Field and Genetic Methodologies for the Study of Felids in the

Selva Lacandona, Chiapas Mexico: A Noninvasive Approach

by

Nashieli Garcia Alaniz

Thesis presented as a partial requirement in the Doctor of Philosophy (Ph.D) in Biomolecular Sciences

School of Graduate Studies Laurentian University Sudbury, Ontario Canada

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Previous work has shown that in most ecosystems, the first species to disappear due to

anthropogenic activity are the large predators and specialists. In tropical ecosystems, the felids

represent this guild. Five species of felids have been reported for the Selva Lacandona, Mexico:

jaguar {Panthera oncd), puma {Puma concolor), ocelot {Leopardus pardalis), jaguarundi {Puma yagouaroundi) and margay {Leopardus wiedii). Currently all these species are threaten by habitat

loss, fragmentation and human persecution and are listed in Appendix I and II by CITES

(Convention on International Trade of endangered Species of Wild Fauna and Flora). Also they

are listed by IUCN (International Union for the Conservation of Nature) that monitors and

protects endangered species, since evidence indicates that all populations of these felids are in

decline. Therefore, it is necessary to generate information on these wild populations that

eventually will lead to the development of appropriate management strategies.

The primary objectives of this study were: (1) to generate information on the population

of the five species of felids present in the Selva Lacandona, Chiapas, Mexico, (2) to develop and

test field and genetic techniques for the study of the populations of felids present in the Selva

Lacandona, and (3) to identify management strategies that will sustain felid populations in

Neotropical ecosystems and prevent extirpation and extinction.

Scale and medullar patterns of the guard hair of each terrestrial mammalian species of

Chiapas were photographed and described and a hair catalogue and a dichotomous key were developed. The scale and medullar patterns of 79 species of terrestrial mammals are presented and described in this work including 22 Families (100% of the Families) and 10 Orders (100% of the Orders). This thesis evaluates the utility of hair-snares to collect mammalian samples with a specific goal of assessing felid populations. We recorded 389 hits on 888 hair-snare stations over the two years of the study which represents a hit rate of 43%. A total of 240 hits over 560 hair-snare stations (42%) were reported the undisturbed areas and a total of 100 hits over 328 hair-snares stations (30%) were reported in the disturbed agricultural areas. From the hair samples obtained using the hair-snare method, we identified margay (Leopardus wiedii, n=2), ocelot (Leopardus pardalis, n=l) and jaguarundi (Puma yaguarundi, n=l).

In addition, primers for species-specific partial sequences of cytochrome b were developed to identify each of the five felid species found in this area. Primers were tested on blood samples obtained from zoo specimens and on hair samples obtained from the field.

Successful amplification was obtained when tested on all blood samples from zoo specimens from the Chiapas region and 3 out of 5 of the target species were identified from field samples.

The primers were especially useful when used on samples with low-yield and degraded DNA and the species-specific sequences allowed for discrimination among species.

The deficit of information on carnivore populations and specifically felids in tropical ecosystems is partially due to the lack of reliable cost effective methodologies and techniques that allow managers to obtain data that will eventually lead to the development of appropriate management strategies. The hair-snare method and results presented in this study in combination molecular markers developed here have a great potential for studying felids in tropical ecosystems. These techniques are valuable for monitoring species of mammalian carnivores and can be used on projects that specifically aim to conserve felid species by supporting ecological research.

iv Dedication

Este trabajo esta dedicado a mis padres Chelo y Fernando y a mi hermano Rafael, con quienes comparti cada momento de esta etapa. Su incansable apoyo y confianza hicieron que pudiera alcanzar este sueno.

v Acknowledgments

I want to express my gratitude to my co-supervisors, Dr. F.F. Mallory and Dr. A. Omri,

who provided me with much support and guidance throughout this project.

I would also like to thank my Research Advisory Committee members, Dr. E.J. Naranjo

and Dr. K. Nkongolo for their constant support, pointed suggestions and comments, which enhanced the writing of this thesis. I would also like to thank to Dr. F.V. Clulow for being my internal examiner and Dr. J. Schaefer (Trent University, Peterborough) for being my external examiner. A very special thank to Dr. A. Kumar, Director of the Biomolecular Sciences Doctoral

Program for his direction and support.

Financial support was provided by The Mexican National Council for Science and

Technology (CONACYT) (Graduate Scholarship 167825 to NGA), Laurentian University

(Waiver of International Tuition), NSERC for the Research Capacity Development Award and the Chair in Cancer Research Stipend. El Colegio de la Frontera Sur, Unidad San Cristobal,

Chiapas, Mexico, provided logistic support.

I would also like to thank the people of the local communities in Chiapas, where this study took place; their hospitality was a key element. To my colleagues at Ecosur; Carlos,

Emilio, Elena, Fatima and Salvador, who helped me during the field work and showing me unconditional support. To Fernando Garcia, who helped me to build all the hair traps and Elsy

Cabrera at Zoologico Miguel Alvarez del Toro and Dr. P. J. Wilson and the people at the Natural

Resources DNA Profiling and Forensic Centre, Peterborough for their help with the DNA analysis.

I also want to thank to my friends at Laurentian University, Kelly, Ashley, Paul, Curtis,

Jesse, Becky and Melanie who were always willing to help me as friends and colleagues.

vi Agradezco a toda mi familia y amigos en Mexico y varias partes del mundo que me expresaron su apoyo continuamente y creyeron en mi. Su confianza y constante presencia me mantuvieron cerca de pesar de la distancia. Quiero agradecer a la familia Gordillo Alaniz, por su apoyo incondicional especialmente durante el transcurso de mi trabajo de campo. A Pablo, Sofia,

Carlos, Elisa, Isaias, Wendy, Ivan, la familia Figueroa y toda esa gente que me ayudo cuando mas lo necesite.

vii List of Abbreviations

Pon Panthera onca Pco Puma concolor Lpa Leopardus pardalis Pya Puma yagouaroundi Lwi Leopardus wiedii cyt b cytochrome b MABR Montes Azules Biosphere Reserve UNESCO United Nations, Educational, Scientific and Cultural Organization IUCN International Union for Conservation of Nature CITES Convention of International Trade of endangered Species of Wild Fauna and Flora UNAM Universidad Nacional Autonoma de Mexico ECOSUR Colegio de la Frontera Sur CONACYT Mexico's National Council of Science and Technology NRDPFC Natural Resources DNA Profiling and Forensic Centre DNA Deoxyribonucleic acid PCR Polymerase chain reaction

viii Table of Contents Abstract iii Dedication v Acknowledgments vi List of Abbreviations viii Table of Contents ix List of Figures and Tables xi

Chapter 1. General Introduction 1 1.1 General Introduction 1

Chapter 2. Taxonomic Guide to the Guard Hairs of Terrestrial Mammals from Chiapas, Mexico 13 2.1 Abstract 13 2.2. Resumen 14 2.3 Introduction 15 2.4 Study Area 16 2.5 Materials and Methods 17 2.6 Results and Discussion 18 2.7 Acknowledgments 23

Chapter 3. Key for the Identification of Guard Hairs of the Terrestrial Mammals from Chiapas, Mexico 25 3.1 Terms of Reference 25 3.2 Key for the Identification of Guard Hairs of the Terrestrial Mammals from Chiapas, Mexico v. 29

Chapter 4. Atlas of the Scale and Medullar Patterns of the Guard Hairs of Terrestrial Mammals from Chiapas, Mexico 47

Chapter 5. Hair-snares: A Noninvasive Method for Monitoring Felid Populations in the Selva Lacandona, Mexico 69 5.1 Abstract 69 5.2 Resumen 70 5.3 Introduction 71 5.4 Study Area 73 5.5 Hair-Snare Surveys 73 5.6 Results 78 5.7 Discussion 79 5.8 Implications for Felid Conservation 82 5.9 Acknowledgments 82

Chapter 6. Species-specific Cytochrome b amplifications for Mexican Neotropical Felids 83 6.1 Abstract 83

ix 6.2 Introduction 6.3. Material and methods 84 6.4. Results and Discussion 85 6.5. Acknowledgements 87

Chapter 7. Conclusions and Future Work 88 7.1 Overview 88 7.2 General Conclusions 89

Literature Cited 90

Appendix

x List of Figures and Tables

Chapter 1 Figure 1.1 Location of the Montes Azules Biosphere Reserve 3

Chapter 2 Figures 2.1 Medullar patterns of guard hairs of the terrestrial mammals from Chiapas, Mexico 21 Figure 2.2 Cortical scale patterns of guard hairs of the terrestrial mammals from Chiapas, Mexico 21

Chapter 5 Figure 5.1 Location of hair-snares transects in the Montes Azules Biosphere Reserve and surrounded Selva Lacandona, Chiapas, Mexico 75 Figure 5.2. Picture showing a jaguar (Panthera onca) rubbing against the hair-snare baited with lures at the zoo in Chiapas and a hair sample obtained from this jaguar 76

Chapter 6 Table 6.1 Nucleotide diagnostic species-specific sequences of 144bp from the cytochrome b gene of five Neotropical felid species from Chiapas, Mexico. Species codes: Pon (Panthera onca), Pco (Puma concolor), Lpa (Leoparduspardalis), Pya (Puma yagouaroundi) and Lwi (Leopardus wiedii) 86

xi Chapter 1. General Introduction

1.1 General Introduction

Biodiversity in tropical ecosystems is greater than most other ecosystems and local species

densities are often patchy (Connell 1978, Gentry 1986). Such patterns of variation complicate

the development of practical strategies for conservation in rain forest ecosystems (Leith and

Werger 1989, Medellin 1994). In addition, human activities have greatly diminished the

original extent of the rainforest area (Anonymous 1992, Medellin 1994) and therefore research

on species in these regions is one of the most urgent priorities for the conservation biologists.

Most studies that have evaluate state of biodiversity and ecosystems support the development

of biological and environmental management strategies for tropical systems.

Similar to other locations in the tropics, the region known as the Selva Lacandona in

Chiapas, Mexico is characterised by high species richness and is facing imminent destruction

due to human encroachment (Soule and Kohm 1989, Medellin 1994). This area is a major portion of tropical rainforest remaining in Mexico and according to reports from the

government of Chiapas (Anonymous 1992), currently just one third of the area remains covered with forest. The Selva Lacandona is still connected with the Guatemalan Peten by a corridor running from Bonampak to the Yaxchilan archaeological site on the Usumacinta River, which

is on the border with Guatemala (Medellin 1994). The Selva Lacandona is a remanent of the

Mesoamerican tropical forest and is considered one of the 25 "hot spots " and one of the most important areas of biodiversity in the world (Mittermeier and Goettsh 1992). With very high numbers of endemic species and serious loss of habitat, these hot spots are key areas for worldwide conservation (Myers et al. 2000).

1 Human encroachment and activity have made severe changes in the distribution and abundances of species in these areas. Recent studies have shown that the greatest threats in this area are habitat loss and fragmentation associated with land ownership, land invasions, forest fires, deforestation and land use changes (Medellin 1994, INE 2000, Naranjo 2002, Naranjo et al. 2004). In addition, floral and faunal extractions for commercial and subsistence purposes and wildlife trafficking and the introduction of exotic species represents significant pressures in the area (Naranjo 2002).

Over three-fifths of the forested area in the Selva Lacandona is within the Montes Azules

Biosphere Reserve (MABR). This reserve is located in the State of Chiapas in southeast

Mexico and it is comprised of 331, 200 hectares of protected forest established in 1978 (Figure

1.1).

The Selva Lacandona is one of the largest areas of humid tropical forest in Mexico and

Central America and is the most biological diverse area in North America and considered a priority for conservation. The reserve has been recognized internationally as part of UNESCO's

Man and Biosphere Program (MAB-UNESCO) since 1979 (INE 2000), represents the last large portion of tropical rain forest in Mexico and urgent solutions are needed to ensure protection and to maintain biodiversity.

2 u>

Figure 1.1. Location of the Montes Azules Biosphere Reserve. Previous work has shown that in most ecosystems, the first species to disappear due to anthropogenic activity are the large predators and specialists (Ceballos and Ehrlich 2002,

Woodroffe and Ginsberg 1998, Laliberte and Ripple 2004). In tropical ecosystems, the felids represent this guild. Five species of felids have been reported for the Selva Lacandona: jaguar

(Panthera onca), puma (.Puma concolor), ocelot (Leopardus pardalis), jaguarundi (Puma yagouaroundi) and margay (Leopardus wiedii) (Aranda 2000, Retana and Lorenzo 2002).

These species of Neotropical felines are sympatric in several areas of the Americas and molecular markers have shown that the jaguar species belongs to the Panthera lineage, the puma and jaguarondi are part of the Puma lineage and the ocelot and margay are sister species that belong to the Ocelot lineage (Masuda et al. 1996, O'Brien and Johnson 2005). Currently all these species are threatened by habitat loss, fragmentation and human persecution and are listed by CITES (Convention of International Trade of endangered Species of Wild Fauna and Flora) which limits trade in these species and their products by ratifying nations and trade is banned for commercial purposes. These species are also listed as threatened by IUCN (International

Union for Conservation of Nature) that monitors and protects endangered species. Under

UICN, all these species were listed under the category of "least concerned" in the 1990; however, the jaguar and the puma are listed now in the category of "near threatened".

According to the Red List, all these felid species have decreasing population trends and could change to more restricted categories if better information on of each species was available

(Nowell and Jackson 1996).

The jaguar is the largest felid in the Americas and historically, this species ranged from the southern USA throughout Central and South America to Argentinean Patagonia. Based on this extended distribution, jaguars are clearly suited to survive in a variety of environments;

4 however, they are most commonly found in areas with high plant cover and abundant water

supply and with a high prey density (Seymur 1989). Jaguars are solitary except during mating

season and when cubs are dependant of their mother. Estimates of home range vary from 10

2 2 km to 100 km depending on the environment type. Females usually have smaller ranges and

neighboring female home ranges often overlapped, while male home ranges seldom overlap

and often include a number of female territories (Seymur 1989). Although many prey species

have been reported in their diet, the prey on which they most frequently specialized are peccaries (Tayassu tajacu and T. pecari) and capybaras (Hydrochoerus hydrochaeris) (Seymur

1989).

The puma is also a large felid with an original distribution throughout the Americas that was much greater than that of the jaguar and it is generally considered a more adaptable species.

The original range of the puma included most of the United States and southern Canada through to Argentina. The puma is the largest member within the Puma genus and its home range varies between season and years, ranging from 50 km2 to 140 km2. Similar to the jaguar, this species is solitary and home ranges of females often overlap while territories of males do not. The density of pumas is strongly dependent on the prey basis and the species taken often are mule deer (Odocoileus hemionus) and white tailed deer (O. virginianus). Pumas and jaguars are sympatric in many areas and a number of studies have reported that they avoid each other under these conditions (Schaller and Crawshaw 1980, Rabinowits 1986, Emmons 1987). Pumas tend to use drier environments, while jaguars will use areas associated with water.

Since European settlement, extermination programs, hunting pressure and habitat loss have all resulted in reduced ranges for both species despite internationally protected programs. At

5 present, the ranges of these two species continue to decrease rapidly, as they have been

extirpated from many areas in recent times (Currier 1983, Kitchener 1991, Hoogesteijn 2003).

The other felid species in the Selva Lacandona are small to medium sized felids whose

populations have had similar declines. The ocelot and margay are spotted cats that resemble

each other and have similar physical characteristics (Eizirik et al. 1998) although ecologically

they vary considerably, as the ocelot can occur in a variety of habitats ranging from tropical-

humid forests to dry scrub lands, while the margay is exclusively a dweller of humid tropical

forests. The ocelot is often active during the day, while the margay is mainly nocturnal (Murray

and Gardner 1997, Oliveira 1998).

The diet of the ocelots is comprised mainly of mammalian prey greater than 1 kg and home

ranges usually increase during the dry season and range from 2 to 11 km2, with male home

ranges sometimes 3 times larger than female home range. Murray and Gardener (1997) noted

that male territories often overlap with several females' territories although female territories

seldom overlap.

