The Pennsylvania State University. The Graduate School. School of Forest Resources KEY AND ATLAS TO THE HAIR OF TERRESTRIAL PENNSYLVANIA MAMMALS

Transcription

1 The Pennsylvania State University The Graduate School School of Forest Resources KEY AND ATLAS TO THE HAIR OF TERRESTRIAL PENNSYLVANIA MAMMALS A Thesis in Wildlife and Fisheries Science by Andrea Nickoloff 2013 Andrea Lee Nickoloff Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2013

2 The thesis of Andrea Nickoloff was reviewed and approved* by the following: Jay R. Stauffer Distinguished Professor of Ichthyology Thesis Advisor Michael G. Messina Head and Professor, Department of Ecosystem Science and Management Matthew D. Hurteau Assistant Professor of Forest Resources *Signatures are on file in the Graduate School

3 iii ABSTRACT Hair is considered one of the synapomorphies (shared derived characters) of extant mammals. Keys and atlases of mammal hairs can be useful for many purposes. Although rare, such keys and atlases can be used for studies of food habits of predators, species identification of material recovered in the illegal trade of wildlife parts and products, determining diet changes, taxonomic and phylogenetic studies, archaeology, research on the contamination of mercury and other metals in mammals, and behavioral studies. These keys and atlases can also be used as a noninvasive method for censusing. In Pennsylvania, there are approximately 70 extant or extinct species of mammals. I determined if: 1) the families of Pennsylvania mammals could be diagnosed based on hair; and 2) whether species within the families Sciuridae (squirrels) and Soricidae (shrews) could be diagnosed by their hair. For this investigation, I examined guard hairs from mammalian species within the Commonwealth of Pennsylvania to 1) determine the synapomorphies of guard hairs from each family; 2) create a taxonomic tree and table to the mammals of Pennsylvania and; 3) create a tool for identifying mammal species that can be utilized with non-lethal sampling approaches.

4 iv TABLE OF CONTENTS List of Figures... v List of Tables... vi Acknowledgements... vii Chapter 1: Introduction... 1 Mammal Hair... 1 Characteristics to Identify Hair... 3 How Hairs are Obtained... 4 Previous Studies... 4 Chapter 2: Purpose... 5 Objectives....6 Hypothesis Chapter 3: Procedure Gross Features of Dorsal Guard Hairs and Making Slides...30 Microscopic Features of Dorsal Guard Hairs I: Cuticular Scale Patterns Microscopic Features of Dorsal Guard Hairs II: Medullar Patterns...31 Part III: MALDI-TOF Mass Spectrometry...32 Results and Discussion...36 Conclusion Effectiveness of Each Method by Order...41 References...207

5 v LIST OF FIGURES Figure 1: Cuticular and Medullar Patterns...34 Figure 2: Dichotomous Key...45 Appendix A: Pennsylvania Mammal Species Cuticular and Medullar Slides with Accompanying Measurement and Counts Data...53 Appendix B: Data Spreadsheet Appendix C: Pennsylvania Mammal Species Mass Spectrometry of Guard Hair Protein Composition Appendix D: Supplies for MALDI-TOF Mass Spectrometry Appendix E: Glossary

6 vi LIST OF TABLES Table 1-1. Family Accounts of Pennsylvania Mammals...7 Table 2-1. List of Pennsylvania Mammals Table 3-1. Semi-Specific Peaks (SEMPs)

7 vii ACKNOWLEDGEMENTS I would, first of all, like to thank my original advisor, Dr. Jacqualine Grant, for designing such a unique and fascinating thesis project for me. I would also like to thank my current advisor, Dr. Jay Stauffer Jr. for taking me in as one of his students, being such a great mentor to me with his support and guidance, and sharing his interesting stories of his fish research and travels around the world. I would like to also thank my other committee members, Dr. Michael Messina and Dr. Matthew Hurteau, for their feedback, and for our weekly conversations during Coffee Hour. I would like to thank Dr. Bruce Stanley, of the Penn State College of Medicine in Hershey, who conducted the mass spectrometry experiments, Dr. Tatiana Laremore, Director of the Proteomics and Mass Spectrometry Core Facility of the Huck Institutes of the Life Sciences, who supplied the chemicals and other equipment, and Denny Coleman, an undergraduate student who coordinated this endeavor for me. I am also grateful for the suggestions and useful information provided to me by Dr Reena Roy, from the Department of Forensic Science. I am thankful for my undergraduate professors at Delaware Valley College, especially Drs. Benjamin Rusiloski and Christopher Tipping, and Mr. Reginald Hoyt, for their support, and for their patience in writing countless letters of recommendation for my graduate school applications. Without all of these people, my thesis would not be possible. I am fortunate to have had several helpful friends, both in the Stauffer Lab and other labs. These include Rich Taylor, who helped me find and purchase equipment, Haskell Sie (Department of Statistics), and Bill Hanson, who were always willing to help me with the challenges of statistics, and Keith Price (Hunter Carrick Lab), who helped with microscope issues. The labmate I am most thankful for is Casey Weathers, who was always ready to lend a hand and assisted me with many things, including formatting my thesis, and develop techniques

8 viii for hair measurement and photography with the microscope. I enjoyed spending time with them, learning about, sharing our enthusiasm for, and discussing our research, and any other topic we felt like talking about. Most of all, I am very thankful for my parents, Diane Nickoloff, and Dr. Edward Nickoloff, D.Sc., Professor of Radiology at the Columbia University College of Physicians and Surgeons, for being very supportive of me and my goals and ambitions. I especially want to thank my dad for teaching me to never give up, for inspiring and encouraging my interest in science and all things related to the outdoors, and for his unending love and guidance.

9 1 Chapter 1: Introduction Mammals (Class Mammalia) include 26 orders, and over 5000 species. Hair is one of the synapomorphies (shared characteristics) that are unique to mammals, and all mammals have hair at some point in their development (University of Michigan Museum of Zoology 2013). There are two layers to the skin of a mammal: the epidermis and the dermis (Teerink 1991). The dermis is the layer that has pain and touch receptors, and gives strength and flexibility to the skin (The Ohio State University Wexner Medical Center). The epidermis, or upper layer, contains both dead and living cells. Part of the epidermis is a basal layer, which has cells that constantly divide to maintain the epidermis. The dermis, or lower layer, has cells that form a connective-tissue sheath around the hair follicle, which has small blood capillaries that supply nutrients to the tissue. A wax gland is formed from outgrowths of the follicle. Some mammal species have an arrector muscle, enabling hairs to stand erect. Central cells of the hair follicle form the hair (Teerink 1991). The color of hair is derived from proteins known as melanins. Most hairs, other than human hairs, have bands of both eumelanin (which is dark), and pheomelanin (which is light) (University of Michigan Museum of Zoology 2013). Mammal Hair Hair is comprised of a protein known as keratin (Linacre 2009) Mammal hair is comprised of α-keratin, while bird feathers consist of β-keratin (Cryan et al. 2004). Other biological structures, such as fingernails, rhino horns, and antler velvet are also made of keratin. The strength of hair and other keratin structures comes from cysteine, an amino acid. Cysteine molecules can bond together in pairs, forming long chains (Linacre 2009). Some mammals are

10 2 hairless; however, this is rare. The only species of mammals that are effectively hairless are elephants, rhinoceroses, hippopotamuses, walruses, pigs, whales, and naked mole-rats. (Pagel and Bodmer 2003). A mammal s fur, or hair, coat is known as pelage (Linacre 2009). Although the terms coat of hair, fur, and pelage are used interchangeably, fur is relatively short and grows only to a certain length. (University of Michigan Museum of Zoology 2013). Most mammals have more than one type of hair, with varying functions. Vibrissae, or whiskers, have sensory and tactile functions; they are thick and stiff (Linacre 2009). Naked mole rats, and some semi-aquatic mammals, such as whales, walruses and hippopotamuses, have no hair except a small number of vibrissae around the mouth (National Geographic 2011). Since vibrissae do not vary much in structure among species, they are not very valuable in species identification. Pigs and wild boars have bristle hairs; these have a flagged or forked tip, a narrow or intruding medulla, or none at all. Species in the family Suidae have forked tips to their hairs, which is useful for identification. Overhairs are longer than the rest of the coat. They are stiff and straight, and have long tips. They do not have much value in species identification. There are many different types of hair, which vary in usefulness for species identification. Guard hairs are the most useful for species identification (Linacre 2009). It is implied that guard hairs are the most often used type of hair for species identification because they are thicker and more deeply pigmented (Hausman 1920). Guard hairs can occur in modified forms, such as spines (i.e., quills of porcupines, where the cuticular scales are modified into barbs), bristles (which are firm, long, and do not stop growing), and awns (which have a weak, narrow shaft, and expanded tip, and do stop growing) (University of Michigan Museum of Zoology 2013). For this investigation, guard hairs will be used to delineate families, genera and species. Most of the coat is composed of this type of hair. It is long and coarse, and can be primary (longer), or secondary (shorter) (Linacre 2009). Guard hairs protect the pelage from abrasion (University of Michigan

11 3 Museum of Zoology 2013). Underhairs are short and very fine. They are visible if the guard hairs are removed or pushed to the side. They do not vary much in thickness from one end to the other (Linacre 2009). The underhair layer consists of wool (which is always growing), fur (which is short, and stops growing once it reaches a certain length), and/or velli (down, of which lanugo, which is found on mammal embryos, is one type). Each mammal hair usually has three layers: the medulla (innermost core), cortex (around the medulla), and outermost layer with a scale pattern (cuticle or cuticula). The cuticula is mainly scales, which are transparent and overlap. Sometimes, they have pigment (i.e., certain bat species). There is much variation in shape, size, margin, and arrangement of scales along the hair shaft; this is helpful in characterizing and identifying species. Each region of a hair (proximal, medial, and distal) also has variation in scale patterns (Linacre 2009). Characteristics to Identify Hair Terrestrial mammal hairs are typically identified by medulla type and shape, cuticular scale patterns, shape of cross-sections, and pigmentation (Hall-Aspland and Rogers 2007). Shape of cross-section, medulla type, and cuticular scale pattern are also examined for identification of mammal hair (Lobert et al. 2001). Waldeyer and Grimm (1884), and Lambert and Balthazard (1910) delimited hair using characteristics of the medulla, and its ratio to the cortex, but did not examine cross-sections (Clement et al. 1980). Some studies have been based on cross-section characteristics only (Glaister, Stoves). Hausman (1920) considered mammalian hair to have four structural elements: the medulla, the cortex, the pigment granules, and the cuticle. Williams (1938) used three structural categories: the medulla, cortex, and cuticle (terms still used today). Sometimes, hairs from two different species are almost identical, and can only be distinguished by cross-sections (Dagnall et al. 1995). Size, shape and color can often be sufficient for identification (Hess et al. 1985). The measurements used by Rosen (1974) to identify primate

12 4 hair were diameter, cross-sectional area, hair index, cuticle scale count, cuticle scale width, and scale index. Hair pattern and width only were analyzed by Sessions et al. (2009). How Hairs are Obtained Hairs can be obtained from scats and hair tubes (Lobert et al. 2001, Mullins et al. 2010), hair tubes alone, without scats (Pauli et al. 2008), hair snares (Castro-Arellano et al. 2008) or museum animals (Seiler 2010, Sessions et al. 2009). Genetic data can be obtained from hairs found in hair tubes and other noninvasive sampling methods (Castro-Arellano et al. 2008). These non-invasive monitoring techniques are replacing other techniques, such as camera stations and track counts, since they require less effort to obtain more data and information (Pauli et al. 2008). Hair is particularly useful for genetically identifying mammals of species that are difficult to observe (Walker et al. 2006). Previous Studies Brewster (1837) and Quekett (1844) performed early studies on mammal hair. Classification of hair structure and hair terminology, as we know it today, may have begun with Hausman (1920). In 1938, Cecil Williams wrote about the identification of mole and shrew hairs, but used drawings, rather than photographs. Wildman (1954) examined the medulla, cuticula, and crosssections of hair, and added to the terminology for describing cuticular and medullar patterns. Dziurdzik (1973) published a key in Polish. Also in 1973, Tupinier studied the cuticle of 29 Chiroptera species from Western Europe with a scanning electron microscope (Teerink 1991). Other keys were published by Mathiak (1938), Mayer (1952), Moore et al. (1974), Stains (1958, 1962), and Williams (1938) (Tumlinson 1983). William V. Mayer published, in 1952, a key to the dorsal guard hairs of California mammals. Another state mammal key is Renn Tumlinson s

13 annotated key of dorsal guard hairs of some Arkansas mammals (game mammals and furbearers). Hess et al. (1985) used a scanning electron microscope to examine and characterize the hair morphology of two families, Suidae and Tayassuidae. A key to scale patterns of guard hairs of Pennsylvania mammals was constructed by Iudica et al. in Sahajpal et al. (2007) characterized the dorsal guard hair of four Indian bear species. An atlas of hair structures of South-African Mammals was constructed by Nicole Seiler (2010). In 2011 Sarkar et al. identified dorsal guard hairs of five species of Cercopithecidae (Primates). Before Hall-Aspland and Rogers key in 2007, there were only light microscopy images of hairs from two fur seal species, but no hair reference keys for pinnipeds. Chapter 2: Purpose Studies of mammal hair are useful in the fields of animal ecology, wildlife biology, gamekeeping, nature management, forensics (Teerink 1991) the fur industry, economic ornithology and mammalogy (Williams 1938) textile testing (Dagnall et al.) archaeology (Dagnall et al., Sessions et al 2009) determining the diet of predators, mammal surveys, and examining raptor and owl pellets (Lobert et al. 2001) taxonomic and phylogenetic studies (Seiler 2010, Debelica and Thies 2009) species identification of material recovered in the illegal trade of wildlife parts and products (Debelica and Thies 2009), including non-labeling and mislabeling in the fur trade (Hollemeyer et al. 2007) diet changes (including short-term changes) (Cerling et al. 2006, Cerling and Viehl 2004, Sponheimer et al. 2003) population dynamics, distribution, and dispersal (Pauli et al. 2008) density and genetic structure of carnivores (Buell and Crooks 2007) contamination of metals (Marcheselli et al. 2010, Malvandi et al. 2010, McLean et al. 2009, Strom 2008, Wenzel et al. 1993), a non-invasive method for censusing (Lobert et al. 2001,

14 6 Castro-Arellano et al. 2008, Mullins et al. 2010) and describing hair morphology in families (Hess et al. 1985). It has been questioned whether or not one can identify certain mammal species accurately by their hair, and it can be difficult if the species are closely related. Identification can be easier if there is skeletal material in addition to the hairs, and a sufficient number of guard hairs (Lobert et al. 2001). Cuticular patterns have been good for identification, but adults and juveniles have a different pattern. Keys can be less useful if it is unknown which part of the pelage the hairs are from (Seiler 2010). Objectives For this investigation, guard hairs from mammalian species within the Commonwealth of Pennsylvania were examined to 1) determine the synapomorphies of guard hairs from each family; 2) create a taxonomic tree and table to the mammals of Pennsylvania and; 3) create a tool for identifying mammal species that can be utilized with non-lethal sampling approaches

