DIETS OF FREE-RANGING MEXICAN GRAY WOLVES

IN ARIZONA AND NEW MEXICO

by

JANET E. REED. B.S.

A THESIS

IN

WILDLIFE SCIENCE

Submitted to the Graduate Faculty

of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

Chairperson of the Committee

Accepjpd

Dean of the Graduate School

May, 2004

ACKNOWLEDGEMENTS

I thank the United States Fish and Wildlife Service (USFWS) for fiinding this

project I appreciated the direction of Mexican wolf recovery leader and committee

member, Brian T Kelly, the assistance of Mexican wolf Interagency Field Team

personnel (i e , USFWS, Arizona Game and Fish Department, New Mexico Department

of Game and Fish, USD A APHIS Wildlife Services, and US Forest Service [USFS])

and all the volunteers who coUected scats Thank you, David R Parsons and Wendy

Brown, for mentoring and believing in me

I thank Dr Warren B Ballard, my major advisor, for providing encouragement

and guidance throughout this study Special thanks go to committee member Dr Robert

J Baker for welcoming me into his genetics lab and to Celine C Perchellet, Adam

Brown, and Drs Jeff Wickliffe and Federico Hoffman for their dedication, assistance,

and humor in the DNA lab I also thank committee members Drs Philip S Gipson, Paul

R. Krausman, and Mark C Wallace for their ad\ice and support A special thank you to

Dr David B Wester for facilitating the statistical analyses

I thank the USFS Alpine and Clifton Ranger Districts for their assistance on the

forest A special thank you to Myron C Burnett, USFS Wilderness Ranger, for rising

above the opposition to teach me to pack burros and believing that I could handle the job

I also thank the residents of Alpine for welcoming me into their community and all the

forest visitors who assisted with scat collecting Thank you Waypoint Enterprises, Show

Low, Arizona, for your donation of UTM and Multi Waypointers

My parents, Roger C and Betty A Stone, my sister, Anita D. Moore, and my

fnend, Alan B Alley, provided unwavering support and encouragement throughout my

education. Thanks to my two burros, Pancho and Lefty, who carried my gear and the

stinky carnivore scats tirelessly over many miles. Finally, my dog. Kaiser von

Stockwerk, provided faithfiil companionship for MVJ years His unconditional love was

greatiy appreciated

111

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT vi

UST OF TABLES viii

USTOFnOURES x

CHAPTER

I INTRODUCTION 1

Literature Cited 2

n fflSTORICAL OVERVIEW OF THE MEXICAN GRAY WOLF

AND ITS RECOVERY 3

Literature Cited 7

in DIFFERENTIATING MEXICAN WOLF AND COYOTE SCATS

USING DNA ANALYSIS 10

Abstract 10

Introduction 11

Study Area 12

Materials and Methods 14

Results 22

Discussion 24

Acknowledgements 31

Literature Cited 32

IV

IV. DIETS OF FREE-RANGING MEXICAN GRAY WOLVES IN

ARIZONA AND NEW MEXICO 45

Abstract 45

Introduction 47

Study Area 48

Methods 51

Resuhs 55

Discussion 57

Acknowledgements 64

Literamre Cited 65

V SUMMARY 82

ABSTRACT

The Mexican gray wolf (Catiis lupus baileyi) was extirpated from the

southwestern United States before any systematic smdies could be conducted, therefore

littie is known about the subspecies' natural history From April 1998 through October

2001, we coUected carnivore scats {n = 1,682) from the Blue Range Wolf Recovery Area

(BRWRA) in .\rizona and New Mexico to study the diets of free-ranging Mexican

wolves We identified the scats to species using traditional field methods (i e , diameter,

location, and sign) and odor, but expected that it would be difficult to separate Mexican

wolf and sympatric coyote {Cams latrans) scats We tested this hypothesis whh fecal

DNA analysis (molecular scatology) to verify the accuracy of identifying Mexican wolf

and sympatric coyote scats Our DNA results showed scats > 28 mm diameter could be

identified as deposited by Mexican wolves, a high overlap in scat diameters for Mexican

wolves and sympatric coyotes, and a difference in the 2 species' scat diameter means

To determine the diets of Mexican wolves, we used our DNA results to refine our

scat identification criteria Scats for diet analysis were identified as deposited by

Mexican wolves by DNA analysis, diameters ^ 28 mm, 2 den site locations, and ttacks.

Diet analysis of 55 scats with diameters ^ 28 mm collected from areas where 6 Mexican

wolf packs received supplemental food items indicated Cienega and Hawks Nest packs

consumed non-supplemental food items during supplemental feeding and Campbell Blue,

Lupine, Mule, and Pipestem packs did not Diet analysis of 251 scats revealed non-

supplememal fed Mexican wolves consumed large-sized food items, primarily elk

VI

{Cerws elaphus caiKhJensis [nelsoni]) adults and calves We compared this diet

composition to that found in 26 scats identified with DNA analysis and found more large-

sized food items appeared in Mexican wolf scats identified by our refined traditional

method than in scats identified with DNA analysis There was no difference between

diameter means or number of food items per scat between the 2 scat identification

methods There was a difference between diet composition for 26 Mexican wolf scats

and that found in 21 sympatric coyote scats identified with DNA analysis, with Mexican

wolves consuming more large-sized food items than sympatric coyotes. There was also a

difference between diameter means and number of food items per scat for the 2 species,

vsith sympatric coyote scats containing more food items per scat than Mexican wolf scats.

Lastly, we found a difference in diet composition of Vtexican wolves when compared to

diet composition reported in 7 previous diet studies of other North American gray

wolves Mexican wolf diet analysis revealed more large-sized food items than the

subspecies' larger, northern counterparts

vn

UST OF TABLES

3.1 Comparison of accuracy of predicting which species (Mexican wolf or coyote) deposited a scat using discriminant analysis based on combination of 3 measurements takoi from scats identified to species with DNA analysis 43

3.2 Locations of scats (n = 47) identified as Mexican wolf or coyote with DNA analysis 44

4.1 Food items found in scats (n = SS) ^ 28 mm diameter collected from areas where Mexican gray wolf packs (n = 6) were fed supplemental food items (i.e., carnivore logs and road-killed elk, deer and jackrabbit) 71

4.2 ConqMurison of diet composition found in scats (n = 55) ^ 28 mm diameter from areas where Mexican gray wolf packs (n = 6) were fed supplemental food items (i.e., carnivore logs and road-killed elk, deer, and jackrabbit) to determine presence of non-supplemental food items (e.g., elk aduhs and calves, deo* adults and £Eiwns, domestic bovine, and insects) 72

4.3 Food items (n = 265) found in free-ranging Mexican gray wolf scats (/i = 251) 73

4.4 Comparison among years (n = 4,1998-2001) of diet composition found in scats {n = 251) of free-ranging Mexican gray wolves collected from April 1998 to October 2001 in Arizona and New Mexico 74

4.5 Comparison between seasons (n = 2, fiill-winter versus s|Hing-summer) of diet composition found in scats (/i = 251) of free-ranging Mexican gray wolves in Arizona and New Mexico (April 1998 - October 2001) 75

4.6 Comparison of diet conq)08ition among packs (n = 4) found in free-ranging Mexican wolf scats (n = 251) collected from AJptii 1998 to October 2001 in Arizona and New Mexico 76

4.7 Food items (n = 33) found in free-ranging Mexican gray wolf scats (n = 26) collected from April 1998 to October 2001 in Arizona and New Mexico 77

4.8 Comparison of diet c<Hiq)Osition of Mexican wolf scats (n = 277) collected from April 1998 to Oct(A>er 2001 in Arizona and New Mexico and identified u«ng 2 methods: DNA analysis (n = 26) and refined traditional (/t = 251; i.e., ^ 28 mm diameter, 2 den sites, and tracks) 78

4.9 Coiiq>ari8(ni of diet composition found in free-ranging Mexican wolf scats (/f = 26) and sympatric coyote scats (/i = 21) collected from hipdX 1998 to October 2001 in Arizona and New Mexico 79

viu

4.10 Food items found in free-ranging Mexican gray wolf scats (n = 26) and sympatric coyote scats (n = 21) coUected from April 1998 to October 2001 in Arizona and New Mexico 80

4. U ConqpariscHi of diet composition found in free-ranging Mexican wolf scats (n = 277) and diet oompositi(» reported in other North American gray wolf diet studies (n = 7) 81

IX

LIST OF FIGURES

2.1 The Bhie Range Wolf Recovery Area (BRWRA) in Arizona and New Mexico where Mexican gray wolves were released beginning April 1998 9

3 1 Agarose gd showing a portion of the mtDN A control region (D-loop) amplified via Pilgrim et al. (1998) canid-specific primers (Mexican wolf and dog, 164 bp; coyote 160 bp) 40

3 2 Agarose gel showing restriction fragment digestion with BstN I of mtDNA control r^on (D-loop) purified polym«-ase chain reaction (PPCR) isolated from Mexican wolf and coyote scats 41

3.3 Comparison of diameters of coyote scats (n = 21, range 17.4 to 27.8 m; X = 22.8 nun) and Mexican wolf scats (n = 26; range 16.3 to 35.8 nun, x = 26.0 nun) identified with fecal DNA analysis 42

CHAPTER I

INTRODUCTION

The following chapters constitute partial fulfillment of the requirements for the

degree of Master of Science in Wildlife Science for the Graduate School at Texas Tech

University These chapters are the resuh of research conducted on Mexican gray wolves

(Cojus lupus baileyi) m Arizona and New Mexico from April 1998 through October

2001 Chapter II is an historical overview of the Mexican gray wolf and its recovery

Chapters ID and I\' are 2 manuscripts intended for submission to peer-reviewed journals.

Chapter III discusses differentiating Mexican wolf and coyote scats using fecal DNA

analysis (molecular scatology) Chapter IV reports the diets of free-ranging Mexican

wolves in .\rizona and New Mexico from April 1998 through October 2001, and

compares the diets of Mexican wolves to sympatric coyotes and to other North American

gray wolves Chapter V is a summary of all chapters ,\11 chapters represent my ideas,

analyses, and writing ability Each chapter has several coauthors, which were determined

using guidelines provided by Dickson and Conner (1978) and the CBE Style Manual

Committee (1994) .Authorships for chapters are as follows

Chapter III Janet E Reed, Robert J Baker, Warren B Ballard, and Brian T Kelly

Chapter IV; Janet E Reed, Warren B Ballard, Philip S Gipson, Brian T Kelly, Paul

R Krausman, Mark C Wallace, and David B Wester

LiteraUire Cited

CBE Style Manual Committee 1994 Scientific style and format: the CBE manual for authors, editors, and publishers Sixth Edition Council of Biology Editors, Cambridge University Press, New York, New York, USA.

Dickson, J. G., and R N Conner 1978 Guidelines for authorship of scientific articles. Wildlife Society Bulletin 6: 260-261

CHAPTER II

HISTORICAL OVERVIEW

OF THE MEXICAN GRAY WOLF AND ITS RECOVERY

Historically, the Mexican gray wolf (Ca7>/5 lupus baileyi), or lobo, lived the

farthest south of all gray wolves (C lupus) on North America and in the most arid

environment at elevations from 1,200 to 3,300 m ranging throughout central and

southeastern Arizona, western Texas, southern New Mexico, and most of Old Mexico

(Young and Goldman 1944, Brown 1983, Parsons 1996) h is believed that the Mexican

wolf comributed to the overall historical biological diversity and ecological functioning

of both the southwestern ecosystems and the continued evolution of species the Mexican

wolf preyed upon (Parsons 1998)

It is uncertain if the Mexican wolf was sympatric with the coyote {Canis latrans)

in the Southwest before humans altered its natural habitat (Brown 1983) Gier (1975)

and Bekoff and Wells (1986) reported that historical coyote distnbutions were confined

primarily to plains and deserts until the spread of civilization and the reduction of gray

wolf ranges

By the early 1880s, humans and associated livestock had moved into the

Southwest in large numbers (Brown 1983, Shumway 1998) Due to overgrazing and

unregulated subsistence and market hunting, native prey species were in decline and

some predators turned to the more abundant and easily caught livestock (United States

Fish and Wildlife Service 1987) Government and private predator comrol programs

designed to protect livestock in the United States and Mexico accelerated the extirpation

of the Mexican wolf from the Southwest by the late 1960s (Gipson and Ballard 1998).