The margay is smaller than the ocelot and the diet consists mainly of small arboreal

mammals. The home range of this species has been calculated from 10 to 15 km2, which is

considered large for a felid with this small body size (Crawshaw 1995, Konecny 1989);

however, little is known of this species and its ecology.

The jaguarundi is another species that coexists in the Selva Lacandona. It is a medium sized non-spotted felid and often considered a cat with relative less feline features. The jaguarondi is less nocturnal than most cats having a crepuscular behavioral pattern and hunting more during the mornings and evenings (Oliveira 1998). Home ranges for the species range from 7 to 17

6 2 km ; however, more studies are needed to corroborate this information. This species preys

mainly on small rodents and birds (Oliveira 1998).

These three medium sized species of felines are sympatric throughout much of their range

although the ocelot reaches more northern areas, including the southern United States. In

contrast, the jaguarondi ranges to more southern areas then the ocelot and margay.

All these five species of felines are currently classified as threatened and it is therefore

essential to develop management strategies to conserve their populations throughout their

remaining ranges and to develop site-specifics conservation strategies. In addition, as top predators in the food chain, conservation of these species would lead to the persistence of many

others species at lower trophic levels (Steneck 2005, Miotto et al. 2007).

The solitary behaviour, low population densities and large home ranges of felids makes information on wild populations difficult to obtain and in some regions their ranges are not easily accessible, often resulting in little data. However, studies using noninvasive research methods in combination with molecular techniques have been shown to be of enormous value and thus these methods should be promoted.

To understand the population ecology of a species, one must document its presence and abundance. However, this can be difficult especially for carnivores (Nowell and Jackson 1996,

Palomares et al. 2002, Kurose et al. 2005).

The study of felids in the Selva Lacandona represents a particular challenge due to the elusive behaviour and low densities of these species and to the intrinsic characteristics of the Selva

Lacandona region. The area is difficult to access especially during the rainy season (June to

November) when much of the forest floods.

7 Combining molecular techniques with noninvasive collection methods to obtain tissue samples can provide the information needed to develop viable management strategies. Recent developments in molecular technology have provide great potential to improve the accuracy of data on wild individuals and populations and is especially important for obtaining information on elusive and endangered species such as New World tropical felids. However, despite the fact that the combination of these methods is of great utility, to use them effectively it is necessary to test them in various environments and ecosystems. Adequate reference databases for the specific species and geographic regions are also important. Once these baselines are established, it is then possible to take advantage of these methods.

Previous work using DNA sequences from hair follicles of wild mammals has been employed to identify species, individuals, gender and genetic relatedness (Foran et al. 1997,

Mills et al. 2000, Sloane et al. 2000, Mowat and Paetkau 2002, Boersen et al. 2003, Fernandes et al. 2008). It also has the potential of proving a more accurate assessment of population richness and determining such parameters as home range size, habitat utilization and genetic variability within populations. To use DNA sequences from hair follicles to obtain information on felid population in the Selva Lacandona, it was necessary to establish effective field methods and appropriate control databases for obtaining and analyzing these data.

Mammalian studies that use noninvasive techniques include methods that vary from assessing tracks and using camera traps to the collection of faeces and hair samples. Each of these techniques has intrinsic pros and cons and been used in studies where results vary from only detecting the presence of species through to the identification of individuals and their gender (Foran et al. 1997, Scott et al. 2004, Kurose et al. 2005, Sharma et al. 2005, Alibhail et al. 2008). Tracks and cameras do not allow for molecular analysis and are relative inaccurate

8 when identifying similar sympatric species. Studies that rely on the collection of faeces or hair

samples can provide opportunity for further molecular analysis. These studies can provide

accurate data and identify species and individuals. However the use of DNA obtained for

noninvasive techniques have always the possibility of being contaminated of not having enough

yield.

The use of hair snags is a method that is a noninvasive and can provide samples for genetic

analysis. This method is less invasive than live trapping which is often logistically difficult,

expensive and detrimental to the animal. In addition, this method of collecting hair samples has

an advantage in tropical ecosystems over the collection of scat samples, due to the high

decomposition rate that occurs in wet and hot environments. In addition, the behavior of felids

makes them an appropriate taxon for hair sampling as this technique uses rub stations that are

sprayed with specific attractants to felids. This technique has been used in other studies of

felids and other mammalian species (McDaniel et al. 2000, Weaver et al. 2005, Schmidt and

Kowalczyk 2006). Some studies had positive results collecting hairs and identifying species

and individuals such as lynx (.Lynx canadensis) in Canada and Poland, and ocelots in southern

Texas (McDaniel et al. 2000, Weaver et al. 2005, Schmidt and Kowalczyk 2006). However, when trying to identify pumas in California and margay in Tamaulipas, Downey et al. (2007) obtained results that were not expected. After 8 surveys, they were unable to detect any of the target species, suggesting that grey fox present in the area affects success on detecting felids using hair snares, however, this supposition needs further examination. In addition, this study suggests that the few are covered when detecting puma was not sufficient due to its low density and large home ranges that this species presents. Therefore, it was necessary to evaluate the

9 value of hair-snares as a noninvasive method for collecting felid hair samples in the Selva

Lacandona.

In order to establish the identity of the hairs of the mammals of Chiapas, Mexico, it was

necessary to establish a key of the mammalian guard hairs in the region based on morphology.

Previous work on the scale and medullar patterns of mammalian guard hairs in different areas

was shown to be successful for the identification of species in the wild. Since hair has a

distinctive microstructure and is relatively resistant to damage, it has been used for the

identification of mammalian species. A number of studies have shown that hair usually

possesses species-specific characteristics. The application of hair identification has been used by many authors (Mayer 1952, Stain 1958, Adorjan and Kolensosky 1969, Moore et al.1974,

Hilton and Kutsha 1978, Kennedy and Carbyn 1981, Kennedy 1982, Wallis 1992, Fernandez

and Rossi 1998, Mowat and Strobeck 2000, Sloane et al. 2000, Baca Ibarra 2002, Rodriguez de la Gala Hernandez 2002).

A number of characteristic have been used for hair identification. Cross sections of hairs have been reported to be useful in the identification species (Moore et al. 1974, Hilton and

Kutsha 1978); however, the shape and diameter of hairs can change easily and such analysis requires microtomes or other sophisticated cutting tools (Harrison 2002). The coloration patterns of hairs have also been used (Wallis 1992, Monroy and Rubio 2003); however, this characteristic varies a lot even in the same individual and must be used with care. In contrast, the scale and medullar patterns of guard hairs are less variable and therefore a better method for identification of mammalian species. Guard hairs from different body areas can have coloration, length and diameter differences, but similar cuticular scale and medullar patterns.

10 For DNA analysis, noninvasive methods such as hair and scat collection often provide

samples with degraded DNA. Therefore, in order to have diagnostic mitochondrial markers for

the Selva Lacandona study where the felid species are sympatric it was necessary to develop a

database of species specific partial sequences and their appropriate primers.

Primers for species specific partial sequences of cytochrome b where developed to identify

each of the five felid species from Chiapas, Mexico and tested on blood samples obtained from

zoo specimens originally from the same region. These primers and species specific sequences

were subsequently used to identify hair samples from felid species in the field.

The primary objectives of this study were:

(1) to generate information on the population of the five species of felids present in the Selva

Lacandona, Chiapas, Mexico, (2) to identify management tools that would sustain felid populations over time in the Selva Lacandona, Mexico, (3) to create a reference collection of guard hairs of the terrestrial mammals from the State of Chiapas, Mexico,

(4) to create a field guide to the guard hairs of the terrestrial mammals from Chiapas, Mexico,

(5) to develop a dichotomous key supplemented with a catalogue of photographs of the scale and medullar patterns of the guard hairs of terrestrial mammals from Chiapas, Mexico, (6) to assess general mammalian activity through the use of hair-snares in the Selva Lacandona,

Chiapas, Mexico and compare this activity in relation to habitat fragmentation and ecological changes associated with human settlement, (7) to assess the assemblage of felid population through the use of hair-snare on the Selva Lacandona as well as the population of each species of felids and compare each one of these categories in relation to habitat fragmentation and ecological changes associated with human settlement, (8) to develop DNA primers with species specific markers to identify each of the five felid species from Chiapas, Mexico, (9) to test

11 these DNA primers with specific markers on blood samples obtained from zoo specimens of the five felid species originating from Chiapas, Mexico, (10) to test the utility of these DNA primers with specific markers to identify hair samples obtained from wild felids in Chiapas,

Mexico.

We hypothesized: (1) that all terrestrial mammalian species from Chiapas can be identified based on the scale and medullar patterns of their guard hairs, (2) That hair snares will allow assessing the general mammalian activity as well as the assemblage of felids on the Selva

Lacandona, (3) that DNA primers with specific genetic markers can be developed to discriminate among the five felid species, (4) that these primers will test positive on blood samples taken from zoo animals originating from Chiapas, Mexico and (5) that individuals of each felid species can be identified from hair samples obtained from the field based on the

DNA markers developed.

12 Chapter 2. Taxonomic Guide to the Guard Hairs of Terrestrial Mammals from Chiapas, Mexico

2.1 Abstract

Previous work on the scale and medullar patterns of mammalian guard hairs has been successful for the identification of species in the wild, since hair has a distinctive microstructure, is easy to obtain and is relatively resistant to damage.

Mexico has the second highest number of mammalian species and the third highest number of endemic mammal species of any country in the world. The State of Chiapas has the second highest mammalian species richness within in the country and research on these wild mammalian species is critically needed as many are threatened with extinction due to human activity. Therefore, information needed to develop viable management strategies is essential and urgent.

The primary objectives of this study were: (a) to develop a guide to the guard hairs for the terrestrial mammals of Chiapas, for taxonomic, ecological and conservation research and (b) to develop a dichotomous key supplemented with a catalogue of micro-photographs of the medullar and scale patterns of the guard hairs of the terrestrial mammals of Chiapas.

An updated taxonomic list of the terrestrial mammals of Chiapas by Retana and Lorenzo

(2002) was used and hair samples were obtained from 4 museum collections. The medullar and cortical scale patterns of each species were photographed and described and a hair catalogue and dichotomous key were developed. The medullar and scale patterns of 79 terrestrial mammalian species are presented and described, which includes each of the 22 families and 10 orders in the region. Of the 79 species, the key identifies 62 to species and 17 to genus and further separation may be possible using subtle features in the photos and geographic parameters. Analysis of the medullar and scale patterns identified 28 different medullar patterns

and 8 different primary scale patterns among these species.

The techniques presented are low cost technology and inexpensive and valuable for

obtaining information on elusive and endangered species that are often logistically and

politically difficult to study. In addition, recent developments in DNA analysis can add

substantially to the utility of these kinds of samples.

Key words: Chiapas, scale patterns, guard hairs, hair identification, mammals, medullar patterns, Mexico.

2.2 Resumen

Estudios anteriores han demostrado que el patron de medula y de escamas del pelo de guardia de los mamiferos presenta una microestructura caracteristica la que permite identificar especies. Ademas, el pelo de guardia presenta la ventaja de ser relativamente facil de colectar y ser resistente. Mexico es el segundo pais con mayor riqueza de especies de mamiferos en el mundo y el tercero en especies endemicas. El estado de Chiapas presenta a su vez el segundo lugar de riqueza dentro del pais. Investigation sobre estas especies es necesaria y urgente ya que se encuentran amenazados principalmente por actividades humanas. Este conocimiento llevara a generar estrategias de manejo viables. Los objetivos de este estudio fueron; (a) desarrollar una gui'a del pelo de guardia de los mamiferos terrestres del estado de Chiapas, para estudios taxonomicos, ecologicos y de conservation, (b) desarrollar una clave dicotomica ilustrada con un catalogo de microfotografias del patron medular y de escamas del pelo de guardia de los mamiferos terrestres de Chiapas. Generamos una lista taxonomica actualizada de los mamiferos de la zona y colectamos muestras de pelo en 4 colecciones cientificas. El patron de medula y de escamas de cada especie fue fotografiado y descrito en la parte proximal y

14 distal. Se elaboro un catalogo y una clave dicotomica. Este trabajo presenta el patron de medula

y de escamas de 79 especies de mamiferos terrestres presentes en el estado, representando 22

Familias (100% de las Familias) y 10 Ordenes (100% Ordenes). De las 79 especies, la clave

dicotomica identifica 62 a nivel de especie, 17 a nivel de genero y posterior separation podria

ser posible tomando en cuenta patrones geograficos. El analisis de medula y escamas

identifico 28 patrones de medula y 8 patrones de escamas diferentes. La tecnica de

identificacion de los mamiferos por medio del pelo de guardia permite un metodo de bajo

costo, confiable y efectivo para poder obtener information de especies amenazadas que

generalmente son dificiles de estudiar en vida silvestre como lo es en el estado de Chiapas.

Ademas, estudios recientes realizados en base a ADN obtenido del pelo, puede incrementar la utilidad de la identificacion de este tipo de muestras.

Palabras clave: Chiapas, escamas, medula, Mexico, pelo de guardia.

2.3 Introduction

Previous work on scale and medullar patterns of mammalian guard hairs in different geographic regions has been successful for the identification of species in the wild, since hair has a distinctive microstructure, is easy to obtain and is relatively resistant to damage even when passed through the digestive tracts of carnivores. Analyses of hair structure has shown that hair possesses species-specific characteristics and the application of hair for the identification of mammalian species has been described by a number of authors (Mayer 1952,

Stain 1958, Adorjan and Kolensosky 1969, Brunner and Coman 1974, Moore et al. 1974,

Hilton and Kutsha 1978, Kennedy and Carbyn 1981, Kennedy 1982, Wallis 1992, Fernandez and Rossi 1998, Mowat and Strobeck 2000, Sloane et al. 2000, Baca Ibarra 2002, Rodriguez de la Gala Hernandez 2002, Boersen et al. 2003).

15 Several authors have identified hairs using characteristics such as; color, total length, cross

sections and medullar and scale patterns (Stain 1958, Moore et al. 1974, Hilton and Kutsha

1978, Kennedy 1982, Wallis 1992, Monroy-Vilchis and Rubio-Rodriguez 2003). However, the

first three characteristics can vary significantly in the same individual or require complicated

methodologies (Harrison 2002). Conversely, the scale and medullar patterns of guard hairs are

less variable and better for the identification of mammalian species. Guard hairs from different body areas often have color, length and diameter differences, but similar scale and medullar patterns.

Chiapas, Mexico has one of the highest mammalian species richness in the world and research on these wild mammal species is critically needed as many species are threatened due to human activity (Medellin 1994, Retana and Lorenzo 2002). The primary objectives of this study were: (a) to develop a guide to the guard hairs of the terrestrial mammals from Chiapas, for taxonomic, ecological and conservation research and (b) to develop a dichotomous key supplemented with a catalogue of photographs, where the medullar and scale patterns are described for each terrestrial mammalian species. Bats were not included in this study as their behavior precludes them from being represented in mammalian stomach contents, scats and hair snare studies (Garcia Alaniz 2004) and their occurrence in owl pellets is rare.

2.4 Study Area

Chiapas is a state located in the southeast region of Mexico with an area of 73, 887 km2. The

State has 3 main climate types: tropical, subtropical and temperate (Sarukan 1968, Rzedwsky

1978, Aranda and March 1987) and the second highest mammalian species richness within

Mexico. Of the 204 mammalian species listed for Chiapas, 66 are endemic: 7 species to

Chiapas, 9 to Mexico and 50 to Mesoamerica (Retana and Lorenzo 2002).

16 2.5 Materials and Methods

A taxonomic list of terrestrial mammals of Chiapas was used from the publication of Retana

and Lorenzo (2002), as it was the most accurate for the region. Hair samples and their voucher numbers were collected from 4 mammalian collections: the National Mammal Collection,

Institute of Biology (UNAM); the Faculty of Science, National Autonomous University of

Mexico (UNAM); the Colegio de la Frontera Sur, Unidad San Cristobal, Chiapas, Mexico; and the Natural History Institute, Tuxtla Gutierrez, Chiapas, Mexico. Dorsal guard hairs from the mid-dorsal region of each specimen were collected.

