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1 This draft manuscript is distributed solely for purposes of scientific peer review. Its content is deliberative and predecisional, so it 2 must not be disclosed or released by reviewers. Because the manuscript has not yet been approved for publication by the U.S. 3 Geological Survey (USGS), it does not represent any official USGS finding or policy. 4 5 09 May 2019 6 Erin Vaughn 7 Commonwealth Scientific and Industrial Research Organisation 8 GPO Box 1700 9 Canberra, ACT 2601 Australia 10 +61 2 6242 1544 11 [email protected] 12 13 RH: Vaughn et al. • Historical Boundaries of Populations

14 Use of Museum Specimens to Refine Historical Pronghorn Subspecies Boundaries

15 ERIN E. VAUGHN,1 2 The University of Arizona, Graduate Interdisciplinary Program in

16 Genetics, Tucson, AZ 85721, USA

17 ANASTASIA KLIMOVA, ACTG Molecular Solutions, La Paz, BCS, 23085, México

18 ADRIÁN MUNGUÍA-VEGA, The University of Arizona, School of Natural Resources and the

19 Environment, Tucson, AZ 85721, USA

20 KEVIN B. CLARK, San Diego Natural History Museum, San Diego, CA 92112-1390, USA

21 MELANIE CULVER, The University of Arizona, School of Natural Resources and the

22 Environment, Tucson, AZ 85721, USA

23

24 ABSTRACT Endangered Sonoran (Antilocapra americana sonoriensis) and Peninsular (A. a.

25 peninsularis) pronghorn persist largely due to captive breeding and reintroduction efforts.

26 Recovery team managers want to re-establish pronghorn in their native range. However, there is

27 currently uncertainty regarding the subspecies status of extinct pronghorn populations that

1 Email: [email protected] 2 Current affiliation: Commonwealth Scientific and Industrial Research Organisation, GPO Box 1700, Canberra, ACT, 2601 Australia Page 3 of 34 Journal of Wildlife Management and Wildlife Monographs

2 Vaughn et al.

28 historically inhabited southern California, U.S., and northern Baja California, Mexico. To address

29 this uncertainty, we genotyped museum specimens and analyzed historical data in the context of

30 three contemporary pronghorn populations. We found that historical northern Baja California

31 pronghorn share the most ancestry with contemporary Peninsular pronghorn while pronghorn in

32 southern California share more ancestry with contemporary American (A. a. americana)

33 pronghorn. We recommend the Peninsular subspecies for reintroductions into northern Baja

34 California. For reintroductions into Southern California, we recommend that ecological factors be

35 considered, as the subspecies most closely related to historical populations (American) may not

36 be well-adapted to the hot, low-elevation deserts of the reintroduction area. Journal of Wildlife Management and Wildlife Monographs Page 4 of 34

3 Vaughn et al.

38 KEY WORDS Antilocapra americana, Arizona, Baja California, California, endangered species,

39 microsatellites, population genetics, pronghorn, reintroduction, subspecies.

40

41 Species conservation increasingly relies on intensive manipulative management activities

42 including captive breeding, habitat restoration, and translocation (Seddon et al. 2014). In North

43 America, translocation has been used with varying success for at least 279 species since the

44 1970s (Brichieri-Colombi and Moehrenschlager 2016). As the list of threatened and endangered

45 species grows, prediction of translocation success is becoming critical to facilitate allocation of

46 limited resources to the most imperiled species (Fischer and Lindenmayer 2000, Seddon et al.

47 2007, Armstrong and Seddon 2008, Batson et al. 2015). Most translocations are conducted with

48 the intent of restoring a species to its historical range (Brichieri-Colombi and Moehrenschlager

49 2016) since translocations into non-native ranges could jeopardize the success of management

50 programs (Webber et al. 2011). For species undergoing captive breeding, there can be limited

51 contemporary data from which to define historical population distributions. In these cases,

52 museum specimens serve as a valuable source of historical genetic and morphological data.

53 Herein, we discuss our refinement of the historical distribution of pronghorn (Antilocapra

54 americana) in southern California, U.S., and northern Baja California, Mexico, from genetic

55 comparison of museum specimens to contemporary populations.

56 Pronghorn are among the many North American species currently undergoing captive

57 breeding and translocation. Pronghorn are the only extant member of the once diverse

58 Antilocapridae family, according to fossil records (McKenna and Bell 1997, Wilson and Reeder

59 2005). Pronghorn numbers are influenced by human activity with many populations having

60 declined due to habitat loss, habitat fragmentation, and, historically, over-hunting (Cancino et al.

61 1998, Laliberte and Ripple 2004). In the face of climatic changes, several populations are Page 5 of 34 Journal of Wildlife Management and Wildlife Monographs

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62 predicted to experience further decline (Gedir et al. 2015). Four subspecies are currently

63 recognized; A. a. americana (American), A. a. sonoriensis (Sonoran), A. a. peninsularis

64 (Peninsular), and A. a. mexicana (Mexican) (Lee Jr et al. 1994). The American pronghorn is the

65 most widespread with Sonoran, Peninsular, and Mexican pronghorn occupying more peripheral

66 distributions. The Sonoran and Peninsular deserts contain the southwestern most tip of pronghorn

67 habitat and it is here that Sonoran and Peninsular pronghorn have historically been observed

68 (Nelson 1925, Hall and Kelson 1959, Lee Jr et al. 1994 Medellin et al. 2005, Figure 1). To a large

69 extent, Peninsular and Sonoran pronghorn are isolated from one another by the Gulf of California

70 but their historical ranges are thought to overlap in the northeastern portion of Baja California

71 (Cancino et al. 1998, Figure 1).