In 1975, the Arizona-Sonora Desert Museum proposed to capture Mexican

wolves from the remaining population in Mexico and to place them in a captive-breeding

program for future re-estabhshment of the subspecies in its historic range (Siminski

1997) The United States government listed the Mexican wolf as endangered in 1976 and

Mexico began protecting this subspecies, as well (United States Fish and Wildlife Service

1996) The last 4 Mexican wolves known to exist were captured in Durango and

Chihuahua, Mexico from 1977 to 1980 (McBride 1978) and 3 of them founded an official

captive-breeding program (Garcia-Moreno et al 1996, Hedrick et al 1997) To increase

genetic diversity in the official Certified (renamed McBride) lineage, 2 other captive

Mexican wolf populations, Arag6n and Ghost Ranch lineages, were certified genetically

pure in July 1995 (Hedrick et al 1997) and incorporated into the official captive-breeding

program The first offspring from cross-lineage pairings were produced in 1997 (Parsons

1998) .\s of October 2001, the global population of Mexican wolves consisted of 227

individuals, the majority of which were held in 40 zoos and wildlife sanctuaries

throughout the United Stales and Mexico (P Suninski, Arizona-Sonora Desert Museum,

personal communication)

Recovery efforts for the Mexican wolf were outlined in the Mexican Wolf

Recovery Plan (United States Fish and Wildlife Service 1982) and research flinded by the

Lmted States Fish and Wildlife Service (USFWS) supported experimental releases of

Mexican wolves imo isolated Southwest habitat where close monitoring could be

conducted (Bednarz 1988) The Final Envu-onmental Impact Statement (FEIS) for the

Mexican wolf proposed reintroduction of the subspecies into a portion of its historical

range in Arizona, New Mexico, and Texas (United States Fish and Wildlife Service

1996) Three areas were considered the Blue Range Wolf Recovery Area (BRWRA) in

.\rizona and New Mexico, White Sands Missile Range Wolf Recovery Area in New

Mexico, and Big Bend National Park in Texas This proposal was formally approved by

Secretar, of the Interior Bmce Babbitt's March 1997 Record of Decision (United States

Fish and Wildlife Service 1997). In April 1998, the first 11 captive-reared Mexican

wol\ es-5 generations removed from the wild-were released within the primary recovery

zone of the BRWRA on the Apache National Forest in east-central Arizona (Figure 2 1).

In the spring 2000, Mexican wolves previously released in Arizona were translocated to

the secondary recovery zone on the Gila National Forest and Wilderness in New Mexico

As of October 2001, at least 37 Mexican wolves were resident within the BRWTIA and

31 of those were radiocollared (United States Fish and Wildlife Service, unpublished

data)

In June 2000, we began a study to determine the diets of free-ranging Mexican

gray wolves in Arizona and New Mexico We collected 1,682 carnivore scats from April

1998 through October 2001 and identified them to species using traditional identification

methods and odor We verified scat identification accuracy with fecal DNA analysis

(molecular scatology) and refined our identification of Mexican wolf scats We then

analyzed Mexican wolf scats identified with DNA analysis and our refined traditional

identification method to determine the diet composition of free-ranging Mexican wolves

in Arizona and New Mexico

Literature Cited

Bednarz,! C 1988 The Mexican wolf biology, history, and prospects for reestabhshment in New Mexico Endangered species report No. 18 United States Fish and Wildlife Service, Albuquerque, New Mexico, USA.

Bekoff, M . and M C Wells 1986 Social ecology and behavior of coyotes Advances in tiie Stiidy of Behavior 16: 251-338

Brown, D E 1983 The wolf in the Southwest: the making of an endangered species. The University of .\rizona Press, Tucson, Arizona, USA

Garcia-Moreno, J , M D Matocq, M S Roy, E Geffen, and R K Wayne 1996 Relationships and genetic purity of the endangered Mexican wolf based on analysis of microsatellite loci Conservation Biology 10 376-389

Gier. H T 1975 Ecology and behavior of the coyotes (Co^iw/a/rans), pages 247-262/>/ M W Fox, editor The Wild Canids their systematics, behavioral ecology and evolution Van Nostrand Reinhold, New York, USA

Gipson, P S , and W B Ballard 1998 Accountsof famous North American wolves Canadian Field-Naturalist 112 724-739

Hednck, P W , P S Miller, E Geffen, and R K Wayne 1997 Genetic evaluation of the three captive Mexican wolf lineages Zoo Biology 16 47-69

McBride, R T 1978 Statusof the gray wolf (ran/5/M^.vZ)a//ev/) in Mexico United States Fish and Wildlife Service Report, Albuquerque, New Mexico, USA.

Parsons, D R 1996 Case Smdy the Mexican wolf Pages 101-123 inE A. Herrera and L F Huenneke, editors New Mexico's natural heritage biological diversity in the Land of Enchantment New Mexico Journal of Science 36 101-123

Parsons, D R. 1998 "Green fire" returns to the Southwest reintroduaion of the Mexican wolf Wildlife Society Bulletin 26: 799-807

Shumway, E W 1998 Alpine, Arizona a stroll through history, Americopy, Mesa, Arizona, USA

Simmski, D P 1997 A history of cooperation in Mexican wolf conservation Proceedings American Zoo and Aquarium Association Regional Conference 1997 384-385

United States Fish and Wildlife Service. 1982. Mexicanwolf recovery plan. United States Fish and Wildlife Service, Albuquerque, New Mexico, USA

United States Fish and Wildlife Service 1987. Restoring America's wildlife 1937-1987: the first 50 years of the Federal Aid in Wildlife Restoration (Pitman-Robertson .\ct) Unhed States Department of the Interior, United States Fish and Wildlife Service, Washington, D C , USA.

United States Fish and Wildlife Service 1996 Reintroduction of the Mexican wolf within its historic range in the southwestern United States Final Environmental Impact Statement. United States Fish and Wildlife Service, Albuquerque, New Mexico, US.\

United States Fish and Wildlife Service 1997. Record of decision and statement of fmdings on the environmental impact statement on reintroduction of the Mexican gray wolf to rts historic range in the southwestern United States United States Fish and Wildlife Service, Albuquerque, New Mexico, USA

Young, S P . andE A Goldman 1944 The wolves of North America The American Wildlife Institute, Washington, D C , USA

CHAPTER III

DIFFERENT1.\TING MEXICAN GRAY WOLF AND

COYOTE SCATS USING DNA ANALYSIS

Abstract

We used fecal DNA analysis (molecular scatology) to test a subsample of scats

(// = 203) identified as deposited by Mexican gray wolves (Ca j/5 lupus baileyi) or

coyotes {Cams latrans) and compared the resuhs to the identification of scats using

traditional methods (i e , diameter, location, and sign) and odor We then used the scats

identified with DN.A. analysis to evaluate discriminant analysis for classifying scats using

3 measurements (i e , diameter, mass, and length) Forty-nine (24° o) of the field-

collected scats {n = 203) tested provided amplifiable DN.A., which identified 28 scats

deposhed by Mexican wolves and 21 deposited by coyotes Scats identified with DNA

analysis for the 2 species had a 79% diameter overiap (Mexican wolf, 16 3 to 35 8 mm,

coyote, 17 4 to 27 8 mm) and scats ^ 28 mm diameter were Mexican wolf scats There

was a difference (/ = -2 428. Z' = 0 019) between diameter means for the 2 species

(Mexican wolf ir = 26 0 mm, coyotex = 22 8 mm) Of 45 scats that would have been

field-identified as deposited by Mexican wolves based on location and odor criteria,

DN.A analysis showed that 19 (42°o) were deposited by coyote, and of 41 scats that

would have been field-identified as deposhed by coyotes based on diameter < 30 mm

criterion, 20 (49%) were deposited by Mexican wolves We then used Halfpenny's

(1986) suggested diameter criterion for field-identification of scats, which identified 3 of

10

the scats as gray {Urocyon cmereoargenleus) or red (Vulpes vulpes) fox (0% correct), 24

as coyote (62o o correct), and 20 as Mexican wolf (75% correct). Discriminant analysis

indicated that diameter and mass offered the best resuhs for accurately classifying coyote

scats (86° o), but provided relatively low accuracy for classifying Mexican wolf scats

(65° o) Molecular scatology appears to be a more definitive scat identification technique

than methods previously utilized (i e , traditional and odor)

Introduction

No scientific smdies were conducted on the Mexican gray wolf (Canw lupus

baileyi) before it was extirpated from the wild, therefore little is known of the subspecies'

natural history (Brown 1983) Previous studies indicate that the Mexican wolf is the

smallest (Hall and Kelson 1959, Bogan and Mehlhop 1983). the most genetically distinct

(Wayne et al 1992, Nowak 1995, Garcia-Moreno et al 1996), and the most endangered

(McBride 1980. Brown 1983, Bednarz 1988, Ginsberg and Macdonald 1990) subspecies

of gray wolf (C lupus) on North America In April 1998, the United States Fish and

Wildlife Service (USFWS) began releasing captive Mexican wolves into the Blue Range

Wolf Recovery .\rea (BRWRA) on the Apache and Gila National Forests and the Gila

Wilderness in Arizona and New Mexico to restore the subspecies into a portion of its

historical range

In June 2000, we began a study to determine the diets of captive-bom and wild-

bom .Mexican gray wolves free-ranging in the southwestern United States From April

1998 through October 2001, we collected carnivore scats {n = 1,682) from the BRWRA

I I

and identified them to species using traditional field methods (i e , diameter, location, and

sign) and odor Mexican wolves are the smallest subspecies of gray wolves found on

North America, therefore we hypothesized that traditional scat identification techniques

might have a high error rate in separating Mexican wolf and coyote (Canis latrans) scats

collected where the 2 species were sympatric

There are no published reports using fecal DNA analysis (molecular scatology) to

differentiate between gray wolf and coyote scats for diet analysis The purpose of this

research was to accurately differentiate Mexican wolf and coyote scats in order to study

the diets of Mexican wolves Accurate identification of scats would be beneficial for

determining the level of dietary overlap of gray wolves and coyotes in areas where they

are s>Tnpatric, managing gray wolf and prey populations, and investigating livestock

depredation incidences in areas where gray wolves have been reintroduced or are

recovering

Study .Area

We conducted research in the BRW'R.A (Figure 2 1), a 17,820 km^ area that

included the Apache National Forest (NF) in east-central Arizona and the Gila NF and

Wilderness in west-central New Mexico Our focus was the 2,600 km^ primary recovery

zone in .Arizona where captive Mexican wolves were released and wild-bom since April

1998 The secondary recovery zone (Gila National Forest and Wilderness, New Mexico)

was included beginning spring 2000 due to translocations of Mexican wolves previously

released in Arizona The BRWRA was bordered by the White Mountain (Fort Apache)

12

and San Carlos Apache Reservations to the west, while private lands were scattered

within and bordered the east, north and south Almost all areas were grazed by domestic

livestock

Elevations within the BRWRA ranged from < 1,220 m along the San Francisco

River to 3,350 m on Mount Baldy, Escudilla Mountain, and the Mogollon Mountains

(United States Fish and Wildlife Service 1996) Rolling hills with moderately steep-

walled canyons and sandy washes characterized the lower elevations, while mgged

slopes, deep canyons, elevated mesas, and rock cliffs typified the higher elevations.

Major vegetation included ponderosa pine (Pittusponderosa), aspen (Populus

iremuloides), fir (Abies spp ), juniper (Juniperus spp ), piflon (Pirrus cembroides),

mesquhe (Prosopis spp ), evergreen oaks {Quercus spp ), and a variety of grasses and

forbs Annual temperatures reported for the BRWHA averaged 16 4-C maximum and

-3 \°C minimum, annual precipitation averaged 52 1 cm, and annual snowfall averaged

139 3 cm (Desert Research Institute, Western Region Climate Center, Reno, Nevada,

impublished data)

From April 1998 through October 2(X)1, > 87 Mexican wolves were either

released from captivity or bom in the wild within the BRWTIA As of October 2001,

> 37 Mexican wolves resided in the BRWRA and 31 of those were fitted with radio

collars (United States Fish and Wildlife Service, unpublished data) Other predators

residem in the BRWRA included humans (Homo sapiens), coyote, gray (Urocyon

cmereoargenleus) and red (Vulpes vulpes) fox, bobcat (Felis rufus), mountain lion (Puma

concolor), and black bear (Ursus americanus) (United States Fish and Wildlife Service

13

1993, Arizona Game and Fish Department 1994) Density estimates for these predators

were unavailable, but the United States Forest Service (USFS) and Arizona Game and

Fish Department (AGFD) reported coyote densities to be relatively high (personal

communication) These sources and the USFWS also reported that feral and stray dogs

or wolf-dog hybrids were rare residents, if at all, within the BRWRA, therefore we

assumed a low likelihood of collecting dog or wolf-dog hybrid scats.