On processing, all samples were initially placed in a Petri dish with water for five minutes, then washed in a sieve with soap and water, and subsequently rinsed with distilled water to remove oils and debris. Each hair was subsequently air-dried in a paper cup. We followed a modified version of the hair imprinting method described by Moore et al. (1974) to obtain medullar and scale patterns. Scale patterns were obtained by making a negative impression of the cuticular scale surface for each hair specimen (Brunner and Coman 1974). In this study, we placed clean and complete hair samples on cleaned microscope slides covered with a thin layer of clear nail polish. Individual hairs were handled with fine forceps and a mounting needle and gently pressed into the medium. After 3-5 minutes and before the nail polish got completely dry, the hair was removed, leaving behind an imprint of the scales.

To obtain medullar patterns, hairs were placed in a xylene solution to reveal the internal structures of the medulla. Time to clearing varied with species ranging from 24 hours to 15 days. In addition, a reference collection of permanent mounts were made by clearing hairs in xylene and transferring them to glass slides. Permanent mounts were made by using

"Permount" (Fisher Scientific Company) in place of the xylene and hairs were then protected

17 with a cover slip (Arita and Aranda 1987). Scale patterns of each species were described at the

proximal and distal ends of the hair and the medullar pattern was then added. Finally, digital

pictures were taken of the scale and medullar patterns of each species. All pictures of hair

structure where taken at magnification (100X). Once the description of the different scale and

medullar patterns was completed for all species, we developed a hair catalogue and a

dichotomous key.

2.6 Results and Discussion

This field guide includes 79 terrestrial mammalian species belonging to 22 families and 10

orders, representing 100% of the families and orders reported for the State. Results of the

analysis of guard hairs of terrestrial mammals from Chiapas identified 28 medullar patterns and

8 different scale patterns (Figures 2.1 and 2.2). Moore et al. (1974) identified 10 medullar

patterns in Wyoming mammals, while Aranda (1991) reported 11 medullar patterns in Mexican

mammals. Additionally, Moore et al. (1974) reported 10 different scale patterns in Wyoming

mammals, while Aranda (1991) reported 13 different scale patterns for Mexican mammals.

Such differences may be due to observer subjectivity and differences in species richness between regions and for this reason it was important to establish a catalogue of pictures of the scale and medullar patterns to supplement the key.

The identification of hair samples can be a valuable taxonomic and wildlife management research tool. Various authors have developed guides for the identification of guard hairs of mammals from different regions (Mayer 1952, Stain 1958, Adoijan and Kolensosky 1969,

Moore et al. 1974, Wallis 1992, Fernandez and Rossi 1998, Baca Ibarra 2002, Rodriguez de la

Gala Hernandez 2002, Monroy-Vilchis and Rubio-Rodriguez 2003). This technique has been used to assess diet, since it allows the identification of prey remains in mammalian feces and

18 avian pellets (Delibes 1980, Kennedy and Carbyn 1981, Kennedy 1982, Nowell and Jackson

1996) and has been used to identify mammalian parts or skins that have been confiscated from

illegal trade or hunting (Aranda 1991).

Clear Medullar Patterns

Clear clear with blotches

clear with vertical broken clear with continuous clear with vertical plus streaks vertical lines lateral broken streaks 1 F ES-.?!.'.! !!-' :':•: f •:-!-! •••.twipJSUJASTT- r I i

Medullar Patterns with Inclusions

1 medullar inclusion 1 and 2 medullar inclusior 2 medullar inclusions

2 and 3 medullar inclusior 3 medullar inclusions 3 and 4 medullar inclusior

19 4 medullar inclusions alternating/touching alternating/non-touching

Medullar Patterns with Lattices light lattice dark lattice

V-like lattice

X-like lattice

Medullar Patterns with Vacuoles, Granules or Special Edges distinct rows embedded larger vacuoles

20 Figure 2.1. Medullar patterns of guard hairs of the terrestrial mammals from Chiapas, Mexico.

coarse mosaic fine mosaic

Figure 2.2. Cortical scale patterns of guard hairs of the terrestrial mammals from Chiapas, Mexico.

21 Of the 79 species described, the descriptions of hair patterns were new for 24 species. There

were 8 species reported for the Order Didelphimorphia and this work includes 7 of them. Of

these species, Chironectes minimus, Caluromys derbianus and Marmosa canescens have not

been described before. This work also described the medullar and scale patterns of the howler

monkey, Alouatta palliata and the spider monkey, Ateles geoffroyi of which no previous

descriptions were found. There were 4 species reported in the Order Xenarthra and all of them

are described. Of these, the anteater, Cyclopes didactylus and the armadillo, Cabassous

centralis had not been described before.

Within the Order Rodentia, the scale patterns of Liomys salvini and Liomys pictus in the

Family Heteromyidae had not been previously described. From the Family Muridae, no

previous descriptions were found for Tylomys spp., Ototylomys phyllotis and Nyctomys

sumichrasti. Also, the Mexican hairy porcupine, Coendu mexicanus, the only species in the

Family Erethizontidae, was first described here and similarly no previous descriptions were

found for Dasyprocta punctata, D. mexicana and Agouti paca, rodents that belong to the

Families Dasyproctidae and Agoutidae.

Within the Order Caraivora, 12 species were reported for Chiapas in the families Mustelidae

and Procyonidae and all of them were described here. No previous description was found for

the ringtail cat, Bassariscus sumichrasti, the tayra, Eira Barbara, the grison, Galictis vittata nor

the river otter, Lontra longicaudis. Of the 4 species of skunks present in the State, no previous

descriptions of scale patterns were found for the hog-nosed skunk, Conepatus mesoleucus nor the hooded skunk, C. semistriatus.

Five of the 6 felines that were reported for Mexico occur in Chiapas and all of them are sympatric and included in this work (Aranda 2000). The jaguarondi, Puma yagouaroundi was

22 the only species with no previous description of the guard hairs. Finally, from the Order

Perissodactyla, the only species present was the tapir, Tapirus bairdii, from which no previous

description of hair was found. '

Chiapas has a great variety of environments (Medellin 1994); therefore, it is important to

note that many species are present only in certain habitats within the state and are not sympatric

with other closely related species. As a result, species with similar guard hair features can often

be separated, if ecological and distribution parameters are taken into consideration.

The methods described in this manuscript provide a useful and cost effective way to study

terrestrial mammalian species, which is vital and urgent for the development of effective wildlife management strategies in this region. This is especially important for obtaining valuable information on elusive and endangered species that are under considerable pressure from habitat loss and human activity. As hair identification is a noninvasive technique, animals do not need to be killed nor captured, but can be studied through the collection of hair samples in scats and hair snares. Recent developments in molecular technology and DNA analysis can also be applied to hair samples and increase the information needed to make informed management decisions (Woods et al. 1999, McDaniel et al. 2000, Mowat and Strobeck 2000,

Sloane et al. 2000, Belant 2003, Boersen et al. 2003, Piggott and Taylor 2003).

2.7 Acknowledgments

We thank the National Mammal Collection at the Institute of Biology, the Mammal

Collection at the Faculty of Science at the National Autonomous University of Mexico

(UNAM), the Mammal Collection at the Colegio de la Frontera Sur, Unidad San Cristobal,

Chiapas, Mexico and the Natural History Institute, Tuxtla Gutierrez, Chiapas, Mexico for providing access to mammal collections and hair samples. Drs. A. Omri and K. Nkongolo

23 reviewed the manuscript and A. Torres provided technical support. Financial aid was provided by the Mexico's National Council of Science and Technology (CONACYT) through a scholarship to NGA and Laurentian University.

24 Chapter 3. Key for the Identification of Guard Hairs of the Terrestrial Mammals from Chiapas, Mexico

3.1 Terms of Reference

Clear - medulla light to gray colored with no regular patter of dark or light colored medullar inclusions

Blotches occasional irregular shaped vertically oriented dark

Medullar inclusions - dark or light inclusions in the medull that appear regularly as spherical or ovoid spots, striations, vacuoles, granular specks or scratches

Distinct - edges of medullar inclusions and/or cortical scal< are well defined and can be distinguished from each othei

1 •mm H

25 Indistinct - edges of medullar inclusions and/or cortical scales are not well defined, often fuzzy and tend to run together

Vertical continuous lines - darker lines that run vertically along the length of the medulla/hair

Vertical broken streaks - darker short streaks that run vertically along the length of the medulla/hair

Concave edges - medullar edges that are composed of a series of concave sections

Granular - the vacuoles are small and appear as small amorphous grains

26 Convex edges - medullar edges that are a series of convex sections

Lattice - where the medullar inclusions connect and form a matrix rather than being individual separate structures

Embedded medullar vacuoles - larger embedded spherical or ovoid vacuoles

Bubble-like - appears as covering of foam of spherical or ovoid vacuoles

Amorphous vacuoles - medullar vacuoles that are not spherical nor ovoid

27 Scratched - irregular scratches with no specific orientation or pattern

Central ridge/furrow - a central ridge/furrow running dowr the centre of the cortex

Serrated edges - medullar edges with small teeth-like projections

Touching, alternating - medullar inclusions touch each oth< laterally and alternate as they project into the center of the medulla

Vertical plus lateral broken medullar streaks

28 3.2 Key for the Identification of Guard Hairs of the Terrestrial Mammals from Chiapas,

Mexico la. Medulla clear with no medullar inclusions or with dark blotches or central vertical streaks or central vertical streaks plus lateral streaks on edges 2

lb. Medulla with numerous dark or light distinct or indistinct inclusions, lattices or medullar vacuoles or granules 12

2a. Medulla clear with no dark blotches nor vertical broken streaks 3

2b. Medulla clear with blotches or vertical and/or lateral streaks 6

3a. Medulla clear with a light line down the center and light edges and indistinct fine mosaic distal scale pattern Cabassous centralis

3b. Medulla clear with no light line down the center with a distinct distal mosaic scale pattern

4

4a. Medulla clear with dark edges and a distinct coarse mosaic distal scale pattern with indistinct double walled cortex Cyclopes didactylus

4b. Medulla clear with light to gray edges or continuous darker vertical lines with a distal mosaic scale pattern 5

5a. Medulla clear with light to gray edges and a distinct coarse mosaic distal scale pattern with an indistinct double walled cortex Coendu mexicanus

5b. Medulla clear with four continuous darker vertical lines and a fine mosaic distal scale pattern with an indistinct double walled cortex Tayassu pecari

6a. Medulla clear with dark blotches 7

6b. Medulla clear with vertical and/or lateral streaks 10

7a. Medulla clear with occasional large distinct dark blotches 8

29 7b. Medulla clear with occasional to many small dark blotches 9

8a. Medulla clear with thin, pointed ended dark blotches and an indistinct fine mosaic distal

scale pattern Alouattapalliata

8b. Medulla clear with thicker, round ended dark blotches and a coarse mosaic distal scale

pattern Tapirus bairdii

9a. Medulla clear with few small dark blotches and indistinct fine mosaic distal scale

pattern Dasypus novemcintus

9b. Medulla clear with many small dark blotches and distinct coarse mosaic distal scale

pattern Philander opossum

10a. Medulla clear with numerous vertical broken streaks and a mosaic scale pattern with

or without a double walled cortex 11

10b. Medulla clear with numerous vertical broken streaks in the center of the medulla and

lateral streaks with fine or coarse streaks in the center of the proximal scale pattern 70

1 la. Medulla clear with numerous vertical broken large streaks and indistinct fine mosaic

scale pattern with a double walled cortex Tamandua mexicana

1 lb. Medulla clear with numerous vertical fine broken streaks/fine granular specks and distinct coarse mosaic scale pattern without a double walled cortex Ateles geoffroyi

12a. Medulla with a single row of distinct or indistinct dark inclusions 13

12b. Medulla with more than a single row of dark inclusions, a lattice or medullary vacuoles or granules 20

13a. Medulla with single row of distinct dark inclusions separated by light spaces or just touching with a distal scale pattern that is pod 14

30 13b. Medulla with a single row of distinct dark inclusions separated by light spaces with a

distal scale pattern that is mosaic 15

14a. Medulla with single row of larger distinct dark inclusions separated by light spaces or

just touching with a distal scale pattern that is pod and a proximal scale pattern that is

pectinate Marmosa canescens

Marmosa mexicana

14b. Medulla with single row of very, very small distinct dark inclusions separated by light

spaces with distal and proximal scale patterns that are pod Glaucomys volans

15a. Medulla with a single row of distinct dark inclusions separated by light spaces with a

distal mosaic scale pattern with a distinct central

ridge/furrow Cryptotis goldmani

Cryptotis goodwini

Cryptotis mexicana

Cryptotis parva

15b. Medulla with a single row of distinct or indistinct dark inclusions separated by light spaces with a mosaic distal scale pattern with no central ridge/furrow 16

16a. Medulla with a single row of distinct dark inclusions separated by light spaces and a mosaic distal scale pattern with distinct dark smooth cortical edges 17

16b. Medulla with a single row of distinct dark inclusions separated by light spaces and a mosaic distal scale pattern with light colored rough cortical edges 19

17a. Medulla with dark bead-like inclusions and a coarse mosaic distal scale pattern Caluromys derbianus

31 17b. Medulla with dark stripe-like inclusions that span the width of the hair with a fine

mosaic distal scale pattern 18

18a. Medulla with dark stripe-like inclusions that span the width of the hair and with

straight medullar edges plus a fine mosaic distal scale pattern ....Conepatus semistriatus

18b. Medulla with dark stripe-like inclusions that span the width of the hair with scalloped

concave medullar edges plus a fine mosaic distal scale pattern Conepatus mesoleucus

19a. Distal cortical edge light colored and rough with medulla having smaller dark single inclusions that do not appear to spiral along hair Sorex saussurei

19b. Distal cortical edge light colored and rough with medulla having larger dark single inclusions that appear to spiral along hair length Sorex veraepacis

20a. Medulla with mixture of one and two dark inclusions across width of hair 21

20b. Medulla with two or more distinct dark inclusions across width of hair, a lattice or medullar vacuoles or granules 25

21a. Medulla with mixture of one and two dark inclusions across width of hair and straight light edges 22

21b. Medulla with mixture of one and two dark inclusions across width of hair and scalloped concave or convex clear light edges 24

22a. Medulla with mixture of one and two dark inclusions across width of hair, straight light edges with single inclusions occurring across entire hair width Mephitis macroura

Spilogale putorius

32 22b. Medulla with mixture of one and two dark inclusions across width of hair, straight light edges with single inclusions occurring in center of hair width 23

23a. Medulla with mixture of one and two dark inclusions across width of hair, straight light edges with single inclusions occurring in center of hair width with a straight edged proximal cortical wall Reithrodontomys microdon

23b. Medulla with mixture of one and two dark inclusions across width of hair with slightly serrated proximal cortical edges Reithrodontomys gracilis

Peromyscus mexicanus

24a. Medulla with scalloped concave edges and a fine mosaic distal scale patterns Bassariscus sumichrasti

24b. Medulla with scalloped convex edges and a coarse mosaic distal scale pattern Reithrodontomys sumichrasti

25a. Medulla with two alternating dark inclusions across width of hair and straight light edges ...26

25b. Medulla with two or more distinct dark inclusions across width of hair or a lattice or medullary vacuoles or granules 29

26a. Medulla with two alternating dark inclusions across width of hair and a coarse or fine mosaic distal scale pattern 27

26b. Medulla with two and/or three dark inclusions across width of hair and mosaic distal scale pattern 29

27a. Medulla with two touching alternating dark inclusions not separated by light colored spaces across width of medulla and coarse mosaic distal and pectinate proximal scale patterns

Peromyscus zarhynchus

33 27b. Medulla with two alternating dark inclusions separated by light colored spaces vertically

and/or laterally across width of hair 28

28a. Medulla with two non-touching alternating dark inclusions separated by light spaces

laterally and vertically across width of medulla and coarse mosaic distal and proximal scale

patterns Reithrodontomys fulvescens

28b. Medulla with two touching alternating dark inclusions across width of medulla

separated by light spaces vertically and fine mosaic distal and proximal scale patterns

Ototylomys phyllotis

29a. Medulla with a mixture of two and three dark inclusions across width of hair separated by light colored spaces 31

29b. Medulla with three or more dark distinct inclusions across width of hair separated by light to gray colored spaces or a lattice or medullar vacuoles or granules 32