15 7 Table 1-1. Family Accounts of Pennsylvania Mammals Didelphidae (opossums): Found in most habitats, some are arboreal, and they are usually omnivorous or carnivorous. They are native to North and South America. Species in this family have a full set of teeth (five upper incisors, four lower incisors, one canine, three premolars, and four molars). Their weight ranges from as little as 10 g, up to 2 kg or more. They have five digits on their feet, with a partially opposable first toe on the hind foot. Claws occur on all digits except for the first toe on the hind foot, which has a nail. Most species in this family have a long, scaly, prehensile tail (University of Michigan Museum of Zoology 2013). Soricidae (shrews): This is the largest insectivore family (over 300 species). Most species are very small, with small eyes, a pointed snout, some are poisonous, and some are believed to use echolocation. Shrews are active during both the day and the night, because they have a high metabolic rate and eat frequently. Most are found in moist habitats, and a few are aquatic. The aquatic species have long, bristly hairs on their toes and feet. Shrew teeth are small and peglike, and can be used in identification. Their skulls are typically long and narrow, with a flat profile, and no zygomatic arches. The tympanic bone is unusual, as it is ring-shaped (University of Michigan Museum of Zoology 2013). Talpidae (moles and desmans): This family consists of about 42 species in North America, Europe, and Asia. Species in this family are either fossorial (about 2/3), aquatic, or forage on the surface. Fossorial moles have small eyes, no external ears, and short legs. The two desman species have a flexible snout and webbed feet. They have short, strong forelimbs with claws. Their fur is velvety and can lie in any direction, for easy movement while in burrows. Moles are found in many different types of habitats, but prefer moist soils. They are active at all times, with

16 8 high metabolic rates. Species in this family usually have a flattened skull, and a narrow rostrum. They have both auditory bullae and zygomatic arches. The humerus and clavicle are broad (University of Michigan Museum of Zoology 2013). Vespertilionidae (evening and vesper bats): This is the largest bat family, which has 318 species. These bats have small eyes, a tragus, and do not have a noseleaf. They have long tails which extend at least to the tail membrane. Their fur is usually black or brown, but can sometimes be orange. They range in size from about 4 g to up to 50 g. They usually live in caves, but are found in other habitats. These species are found throughout the world, except for the polar regions, and some remote islands. They can live in colonies, pairs, or be solitary. In temperate zones, they either migrate or hibernate, due to the shortage of insects in cold weather (University of Michigan Museum of Zoology 2013). Nearly all species are insectivorous, and use echolocation (Skulls Unlimited 2013). They can be identified by their skulls, which have an unfused premaxilla, short jaws, and lack of postorbital processes. They have small incisors, and the total number of teeth is (University of Michigan Museum of Zoology 2013). Leporidae (hares and rabbits): There are 50 species in this family, which are either native or introduced, and inhabit all continents except Antarctica (Skulls Unlimited 2013). The size of species in this family ranges from 300 g (pygmy rabbits) to 5 kg (European Hares). Females are larger than males, which is uncommon in mammals. The hind limbs, feet, and ears of leporids are long. Their color can be brown, black, or white. They are not striped, except for two species. Some species have white winter coats. Their tails are short and bushy, and have hair on the soles on their hind feet. Their hair can be thick and soft, or coarse and woolly, depending on the species, but is sparse on the ears. Usually, they are dark dorsally, and lighter ventrally. Their

17 9 skulls can be easily recognized, since they are arched, and are slightly constricted between the orbits. They have enlarged primary incisors, which are fully encased in enamel, and small, peglike secondary ones. They lack canines, and their incisors are separated by a diastema from the molars and premolars. The molars and premolars have two transverse ridges (University of Michigan Museum of Zoology 2013). Sciuridae (squirrels): Squirrels live on every continent, except Australia and Antarctica (National Geographic, 2012). There are three basic morphologies of squirrels: ground squirrels, tree squirrels, and flying squirrels. They have long bodies, soft fine hair (sometimes thick hair), and large eyes. They have four digits on their forefeet but five on their hind feet. They have claws, except on the thumb (it has a nail). They have many vibrissae all over the body. The size of species in this family ranges from about 10 g for African Pygmy Squirrels, to 8 kg for some marmots. Their fur can be a wide variety of colors, such as black, white, red, and brown. All squirrels have their skull structure in common. Their skulls are short, with a primitive jaw structure. The zygomatic plate is tilted, and the masseter muscle attaches to it. They have long jugals, and the postorbital processes are well-developed. They have four chisel-like incisor teeth, which do not stop growing, and the roots extend far back (University of Michigan Museum of Zoology 2013). Castoridae (beavers): There are only two species of beavers in North America, Europe and Asia. They can weigh over 30 kg, and are semiaquatic. They have long guard hairs and dense underfur. Their hind feet are webbed, and their tail is flattened. They are able to use muscles to close their ears and nostrils while swimming. Their skulls are strong and flat, and they have powerful jaws.

18 10 They have heavy incisors, and their cheekteeth have folds. It is unusual that they have their epiglottis above their soft palate (University of Michigan Museum of Zoology 2013). Muridae (Old world mice, rats, and relatives): The diagnostic characters of this family vary. The digits of their forefeet have claws, except the thumb, which has a nail. Their hind foot digits usually all have claws, except the first toe sometimes has a nail. There are about 1150 living species in 260 genera. They inhabit all continents except Antarctica, and numerous types of ecosystems. The foramen of the skull is typically shaped like a keyhole. They have a broad zygomatic plate, and no postorbital process. No species in this family has canines or premolars, but they have up to three molars. They all have two upper and two lower incisors. There is a diastema after each incisor (University of Michigan Museum of Zoology, 2013). Dipodidae (jumping mice): This is the same family that includes Old World desert rodents, such as jerboas. They are not closely related to New World mice. Their tails are about one-half times longer than the head and body length combined, and their hind feet are also very long. There are deep grooves in their upper incisors. Their sides are very colorful (orange or yellowish), and their backs are dark. A number of jumping mice species can jump 3 m in a single bound (Reid 2006). Erethizontidae (New World porcupines): There are four genera and twelve species in this family, which has a range from the Arctic coast, south to northern Argentina. They can weigh as much as 18 kg. Some species are arboreal, with long prehensile tails, but some are not as arboreal, and have shorter tails. All species have four digits on their forefeet, and most have a short hallux on their hind feet. All have hairs modified into barbed spines. They have rooted cheekteeth with wide folds. Their skulls have strong zygomatic arches. Both the infraorbital canal and auditory

19 11 bullae are large. Their second and third cervical vertebrae are fused (University of Michigan Museum of Zoology 2013). Canidae (dogs, foxes, coyotes, jackals, and wolves): This family consists of 34 species in 14 genera, which are found on every continent except Antarctica. They have deep chests and long muzzles, and are unusually omnivorous, compared to most other carnivores. They have long legs and feet, typically with five toes on the forefeet, and four on the hind feet. Their claws do not retract. Their skulls have an elongated facial region, and teeth. Their canine teeth are unspecialized, and the molars are for crushing (University of Michigan Museum of Zoology 2013). Ursidae (bears): There are eight species of bears in five genera, found on all continents except Australia and Antarctica. They vary in size from 25 kg to 800 kg. They have long, rough unicolor fur, which is usually brown, black, or white. Their ears and eyes are small, and tails are short. Males can weigh more than twice the size of females. They have claws that do not retract. Their feet have hairy soles, except species that climb trees. Bears have elongated skulls, with unspecialized incisors, flattened carnassials for crushing, and canines that are long, with a slight hook (University of Michigan Museum of Zoology 2013). Procyonidae (raccoons, coatis, and relatives): This family consists of 18 species in six genera, which can be found from southern Canada to northern Argentina. They weigh from less than 1 kg to over 20 kg. Every species in this family has a medium or long tail, and gray or brown fur, with markings on the face and rings on the tail. Their faces are usually short and broad, with short, erect ears that are round or pointy. They have five digits on each foot, with short, curved claws that are partially retractile in certain species. Their skulls typically have short rostrums.

20 12 They have unspecialized incisors, wide molars, and canines that are somewhat long and look ovate in cross-section (University of Michigan Museum of Zoology 2013). Mustelidae (badgers, otters, weasels, and relatives): This is the largest family of carnivores, which consists of 22 genera with 56 species. They are found worldwide, except Australia, Antarctica, and some islands. They can weigh as little as 25 g, or as much as 45 kg or more. They usually have slender bodies. They have five digits on their feet, with non-retractile claws. The ears and legs are both short. Their skulls are elongated, with a short rostrum. They can have from 28 up to 38 total teeth. They have long canines, and well-developed carnassials. Their upper molars are shaped like an hourglass. Their bite is powerful, and numerous species do not have rotary motion in their lower jaw (University of Michigan Museum of Zoology 2013). Felidae (cats): There are 18 genera in this family, with 36 species. They are grouped as either large cats or small cats. Small cats cannot roar, because the hyoid bone is hardened. They can weigh from 2 kg to 300 kg. They can be a wide variety of colors, and have stripes, spots, or rosettes for camouflage. In some species, kittens have spots, while adults do not. Many species have large, semi-rotating ears, and a sandpaper-like tongue. Their skulls have a short rostrum. They usually have 30 teeth. Their cheek teeth are small or absent, and may have a small upper premolar or no upper premolar. The carnassials are well-developed, and they have cheekteeth for shearing. Their canines are typically long and conical. Felids have five toes on their forefeet, but four on their hind feet. Their claws are retractile (University of Michigan Museum of Zoology 2013). Cervidae (deer): This family has 47 species in 23 genera. All species have antlers, except the Chinese Water Deer. Only males have antlers, except for caribou. They can weigh from 9 kg to

21 kg. Their torsos are compact, and legs are elongated. Their cheekteeth have low or medium crowns, and are specialized for browsing. They have no upper incisors, but do have a hard palate. Males of some species have long, fang-like upper canines, which are used to attract mates. Their skulls are unique in that they do not have a sagittal crest, and have a postorbital bar (University of Michigan Museum of Zoology 2013). Bovidae (cattle, antelopes, gazelles, goats, sheep, and relatives): This is the largest family in Artiodactyla, with at least 140 extant species. All are ruminants, with a four chambered stomach, and all have horns, usually in both sexes. The horns are unbranched and do not shed. Typically, there are two horns, with the only exception being the Indian Four-Horned Antelope. They are comprised of bone covered in a keratinized sheath. Bovids range in size from 3 kg to over 1000 kg. They lack upper incisors, and have small or absent upper canines. They have a dental pad in place of the incisors. The lower incisors are projected forward, and the lower canines are modified (University of Michigan Museum of Zoology 2013).

22 14 Table 2-1. List of Pennsylvania Mammals (Joseph Merritt, 1987, from American Society of Mammalogists website) *Corrections made to incorrect (outdated) scientific names *Other common names added MAMMALS OF PENNSYLVANIA ORDER/Family Common Name Species Name Status Distribution DIDELPHIMORPHIA: Largest order of marsupials in New World, one family, Didelphidae (Reid 2006). Didelphidae: one species in Virginia Opossum: Only marsupial in Didelphis virginiana Common State-wide this family, Virginia Opossum North America, much larger than House Rat, with longer fur, and fur on the base of the tail (Reid 2006). (Reid 2006). SORICOMORPHA: Small, long and narrow snouts, five digits on each foot (Reid 2006). Soricidae: North America s smallest mammals, mouselike, narrow, long Masked Shrew or Cinereous Shrew: Small, long tail, back is typically dark brown, sometimes has pale sides Sorex cinereus Common State-wide snouts, row of small teeth, five and a dark dorsal line, pale belly toes on each foot, molt in spring and fall, if from northern climates, all American shrews have teeth which is grayish brown and silvery, bicolored tail which has a black tuft: 5 upper unicuspids, first 4 are similar in size, 5 th is smaller (Reid 2006). with reddish tips, (Reid 2006). Rock Shrew or Long-Tailed Shrew : Small, but long tail, slate gray back, gray belly, lighter than back, long muzzle, pale tops of feet, tail is at least 90 percent of body length, longer and narrower teeth than other shrews, does not have white belly or Sorex dispar Uncommon Appalachian Uplands

23 15 fringed hind feet (Reid 2006). Maryland Shrew: Similar to Masked Shrew, but smaller, with shorter tail Sorex fontinalis Common Piedmont and Valley and Ridge *Sometimes considered to be the same species as the Masked Shrew(Reid 2006). Smoky Shrew: Summer coat has a grayish brown back and pale brown belly, winter coat has a pale gray belly and dark gray to blackish back, bicolored tail with tufted tip, white tops of feet, larger than Masked Shrew, smaller, with shorter tail than American Water Shrew, hind feet not fringed (Reid 2006). Sorex fumeus Common State-wide Pygmy Shrew: Very small, has a short tail, back ranges in color from smoky brown to copper brown, belly is grayish brown in summer, but whitish in winter, bicolor tufted tail, feet with pale tops, 3 unicuspids are visible from side view, usually the smallest shrew in its regions, slightly shorter tail than Masked Shrew, which has 4-5 unicuspids visible (Reid 2006). Sorex hoyi Uncommon Presumed Statewide Water Shrew or American Water Shrew: Dark, has a long tail, various colors, tuft on tip of tail, large feet, partially webbed toes on hind feet, which are fringed with white hair, larger, with a loner tail, than most shrews in its range, Marsh Shrew has fringes on feet and unicolor tail (Reid 2006). Sorex palustris Uncommon Mountain streams Northern Short-Tailed Shrew: North America s largest shrew, varies from silvery gray to charcoal gray, silvery belly, skin is bare around eyes, small ears, broad front feet, long claws, Blarina brevicauda Common State-wide short bicolored, tufted tail at tip, much larger than most other shrews in its range, shorter tail than American Water Shrew (Reid 2006).