72 Due to long-term population decline and habitat loss, both the Peninsular and Sonoran

73 subspecies are listed as endangered and provided protection in the U.S. and Mexico (United

74 States Fish and Wildlife Service [USFWS] 1967, Secretaría de Medio Ambiente y Recursos

75 Naturales 2009). Captive breeding of Peninsular pronghorn commenced in 1998 at the Vizcaino

76 Biosphere Reserve (VBR), Baja California, with 25 wild individuals introduced over the course

77 of the first 5 years of the program (Cancino et al. 2005). Captive breeding of Sonoran pronghorn

78 commenced in 2004 at the Cabeza Prieta National Wildlife Refuge (CPNWR), Arizona, with the

79 introduction of 14 wild individuals over the course of 2 years (Otte 2006, USFWS 2010). In

80 2011, a second captive Sonoran herd was established at Kofa National Wildlife Refuge, Arizona

81 (Kofa NWR), with the transfer of 13 individuals from the CPNWR herd.

82 Three separate self-sustained captive populations of Peninsular pronghorn have been

83 established in the central Baja California peninsula and are distributed between the states of Baja

84 California and Baja California Sur: estación La Choya, Llano del Berrendo, and ejido Benito

85 Juárez with approximately 261, 105 and 34 animals, respectively, as of 2019 (Sanchez Journal of Wildlife Management and Wildlife Monographs Page 6 of 34

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86 Sotomayor, Área de Protección de Flora y Fauna - Comisión Nacional de Áreas Naturales

87 Protegidas, personal communication). The most recent aerial census of the VBR, performed in

88 2016, estimated 3550 wild Peninsular individuals occupying habitat outside of the captive pens,

89 likely representing escaped captive-bred individuals (Sanchez Sotomayor, unpublished report).

90 Population numbers of wild Peninsular pronghorn outside of the reserve are believed to be less

91 than 250 individuals (Cancino et al. 2010). As of June 2015, there were also 31 Peninsular

92 individuals in 6 U.S. institutions including the Los Angeles , , The Living

93 Desert, El Paso Zoo, Sedgwick County Zoo, and San Diego Zoo Safari Park.

94 The Sonoran pronghorn population has grown since its establishment in 2006 from 25

95 individuals to a stable population of 100-110 individuals maintained for reintroduction purposes

96 (USFWS 2015). Reintroduction of captive individuals has boosted the wild population in Arizona

97 from a low of 21 individuals in 2002 to an estimated 202 individuals in 2014 (USFWS 2015).

98 Two wild Sonoran herds have persisted without captive breeding in Sonora, Mexico (one at

99 Pinacate Biosphere Reserve and the other at Quitovac). Recent population estimates for the

100 Pinacate and Quitovac herds are 122 and 434 individuals, respectively (USFWS 2015). With

101 captive breeding protocols established and captive population numbers stabilized, managers are

102 planning to expand reintroduction efforts. Encouraged by recent re-establishment of a wild herd

103 of Sonoran pronghorn at Kofa NWR, managers are searching for suitable release sites for future

104 restoration efforts.

105 Pronghorn were once abundant in California but were extirpated from all but the

106 northeastern most tip of the state by the 1930s (Brown et al. 2006). Despite the diversity of

107 habitat types and complex geographical boundaries in California, all pronghorn in California

108 have traditionally been classified as American pronghorn (Hall and Kelson 1959, O’Gara et al.

109 2004). The historical range of Peninsular pronghorn is traditionally restricted to Baja California Page 7 of 34 Journal of Wildlife Management and Wildlife Monographs

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110 (Hall and Kelson 1959), however, the placement of the subspecies boundary has not been

111 confirmed by genetic analyses. Likewise, no genetic analyses have confirmed the restriction of

112 Sonoran pronghorn to Arizona and Sonora, Mexico. Should recovery team managers seek to

113 reintroduce pronghorn to southern California and northern Baja California, it is unclear which

114 subspecies should be released. To investigate the historical subspecies status of extinct

115 populations in the Sonoran Desert flanking the U.S.-Mexico border (Fig. 1) and guide

116 reintroduction of regionally-adapted pronghorn to their native range, we performed genetic

117 analyses of museum specimens collected in southern California and Baja California. To clarify

118 the historical distribution of pronghorn subspecies in this region, we described museum specimen

119 genotypes and their relationship to contemporary genotypes from Sonoran, Peninsular, and

120 American pronghorn.

121

122 STUDY AREA

123 Our study area (Fig. 1) encompassed a total area of approximately 700,000 km2 including the

124 southwestern United States in Arizona and California and northwestern Mexico in Baja

125 California and Baja California Sur. Climate in the study area is semi-arid to arid with

126 temperatures commonly falling below 0 C in winter and exceeding 40 C in summer. Pronghorn