31a. Medulla with a mixture of two and three small thin dark inclusions across width of hair separated by light colored spaces, a fine mosaic distal and coarse mosaic proximal scale pattern Tylomys bullaris

31b. Medulla with a mixture of two and three large bulbous dark inclusions across width of hair separated by light colored spaces, with fine mosaic distal and pectinate proximal scale patterns Oryzomys alfaroi

32a. Medulla with three thin dark inclusions across width of hair separated by light colored spaces with single walled distal cortex and coarse mosaic distal scale pattern Neotoma mexicana

34 32b. Medulla with three or more thin or bulbous dark inclusions across width of hair

separated by light to gray colored spaces with a mosaic distal scale pattern or the medulla has a

lattice or medullar vacuoles or granules 33

33a. Medulla with three thin dark inclusions across width of hair separated by light colored

spaces with a coarse mosaic distal scale pattern and pectinate proximal scale pattern

Peromyscus gymnotis

33b. Medulla with three or more bulbous dark inclusions across width of hair with double

walled cortex and mosaic distal scale pattern or medulla has a lattice or medullar vacuoles or

granules 34

34a. Medulla with three bulbous dark inclusions across width of hair not distinctly separated by light to gray colored spaces with double walled cortex and mosaic distal scale pattern Scurius variegatoides

Scurius yucatanensis

Scurius deppei

34b. Medulla with both three and/or four thin dark inclusions across width of hair distinctly separated by light colored spaces and a mosaic distal scale pattern or medulla has a lattice or medullar vacuoles or granules 35

35a. Medulla with both three and four thin dark inclusions across width of hair distinctly separated by light colored spaces with double walled cortex, coarse mosaic distal scale pattern and a pectinate proximal scale pattern Reithrodontomys tenuirostris

35 35b. Medulla with both three and four dark inclusions across width of hair distinctly

separated by light spaces with distal mosaic scale pattern or medulla has a lattice or medullar

vacuoles or granules 36

36a. Medulla with three and four dark inclusions across width of hair distinctly separated by

light spaces with a fine mosaic distal scale pattern and a pectinate proximal scale

pattern Sigmodon hispidus

36b. Medulla with four dark inclusions across width of hair distinctly separated by light

spaces with a mosaic distal scale pattern and with or without a pectinate proximal scale

pattern or medulla has a lattice or medullar vacuoles or granules 37

37a. Medulla with four dark inclusions across width of hair distinctly separated by light

spaces with a coarse mosaic distal scale pattern and with a pectinate proximal scale

pattern Baiomys musculus

37b. Medulla with mainly four dark inclusions across width of hair distinctly separated by

light spaces with a fine mosaic distal scale pattern and with no pectinate proximal scale pattern or medulla has a lattice or medullar vacuoles or granules 38

38a. Medulla with four dark inclusions across width of hair distinctly separated by light spaces with a fine mosaic distal scale pattern and no pectinate proximal scale pattern Nyctomys sumichrasti

38b. Medulla with lattices or medullar vacuoles or granules 39

39a. Medulla with dark lattice with distinct Y-like inclusions 40

39b. Medulla with dark or light lattices with no Y-like inclusions or medulla with medullar vacuoles or granules 42

36 40a. Medulla with dark lattice with Y-like inclusions, a distal mosaic scale pattern with a

central light stripe Reithrodontomys mexicanus

40b. Medulla with a dark lattice with Y-like inclusions, a distal mosaic scale pattern without

a central light stripe 41

41a. Medulla with a dark lattice with Y-like inclusions, a coarse mosaic distal scale pattern without a central light stripe and a mosaic proximal scale pattern Tylomys nudicaudus

41b. Medulla with a dark lattice with Y-like inclusions, a fine mosaic distal scale pattern without a central light strip and a pectinate proximal scale pattern Sigmodon mascotensis

42a. Medulla with distinct dark lattice with a series of connected V-like inclusions, a fine mosaic distal scale pattern and a pectinate proximal scale pattern.. .Peromyscus guatemalensis

42b. Medulla with dark or light lattices without a series of connected V-like inclusions or a medulla with medullar vacuoles or granules 43

43a. Medulla with indistinct dark lattice without a series of connected V-like inclusions, a fine mosaic distal scale pattern with thick double walls and a mosaic proximal scale pattern with thin single cortical walls Potos flavus

43b. Medulla with indistinct dark or light lattices without a series of connected V-like inclusions and a mosaic distal scale pattern or with medullar vacuoles or granules 44

44a. Medulla with indistinct dark lattices without a series of connected regular V-like inclusions and a distal and proximal scale patterns that are coarse mosaic with one or maximally two scales crossing entire width Orthogeomys hispidus

44b. Medulla with dark or light lattices without V-like inclusions or with medullary vacuoles or granules 45

37 45a. Medulla with indistinct dark lattice that appears as thick streaks crossing width of the hair without a series of connected regular V-like inclusions with a distal fine mosaic scale pattern and a proximal scale pattern that is pectinate Lontra longicaudis

45b. Medulla with dark or light lattices with no thick streaks crossing width of hair or medulla with medullar vacuoles or granules 46

46a. Medulla with indistinct dark lattice with fine serrated edges of the medulla, a fine mosaic distal scale pattern and a pectinate proximal scale pattern Leopardus weidii

46b. Medulla with an indistinct dark lattice with fine serrated edges of the medulla, a fine mosaic distal scale pattern and no pectinate proximal scale pattern or a medulla with a light to gray colored lattice with or without serrated edges and a mosaic distal scale pattern or with medullar vacuoles or granules 47

47a. Medulla with indistinct dark lattice with fine serrated edges of the medulla, a coarse mosaic distal scale pattern and no pectinate proximal scale pattern Leopardus pardalis

47b. Medulla with light to gray colored lattice with or without serrated edges and a mosaic distal scale pattern or with medullar vacuoles or granules 48

48a. Medulla with light to gray colored lattice with distinct X-like inclusions, a fine mosaic distal scale pattern and a pectinate proximal scale pattern where single scales span the width of the cortex Mustela frenata

48b. Medulla with light to gray colored lattices with no distinct X-like inclusions or with medullar vacuoles or granules 49

49a. Medulla with light to gray colored multi-worm-like lattice with no distinct X-like inclusions, no embedded larger vacuoles and an absence of the medullar wall with a fine mosaic distal scale pattern Dasyprocta punctata

38 49b. Medulla with light to gray colored lattices, no distinct X-like inclusions and a distinct dark medullar wall or with a covering of bubble-like vacuoles that form the entire lattice or with larger embedded medullar vacuoles or granules 50

50a. Medulla with light to gray colored lattice, no distinct X-like inclusions, no embedded larger vacuoles and a distinct dark medullar wall with a coarse mosaic distal scale pattern where only one or maximally two scales span the width of the hair Chironectes minimus

50b. Medulla with light to gray colored lattice, no distinct X-like inclusions and a distinct dark edged medullar wall with a mosaic distal scale pattern or with medullar vacuoles or granules 51

51a. Medulla with light to gray colored lattice, no distinct X-like inclusions, no embedded larger vacuoles and a distinct dark edged medullar wall which is serrated and a coarse mosaic distal scale pattern where three or more scales span the width of the hair Didelphis marsupialis

51b. Medulla with light to gray colored lattice, no distinct X-like inclusions and a distinct dark medullar wall with a coarse mosaic distal scale pattern where only one or maximally two scales span the width of the hair or with medullar vacuoles or granules 52

52a. Medulla with light to gray colored lattice, no distinct X-like inclusions, no embedded larger vacuoles and a light serrated medullar wall with a fine mosaic distal scale pattern where three or more scales span the width of the hair Didelphis virginiana

52b. Medulla with light to gray colored lattice with embedded light colored larger vacuoles or a continuous covering of smaller medullar vacuoles or granules over the entire inner portion of the medulla 53

39 53a. Medulla with light to gray colored lattice with embedded light colored larger vacuoles that span the inner medulla and are separated by striations 54

53b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored small bubble-like medullar vacuoles or granules 58

54a. Medulla with light to gray colored lattice with occasional large embedded irregular shaped light colored vacuoles that span the entire width of the inner portion of the medulla which has dark colored edges and distal and proximal scale patterns that are coarse mosaic Eira barbara

54b. Medulla with light to gray colored lattice with large embedded ovoid light colored vacuoles that span the entire width of the inner portion of the medulla, which has dark colored edges and a mosaic distal scale pattern 55

55a. Medulla with light to gray colored lattice with occasional large ovoid embedded light colored vacuoles that span the entire width of the inner portion of the medulla, which has dark colored non-serrated medullar edges, a fine mosaic distal scale pattern and a distinct two walled coarse mosaic proximal scale pattern Panthera onca

55b. Medulla with light to gray colored lattice with large ovoid embedded light colored vacuoles that partially span the entire width of the inner portion of the medulla and a coarse mosaic proximal scale pattern and fine mosaic distal scale pattern with or without a two walled edge 56

56a. Medulla with light to gray colored lattice with regular large ovoid embedded light colored vacuoles that span the entire width of the inner portion of the medulla, which has dark colored medullar edges and a fine mosaic distal scale pattern and a coarse mosaic proximal scale pattern without a two walled edge Puma yagouaroundi

40 56b. Medulla with light to gray colored lattices with a continuous mixture of large and medium sized ovoid embedded light colored vacuoles that span and partially span the width of the inner portion of the medulla or occasional large light colored vacuoles that partially span the inner medulla 57

57a. Medulla with light to gray colored lattice and serrated dark outer wall with occasional medium sized ovoid embedded light colored ovoid vacuoles that partially span the width of the inner portion of the medulla and a fine distal and coarse mosaic proximal scale pattern. Puma concolor

57b. Medulla with light to gray colored lattices with a continuous mixture of large and medium sized ovoid embedded light colored vacuoles that span and partially span the width of the inner portion of the medulla with coarse mosaic distal and pectinate proximal scale patterns Canis latrans

58a. Medulla with light to gray colored lattice with a continuous covering of relatively small bubble-like medullar vacuoles, some slightly and distinctly larger than others with coarse mosaic distal and pectinate proximal scale pattern Urocyon cinereoargenteus

58b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles or granules 59

59a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that occur in distinct rows 60

59b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles or granules that do not occur in distinct rows 63

41 60a. Medulla with light to gray colored lattice with a continuous covering of light to gray

colored equal sized bubble-like medullar vacuoles that occur in three distinct

rows Pecari tajacu

60b. Medulla with light to gray colored lattice with a continuous covering of light to gray

colored equal sized bubble-like medullar vacuoles that occur in four or more distinct

rows 61

61a. Medulla with light to gray colored lattice with a continuous covering of light to gray

colored equal sized bubble-like medullar vacuoles that occur in four or more distinct rows with dark vacuoles occurring on each edge of the medulla 62

61b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that occur in four or more distinct rows with light colored vacuoles occurring on each edge of the medulla and fine mosaic distal scale pattern Lepus flavigularis

62a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that occur in four or more distinct rows with dark vacuoles occurring on each edge of the medulla, a regular fine mosaic distal scale pattern with one or two scales across the width of the hair Sylvilagus brasiliensis

62b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that occur in four or more distinct rows with dark vacuoles occurring on each edge of the medulla, a irregular fine mosaic distal scale pattern tending to be in the form V-shapes with often more than two scales across the width of the hair Sylvilagus floridanus

42 63a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct rows but are spherical or ovoid 64

63b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored amorphous (not spheroid nor ovoid) small medullar vacuoles or granules that do not occur in distinct rows 68

64a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct rows but are spherical or ovoid with a tendency for the vacuoles to form a row along each medullar edge with a fine mosaic distal scale pattern Sciurus aureogaster

64b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct rows but are spherical or ovoid without a tendency for the vacuoles to form a row along each medullar edge with a fine mosaic distal scale pattern 65

65a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct rows but are distinctly spherical or ovoid without a tendency for the vacuoles to form a row along each medullar edge with a coarse mosaic distal scale pattern Nasua narica

65b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct rows but are spherical or ovoid without a tendency for the vacuoles to form a row along each medullar edge with a fine mosaic distal scale pattern 66

43 66a. Medulla with light to gray colored lattice with a continuous covering of large distinct

light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct

rows but are distinctly spherical or ovoid without a tendency for the vacuoles to form a row

along each medullar edge with fine mosaic distal and proximal scale

patterns Mazama americana

Odocoileus virginianus

66b. Medulla with light to gray colored lattice with a continuous mixture of small indistinct

light to gray colored bubble-like medullar vacuoles and granules that do not occur in distinct

rows but are indistinctly spherical and ovoid without a tendency for the vacuoles to form a

row along each medullar edge 67

67a. Medulla with light to gray colored lattice with a continuous covering of small indistinct

light to gray colored equal sized bubble-like medullar vacuoles that do not occur in distinct rows but are spherical or ovoid without a tendency for the vacuoles to form a row along each medullar edge with a coarse mosaic distal scale pattern Dasyprocta mexicana

67b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored bubble-like medullar vacuoles and granules that do not occur in distinct rows 68

68a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored bubble-like medullar vacuoles and small granules that do not occur in distinct rows, with coarse mosaic distal and pectinate proximal scale patterns Procyon lotor

44 68b. Medulla with light to gray colored lattice with a continuous covering of light to gray

colored amorphous (not spheroid nor ovoid) small medullar vacuoles and granules that do

not occur in distinct rows with fine mosaic distal scale patterns 69

69a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored amorphous (not spheroid nor ovoid) small medullar vacuoles that do not occur in distinct rows with a fine mosaic distal scale patterns and a medulla with thick walls Galicitis vittata

69b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored amorphous (not spheroid nor ovoid) small medullar vacuoles or granules with a medulla with thin or no apparent medullar walls 71

70a. Medulla clear with numerous vertical broken fine streaks in the center of the medulla with lateral streaks along edges and coarse streaks in the center of the proximal scale pattern Liomys salvini

70b. Medulla clear with numerous vertical broken fine streaks in both the center of the medulla and the center of the proximal scale pattern and lateral streaks along the medulla edges Liomys pictus

Heteromys desmarestianus

71a. Medulla with light to gray colored lattice with a continuous covering of light to gray colored amorphous (not spheroid nor ovoid) small granular medullar vacuoles that look like specks that do not occur in distinct rows with distal and proximal scale patterns that appear to have no pattern (appear as scratched surfaces) and thin medullar walls Agouti paca

45 71b. Medulla with light to gray colored lattice with a continuous covering of light to gray colored amorphous (not spheroid nor ovoid) medullar vacuoles with a medulla with no apparent medullar walls Orthogeomys grandis

46 Chapter 4. Atlas of the Scale and Medullar Patterns of the Guard Hairs of Terrestrial Mammals from Chiapas, Mexico

Order Didelphimorphia Family Marmosidae Subfamily Marmosinae

Marmosa canescens (Fsc, 04408) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Single Row-Dark Pectinate Pod Inclusions

Marmosa mexicana (Fsc, 04258) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Single Row-Dark Pectinate Pod Inclusions

Family Caluromyidae Subfamily Caluromyinae

Caluromys derbianus (lb, 5828) Scale pattern Medullar pattern

Coarse Mosaic Single Row- Dark Inclusions

47 Familiy Didelphidae Subfamily Didelphinae

Chironectes minimus (lb, 22189) Scale pattern Medullar pattern

Coarse Mosaic Lattice

Didelphis marsupialis (Fsc, 06285) Scale pattern Medullar pattern

l|'i

Coarse Mosaic i Lattice

Didelphis virginiana (Fsc, 03467) Medullar pattern 9R ' S* iV' f

Fine Mosaic Lattice

Philander opossum (Fsc, 06287) Scale pai Medullar pattern % 1

|.?:4

Coarse Mosaic Clear with Blotches

48 Order Xenarthra Family Dasypodidae Subfamily Dasypodinae Cabassous centralis (lb, 23898) Scale pattern Medullar pattern

i. .taili

Fine Mosaic Clear with Central Line

Dasypus novemcintus (Fsc, 7905) Scale pattern Medullar pattern '1 % M'. •f »t» fjii'jSif^-^S'iPflf Fine Mosaic Clear with Blotches

Family Myrmecophagidae

Cyclopes didactylus (lb, 14576)

UUttlC^olWa wnftaUttllWliwl ITlVUUliUA/fa/liillalr nattorUUllVinU

Coarse Mosaic Clear with Dark Edges

Tamandua mexicana (lb, 4043) Scale pattern Medullar pattern

Fine Mosaic Clear with Broken Streaks

49 Order Insectivora Familiy Soricidae Subfamily Soricinae

Cryptotis goldmani (Fsc, 03482) Scale pattern Medullar pattern 1

\

• \ ... > Proximal Cortex Distal Cortex Single Row-Dark Pectinate Coarse Mosaic with Rid^ Inclusions

Cryptotis goodwini (lb, 22784) Scale pattern Medullar pattern 1 1 1' Proximal Cortex Distal Cortex Single Row-Dark Pectinate Coarse Mosaic with Ridge Inclusions

Cryptotis mexicana (Fsc, 8048) Scale pattern Scale pattern Medullar pattern 1 — a ^if Proximal Cortex Distal Cortex Single Row-Dark Pectinate Coarse Mosaic with Ridge Inclusions

50 Cryptotisparva (Fsc, 0668) Scale pattern Medullar pattern H

Proximal Cortex Distal Cortex Single Row Pectinate Coarse Mosaic with Ridj Inclusions

Sorex saussurei (Fsc, 1251) Scale pattern Medullar pattern

?!