24 16 Least Shrew: Small, has a very short tail, back varies from dull brown to nearly black, gray-brown to silvery belly, very small ears hidden by fur, bicolored tufted tail, 4 upper unicuspids, almost always smaller than Short-Tailed Shrews, which have 5 upper unicuspids, shorter tail than Long-Tailed Shrew, which has more visible ears (Reid 2006). Cryptotis parva Endangered Southcentral Talpidae: Large, small eyes, Hairy-Tailed Mole: Snout is long and no external ears, very broad narrow, blackish or charcoal grey, front feet which are turned belly is silvery gray, long, coarse hair outward, scoop-shaped claws, on tail, does not have tentacles on velvety hair, very flexible snout (Reid 2006). spine Parascalops breweri Uncommon State-wide (Reid 2006). Eastern Mole: Snout is narrow, graybrown, paler belly, tail is almost hairless, does not have a hairy tail or tentacles on snout (Reid 2006). Scalopus aquaticus Uncommon Piedmont and Valley and Ridge Star-Nosed Mole: Snout has 22 Condylura cristata Uncommon State-wide tentacle-like appendages, varies from chocolate brown to blackish, long hairy tail, front feet have black scaly skin (Reid 2006). CHIROPTERA: Only flying mammals, have a wing membrane Reid 2006). Vespertilionidae: All Northeastern and most American bats, plain-nosed, long tails enclosed by a membrane, sometimes a tail tip beyond the membrane, Keen's Myotis: Color ranges from yellowish brown to dark brown on back, blackish snout and ears, long ears, long pointy narrow tragus, 6-9 mm hair on midback with pale 2-3 mm tips, does not have a fringe on Myotis keenii Rare State-wide most use their wing tips or tail the tail membrane, almost membrane to capture insects indistinguishable from Long-Eared Myotis, which has larger, darker ears, (Reid 2006). and midback fur is 9-11 mm long with 4-5 mm pale tips (Reid 2006). Small-Footed Myotis: Smallest myotis species in the Eastern U.S., hair on back is pale yellow-golden Myotis leibii Threatened State-wide

25 17 brown, belly is cream, hairs have black roots, ears and mask are black, narrow pointy tragus, small feet, shorter forearm and hind foot than Little Brown Myotis, does not have pinkish face and brown fur like Indiana Bat, does not have pale face or ears, or pinkish forearms like Tri- Colored Bat (Reid 2006). Little Brown Myotis or Little Brown Bat: small, with glossy fur on back, yellowish brown back, buff yellow or gray-white belly, hairs have dark roots and pale tips, dark brown-black ears and snout, medium ears with straight narrow tragus, much smaller than big brown bat, without blunt curved tragus, hairs extend beyond claws, unlike in Indiana Bat, does not have woolly gray fur or a pinkish face (Reid 2006). Myotis lucifugus Common State-wide Indiana Bat or Indiana Myotis: Small, Myotis sodalis Endangered Central back is dull pinkish brown-graybrown, each hair has dark roots, is pale in the middle, and the tip is dark, buff belly, pale brown snout, has pinkish skin around eyes, brown snout, brown medium ears, narrow tragus, small feet with not much hair on toes (Reid 2006). Red Bat or Eastern Red Bat: Males are bright reddish orange, females are dull orange-brown, short round ears, shoulders and thumbs have white patches of hair, tail membrane is very hairy, has brighter hair than Western Red Bat, not dark brown like Seminole bat (Reid 2006). Lasiurus borealis Uncommon State-wide Hoary Bat: Large, each hair has 4 bands: dark roots middle is cream brown outer layer which is frosted with white, round ears, nose and edge of ears are black yellow hair around face base of thumbs and shoulders have white patches of fur, pinkish arms and fingers, black wing membranes, upper surface of tail membrane has lots of hair, larger Lasiurus cinereus Uncommon State-wide

26 18 than Silver-Haired Bat, which has unpatterned wings and two-tone fur (Reid 2006). Seminole Bat: Medium in size, back is rich mahogany brown, frosted with white, shoulders and base of thumbs have white patches of fur, upper tail membrane has lots of hair, female Eastern Red Bat has paler and redder fur (Reid 2006). Lasiurus seminolus Rare Piedmont Silver-Haired Bat: Blackish fur on back with silvery frosting, dark face, round black ears with a pale patch of fur, half of tail has fur on top, smaller than Hoary Bat, which has yellow fur around its face (Reid 2006). Lasionycteris noctivagans Uncommon State-wide Tri-Colored Bat: Small, brown or reddish back, pinkish brown face and ears, medium ears, dark brown wing membranes with pinkish forearms, tricolor fur which has dark roots, pale in the middle, and brown tips, myotis spp. in the east have darker fur on their ears and forearms, and narrow tragi (Reid 2006). * Former name: Eastern Pipistrelle Perimyotis subflavus Uncommon State-wide (Bat Conservation International 2013) Big Brown Bat: Large, back is glossy, Eptesicus fuscus Common State-wide yellowish-dark brown face and ear skin blackish, medium ears with round tips, broad, curved tragus, larger than Little Brown Myotis, which has a straight narrow tragus (Reid 2006). Evening Bat: Small, glossy hair yellow brown-dark brown, medium ears, short curved tragus, does not have a keeled calcar, short, stocky legs, similar to Big Brown Bat, but smaller (Reid 2006). Nycticeius humeralis Rare Southeastern LAGOMORPHA: Born with 3 pairs of upper incisors, eat vegetation

27 19 (Reid 2006). Leporidae: Long ears, long hind feet, short, cotton-like tail, rabbits have altricial young, while hares have precocial young (Reid 2006). Eastern Cottontail: Back is orange grizzled with black, sides are paler and grayer in color, has a deep orange nape, ears have whitish edges and black tips, cream eye rings, tail is grayish brown on top, rusty at base, narrow white edge, bottom is cottony white, usually has deep orange legs, large whitish or pale orange feet, ear length can distinguish this species from Desert, New England, and Mountain Cottontails, but ear length can vary with habitat and elevation (Reid 2006). Sylvilagus floridanus Common State-wide New England Cottontail: Has an Sylvilagus transitionalis Rare orange-brown back heavily grizzled with black, pale sides, white belly, and orange chest, short ears with black tips, cream fringe, orange nape, black spot between ears, orange eye rings, top of tail is brown, and bottom is white, orange-brown legs, pale orange or white feet, almost identical to Appalachian Cottontail, but differences in genetics and range, not as pale or gray as Eastern Cottontail, which has cream eye rings, and usually a white spot on its forehead (Reid 2006). Eastern Mts. Snowshoe Hare: Much smaller than other hares, much larger hind feet than cottontails, which have noticeable white tails (Reid 2006). Lepus americanus Rare Appalachian Uplands RODENTIA: World s largest order of mammals, usually small and ratlike, incisors grow constantly (Reid 2006). Sciuridae: Diurnal (except flying squirrels), common (Reid 2006). Eastern Chipmunk: Only chipmunk in Tamias striatus Common State-wide Eastern U.S., larger than Least Chipmunk, with a shorter tail, and less extensive stripes on its back and face (Reid 2006).

28 20 Woodchuck or Groundhog: Large, grizzled blackish brown back, rusty brown belly, head dark brown on top, whitish sides of muzzle, pale creambrown cheeks, blackish legs, tail is black with cream edges, color ranges from black to albino, depending on population, shoulders are not as pale as Hoary Marmot, and does not have black-and-white markings on its head, Yellow-Bellied Marmots have yellowish-orange underparts, white spots between their eyes, and a reddish tail (Reid 2006). Marmota monax Common State-wide Gray Squirrel or Eastern Gray Squirrel: Gray with yellow-brown on upper back and head, white or pale orange eye rings, gray or rusty brown ears, white belly, gray or rust-colored legs and feet, tail has a yelloworange center, mixed with black, and white edges, shorter tail than Eastern Fox Squirrel, does not have orange or cream on edges, smaller than Fox Squirrels, has rust on back and tail, unlike Western Gray Squirrel (Reid 2006). Sciurus carolinensis Common State-wide Fox Squirrel or Eastern Fox Squirrel: Largest and most widespread Eastern squirrel, grizzled yellowish brown, has pale orange-rusty brown belly, cheeks, eye ring and feet, tail has orange-brown edges, frosted whitish tail, larger than Gray Squirrel (Reid 2006). Sciurus niger Uncommon Southern & Western Red Squirrel or Pine Squirrel: Small, Tamiasciurus short tail, deep orange upper back, hudsonicus brownish sides, white belly and eye rings, tufted ears in winter, orange feet, tail is orange on top, sides and bottom are grizzled black and yellow, shorter, with a shorter tail, than most other tree squirrels, no orange eyerings or belly, no white edges on tail, unlike Douglas Squirrel (Reid 2006). Common State-wide Northern Flying Squirrel: Smaller and Glaucomys sabrinus Rare Northern fluffier than southern Flying Squirrel, usually has a gray face and a brown

29 21 body, and tail is darker at the tip (Reid 2006). Southern Flying Squirrel: Larger, hair Glaucomys volans Common State-wide is short and smooth, fur contrasts sharply from its back to its belly (Reid 2006). Castoridae: Largest rodents in Beaver or American Beaver: Largest Castor canadensis Reintroduced State-wide N. America, have castor glands rodent in U.S., world s second largest after Capybara, flat tail which is scaly and paddle-shaped, easily (Reid 2006). recognizable on land, while swimming, only the head and upper back are exposed (Reid 2006). Muridae: Molars have patterns on biting surfaces, usually nocturnal (Reid 2006). Marsh Rice Rat: Longer tail and lighter feet than Hispid Cotton Rat, smaller than House Rat, which has a unicolor tail and dusky tops of feet (Reid 2006). Oryzomys palustris Extinct Coastal Plain White-Footed Mouse: Hard to distinguish from woodland form of American Deer Mouse, has a white chin patch extending more than 1 cm from mouth, lightly haired tail with short hairs at the tip, two-tone hair on back, which is smooth and slightly shiny, fur and belly and sides is gray at base, and the other ¾ is white (Reid 2006). Peromyscus leucopus Common State-wide Deer Mouse or American Deer Mouse: Has a white chin patch extending less than 1 cm from mouth, well-haired tail with tuft at the tip, uniform hair on back, which is smooth and slightly shiny, fur and belly and sides is gray at base to ½ of total length and the other 1/2 is white (Reid 2006). Peromyscus maniculatus Common State-wide Allegheny Woodrat: Similar to Eastern Woodrat (N. floridana), except for skull and biochemical differences (Reid 2006). Neotoma magister Threatened Mt. Ridges Norway Rat or Brown Rat: Similar to Black Rat, tail is less hairy than woodrats, larger than rice rats, which have long, narrow feet and longer Rattus norvegicus Introduced State-wide

30 22 tails (Reid 2006). House Mouse: Brown naked-looking tail, linger tail than Northern Pygmy Mouse, Deer Mouse has white belly, Harvest Mice have bicolor tails, white or buff bellies, and grooved upper incisors (Reid 2006). Mus musculus Introduced State-wide Southern Red-Backed Vole: Upper back and crown are dark reddish brown or chestnut, or occasionally gray-brown or yellow-brown, bicolor almost naked tail, Northern Red- Backed Vole has tail with thick hair and bright orange back (Reid 2006). Myodes gapperi Common State-wide Rock Vole: Orange snout (distinctive in adult) and eye-rings, larger eyes and ears than Woodland Vole (Reid 2006). Microtus chrotorrhinus Rare Northeast Meadow Vole: Dark brown back, grayish white belly, long tail, similar to Montane Vole, but longer tail, and no white tops of hind feet, has longer tail and shorter whiskers than Heather Vole, and tops of hind feet not white (Reid 2006). Microtus pennsylvanicus Common State-wide Woodland Vole or Pine Vole: Reddish brown back, short, velvety fur, very short, bicolor, almost naked tail, shorter tail than other voles, which do not have velvety fur (Reid 2006). Microtus pinetorum Common State-wide Muskrat or Common Muskrat: Semiaquatic, with tail that is scaly and almost naked, compressed, smaller than Beaver and Nutria, does not have a paddle-shaped tail like the Beaver, tail is not rounded, and fur on nose and mouth is not white like the Nutria (Reid 2006). Ondatra zibethicus Common State-wide Southern Bog Lemming: Large head, Synaptomys cooperi Uncommon State-wide hair on back is dark brown grizzled with buff, very short tail, grooved upper incisors, voles usually have longer tails, Northern Bog Lemming s lower incisors are narrower, has

31 23 orange hair in front of its ears (Reid 2006). Dipodidae: Long and narrow Meadow Jumping Mouse: Long tail Zapus hudsonius Common State-wide tails, long hind feet, orange or that is very narrow, Western Jumping yellow sides, dark backs, deep grooves in upper Mouse is yellow on its sides, and more grizzled (Reid 2006). incisors (Reid 2006). Woodland Jumping Mouse: Back and top of head are dark brown, sides and cheeks are yellowish orange or reddish brown, long, narrow bicolor tail with white tip, Meadow Jumping Mouse does not have a white tail tip (Reid 2006). Napaeozapus insignis Common Northern & Western Erethizontidae: Long, sharp spines, quills have barbs on tips (Reid 2006). Porcupine or North American Porcupine: Smaller in east, quills are yellowish on head, rump, and top of tail, blackish guard hairs, short thick tail, no hair on soles of feet, only mammal with quills in its area (Reid 2006). Erethizon dorsatum Uncommon Northern & Central CARNIVORA: Catch and kill prey, generalist diet, typically terrestrial or arboreal (Reid 2006). Canidae: Long, narrow muzzle, triangular ears, flat backs, bushy tails, 5 toes on front feet and 4 on hind feet, claws do not retract (Reid 2006). Coyote: Legs long, ears narrow, large muzzle, gray fur, rusty colored muzzle and legs, bushy tail with black tip, much smaller than the Gray Wolf, which has larger feet, shorter ears, and a nose pad at least 1 in wide (Reid 2006). Canis latrans Common State-wide Gray Wolf or Timber Wolf: Large, long legs, big feet, small ears, long muzzle, varies in color, larger than coyote, which has longer ears and a more tapered muzzle (Reid 2006). Canis lupus Extinct

32 24 Red Fox: Medium-sized, long legs, long, bushy tail, usually orange-red, black stockings, black ears, white Vulpes vulpes Common State-wide belly and tail tip, only fox species with a white tail tip (Reid 2006). Gray Fox or Common Gray Fox: Urocyon Medium in size with short legs, cinereoargenteus grizzled gray back, rusty orange on ears, legs, and neck, white throat and belly, tail is bushy, black on top and at tip, shorter legs than Red Fox, which has black stockings and a white tail tip, Kit and Swift Foxes are smaller, have less grizzled fur, and a black-tipped tail (Reid 2006). Common State-wide Ursidae: Largest terrestrial Black Bear: Straight or convex snout, Ursus americanus Uncommon Appalachian carnivores, stocky with small buff brown-colored muzzle, black, Uplands tails, 5 toes on each foot, walk some have a white v on chest, on soles smaller than Brown Bear, which has a concave face, and a shoulder (Reid 2006). hump (Reid 2006). Procyonidae: 5 toes on each Raccoon or Northern Raccoon: Nose Procyon lotor Common State-wide foot, omnivorous, all except and mask are black, sides of muzzle Ringtail walk with soles flat on and hair above eyes are white, long ground grizzled gray hair, short tail with cream or orange and black bands, (Reid 2006). has a shorter tail than the Ringtail and White-Nosed Coati (Reid 2006) Mustelidae: Most are long and Marten or American Marten: Long Martes americana slender, have short legs, 5 body, usually chocolate brown, has a toes on each foot, anal scent bushy tail and white, cream, or glands, musky odor orange throat, smaller than Fisher, does not have a grizzled head or (Reid 2006). neck (Reid 2006). Extinct Fisher: Tail is bushy, yellowish brown Martes pennanti Reintroduced North Central or grayish yellow head, neck, and shoulders, body is dark brown with dark, long guard hairs, smaller than the Wolverine, which has yellowish bands from its shoulder to its rump (Reid 2006). Ermine or Short-Tailed Weasel: Tail is shorter than half of head and body length, has a whitish, cream, or Mustela erminea Uncommon Absent from Southwest