Materials and Methods

Sample collection

Carnivore scats (/; = 1,682) were collected opportunistically from the BRWRA by

Mexican wolf Interagency Field Team (IFT, i e , U S Fish and Wildlife Service, Arizona

Cjame and Fish Department, New Mexico Department of Game and Fish, USDA APHIS

Wildlife Services, and U S Forest Service) personnel from April 1998 through October

2001 We actively collected scats from June through August 2000 and March through

October 2(X)1 in areas where captive-released, translocated, and wild-bom Mexican

wolves were known to frequent The scat collection process employed an opportunistic

sampling strategy (Frenzel 1974) in which the sampling areas followed Mexican wolves

as they moved within the study area as reported by the USFWS Our search methods

included driving forest roads, hiking or horseback riding forest trails, ridgelines, and

npanan areas, and investigating forest campsites, opened release pens, den sites and kill

or carcass sites

14

Scats were collected using disposable mbber or food preparation gloves and

placed in brown paper bags, which were labeled with date, location, and scat number.

We aged scats as old, recent, or fresh according to appearance, exposure of deposition

site, and weather conditions (Ciucci et al 1996) Scats were identified initially to species

using traditional identification techniques (i e , location, diameter, and sign) and odor as

described below We did not autoclave the scats, as there were no reports of

Echinoci\:cus granulosus in the Southwest The scats were allowed to ah dry in the

brown paper bags before storing in large plastic containers at room temperature until

analyzed

The location criterion was established as areas frequented by Mexican wolves,

such as opened release pens, den sites, and kill or carcass sites We measured the

maximum diameter of each air-dried scat as described by Scott (1943). Weaver and Fritts

(1979), Green and Flinders (1981), and Danner and Dodd (1982). however, we took 2

measurements at the maximum diameter to the nearest 0 1 mm with a 152 mm dial

caliper (General Tools Manufacturing Co , LLC, New York, New York) and used the

average for diameter size Thompson (1952) established the minimum diameter for

identifying northern gray wolf scats in the field as > 24 mm and has been the accepted

critenon for several smdies (Mech 1970, Stephenson and Johnson 1972, Peterson 1974,

Van Ballenberghe et al 1975) However, Weaver and Fritts (1979) suggested > 30 mm

minimum diameter be used for identifying gray wolf scats, which was the diameter

critenon used by Arjo et al (2002) Halfpenny (1986) reviewed 3 stiidies and suggested

> 25 mm diameter for identifying gray wolf scats Initially, we used the more

15

conservative > 30 mm diameter criterion for classifying scats as deposited by Mexican

wolves Sign criterion was tracks of Mexican wolves Mexican wolf IFT personnel

provided instmction for identifying Mexican wolf scats by odor We were provided with

scats identified as Mexican wolf scats by the IFT personnel and were instmcted that the

odor (i e , sweet, musky) of those scats was that of wolf No odor description was

available for identify ing coyote scats.

Genetic identification

We used fecal DN.A analysis to identify scats deposited by either Mexican wolves

or coyotes Our design was to isolate and analyze Mexican wolf and coyote DNA from

shed epithelial cells from the intestinal lining found on scats (Kohn et al 1995, Foran et

al 1997, Kohn and Wayne 1997, Reed et al 1997, Frantzen et al 1998) We examined a

portion of the mhochondrial DNA (mtDNA) control region (D-loop, Pilgrim et al. 1998)

to ditTerentiate Mexican wolf scats from s\mpatric coyote scats collected from the field

We subsampled 203 of the field-collected scats by taking 2 to 6 fecal surface

samples from each scat for DNA analysis The surface of the scats were scraped to

decrease the possibility of removing undigested prey parts from within the scats and to

increase the probability of obtaining sloughed epithelial cells (Reed et al. 1997) Each of

the subsamples, comprising ^ 1 g of fecal material taken from each scat, was placed in a

1 5 mL micro centrifuge tube and stored at room temperature (Taberlet et al 1997) The

remainder of each scat was used for diet analysis To optimize laboratory efforts, scats

were not subsampled randomly but were selected primarily from scats field-identified as

16

deposited by Mexican wolves (/»= 169) We sorted the master database for scats

identified as probably deposited by Mexican wolf scats (/»= 1.111) based on traditional

field metiiods and odor, tiien selected every 5* scat to be subsampled for DNA analysis.

DN.A isolation

Fecal material is likely to contain relatively low concentrations of DNA much of

which is degraded (Gerloff et al 1995, Kohn et al 1995), yet mtDNA is likely to be

present on a scat m greater quantity than single-copy nuclear DNA (Reed et al 1997,

Woods etal 1999) Polymerase chain reaction (PCR) amplification of short mtDNA

fragments results in more consistem amplifications than longer fragments (Kohn et al

1995) Therefore, it was necessary to identify and establish a simple, rapid and reliable

protocol that extracted as much DNA as possible from degraded samples and removed

any potential PCR inhibitors (Boom et al 1990, Deuter et al 1995. Kohn et al 1995.

Reed et al 1997) We tried 4 DNA isolation methods Proteinase K method (Sambrook

etal 1989). salt extraction method (Miller et al 1988), Qiagen DNeasy* Tissue Kit

(Qiagen, Valencia, California, USA), and guanidinium thiocyanate (GuSCN) and silica

method (Boom et al 1990, Hoss et al 1992, Hbss & Piabo 1993) Our resuhs were

similar to those of Hoss and Paabo (1993) and Reed et al (1997), with the GuSCN and

silica method having the highest isolation efficiency and providing the most consistent

resuhs We used this method as our DNA isolation protocol and optunized it for our

application, as described below

17

We isolated genomic DNA from: whole blood (n = 38) previously collected from

released Mexican wolves, museum specimen coyote tissue (liver, n = 4), and Mexican

wolf scats (n = 13) collected from captive individuals, coyote scats (n = 5) collected

where no wx)l\ es occurred along the New Mexico and Texas border, and domestic dog

scats (/; = 5) The DNA isolated from tiiese sources provided the reference collection for

genetically identifying target species' (Foran et al 1997) Addhionally, we attempted to

isolate DN.A from museum specimen rodent tissue (liver, /; = 8), and hair (n = 4) and

grass {n ^ 2) from field-collected scats to test for possible amplication of food items

ingested

We modified an extraction buffer (5 M GuSCN, 0 1 M Tns-HCl pH 6 4, 0 2 M

EDT.A pH 8 0, 13% Triton X-100), bringing the volume to 50 mL with ddHjO (Boom et

al 1990, Hoss and Paabo 1993, Hoss 1994) One millimeter of extraction buffer was

added to each <. 1 g of dry fecal material in a 1 5 mL micro centrifiige tube The products

were vortexed, then incubated overnight at 55°C with constant agitation Samples were

then centnfiiged for 10 min at 13.000 rpm in a Microfuge* 18 Centrifuge (Beckman

Couher^, Inc , Palo Alto, California, USA) Approximately 7(X) [iL of the supernatant

was transferred to a sterile 1 5 mL micro centrifuge tube and 300 (iL of fresh extraction

buffer and 50 nL of silica matrix were added (Boom et al. 1990) The mixture was

vortexed before incubated at 55°C for 10 min with constant agitation After

centrifligation (1 min, 5,000 rpm), the supernatant was decanted and discarded. The

remaining silica pellet was washed twice with 1 mL of a modified wash buffer (5 M

GuSCN, 0 1 M Tns-HCl pH 6 4, Boom et al 1990) and once with 1 mL of 70% ethanol,

18

centrifiiging each time for 1 min at 5,000 rpm The pellet was dried at 55°C and then the

extracted DNA was eluted at 55°C with constant agitation for 30 min in 100 ^L elution

buff'er (10 mM Tris-CI, pH 8 5) The product was centrifliged (5 min, 13,000 rpm) and

75 [iL of the supernatant was removed carefully to avoid transferring silica particles that

could inhibit the PCR (Hoss and PMbo 1993) and placed in a sterile 15 mL micro

centrifuge tube These samples were stored at -20°C until PCR amplification

DN A isolation procedures were performed under a fume hood in a laboratory

exclusiN ely dedicated to DNA isolation and PCR amplification Care was taken

throughout all procedures to ensure no cross-contamination To monhor contamination

during DN.A isolations, negative controls (no fecal material added to the extraction

buffer) were treated identically through both the isolation procedure and the subsequent

PCR amplifications (Handt et al 1994)

PCR amplification

We used the PCR to amplify < 200 base pairs (bp) of the mtDNA control region

(D-loop) using canid-specific mtDNA primers (F-4479679, R-4479680, Integrated DNA

Technologies, Inc , Coralville, Iowa, USA) designed by Pilgrim et al (1998) to produce

species-specific bands of 164 bp for wolves and dogs and 160 bp for coyotes

Four microliters of excremental DNA extract were placed in a sterile 0 5 mL

micro centrifiige tube with a 46 nL volume containing 0 2 mM each dNTP, 3 25 mM

.MgCIj, IX buff'er, 0 5 piM each pnmer, 0 5 U Tag polymerase (Ml865, Promega

Corporation, .Madison, Wisconsin, USA), and 25 0 nL ddH20, then topped with 2 drops

19

of autoclaved mineral oil to prevent evaporation. The DNA and reagent mixtiires were

amplified in a programmable Perkin Elmer CeUis 480 DNA thermal cycler (Perkin

Elmer, Norwalk, Connecticut, USA) for 32 cycles, each cycle consisting of a

denaturation step for 20 sec at 95°C, an annealing step for 40 sec at 50°C, and an

extension step of 30 sec at 72°C Three microliters of tiie amplification reaction were

mixed with 3 |iL standard tracking dye and were separated at 60 milliamps on a 2%

agarose gel containing etiiidium bromide in TAE buff'er (Ausubel et al 1989) Gels were

visualized under ultraviolet (LTV) excitation on an Eagle Eye n (Stratagene*, La Jolla,

California, US.A) Amplicoms were sized using a 50 bp DNA ladder (Fisher Scientific

BP 2550-100)

Negative extraction and PCR controls were used alongside the excremental DNA

samples to monitor contamination of the PCR reagents As positive controls, we used the

DN.A extracted from blood samples collected from previously released Mexican wolves.

DNA analysis

Pilgrim et al (1998) identified a control region length difference between coyotes

and wolves and dogs, which also results in a restriction-site polymorphism present in

wolves and dogs but lacking in coyotes Using the restriction enzyme Mva I (BstN I,

New England BioLabs Inc , Beverly, Massachusetts, USA), Pilgrim et al (1998)

distinguished coyote amplicons from those of wolf and dog Twenty-five PCR products

were digested with 0 35 ^L restriction enzyme BstN I, 3 fiL lOX buff'er, 3 ^L BSA and

15 95 ^L ddH20 at 37°C for 3 hr Digested amplicons were analyzed on a 2 5% agarose

20

gel containing ethidium bromide in TAE buff'er, then visualized under UV excitation on

an Eagle Eye n Ehgested products were sized using a 50 bp ladder Products that

remained undigested at 160 bp were identified as coyote and fragments that digested into

2 fi^gments of 49 and 113 bp were identified as wolf

To confirm the results of the mtDNA restriction fragment analysis, we sequenced

tiie same fragment used for the digestions of 31 scats PCR products were purified using

the QLAquick PCR Purification Kit (Qiagen) following the supplier's instmctions

Punfied PCR (PPCR) products were cycle-sequenced in both directions (2 reactions)

using a programmable PTC-200 Pehier Thermal Cycler (MJ Research, Inc , Watertown,

Massachusetts, USA). 1 5 ^L PPCR, 1 0 nL 2 mM forward or reverse primer. 4 ^L Big

D\e Terminator v3 0 (4390244, Applied Biosystems, Foster City, California, USA), and

3 5 (iL ddHjO Each cycle-sequenced product was purified with 3 0 ^L 3 M NaOAc pH

4 6, b2 5 ^L 100% EtOH, and 14 5 nL ddHjO for 15 min at room temperaUire, then

centrifliged for 20 min at 13,(XX) rpm The supernatant was decanted and discarded, then

the pellet was washed with 250 |iL 70% EtOH followed by centrifligation for 5 min at