Proximal Cortex Distal Cortex Single Row of Dark Pectinate Coarse Mosaic Inclusions

Sorex veraepacis (Fsc, 03493) Scale pattern Scale pattern Medullar pattern 1 1TT-TS3 \

"w *

Proximal part of the haii Distal Cortex Single Row of Dark Pectinate Coarse Mosaic Inclusions Order Primates Family Cebidae Subfamily Alouattinae

Alouattapalliata (lb, 37199) Scale pattern Medullar pattern

Fine Mosaic Clear with Blotches

51 Subfamily Atelinae Ateles geoffroyi (lb, 337) Scale pattern Medullar pattern

Coarse Mosaic Clear with Broken Streaks Order Carnivora Family Canidae

Canis latrans (Fsc, 1429) Scale pattern Scale pattern Medullar pattern •1

• ) r' «t illiiifi \ * liflili^ I mi Lattice with Proximal Cortex Distal Cortex Large/Medium Sized Pectinate Coarse Mosaic Vacuoles

Urocyon cinereoargenteus (Fsc, 1321) acaie pattern Medullar pattern

i

Proximal Cortex Distal Cortex Lattice with Bubble-lil Pectinate Coarse Mosaic Vacuoles

52 Family Felidae Subfamily Felinae

Puma yagouaroundi (lb, 8475) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Large Ovoii Coarse Mosaic Fine Mosaic Vacuoles

Leopardus pardalis (lb, 6612) Scale pattern Medullar pattern

Lattice with Fine Serrated Edges Coarse Mosaic

Leopardus weidii (Fsc, 1409) Scale pattern Scale pattern Medullarpattern

Proximal Cortex Distal Cortex Lattice with Fine Serrate Pectinate Fine Mosaic Edges

Puma concolor (lb, 26622) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Medium Coarse Mosaic Fine Mosaic Sized Vacuoles

53 Subfamily Pantherinae Panthera onca (lb, 3995) Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Large Size( Coarse Mosaic Fine Mosaic Vacuoles

Family Mustelidae Subfamily Luterinae

Lontra longicaudis (lb, 3952) Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Thick Strea Pectinate Fine Mosaic

Owuiuillll^ iTlVplllMllUW

Conepatus mesoleucus (Fsc, 1404) Scale pattern Medullary pattern

Fine Mosaic Dark Stripe-like Inclusions

54 Conepatus semistriatus (lb, 144) Scale pattern Medullar pattern

Fine Mosaic Dark Stripe-like Inclusions

Mephitis macroura (Fsc, 1402) Scale pattern Medullar pattern

Fine Mosaic One and Two Dark Inclusions

Spilogaleputorius (Fsc, 03458) Scale pattern Medullar pattern

Fine Mosaic One and Two Dark Inclusions

Subfamily Mustelinae

Eira Barbara (lb, 1262) Scale pattern Medullary pattern

Coarse Mosaic Lattice with Large Vacuo

55 Galictis vittata (Fsc, 3954) Scale pattern Medullar pattern

Fine Mosaic Lattice with Amorphous Vacuoles

Mustela frenata (Fsc, 0292) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with X-like Pectinate Fine Mosaic Inclusions

Family Procyonidae Subfamily Potosinae Potos flavus (Fsc, 6616) Scale pattern Medullar pattern •-•J

Proximal Cortex Distal Cortex Lattice without V-like Coarse Mosaic Fine Mosaic Inclusions

Subfamily Procyoninae Bassariscus sumichrasti (lb, 6402) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex One and Two Dark Pectinate Fine Mosaic Inclusions

56 Nasua narica (lb, 186) Scale pattern Medullar pattern

Coarse Mosaic Lattice with Bubble-like Vacuoles Procyon lotor (Fsc, 6765) Scale pattern Scale pattern Medullar pattern V iiS

-J Proximal Cortex Distal Cortex Lattice with Granules Pectinate Fine Mosaic Order Perissodactyla Family Tapiridae

Tapir us bairdii (lb, 16574) Scale pattern Medullar pattern Iit iRi"v Smp J Coarse Mosaic Clear with Blotches Order Artiodactyla Family Tayassuidae

Pecari tajacu (Fsc, 2766) Scale pattern Medullar pattern

Fine Mosaic Lattice with Bubble-like Vacuoles

57 Tayassu pecari (lb, 38275) Scale pattern Medullar pattern

Fine Mosaic Clear with Continuous Vertical Lines

Family Cervidae Subfamily Odocoileinae

Mazama americana (Fsc, 6617) Scale pattern Medullar pattern f> -""i-i-V • * •

^ v Mw " t f 4 \ J? 1 J-/ Fine Mosaic Lattice with Bubble-like Vacuoles

Odocoileus virginianus (lb, 2614) Scale pattern ivieaunar pattern

Fine Mosaic Lattice with Bubble-like Vacuoles

58 Order Rodentia Family Sciuridae Subfamily Sciurinae

Sciurus aureogaster (Fsc, 4433) Scale pa Medullar pattern

Lattice with Bubble-like Vacuoles Fine Mosaic Sciurus deppei (Fsc, 0681) Scale pattern Medullar pattern

Fine Mosaic Three Dark Inclusions Sciurus variegatoides (Fsc, 0097) Scale pattern Medullar pattern

Fine Mosaic Three Dark Inclusions

Sciurus yucatanenses (Fsc, 1051) Scale pattern Medullar pattern

Three Dark Inclusions Fine Mosaic

59 Subfamily Petauristinae

Glaucomys volans (Fsc, 7388) Scale pattter n Medullar pattern >• Jf § i «i if '•mM a I F

Pod Single Dark Inclusions

Family Geomyidae

Orthogeomys grandis (lb, 8497) Scale pattern Medullar pattern

Fine Mosaic Lattice with Amorphous Vacuoles

Orihogeomys hispiuua (Fsi;, 7781) Scale pattern Medullar pattern M

Coarse Mosaic Lattice with V-like Inclusions

60 Family Heteromydae Subfamily Heteromyinae Heteromys desmarestianus (Fsc, 7169) Scale pattern Scale pattern Medullar pattern

Proximal Cortex with Distal Cortex Clear with Vertical and Vertical and Lateral Clear Lateral Streaks Streaks Liomys pictus (Fsc, 0528) Scale pattern Scale pattern Medullar pattern

Proximal Cortex with Distal Cortex Clear with Vertical and Vertical and Lateral Clear Lateral Streaks Streaks

Liomys salvini (lb, 7987) Cnnln — * * A ij. joaiw uaiiA^iii "fl ivieuunai pauci n fJ[M Wjfii

Proximal Cortex with Distal Cortex Clear with Vertical and Vertical and Lateral Fine Mosaic Lateral Streaks Streaks

61 Family Muridae Subfamily Sigmodontinae

Baiomys musculus (Fsc, 02356) ] Scale pattern Medullar pattern t <11 sf 1s Proximal Cortex Distal Cortex Four Dark Inclusions Pectinate Coarse Mosaic

Neotoma mexicana (Fsc, 80888) Scale pattern Medullar pattern

Coarse Mosaic Three Dark Inclusions

Nyctomys sumichrasti (Fsc, 06876) Scale pattern Medullar pattern

; J!'

.^i^iif'j

Fine Mosaic Four Dark Inclusions

Oryzomys alfaroi (Cfs, 1345) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Two and Three Dark Pectinate Fine Mosaic Inclusions

62 Ototylomys phyllotis (Fsc, 7004) Scale pattern Medullar pattern

Fine Mosaic Two Alternating Dark Inclusions

Peromyscus guatemalensis (Fsc, 04495) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with V-like Pectinate Fine Mosaic Inclusions

Peromyscus gymnotis (lb, 240) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Three Dark Inclusions Pectinate Fine Mosaic Peromyscus mexicanus (Nhi, 01845) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex One and Two Dark Pectinate Fine Mosaic Inclusions

63 Peromyscus zarhynchus (lb, 31597) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Two Alternating Dark Pectinate Coarse Mosaic Inclusions

Reithrodontomys fulvescens (Fsc, 02945) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Two Alternating Dark Coarse Mosaic Coarse Mosaic Inclusions

Reithrodontomys gracilis (lb, 30769) Scale pattern Scale pattern Medullarjjattern

Proximal Cortex Distal Cortex One and Two Dark Pectinate Coarse Mosaic Inclusions

Reithrodontomys mexicanus (Fsc, 05261) Scale pattern Scale pattern

Proximal Cortex Distal Cortex Lattice with Y-like Pectinate Coarse Mosaic Inclusions

64 Reithrodontomys microdon (Fsc, 03797) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex One and Two Dark Pectinate Coarse Mosaic Inclusions

Reithrodontomys sumichrasti (Fsc, 03802) Scale pattern Medullar pattern f! *

I

I '/K u Proximal Cortex Distal Cortex Two Alternating Dark Pectinate Coarse Mosaic Inclusions

Reithrodontomys tenuirostris (lb, 35516) Scale pattern Medullar pattern

1-" i f

t- 1/ Proximal Cortex Distal Cortex Three and Four Dark Pectinate Coarse Mosaic Inclusions

Sigmodon hispidus (Fsc, 7719) Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Three and Four Dark Pectinate Fine Mosaic Inclusions

65 Sigmodon mascotensis (Fsc, 02368) Scale pattern e pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Y-like Pectinate Coarse Mosaic Inclusions

Tylomys bullaris (lb, 3096) Scale pattern Scale pattern Medullar pattern

Distal Cortex Proximal Cortex Two and Three Dark Fine Mosaic Pectinate Inclusions

Tylomys nudicaudus (Fsc, 6796) Scale pattern Medullar pattern rrs^asHapw—

^dfi Proximal Cortex Distal Cortex Lattice with Y-like Coarse Mosaic Fine Mosaic Inclusions Suborder Histricognathi Family Erenthizontidae

Coendu mexicanus (Fsc, 0096) Scale pattern Medullar pattern

Coarse Mosaic Clear

66 Family Dasyproctidae

Dasyprocta mexicana (lb, 3590) Scale pattern Medullar pattern

Coarse Mosaic Lattice with Bubble-like Vacuoles

Dasyprocta punctata (lb, 1529) Scale pattern Medullar pattern

'jif/^J"

I !_ Fine Mosaic Lattice with Worm-like Inclusions

Family Agoutidae

Agouti paca (lb, 334) Scale pattern Medullar pattern

With Scratches Lattice with Granules

67 Order Lagomorpha Family Leporidae Subfamily Leporinae

Sylvilagus brasiliensis (Fsc, 0043) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Bubble-like Pectinate Fine Mosaic Vacuoles

Sylvilagus floridanus (Fsc, 6618) Scale pattern Medullar pattern If* - ' \ pV; m I T' £ ; f^v; J - ft' •""»* Distal Cortex Proximal Cortex Lattice with Bubble-like Fine Mosaic Pectinate Vacuoles

Lepus flavigularis (lb, 34854) Scale pattern Scale pattern Medullar pattern

Proximal Cortex Distal Cortex Lattice with Bubble- Fine Mosaic Fine Mosaic like Vacuoles

68 Chapter 5. Hair-snares: A Noninvasive Method for Monitoring Felid Populations in the Selva Lacandona, Mexico

5.1 Abstract

Noninvasive techniques such as hair-snares have been used in conjunction with molecular

methods to study species that occur at low densities and have elusive behavior, as an alternative

to invasive methods such as trapping and hunting. This study was designed to evaluate the use

of hair-snares as a noninvasive method for the collection of mammalian samples in the tropical

forest ecosystem of the Selva Lacandona, Chiapas, Mexico, with the goal of studying felid

populations.

Hair-snares were placed along transects for 4 months in both 2005 and 2006 and changed

every month. Hairs were selected based on morphological characteristics and identification of

species was done based on a diagnostic portion of mtDNA cytochrome b region. A total of 389

hits on 888 hair-snare checks were recorded representing a capture rate of 43 percent.

The species identified and the number of individuals (n) included: margay (Leopardus

wiedii, n=2), ocelot (Leoparduspardalis, n=l), jaguarundi (Pumayaguarundi, n=l), grey fox

(Urocyon cinereoargenteus, n=l), tayra (Eira Barbara, n=3), coati (Nasua narica, n=l), four

eyed opossum (Metachirus nudicaudatus, n=6) and common opossum (Didelphis marsupialis,

n=16).

The present study is the first to report the successful collection of hair samples from jaguarondi and margay in the wild and hair samples from ocelots in tropical areas. The deficit

of information on carnivore populations in tropical ecosystems is due mainly to the lack of

appropriate methodologies that are reliable and cost effective. This study supports the

conclusion that this technique is viable and inexpensive in tropical ecosystems. It is also

69 valuable when monitoring carnivore populations that have wide geographic distributions and

low densities.

5.2 Resumen

Las tecnicas no invasivas tales como las trampas de pelo han sido utilizadas junto con metodos moleculares para estudiar especies de mamiferos que ocurren en bajas densidades o que presentan un comportamiento elusivo, tratando de encontrar una alternativa a metodos invasivos como la colecta. Este estudio evalua el uso de las trampas de pelo para la obtencion de muestras de mamiferos, con especial enfasis en las poblaciones de felinos, en ecosistemas tropicales como la Selva Lacandona, Chiapas, Mexico.

Las trampas de pelo se colocaron en transectos durante cuatro meses en 2005 y 2006. Las trampas fueron reemplazadas cada mes y las muestras obtenidas se seleccionaron en base a las caracteristicas morfologicas del pelo. La identificacion de las especies se realizo en base a una portion del gen mitocondrial citocromo b. Un total de 389 hits fueron registrados en 888 visitas a las trampas. Las especies reportadas incluyen: margay (Leopardus wiedii, n=2), ocelote

(Leoparduspardalis, n=l), jaguarundi (Pumayaguarundi, n=l), zorra gris (Urocyon cinereoargenteus, n=l), tayra (Eira barbara, n=3), coati (Nasua narica, n=l), tlacuache cuatro ojos (Metachirus nudicaudatus, n=6) y tlacuache comun (Didelphis marsupialis, n=16).

La falta de information de poblaciones de carnivoros en ecosistemas tropicales, se debe en gran medida a la carencia de un metodo confiable y de bajo costo. Este estudio demuestra que esta tecnica es viable, de bajo costo y efectiva en ecosistemas tropicales, especialmente para monitorear poblaciones de carnivoros los cuales presentan bajas densidades y gran distribution geografica.

70 Key words: Leoparduspardalis, Leopardus wiedii, Panthera onca, Puma concolor, Puma yagouaroundi

5.3 Introduction

To understand the population ecology of a species, one must document its presence and abundance. However, this can be difficult especially for carnivores (Nowell and Jackson 1996,

Kurose et al. 2005, Palomares et al. 2005). Noninvasive techniques have been used to study species that occur at low densities and have elusive behavior. These technologies provide an alternative to invasive methods such as trapping and hunting.