127 habitat within this area is characterized by short grass, mixed grass-shrub, and desert habitats

128 (Yoakum 1972). We collected contemporary American samples between 2010 and 2014 at sites

129 in central and northern Arizona where the climate is more varied and milder compared to

130 southwestern Arizona. We collected contemporary Sonoran samples between 2009 and 2014

131 from the semi-captive breeding population located within the CPNWR in southwestern Arizona

132 (USFWS 2015). The semi-captive Sonoran herds and wild herds established from released

133 individuals occupy habitat within the Arizona Upland Subdivision which is predominantly Journal of Wildlife Management and Wildlife Monographs Page 8 of 34

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134 creosote bush, (Larrea tridentata), triangle leaf bursage (Ambrosia deltoidea), palo verde

135 (Parkinsonia spp.), mesquite (Prosopis juliflora), ironwood (Olneya tesota), and ocotillo

136 (Foucmieria splendens) (Hughes 1991). Pronghorn prefer flat valleys and bajadas in this region

137 (Hervert et al. 2005). The climate is arid with a bimodal precipitation pattern characterized by

138 summer (July–September) and winter (December–March) rains ranging from an average of

139 1020 cm annually (Morgart et al. 2005). Sonoran pronghorn habitat is described in greater detail

140 by Hervert et al. (2005). We collected contemporary Peninsular samples between 2012 and 2014

141 from captive management facilities at the VBR and Valle de Los Cirios wildlife protection area

142 in northern Baja California Sur, Mexico (Fig. 1). Topography in this region is flat with a hot and

143 arid climate; average temperature range is 1822 C and annual precipitation averages 10 cm.

144 Vegetative ground cover is usually less than 50% and dominated by xerophytic scrub including

145 alkali heath (Frankenia palmeri), datilillo (Yucca valida), saltbush (Atriplex julacea), brittle-bush

146 (Encelia spp.), and Adam's tree (Fouquieria digueti) (Cancino et al. 2002, Cancino 2005,

147 Cancino et al. 2005). Aerial survey of Peninsular pronghorn habitat indicated presence of cows,

148 horses, coyotes and rabbits (Raymond Lee, VBR, unpublished report). An exhaustive description

149 of historical and current Peninsular pronghorn habitat can be found in Cancino (2005). We

150 received samples of museum specimens (Fig. 1, Table 2) from the Field Museum of Natural

151 History, the University of Washington Burke Museum, the U.S. National Museum (Smithsonian),

152 Museum of Vertebrate Zoology at Berkeley, the Anza-Borrego Desert State Park, and the San

153 Diego Natural History Museum.

154

155 METHODS

156 We included 17 pronghorn museum specimens (Table 1), originating from sites in southern

157 California and northern Baja California (Fig. 1). Of the specimens with known age, the most Page 9 of 34 Journal of Wildlife Management and Wildlife Monographs

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158 recent were collected in 1971 and the oldest were collected in 1903. In collaboration with the

159 Sonoran pronghorn recovery team, we obtained blood samples from 176 captive Sonoran

160 pronghorn (Fig. 1) collected during routine herd management operations approved by the Arizona

161 Game and Fish Department (AGFD). The AGFD provided blood samples from 92 American

162 pronghorn collected at sites in central and northern Arizona (Fig. 1). VBR personnel provided ear

163 cartilage samples from 40 Peninsular pronghorn during routine tagging of captive animals.

164 Sampling procedures for the Peninsular pronghorn were part of an approved management plan

165 associated with the Management Unit (UMA) permit with registration key DGVS-UMA-VL-

166 3755-BC.

167 We extracted DNA from blood, ear punches, hide, tooth, horn, and bone using standard

168 protocols (Supplementary Methods, available online in Supporting Information). Anticipating

169 recovery of highly fragmented DNA from the museum specimens, we used Primer3web v4.0.0

170 (Untergasser et al. 2012, Koressaar and Remm 2007) to design primers to amplify 3 short (< 300

171 bp) sections of the pronghorn mitochondrial control region (mtCR) (Hassanin et al. 2012, Table

172 S1, available online in Supporting Information). The 3 amplicons covered 6 sites known to be

173 variable between Sonoran and Peninsular pronghorn (Klimova et al. 2014). We developed 2 high-

174 throughput multiplex assays to genotype samples at 13 previously described microsatellite loci

175 (Lou 1998, Carling et al. 2003, Dunn et al. 2010, Munguia-Vega et al. 2013, Woodruff et al.

176 2016, Table S2, available online in Supporting Information). To account for allelic drop out

177 within our museum samples due to fragmentation and low DNA concentrations, we performed

178 microsatellite PCRs for all museum samples in triplicate. We performed microsatellite fragment

179 analyses on the ABI3730 DNA Analyzer platform and called alleles in GENEMARKER

180 (SoftGenetics, State College, PA USA). We employed a simple “majority rule” method to call

181 alleles when visually inspecting peak profiles for triplicate sets of museum samples. To test for Journal of Wildlife Management and Wildlife Monographs Page 10 of 34

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182 large allele drop out and the presence of null alleles, we ran MICRO-CHECKER v2.2.3 (Van

183 Oosterhout et al. 2004) .

184 To visualize differences between haplotypes, we used a minimum-spanning network

185 model (Bandelt et al. 1999) to create a haplotype network in POPART (Leigh and Bryant 2015).