13,(XX) rpm The EtOH was decanted and discarded, then the pellet was air-dried at room

temperature overnight in the dark Twenty microliters of Template Suppression Reagent

(Applied Biosystems) were added to the dried pellet, then vortexed The pellet was

shaken down by hand and incubated at 95°C for 3 min, then quick spun Raw sequence

data were collected using a 310 Genetic Analyzer (Applied Biosystems) Resuhing

sequences were aligned and analyzed using Sequencher v3 1 (Gene Codes Corporation,

Inc , Arm Arbor, Michigan, USA) Aligned, proofed sequences were compared to

homologous wolf and coyote sequences ((jenbank No AF487754, AF020700) and

domestic dog mtDNA sequences (Genbank No AF487751, Slade et al. 1994) The

difference between scat diameter means for Mexican wolf and coyote was calculated

using a standard /-test (Ott 1998) and deemed significant if P <. 0 05

Discriminant analysis

Based on the measurement values (i e , diameter, mass, and length) of the scats,

we used discriminant analysis (Williams 1982) to classify scats as either Mexican wolf or

coyote Forty-seven of the scats identified with DNA analysis as deposited by Mexican

wolf or coyote were weighed to the nearest 0 1 g on a OHAUS Precision Plus TP4000

scale (OHAUS Corporation, Florham Park, New Jersey, USA), and the total length of

each scat was measured with a metric straight edge to the nearest 0 1 cm We used

discriminant analysis to classify scats based on (1) diameter, (2) diameter and mass,

(3) diameter and length, and (4) diameter, mass, and length Since coyote density was

unknown within the smdy area, we accepted prior probability (q,) = 0 50

Resuhs

Genetic identification

Forty-nine (24°o) of 203 scats provided amplifiable DNA (Figure 3 1) Twenty-

eight were identified as Mexican wolf scats and 21 were identified as coyote scats with

restriction fragment analysis (n = IS, Figure 3 2), sequence analysis (n = 24), or both

(/J = 7) Two of the scats identified as deposited by Mexican wolves were uncollectibles,

which are loose, liquid feces containing little idemifiable prey material (Floyd et al

22

1978) Uncollectible scats cannot be measured for diameter or length and traditionally

have not been included in diet stiidies Therefore, the 2 uncollectible scats were

discarded, leaving 26 Mexican wolf scats for further analysis

Mexican wolf scats (n = 26) ranged in diameter from 16 3 to 35.8 mm, while

coyote scats (n = 21) ranged in diameter from 17 4 to 27 8 mm (Figure 3.3). We found a

79«/o o%erlap in scat diameter for Mexican wolf (AJ = 16) and coyote (n = 2l) There was

a difference (t = -2 428, P = 0.019) between scat diameter means for the 2 species

(Mexican wolf x = :6 0 mm, coyote r = 22 8 mm. Figure 3 3) Scat diameters > 28 mm

appeared adequate for identifying Mexican wolf scats without other identification criteria

(i e , location and sign)

Of the 45 scats that would have been identified as deposited by Mexican wolves

based on location and odor criteria, 19 (42° o) were acmally deposited by coyotes, and of

the 41 scats that would have been identified as deposited by coyotes based on diameter

< 30 mm criterion, 20 (49%) were actually deposited by Mexican wolves Using

Halfijenny's (1986) suggested diameter criterion for identifying the scats of 3 carnivore

species, scat diameters < 18 mm would have been identified as fox (n = 3, 0% correct),

diameters 18 mm to 25 mm would have been identified as coyote (n = 24, 62% correct),

and diameters > 25 mm would have been identified as Mexican wolf (/? = 20, 75%

correct)

Foran et al (1997) reported that ingested prey tissue did not produce erroneous

resuhs when targeting species-specific DNA and our resuhs supported this claim Chir

attempts to isolate DNA from rodent tissue (liver) and ingested grass and prey hair taken

23

from scats provided no amplifiable PCR products using the canid-specific primers

designed by Pilgrim et al. (1998)

Discriminant analysis

For scats identified as Mexican wolf or coyote wrth DNA analysis, we used

discriminant analysis for 4 classifications (Table 3 1) Scat diameter (Classification 1)

accuratdy identified 81% of coyote scats, but only 50% of Mexican wolf scats Scat

diameter and mass (Classification 2) accurately identified 86% of coyote scats and 65%

of Mexican wolf scats Scat diameter and length (Classification 3) accurately identified

68% of coyote scats and 59% of Mexican wolf scats Lastly, a combination of scat

diameter, mass, and length (Classification 4) accurately identified 79% of coyote scats

and 55% of Mexican wolf scats

Discussion

Fecal material is often the most common sign and easily collected source of

information for rare, secretive carnivores (Putman 1984), and scat analysis appears to

provide the best noninvasive sampling method for determining diets of free-ranging

carnivore species However, which species actually deposited a scat must be accurately

determined for the method to be vahd Traditional scat identification criteria have been

based primarily on morphology (Halfpenny 1986, Foran et al 1997), which can be

subjective and confounded by sympatric species that are comparably sized and share

similar diets (Weaver and Fritts 1979, Green and Flinders 1981, Danner and Dodd 1982,

24

Foran et al 1997). Halfpenny (1986) reported that visual identification of scat to species

by experienced naturaUsts had error rates approaching 50 to 66%. Scott (1941, 1943)

reported tiiat fecal passage sizes varied approximately in proportion to the amount of

food consumed Weaver and Fritts (1979) reported tiiat scat diameter might be

influenced by diet composition and suggested that canid scats could not be identified to

species based on diameter alone Previous dietary analysis based on scat identification

using diameter alone may have biased results in favor of prey items that produce larger

scat diameters (Danner and Dodd 1982)

Halfjpenny (1986) reported that scat diameter and length values do not provide

positive identification of a species, because both values are too variable to be used as

adequate diagnostic criteria Green and Flinders (1981) reported that the dry mass of

scats \aried considerably for coyotes and red foxes and suggested that diameter used in

conjunction with mass could be used for identifying scats of the 2 species Discriminant

analysis of diameter and mass values of scats (Classification 2) provided the most

accurate identification for both species (coyote, 86%, Mexican wolf, 65%) However,

14°'o of the scats deposited by coyotes were classified incorrectly as Mexican wolf scats

and 35° 0 of the Mexican wolf scats were classified incorrectly as coyote scats Although

diameter and mass values provided a relatively high percentage of accuracy for

identifying coyote scats, we considered the resuhs unsatisfactory for identifying Mexican

wolf scats

Uncollectible scats, as defined by Floyd et al (1978), have traditionally been

discarded because it is difficult to identify them to species and uncollectibles usually

25

contain little identifiable prey material We were able to isolate DNA and identify the

species of 2 uncollectibles subsampled When using the appropriate primers, it is also

possible to amplify any prey DNA found in uncollectibles so that they can now be

included in cami\ore diet studies, if desired

Location of where scats have been deposited has also been used to identify wolf

scats for diet analysis smdies Scats collected from wolf den and rendezvous sites

undoubtedK have provided accurate diet information because coyotes rarely attend these

areas (Ballard et al 2003) However, the information only provides wolf diet data for

late spring through summer (Murie 1944, Mech 1966, Ballard et al 1987, Fuller 1989,

Spaulding et al 1997) Identification of scats collected from kill and carcass sites

(Thompson 1952, .Arjo et al 2(X)2) may be more problematic in areas where wolves and

coyotes are svmpatric because coyotes often scavenge from wolf kills (Paquet 1992,

Phillips and Smith 1996) The scats (n = 47, Table 3 2) from which we were able to

isolate DN.A were collected from forest trails (21 scats), forest highways, roads, and

2-tracks (16 scats), an elk carcass (1 scat), a wolf den (1 scat), an opened release pen

(2 scats), and other locations (i e , stream bed, cabin area, crater, canyon, mesa, and ridge,

6 scats)

Odor has also been reported as a scat identification technique, but it is subjective

(Stokes and Stokes 1986, Bang 2001) and currently cannot be quantified The only

mention of odor in the literature as an identification technique was for fox (Scott 1943,

Murie 1954, Wilcomb 1956, Korschgen 1980, Turkowski 1980), not for wolves or

coyotes Halfpenny (1986) suggested that odor resuhed from the camivore's diet

26

However, some wolf biologists have reported through personal communications that they

can identify wolf scats by odor, but tiiis claim has not been scientifically substantiated

and our resuhs suggest that odor was not a reliable method for this stiidy

Another attempt to identify carnivore scats has employed bile acids (Eneroth and

Sjovall 1969, .Alfred 1980, Major et al 1980, Clinite 1981, Johnson et al 1984, Quinn

and Jackman 1994) or pH (Green and Fhnders 1981) with httle or no success According

to Halfpenny (1986), these methods have proven more successful for herbivores than

carnivores

Recent noninvasive sampling studies of free-ranging mammals (Foran et al 1997,

Kohn and W ayne 1997, Reed et al 1997, Ernest et al 2000, Lucchini et al 2002) have

confirmed that fecal DNA analysis provides a more accurate assignment of the species

that deposited a scat rather than morphology of scats With noninvasive sampling and

fecal DN.A analysis, biologists can collect individual fecal samples at any time, from any

location, to study free-ranging species without having to disturb them (Hoss et al 1992,

Taberlet and Bouvet 1992. Morin et al 1993, Kohn and Wayne 1997, Taberlet et al

1999) We were able to collect scats from the field and isolate DNA for species

identification without ever seeing or dismpting the individuals

DNA from sloughed intestinal epithelial cells found on fecal material provides an

objective marker for species identification, and potentially for individuals, that remains

invariable during the life of an animal (Foran et al 1997) Mitochondrial DNA is

inherited maternally and is non-recombining (Brown 1985), making it well suited for

species identification of fecal samples (Moritz 1994, Kohn and Wayne 1997) and

27

mammalian population genetic stiidies (Randi et al. 1994, Slade et al. 1994, Taberlet et

al 1995) Mitochondrial DNA's extia-nuclear genome provides several hundred copies

per cell (Kohn et al 1995) and is more amenable to genetic manipulation than single-

copy nuclear genes (Kohn and Wayne 1997, Frantten et al 1998). The control region

(D-loop) is usually more polymorphic and informative for differentiating species (Pilgrim

et al 1998) because it evolves faster in mammals than either the rest of the mtDNA

molecule or most single-copy nuclear DNA (Avise 1994) Beginning with small

quantities of relatively short target sequences of DNA (Walsh et al. 1991. Mullis et al

1994. Reed et al 1997), the PCR is an enzymatic process that can experimentally

synthesize a large number of copies of specific DNA sequences from degraded or impure

samples (Saiki et al 1985, Mullis and Faloona 1987, Saiki et al 1988, Amheim et al

1990, Walsh et al 1991) When applied to mtDNA sequences useful for estimating

genetic differences between closely related species can be amplified (Kocher et al 1989,

.A\ise 1994) Species identification of scats using DN.A-based assays provides an

accurate method that is rapid (3-4 days), repeatable, and relatively inexpensive (The cost

is approximately $5 00 [USJ/sample for the disposable items, enzymes, and chemicals

required for sequencing Other costs include the sequencer, non-disposable lab

equipmem [e g , pipetors, cameras, and computers] and salaries of students and

technicians ) Furthermore, the resuhs are definitive and not subject to confidence

intervals or probabilistic estimation (Foran et al 1997) However, our low DNA isolation

success (24%) may be cause for concem

28

DNA isolated from fecal material is often of low quantity and quality (Taberlet et

al 1996) In addition, epitiielial cells usually are distributed unevenly (Kohn et al 1995)

and our fecal subsamples may not have included tiie cells required for DNA isolation.

Fecal material may also contain Taq DNA polymerase or PCR inhibitors, however, tiiis

problem can be reduced by using tiie silica-based extraction method (Boom et al 1990,

H6SS and Paabo 1993, Kohn et al 1995) Our modest DNA isolation success was

attnbuted to low-quality and low-quamity DNA found on scats Furthermore, the scats

we tested were dry-stored for up to 5 years at the time we conducted DNA analysis.