Noninvasive techniques include tracking, automated camera systems, and feces and hair sample collection. Each of these techniques has intrinsic pros and cons and have been used in various studies where results varied from the detection of a species to the identification of individuals and gender (Foran et al. 1997, Moruzzi et al. 2002, Scott et al. 2004, Kurose et al. 2005,

Sharma et al. 2005, Alibhali et al. 2008).

Due to the elusive behavior and large home ranges of felids, information on the abundance and ecology of wild populations is difficult to obtain and often ranges are not easily accessible to researchers. Combining molecular techniques with noninvasive sample collection should lead to the generation of information required to develop viable management strategies.

Considering the natural history and behavior of these species, studies using noninvasive methods such as hair-snares in combination with molecular DNA techniques have enormous potential.

Tropical rain forest areas represent a particular challenge for the study of felid populations which have elusive behavior, occur in low densities and whose ranges are often difficult to access. Hair-snares are a method that can be used as a noninvasive technique to obtain hair

71 samples in these areas. This method can be an alternative to live trapping that is often logistically difficult, expensive and invasive. In addition, this method is more successful in tropical ecosystems than scat collection, due to the high decomposition rates of feces in the tropical rain forest. Also, the use of hair-snares can be of particular value in studying felids due to behavioral characteristics that make them an appropriate taxon for hair sampling. This technique used rub pads sprayed with specific scents to encourage individual animals to rub and leave hairs. Samples coupled with DNA identification allow researchers to assess the ecological aspects of carnivore communities and relate these data to issues such as occurrence and distribution, relative abundance, monitoring, habitat fragmentation and human disturbance

(Ruell and Crooks 2006, Kendall and Mckelvey 2008). This approach has been undertaken previously in temperate areas and included a variety of species including; lynx (McDaniel et al.

2000, Mills et al. 2000, Schmidt and Kowalczyk 2006), bobcat (Ruell and Crooks 2006), pumas (Mills et al. 2000, Ruell and Crooks 2006) and ocelots (Weaver et al. 2005). Other researchers targeting pumas and margay using this technique were unsuccessful in obtaining results (Downey et al. 2007).

This study evaluated the utility of hair-snares for the collection of mammalian hair samples with a specific goal of assessing felid populations in a tropical ecosystem. The Selva

Lacandona is a humid tropical rain forest ecosystem that is inhabited by five species of

Neotropical felids; jaguar (Panthera onca), puma (Puma concolor), ocelot (Leopardus pardalis), jaguarundi (Puma yagouaroundi) and margay (Leopardus wiedii). The primary goals of this study were: (1) to assess mammalian activity through the use of hair-snares in the Selva

Lacandona, Chiapas, Mexico, (2) to compare felid occurrence by species in the undisturbed

Montes Azules Biosphere Reserve (MABR) in the Selva Lacandona and adjacent disturbed

72 areas with habitat fragmentation and ecological disturbance associated with human settlement

and (3) to assess the utility of hair-snares with our specific lure for attracting and obtaining

samples from the 5 sympatric felid species in this region.

5.4 Study Area

The Selva Lacandona is located in the southeast portion of Chiapas (16° 05'-17°15' N,

90°30'-91° 30' W) and is delimited by Guatemala on the east, north and south and with the

Chiapas Highlands in the west (Naranjo et al. 2004). The Montes Azules Biosphere Reserve

(MABR) is located in the Selva Lacadona region. It is comprised of 331,200 hectares of protected forest established in 1978 and has been recognized internationally as part of

UNESCO's Man and Biosphere Program (MAB-UNESCO) since 1979 (Anonymus 2000). The reserve constitutes the largest protected area in the Selva Lacandona. Average annual temperature ranges from 24° to 26° C, with maximum and minimum values in May (28°C) and

January (18°C), respectively. Mean annual rainfall is 2,500 to 3,500 mm, with 80% of rains falling between June and November (Anonymous 2000).

5.5 Hair-snare surveys

We sampled for felids in the Selva Lacandona using hair-snare stations and conducted surveys for 4 months during the dry season in 2005 (March-June) and again in 2006 (February-

May). One hundred and eleven stations were set up each month at 150 m intervals along 12 transects located inside and outside the MABR (Figure 5.1) each transect contained between 10 to 12 hair-snare stations. Habitat type in the study area was originally tropical rain forest.

However outside the conservation area, habitat types varied and hair-snares were set up in remaining patches of rain forest surrounded by disturbed habitat, mainly associated with

73 agriculture. All traps were placed along transects that followed game trails based as evident by

the signs left by the animals inhabiting the area.

Hair-snares were made from 25 x 15 cm pieces of carpet with 2 velcro strips and carpet nails. Nails were placed through the carpet in a circular arrangement with 2 velcro strips on each side. Each trap was nailed to the base of a tree approximately 30 cm from the ground and flagging tape was placed 2 m above each trap. To facilitate rubbing, when necessary lower branches were cleared from trees where the hair-snare was nailed. Each trap was sprayed with liquid catnip (Napeta catarid) mixed with pieces of dried plant. In addition, commercial carnivore bait for felids "Wildcat Lure # 2" (Hawbaker's Wildcat Lures, Mansfield, Louisiana) was added. Each trap location was identified using a Garmin GPS unit (Model # 12 XL) and locations were downloaded onto a digital mapping system using Arc View.

Prior to the study, hair-snares and commercial baits (Wildcat Lure # 2 and catnip) were tested on captive felid species from the Zoologico Miguel Alvarez del Toro. This study of captive animals induced rubbing behavior and resulted in clusters of hairs being left on the hair-snare (Figure 5.2).

74 —1

Figure 5.1. Location of hair-snares transects in the Montes Azules Biosphere Reserve and

surrounded Selva Lacandona, Chiapas, Mexico.| Figure 5.2. Picture showing a jaguar {Panthera onca) rubbing against the hair-snare baited with lures at the zoo in Chiapas and a hair sample obtained from this jaguar After being placed along transects, hair-snares were collected and replaced each month. On

removal, hair-snares were placed individually into plastic bags and labeled with the station

number and the date. Hairs were subsequently collected from each hair-snare using a

magnifying glass and stored in envelopes at room temperature with a silica gel desiccant until

scale patterns were analyzed for species identification. Hair samples were analyzed using a

stereoscopic microscope and field guides (Baca Ibarra 2002, Rodriguez de la Gala Hernandez

2002, Monroy-Vilchis and Rubio-Rodriguez 2003) plus a slide reference collection prepared

from museum specimens (National Mammal Collection, Institute of Biology [UNAM], the

Faculty of Science, National Autonomous University of Mexico [UNAM], the Colegio de la

Frontera Sur, Unidad San Cristobal, Chiapas, Mexico and the Natural History Institute, Tuxtla

Gutierrez, Chiapas, Mexico). Only hair with scale patterns similar to felids were selected for

DNA analysis.

All DNA samples were analyzed at the Natural Resources DNA Profiling and Forensic

Centre, Trent University, Peterborough, Ontario, Canada. All DNA extractions were carried out in a dedicated room using a Qiagen kit for tissue following a standard extraction protocol.

Identification to species was done based on a diagnostic portion of mtDNA cytochrome b region obtained with primers developed for these 5 Neotropical feline species. References sequences were obtained from blood samples obtained from zoo specimens of all 5 felid species that had originated from the Chiapas region. Wild species identification was based on analysis and comparison to the reference and wild samples to sequences from Genbank using the computer program Mega 3.1.

77 5.6 Results

We recorded 389 hits on 888 hair-snare stations over the two years of the study, which represented a success rate of 43 percent. A total of 240 hits over 560 hair-snare stations (42%) were reported inside the MABR and a total of 100 hits over 328 hair-snares stations (30%) were reported outside the MABR in the disturbed agricultural areas. An assessment of mammalian activity (number of hits/number of traps x number of nights = number of hits/1 night x 1000 = number of hits/1000 trap-nights) indicated a total rate of 14.6 hits/1000 trap- nights, with 14.2 hits/1000 trap-night occurring inside the conservation area and 10.1 hits/1000 trap-night occurring outside the conservation area with a mean hit rate of 0.15 hits/ 1000 trap- nights for the entire study. When comparing the data from inside and outside the conservation area, there were 0.23 felid hits/ 1000 trap-nights inside the conservation area and 0.00 felid hits/1000 trap-nights felid outside in the disturbed areas. Results per felid species were: margay

0.075 hits/ 1000 trap-nights, 0.07 hits/ 1000 trap-nights for ocelot and 0.07 hits/ 1000 trap- nights for jaguarondi. When comparing these data based on location, 0.119 hits/ 1000 trap- nights were recorded for margay inside the conservation area, while 0.059 hits/ 1000 trap- nights were recorded for the other two felid species. Zero hits for felid species were reported outside the MABR.

Based on scale pattern, only 72 hair samples were selected for DNA analysis of which 57 were collected inside the conservation area and 15 from outside. Hairs were selected based on presence of scale patterns that approximate those of felids and that had complete hair follicles at their root. From these samples, a total of 41 produced DNA, while the rest failed to yield adequate amounts of non-degraded DNA. From these hair samples, we identified margay

(Leopardus wiedii, n=2), ocelot (Leopardus pardalis, n=l) and jaguarundi (Puma yaguarundi,

78 n=l). Other species identified were grey fox (Urocyon cinereoargenteus, n=l), tayra (Eira

barbara, n=3), coati (Nasua narica, n=l) and two species of marsupials; four eyed opossum

(Metachirus nudicaudatus, n=6) and common opossum (Didelphis marsupialis, n=16). Eight

samples were of unknown identity and two samples contained evidence of mustelids. However,

these were excluded due the low confidence of identification. From the 41 samples that produced DNA, only one was from outside the conservation area, but the species was not identifiable.

5.7 Discussion

Our hair sample protocol proved useful for assessing activity and distribution of a variety of mammalian species. Results on the percentage of hits reported were similar to other studies that have reported 46% to 49% success, respectively (Ruell and Crooks 2006, Downey et al. 2007).

The data obtained in all categories analyzed in this study, showed fewer hits outside the conservation area, suggesting that this technique can in fact address questions related to changes in mammalian communities influenced by habitat fragmentation and human disturbance. Even though expensive, we recommend that future studies identify all samples using DNA techniques and not just target species, since anthropogenic activities including the introduction of domestic animals influence the results.

The hair-snare protocol used in this study allowed for the identification of three of the five target felid species, but failed to detect high numbers of any individual species. Attraction to hair-snares by ocelot, jaguarondi and margay has been previously reported for captive animals

(Reiger 1979, Harrison 1997); however, the present study is the first to report the successful collection of hair samples from jaguarondi and margay in the wild and hair samples from ocelots in a tropical rainforest ecosystem. Hair samples from ocelots were low in this study;

79 however, other researchers have obtained higher numbers of ocelot hair samples using hair-

snares in the drier grassland ecosystems of Texas (Shinn 2002, Weaver et al. 2005).

Differences in the number of hair samples collected for this species may be due to lower densities in populations in tropical ecosystems (Connell 1978, Leith and Werger 1989). The other two species targeted in this study, the jaguar and puma were not collected. Collection of jaguar hair samples using hair-snares in the wild has not been reported to date, while data on the puma have reported low returns or no returns at all (Shinn 2002, Ruell and Crooks 2006,

Downey et al. 2007). McDaniel et al. (2000), Mills et al. (2000) and Schmidt and Kowalczyk

(2006) successfully obtained hair samples from the field when targeting lynx (Lynx canadensis) in boreal ecosystems. Detection rates were also high in hair-snare studies of ocelots in Texas, as Shinn (2002) and Weaver et al. (2005) obtained high numbers of samples when working with ocelot populations that had radio-collared individuals. However, attempts using the hair- snares to study larger felid species remain relatively unsuccessful. Shinn (2002) identified only

1 puma hair sample despite the fact that a relative large population was known to exist in the study area. Similarly, Ruell and Crooks (2006) obtained relative low felid results for bobcat and puma compared with non-target species, while Downey et al. (2007) did not detect margays and pumas in prime habitat for these species, although a large variety of non-target species were represented in their data.

Previous studies have reported the use of hair-snares primarily to assess populations of the

Order Caraivora. However in this study, we report the presence of 2 species of opossums, which belong to the Order Marsupialia and are omnivorous. The use of the hair-snares by opossums seems to be common and needs to be considered when researchers are using this method in areas where opossums are present. Despite the fact that the hair-snares in this study

80 were designed to target carnivores with special emphasis on felids, it is clear that they attracted

other species of mammals. Schmidt and Kowalczyk (2006) were able to successfully avoid

non-target species in their study by setting of hair-snares beyond the reach of most other

species. Also, as their study occurred in a more temperate region with lower species richness,

the chances that non-target species would be involved were reduced. The sampling of non- target species is problematic, as DNA analysis is time consuming and costly. However,

sometimes this "bycatch" can provide useful information and is often of interest and especially useful in areas such as the tropics where inventories and information on most species is limited.

This method could also be useful for diversity and abundance studies or if a method or lures can be developed to consistently collect hairs from a single individual species. To reduce the number of non-target species samples obtained at each station shorter collection intervals have been recommended or the design of a trap that could limit sampling to a single visit by the targeted species. Kendall and Mckelvey (2008) have suggested developing a hair snare trap that has a single serving size of bait that is consumed during the first visit or a modified box trap in which the door is non-locking for the first captured animal, but locks after the animal escapes and prevents other animals from entering.

Results of this and other studies support the conclusion that existing hair-snare protocols have particular challenges associated with them when studying felids and often results in many samples from other mammalian species. This could be due to the nature of felid hairs, which are very short and fine compared to the coarser hair found in canids, bears and mustelids

(Woods et al. 1999, Mowat and Strobeck 2000, Mowat and Paetkau 2002, Kendall and

Mckelvey 2008) The establishment of specific molecular techniques for low yield felid DNA would also increase success (Gagneux 1997, Paetkau 2003).

81 5.8 Implications for Felid Conservation

The deficit of information on carnivore populations and specifically felids in tropical

ecosystems is partially due to the lack of reliable cost effective methodologies and techniques

that allow managers to obtain data that will eventually lead to the development of appropriate

management strategies. The hair-snare method and results presented in this study indicate the

great potential that this technique has for studying mammals in tropical ecosystems. This

technique is valuable for monitoring species of mammalian carnivores and suggested further refinements are needed to increase the utility of this population assessment methodology to study felid populations.

This methodology can be used on projects that specifically aim to conserve felid species by supporting ecological research. It can assist in research that will provide base-line data regarding the behavior and ecology of these five species of wild cats, providing an insight into their relative abundance, population status, distribution, movement patterns and genetic variability. Knowledge that is required to apply successful and appropriate management and protection strategies.

5.9 Acknowledgments

We thank E. A. Cabrera at the Zoologico Miguel Alvarez Del Toro for providing blood samples and T. Chong (NRDPFC) for laboratory support, F. Garcia for building the hair-snares and Dr. A. Omri and Dr. K. Nkongolo for reviewing the manuscript. Financial support was provided by Laurentian University and Mexico's National Council of Science and Technology

(CONACYT) scholarship to NGA. El Colegio de la Frontera Sur, Unidad San Cristobal,

Chiapas, Mexico, provided logistic support.

82 Chapter 6. Species-specific cytochrome b amplifications for Mexican Neotropical Felids

6.1 Abstract

Primers for species-specific partial sequences of cytochrome b were developed to identify

five Neotropical felid species found in Mexico. Amplification and diagnostic sites were

surveyed on control blood samples obtained from Mexican zoo specimens and on hair samples

obtained from the field, using a noninvasive hair snare method. Successful amplification was

obtained when tested on all blood samples and 3 out of 5 of the target species were identified

from hair samples collected in the field. The amplification of the species diagnostic region of

the cytochrome b was especially useful when used on samples with low quantities of degraded

DNA associated with the noninvasive sampling method.