33 25 yellow belly, tail has black tip, slightly smaller than the Long-Tailed Weasel, which has a tail longer than half of head and body length (Reid 2006). Long-Tailed Weasel: Largest species Mustela frenata Common State-wide of weasel, tail length is more than half of head and body length, in summer, usually has brown fur on top and white or orange on bottom, smaller than Mink, which has a dark belly (Reid 2006). Least Weasel: Smallest carnivore, long body, but very short tail, tail is tipped with a few black hairs, smaller than other weasels, which have much longer tails with black tips (Reid 2006). Mustela nivalis Rare Western 2/3's Mink or American Mink: Much larger than weasels, which have white or orange bellies, does not have a pale throat or long, bushy tail, like the Marten (Reid 2006). Neovison vison Common State-wide Wolverine or Glutton: Dark brown body, which has a broad yellowish band on side, extending from the shoulder to the base of tail, larger than the Fisher, which does not have the band on side (Reid 2006). Gulo gulo Extinct Badger or American Badger: Legs Taxidea taxus are very short, top of head has white stripe, black sides of muzzle, black spot in front of ears, only mammal with a broad, low shape, unique head pattern (Reid 2006). Extinct Eastern Spotted Skunk: Small, has white triangle-shaped fur between eyes, white narrow stripe behind eyes (Reid 2006). Spilogale putorius Rare Southcentral Striped Skunk: Has two broad white stripes, which meet at the shoulders and crown of the head, and extend to the sides of the rump (Reid 2006). Mephitis mephitis Common State-wide River Otter: Has webbed feet, semiaquatic, much larger than the Lontra canadensis Rare Northern

34 26 Mink and Muskrat, which has a thinner, naked tail (Reid 2006). Felidae: Short faces, small ears, walk on toes, retractile claws (Reid 2006). Mountain Lion, Cougar, or Puma: Puma concolor Has a small head and long tail, sandy brown or reddish brown fur on top, whitish fur on bottom, larger than the Jaguarundi, with longer legs (Reid 2006). Extinct Lynx or Canada Lynx: Has very long legs, and large feet, has a blacktipped bobbed tail, grayish mottled fur on back, ears have long, black tufts, larger legs, feet, and ear tufts than Bobcat, which has white on bottom of tail tip (Reid 2006). Lynx lynx Extinct Bobcat: Has a bobbed tail, which is Lynx rufus Rare Appalachian black on top and white on the bottom, Uplands slightly smaller than Lynx, which has a black-tipped tail (Reid 2006). ARTIODACTYLA: hoofed, 2 functional toes, herbivorous, walk on tiptoes (Reid 2006). Cervidae: Males have antlers, Elk or Wapiti: Male s antlers have young are spotted, ruminants, one main beam, usually with 6 no upper incisors points, has a rump patch and short tail, which are buff, Moose have (Reid 2006). flattened antlers and no rump patch, larger, and less uniformly colored than Mule Deer (Reid 2006). Cervus elaphus Reintroduced North Central White-Tailed Deer or Whitetail Deer: Has a relatively long tail which is white on bottom, males have antlers with one main beam, and several vertical points, has longer tail and shorter ears than Mule Deer, and no black-tipped tail, male has more than one main branch to the antlers (Reid 2006). Odocoileus virginianus Common State-wide Moose: Has a Roman nose and long dewlap on chin, male s antlers are flattened, very large, and widely spread, easily recognizable, much Alces alces Extinct

35 27 taller than other deer (Reid 2006). Bovidae: True horns which do Bison or American Bison: Large, Bison bison not shed and are unbranched, head is massive, shoulder hump, all males and most females short horns are curved, dark brown have horns or blackish, hair on head and shoulders is woolly, larger than (Reid 2006). domestic cattle, which are not woolly, and usually not dark brown (Reid 2006). Extinct

36 28 Table 3-1. Semi-Specific Peaks (SEMPs) *Semi specific peaks are mass spectrometry peaks that are found in less than 80% of mammal species tested *For this table, the one to four largest peaks for each species were chosen *Species with noticeable similarities or differences in peaks were chosen *MALDI-TOF mass spectrometry peaks (one-four largest peaks) for selected species of six different orders (From top to bottom: Lagomorpha, Artiodactyla, Carnivora, Rodentia, Insectivora, Chiroptera) Eastern Cottontail Snowshoe Hare White-Tailed Deer Moose Red Fox Gray Fox Domestic Dog Raccoon House Mouse Norway Rat White-Footed Mouse Deer Mouse Allegheny Woodrat Masked Shrew Smoky Shrew Maryland Shrew Northern Short-Tailed Shrew

37 29 Hairy-Tailed Mole Star-Nosed Mole Red Bat Tri-Colored Bat Big Brown Bat

38 30 Hypothesis My goal was to determine if Pennsylvania mammals can be identified using an atlas and dichotomous key based on characteristics of hair. I also investigated whether the species of Sciuridae and Soricidae can be diagnosed based on hair. In previous studies, Soricidae (shrew) hairs have been identified by using keys, reference specimens, or photographs (Pocock and Jennings 2006) Chapter 3: Procedure My key and atlas are modeled after the Atlas and Key to the Hair of Terrestrial Texas Mammals published by Debelica and Thies (2009) of Texas Tech University. The hairs were taken from specimens in the Penn State Department of Ecosystem Science and Management Bird and Mammal Collection. Each specimen has a Forest Resources (FR) number, beginning with the number 1. I used the American Society of Mammalogists list of species of Pennsylvania mammals to determine from which specimens to collect. I collected at least ten guard hairs from three specimens of each species. Gross Features of Dorsal Guard Hairs and Making Slides To collect the hairs, I used tweezers, which I placed close to the skin, and then pulled. The scale pattern can change along the length of the hair, so the entire hair is necessary (Alaska Fur ID Project 2013). Before making the slides, I measured two to three hairs of each specimen of each species with a small tape measure, as it is difficult to make a length measurement of greater than 10 µm under the microscope, since the graticule is only 10 µm long. To prepare all slides, I used a hot plate, a Pyrex glass measuring cup, and Knox gelatin. Then, about three drops of blue dye were added to improve visibility under the microscope. The measuring cup was placed in a water bath on a Corning PC-400D hot plate, which was heated to approximately 100 degrees C. When left overnight, it was heated to about degrees C the next morning, to dissolve the gelatin. Sometimes, a small amount of water was added to the gelatin,

39 31 because the gelatin may be too solid or sticky. To add the gelatin to the slides, I held the slides vertically, and used a glass dropper about one-fifth of the way from the top, leaving that top 20% to write the species name and FR number. The gelatin would be a thin film, and I wiped the excess of the bottom of the slide on the edge of the measuring cup. Microscopic Features of Dorsal Guard Hairs I: Cuticular Scale Patterns First, I prepared a scale cast of the cuticula of each hair on a slide coated with the gelatin solution. I used two to three hairs from each specimen. The hairs were placed on the slide with the tip (apical) end attached, to make the hairs easier to remove. The slides were left for the gelatin to dry for about 5-10 minutes before the hairs were removed. To have an impression of all parts of the cuticula, the hairs were reattached by the root end either above, or next to, where the hair was originally placed. My data were collected from all two or three hairs on each slide, if the scale casts came out well, beginning with the first hair (the one closest to the species name and FR number). If not, I would use the second and/or third hair. Microscopic Features of Dorsal Guard Hairs II: Medullar Patterns The second part was to examine medullar patterns. This was done by using a glass dropper to add xylene on top of the hairs, under a chemical hood, and not using gelatin. I used one hair (or more, if they were from a species with smaller hairs) from one specimen of each species, with the only exception being species with different summer and winter pelage, and albino specimens; in these cases, a second hair and slide were used. This does not seem to have made a difference in terms of medulla type. Larger hairs were cut in half with a razor blade to enhance absorption of the xylene. Unlike the cuticular slides, the hairs were not removed. After the xylene dried, I added Duco cement to each end of each hair, to prevent the hairs from falling off the slide. For species with darkly pigmented fur, I placed the hairs in hydrogen peroxide in a glass Pyrex dish, to improve visualization of the medulla. (Sahajpal and Goyal, in Forensic Science in Wildlife Investigations, by Adrian Linacre). The hydrogen peroxide appears to have worked very little, or not at all.

40 Part III: MALDI-TOF Mass Spectrometry 32 The third part was using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, performed at the Penn State College of Medicine in Hershey. This is also known as the SIAM method (Species Identification of Animals with MALDI-TOF mass spectrometry), and works by pretreating hairs by cooking them in a chemically reducing solvent. A trypsin solution cuts the keratin chains into peptides. These peptides are of different lengths and molecular weights. Mass spectrometry arranges them according to their molecular weights, which forms a peptide mass fingerprint (pmf) that is specific to each species. Species can be identified, and relationships determined, by this spectrum pattern (Hollemeyer and Heinzle 2012). This is an easy method to learn and use, with both dyed and nonprocessed hair, and can distinguish closely-related species with less difficulty than with using a microscope. The SIAM method of hair identification is currently accepted in the European Union for commercial hair analysis, and by German customs. In 2007, in the United States, this method exposed a scandal in which real furs, mainly from raccoon dogs were being sold as faux furs (Hollemeyer and Heinzle 2012). (The raccoon dog is a species of canid native to Eastern Asia, which has a raccoon-like fur pattern) (World Association of Zoos and Aquariums 2013) mg of hair of each species was used. A weigh boat was placed on an OHAUS Explorer scale. The weigh boat was zeroed. The hairs were weighed to the nearest 10 th of a milligram. The hairs were then transferred to.65ml centrifuge tubes with tweezers. After each transfer, the tweezers were cleaned with 95% EtOH, 2% bleach, and then rinsed in reverse osmosis (RO) water. The centrifuge tubes were labeled 1-53, correspondingly with each sample. All slides were labeled, and then viewed, mainly under 200x magnification with a Leica DMR light microscope. I photographed the microscopic images provided by each cast with a Nikon COOLPIX 5000 digital camera on the microscope, and kept track of the electronic files on a spreadsheet and in a notebook. Often, these studies are conducted using a scanning electron microscope (SEM), transmission electron microscope, or both (Hess et al. 1985, Clement et al. 1980, Sessions et al. 2009). A recent technique is to view the cuticle with a light microscope first, and then with a scanning electron

41 33 microscope (Clement et al. 1980). Light microscopy has been used in earlier studies, such as Hausman (1920) (Sessions et al. 2009), and also by Sahajpal et al. (2007). After I had prepared all of my slides, I compiled a table of results for each method. My data were recorded on two Excel spreadsheets. The columns listed the common name of the species, the FR number, Cuticula Type, Comments, Medulla Type, Total Length (cm), Salient Features, and two extra measurements for bat species: Scale Index (SI) and Width Index (WI). Total Length was not measured for the hairs of smaller species, because it is too difficult to separate one hair from the rest, and find it on the slide, so those spaces were left blank. Data on width (µm), number of scales across width (Scale Count), and photo number were also recorded for each the three parts along the length of each hair (tip, middle shaft, and root). One to three specimens of each species were used, and up to three hairs for each species. Measurements across the row of the spreadsheet correspond to each hair. The FR (Forest Resources) column has an asterisk (*) if the number of the specimen was missing, or if I did not record which specimen the hair came from. If a date is included on the spreadsheet, that specimen only had a date written on the tag, and no FR number. Sometimes, the scale pattern would change along the length of the hair, thus requiring a fourth photo, and sometimes a fourth width measurement. With my data, I constructed a quantitative key to the hair of Pennsylvania mammals.

42 Figure 1: Cuticular and Medullar Patterns 34 Different scientists have come up with different classification criteria for cuticular and medullar patterns. Cuticular scale patterns and medullar patterns are classified using terms from Debelica and Thies (2009), and terms from other scientists (ladder), as well as some of my own (symmetrical, asymmetrical). I also used some of Hausman s classifications (1924) as Discontinuous (Fig. C Simple, Fig. D Compound, Fig. E Fragmental) or Continuous (Fig. F Nodose, Fig. G Homogeneous). Drawings from Debelica and Thies (2009). a b c d e Figure A. Imbricate Cuticula Types: (a) Ovate, (b) Acuminate, (c) Elongate, (d) Crenate, and (e) Flattened. Figure B. Coronal Cuticula Types a b c Figure C. Simple Medulla Types: (a) Ovate, (b) Elongate, and (c) Ladder

43 35 a b Figure D. Compound Medulla Types: (a) Ovate and (b) Flattened Figure E. Fragmental Medulla Types Figure F. Nodose Medulla Types Figure G. Homogeneous Medulla Types

44 Results and Discussion 36 The purpose of this research was to 1.) determine the synapomorphies of guard hairs from each family; 2.) create a taxonomic key and table to the mammals of Pennsylvania and; 3.) create a non-lethal approach to sample mammal species across their range. It seems that all of the three, methods work for these purposes, with varying effectiveness. Of the three methods used, I found the MALDI-TOF mass spectrometry to be the most reliable, followed by medullar patterns, and then cuticular patterns. The MALDI-TOF method gives the most accurate, consistent results. Unlike other methods, MALDI-TOF eliminates the issues with identifying scale patterns or medullar types, changes in scale patterns along the hair, and use of scale counts, all of which can be difficult to determine with light microscopy (Although it can be more easily accomplished with scanning electron microscopy). Also, there can be differences in hair appearance between juveniles and adults, and summer vs. winter pelage, and domestic vs. wild mammals. Some species had a medullar pattern that did not appear under the microscope or in the photographs, because it was too dark. It gives objective, numerical results, as opposed to using a subjective classification system (there is also more than one way to categorize cuticular and medullar patterns, by different scientists and authors). Appendix A contains the photos of the mammal hairs. In the photos, it is easy to see the cuticula and medulla, with a few exceptions. When the cuticula or medulla was not visible, this problem could sometimes be solved by looking at the page for that species in the key and atlas by Debelica and Thies (if the species occurs in Texas), or using a photo of a hair from an albino specimen, when possible. The average widths and lengths were predictable, for the most part, with some exceptions. (i.e., the Southern Flying Squirrel having a wider tip, middle shaft, and root than the Northern Flying Squirrel) but this could be explained by human error in measurement, labeling, or calculation, or due to seasonal pelage length (i.e., shorter in summer, and longer in winter). With few exceptions, when certain species hairs that appeared to be longer and wider than those of other species without the microscope, that was actually true, according to my measurements. The cuticula type is listed using the terms and classification of the Key and Atlas to the Hair of Terrestrial Texas Mammals, by Debelica and Thies (2009). Again, I placed an asterisk in the space if I was unable to determine the cuticular type, subtype, or both by either looking at the hair under the light

45 37 microscope, or using the key and atlas by Debelica and Thies (finding a photo of a hair from the same species, or a different species with a similar cuticular pattern). Length was measured in cm with a small tape measure. Approximate length was indicated with ~. Length was not measured for species whose hairs were too short to easily and accurately measure. Width at tip, middle shaft, and root were measured in µm with the graticule under the microscope, across the width of the hair. The magnification is included, if it is not 200x. If one of the three sections (tip, middle shaft, root) was measured from a different hair, that is indicated in the Comments section of the spreadsheet. Photo # is the photo number, which appears on the screen on the camera. Scale Count is the number of scales across the width of the hair, at the tip, middle shaft, and root. If the scale count, cuticular pattern, or width was different at the tip or root from the middle shaft, a. f. c. (away from center), or p. c. (pattern changes) appears. It can be difficult to determine the exact number of scales using a light microscope, so some of these values are ranges, i.e., 1-2. The Salient Features column is where the features of the hairs that are noticeable without a microscope are recorded. The types of features listed are length, described in words, sometimes as a range, and color. The colors of some hairs are described using Mammals of North America (Reid 2006). Color is described as varies if there are many possible colors or patterns for the species. The second page of the spreadsheet contains Species, FR #, and Photo #, like the first page, but also Medulla Type. Only one photo was taken of one hair of each species, except in the case of albino specimens, where a second photo was taken in addition to the one from the specimen with the typical pelage color. The medulla types were described using terms from the key and atlas by Debelica and Thies, like the cuticula, but also terms from other scientists, and original terms. The percentage of the shaft which the medulla makes up was also included, if it could be determined under the microscope, or by using Debelica and Thies (2009) as a reference. All of the bat species had medulla absent listed, as they do not have a medulla. Since bat hairs have no medulla, two measurements: Scale Index (SI), and Width Index (WI) are used instead. Although the widths of the hairs differed, there was a noticeable pattern: the tip usually had the smallest width, the middle shaft had the largest width, and the width of the root was in between. Most species seem to have a cuticula that is either imbricate, crenate or imbricate, flattened. All shrew and mole species in this key and atlas have an imbricate, elongate cuticula. Similarly, all bat species had