186 We used PEGAS (Paradis and Potts 2010) in R (version 3.2.4, http://cran.r-project.org/, accessed

187 15 March 2016) to compute nucleotide diversity () and calculated haplotype diversity (h) as:

푁 2 ℎ = (1 ― 푥푖 ) 188 푁 ― 1 ∑ where N is the sample size and 푥푖 is the relative haplotype frequency of 푖

189 each haplotype. To compare diversity within the three subspecies, we incorporated haplotype

190 frequencies from Klimova et al. (2014) and re-calculated  for the section of the mtCR sequenced

191 in this study. We calculated pairwise genetic distances from mitochondrial sequences as PT via

192 Analysis of Molecular Variance (AMOVA) with 9,999 permutations in GENALEX v6.502

193 (Peakall and Smouse 2006, 2012). We used PHYML v.3.0 (Guindon et al. 2010) to infer a

194 maximum-likelihood phylogenetic tree from the mitochondrial sequence data. To root our tree,

195 we included mtCR sequence from roe deer, Capreolus capreolus, obtained from GenBank

196 (accession number Z70318). We selected default parameters for PHYML under a HKY85

197 substitution model (Hasegawa et al. 1985) and calculated branch support with an approximate

198 likelihood-ratio test (Anisimova and Gascuel 2006) in addition to bootstrapping from 100

199 replicates. We visualized the resulting tree with FIGTREE (Version1.4.2,

200 http://tree.bio.ed.ac.uk/software/figtree/, accessed August 2016).

201 We calculated number of alleles (NA), allelic richness (AR), expected (HE) and observed

202 (HO) heterozygosities in GENODIVE (Meirmans and Van Tienderen 2004) and GENALEX.

203 Following methods by Paetkau et al. (1995, 2004), we performed population assignment tests in

204 GENODIVE setting significance for each population separately at 0.01 and the number of Page 11 of 34 Journal of Wildlife Management and Wildlife Monographs

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205 permutations at 10,000. We implemented a Bayesian clustering algorithm in STRUCTURE

206 v2.3.4 (Pritchard et al. 2000) to assign samples to putative populations. We employed the

207 admixture model in STRUCTURE and assumed correlated allele frequencies to test for K=18

208 with 5 iterations of each possible K value. After an initial 100,000 burn-in generations, we ran

209 the STRUCTURE analysis for 1,000,000 generations. We then used STRUCTURE

210 HARVESTER v.0.6.94 (Earl and von Holdt 2012) to compute delta K and determine the most

211 plausible base value for K clusters (Evanno et al. 2005). We used CLUMPP v.1.1.2 (Jakobsson

212 and Rosenberg 2007) to determine the optimal alignment for replicate STRUCTURE analyses

213 and mean membership coefficients across replicate runs. To estimate genetic distance between

214 each pair of individuals we used Cavalli-Sforza and Edwards (1967) chord distance, DC. DC is the

215 most effective distance measure in recovering the correct tree topology from microsatellite data

216 under a variety of evolutionary scenarios (Takezaki and Nei 1996). We created a neighbour-

217 joining (NJ) algorithm as implemented in SPLITSTREE (Huson and Bryant 2006) with the

218 estimated DC to reconstruct phylogenetic relationships between pronghorn individuals. To gain

219 perspective on the overall genetic relationships among the large number of individuals screened,

220 we used ADEGENET (Jombart 2008) in R to carry out a principal component analysis (PCA) on

221 microsatellite frequency data. Finally, we measured pairwise population differences between

222 subspecies by estimating RST in ARLEQUIN v3.5.2.2 (Excoffier and Lischer 2010) and assessing

223 statistical significance by 10,000 permutations of the data.

224 We used 3 independent analyses to assign museum specimens with complete genotypes to

225 subspecies. From the haplotype network, we assigned specimens to the subspecies occupying the

226 node nearest the specimen’s haplotype. From our STRUCTURE results, we assigned specimens

227 according to mean membership coefficients (probability threshold of > 94%) across 5 replicate Journal of Wildlife Management and Wildlife Monographs Page 12 of 34

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228 analyses with the putative populations that corresponded to the 3 subspecies. From our NJ tree,

229 we assigned specimens by association of subspecies with clusters of genetic distances.

230

231 RESULTS

232 Of the 17 museum samples we processed, we successfully assembled sequence covering all 3 of

233 the targeted mtCR sections for 10 samples. We discarded 7 contemporary American samples that

234 failed to amplify with all 3 primer pairs. After sequence quality trimming, our final concatenated

235 alignment covered a total of 423 bases of the mtCR. We restricted our analyses of the

236 microsatellite data to 8 loci that amplified consistently across all samples. We removed samples

237 that failed to amplify at more than 4 microsatellite loci, which reduced our final microsatellite

238 data set to 308 pronghorn individuals, including 10 museum samples.