6.2 Introduction

The jaguar (Panthera onca), puma (Puma concolor), ocelot (Leopardus pardalis), jaguarundi (Puma yagouaroundi) and margay (Leopardus wiedii) are Neotropical felids present in Mexico and are sympatric in several areas. All are impacted by habitat loss, habitat fragmentation and human persecution and are listed in Appendix I or II by the Convention on the International Trade of Endangered Species (CITES) and by IUCN that monitors and protects endangered species. Evidence indicates that all populations of these felids are in decline and their status may require reconsideration if more data were available (Nowell and

Jackson 1996). Considering the solitary and elusive behavior of these species, information on wild populations is not easily obtained. Studies using noninvasive sampling methods in combination with molecular techniques are of enormous value. However, these methods often provide samples with low quantities of degraded DNA (Piggott & Taylor 2003, Kurose et al.

83 2005, Fernandes et al. 2008). Therefore, it is important to develop efficient and effective

molecular tools for the noninvasive study of such populations.

Primers for a diagnostic mitochondrial DNA marker were developed to amplify all the wild

Neotropical felid species from Mexico. The primers were tested on blood samples obtained

from zoo specimens and DNA from hair samples obtained from the field.

6.3 Material and methods

Blood samples were from zoo specimens housed at ZOOMAT, Chiapas, Mexico. Samples

were preserved on Whatman filter paper and kept in sealed envelopes. All extractions were

carried out at the Natural Resources DNA Profiling and Forensic Center, Peterborough,

Ontario, Canada. DNA was extracted using Qiagen kits for tissue. A portion of the mtDNA cytochrome b region was initially amplified using universal primers LI4724 and HI 5149

(Kocher et al. 1989). The reaction volume (25/j.l) contained lx PCR reaction buffer

(invitrogen), 0.3 ^M of each primer, 2.5mM of dNTPs, 2mM MgCl2, 1 U Taq DNA polymerase (invitrogen), and 5ul of genomic DNA mixture. After an initial incubation at 94°C for 5 min, the PCR profile was, 35 cycles at 94°C for 1 min, 55° C for 1 min, and 72° C for 1.5 minutes (Mills et al. 2000). All products were electrophoresced on a 1.5 % gel with ethidium bromide stain using a low size/mass ladder. Isolations and PCR amplifications included positive and negative controls. Subsequently, 5|il of PCR product and 2|il of EXOSAP was used to purify the samples and prepared them for sequencing with the same PCR primers in a

MegaBACE sequencer, which resulted in sequences from 430bp to 470bp.

To create mitochondrial markers for these Neotropical feline species exiting in Mexico when using degraded DNA, primers were developed for species-specific regions of the mitochondrial cytochrome b gene. For each species, forward and reverse sequences were

84 obtained and assessed manually for ambiguities based on the electropherograms. Sequences of

tiger (.Panthera tigris) and a sequence of domestic cat (Felis catus) were also included in this

analysis for comparison (Lopez et al. 1996). Revised sequences were aligned using MEGA 3.1

and assessed for differences. Subsequently, species diagnostic sequences of a region of the

cytochrome b gene were identified to differentiate among species. Primers were developed at

the opposite ends of the sequences at sites of low variability.

The newly designed primers were tested on blood samples of the five species and the same

protocol for PCR amplification and sequencing was performed. Sequences for each species

were compared with available data from Genbank and the literature (Masuda et al. 1996). To

test if the designed DNA primers were useful for identifying each of the species in the wild,

primers were used to amplify DNA from hair samples obtained from the field from a study in

the Selva Lacandona, Mexico, where all species are sympatric. DNA from the hairs was

extracted by cutting them in pieces. Initially, 50jil (600U/ml) of Proteinase K was added and

samples were placed in an incubator at 65°C for 2 hours. An additional 50jj.1 of Proteinase K was subsequently added and samples were then placed for 12 hours in the incubator with an initial temperature of 65°C and a final temperature of 37°C. To isolate the DNA, a Qiagen kit for tissue was used. After applying the same PCR protocol using the newly developed primers, sequences were obtained and compared to reference sequences identified in the blood samples.

Wild species assignment was based on > 98% similarity to the reference sequences from the blood samples.

6.4 Results and Discussion

The newly designed primers (MxCtF 5' -CCATCCAACATCTCAGCATGATG-3' and

MxCtR 5 '-GAGGCTCCGTTGGCATGTAT-3') provided amplification of the blood DNA

85 from the five felid species. A band of expected size was attained for all species and the amplified product was 170 bp. Species diagnosis was based on 10 to 26 variable sites in a 144 bp region of this sequence (Table 6.1). Reference sequences obtained were placed in GenBank

(accession no.FJ490205-FJ490209). All reference sequences obtained had at least > 95% similarity to previously published sequences except for those of the jaguarondi, which was 85% similar (Masuda et al. 1996) (GenBank accession no. AY886751).

Table 6.1. Nucleotide diagnostic species-specific sequences of 144bp from the cytochrome b gene of five Neotropical felid species from Chiapas, Mexico. Species codes: Pon (Panthera onca), Pco (Puma concolor), Lpa (Leopardus pardalis), Pya (Puma yagouaroundi) and Lwi (Leopardus wiedii).

Species code Lpa GGCTCCTTAT TAGGAGTTTG CCTAATTTTA CAAATTCTCA CCGGCCTCTT Pco C...... G..C.. . . CC . . C..A. Pya . .T C. . CC. . Lwi CC. . . T T . . Pon C. . . c.. T CC . .

Lpa TCTAGCCATA CACTATACAT CAGATACAAC AACCGCCTTT TCATCAGTTA Pco C. .G ....C....T G. .T C. Pva c . .T ....C C A. Lwi c C...... C T. .C Pon .T. . .C. . . . .,,CA...GT T. . .

Lpa CCCACATCTG CCGCGACGTC AACTATGGCT GAATCATCCG ATAC Pco .T C Pya T. . T. .A c...... T Lwi . . . . T Pon T. . A

86 When primers were used to amplify the DNA samples from the field, it was possible to

identify three of the five target species including ocelot, jaguarundi and margay. The similarity of the field sequences to reference sequences obtained from blood were 98%, 99% and 98%, respectively and varied by 4, 1, and 3 base pairs, respectively. The other two species, jaguar and puma, were not represented in the hair samples obtained.

To establish adequate management strategies and assign species to the appropriate status categories, we need to document the presence and abundance. The primers developed in this study should be of considerable value as they can identify the felid species studied using DNA obtained from a noninvasive sampling method. The markers obtained allowed for the discrimination among the five sympatric species in Mexico. The results support this conclusion as the reference sequence from the zoo animals and samples from the field were 98% similar, while when comparing the jaguarondi sample from Mexico with other geographic region it was only 85% similar. In addition, the primers developed in this study proved to be useful with low- yield and degraded DNA. This combination of methodologies allowed for the rapid and reliable identification of felid species from Mexico.

6.5 Acknowledgements

We thank E. Cabrera at the ZOOMAT for providing blood samples and T. Chong for laboratory support. This project was financed by Laurentian University and a doctoral scholarship to N.G.A. from The Mexico's National Council of Science and Technology

(CONACYT).

87 Chapter 7. Conclusions and Future Work

7.1 Overview

Five species of felids inhabit the Selva Lacandona and evidence indicates that all

populations of these felids are in decline in disturbed areas. This study developed and tested

genetic and field techniques designed to help expand the limited information available for these

species in this region in the hope that this information will facilitate the development

sustainable management strategies that are appropriate for the area.

Based on scale and medullar patterns of guard hair, it was possible to identify the terrestrial

mammalian species that exist in Chiapas. These methods have special utility as they employ

noninvasive techniques to obtain mammalian hair samples. The hair-snare method tested shows

that one is able to obtain mammalian hair samples that can be used to assess felid populations.

However when used on tropical areas that have a great variety of mammals, this technique attracts many non-target species and needs to be improved to avoid such interference.

Primers and species-specific sequences developed m this study allowed me to discriminate among all five felid species and proved to be useful for identifying three out of the five sympatric Selva Lacandona felid species in the wild. The primers were especially useful for samples with low-yield and degraded DNA, a condition that is common when using samples obtained by noninvasive methods.

The combination of genetic and field methodologies presented in this study could be used to assess felid populations in tropical areas and help to develop management strategies which may sustain felid populations and should be encouraged by government and conservation organizations.

88 7.2 General Conclusions

The results of this study support the conclusions that:

(1) guard hairs are useful for the identification of terrestrial mammals in Chiapas, Mexico, (2) scale and medullar patterns of the guard hairs of terrestrial mammals from Chiapas, Mexico, did not differ from the same species located in different geographic areas, (3) the hair-snare method has potential for studying mammals in tropical ecosystems, (4) the hair-snare method is less successful for felids and often more successful for other non-target carnivore species, (5) the hair-snare method is cost effective and can allow for reliable identification of target species, (6) the hair-snare method was valuable for monitoring mammalian carnivores; however, further research is needed to expand the utility of this population assessment methodology, (7) the primers developed successfully amplified low-yield and degraded DNA obtained from hair samples for the 5 felid species, (8) the species-specific sequences allowed for the discrimination among the five sympatric species, (9) the sequences obtained for the identification of the five felid species from Chiapas allow comparison among specimens from different geographic regions, (10) the primers and species-specific sequences may be useful for the identification of these species throughout Central America and northern South America, (11) the hair-snare methodology combined with the developed molecular DNA tools has the considerable potential for the study of rare and endangered felid species, (12) further refinement of these methodologies (design and lures) should allow researchers to target specific species and reduce the incidence of non-target species,

89 Literature Cited

Adorjan, A.S. and G.B. Kolensosky. 1969. A Manual for the Identification of Hairs of Selected

Ontario Mammals. Ontario Ministry of Natural Resources, Toronto.

Alibhail, S.K., Z.C. Jewell and P.R. Law. 2008. A footprint technique to identify white rhino

Ceratotherium simum at individual and species levels. Endangered Species Research 4: 205-

218.

Anonymous. 1992. Propuesta de plan de manejo para la Reserva Integral de la Biosfera Montes

Azules. Edition del Gobierno del Estado de Chiapas, Chiapas, Mexico.

Anonymous. 2000. Programa de manejo de la Reserva de la Biosfera Montes Azules. Institute

Nacional de Ecologia, Distrito Federal.

Aranda, M. 1991. Wild Mammals Skin Trade. Pp. 174-177, in Neotropical Wildlife Use and

Conservation. J. Robinson (ed). Fondo de Cultura Economica, Distrito Federal.

Aranda, M. 2000. Huellas y otros rastros de los mamiferos grandes y medianos de Mexico.

Instituto de Ecologia A.C, Veracruz.

Aranda, M. and I. March. 1987. Guia de los Mamiferos Silvestres de Chiapas. Instituto Nacional

de Investigaciones Sobre Recursos Bioticos, Xalapa,Veracruz.

Arita, H.T. and M. Aranda. 1987. Tecnicas para el estudio y clasificacion de los pelos. Instituto

Nacional de Investigaciones Sobre Recursos Bioticos, Veracruz.

90 Baca Ibarra, I.I. 2002. Catalogo de pelos de guardia dorsal en mamiferos terrestres del estado de

Oaxaca, Mexico. Undergraduate Thesis. Universidad National Autonoma de Mexico,

Distrito Federal, Mexico.

Belant, J.L. 2003. A hair snare for forest carnivores. Wildlife Society Bulletin 31: 482-485.

Boersen, M.R., J.D. Clark and T.L. King. 2003. Estimating black bear population density and

genetic diversity at Tensas River, Louisiana using microsatellite DNA markers. Wildlife

Society Bulletin 31:197-207.

Brunner, H. and B.J. Coman. 1974. The Identification of Mammalian Hair. Intaka Press,

Melbourne.

Ceballos G and P. R. Ehlich. 2002. Mammal population losses and the extinction crisis. Science

296: 904-907.

Connell, J.H. 1978. Diversity in tropical rain forests and coral reefs. Science 199: 1302-1310.

Crawshaw, P.G. 1995. Comparative ecology of ocelot (Felis pardalis) and jaguar (Panthera

onca) in a protected subtropical forest in Brazil and Argentina. Doctoral Thesis. University

of Florida, Gainesville.

Currier, M.J.P. 1983. Felis concolor. Mammalian Species 200: 1-7.

Delibes, M. 1980. El Lince Iberico: ecologia y comportamiento alimenticio en el Coto Donana,

Huelva. Revista de Vertebrados de la Estacion Biologica de Donana 7: 1-109.

Downey, P.J., E.C. Hellgren, A. Caso, S. Carvajal and K. Frangioso. 2007. Hair snares for

noninvasive sampling of felids in North America: do grey foxes affect success? Journal of

Wildlife Management 71: 2090-2094.

91 Eizirik, E., S.L. Bonatto, W.E. Johnson, P.G. Crawshaw Jr., J.C. Vie', D.M. Brousset, S.J.

O'Brien and F.M. Salzano. 1998. Phylogeographic patterns and evolution of the

mitochondrial DNA control region in Two Neotropical Cats (Mammalia, Felidae). Journal

of Molecular Evolution 47: 613-624.

Emmons, L.H. 1987. Comparative feeding ecology of felids in a Neotropical rainforest.

Behavior Ecology and Sociobiology 20: 271-283.

Fernandes, C.A., C. Ginja, I. Pereira, R. Tenreiro, M.W. Bruford and M. Santos-Reis. 2008.

Species-specific mitochondrial DNA markers for identification of noninvasive samples from

sympatric carnivores in the Iberian Peninsula. Conservation Genetics 9: 681-690.

Fernandez, G.J. and S.M. Rossi. 1998. Medullar type and cuticular scale patterns of hairs of

rodents and small marsupials from the Monte Scrubland (San Luis Province, Argentina).

Mastozoologia Neotropical 5: 109-116.

Foran, D.R., S.C. Minta and K.S. Heinemeyer. 1997. DNA-basea analysis of hair to identify

species and individuals for population research and monitoring. Wildlife Society Bulletin

25: 840-847.

Gagneux, P., C. Boesch and D.S. Woodruff. 1997. Microsatellite scoring errors associated with

noninvasive genotyping based on nuclear DNA amplified from shed hair. Molecular

Ecology 6: 861-868.

Gentry, A. 1986. Endemism in tropical versus temperate plant communities. Pp. 153-181, in

Conservation Biology, the Science of Scarcity and Diversity. M.E. Soule (ed). Sinauer,

Sunderland, Masashuse.

92 Goloboff, P., J. Farris and K. Nixon. 2003. Tree analysis using new technology. Program and

documentation available from the authors at www.zmuc.dk/pubiic/phvlogeDy

Harrison, R.L. 1997. Chemical attractants for Central American felids. Wildlife Society Bulletin

25: 93-97.

Harrison, R.L. 2002. Evaluation of microscopic and macroscopic methods to identify felid hair.

Wildlife Society Bulletin 30: 412-419.

Hilton, H. and N.P. Kutsha. 1978. Distinguishing characteristics of the hairs of eastern coyote,

domestic dogs, red fox and bobcat in Maine. American Midland Naturalist 100: 223-227.

Hoogesteijn, R. 2003. Manual sobre problemas de depredation causados por jaguares y pumas

en hatos ganaderos. WCS Report, www.savetheiaguar.com/iag-conservation

(verified September 19, 2008).

Kendall, K.C. and K.S. Mckelvey. 2008. Hair collections. Pp. 135-176, in Noninvasive survey

methods for North American carnivores. R.A. Long, P. MacKay, J.C. Ray, and W.J.

Zielinski (eds). Island Press, Washington D.C.

Kennedy, A.J. 1982. Distinguish characteristics of the hairs of wild and domestic canids from

Alberta. Canadian Journal of Zoology 60: 536-541.

Kennedy, A.J. and L.N. Carbyn. 1981. Identification of wolf prey using hair and feather

remains, with special reference to western Canadian National Parks. Canadian Wildlife

Services, Edmonton.

Kitchener, A. 1991. The Natural History of Wild Cats. Costmock, Cornell, Ithaca.

93 Kocher, T.D., W.K. Thomas, A. Meyer, S.V. Edwards, S. Paabo, F.X. Villablanca and A.C.

Wilson. 1989. Dynamics of mitochondrial DNA evolution in animals: amplification and

sequencing conserved primers. Proceedings of the National Academy of Sciences of the

USA 86: 6196-6200.