46 a coronal cuticula, but the specific type of coronal was not able to be determined with the light 38 microscope. The porcupine hairs were measured and photographed under 50x magnification, because otherwise the entire hair would not be visible (they appear very large under the microscope). While a flattened cuticula is defined as having two or less scales, this was not always the case. Albino hair had no effect on medullar type, but the medulla was more easily visible under the microscope, and in photos. Since hair color is mainly due to pigment granules (Hausman 1924) albino hairs would be no different in structure than typical mammal hairs. It would be difficult to distinguish the bat species based solely on scale index and width index, since the scale index measurements were not very different between species, and all had the same width index, which was 2, except the Keen s Myotis, which had a width index of 3 (its hair is noticeable, microscopically, to be wider than hairs of the other bats). The cuticular and medullar types of all of the species could probably be determined with a scanning electron microscope, which was not available. The House Mouse and Norway Rat hairs both have a distinct cuticula (imbricate, ovate and imbricate, acuminate, respectively). The MALDI-TOF mass spectrometry seems to be the most accurate and reliable method to distinguish mammals by their hair, of all of the methods that I used. This is especially true without a scanning electron microscope. There is no missing information, unlike for the medullar and cuticular patterns, some of which I was unable to identify (with the exception of the data from the Little Brown Myotis; the researcher conducting the mass spectrometry accidentally used another species twice). It is less, or not at all, subjective (i.e., one cuticular or medullar type might be given a different name by different scientists, or what one scientist considers two different cuticular types may be classified as two different subtypes of the same type). Some species can be separated out by gross features, such as hair color or pattern. When a species has a different summer and winter pelage, the species must appear more than once in the key. MALDI- TOF mass spectrometry may be the only way to distinguish some species in a dichotomous key, because the cuticular and medullar type may be the same for all species in an order, and they may also have the same hair color or pattern. This is also true for domestic animals and humans, because their hair can vary greatly.

47 39 Appendix C contains mass spectrometry data of guard hair protein composition. Each graph has an x axis, which is Daltons, and a y axis, which is concentration of ions. The graphs are line graphs, with various amounts of data points, depending on how much raw data is in the table. The graphs and tables are labeled for each species. According to a Hollemeyer et al. study, some MALDI-TOF mass spectrometry peaks are found in at least 80% of the mammal species tested. Those that are not are referred to as semi-specific peaks, or SEMPs (which can be further divided into unique species-specific peaks, or USSPs, and more frequent peaks occurring in several species, or MFGs). These can be used to identify species relationships (2002), with the largest peaks being the most useful. If all of the peaks are the same, the two or more specimens are the same species. Ancient mammal specimens have been classified by their dental pulp with MALDI- TOF mass spectrometry, and this method can be used along with DNA sequencing, especially with ancient specimens, or for forensic purposes (Tran et al. 2011). Three of the four shrew species above (the Smoky Shrew, Maryland Shrew Northern Short-Tailed Shrew have MALDI-TOF peaks at 1504, 1503, and 1504, respectively had similar peaks, but the peak from one was very different (Masked Shrew, with a high peak at 805). As one might expect, the two moles included above, the Hairy-Tailed and Star- Nosed Mole share a peak at 1263, and the Eastern Cottontail and Snowshoe Hare both have a peak at The two introduced Muridae species, the House Mouse and Norway Rat, both have a peak in common at 1164, while two of the three native species, the White-Footed Mouse and Deer Mouse share a peak at 805. The Allegheny Woodrat s 2087 peak is similar to that of the White-Footed Mouse, at While the domestic dog does not share any peaks at the exact same number with either of the two fox species, the Red and Gray foxes both have a peak at 1011, and the domestic dog, surprisingly, shares a peak at 1038 with the raccoon. I chose these carnivore species for this SEMP comparison mainly because of these unusual results. While the White-Tailed Deer has peaks at 1109 and 1505, the Moose has two peaks at 1107 and The peaks of the bats of Pennsylvania are not very far apart (1435, 1459, and 1453, for the Red Bat, Tri-Colored Bat, and Big Brown Bat, in that order). The Red Bat and Tri-Colored Bat also have similar peaks at 1546 and 1548, respectively.

48 Conclusion 40 MALDI-TOF mass spectrometry was the most useful for the dichotomous key, followed by gross features (Although I had to take seasonal pelage differences into account for the latter). Cuticular and medullar patterns/types ( pattern and type both have the same meaning), or the absence of a medulla, however, could be used to distinguish one order or one family (not as easy as order) from another. It was difficult to distinguish species of Soricidae from each other with medullar and cuticular patterns, and gross features. This was also the case with Sciuridae. Often, MALDI-TOF was the only way to distinguish species or orders, if I was unable to determine the cuticular or medullar pattern, or if they were the same for two or several species. This was especially true for Chiroptera (all have the same cuticula and absence of medulla), and Insectivora (same cuticula, and most have the same medulla). The only problem with this method was that the number of the highest peak could be the same for two species, but I could solve that problem by also indicating the second largest peak. Some of my results for hair width measurement were unusual (i.e., the Southern Flying Squirrel having a wider tip, middle shaft, and root than the Northern Flying Squirrel, and the root of the moose hair being 11.5 um wide), but this could be explained by human error in measurement, labeling, or calculation (all three of these species), this could be correct due to the season in which the specimens were collected (in the case of the squirrels), or not recording which magnification under the microscope I used (in the case of the moose). While MALDI-TOF mass spectrometry was the most effective method for mammal hair identification, there were some species where its use was not necessary, and other methods were sufficient.

49 Effectiveness of Each Method by Order 41 Didelphidae: The only Pennsylvania (PA) species in this order is the Virginia Opossum. MALDI-TOF mass spectrometry (MALDI-TOF MS) was necessary, since the hair did not have any distinct features not found in other species hair. Insectivora: All species hairs had the same cuticular (imbricate, elongate), and medullar pattern (discontinuous, simple, ladder), with the possible exception of the Star-Nosed Mole, which may not have a ladder medulla). Every species in this order has hair which is similar in gross features, except for the color of the tip of the hair. The water shrew hairs had a darker rust-colored tip than the tips of the other species hairs, but this may not be noticeable to everyone. Again, MALDI-TOF MS is the most effective method. Chiroptera: Since hairs of all PA species in this order do not have a medulla, and the same cuticular type (the subtype of coronal could be determined with a scanning electron microscope, if available) these methods are ineffective. The scale index and width index were similar, or exactly the same for all species, except the Keen s Myotis, which had a width index of three (as opposed to two for the other species). However, this may not be a noticeable enough difference for all people to identify this species, or distinguish it from the others. Therefore, MALDI-TOF MS is very useful for this family. Lagomorpha: If a Snowshoe Hare specimen has its winter pelage, that is enough to distinguish it from PA s other lagomorph species used in this study, the Eastern Cottontail, but not from all PA mammal species. If not, the hairs are identical in cuticular and medullar pattern, and similar in gross features, so MALDI-TOF MS would be the best method. Rodentia: Although this is a large family, the hairs of some species have unique features that can used to identify the species and distinguish them from other PA mammals. Since the Porcupine is the only

50 42 mammal in this study with quills, no more methods would be required to identify it, except if the hair is one that does not look as much like a quill (the Porcupine appears in the dichotomous key twice for this reason). The hair of the House Mouse has a distinct cuticular pattern (imbricate, ovate) found in no other mammal in the Commonwealth. The same is true for the Norway Rat (cuticula imbricate, acuminate), so these species are easy to identify, whether it is known that a hair is from a rodent, or not. With the absence of the Beaver in this study, the Muskrat has easy-to-identify hair among PA mammals based on gross features, if one knows that the hair came from a rodent. Besides these exceptions, PA rodent hairs seem to be difficult to identify and distinguish from other species, since there are many species in this family. Some MALDI-TOF graphs had noticeable results, such as having two peaks of about equal size, one at the right end, and one at the left end of the graph, which can be useful in studies of large families of mammals. Carnivora: Like Rodentia, gross features had some uses, despite the fact that MALDI-TOF MS still turned out to be the most effective method. Badger hair had both a unique medulla (occupies 1/3 of shaft), unique color band pattern, and was noticeably long, and therefore can easily be separated out from the other PA mammal species without making cuticular slides or using MALDI-TOF MS. Likewise, the Red Fox was the only PA species (carnivore or not) to have long, orange hair. If a hair from a PA mammal is entirely black, it is from a carnivore, but other methods would need to be used to know exactly what species. If a carnivore hair is entirely white, there are a few species possibilities, but one would need to use MALDI-TOF MS to know which one it is from. This method would also be required for the domestic dog hair, due to variation in between and among different breeds. The occurrence of winter pelage further complicates studies including this family, but species can appear more than once in a dichotomous key. Artiodactyla: Like the lagomorph species the two PA species in this study have hair that is identical in cuticular and medullar pattern, and similar in gross features, so one would have to resort to MALDI-TOF

51 MS. Also, the Moose hairs came from an older specimen, and were very dry, but this did not seem to 43 affect the analysis Primates: Since human hair can vary greatly in length and color, especially if it comes from different parts of the body, a human hair probably cannot be identified as such from gross features alone, unless it is many times longer than the longest hair of another species, or if one uses MALDI-TOF MS. There are several possible shortcomings of using hair to identify species. For example, the Allegheny Woodrat and Eastern Woodrat are almost identical, with the exception of skull and biochemical differences (Reid 2006). In cases such as this, examining hair would not be enough, and lethal sampling (for skull examination), and/or a biochemical study would be necessary. The hair approach would also not be sufficient for distinguishing the Eastern Cottontail from the New England Cottontail, which are very similar in appearance, but have genetic and range differences. In this situation, one may need an individual rabbit, not just hair samples, of each species. Underfur is rarely included in mammalian hair keys, because it has less diagnostic value, but it may be useful for orders such as Chiroptera, in which it is difficult to distinguish the guard hair from the underfur, as shown by Nason (1948) and Benedict (1957) (Sessions et al. 2006). Hausman, on the other hand, believed that guard hairs were more difficult to work with than underhairs, since they are thicker, more deeply pigmented, and the scales were often worn off. However, the two types of hair differ so much in structure and other characteristics that both should be used (1920). Researchers often use more than one method for conducting these studies, for example, DNA sequencing and MALDI-TOF mass spectrometry, or both light microscopy and scanning electron microscopy. There were some methods that I used originally, that turned out to be ineffective. Although a sonicator may be useful for cleaning hairs for SEM, it did not appear to make a difference for light microscopy. I was unable to make cross-sections of the hairs, because the wax either did not cut, or shattered when I cut it. Cross-sections may not be consistent, especially if the hair is thinner than the

52 44 blade, according to Hollemeyer and Heinzle (2012). Measuring hairs on index cards with the computer program ImageJ was not successful, either. Cuticular scale casts can be unreliable. The medulla may be visible on a cuticular slide, thus obscuring part or all of a hair s scale pattern. Conversely, the medulla may be unintentionally torn out, along with the scale pattern down the center of the hair. The tip can be mistaken for the root, or vice versa. However, if measured correctly, the root should be wider than the tip, and the center of the hair has the largest width of all three sections. This is less noticeable in Talpidae. Longer hairs (badger, etc.) tend to break or split at the tip. If part of the tip is missing, the scale count and width from what is assumed to be the tip will not actually be the width and scale count of the tip. This is also true about the root. Some scale patterns, such as crenate and flattened, can be mistaken for each other under a light microscope, especially at low power. The scale pattern can also change as one looks up or down the shaft. A different method of observing scale patterns was used by Cavia et al. (2008), which consisted of starting at the root of the hair, and observing the pattern at 1/4, 3/8, ½, 5/8, and ¾ of the hair length, while I only observed one scale pattern at the middle shaft, with a few exceptions, which I indicated with the words pattern changes. Hairs can also come from different parts of the body. Other types of microscopes, such as a scanning electron microscope (SEM), may not be as widely available, more expensive, difficult to use, or all three of these factors. Some hairs, such as the porcupine, are too wide for 200x, leading to a less consistent key, as not all hairs are measured and photographed under 200x. My key and atlas did not include cross-sections, but cross-sections might be helpful when two or several species have the same cuticular or medullar type. Also, some common species, such as the black bear and coyote, were missing from the key, as I was unable to obtain hairs. In such a situation, the usefulness of the key can be affected. Microscopic hair identification, according to some, should be reserved for experts (Hollemeyer and Heinzle 2012). According to Dr. Carlos Iudica, Assistant Professor in the Department of Biology, Susquehanna University, these keys and atlases are, or should be, constantly updated, as people discover new features of the hairs to be observed or measured. Therefore, a website would be more suitable for such a key than a book (personal communication).