239 We observed 12 variable mtCR sites (Table S3, available online in Supporting

240 Information), including the 6 previously observed in Sonoran and Peninsular pronghorn

241 (Klimova et al. 2014). Of the 10 haplotypes we observed within American pronghorn, 3 were

242 previously observed in Sonoran pronghorn (Aas2, Aas3, and Aas4) and 2 were previously

243 observed in Peninsular pronghorn (Aap1 and Aap2) (Fig. 2). Five museum samples shared

244 haplotypes with contemporary reference (Fig. 2). Of these 5 samples, 2 (Parker Dam A and

245 Parker Dam B) were haplotype Aaa4, otherwise observed in American pronghorn, and 3 were

246 haplotype Aap1, otherwise observed in both Peninsular and American pronghorn. Five museum

247 samples exhibited 3 new distinct haplotypes (MS13, Fig. 2, Table S3, available online in

248 Supporting Information). American pronghorn were the most diverse and Peninsular pronghorn

249 were the least diverse as measured by AR, HO, h, and π (Tables S5 and S6, available online in

250 Supporting Information). Page 13 of 34 Journal of Wildlife Management and Wildlife Monographs

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251 The topology of our maximum likelihood tree supports a distinction between Sonoran and

252 Peninsular pronghorn (Fig. S1, available online in Supporting Information). Branch support,

253 however, was low and several polytomies are present in the tree. Poor support within our tree is

254 likely the result of sampling so few variable sites within the highly-degraded DNA of the

255 museum specimens. Our tests of subspecies differentiation based on haplotype and genotype

256 frequencies resulted in a strong signature of differentiation between all three subspecies.

257 American pronghorn were more differentiated from Peninsular pronghorn (PT = 0.301, RST =

258 0.240) than from Sonoran pronghorn (PT = 0.165, RST = 0.120). Peninsular and Sonoran

259 pronghorn were highly differentiated (PT = 0.610, RST = 0.290).

260 In our sampling of American pronghorn, we observed 5 of the 6 haplotypes previously

261 observed in either Peninsular or Sonoran pronghorn at frequencies between 0.024 and 0.277

262 (Table S4, available online in Supporting Information). Of the 5 haplotypes unique to American

263 pronghorn, 3 (Aaa3, Aaa4, and Aaa5) were 1 mutational step from a haplotype observed in

264 Sonoran pronghorn while only 1 (Aaa2) was 1 step from a haplotype observed in Peninsular

265 pronghorn (Fig. 2). Haplotype Aaa1 was within 2 steps of both Sonoran and Peninsular

266 pronghorn. We observed both Aaa1 and Aaa2 at relatively low frequency (0.012) compared to

267 the other American pronghorn haplotypes (Table S4, available online in Supporting Information).

268 Of the haplotypes observed in both Sonoran and American pronghorn, Aas2 had the highest

269 frequency in American pronghorn (0.277) but relatively low frequency in Sonoran pronghorn

270 (0.078). Of the haplotypes observed in both Peninsular and American pronghorn, Aap2 had the

271 higher frequency. The Aap1 haplotype observed in 3 museum specimens (Tres Pozos E, Tres

272 Pozos G and Laguna Chapala) had a frequency of 0.267 in Peninsular pronghorn and 0.048 in Journal of Wildlife Management and Wildlife Monographs Page 14 of 34

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273 American pronghorn. The Aaa4 haplotype, observed in 2 museum samples (Parker Dam A and

274 B), had a frequency of 0.157 in American pronghorn.

275 In general, all of our analyses of differentiation confirmed the current subspecies

276 partitioning scheme. Nonetheless, some analyses were more sensitive in detecting and resolving

277 phylogenetic relationships between studied groups. STRUCTURE clearly supported 3

278 differentiated genetic groups (Fig. 3C) with the 3 clusters corresponding to the 3 referenced

279 subspecies (Fig. 3A). STRUCTURE assigned the museum specimens collected in the United

280 States to the American cluster and the specimens collected in Mexico to the Peninsular cluster

281 (Fig. 3B). Our NJ tree revealed clear separation of a Peninsular cluster (Fig. 4). American and

282 Sonoran specimens formed isolated clusters within the NJ tree except for a small subset of

283 individuals. The NJ tree placed the museum specimens collected at Laguna Chapala, and 5 of the

284 Tres Pozos samples within Peninsular cluster while it placed the specimens collected at Parker

285 Dam and one of the Los Angeles county sites within the American cluster. We observed similar

286 subspecies clustering from PCA (Fig. S2, available online in Supporting Information) with the

287 first component separating out Peninsular pronghorn and the second component further dividing

288 Sonoran and American pronghorn. The PCA clustered the museum samples in a similar manner

289 as the NJ Tree only with 2 of the Tres Pozos samples sitting between the Peninsular and Sonoran

290 clusters. Placement of the individual specimens within the PCA plot is influenced by principal

291 component (PC) selection. However, we did not plot further PCs as the first and second PCs

292 summarized just 6.84% and 5.93% of observed variance in the microsatellite frequency data.