Reed et al (1997) and Lucchini et al (2002) reported that fecal sample age affected DNA

isolation success and suggested that fresh scats would be more suitable for DNA analysis

-Although our DNA isolation success (24%) was low, we found, as did Foran et al

(1997), that a scat's physical appearance was not a definitive guide to the DNA quality

a\ailable Another possibility influencing our low DNA isolation success could have

been that some of the scats were deposited by non-target species whose DNA could not

be amplified with the canid-specific primers designed for wolf and coyote that we used

We found, however, that the targeted species could be identified if a PCR product could

be obtained, since DNA too degraded to amplify produced no results as opposed to

incorrect results (Foran et al 1997) Our results were consistent with the findings of

Pilgrim et al (1998) that wolf and coyote mtDNAs were distinct and could be

differentiated by a single restriction site and length polymorphism We recognize that

there is a possibility that a low percentage of the scats identified as wolf could have been

deposited by feral dogs or wolf-dog hybrids if they existed within the BRWRA.

29

Therefore, we recommend that ftiture wolf DNA research utilize primers (Foran et al.

1997) that differentiate between wolf and dog DNA,

With a sample size of 47 scats identified to species using DNA analysis, we

suggest that our results be interpreted with caution Our resuhs demonstrated, however,

that identification of Mexican wolf and coyote scats using DNA analysis was more

accurate than identification methods previously available Molecular scatology can

facilrtate the identification of species, individuals, their gender, food habhs, and

pathology This would require an experimental design with extended systematic transects

from which fresh fecal samples are obtained, coupled with an appropriate preservation of

fecal material and DN.A isolation method (Reed et al 1997, Wasser et al 1997, Frantzen

et al 1998, Taberlet et al 1999, Lucchini et al 2(X)2), and using appropriate species-

sp>ecific primers (Foran et al 1997). These data could then be used further to estimate

home range (Adams et al 2(K)3), reproductive patterns, kinship stmcture and population

size (Kohn and W ayne 1997) Molecular scatology also has potential to detect

hybndization (Lehman et al 1991, Wayne et al 1992, Gondii et al 1994, Pilgrim et al

1998. Vila and Wayne 1999, Adams et al 2003) Finally, these data could be used for

validating the presence of wolves in livestock depredation incidences

Our resuhs suggest that previous diet studies using traditional scat identification

methods may have misrepresented the diets of both the North American gray wolf and

coyote where the 2 species were sympatric Fecal DNA analysis provides an accurate

method for assessing the visual identification of scat samples collected from the field and

improves diet analysis (Reed et al 1997) Molecular scatology appears to have

30

significant potential as a noninvasive sampling technique to momtor and manage free-

ranging Mexican wolves where the subspecies is sympatric with other carnivore species

Acknowledgements

The United States Fish and Wildlife Service funded this research and provided

scats from captive Mexican wolves The University of New Mexico and Arizona

Veterinary Diagnostic Lab provided blood samples from free-ranging Mexican wolves

The Museum of Texas Tech University provided coyote and rodent tissue samples.

Kaiser von Stockwerk generously provided domestic dog scats A Brown, C C.

Perchellet, and Drs F Hoffinan and J Wickliffe contributed invaluable assistance and

dedication in the DN.A lab Dr D B Wester facilitated the statistical analysis This is a

Texas Tech University. College of Agriculmral Sciences and Natural Resources technical

fwjblication T-X-.\XX

31

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39

FIELD SCATS TISSUE Wl _Ci W2 C2

KNOWN SCATS Wl DJ £ 1

250 bp ^

150 b p ^

Figure 3 1 Agarose gel showing a pxDrtion of the mtDNA control region (D-loop) amplified via Pilgrim et al (1998) canid-specific primers (Mexican wolf and dog, 164 bp; coyote 160 bp) Lanes 1 and 2 are field-collected scats Mexican wolf (WI) and coyote (CI) Lanes 3 and 4 are tissues Mexican wolf blood (W2) and coyote hver (C2) Lane 5 (S) is a 50 bp DNA size standard Lanes 6 through 8 are known scats: Mexican wolf (W I), domestic dog (Dl), and coyote (CI)

40

DIGESTED Wl W2

UNDIGESTED CI C2 C3

250 bp ^

150bp->

100bp->

Figure 3 2 Agarose gel showing restriction fragment digestion with BstN I of mtDNA control region (D-loop) purified polymerase chain reaction (PPCR) isolated from Mexican wolf and coyote scats The first lane is a 50 bp DN.A size standard Lanes 2 and 3 show digested Mexican wolf mtDNA (W1-W2, 113 bp), and the last 3 lanes show undigested coyote (C1-C3, 160 bp)

41

V5

« -

35 -

X -

25 -

2D -

35 S mm

27 8 ^u^_r-

r =22 8mm

174 mm

x = 26.0 mm

16.3 nun

Coyote Species

Mexican wolf

Figure 3 3 Comparison of diameters of coyote scats ((// = 21. range 17 4 to 27 8 m, X = 22 8 mm) and Mexican wolf scats {n = 26, range 16 3 to 35 8 mm, x = 26 0 mm) idemified with fecal DN.A analysis

42

Table 3 1 Comparison of accuracy of predicting which species (Mexican wolf or coyote) deposited a scat using discriminant analysis based on combination of 3 measurements taken from scats identified to species with DNA analysis Scats were collected in southeastern Arizona and southwestem New Mexico from April 1998 to October 2001

I 2 3 4

Classification Diameter (mm) Diameter and mass (g) Diameter and length Diameter, mass and length (cm)

Coyote (%) 81 86 68 79

Mexican wolf (%) 50 65 59 55

43

Table 3.2. Locations of scats (n = 47) identified as Mexican wolf or coyote with DNA analysis Scats were collected in southeastern Arizona and southwestem New Mexico from April 1998 to October 2001

Location Forest trails Forest roads and highways

Forest roads Forest 2-tracks Highway

Elk carcass Mexican wolf den Opened release pen Otiier Total

Mexican wolf 10 10

1 1 0 4 26

6 3 1

11 6

0 0 2 2

21

Coyote

4 2 0

Total 21 16

1 1 2 6 47

44

CHAPTER IV

DIETS OF FREE-RANGING MEXICAN GRAY WOLVES

IN ARIZONA AND NEW MEXICO

Abstract

There were no systematic diet smdies of Mexican gray wolves (Canis lupus

baileyi) before their extupation by the late 1960s from the southwestem United States.

We collected carnivore scats (n = 1,682) from the Apache and Gila National Forests of

Arizona and New Mexico from April 1998 through October 2001 and identified the scats

to species using traditional field methods (i e , diameter, location, and sign) and odor

We verified the accuracy of scat identification with fecal DNA analysis (molecular

scatology, n = 26, Reed 2004) to determine the diets of free-ranging Mexican wolves in

Arizona and New Mexico We analyzed scats > 28 mm diameter (n = 55) collected from

areas where Mexican wolf packs (n = 6) were fed supplemental food items and

determined if the packs consumed non-supplemental food items to detect a shift in diet as

they acclimated to a wild existence We foimd a difference (Go, = 12 995, P = 0 023) m

diet composition among packs, with the Cienega and Hawks Nest packs consuming more

non-supplememal food hems than the Lupine, Campbell Blue, Mule and Pipestem packs.

We analyzed Mexican wolf scats (w = 251) identified with our refined traditional field

methods established with DNA analysis (Reed 2004) and found the diet composition of

tiiose scats consisted of large-sized food items (92 8% percent frequency of occurrence

[PFO]), primarily elk (Cervus elaphus canadensis [nelsonij) adults (36 6% PFO) and

45

calves (36 2% PFO). We found no difference in Mexican wolf diets among years (n = 4,

God, = 2 588, P = 0 460). between seasons (n - 2. G^j, = 0 490, P = 0 484), or among

packs (n = 4. G.^= 7.719, P = 0.052) We analyzed Mexican wolf scats (n = 26)

identified witii DNA analysis and compared the resuhs to tiie diet composition of

Mexican wolf scats (n = 251) identified witii our refined ti-aditional methods. There was

a difference (G*^ = 9 761, P = 0 002) in diet composition between the 2 methods, with

tiie scats idemified witii DNA analysis composed of fewer large-sized food items (72.7%

PFO) than found in the scats identified with our refined tiaditional methods (92.8%

PFO) However, we found no difference (/ = - I 95, f = 0 001) between diameter means

(refined tt-aditional method x = 29 3 mm, DNA analysis r = 26 0 mm) and no difference

(/ = 1 50,P = 0 005) between number of food items per scat for the 2 identification

methods (refined traditional method = 1 06 food items per scat, DNA analysis 1 27 food

items per scat) We then compared diet composition found in scats identified with DNA

analysis as Mexican wolf scats (n = 26) and sympatric coyote scats (C. latrans: n = 2l)

and found a difference (Gajj = 9 647, P = 0002) Mexican wolf diets consisted primarily

of large-sized food items (72 7% PFO), while coyote diets were more variable and

composed of more medium- and small-sized food items (63 4% PFO) There was a

difference (/ = -2 428, P = 0 019) between diameter means for the 2 species (Mexican

wolf X = 26 0 mm, coyote x = 22 8 mm. Reed 2004) There was also a difference (/ =

2 849, P = 0 007) between number of food items per scat for the 2 species (Mexican wolf

X = 1 27 food hems per scat, coyote x = I 95 food items per scat) We compared the diet

composition of Mexican wolf scats (n = 277) identified with our refined traditional field

46

methods (n = 251) and DNA analysis (n = 26) to tiie diets reported in previous North

American gray wolf (C. lupus) stiidies (n = 7) and found a difference (Gadj = 462,492,

P = < 0 0001) Mexican wolf diet consisted primarily of large-sized food items (90.6%

PFO), which differed from late spring and summer- and carcass-based diets previously

reported for North American gray wolves Our results suggest that free-ranging Mexican

wolves consumed more large-sized food items than either sympatric coyotes or other

North .American gray wolves

Introduction

Little is known of the Mexican gray wolfs (Ccaiis lupus baileyi) natural history

because systematic studies were not conducted on the subspecies before it was extirpated

from the wild (Brown 1983) The Mexican wolf is the smallest and southern-most

occurring gray wolf (C lupus) of North America (Young and (joldman 1944, Hall and

Kelson 1959, Bogan and Mehlhop 1983, Nowak 1995) The subspecies is also the most

genetically distinct (Wayne et al 1992, Garcia-Moreno et al 1996) and the most

endangered (McBride 1980, Brown 1983, Bednarz 1988, Ginsberg and Macdonald 1990)

North .American gray wolf The United States Fish and Wildlife Service (USFWS) began

releasing captive-reared Mexican wolves in April 1998 into the Blue Range Wolf

Recovery Area (BRWRA) in Arizona and New Mexico

The purpose of our smdy was to determine the diets of free-ranging Mexican gray

wolves in Arizona and New Mexico and to determine if there were any differences

among years, between seasons, or among packs We also determined if non-

47

supplemental food items were consumed by Mexican wolves while they were fed

supplemental food items From the resuhs of our fecal DNA analysis (molecular

scatology. Reed 2004). we were able to compare the diet composhion of Mexican wolves

with that of sympatric coyotes (Ca/iis latrans) Lastiy we compared the diet composition

of Mexican wohes to that reported in 7 previous diet studies of northern gray wolves

This information could prove beneficial for managing Mexican wolf and prey

populations and investigating depredation incidences in areas where Mexican wolves are

recovering in the southwestem United States

Study Area

We conducted our research within the BRWTIA, which encompassed 17,700 km

and included all of the .Apache and Gila National Forests in east-central Arizona and

west-central New Mexico The primary recovery zone (2,600 km') in Arizona, in which

capti\e-reared Mexican wolves were released since April 1998. was our principal focus

In Spring 2000, our research efforts extended to the secondary recovery zone (Gila

National Forest and Wilderness, New Mexico) due to translocations of Mexican wolves

previously released in .Arizona The White Mountain (Fort Apache) and San Carlos

Indian Reservations bordered the BRWRA on the west and private lands bordered the

area to the east, north and south, and were scattered within public lands Domestic cattle

grazed almost all areas (USFS, unpublished data)

The BRWRA s elevations ranged from about 1,200 m along the San Francisco

River to the 3,350 m mountain ranges of Mount Baldy. Escudilla, and Mogollon (United

48

States Fish and Wildlife Service 1996) Lower elevations were characterized by rolling

hills with moderately steep-walled canyons and sandy washes, while higher elevations

were typified by mgged slopes, deep canyons, elevated mesas, and rock cliffs (Umted

States Fish and Wildlife Service 1996) Dominant vegetation included ponderosa pine