53 Figure 2: Dichotomous Key 45 *Hair refers to a single hair, not the entire pelage *Species names and number of species listed, if there are only one or two species in Pennsylvania 1. Largest MALDI-TOF peaks at , and between 1239 and Didelphimorphia Largest MALDI-TOF peaks not at , or between 1239 and Medulla visible in cuticular photos Insectivora Medulla not visible in cuticular photos.3 3. Medulla not present Chiroptera Medulla present.4 4. Largest MALDI-TOF peak around 1848 Lagomorpha Largest MALDI-TOF peak not around Largest MALDI-TOF peaks around and Artiodactyla Largest MALDI-TOF peaks not around or Largest MALDI-TOF peaks at , large peak between and , many small peaks...primates Largest MALDI-TOF peaks not at , no large peak between and Hair has a white-yellow-black-yellow-white pattern.. Carnivora Hair does not have a white-yellow-black-yellow-white pattern Hair is entirely black... Carnivora Hair is not entirely black Hair is long and orange.carnivora Hair is not long and orange Medulla is continuous and occupies ½ or entire shaft Carnivora Medulla is not continuous and does not occupy ½ or entire shaft Hair is a quill. Rodentia Hair is not a quill Cuticula is ovate..rodentia Cuticula is not ovate Cuticula is acuminate Rodentia Cuticula is not acuminate Medulla is continuous and ladder, continuous and asymmetrical, or discontinuous.rodentia

54 46 Order Didelphimorphia One species-didelphis virginiana (Virginia Opossum). Cuticula imbricate, crenate, medulla continuous, nodose, occupies more than ½ of shaft. 1. Largest MALDI-TOF peaks at , and between 1239 and Didelphis virginiana (Virginia Opossum) Order Insectivora All have cuticula imbricate, elongate and medulla discontinuous, simple. 1. Largest MALDI-TOF peak at 805 (also, few peaks)...sorex cinereus (Masked Shrew) Largest MALDI-TOF peak not at Largest MALDI-TOF peak between and , and at Sorex fontinalis (Maryland Shrew) Largest MALDI-TOF peak not between and or at Largest MALDI-TOF peaks at and Sorex fumeus (Smoky Shrew) Largest MALDI-TOF peak not at and Largest MALDI-TOF peaks between and and around Sorex hoyi (Pygmy Shrew) Largest MALDI-TOF peaks not between and or at Largest MALDI-TOF peaks between and and at Sorex palustris (Water Shrew) Largest MALDI-TOF peaks not between and or at Largest MALDI-TOF peaks around and Blarina brevicauda (Northern Short-Tailed Shrew) Largest MALDI-TOF peaks not at or Largest MALDI-TOF peaks at and between and Cryptotis parva (Least shrew) Largest MALDI-TOF peaks not at or between and Largest MALDI-TOF peaks at and Parascalops breweri (Hairy-Tailed Mole) Largest MALDI-TOF peaks not at and

55 9. Largest MALDI-TOF peaks between 1840 and 1968, and between 1038 and Scalopus aquaticus (Eastern Mole) Largest MALDI-TOF peaks not between 1840 and 1968, or between 1038 and Largest MALDI-TOF peaks around and Condylura cristata (Star-Nosed Mole) 47 Order Chiroptera All have cuticula coronal and no medulla *Myotis lucifugus (Little Brown Myotis) is not included because MALDI-TOF mass spectrometry data was not available 1. Largest MALDI-TOF peaks at 1453 and between 2669 and Eptesicus fuscus (Big Brown Bat) Largest MALDI-TOF peaks not at 1453 or between 2669 and Largest MALDI-TOF peaks between and , and Lasiurus borealis (Red Bat) Largest MALDI-TOF peaks not at 1453 or between 2669 and Largest MALDI-TOF peaks between and Myotis Keenii (Keen s Myotis) Largest MALDI-TOF peaks not between and Largest MALDI-TOF peaks between and Perimyotis subflavus (Tri-Colored Bat) Largest MALDI-TOF peaks not between and Largest MALDI-TOF peaks between 1038 and Lasionycteris noctivagans (Silver-Haired Bat) Order Lagomorpha Two species-sylvilagus floridanus (Eastern Cottontail) and Lepus americanus (Snowshoe Hare). Both have cuticula imbricate, flattened and medulla continuous, nodose, symmetrical, occupies entire shaft. 1. Hair is white (winter pelage)... Lepus americanus (Snowshoe Hare) Hair is not white.... 2

56 2. Largest MALDI-TOF peaks between 1848 and 1956 Lepus americanus (Snowshoe Hare) Largest MALDI-TOF peaks are not between 1848 and Largest MALDI-TOF peaks between 1848 and 2064 Sylvilagus floridanus (Eastern Cottontail) 48 Order Rodentia 1. Hair is a quill... Erethizon dorsatum (Porcupine) Hair is not a quill Cuticula imbricate, ovate...mus musculus (House Mouse) Cuticula not imbricate, ovate Cuticula imbricate, acuminate....rattus norvegicus (Norway Rat) Cuticula not imbricate, acuminate Hair is long and dark brown... Ondatra zibethicus(muskrat) Hair is not long or dark brown Has largest MALDI-TOF peaks of about equal size at right and left ends of graph Does not have largest MALDI-TOF peaks of about equal size at right and left ends of graph Largest MALDI-TOF peaks are between and , and at Tamias striatus (Eastern Chipmunk) Largest MALDI-TOF peaks are not between and , or at Largest MALDI-TOF peaks are at , and to the right of Peromyscus leucopus (White-Footed Mouse) Largest MALDI-TOF peaks are not at , or to the right of Largest MALDI-TOF peaks are between and , and between and Erethizon dorsatum (Porcupine) Largest MALDI-TOF peaks are not between and , or between and Largest MALDI-TOF peaks are at , and between and Marmota monax (Woodchuck) Largest MALDI-TOF peaks are not at , or between and Largest MALDI-TOF peaks are between 1153 and 1198, and between 1635 and Sciurus carolinensis (Gray squirrel)

57 Largest MALDI-TOF peaks are not between 1153 and 1198, or between 1635 and Largest MALDI-TOF peaks are at and between and Sciurus niger (Fox Squirrel) Largest MALDI-TOF peaks are not at or between and Largest MALDI-TOF peaks are at 1518, and between 2162 and Tamiasciurus hudsonicus (Red Squirrel) Largest MALDI-TOF peaks are not at 1518, or between 2162 and Largest MALDI-TOF peaks are at 1805, and between 2074 and Glaucomys sabrinus (Northern Flying Squirrel) Largest MALDI-TOF peaks are not at 1805, or between 2074 and Largest MALDI-TOF peaks are between 1153 and 1198, and between 1559 to Glaucomys volans (Southern Flying Squirrel) Largest MALDI-TOF peaks are not between 1153 and 1198, or between 1559 to Largest MALDI-TOF peaks are at 805 and 2272, (also, many small peaks)...peromyscus maniculatus (Deer Mouse) Largest MALDI-TOF peaks are not at 805 or Largest MALDI-TOF peaks are at , and between and Neotoma magister (Allegheny Woodrat)* *May be N. floridana, but all N. magister specimens were originally labeled N. floridana Largest MALDI-TOF peaks are not at , or between and Largest MALDI-TOF peaks are at 1518 and (also, lots of peaks)...myodes gapperi (Southern Red-Backed Vole) Largest MALDI-TOF peaks are not at 1518 or Largest MALDI-TOF peaks are at and Microtus pennsylvanicus (Meadow Vole) Largest MALDI-TOF peaks are not at or Largest MALDI-TOF peaks are between 1427 and 1548, and to the right of Microtus pinetorum (Woodland Vole) Largest MALDI-TOF peaks are not between 1427 and 1548, or to the right of Largest MALDI-TOF peaks are at 1164 and 1240 Synaptomys cooperi (Southern Bog Lemming) Largest MALDI-TOF peaks are not at 1164 or

58 Largest MALDI-TOF peaks are at 1348, and between 2242 and Zapus hudsonius (Meadow Jumping Mouse) Largest MALDI-TOF peaks are not at 1348, or between 2242 and Largest MALDI-TOF peaks are between and , and to the right of Napaeozapus insignis (Woodland Jumping Mouse) Order Carnivora 1. Medulla occupies 1/3 of shaft Medulla does not occupy 1/3 of shaft Hair has a white-yellow-black-yellow-white pattern.taxidea taxus (Badger) Hair does not have white-yellow-black-yellow-white pattern Hair is orange... Vulpes vulpes (Red Fox) Hair is not orange Largest MALDI-TOF peaks are between 1038 and 1087 Procyon lotor (Raccoon) Largest MALDI-TOF peaks are not between 1038 and Largest MALDI-TOF peaks are between and and at Canis familiaris (Domestic Dog) Largest MALDI-TOF peaks are not between and or at MALDI-TOF graph has few (16) peaks Mustela frenata (Long-Tailed Weasel) MALDI-TOF graph does not have few (16) peaks Hair is entirely black... Mephitis mephitis (Striped Skunk) Hair is not entirely black Hair is entirely white Hair is not entirely white Hair is brown, brownish or reddish brown Largest MALDI-TOF peaks are at 1011 and Urocyon cinereoargenteus (Gray Fox) Largest MALDI-TOF peaks are not at 1011 or Largest MALDI-TOF peaks are between and and at Mephitis Mephitis (Striped Skunk)

59 Largest MALDI-TOF peaks are not between and or at Largest MALDI-TOF peaks are at and between and Mustela erminea (Ermine) Largest MALDI-TOF peaks are not at or between and Largest MALDI-TOF peaks are between 801 and 807, and at 1159 Mustela nivalis (Least Weasel) Largest MALDI-TOF peaks are between 801 and 807, and at Largest MALDI-TOF peaks are between and and between and Vulpes vulpes (Red Fox) Largest MALDI-TOF peaks are not between and or between and Largest MALDI-TOF peaks are between 801 and 807, and at Mustela nivalis (Least Weasel) Largest MALDI-TOF peaks are not between 801 and 807, or at Largest MALDI-TOF peaks are at 1159 and 2272.Neovison vison (Mink) Largest MALDI-TOF peaks are not at 1159 and Largest MALDI-TOF peaks are at and between and Martes Americana (Marten) Largest MALDI-TOF peaks are not at or between and Largest MALDI-TOF peaks are at and between and Mustela erminea (Ermine) Order Artiodactyla Two species-odocoileus virginianus (White-Tailed Deer) and Alces alces (Moose). Both have cuticula imbricate and medulla continuous, nodose. 1. Largest MALDI-TOF peaks around 1109 and between 1484 and Odocoileus virginianus(white-tailed Deer) Largest MALDI-TOF peaks are not around 1109 and between 1484 and

60 2. Largest MALDI-TOF peaks between 1107 and 1140, 1407 and Alces alces (Moose) 52 Order Primates One species-homo sapiens (Human). Has cuticula imbricate, crenate and a medulla discontinuous, fragmental, can be absent in some places 1. Largest MALDI-TOF peaks at , large peak between and , many small peaks.homo sapiens (Human)

61 53 Appendix A: Pennsylvania Mammal Species Cuticular and Medullar Slides with Accompanying Measurement and Counts Data *One specimen from each species was used, with four photos for each species (cuticular tip, middle shaft, and root scale patterns, and medullar pattern), except for the bats, whose hairs lack a medulla, and therefore only three photos were necessary *Photos are labeled, starting with Figure 1, and the magnification is included *Below the photos: common and scientific names of each species, in bold, descriptions of the cuticula and medulla, salient features, Average Total Length, Average Width at Tip, Average Width at Middle Shaft, and Average Width at Root *Cuticula and medulla type not indicated if unable to be determined from these photos or other mammal hair keys/atlases *Bat hairs do not have a medulla, so scale index and width index are used to distinguish species *Total length was not measured from some species with small hairs *Average Total Length was calculated by adding the lengths of every hair of every specimen of a species, and diving by the number of hairs (up to 9) *Less than 0.05 and less than 0.1 were rounded to 0.05 and 0.1, respectively, for calculations *Pointed edges noticeable in photos of hairs of shrew and mole species in this key and atlas, but not indicated in other keys/atlases

62 54 Figure 1. Virginia Opossum (Didelphis virginiana) Cuticular Tip Scale Pattern at 200x. Figure 2. Virginia Opossum (Didelphis virginiana) Cuticular Middle Shaft Scale Pattern at 200x. Figure 3. Virginia Opossum (Didelphis virginiana) Cuticular Root Scale Pattern at 200x. Figure 4. Virginia Opossum (Didelphis virginiana) Medullar Pattern at 200x Virginia Opossum-Didelphis virginiana Cuticula imbricate, crenate. Medulla continuous, nodose, occupies more than 1/2 of shaft. Salient Features: Long, white, gray, or sometimes yellowish. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: 3 µm.

63 55 Figure 5. Masked Shrew (Sorex cinereus) Cuticular Tip Scale Pattern at 200x. Figure 6. Masked Shrew (Sorex cinereus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 7. Masked Shrew (Sorex cinereus) Cuticular Root Scale Pattern at 200x. Figure 8. Masked Shrew (Sorex cinereus) Medullar Pattern at 200x. Masked Shrew-Sorex cinereus Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder,* may have pointed edges. Salient Features: Very short, gray with yellow tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

64 56 Figure 9. Maryland Shrew (Sorex fontinalis) Cuticular Tip Scale Pattern at 200x. Figure 10. Maryland Shrew (Sorex fontinalis) Middle Shaft Scale Pattern at 200x. Figure 11. Maryland Shrew (Sorex fontinalis) Cuticular Root Scale Pattern at 200x. Figure 12. Maryland Shrew (Sorex fontinalis) Medullar Pattern at 200x. Maryland Shrew-Sorex fontinalis Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges. Salient Features: Very short, gray with light or dark rust-colored tip. Average Total Length: *not measured. Average Width at Tip: 0.15 µm. Average Width at Middle Shaft: µm. Average Width at Root: 0.4 µm.

65 57 Figure 13. Smoky Shrew (Sorex fumeus) Cuticular Tip Scale Pattern at 200x. Figure 14. Smoky Shrew (Sorex fumeus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 15. Smoky Shrew (Sorex fumeus) Cuticular Root Scale Pattern at 200x. Figure 16. Smoky Shrew (Sorex fumeus) Medullar Pattern at 200x. Smoky Shrew-Sorex fumeus Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges. Salient Features: Very short, tip is yellow or light rust color, Summer pelage: grayish brown, Winter pelage: dark gray. Average Total Length: not measured. Average Width at Tip: µm. Average Width at Middle Shaft: 0.6 µm. Average Width at Root: 0.4 µm.

66 58 Figure 17. Pygmy Shrew (Sorex hoyi) Cuticular Tip Scale Pattern at 200x. Figure 18. Pygmy Shrew (Sorex hoyi) Cuticular Middle Shaft Scale Pattern at 200x Figure 19. Pygmy Shrew (Sorex hoyi) Cuticular Root Scale Pattern at 200x. Figure 20. Pygmy Shrew (Sorex hoyi) Medullar Pattern at 200x. Pygmy Shrew-Sorex hoyi Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges. Salient Features: Very short, gray or grayish brown with yellow or light rust-colored tip. Average Total Length: not measured. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

67 59 Figure 21. Water Shrew (Sorex palustris) Cuticular Tip Scale Pattern at 200x. Figure 22. Water Shrew (Sorex palustris) Cuticular Middle Shaft Scale Pattern at 200x. Figure 23. Water Shrew (Sorex palustris) Cuticular Root Scale Pattern at 200x. Figure 24. Water Shrew (Sorex palustris) Medullar Pattern at 200x. Water Shrew-Sorex palustris Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges. Salient Features: Very short, gray or black with dark rust-colored tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 0.4 µm. Average Width at Root: 0.3 µm.

68 60 Figure 25. Northern Short-Tailed Shrew (Blarina brevicauda) Cuticular Tip Scale Pattern at 200x. Figure 26. Northern Short-Tailed Shrew (Blarina brevicauda) Cuticular Middle Shaft Scale Pattern at 200x. Figure 28. Northern Short-Tailed Shrew (Blarina brevicauda) Cuticular Root Scale Pattern at 200x. Figure 27. Northern Short-Tailed Shrew (Blarina brevicauda) Medullar Pattern at 200x. Northern Short-Tailed Shrew-Blarina brevicauda Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges. Salient Features: Very short, gray with yellow tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: µm. Average Width at Root: 0.3 µm.

69 61 Figure 29. Least Shrew (Cryptotis parva) Cuticular Tip Scale Pattern at 200x. Figure 30. Least Shrew (Cryptotis parva) Cuticular Middle Shaft Scale Pattern at 200x. Figure 31. Least Shrew (Cryptotis parva) Cuticular Root Scale Pattern at 200x. Figure 32. Least Shrew (Cryptotis parva) Medullar Pattern at 200x. Least Shrew-Cryptotis parva Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges, occupies more than 1/2 of shaft. Salient Features: Very short, brown or gray with yellow tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.2 µm.

