to individual size differences among the samples. Each data file was reformatted into a text file using Microsoft Word 2011, then imported into Microsoft Excel 2011 to be saved as a native Excel file for import and opening into PASW (also called SPSS). Statistical analyses consisting of Principal Component Analysis, Discriminant Analysis and Step-wise Discriminant Analysis were performed both in the fossil identifications and in the search for patterns based on specimen sex and geographic origin. This project began with the goal of identifying three fossil jaw fragments from Guy Wilson Cave using landmark analysis of the lower carnassial. There has been a rather surprising success in that effort, confirming the initial assignments of species given to the GWC fossils. However, additional and improved trials with other species are needed to confirm the usefulness of the lower carnassial as a tool for identifying carnivorans. It may also be useful to compare combinations of teeth against single ones to assess accuracy in identifications. Ongoing work on the noble marten and its status as a species or subspecies might benefit from applying the techniques developed here, particularly since much of the comparison to date has included linear measurements of marten and fisher lower carnassials (Anderson, 1970; Hughes, 2009; Lyman, 2010). Dental characteristics have also been used to investigate the potential species status of the sea mink (Sealfon, 2007); a follow-up of that research with the techniques used here could be very interesting and informative. Given recent finds of late Miocene and early Pliocene skunk fossils in North America having at least one m1 for comparison (Wang et al, 2005; Wang and Carranza-Castañeda, 2008), this procedure might prove useful in learning more about the evolutionary history of and modern relationships among skunks. It could even prove useful in studies of the mephitidae in South America, a topic in need of further study (Wang and Carranza-Castañeda, 2008). The secondary goal involved sifting through the reference data set for patterns in the variance relating to sex and geography. From this study the case for separation by sex is a little uncertain because of the missing data for many of the specimens used. Nevertheless, based on the results the use of the lower carnassial to identify musteloids by sex deserves a second look with a larger and better sample collection. There is the additional question of why males and females would evolve differences in post-canine dentition, which is still being studied (Guilianoet al, 1989; Loy et al, 2004; Moors, 1980; Powell and Leonard, 1983; Thom et al, 2004; Zalewski, 2007). Evidence for geographic variation was also demonstrated here and revealed several interesting patterns of regional variation that need explanation. Are the differences the result of natural barriers to interbreeding, or regional variations in diet? An established body of work on the history and distribution of musteloids in North America already exists (e.g., Anderson, 1970; Graham and Graham, 1994). If confirmed, the patterns found in this thesis might lead to deeper understanding of these animals’ paleogeography. Current research on non-analog faunas, as well as ecological studies, could benefit from applying our procedures to a wider range of fossil and recent specimens. With all of these conclusions comes a set of reservations. There is clear evidence for noise in the data. This may be both from the sample sizes being very close to acceptable minimums for statistical validity, from human error at any number of places in the execution of the study procedures, and even from the uneven numbers of represented locations and sexes in the sample populations. Particularly in the geographical inquiry, there needs to be both more specimens in total and a more equal number from each region. Improving upon the photographic techniques used here could provide greater image consistency and quality, and provide a more efficient methodology for future use. The geographic regions that were defined for the regional part of the study were as follows: ‘West Canada’ consisted of Manitoba, Saskatchewan, Alberta, British Columbia, Nunavut, Northwest Territories, and Yukon Territory; ‘East Canada’ was Ontario, Quebec, New Brunswick, Newfoundland and Labrador; ‘West US’ was composed of all of the lower 48 states west of the Mississippi River, minus Minnesota because the Mississippi’s point of origin is in that state; ‘East US’ was Minnesota plus all of the lower 48 east of the Mississippi River. Alaska and Mexico were treated as separate from both Canada and the lower 48 US states (Figure 18). The initial assumption was that there would be much more distinction between eastern specimens than western ones because of the major barrier formed by the Great Lakes and the St. Lawrence River. Mountains were not expected to be as important, at least for martens and fishers since they are known to inhabit higher elevations at lower latitudes (e.g., Anderson, 1970; Graham and Graham, 1994). In each of the geographical DA plots there is a clear separation into north-to-south and east-to-west groups(Figures 15, 16 and 17). However, the exact patterns are different among the three species. The overall patterns for all three species of musteloid imply some force driving regionally distinct variations in the lower carnassial. Multiple barriers to gene flow across the North American continent could be an explanation, alone or in combination with regionally unique diets. Assuming for instance that Martes filled in recovering ecosystems from west to east after the last major glaciation, it would be reasonable to find more variance in the western populations. But if the formation of