(Pittus ponderosa), aspen (Popuhs tremuloides), fir (Pseudotsuga menziesii and Abies

spp ), juniper (Juniperus spp ), piflon (Pinus cembroides), mesquhe (Prosopis spp ),

evergreen oaks (Quercus spp ), and a variety of grasses and forbs (United States Fish and

Wildlife Service 1996) Annual temperatures averaged 16 4°C maximum and-3.1°C

minimum and most precipitation fell during thunderstorms (annual average 52.1 cm)

from July through September and snow (annual average 139 3 cm) from December

through March (Desert Research Institute, Western Region Climate Center, Reno,

Nevada, unpublished data)

From April 1998 through October 2001, USFWS reported > 87 Mexican wolves

either released from captivity or bom in the wild within the BRWRA Approximately 37

Mexican wolves were free-ranging as of October 2(X)1 and 31 of those individuals were

fitted with radio collars Other predators within the study area included humans (Homo

sapiens), mountain lion (Puma concolor), black bear (Ursus americanus), coyote, bobcat

(Felis rufus), and gray (Urocyon cmereoargenleus) and red (Vulpes vulpes) fox (United

States Fish and Wildlife Service 1993, Arizona Game and Fish Department 1994)

Density estimates were unavailable for these species

Potential large-sized prey within the BRWRA included Rocky Mountain elk

(Cerxvs elaphus canadensis [nelsoni]), Coues white-tailed (Odocoileus virginianus

49

couesi) and desert mule (O. hemionus eremicus) deer, pronghom (Antilocapra

americana\ and Rocky Mountain bighorn sheep (Ovis canadensis. United States Fish

and Wildlife Service 1996). It was estimated that tiie BRWRA supported 57.000 deer

and 16,000 elk (Parsons 1998); however, the densities of these ungulates were unknown

during our study. Potential medium- and small-sized prey included javelina (Dicotyles

kyacu. United States Fish and Wildlife Service 1996), beaver (Castor canadensis),

jackrabbit (Lepus spp ), cottontail rabbit (Syhnlagus spp ), skunk (Mephitis spp), various

tree (Schmts and Tamiasciurus spp ) and ground squirrels (Spermophilus spp), chipmunk

(Eutamiasspp),rat(Neotomaspp),mice(Per<miyscusspp), vole(Microtusspp), and

other small mammals (Hofifmeister 1986, Southwest Region US Forest Service 1992),

porcupines (Erethizon dorsatum), and Merriam's turkey (Meleagris gaJlopavo, Groebner

etal. 1995).

Elk calving occurred during May and June, mule deer fawning occurred June

through August, and white-tailed deer fiiwning occurred during August (AGFD,

unpublished data. New Mexico Department of Game and Fish 1998—2001). Native

ungulate harvesting occurred intermittently from late August through January for white-

tailed and mule deer and from September through mid-December for elk (Arizona Game

and Fish Departmem 1998—2001, New Mexico Department of Game and Fish 1998—

2001), which could have resulted in carrion for Mexican wolves.

50

Methods

We collected 1.682 carnivore scats from April 1998 through October 2001 from

areas where captive-released, translocated, and wild-bom Mexican wolves were known to

frequent within the BRWRA on the Apache and Gila National Forests of Arizona and

New Mexico Personnel of the Mexican wolf Interagency Field Team (IFT, i e , US

Fish and W ildlife Service, .Arizona Game and Fish Department, New Mexico Department

of Game and Fish, USDA APHIS Wildlife Services, and U S Forest Service) collected

scats opportumstically from April 1998 through October 2001 and we collected scats

actively from June through August 20(X) and March through October 2001 An

opportunistic sampling strategy (Frenzel 1974) was employed as the sampling areas

followed Mexican wolves as they moved within the study area We collected scats along

forest roads, trails, ridgelines, and riparian areas, and from opened release pens,

campsites, den sites, and kill and carcass sues Scats collected by 4 Drag Ranch

personnel in areas where Mexican wolves were repyortedly depredating cattle were not

available for this smdy

Scat collectors wore disposable mbber or food preparation gloves and placed the

scats in brown paper bags, which were labeled with date, location, and scat number

Scats were aged as old, recent, or fresh based on appearance, exposure of deposition site,

and weather conditions (Ciucci et al 1996) We identified scats to carnivore species

using traditional field methods (i e , diameter, location, and sign) reported in the hterature

and odor There were no reports of Echinococcus granulosus in Arizona or New Mexico,

therefore we did not autoclave the scats The bagged scats were ah dried before storing

51

at room temperatiire in large plastic comainers until fiirther analysis We used DNA

analysis to verify traditional scat idemification methods for a subsample of scats (Reed

2004). From our DNA analysis resuhs, we refined the tiaditional scat identification

techniques as described below

The location criterion was defined as den sites (n = 2) only We measured the

maximum diameter of each dried scat (Scott 1943, Weaver and Fritts 1979, Green and

Flinders 1981. Danner and Dodd 1982) using 152 mm dial calipers (General Tools

Manufacturing Co . LLC. New ^ ork. New York) However, we took 2 measurements to

the nearest 0 I mm and used the average for diameter size The diameter criterion for

identifying gray wolf scats was > 24 mm diameter established by Thompson (1952) and

has been accepted for several smdies (Mech 1970. Stephenson and Johnson 1972.

Peterson 1974, Nan Ballenberghe et al 1975) Weaver and Fritts (1979) suggested scat

diameters > 30 mm be used to identify gray wolf scats Halfpenny (1986) proposed > 25

mm diameters for identifying gray wolf scats after reviewing 3 studies For identifying

Mexican wolf scats in the field, we chose the more conservative > 30 mm diameter

criterion, then redefined it as > 28 mm based on results from DNA analysis (Reed 2004).

We used wolf tracks and visual observations for sign criterion Some wolf biologists

believe that they can identify wolf scats by odor (Halfpenny 1986) We attempted to

idemify Mexican wolf scats based on odor (eg , a sweet, musky odor) as instmcted by

.Mexican wolf IFT personnel No distinguishing odor for coyote scats was reported.

We established a reference collection of potemial prey items from the BRWRA

We trapped and euthanized small prey and collected road-killed small prey, which were

52

prepared as museum specimens Hair samples were taken from various body areas (eg.,

mmp, hind legs, belly, back, and neck) of road-killed elk and deer Elk calf hair samples

were taken from the carcass remains of a known Mexican wolf kill. Fawn hair samples

were taken from a 2-month-old mule deer orphan residing at Sipes White Mountain

Wildlife Refijge, Springerville, Arizona.

We estimated the diets of Mexican wolves from 332 of the 1,682 scats. Twenty-

one additional scats were positively identified as coyote scats with DNA analysis (Reed

2004) The remaining 1,329 scats were < 28 mm diameter and could not be positively

identified to carnivore species These scats were excluded from this diet study and were

analyzed for another

We analyzed 55 scats with diameters > 28 mm collected from areas where

Mexican wolves were fed supplemental food items (i e , 11 1 kg carnivore logs [zoo diet

formulation for wild canids. Central Nebraska Packing. Inc . North Platte, Nebraska,

USA] and road-killed elk, deer, and jackrabbit) By comparing food items found in these

scats with supplemental food records (United States Fish and Wildlife Service [USFWS],

unpublished data), we were able to determine if any non-supplemental food (i e , elk

aduhs and calves, deer adults and fawns, domestic bovine, and insects) were consumed.

We determined overall diet of non-supplemental fed Mexican wolves by

analyzing scats (n = 25\) identified as deposhed by the subspecies based on > 28 mm

diameter (Reed 2004), den site location, and sign criteria, plus the scats (n = 26)

identified as deposhed by Mexican wolves with DNA analysis (Reed 2004). The 251

scats identified by our refined traditional methods were used to determine differences

53

among years, between seasons, and among packs. We then compared the diet

composition of the Mexican wolf scats identified with our refined fradhional methods to

scats identified with DNA analysis We also compared the diet composition found in

Mexican wolf scats (n = 26) to coyote scats (n = 21) identified with DNA analysis (Reed

2004) to determine a difference between the 2 species Mexican wolf scats identified

with our refined tt^ditional methods (n = 251) and DNA analysis (n = 26) were combined

for comparison with 7 North American gray wolf diet studies to determme any

differences in diet composition

Scats were broken apart by hand and undigested food items (i e , hak, bone, teeth,

claw s, and hooves) were separated We identified the undigested food items

macroscopically by comparing to our reference collection

We calculated frequency of occurrence (FO) of prey items (i e . number of

occurrences of a prey item divided by the total number of scats) and percent frequency of

occurrence (PFO) of prey items (i e , number of occurrences of a prey item divided by the

total number of occurrences of all prey items) We used PFO for all statistical analyses.

To fiacilitate statistical comparisons, we pooled ungulate prey (i e , adult and neonate elk

and deer, and domestic bovine) and considered them large-sized food hems We then

pooled smaller food items (i e , medium- and small-sized mammals, birds, reptiles,

insects, and vegetation) and considered them medium- and small-sized food hems. We

did not include non-food hems (e g , rocks, sticks, pine needles, string, plastic, mbber,

paper, and ahiminum foil) We used /-tests to examine differences in diameter means and

number of food items per scat between Mexican wolf scats identified whh our refined

54

tt^ditional identification methods and DNA analysis, and between Mexican wolf and

sympatric coyote scats identified with DNA analysis We used chi-square likelihood

ratio contingency-table analysis (G-test. Ott 1988) corrected for continuity (Williams

1976) to determine the presence of non-supplemental food items found in scats identified

with our refined traditional techniques collected where Mexican wolves were fed

supplemental food items and to determine differences in diet composition among years

(1998—2001), between 2 seasons (spring-summer March—August, fall-winter:

September—Febmary), between packs (n = 4). between scat identification methods

(refined traditional and DN.A analysis), between species (Mexican wolves and sympatric

coyotes), and between Mexican wolves and northern gray wolves All tests were

considered significant at the probability level of/* ^ 0 05

Results

We found a difference {Godj - 12 995. P = 0 023. Table 4 1) in diet composhion

of scats (n = 55) > 28 mm diameter collected from areas where Mexican wolf packs

(n = 6) were fed supplemental food items Two packs, Cienega and Hawks Nest,

consumed non-supplemental food items while being fed supplemental food items, and

Lupine, Campbell, Mule and Pipestem packs did not (Table 4 2)

Diet composition of Mexican wolf scats (n = 251) identified with our refined

traditional field methods consisted mainly of native ungulates (88 6% PFO, Table 4 3).

Smaller mammalian prey (5 3% PFO), birds (0 4% PFO), insects (0,8% PFO), and

vegetation (0 8% PFO) were of lesser importance Cattle comprised 4 2% PFO We

55

found no differences in diet among years (n = A,G^ = 2 588, P = 0 460, Table 4.4),

between seasons (n = 2. G«* = 0 490, P = 0 484. Table 4 5), or among packs (// = 4,

C/a4=7 719, P =0 052. Table 4 6) All 3 comparisons indicated Mexican wolf diets

were composed mainly of large-sized food hems

Diet composhion of Mexican wolf scats (n = 26) identified with DNA analysis

consisted of a variable diet composed of large-sized food items (72 7% PFO; Table 4 7)

and medium- and small-sized food hems (27 3°'o PFO) We compared these results with

the diet composition of the scats identified with our refined traditional methods and found

a difference (Ga^ = 9 761. P = 0 002, Table 4 8) in diets based on scat identification

techniques Diet composhion of scats identified with our refined tradhional methods

consisted of more large-sized food items (92 8°o PFO) and fewer medium- and small-

sized food Items (7 2°o PFO) than found in the scats identified by DN.A analysis We

found no difference between diameter means (I = -\ 95, P =0 052) for the 2 scat

idemification methods (refined traditional method x = 29 3 mm diameter, DNA analysis

X = 26 0 mm diameter) We also found no difference between number of food hems per

Mexican wolf scat (t = I 50, P = 0 135) for the 2 scat identification methods (1 06 food

hems per scat identified with our refined traditional methods. Table 4 3, 1 27 food hems

per scat identified with DN.A analysis. Table 4 7)

We found a difference (G^d, = 9 647, P = 0 002, Table 4 9) in diet composhion

found in Mexican wolf (n = 26) and sympatric coyote (/»= 21) scats when the 2 species

were poshively identified with DNA analysis (Reed 2004). Mexican wolf diets consisted

mainly of large-sized food rtems (72 7% PFO), while coyote diet had a more variable diet

56

consisting of medium- and small-sized food items (63.4% PFO). We also found a

difference (t = 2.849, P = 0.007) in number of food hems per scat for the 2 species (1.27

food items per Mexican wolf scat; 1 95 food items per coyote scat. Table 4.9).