Kurose, N., R. Masuda and M. Tatara. 2005. Fecal DNA analysis for identifying species and sex

of sympatric carnivores: A noninvasive method for conservation on the Tsushima Islands,

Japan. Journal of Heredity 96: 688-697.

Laliberte A. S. And W. J. Ripple. 2004. Range Contractions of North American Carnivores and

Ungulates. Bioscience 54: 123- 138.

Leith, H. and M.J. A. Werger. 1989. Ecosystems of the world. Tropical rain forest ecosystem.

Biogeographical and ecological studies. Elsevier Publishing, New York.

Lopez, J. V., S. Cevario and S.J. O'Brien. 1996. Complete nucleotide sequences of the domestic

cat (Felis catus) mitochondrial genome and a transposed mtDNA tandem repeat (Numt) in

the nuclear genome. Genomics 33: 229-246.

Masuda, R., J.V. Lopez, J.S. Slattery, N.Y. Yuhki and S.J. O'Brien. 1996. Molecular phylogeny

of mitochondrial cytochrome b and 12S rRNA sequences in the Felidae: ocelot and domestic

cat lineages. Molecular Phylogeny and Evolution 6: 351-365.

Mayer, W.V. 1952. The hair of California mammals with keys to the dorsal guard hairs of

California mammals. The American Midland Naturalist 48: 480-512.

McDaniel, G.W., K.S. Mckelevry, J.R. Squires and L.F. Ruggiero. 2000. Efficacy of lures and

hair snares to detect lynx. Wildlife Society Bulletin 28: 119-123.

94 Medellin, R. 1994. Mammal diversity and conservation in the Selva Lacandona, Chiapas,

Mexico. Conservation Biology 85: 2780-2798.

Mills, L.S., K.L. Pilgrim, M.K. Schwartz and K. McKelvey. 2000. Identifying lynx and other

North American felids based on mtDNA analysis. Conservation Genetics 1: 285-288.

Miotto, R.A., F. Pacheco Rodriguez, G. Ciocheti and P. Manoel Galetti Jr. 2007. Determination

of the minimum population size of pumas (Puma concolor) through fecal DNA analysis in

two protected cerrado areas in the Brazilian southeast. Biotropica 39: 647-654.

Mittermeier, R.A. and C. Goettsh. 1992. La importancia de la diversidad biologica de Mexico

Pp. 62-73, in Mexico ante los retos de la biodiversidad J. Sarukan, and R. Dirzo (eds).

Comision Nacional para el Conocimiento y Uso de la Biodiversidad, Distrito Federal.

Monroy-Vilchis, O. and R. Rubio-Rodriguez. 2003. Guia de identification de mamiferos

terrestres del Estado de Mexico, a traves del pelo de guardia. Universidad Autonoma de

Estado de Mexico, Toluca.

Moore, T.D., L.E. Spence and C.E. Dugnolle. 1974. Identification of the dorsal hairs of some

mammals of Wyoming. Wyoming Fish and Game Department, Wyoming.

Moruzzi, T.L., T.K. Fuller, R.M. DeGraaf, R.T. Brooks and W. Li. 2002. Assesing remotely

triggered cameras for surveying carnivore distribution. Wildlife Society Bulletin 30: 380-

386.

Mowat, G. and D. Paetkau. 2002. Estimating marten Martes americana population size using

hair capture and genetic tagging. Wildlife Biology 8: 201-209.

95 Mowat, G. and C. Strobeck. 2000. Estimating population size of grizzly bears using hair

capture, DNA profiling, and mark-recapture analysis. Journal of Wildlife Management 64:

183-193.

Murray, J.L. and G.L. Gardner. 1997. Leopardus pardalis. Mammalian Species 548: 1-10.

Myers, N., R.A. Mittermeier, C.G. Mittermeier, G.A.B. da Fonseca and J. Kent. 2000.

Biodiversity hotspots for conservation priorities. Nature 403: 853-858.

Naranjo, E.J., M.M. Guerra, R.E. Bodmer and J.E. Bolanos. 2004. Subsistence hunting by three

ethnics groups of the Lacandon Forest, Mexico. Journal of Ethnobiology 24: 233-253.

Naranjo, E.J., C. Lorenzo Monterrubio and A. Horvat. 2005. La diversidad de mamiferos en

Chiapas Pp. 221-252, in Diversidad Biologica en Chiapas. M. Gonzalez-Espinoza, N.

Neptali Ramirez-Martial, and L. Ruiz-Montoya (eds). El Colegio de la Frontera Sur,

Chiapas, Mexico.

Naranjo, J.E. 2002. Population ecology and conservation of ungulates in the Lacandon Forest,

Mexico. Doctoral Thesis. University of Florida, Gainesville.

Nowell, K. and P. Jackson. 1996. Wild cats, status survey and conservation action plan,

IUCN/SSC Cat Specialist Group. International Union for Conservation of Nature, Gland.

O'Brien, S.J. and W.E. Johnson. 2005. Big Cat Genomics. Annual Review of Genomics and

Human Genetics 6: 407-429.

Oliveira, T.G. 1998a. Herpailurus yagouaroundi. Mammalian Species 578: 1-6.

Oliveira, T.G. 1998b. Leopardus wiedii. Mammalian Species 579: 1-6.

96 Paetkau, D. 2003. An empirical exploration of data quality in DNA-based population

inventories. Molecular Ecology 12: 1375-1387.

Palomares, F., J.A. Godoy, A. Piriz, J. O'Brien and W.E. Johnson. 2005. Faecal genetic analysis

to determine the presence and distribution of elusive carnivores: design and feasibility for

the Iberian lynx. Molecular Ecology 11: 2171-2182.

Piggott, P. and A. Taylor. 2003. Remote collection of animal DNA and its applications in

conservation management and understanding the population biology of rare and cryptic

species. Wildlife Research 30: 1-13.

Rabinowits, A.R. 1986. Jaguar predation on domestic livestock in Belize. Wildlife Society

Bulletin 85: 170-174.

Reiger, I. 1979. Scent rubbing in carnivores. Carnivores 2: 17-25.

Retana, O.G. and C. Lorenzo. 2002. Lista de los Mamiferos terrestres de Chiapas: Endemismo y

Estado de Conservation. Acta Zoologica Mexicana 85: 25-49.

Rodriguez de la Gala Hernandez, R. 2002. Catalogo de pelos de guardia de los mamiferos del

estado de Baja California, Mexico. Undergraduate Thesis. Universidad Nacional Autonoma

de Mexico, Distrito Federal, Mexico.

Ruell, E.W. and K.R. Crooks. 2006. Evaluation of noninvasive genetic sampling methods for

felid and canid populations. Journal of Wildlife Management 71: 1690-1694.

Ryder, M.L. 1973. Hair. Edward Arnold, London.

Rzedwsky, J. 1978. Vegetation de Mexico. Limusa, Distrito Federal.

97 Sarukan, K. 1968. Los tipos de vegetation arborea en la zona calido-humeda de Mexico. Pp.

204-235, in Manual para la Identificacion de campo de los principales arboles tropicales de

Mexico. T. D. Pennington, and J. Sarukan (eds). Instituto Nacional de Investigaciones

Forestales, Distrito Federal.

Schmidt, K. and R. Kowalczyk. 2006. Using scent-marking stations to collect hair samples to

monitor Eurasian lynx populations. Wildlife Society Bulletin 34: 462-466.

Scott, C.S., L.E.T. Ostro, L.K. Marsh, L. Maffei, A.J. Noss, M.J. Kelly, R.B. Wallace, H.

Gomez and G. Ayala. 2004. The use of camera traps for estimating jaguar Panthera onca

abundance and density using capture/recapture analysis. Oryx 38: 148-154.

Seymur, L.K. 1989. Pantera onca. Mammalian Species 340: 1-9.

Sharma, S., Y. Jhala and V.B. Sawarkar. 2005. Identification of individual tigers (Panthera

tigris) from their pugmarks. Journal of Zoology (London) 267: 9-18.

Shinn, K.J. 2002. Ocelot distribution in the lower Rio Grande Valley national refuge. Doctorate

Thesis. Texas-Pan American, Edinburg, USA.

Sloane, M.A., P. Sunnucks, D. Alpers, B. Beheregaray and C. Taylor. 2000. High reliable

genetic identification of individual northern hairy-nosed wombats from single remotely

collected hairs: a feasible censusing method. Molecular Ecology 9: 1233-1240.

Soule, M.E. and K.A. Kohm. 1989. Research priorities for conservation biology. Island Press,

Washington, D.C.

Stain, H.J. 1958. Field Key to Guard Hair of Middle Western Furbearers. Journal of Wildlife

Management 22: 95-97.

98 Steneck, R. 2005. An ecological context for the role of large carnivores in conserving

biodiversity. Pp. 9-32, in Large Carnivores and the Conservation of Biodiversity. J. Ray, R.

Redford, J. Steneck, and J. Berger (eds). Island Press, Washington.

Wallis, R.L. 1992. A key for the identification of guard hairs of some Ontario mammals.

Canadian Journal of Zoology 71:589-591.

Weaver, J.L., P. Wood, D. Paetka and L.L. Laack. 2005. Use of scented hair snares to detect

ocelots. Wildlife Society Bulletin 33: 1384-1391.

Woodroffe R. and J. R. Ginsberg. 1998. Edge effects and the extinction of populations inside

protected areas. Science 280:2126-2128.

Woods, J.G., D. Paetkau, D. Lewis, B.N. Mclellan, M. Proctor and C. Strobeck. 1999. Genetic

tagging of free-ranging black and brown bears. Wildlife Society Bulletin 7: 616-627.

99 Appendix

Reference sequences obtained from this work entered in GenBank (accession no. FJ490205-FJ490209).

Jaguar (Panthera onca)

LOCUS bankitl 158223 165 bp DNA linear MAM 26-NOV-2008 DEFINITION cytochrome b gene, partial cds; mitochondrial gene. ACCESSION 1158223 VERSION KEYWORDS . SOURCE mitochondrion Panthera onca ORGANISM Panthera onca Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Pantherinae; Panthera. REFERENCE 1 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Primers for cytochrome b species-specific partial sequences to identify five sympatric felids from Chiapas, Mexico JOURNAL Unpublished REFERENCE 2 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Direct Submission JOURNAL Submitted (26-NGV-2008) Biology, Laurentian University, Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada COMMENT Bankit Comment: [email protected]. FEATURES Location/Qualifiers source 1 ..165 /organism-'Panthera onca" /organelle-'mitochondrion" /mol_type="genomic DNA" /db_xref="taxon:9690" /note="Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi ;Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Pantherinae; Panthera" BASE COUNT 41a 52 c 27 g 451 ORIGIN 1 ggctccctat taggagtctg tctaatccta caaattctca ccggcctctt tctagccata 61 ctctacacat cagcaacagt aaccgctttt tcatcagtta cccacatttg ccgcgacgta 121 aactatggct ggattatccg atacatgcac gccaatggag cctcc //

100 Puma (Puma concolor)

LOCUS bankitl 158240 165 bp DNA linear MAM 26-NOV-2008 DEFINITION cytochrome b gene, partial cds; mitochondrial gene. ACCESSION 1158240 VERSION KEYWORDS . SOURCE mitochondrion Puma concolor ORGANISM Puma concolor Eukaiyota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Puma. REFERENCE 1 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Primers for cytochrome b species-specific partial sequences to identify five sympatric felids from Chiapas, Mexico JOURNAL Unpublished REFERENCE 2 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Direct Submission JOURNAL Submitted (26-NOV-2008) Biology, Laurentian University, Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada COMMENT Bankit Comment: [email protected]. FEATURES Location/Qualifiers source 1..165 /organism-'Puma concolor" /uigaucnc/ ii — Miiinutiiuiiuiiui i i/ —i ii /moltype-'genomic DNA" /db_xref="taxon:9696" /note="Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Puma." BASE COUNT 41 a 52 c 28 g 441 ORIGIN 1 ggctccctat taggggtctg cctaatccta caaatcctaa ccggcctctt cctggccata 61 cactatacat cagacacaat gactgccttt tcatcagtca ctcacatctg tcgtgacgtt 121 aactacggct gaattattcg gtacatacat gccaacggag cctcc //

101 Ocelot (Leopardus pardalis)

LOCUS bankitl 158243 165 bp DNA linear MAM 26-NOV-2008 DEFINITION cytochrome b gene, partial cds; mitochondrial gene. ACCESSION 1158243 VERSION KEYWORDS . SOURCE mitochondrion Leopardus pardalis ORGANISM Leopardus pardalis Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Leopardus. REFERENCE 1 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Primers for cytochrome b species-specific partial sequences to identify five sympatric felids from Chiapas, Mexico JOURNAL Unpublished REFERENCE 2 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Direct Submission JOURNAL Submitted (26-NOV-2008) Biology, Laurentian University, Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada COMMENT Bankit Comment: [email protected]. FEATURES Location/Qualifiers source 1..165 /organism-'Leopardus pardalis" /organelle-'mitochondrion" /mol_type-'genomic DNA" /db_xref="taxon:32538" /note-'Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Leopardus." BASE COUNT 44 a 52 c 24 g 451 ORIGIN 1 ggctccttat taggagtttg cctaatttta caaattctca ccggcctctt tctagccata 61 cactatacat cagatacaac aaccgccttt tcatcagtta cccacatctg ccgcgacgtc 121 aactatggct gaatcatccg atacatacat gccaacggag cctcc //

102 Jagurundi (Puma yaguarondi)

LOCUS bankitl 158245 165 bp DNA linear MAM 26-NOV-2008 DEFINITION cytochromc b gene, partial cds; mitochondrial gene. ACCESSION 1158245 VERSION KEYWORDS . SOURCE mitochondrion Herpailurus yaguarondi ORGANISM Herpailurus yaguarondi Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Herpailurus. REFERENCE 1 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Primers for cytochrome b species-specific partial sequences to identify five sympatric felids from Chiapas, Mexico JOURNAL Unpublished REFERENCE 2 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Direct Submission JOURNAL Submitted (26-NOV-2008) Biology, Laurentian University, Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada COMMENT Bankit Comment: [email protected]. FEATURES Location/Qualifiers, source 1..165 /organism-'Herpailurus yaguarondi" /organelle="mitochondrion" /mol_type="genomic DNA" /db_xref="taxon:61402" /note-'Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae;Felinae; Puma" BASE COUNT 46 a 48 c 27 g 441 ORIGIN 1 ggttccttat taggagtctg cctaatccta cagattctga caggcctatt cctagccata 61 cattatacat cagacacaac aaccgccttc tcatcagtaa cccacatttg ccgcgatgta 121 aactacggct gaatcatccg atatatacat gccaacgggg cttct //

103 Margay (Leopardus wiedii)

LOCUS bankitl 158247 165 bp DNA linear MAM 26-NOV-2008 DEFINITION cytochrome b gene, partial cds; mitochondrial gene. ACCESSION 1158247 VERSION KEYWORDS . SOURCE mitochondrion Leopardus wiedii ORGANISM Leopardus wiedii Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Leopardus. REFERENCE 1 (bases 1 to 165) AUTHORS Garcia Alaniz,N„ Wilson,P.J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Primers for cytochrome b species-specific partial sequences to identify five sympatric felids from Chiapas, Mexico JOURNAL Unpublished REFERENCE 2 (bases 1 to 165) AUTHORS Garcia Alaniz,N., Wilson,P. J., Mejia Guerrero,O.H. and Mallory,F.F. TITLE Direct Submission JOURNAL Submitted (26-NOV-2008) Biology, Laurentian University, Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada COMMENT Bankit Comment: [email protected]. FEATURES Location/Qualifiers source 1..165 /organism-'Leopardus wiedii" /organelle-'mitochondrion" /mol_type-'genomic DNA" /db_xref="taxon:61382" /note="Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;Mammalia; Eutheria; Laurasiatheria; Carnivora; Feliformia; Felidae; Felinae; Leopardus." BASE COUNT 43 a 55 c 24 g 431 ORIGIN 1 ggctccttat taggagtttg cctaatccta caaattctca ctggcctttt cctagccata 61 cactacacat cagacacaac aaccgctttc tcatcagtta cccacatctg ccgcgacgtc 121 aactatggct gaattatccg atacctacat gccaacggag cctcc //

104