293 Combining our 3 analyses, we assigned 6 of the 8 museum samples collected in Baja

294 California to the Peninsular subspecies based on the microsatellite data (Table 2). One of the Baja

295 California samples (Tres Pozos A) failed to amplify. The remaining Baja California sample, Tres

296 Pozos B, yielded only mtCR sequence and its haplotype (like those of the other samples from Page 15 of 34 Journal of Wildlife Management and Wildlife Monographs

14 Vaughn et al.

297 Mexico) was equidistant from Peninsular and American haplotypes, preventing us from making a

298 firm subspecies assignment. We assigned both specimens from Arizona (Parker Dam A and B) to

299 the American subspecies (Table 2). None of the California samples amplified with both nuclear

300 and mitochondrial primers. We assigned Fresno Co and LA Co B to the American subspecies

301 based on isolated haplotype and genotype data, respectively (Table 2).

302

303 DISCUSSION

304 We performed genetic analyses of museum and contemporary specimens to investigate the

305 historical distribution of pronghorn subspecies in northern Baja California and southern

306 California. We assigned 6 museum specimens collected in Baja California to the Peninsular

307 subspecies, which suggests that the historical range of this subspecies extended further north than

308 previously estimated (Hall and Kelson 1959). Unfortunately, we could not further refine the

309 subspecies boundary as we had poor genotyping success with the samples collected in California

310 due to the highly-degraded state of the DNA. With the limited data that we obtained from the

311 U.S. samples, we assigned those specimens to the American subspecies.

312 Clustering of the southern California and western Arizona museum specimens with the

313 American subspecies is consistent with evolution of 3 distinct clades through vicariant events

314 associated with delineations of the Mojave, Peninsular, and Sonoran deserts (Fig. 1) as described

315 by Riddle and Hafner (2006). Major barriers to gene flow in the international border region

316 include the Colorado River and the Gulf of California. Numerous studies have documented

317 genetic differentiation among populations on either side of the Colorado River, in species such as

318 mountain (Puma concolor), kit fox (Vulpes macrotis), pocket gopher (Thomomys bottae),

319 and desert tortoise (Xerobates agassizi) (Smith and Patton 1980, Lamb et al. 1989, Mercure et al.

320 1993, McRae et al. 2005). Both American and Sonoran pronghorn are found east of the Colorado Journal of Wildlife Management and Wildlife Monographs Page 16 of 34

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321 River in Arizona. Our assignment of LA Co B and Fresno Co (both sampled west of the Colorado

322 River) and Parker Dam A and B (sampled east of the Colorado River) to the American pronghorn

323 species could reflect a shifting barrier to gene flow created by changes in the course of the

324 Colorado River over time. The Colorado River changed course and flowed west into the Salton

325 Sink (Fig. 1) 5 to 6 times between the years 800 and 1700 AD (Philibosian et al. 2011). The

326 movement of the river would have opened a gap of at least 80 km across the now dry riverbed,

327 north of the Gulf of California, potentially allowing pronghorn from east and west of the river to

328 come into contact. Our results do not exclude the possibilities that the 3 subspecies ranges

329 historically overlapped or were distributed clinally across southern California. Unfortunately, we

330 could not determine the historical subspecies boundary with greater certainty as the specimens

331 collected at the most southerly sites in California did not yield usable genetic information.

332 We placed the genotypes of historical samples into the landscape of contemporary allelic

333 frequencies. One caveat to our findings is that temporal changes in allelic distributions within and

334 between the pronghorn subspecies could confound our inference of population membership of the

335 historical samples. All 3 subspecies have undergone recent local extirpations and population

336 bottlenecks, and current genetic diversity levels in Sonoran and Peninsular pronghorn have

337 declined relative to historical levels (Klimova et al. 2014, Vaughn 2016). Additionally, our

338 sampling of American pronghorn was limited to herds in central and northern Arizona, which are

339 less differentiated from Sonoran pronghorn than other populations (Stephen et al. 2005). If we

340 had conducted more extensive sampling, including historical samples from surrounding regions,

341 we could have possibly pieced together a more fine-scale picture of the historical genotypic

342 landscape. Alternatively, if we had conducted deeper sequencing of the samples collected in this

343 study, we could have potentially generated more data with which to assign museum specimens to

344 subspecies. Page 17 of 34 Journal of Wildlife Management and Wildlife Monographs

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345 This study is the first to compare American, Sonoran, and Peninsular pronghorn with the

346 same set of genetic markers. Our estimates of differentiation between American and Sonoran

347 pronghorn were greater than earlier estimates (Stephen et al. 2005), likely due to changes in

348 population structure within the captive Sonoran pronghorn herd over the course of the last decade

349 of semi-captive breeding. Our estimations of genetic differentiation between Sonoran and

350 Peninsular pronghorn differed slightly from that made by Klimova et al. (2014) despite using the

351 same samples due to having surveyed fewer loci in our final analyses. Our tests of population

352 differentiation with mtCR sequence data indicated that both endangered subspecies are distinct

353 from the American subspecies. Our results supported recent and independent divergence of the

354 Sonoran and Peninsular lineages from the American lineage following fragmentation of a once

355 contiguous population in conjunction with subsequent drift in small populations.