We pooled scats identified with DNA analysis (n = 26) and those identified with

our refined traditional field techniques (n = 251) to compare witii previous North

American gray wolf diet studies (n = 7). We found a difference (Gad/ = 462.492,

P = < 0.0001, Table 4.11) between the diets of Mexican wolves and other North

American gray wolves, with Mexican wolves consuming a higher percentage of large-

sized fixKl items (90.6% PFO) than reported for North American gray wolves (range

58 5% to 82 9% PFO).

Discussion

Most early researchers of the Mexican wolf based their assessment of the

subspecies' diet on field observations and stomach analysis (Young and Goldman 1944).

Bailey (1931) reported the subspecies fed primarily on cattie, as determined from

undigested hair found in scats. Leopold (19S9) hypothesized that the Mexican wolfs diet

consisted mainly of deer, but included bighorn sheep, pronghom, javelina, rabbits,

rodents, and some plam foods. McBride (1980) reported that deer were the principal

native prey of Mexican wolves, but they also consumed pronghom, rabbhs, and mice.

Brown (1983) speculated that the Mexican wolf historically preyed almost entu-ely on the

diminutive Coues white-tailed deer Parsons (1996) acknowledged that the Mexican

wolfs historic diets were not well documented, but suggested that the subspecies preyed

57

primarily on whrte-tailed and mule deer, and ahematively on elk, pronghom, javelina,

beaver, rabbits, hares, and other small mammals

Introduced domestic livestock increased in Arizona and New Mexico from 1880

to 1890 and native prey populations decreased due to unregulated subsistence and market

hunting (Unhed States Fish and Wildlife Service 1987) Historically, the BRWRA had a

plentiftil supply of elk, deer, and otiier wild game (Shumway 1998). By 1890 the native

elk (Cer\tis merriami) were extinct and current populations of Rocky Mountain elk were

tt^nslocated to tiie area around 1925 (Nelson 1902, Shumway 1998) With the reduction

of the large native ungulate prey base, the Mexican wolf reportedly turned to the more

abundam and more-easily caught livestock (Young and Goldman 1944, Brown 1983,

Parsons 1996) Leopold (1959) indicated that not all wolves were livestock killers and

that such behavior was that of particular packs or individuals Historical records of the

Mexican wolfs diets during the extirpation campaign focused primarily on wolves that

depredated domestic cattle and the reports may have been exaggerated (Gipson and

Ballard 1998)

To smdy the diets of free-ranging Mexican wolves, it was necessary to select a

sampling technique that would not interfere with recovery efforts Scat analysis is a

noninvasive field technique widely used in determining carnivore diets, and scats are

readily available and easily collected (Scott 1941, Putman 1984) However, carnivore

scats must be identified accurately to species for scat analysis to be a valid method of

determining the diet of the species under smdy (Reed 2004)

58

Frequency of occurrence of food hems per scat is the most commonly used and

easily applied method of diet analysis (Leopold and Krausman 1986, Spaulding et al

1997). although this method may overemphasize tiie frequency of small prey (Corbett

1989) We used the PFO method for this study because our objective was to determine

the diet composhion and relative amounts of food items consumed by Mexican wolves to

compare with previous North American gray wolf diet studies If PFO did over

emphasize smaller food rtems in the diet composhion of Mexican wolves, h was not

apparent in our resuhs

Diet analysis based upon scats identified with DNA analyses as deposhed by

Mexican wolves and sympatric coyotes revealed a difference between the 2 species'

diets, which was consistent with Thurber et al (1992) and Arjo et al (2002) who found

that coyote diets contained more small-sized food items than sympatric gray wolf diets

This could be contributed to morphological and social differences between the 2 species.

Coyotes are medium-sized carnivores that usually prey on smaller animals (Mech 1970,

Pilgrim et al 1998), and the presence of ungulate remains in their scats may resuh from

feeding on carrion (Ozoga and Harger 1966, Berg and Chesness 1978, Weaver 1979)

Wolves, on the other hand, are 3 to 4X the mass of coyotes and are more likely to hunt in

cooperative packs than coyotes, which allows preying on larger animals (Mech 1970,

Ballard et ai 1987, .Moehlman 1989, Thurt)er and Peterson 1993, Dale et al 1995) In

our smdy area, Mexican wolves weighed 22 7 to 40 9 kg (Unhed States Fish and Wildlife

Service 1996) and southwestem coyotes averaged 9 5 to 10 6 kg (Woolsey 1985)

59

Although our sample sizes were small, DNA analysis of scats may provide a more

comprehensive representation of wolf and coyote diets than analysis of scats identified to

species by ttadhional methods used by previous diet studies. Early northern gray wolf

diet smdies (Murie 1944. Cowan 1947. Thompson 1952, Mech 1966, Pimlott et al. 1969)

supported the conclusion that the wolfs diet consisted mainly of ungulate prey either

killed or scavenged (acmal predation and carrion feedmg cannot be distinguished by scat

analysis [Pimlott 1967]), as did more recent studies (Ballard et al 1987, Arjo et al. 2002).

Although Mech (1970) reported that wolves prey on sick, weak, and unfit prey.

Brown (1983) reported that there was no evidence in southwestem historic records that

indicated such hunting behavior by Mexican wolves However, during the first year of

Mexican wolf recovery efforts, all confirmed Mexican wolf prey carcasses were elk and

most remains were young of the year and old or injured individuals (Parsons 1998), and

the first documented native prey kill was by the Hawks Nest pack whhin 2 weeks after

tiieir release (USFWS, unpublished data) From May 1998 tiirough October 2001,

Defenders of Wildlife-Bailey Wildlife Foundation Wolf Compensation Tmst

(unpublished data) paid $16,612 (US) for confirmed and unconfirmed Mexican wolf-

related injuries to 2 horses, 1 calf, and 1 guard dog, and deaths of 1 bull, 2 cows, 1 heifer,

11 calves, and 1 herding dog within Arizona and New Mexico Our resuhs combined

with USFWS kill reports and Defenders of Wildlife-Bailey Wildlife Foundation Wolf

Compensation Trust reports indicated that Mexican wolves consumed more large-sized

native ungulates than domestic livestock

60

Mech (1970) and Messier and Crete (1985) indicated wolves concentrated on the

smallest or easiest to catch large prey species in areas where > 2 large prey species

inhabrt the same area Our resuhs suggest that Mexican wolves were consuming

primarily adult elk (males ^ 454 5 kg, females ^ 227 3 kg, AGFD, unpublished data) and

calves, the larger of tiie native ungulate species available within the BRWRA, and not the

smaller white-tailed deer (males < 56 8 kg, females <. 36 4 kg; AGFD, unpublished data)

or mule deer (males < 102 3 kg, females ^ 56 8 kg, AGFD, unpublished dau). When

compared to previous diet studies for North American gray wolves, our resuhs suggest

that Mexican gray wolves consumed a higher proportion of large-sized native ungulates

than their northern counterparts This may have been the result of our conservative

identification of Mexican wolf scats using diameters ^ 28 mm, which may have biased

our diet analysis results towards the larger prey commonly found in large diameter scats

(Daimer and Dodd 1982) Additionally, previous gray wolf diet smdies included scats

collected from den and rendezvous sites and kill or carcass shes, while our Mexican wolf

scats were collected from all locations frequented by the subspecies, during all seasons,

and 2 den srtes

Our results indicate that elk aduhs and calves were the primary food source for

Nfexican wolves in Arizona and New Mexico from April 1998 through October 2001

Gray wolves in differem areas rely on different prey, and usually the wolfs diet is

compnsed of 1 or 2 species (Mech 1970) Elk were the most abundant and largest prey

available whhin the BRWRA (AGFD, unpublished data, USFS, unpubhshed data)

Although we did not study prey selection, we were curious why Mexican wolves

61

consumed mostiy elk aduhs and calves We can only hypothesize based on previous

lueramre of prey selection studies of gray wolves First, the density of elk within

Arizona and New Mexico was unknown, however, elk densrty was reported to be high

(Ballard et al 1998. AGFD, unpublished data) Therefore, h is unknown if the elk herd

within the BRWRA had neared or reached ecological carrying capacity. Furthermore, we

did not know if elk were subjected to winter severity (Mech et al 2001), malnutrition,

disease, or injunes or death from human activity ( e g , hunting and vehicles) that possibly

played a role in the Mexican wolfs consumption of elk One reason that a large

proportion of elk remains were found in Mexican wolf scats may have been because of

the low-densrty, early colonizing stage of wolves (Fritts and Mech 1981. Boyd et al

1994) These authors, as did Carbyn (1974. 1983) and Huggard (1992), found that

wolves primarily preyed upon the most vulnerable ungulates juvenile, old, or post-mt

males These data and ours suggest low-density, colonizing Mexican wolves may have

preyed upon vulnerable elk at a higher rate than other wolves in established northern

populations

Another reason why Mexican wolves consumed primarily elk may be because of

wolf-naive elk Although the resident elk population had been exposed to other predators

(i e , mountain lions, black bears, and coyotes), the elk had not been exposed to wolves.

This may have increased the vulnerability of resident elk that had never seen wolves until

confronted with them in 1998 Naive prey confronted with new predators have been less

wary than prey previously exposed to such danger (Byers 1998, Berger 1999, 2001)

62

To answer why Mexican wolves consumed primarily elk, ftuther study would be

required Mexican wolves are 1 of the 4 major predator species in Arizona and New

Mexico, and little research has been conducted on the other wild carnivores (i.e.,

mountain lion, black bear, and coyote) that now share the same prey base A muhi-

carmvore prey selection study would provide the information necessary for optimal

management of predators and then prey within Arizona and New Mexico

Our estimates of the diets of free-ranging Mexican gray wolves in Arizona and

New Mexico were based on a small sample of scats This is a result of our conservative

approach to identif>ing Mexican wolf scats based on DNA analysis and refined

traditional identification methods (i e , > 28 mm diameter, den site locations, and tracks)

Our results suggest that our refined traditional scat identification methods may have

biased estimates of the diets of Mexican wolves toward large ungulate food items WTien

researching the diets of colonizing and established wolf populations, h is important to

provide wildlife managers and the public whh accurate information We recommend that

future gray wolf diet studies incorporate DNA analysis of scats to ensure that only gray

wolf scats are included in diet analyses for the species This approach could prove

beneficial in resolving human-wolf conflicts in areas where gray wolves are reintroduced

or recovering

63

Acknowledgements

The Unhed States Fish and Wildlife Service funded this research The Mexican

wolf Interagency Field Team (IFT, i e , US. Fish and Wildlife Service, Arizona Game

and Fish Department, New Mexico Department of Game and Fish, USDA APHIS

Wildlife Services, and U S Forest Service) p)ersonnel assisted with the collection of scats

and provided Mexican wolf location data The United States Forest Service Alpine and

Clifton Ranger District personnel assisted whh logistics This is a Texas Tech

University, College of Agricultural Sciences and Natural Resources technical publication

T-X-XXX

64

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70

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71

Table 4 2 Comparison of diet composhion found in scats (n = 55) ^ 28 mm diameter from areas where Mexican gray wolf packs (n = 6) were fed supplemental food hems (i e , carnivore logs and road-killed elk, deer, and jackrabbrt) to determine presence of non-supplemental food items ( eg , elk adults and calves, deer adults and fawns, domestic bovine, and insects) Scats were collected from April 1998 to October 2001 in Arizona and New Mexico and compared to Mexican wolf Interagency Field Team dated supplemental food records Comparison values are express as percent frequency of occurrence (PFO)

Pack Lupine Campbell Blue Mule Pack Pipestem Cienega Hawks Nest

Total

No food rtems 3

- I T

8 5 5 12 55

SF' 3 16 5 2 1 2 29

Food hems NSF^

1 6 3 3 4 10 27

NSF PFO (%)' 25 0 a 27.3 a 37.5 a 60 0ab 80 0ab 833 b 49 1

'SF = supplemental food items * NSF = non-supplemental food hems *PFO (%) = percent frequency of occurrence Percentages followed by the same lower

case letter were not significantly different (P>0 05, G-test. adjusted for continuity)