the Great Lakes/St. Lawrence River system was any sort of obstacle to travel by small to medium sized animals, then one would expect to find the eastern specimens to be significantly different from each other. Genetic barriers do not have to physical; simply having individuals maintain a limited range size could restrict breeding across larger areas. The common preference of both Martesspp. for boreal habitat (Anderson, 1970; Graham and Graham, 1994) does not appear to preclude differences between populations. On the other hand, the striped skunk has a much wider range than either martens or fishers (Figures 19, 20 and 21); this can and even should encourage distinctive regional characteristics. Figure 3: Adult marten (Martesamericana) in the wild. Photo source is http://upload.wikimedia.org/wikipedia/commons/f/ff/Martes_martes_crop.jpg on 10/13/2011. Figure 4: Adult fisher (Martespennanti) in the wild. Photo source is http://upload.wikimedia. org/wikipedia/en/f/f7/Fisher_005.jpg on 10/13/2011. Figure 5: Adult striped skunk (Mephitis mephitis) in the wild. Photo source is http://upload.wikimedia.org/wikipedia/commons/0/0c/Striped_Skunk_%28Mephitis_mephitis%29_DSC_0030.jpg on 10/13/2011. Figure 1: Left lower carnassial for M. pennanti specimen ETMNH 497 in occlusal view. The three anterior cusps are collectively the trigonid, the bowl-shaped feature behind the trigonid is the talonid. Results The original identifications for the GWC fossils appear to be confirmed by the landmark analyses (Figures 9, 10 and 11). Given how very physically similar the carnassial is between the marten and fisher on one hand, and between all of the North American skunks on the other, this is especially encouraging. But while these three results seem very clear, there needs to be a series of follow-ups with larger sample sizes and with additional species from within the Carnivora. Multiple different sets of landmarks can and should be devised and tested, as well. Even comparing combinations of post-canine teeth to single teeth could be done, assessing the relative accuracy and precision of each arrangement. Perhaps then the lower carnassial can be considered a useful tool in identifying fossil and recent carnivoran specimens, with potential applications for research in paleontology and archeology The search for patterns based on sex and geographical was done individually for martens, fishers and striped skunks, taking advantage of the wider geographic ranges of these taxa (Figures). The Guy Wilson Cave fossils were included with their respective species. About a third each of the Smithsonian’s martens and fishers and roughly a fifth of their striped skunk specimens lacked any record as to their sex. Musteloids are well known for their size-based sexual dimorphism; depending upon the species, males can be as little as ten percent larger than or as much as two times the size of females (Anderson, 1970; Thom et al, 2004; Wolsanet al, 1985; Zakrzewski, 1967). Sex-based differences were initially examined in this study to determine if they might be a significant source of variance. If so, it could potentially hinder understanding any observed geographic patterns in the data. On the other hand, the presence of such variances could be interesting in and of themselves. Some authors have explained musteloid sexual dimorphism as a way to reduce male-vs-female competition for prey (Guilianoet al, 1989; Loy et al, 2004; Moors, 1980; Thom et al, 2004; Zalewski, 2007), while others cited the reduced energy requirements of smaller females (Moors, 1980; Powell and Leonard, 1983). There seems to be some degree of sex-based variance within martens, fishers and striped skunks, but the unknowns for each taxon are spread out across the x-axis without even clustering together into a third bell curve, let alone grouping around either of the known sexes (Figures 12, 13 and 14). Repeated checks of both the data and the procedure revealed no obvious errors, so it is uncertain what is causing this result. The lack of data on the sex of all of the specimens for each taxon makes it more difficult to be certain about any observed patterns. But the apparently good separation of the known samples does suggest some process might be generating variation between male and females. Since carnassial teeth process food (Meiriet al, 2005), the process could simply be dietary. Larger males might be accessing larger (and perhaps tougher) prey items; females could be going after a greater amount of smaller and more abundant food items like smaller rodents, insects, fruits and seeds. Analysis of wear patterns on the carnassials of males and females could help answer this question, not only by demonstrating that there is a sex-based difference but also by linking wear patterns to likely food sources. Figure 18: Map of North America displaying the regions defined for the geographical pattern search. Alaska and Mexico were treated as separate ‘countries’ in this study. East and West US were defined by bordering the Mississippi River, except for Minnesota (where the Mississippi begins). East and West Canada were defined by bordering the Great Lakes and St. Lawrence River system. Adapted from http://www.zonu.com/fullsize-en/2009-11-08-10949/North-America-Political-Outline-Map.html, accessed on 10/28/2011. Figure 19: Current range for American marten (Martesamericana), covering boreal regions throughout North America. Adapted from http://upload.wikimedia.org/wikipedia/commons/8/86/American_Marten_area.png; accessed 10/28/2011. Figure 20: Current range for the fisher (M. pennanti), covering boreal regions across parts of northern and western North America. Source is http://en.wikipedia.org/wiki/File:Fisher_area.png; accessed on 10/28/2011. Materials & Methods