70 62 Figure 33. Hairy-Tailed Mole (Parascalops breweri) Cuticular Tip Scale Pattern at 200x. Figure 34. Hairy-Tailed Mole (Parascalops breweri) Cuticular Middle Shaft Scale Pattern at 200x. Figure 35. Hairy-Tailed Mole (Parascalops breweri) Cuticular Root Scale Pattern at 200x. Figure 36. Hairy-Tailed Mole (Parascalops breweri) Medullar Pattern at 200x. Hairy-Tailed Mole-Parascalops breweri Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges. Salient Features: Very short, dark gray. Average Total Length: not measured. Average Width at Tip: 0.05 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.1 µm.

71 63 Figure 37. Eastern Mole (Scalopus aquaticus) Cuticular Tip Scale Pattern at 200x. Figure 38. Eastern Mole (Scalopus aquaticus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 39. Eastern Mole (Scalopus aquaticus) Cuticular Root Scale Pattern at 200x. Figure 40. Eastern Mole (Scalopus aquaticus) Medullar Pattern at 200x. Eastern Mole-Scalopus aquaticus Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder, may have pointed edges, occupies entire shaft. Salient Features: Very short, grayish brown with light yellow tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.2 µm.

72 64 Figure 41. Star-Nosed Mole (Condylura cristata) Cuticular Tip Scale Pattern at 200x. Figure 42. Star-Nosed Mole (Condylura cristata) Cuticular Middle Shaft Scale Pattern at 200x. Figure 43. Star-Nosed Mole (Condylura cristata) Cuticular Root Scale Pattern at 200x. Figure 44. Star-Nosed Mole (Condylura cristata) Medullar Pattern at 200x. Star-Nosed Mole-Condylura cristata Cuticula imbricate, elongate. Medulla discontinuous, simple, ladder. Salient Features: Very short, brown or black, may have yellow or rust-colored tip. Average Total Length: not measured. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

73 65 Figure 45. Eastern Cottontail (Sylvilagus floridanus) Cuticular Tip Scale Pattern at 200x. Figure 46. Eastern Cottontail (Sylvilagus floridanus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 47. Eastern Cottontail (Sylvilagus floridanus) Medullar Pattern at 200x. Figure 48. Eastern Cottontail (Sylvilagus floridanus) Cuticular Root Scale Pattern at 200x. Eastern Cottontail- Sylvilagus floridanus Cuticula imbricate, flattened. Medulla continuous, nodose, symmetrical, fewer than 6 rows, occupies entire shaft. Salient Features: Medium, three bands of color: gray-orange-gray. Average Total Length: cm. Average Width at Tip: 0.25 µm. Average Width at Middle Shaft: 2.6 µm. Average Width at Root: 0.5 µm.

74 66 Figure 49. Snowshoe Hare (Lepus americanus) Cuticular Tip Scale Pattern at 200x. Figure 50. Snowshoe Hare (Lepus americanus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 51. Snowshoe Hare (Lepus americanus) Cuticular Root Scale Pattern at 200x. Figure 52. Snowshoe Hare (Lepus americanus) Medullar Pattern at 200x. Snowshoe Hare-Lepus americanus Cuticula imbricate, flattened. Medulla continuous, nodose, symmetrical, occupies entire shaft. Salient Features: Winter pelage: long and white, Summer pelage: similar in color to Eastern Cottontail, but can be longer. Average Total Length: cm. Average Width at Tip: 0.3 µm. Average Width at Middle Shaft: 2.45 µm. Average Width at Root: 2 µm.

75 67 Figure 53. Woodchuck (Marmota monax) Cuticular Tip Scale Pattern at 200x. Figure 54. Woodchuck (Marmota monax) Cuticular Middle Shaft Scale Pattern at 200x. Figure 55. Woodchuck (Marmota monax) Cuticular Root Scale Pattern at 200x. Figure 56. Woodchuck (Marmota monax) Medullar Pattern at 200x. Woodchuck-Marmota monax Cuticula imbricate. Salient Features: Long, 3 or 4 bands of color: brown or gray, small area of orange or yellow, brown or gray, lighter brown. Average Total Length: cm. Average Width at Tip: 0.35 µm. Average Width at Middle Shaft: 3.9 µm. Average Width at Root: 2.3 µm.

76 68 Figure 57. Eastern Chipmunk (Tamias striatus) Cuticular Tip Scale Pattern at 200x. Figure 58. Eastern Chipmunk (Tamias striatus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 59. Eastern Chipmunk (Tamias striatus) Cuticular Root Scale Pattern at 200x. Figure 60. Eastern Chipmunk (Tamias striatus) Medullar Pattern at 200x. Eastern Chipmunk-Tamias striatus Cuticula imbricate, crenate. Medulla continuous, nodose, asymmetrical, occupies more than 1/2 of shaft. Salient Features: Short to medium, 3 or 4 bands of color: gray or black, small area of orange or yellow, gray or black, light brown. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: 1.1 µm.

77 69 Figure 61. Gray Squirrel (Sciurus carolinensis) Cuticular Tip Scale Pattern at 200x. Figure 62. Gray Squirrel (Sciurus carolinensis) Cuticular Middle Shaft Scale Pattern at 200x. Figure 63. Gray Squirrel (Sciurus carolinensis) Cuticular Root Scale Pattern at 200x. Figure 64. Gray Squirrel (Sciurus carolinensis) Medullar Pattern at 200x. Gray Squirrel-Sciurus carolinensis Cuticula imbricate, crenate. Medulla continuous, nodose, asymmetrical, occupies more than 1/2 of shaft. Salient Features: Short to long, 3 or 4 bands of color, amount of gray/black and orange/yellow varies. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

78 70 Figure 65. Fox Squirrel (Sciurus niger) Cuticular Tip Scale Pattern at 200x. Figure 66. Fox Squirrel (Sciurus niger) Cuticular Middle Shaft Scale Pattern at 200x. Figure 67. Fox Squirrel (Sciurus niger) Cuticular Root Scale Pattern at 200x. Figure 68. Fox Squirrel (Sciurus niger) Medullar Pattern at 200x. Fox Squirrel-Sciurus niger Cuticula imbricate, crenate. Medulla continuous, nodose, asymmetrical, occupies more than 1/2 of shaft. Salient Features: Short to long, but most often long, 3 or 4 bands of color, amount of gray/black and orange/yellow varies. Average Total Length: 2.2 cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

79 71 Figure 69. Red Squirrel (Tamiasciurus hudsonicus) Cuticular Tip Scale Pattern at 200x. Figure 70. Red Squirrel (Tamiasciurus hudsonicus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 71. Red Squirrel (Tamiasciurus hudsonicus) Cuticular Root Scale Pattern at 200x. Figure 72. Red Squirrel (Tamiasciurus hudsonicus) Medullar Pattern at 200x. Red Squirrel-Tamiasciurus hudsonicus Cuticula imbricate, crenate. Salient Features: Short to long, but most often long, 3 or 4 bands of color, amount of gray/brown and orange/yellow varies. Average Total Length: cm. Average Width at Tip: 0.15 µm. Average Width at Middle Shaft: 1.75 µm. Average Width at Root: 0.5 µm.

80 72 Figure 73. Northern Flying Squirrel (Glaucomys sabrinus) Cuticular Tip Scale Pattern at 200x. Figure 74. Northern Flying Squirrel (Glaucomys sabrinus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 75. Northern Flying Squirrel (Glaucomys sabrinus) Cuticular Root Scale Pattern at 200x. Figure 76. Northern Flying Squirrel (Glaucomys sabrinus) Medullar Pattern at 200x. Northern Flying Squirrel-Glaucomys sabrinus Cuticula imbricate, crenate. Medulla continuous, nodose, 2-3 columns, asymmetrical, occupies most of shaft. Salient Features: Short, gray or grayish brown, yellow tip. Average Total Length: not measured. Average Width at Tip: 0.05 µm. Average Width at Middle Shaft: 0.4 µm. Average Width at Root: 0.4 µm.

81 73 Figure 77. Southern Flying Squirrel (Glaucomys volans) Cuticular Tip Scale Pattern at 200x. Figure 78. Southern Flying Squirrel (Glaucomys volans) Cuticular Middle Shaft Scale Pattern at 200x. Figure 79. Southern Flying Squirrel (Glaucomys volans) Cuticular Root Scale Pattern at 200x. Figure 80. Southern Flying Squirrel (Glaucomys volans) Medullar Pattern at 200x. Southern Flying Squirrel-Glaucomys volans Cuticula imbricate, crenate. Medulla continuous, nodose, 2-3 columns, asymmetrical, occupies most of shaft. Salient Features: Short, gray or grayish brown, yellow tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: µm. Average Width at Root: 0.4 µm.

82 74 Figure 81. White-Footed Mouse (Peromyscus leucopus) Cuticular Tip Scale Pattern at 200x. Figure 82. White-Footed Mouse (Peromyscus leucopus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 83. White-Footed Mouse (Peromyscus leucopus) Cuticular Root Scale Pattern at 200x. Figure 84. White-Footed Mouse (Peromyscus leucopus) Medullar Pattern at 200x. White-Footed Mouse-Peromyscus leucopus Cuticula imbricate, crenate. Medulla discontinuous, ovate, 2 columns. Salient Features: Short, dark brown and orange-brown, in various combinations. Average Total Length: not measured. Average Width at Tip: 0.05 µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

83 75 Figure 85. Deer Mouse (Peromyscus maniculatus) Cuticular Tip Scale Pattern at 200x. Figure 86. Deer Mouse (Peromyscus maniculatus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 87. Deer Mouse (Peromyscus maniculatus) Cuticular Root Scale Pattern at 200x. Figure 88. Deer Mouse (Peromyscus maniculatus) Medullar Pattern at 200x. Deer Mouse-Peromyscus maniculatus Cuticula imbricate, crenate. Medulla discontinuous, simple, rectangular cells, occupies entire shaft. Salient Features: Short, dark brown, grayish brown or orange-brown. Average Total Length: not measured. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

84 76 Figure 89. Allegheny Woodrat (Neotoma magister) Cuticular Tip Scale Pattern at 200x. Figure 90. Allegheny Woodrat (Neotoma magister) Cuticular Middle Shaft Scale Pattern at 200x. Figure 91. Allegheny Woodrat (Neotoma magister) Cuticular Root Scale Pattern at 200x. Figure 92. Allegheny Woodrat (Neotoma magister) Medullar Pattern at 200x. Allegheny Woodrat-Neotoma magister (May be N. floridana, but all N. magister specimens were originally labeled N. floridana) Cuticula imbricate, flattened. Medulla discontinuous, compound, no columns, most rows fused. Salient Features: Short to long, but most often long, gray or brown with a light yellow tip, may have a lighter root than middle shaft. Average Total Length: 2.03 cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

85 77 Figure 93. Norway Rat (Rattus norvegicus) Cuticular Tip Scale Pattern at 200x. Figure 94. Norway Rat (Rattus norvegicus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 95. Norway Rat (Rattus norvegicus) Cuticular Root Scale Pattern at 200x. Figure 96. Norway Rat (Rattus norvegicus) Medullar Pattern at 200x. Norway Rat-Rattus norvegicus Cuticula imbricate, acuminate. Medulla discontinuous, fragmental, cortical intrusions. Salient Features: Medium to long, brown and yellow, amounts of colors varies. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: 1.8 µm. Average Width at Root: µm.

86 78 Figure 97. House Mouse (Mus musculus) Cuticular Tip Scale Pattern at 200x. Figure 98. House Mouse (Mus musculus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 99. House Mouse (Mus musculus) Cuticular Root Scale Pattern at 200x. Figure 100. House Mouse (Mus musculus) Medullar Pattern at 200x. House Mouse-Mus musculus Cuticula imbricate, ovate. Medulla discontinuous, compound, ladder, no columns, cortical intrusions. Salient Features: Short, gray or brown with a dark yellow tip (similar to Eastern Woodrat, except shorter and darker. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

87 79 Figure 101. Southern Red-Backed Vole (Myodes gapperi) Cuticular Tip Scale Pattern at 200x. Figure 102. Southern Red-Backed Vole (Myodes gapperi) Cuticular Middle Shaft Scale Pattern at 200x. Figure 103. Southern Red-Backed Vole (Myodes gapperi) Cuticular Root Scale Pattern at 200x. Figure 104. Southern Red-Backed Vole (Myodes gapperi) Medullar Pattern at 200x. Southern Red-Backed Vole-Myodes gapperi Cuticula imbricate, flattened. Medulla discontinuous, simple, ladder. Salient Features: Short to medium, gray and dark yellow, amounts of colors vary. Average Total Length: 0.62 cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

88 80 Figure 105. Meadow Vole (Microtus pennsylvanicus) Cuticular Tip Scale Pattern at 200x. Figure 106. Meadow Vole (Microtus pennsylvanicus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 107. Meadow Vole (Microtus pennsylvanicus) Cuticular Root Scale Pattern at 200x. Figure 108. Meadow Vole (Microtus pennsylvanicus) Medullar Pattern at 200x. Meadow Vole-Microtus pennsylvanicus Cuticula crenate, flattened. Medulla discontinuous, simple. Salient Features: Short to medium, mainly dark brown with a yellow tip. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: 1 µm. Average Width at Root: 0.8 µm.

89 81 Figure 109. Woodland Vole (Microtus pinetorum) Cuticular Tip Scale Pattern at 200x. Figure 110. Woodland Vole (Microtus pinetorum) Cuticular Middle Shaft Scale Pattern at 200x. Figure 111. Woodland Vole (Microtus pinetorum) Cuticular Root Scale Pattern at 200x. Figure 112. Woodland Vole (Microtus pinetorum) Medullar Pattern at 200x. Woodland Vole-Microtus pinetorum Cuticula imbricate, elongate. Medulla discontinuous, compound, 4 columns, some rows, occupies entire shaft. Salient Features: Short to medium, dark brown, grayish brown, orange-brown, and reddish brown, in various combinations. Average Total Length: not measured. Average Width at Tip: µm. Average Width at Middle Shaft: 0.5 µm. Average Width at Root: µm.

90 82 Figure 113. Muskrat (Ondatra zibethicus) Cuticular Tip Scale Pattern at 200x. Figure 114. Muskrat (Ondatra zibethicus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 115. Muskrat (Ondatra zibethicus) Cuticular Root Scale Pattern at 200x. Figure 116. Muskrat (Ondatra zibethicus) Medullar Pattern at 200x. Muskrat-Ondatra zibethicus Cuticula imbricate, crenate. Medulla discontinuous, compound, ovate, columns absent, fused rows, occupies 1/2 of shaft. Salient Features: Long, 1/3 to 2/3 dark brown, the rest lighter brown or yellow. Average Total Length: cm. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 2.4 µm. Average Width at Root: 0.7 µm.

91 83 Figure 117. Southern Bog Lemming (Synaptomys cooperi) Cuticular Tip Scale Pattern at 200x. Figure 118. Southern Bog Lemming (Synaptomys cooperi) Cuticular Middle Shaft Scale Pattern at 200x. Figure 119. Southern Bog Lemming (Synaptomys cooperi) Cuticular Root Scale Pattern at 200x. Figure 120. Southern Bog Lemming (Synaptomys cooperi) Medullar Pattern at 200x. Southern Bog Lemming-Synaptomys cooperi Cuticula imbricate, flattened. Salient Features: Short, dark brown with dark yellow tip. Average Total Length: 1.1 cm. Average Width at Tip: 0.05 µm. Average Width at Middle Shaft: 1.3 µm. Average Width at Root: 0.7 µm.