356

357 MANAGEMENT IMPLICATIONS

358 Based on genetic comparisons to contemporary references, we conclude that release of captive

359 bred Peninsular pronghorn at sites in northern Baja California, Mexico, is justified. Given that

360 our sampling of contemporary American pronghorn was restricted to populations in Arizona, it is

361 most accurate to say that the California museum specimens most closely resemble American

362 pronghorn populations in Arizona. Extinct southern California pronghorn likely possessed unique

363 genetic adaptions to the hot, dry desert environment that may now be lost. The American

364 subspecies’ native habitat is cold, high-elevation grasslands and forested areas while southern

365 California is characterized by low-elevation desert. Therefore, despite our findings that the

366 extinct southern California population is most genetically similar to the American subspecies,

367 introduction of this subspecies may be ill-advised as it would not constitute re-introduction into

368 appropriate habitat. Modern Sonoran pronghorn populations occur in habitat most similar to Journal of Wildlife Management and Wildlife Monographs Page 18 of 34

17 Vaughn et al.

369 southern California deserts. Therefore, ecological aspects and conservation goals surrounding the

370 two endangered desert subspecies should be considered in any future reintroduction program.

371 Future release locations should also be selected to preserve the subspecies distinction and

372 managers should carefully monitor gene flow if released herds function to establish contact

373 between the subspecies.

374

375 ACKNOWLEDGMENTS

376 Any use of trade, firm, or product names is for descriptive purposes only and does not imply

377 endorsement by the U.S. Government. We thank the VBR, Valle de los Cirios staff and V. S.

378 Sotomayor for cooperation and Peninsular pronghorn samples. We thank A. Justice-Allen for

379 providing American pronghorn blood samples. We thank D. E. Brown for assistance acquiring

380 museum specimens. We thank K. Vargas for genotyping assistance. Funding for this project was

381 provided by DOI USGS RWO#61.

382

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553 Figure Captions

554 Figure 1. Museum samples and contemporary pronghorn specimens included in an analysis of

555 subspecies distribution in the western U.S.-Mexico border region, collected between 2009 and

556 2014. The historical and current distributions for Sonoran and peninsular pronghorn are depicted

557 as well as the collection sites for all samples included in this study. Museum specimen sampling

558 locations are numbered with the associated sample names appearing in the legend. The Mojave,

559 Sonoran and Peninsular deserts are shaded and the Colorado River is highlighted in blue.

560 Abbreviations: ABDSP – Anza-Borrego Desert State Park, LA Co – Los Angeles County.

561

562 Figure 2. A haplotype network of observed mitochondrial haplotypes in pronghorn museum

563 specimens and contemporary specimens collected in the U.S. and Mexico between 2009 and

564 2014. Each square node represents an observed haplotype. Black circles represent inferred

565 haplotypes. Solid lines indicate a relationship between haplotypes. Each tick mark on the solid

566 line indicates a single DNA sequence difference between haplotypes. The squares are colored

567 according to the subspecies in which they were found. Museum sample haplotypes are indicated

568 in black and the identities of the museum samples are indicated within the accompanying boxes.

569 The proportion of the square node occupied by a color is not intended to represent relative

570 haplotype frequency.

571

572 Figure 3. STRUCTURE results assessing population membership of pronghorn museum

573 specimens relative to contemporary samples from three subspecies collected in the U.S. and

574 Mexico between 2009 and 2014. (A) Population membership coefficients for all samples, (B)

575 map of study area showing proportional population membership (represented by pie charts) for Journal of Wildlife Management and Wildlife Monographs Page 28 of 34

27 Vaughn et al.

576 museum samples, and (C) delta K versus K showing strong support for 3 populations. The colors

577 used in B correspond to the populations identified in A.

578

579 Figure 4. Neighbor joining tree of individual genetic distances based on microsatellite genotypes

580 of pronghorn museum specimens and contemporary samples from three subspecies collected in

581 the U.S. and Mexico between 2009 and 2014. Samples are colored by subspecies: orange-

582 American; blue – Peninsular; green – Sonoran. Museum specimens are indicated with black

583 squares. Abbreviations: LA Co – Los Angeles County.

584 Page 29 of 34 Journal of Wildlife Management and Wildlife Monographs

28 Vaughn et al.

585 Table 1. Sampling details of pronghorn museum specimens originating from the U.S. and Mexico

586 dating from 1903 – 1971. Abbreviations: FMNH – Field Museum of Natural History, UWMB –

587 University of Washington Burke Museum, USNM – U.S. National Museum (Smithsonian), MVZ

588 – Museum of Vertebrate Zoology at Berkeley, ABDSP – Anza-Borrego Desert State Park,