72

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73

Table 4 4 Comparison among years (n = 4, 1998-2001) of diet composhion found in scats (n = 251) of free-ranging Mexican gray wolves collected from April 1998 to October 2001 in Arizona and New Mexico Scats were identified to species by refined tiaditional field methods (i e, diameter, location, and sign; Reed 2004). Food rtems were combined as large-sized (i e . aduh and young ungulates) food items and medium- and small-sized food items (i e . medium- and small-sized mammals, birds, insects, and vegetation). Non-food rtems were not included. Comparison values are expressed as percent frequency of occurrence (PFO)

Year 1998 1999 20OO 2001 Total

Large-sized food hems

135 19 53 39

246

Medium - and small-sized food rtems

14 1 2 2 19

Large-sized food hems PFO (%)'

90.6 95.0 964 95 1

'PFO ("*/o) = percent frequency of occurrence, none differed significantly (P>0 05, G-test, adjusted for cominuity)

74

Table 4 5 Comparison between seasons (;i = 2, fall-winter versus spring-summer) of diet composhion friund in scats (/i = 251) of free-ranging Mexican gray wolves in Arizona and New Mexico (April 1998 - October 2001) Scats were identified to species by refined traditional field methods (i e , diameter, location, and sign. Reed 2004) Food items were combined as large-sized (aduh and young ungulates) food hems and medium-and small-sized food rtems (i e , medium- and small-sized mammals, birds, msects, and vegetation) Non-food rtems were not included Comparison values are expressed as percent frequency of occurrence (PFO)

Season fall-winter spring-summer Total

Large-sized food items

36 210 246

Medium- and small-sized food hems

4 15 19

Large-sized food rtems PFO (%)'

900 933

'PFO (%) = percent frequency of occurrence, none differed significantly (P > 0 05, G-test. adjusted for continuity)

75

Table 4 6 Comparison of diet composhion among packs (n = 4) found in free-ranging Mexican wolf scats (n = 251) collected from April 1998 to October 2001 in Arizona and New Mexico Scats were identified by refined traditional field methods (i.e., diameter, location, and sign. Reed 2004) Food items were combined as large-sized (i.e., adult and young ungulates) food rtems and medium- and small-sized food hems (i e., medium- and small-sized mammals, bhds, insects, and vegetation) Non-food items were not included. Comparison values are expressed as percent frequency of occurrence (PFO).

Pack Hawks Nest Campbell Blue Francisco Cienega Total

Large-sized food rtems

66 98 20 26

210

Medium- and small-sized food rtems

8 9 0 0 17

Large-sized food hems PFO (%)'

892 91 6

100 0 1000

'PFO ("o) = percent frequency of occurrence, none differed significantly (P > 0 05, G-test, adjusted for continuity)

76

Table 4.7. Food items (n = 33) found in free-ranging Mexican gray wolf scats (n = 26) collected from April 1998 to October 2001 in Arizona and New Mexico. Scats were identified by DNA analysis (Reed 2004). Non-food items were not included. Comparison values are expressed as percent frequency of occurrence (PFO).

Food rtems Large-sized food items

Elk (Cervus elaphus canadensis fnels<mij) Aduh Calf

Deer (Odocoileus virginianus and 0. hemionus) aduh Unknown native ungulate Domestic cattle (Bos taunts)

Medium- and Small-sized food rtems Javelina (Dicotyles tajacu) Red squirrel (Tamiasciurus hudsonicus) Mouse (Peromyscus spp.) Unknown rodent Birds Insects

Total number of food items Total number of scats Number food items per scat

No.

5 14 2 I 2

1 I 2 2 2 1

33 26

I 27

PFO (%)'

15.2 42.4 6.1 3.0 6.1

3.0 3.0 6 1 6.1 6 1 30

'PFO (%) = percent frequency of occurrence

77

Table 4 8 Comparison of diet composhion of Mexican wolf scats (n = 277) collected from .April 1998 to October 2001 in Arizona and New Mexico and identified using 2 methods DN.A analysis (n = 2b) and refined traditional (n = 251, i e , > 28 mm diameter, 2 den srtes, and ttacks) Food items were combmed as large-sized (i e , aduh and young ungulates) food items and medium- and small-sized food hems (i e., medium- and small-sized mammals, bhds, insects, and vegetation) Non-food hems were not mcluded. Comparison \^ues are expressed as percent frequency of occurrence (PFO)

Method DN.A analysis Refined traditional

identification Total

Large-sized food hems

24 246

270

Medium- and small-sized food hems

9 19

28

Large-sized food hems PFO (%)'

72.7 a 92 8 b

'PFO (°'o) = percent frequency of occurrence Percentages followed by the same lower case letter are not significantly different (P > 0 05, G-te^, adjusted for continuity)

78

Table 4 9, Comparison of diet composhion found in free-ranging Mexican wolf scats (n = 26) and sympatric coyote scats (/»= 21) collected from April 1998 to October 2001 in Arizona and New Mexico Scats were identified by DNA analysis (Reed 2004). Food hems were combmed as large-sized (i e . aduh and young ungulates) food hems and medium- and small-sized food hems (i e , medium- and small-sized mammals, birds, insects, and vegetation) Non-food hems were not included Comparison values are expressed as percent frequency of occurrence (PFO)

Species Mexican gray wolf Coyote Total

Large-sized food hems

24 15 39

Medium- and small-sized food hems

9 26 35

Large-sized food rtems PFO (%)'

72.7 a 36 6 b

'PFO l" o) = percem frequency of occurrence Percertages followed by the same lower case letter are not significantly different (P > 0 05, G-test. adjusted for continuity)

79

Table 4.10. Food items found in free-rangmg Mexican gray wolf scats (n = 26) and sympatric coyote scats (n = 21) collected from April 1998 to October 2001 in Arizona and New Mexico. Scats were identified by DNA analysis (Reed 2004). Non-food hems were not mcluded. Comparison values are expressed as percent frequency of occiurence (PFO).

Food items Large-sized food items

Elk (Cervus ekphia cxmadensis [nelsoni]) Adult Calf

Deer (Odocoileus virginiamis and O. hemionus) Adult Fawn

Unknown nati\<e nngiiiat^ Domesbc cattle (Bos Uwrns)

Medium- and SnuO-sized food items Ja 'dina {Dicotyles tajacu) Nunall's (mountain) cottomail (Sytvilagus nuttallii) Red squind {Taniasdums hudsonicus) Golden-ffiantled ground squiirel (Spermophilus lauratis) Gny-coUared chipmunk Mouse {Peromyscus spp.) Unknown rodeot Shrew Bink Reptiles Insects VegEtatioa

Total nomter of food items Total mmter of scats Noraber food items per scat

Mexicanwolf No. PFO(%)'

5 15.2 14 42.4

2 6.1 — — 1 3.0 2 61

1 30 — — 1 30 — — — — 2 61 2 6 1 — — 2 6 1

1 3.0 _ _ 33 26

1.27

No.

2 7

1 2 -3

1 4 1 4 1 1 — 1 — 1 7 5

41 21

1.95

Coyote HfO(%)

4.9 17.1

2.4 4.9 — 7.3

2.4 9.8 2.4 9.8 2.4 2.4 — 2.4 — 2.4

17.1 12.2

'PFO (•/•) = percem frequency of occurrence

80

Table 4 11 Comparison of diet composhion found in free-ranging Mexican wolf scats (n = 277) and diet composition reported in other North American gray wolf diet studies (n = 7) Mexican wolf scats (/; = 270) were identified by refined traditional field methods (" = 251) and DNA analysis (n = 26. Reed 2004) Scats were collected from April 1998 to October 2001 in Arizona and New Mexico Food items were combined as large-sized (i e , adult and young ungulates) food items and medium- and small-sized food hems. Non-food items were not included Comparison values are expressed as percent frequency of occurrence (PFO)

Source Ballard etal (1987) Thompson (1952) Spaulding et al (1997) Murie (1944) Mech (1966) Cowan (1947) .Arjo etal (2002) This study

Large-sized food items

3,263 421

2,402 935 392 353 753 270

Medium- and small-sized food hems

2,316' 292'

1,082 406 124 94

155 28

Large-sized food hems PFO (%)'

58 5 a 59 0 a 68 9 b 697 b 76 0 c 79 0 cd 829 d 90 6 e

'PFO (" o) = percent frequency of occurrence Percentages followed by the same lower case letter are not significantly different (P > 0 05. G-test, adjusted for continuity)

^Data included unidentified ungulates and undefined "unidentified" food hems 'Data mcluded unidentified non-food items

81

CHAPTER V

SUMMARY

This was the first systematic stiidy to determine the diets of free-ranging Mexican

gray wolves (Carus hipus baileyi) in tiie soutiiwestem United States. We collected

carnivore scats (n = 1,682) from tiie Bhie Range Wolf Recovery Area (BRWRA) in

Arizona and New Mexico from April 1998 tiirough October 2001. We identified tiie

scats to species using ti^dhional field methods (i.e., diameter, location, and sign) and

odor. We then used fecal DNA analysis (molecular scatology) to verify the accuracy of

identifying Mexican wolf scats with tradhional methods and odor. DNA analysis of

Mexican wolf (n = 26) and sympatric coyote (» = 21) scats showed a 79% overlap in

diameto- size (Mexican wolf; 16 3 to 35 8 mm, coyote, 17 4 to 27 8 mm) and scats ^ 28

mm diameter were deposhed by Mexican wolves We found a difference (/ = -2.428,

P = 0 019) between diameter means for the 2 species (Mexican wolf F= 26 0 mm, coyote

X = 22.8 mm). From our DNA analysis resuhs, we refined the traditional methods for

identifying Mexican wolf scats as diameters ^ 28 mm, den site locations, and tracks, as

well as scats identified whh DNA analysis Diet analysis of Mexican wolf scats (n = 55)

with diameters > 28 mm collected from areas where 6 packs received supplemental

carnivore logs and road-killed elk, deer, and jackrabbit showed that non-supplemental

food items were consumed by Hawks Nest and Cienega packs (G«^ = 12.995, P = 0.023),

but not by Lupine, Campbell Blue, Mule or Pipestem packs. Diet analysis of Mexican

wolf scats (n = 251) identified according to > 28 mm diameter, den she location, and

82

tracks revealed a diet composhion consisting mainly of large-sized food hems (92.8%

PFO). primarily elk aduhs (36 6% PFO) and calves (36,2% PFO). There was no

difference in diets among years (n = 4. Gad/ = 2 588, P = 0.460), between seasons (n = 2,

Gad, = 0 490. P = 0 484), or among packs (n = 4, Gad, = 7 719, P = 0.052). We found a

difference (G^^ = 9 761, P = 0 002) in diet composhion of Mexican wolf scats identified

with our refined ttaditional methods (n = 251) when compared to that of DNA analysis

identified scats (n = 26) There were more large-sized food items in the scats identified

with our refined ttaditional method than found in scats identified with DNA analysis.

There was no difference (/ = -1 95, P = 0 001) between diameter means (refined

traditional method x = 29 3 mm, DNA method x = 26 0 mm) There was also no

difference (t = \ 50, P = 0 005) in number of food items per scat when comparing the 2

methods (refined tradhional method x 1 06 food items per scat, DNA method x = 1 27

food hems per scat) We found a difference ((}ad, = 9 647, Z' = 0 002) in diet composhion

of Mexican wolves and sympatnc coyotes, with Mexican wolf diets consisting primarily

of large-sized food hems (72 7°o PFO) and sympatnc coyote diets consisting primarily of

medium- and small-sized food items (63 4% PFO) There found a difference between the

2 species' scat diameter means {t = -2 428. P = 0 019, Mexican wolf x = 26 0 mm,

coyote X = 22 8 mm) There was also a difference (/ = 2 849, P = 0 007) between number

of food items per scat for the 2 species (Mexican wolf F = 1 27 food items per scat,

coyote x = 1 95 food items per scat) Finally, we found a difference (Gad, = 462 492,

/> = < 0 0001) in diet composhion of Mexican wolves when compared to diets reported

previously for other North American gray wolves Mexican wolf diet analysis revealed

83

more large-sized food hems (90 6% PFO) than that reported in 7 northern gray wolf diet

smdies (range 58 5% to 82 90/0 PFO) Our results imply that free-ranging Mexican gray

wolves in .\nzona and New Mexico consumed more large-sized food hems, primarily elk

aduhs and calves, than sympatric coyotes and the larger, northern gray wolves. This

information could prove useftil in managing both Mexican wolf and prey populations, as

well as determining the presence of Mexican wolves in depredation incidences in Arizona

and New Mexico

84

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