92 84 Figure 121. Meadow Jumping Mouse (Zapus hudsonius) Cuticular Tip Scale Pattern at 200x. Figure 122. Meadow Jumping Mouse (Zapus hudsonius) Cuticular Middle Shaft Scale Pattern at 200x. Figure 123. Meadow Jumping Mouse (Zapus hudsonius) Cuticular Root Scale Pattern at 200x. Figure 124. Meadow Jumping Mouse (Zapus hudsonius) Medullar Pattern at 200x. Meadow Jumping Mouse-Zapus hudsonius Medulla discontinuous, compound. Salient Features: Short to medium, dark brown, orange, yellow, and white in various combinations. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: 2.1 µm. Average Width at Root: 1.35 µm.

93 85 Figure 125. Woodland Jumping Mouse (Napaeozapus insignis) Cuticular Tip Scale Pattern at 200x. Figure 126. Woodland Jumping Mouse (Napaeozapus insignis) Cuticular Middle Shaft Scale Pattern at 200x. Figure 127. Woodland Jumping Mouse (Napaeozapus insignis) Cuticular Root Scale Pattern at 200x. Figure 128. Woodland Jumping Mouse (Napaeozapus insignis) Medullar Pattern at 200x. Woodland Jumping Mouse-Napaeozapus insignis Salient Features: Short to medium, dark brown, orange, yellow, and white, in various combinations. Average Total Length: cm. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

94 86 Figure 129. Porcupine (Erethizon dorsatum) Cuticular Tip Scale Pattern at 50x. Figure 130. Porcupine (Erethizon dorsatum) Cuticular Middle Shaft Scale Pattern at 50x. Figure 131. Porcupine (Erethizon dorsatum) Cuticular Root Scale Pattern at 50x. Figure 132. Porcupine (Erethizon dorsatum) Medullar Pattern at 50x. Porcupine-Erethizon dorsatum Cuticula imbricate, crenate. Medulla discontinuous, fragmental, cortical intrusions, occupies more than 1/2 of shaft. Salient Features: Quills of various lengths and widths, can be brown (light or dark) and yellowish or all yellowish. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

95 87 Figure 133. Red Fox (Vulpes vulpes) Cuticular Tip Scale Pattern at 200x. Figure 134. Red Fox (Vulpes vulpes) Cuticular Middle Shaft Scale Pattern at 200x. Figure 135. Red Fox (Vulpes vulpes) Cuticular Root Scale Pattern at 200x. Figure 136. Red Fox (Vulpes vulpes) Medullar Pattern at 200x. Red Fox-Vulpes vulpes Cuticula imbricate, crenate. Medulla continuous, vacuolated, occupies entire shaft. Salient Features: Long, orange, brown. Average Total Length: cm. Average Width at Tip: 0.4 µm. Average Width at Middle Shaft: 3 µm. Average Width at Root: 1.3 µm.

96 88 Figure 137. Gray Fox (Urocyon cinereoargenteus) Cuticular Tip Scale Pattern at 200x. Figure 138. Gray Fox (Urocyon cinereoargenteus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 139. Gray Fox (Urocyon cinereoargenteus) Cuticular Root Scale Pattern at 200x. Figure 140. Gray Fox (Urocyon cinereoargenteus) Medullar Pattern at 200x. Gray Fox-Urocyon cinereoargenteus Cuticula imbricate, flattened. Medulla continuous, nodose, occupies more than 1/2 of shaft. Salient Features: Long, white. Average Total Length: cm. Average Width at Tip: 0.3 µm. Average Width at Middle Shaft: 3 µm. Average Width at Root: 2.4 µm.

97 89 Figure 141. Raccoon (Procyon lotor) Cuticular Tip Scale Pattern at 200x. Figure 142. Raccoon (Procyon lotor) Cuticular Middle Shaft Scale Pattern at 200x. Figure 143. Raccoon (Procyon lotor) Cuticular Root Scale Pattern at 200x. Figure 144. Raccoon (Procyon lotor) Medullar Pattern at 200x. Raccoon-Procyon lotor Cuticula imbricate, flattened. Medulla continuous, nodose, vacuolated, occupies 1/2 of shaft. Salient Features: Long, black, gray, and white, in various combinations. Average Total Length: 5.7 cm. Average Width at Tip: µm. Average Width at Middle Shaft: 3.23 µm. Average Width at Root: µm.

98 90 Figure 145. Marten (Martes americana) Cuticular Tip Scale Pattern at 200x. Figure 146. Marten (Martes americana) Cuticular Middle Shaft Scale Pattern at 200x. Figure 147. Marten (Martes americana) Cuticular Root Scale Pattern at 200x. Figure 148. Marten (Martes americana) Medullar Pattern at 200x. Marten-Martes Americana Cuticula imbricate. Salient Features: Medium, dark brown and light yellow. Average Total Length: cm. Average Width at Tip: 0.2 µm. Average Width at Middle Shaft: 1.9 µm. Average Width at Root: 0.7 µm.

99 91 Figure 149. Ermine (Mustela erminea) Cuticular Tip Scale Pattern at 200x. Figure 150. Ermine (Mustela erminea) Cuticular Middle Shaft Scale Pattern at 200x. Figure 151. Ermine (Mustela erminea) Cuticular Root Scale Pattern at 200x. Figure 152. Ermine (Mustela erminea) Medullar Pattern at 200x. Ermine-Mustela erminea Cuticula imbricate. Medulla continuous, nodose, vacuolated, occupies more than 1/2 of shaft. Salient Features: Medium, reddish brown or dark brown, sometimes some light yellow. Average Total Length: cm. Average Width at Tip: 1.8 µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

100 92 Figure 153. Long-Tailed Weasel (Mustela frenata) Cuticular Tip Scale Pattern at 200x. Figure 154. Long-Tailed Weasel (Mustela frenata) Cuticular Middle Shaft Scale Pattern at 200x. Figure 155. Long-Tailed Weasel (Mustela frenata) Cuticular Root Scale Pattern at 200x. Figure 156. Long-Tailed Weasel (Mustela frenata) Medullar Pattern at 200x. Long-Tailed Weasel-Mustela frenata Cuticula imbricate, flattened. Medulla continuous, nodose, vacuolated, occupies more than 1/2 of shaft. Salient Features: Medium, Summer pelage: brown, Winter pelage: white. Average Total Length: 1.5 cm. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 1.7 µm. Average Width at Root: 1 µm.

101 93 Figure 157.Least Weasel (Mustela nivalis) Cuticular Tip Scale Pattern at 200x. Figure 158. Least Weasel (Mustela nivalis) Cuticular Middle Shaft Scale Pattern at 200x. Figure 159. Least Weasel (Mustela nivalis) Cuticular Root Scale Pattern at 200x. Figure 160. Least Weasel (Mustela nivalis) Medullar Pattern at 200x. Least Weasel-Mustela nivalis Medulla continuous, nodose, vacuolated, occupies more than 1/2 of shaft. Salient Features: Short, especially compared to hairs of other weasel species, brown, lighter at root. Average Total Length: cm. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 1.95 µm. Average Width at Root: 1.8 µm

102 94 Figure 161. Mink (Neovison vison) Cuticular Tip Scale Pattern at 200x. Figure 162. Mink (Neovison vison) Cuticular Middle Shaft Scale Pattern at 200x. Figure 163. Mink (Neovison vison) Cuticular Root Scale Pattern at 200x. Figure 164. Mink (Neovison vison) Medullar Pattern at 200x. Mink-Neovison vison Cuticula imbricate, flattened. Medulla continuous, nodose, vacuolated, occupies more than 1/2 of shaft. Salient Features: Long, dark brown, with light yellow root. Average Total Length: cm. Average Width at Tip: µm. Average Width at Middle Shaft: µm. Average Width at Root: µm.

103 95 Figure 165. Badger (Taxidea taxus) Cuticular Tip Scale Pattern at 200x. Figure 166. Badger (Taxidea taxus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 167. Badger (Taxidea taxus) Cuticular Root Scale Pattern at 200x. Figure 168. Badger (Taxidea taxus) Medullar Pattern at 200x. Badger-Taxidea taxus Cuticula imbricate. Medulla continuous, nodose, vacuolated, occupies 1/3 of shaft. Salient Features: Longer than hairs of most other species, color pattern from tip to root is: white, yellow, black, yellow, white. Average Total Length: cm. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 2.7 µm. Average Width at Root: 0.8 µm.

104 96 Figure 169. Striped Skunk (Mephitis mephitis) Cuticular Tip Scale Pattern at 200x. Figure 170. Striped Skunk (Mephitis mephitis) Cuticular Middle Shaft Scale Pattern at 200x. Figure 171. Striped Skunk (Mephitis mephitis) Cuticular Root Scale Pattern at 200x. Figure 172. Striped Skunk (Mephitis mephitis) Medullar Pattern at 200x. Striped Skunk-Mephitis mephitis Cuticula imbricate, flattened. Medulla continuous, vacuolated, occupies more than 1/2 of shaft. Salient Features: Long, solid colors: black, white. Average Total Length: cm. Average Width at Tip: 0.2 µm. Average Width at Middle Shaft: 2.8 µm. Average Width at Root: µm.

105 97 Figure 173. White-Tailed Deer (Odocoileus virginianus) Cuticular Tip Scale Pattern at 200x. Figure 174. White-Tailed Deer (Odocoileus virginianus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 175. White-Tailed Deer (Odocoileus virginianus) Cuticular Root Scale Pattern at 200x. Figure 176. White-Tailed Deer (Odocoileus virginianus) Medullar Pattern at 200x. White-Tailed Deer-Odocoileus virginianus Cuticula imbricate, crenate. Medulla continuous, nodose, undisturbed, occupies more than 1/2 of shaft. Salient Features: Long, reddish brown in summer, gray in winter, can be distinguished from dog hair, as it will break if bent (personal communication, Pennsylvania Game Commission Wildlife Conservation Officer). Average Total Length: 5.6 cm. Average Width at Tip: 0.3 µm. Average Width at Middle Shaft: 2.7 µm. Average Width at Root: 0.7 µm.

106 98 Figure 177. Moose (Alces alces) Cuticular Tip Scale Pattern at 200x. Figure 178. Moose (Alces alces) Cuticular Middle Shaft Scale Pattern at 200x. Figure 179. Moose (Alces alces) Cuticular Root Scale Pattern at 200x. Figure 180. Moose (Alces alces) Medullar Pattern at 200x. Moose-Alces alces Cuticula imbricate. Medulla continuous, nodose, round cells. Salient Features: Long, color pattern from tip to root: dark brown, light brown, white. Average Total Length: 5.5 cm. Average Width at Tip: 0.6 µm. Average Width at Middle Shaft: 4.3 µm. Average Width at Root: 11.5 µm.

107 99 Figure 181. Domestic Dog (Canis familiaris) Cuticular Tip Scale Pattern at 200x. Figure 182. Domestic Dog (Canis familiaris) Cuticular Middle Shaft Scale Pattern at 200x. Figure 183. Domestic Dog (Canis familiaris) Cuticular Root Scale Pattern at 200x. Figure 184. Domestic Dog (Canis familiaris) Medullar Pattern at 200x. Domestic Dog-Canis familiaris Cuticula imbricate. Medulla discontinuous, compound, flattened, 4 columns, some rows, occupies entire shaft. Salient Features: Variable, depending on breed. Average Total Length: cm (varies). Average Width at Tip: 0.4 µm (varies). Average Width at Middle Shaft: 1.9 µm (varies). Average Width at Root: 2.9 µm (varies).

108 100 Figure 185. Human (Homo sapiens) Cuticular Tip Scale Pattern at 200x. Figure 186. Human (Homo sapiens) Cuticular Middle Shaft Scale Pattern at 200x. Figure 187. Human (Homo sapiens) Cuticular Root Scale Pattern at 200x. Figure 188. Human (Homo sapiens) Medullar Pattern at 200x. Human-Homo sapiens Cuticula imbricate, crenate. Medulla discontinuous, fragmental, can be absent in some places. Salient Features: Variable. Average Total Length: cm (varies). Average Width at Tip: µm (varies). Average Width at Middle Shaft: 1.7 µm (varies). Average Width at Root: µm (varies).

109 Bat Species 101 Figure 189. Keen s Myotis (Myotis keenii) Cuticular Tip Scale Pattern at 200x. Figure 190. Keen s Myotis (Myotis keenii) Cuticular Middle Shaft Scale Pattern at 200x. Figure 191. Keen s Myotis (Myotis keenii) Cuticular Root Scale Pattern at 200x. Keen s Myotis-Myotis keenii Cuticula coronal. Medulla not present. Scale Index: *0.4. Width Index: *3. Salient Features: Short, dark brown. Average Total Length: not measured. Average Width at Tip: 0.05 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.2 µm.

110 102 Figure 192. Little Brown Myotis (Myotis lucifugus) Cuticular Tip Scale Pattern at 200x. Figure 193. Little Brown Myotis Myotis (Myotis lucifugus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 194. Little Brown Myotis (Myotis lucifugus) Cuticular Root Scale Pattern at 200x. Little Brown Myotis-Myotis lucifugus Cuticula coronal. Medulla not present. Scale Index: 0.5. Width Index: 2. Salient Features: Short, brown and yellow. Average Total Length: not measured. Average Width at Tip: 0.15 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.25 µm.

111 103 Figure 195. Red Bat (Lasiurus borealis) Cuticular Tip Scale Pattern at 200x. Figure 196. Red Bat (Lasiurus borealis) Cuticular Middle Shaft Scale Pattern at 200x. Figure 197. Red Bat (Lasiurus borealis) Cuticular Root Scale Pattern at 200x. Red Bat-Lasiurus borealis Cuticula coronal. Medulla not present. Scale Index: 0.4. Width Index: 2. Salient Features: Short, dark orange-red. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 0.2 µm. Average Width at Root: 0.3 µm.

112 104 Figure 198. Silver-Haired Bat (Lasionycteris noctivagans) Cuticular Tip Scale Pattern at 200x. Figure 199. Silver-Haired Bat (Lasionycteris noctivagans) Cuticular Middle Shaft Scale Pattern at 200x. Figure 200. Silver-Haired Bat (Lasionycteris noctivagans) Cuticular Root Scale Pattern at 200x. Silver-Haired Bat-Lasionycteris noctivagans Cuticula coronal. Medulla not present. Scale Index: Width Index: 2. Salient Features: Short, black with silver or white. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.2 µm.

113 105 Figure 201. Tri-Colored Bat (Perimyotis subflavus) Cuticular Tip Scale Pattern at 200x. Figure 202. Tri-Colored Bat (Perimyotis subflavus) Cuticular Middle Shaft Scale Pattern at 200x. Figure 203. Tri-Colored Bat (Perimyotis subflavus) Cuticular Root Scale Pattern at 200x. Tri-Colored Bat-Perimyotis subflavus Cuticula coronal. Medulla not present. Scale Index: Width Index: 2. Salient Features: Short, three bands of color: dark root, pale middle shaft, brown tip. Average Total Length: not measured. Average Width at Tip: 0.1 µm. Average Width at Middle Shaft: 0.3 µm. Average Width at Root: 0.3 µm.