589 SDNHM – San Diego Natural History Museum, LA Co – Los Angeles County, CA – California,

590 AZ – Arizona, BC – Baja California.

Sample name Institution Sampling location Sampling Sample type year Fresno Co MVZ Mendota, Fresco Co, CA, U.S. 1920 bone LA Co A FMNH Neenach, Los Angeles Co, CA, U.S. 1903 skin LA Co B FMNH Neenach, Los Angeles Co, CA, U.S. 1903 skin LA Co C MVZ Antelope Valley, Los Angeles Co, CA, U.S. 1935 bone La Jolla SDNHM Rinocanada archeological site, Mt. Soledad; La unknown bone Jolla, San Diego Co, CA, U.S. ABDSP A ABDSP Short Wash near Fonts Point Wash, San Diego unknown tooth Co, CA, U.S. ABDSP B ABDSP Fish Creek Wash, San Diego Co, CA, U.S. unknown bone Parker A USNM Parker Dam, AZ, U.S. 1971 horn Parker B USNM Parker Dam, AZ, U.S. 1971 skin and bone Tres Pozos A USNM Tres Pozos, BC, Mexico 1914 skin Tres Pozos B USNM Tres Pozos, BC, Mexico 1914 skin Tres Pozos C USNM Tres Pozos, BC, Mexico 1914 bone Tres Pozos D USNM Tres Pozos, BC, Mexico 1914 bone Tres Pozos E USNM Tres Pozos, BC, Mexico 1914 bone Tres Pozos F USNM Tres Pozos, BC, Mexico 1914 bone Tres Pozos G UWBM Tres Pozos, BC, Mexico 1922 skin Laguna Chapala MVZ Laguna Chapala, BC, Mexico 1931 bone 591

592 Journal of Wildlife Management and Wildlife Monographs Page 30 of 34

29 Vaughn et al.

594 Table 2. Subspecies assignment of pronghorn museum specimen resulting from comparison to

595 contemporary samples from three subspecies sampled in the U.S. and Mexico between 2009 and

596 2014. Subspecies assignment is indicated as A - American or P - Peninsular. No specimens were

597 assigned to the Sonoran subspecies. Insufficient mtCR sequence or microsatellite allele data

598 prohibited assignment where indicated with an ‘-’. The consensus column contains our

599 determination of subspecies status combining 3 analyses (A = American; P = Peninsular; I =

600 indeterminate). The haplotype network analysis resulted in our observation of the nearest node’s

601 haplotype in more than one subspecies as indicated with ‘P or A’ for multiple specimens.

602 Abbreviations: LA Co – Los Angeles County; ABDSP – Anza-Borrego Desert State Park

Collection region Sample name Haplotype Structure NJ tree Consensus California, U.S. Fresco Co. A - - A California, U.S. LA Co A - - - I California, U.S. LA Co B - A A A California, U.S. LA Co C - - - I California, U.S. La Jolla - - - I California, U.S. ABDSP A - - - I California, U.S. ABDSP B - - - I Arizona, U.S. Parker A A A A A Arizona, U.S. Parker B A A A A Baja California, Mexico Tres Pozos A - - - I Baja California, Mexico Tres Pozos B P or A - - P or A Baja California, Mexico Tres Pozos C P or A P P P Baja California, Mexico Tres Pozos D P or A P P P Baja California, Mexico Tres Pozos E P or A P P P Baja California, Mexico Tres Pozos F P or A P P P Baja California, Mexico Tres Pozos G P or A P P P Baja California, Mexico Laguna Chapala P or A P P P 603 Page 31 of 34 Journal of Wildlife Management and Wildlife Monographs

30 Vaughn et al.

604 Summary for online Table of Contents

605 Extinct pronghorn populations in northern Baja California, Mexico, share ancestry with the

606 contemporary Peninsular subspecies, justifying the release of captive bred endangered Peninsular

607 pronghorn at sites in this area. Ancestry of extinct populations in southern California, U.S. is less

608 certain, therefore, ecological data and conservation goals should be prioritised in determining

609 which subspecies to release into this area. Journal of Wildlife Management and Wildlife Monographs Page 32 of 34

Figure 1. Museum samples and contemporary pronghorn specimens included in an analysis of subspecies distribution in the western U.S.-Mexico border region, collected between 2009 and 2014. The historical and current distributions for Sonoran and peninsular pronghorn are depicted as well as the collection sites for all samples included in this study. Museum specimen sampling locations are numbered with the associated sample names appearing in the legend. The Mojave, Sonoran and Peninsular deserts are shaded and the Colorado River is highlighted in blue. Abbreviations: ABDSP – Anza-Borrego Desert State Park, LA Co – Los Angeles County.

382x289mm (300 x 300 DPI) Page 33 of 34 Journal of Wildlife Management and Wildlife Monographs

Figure 2. A haplotype network of observed mitochondrial haplotypes in pronghorn museum specimens and contemporary specimens collected in the U.S. and Mexico between 2009 and 2014. Each square node represents an observed haplotype. Black circles represent inferred haplotypes. Solid lines indicate a relationship between haplotypes. Each tick mark on the solid line indicates a single DNA sequence difference between haplotypes. The squares are colored according to the subspecies in which they were found. Museum sample haplotypes are indicated in black and the identities of the museum samples are indicated within the accompanying boxes. The proportion of the square node occupied by a color is not intended to represent relative haplotype frequency.

190x137mm (300 x 300 DPI) Journal of Wildlife Management and Wildlife Monographs Page 34 of 34

A B

C Page 35 of 34 Journal of Wildlife Management and Wildlife Monographs

Figure 4. Neighbor joining tree of individual genetic distances based on microsatellite genotypes of pronghorn museum specimens and contemporary samples from three subspecies collected in the U.S. and Mexico between 2009 and 2014. Samples are colored by subspecies: orange-American; blue – Peninsular; green – Sonoran. Museum specimens are indicated with black squares. Abbreviations: LA Co – Los Angeles County.