Journal of Wildlife Management and Wildlife Monographs Page 2 of 34
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 Pronghorn 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
4 Vaughn et al.
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
5 Vaughn et al.
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 3550 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 Zoo, San Diego Zoo, 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
6 Vaughn et al.
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
7 Vaughn et al.
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 1020 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 1822 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
8 Vaughn et al.
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
9 Vaughn et al.
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
10 Vaughn et al.
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=18
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
11 Vaughn et al.
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 (MS13, 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
12 Vaughn et al.
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
13 Vaughn et al.
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 lion (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
15 Vaughn et al.
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
16 Vaughn et al.
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
383 LITERATURE CITED
384 Anisimova, M., and O. Gascuel. 2006. Approximate likelihood-ratio test for branches: A fast,
385 accurate, and powerful alternative. Systematic Biology 55: 539552.
386 Armstrong, D. P., and P. J. Seddon. 2008. Directions in reintroduction biology. Trends in
387 Ecology and Evolution 23: 2025.
388 Bandelt, H. J., P. Forster, and A. Rohl. 1999. Median-joining networks for inferring intraspecific
389 phylogenies. Molecular Biology and Evolution 16: 3748.
390 Batson, W. G, I. J. Gordon, D. B. Fletcher, and A. D. Manning. 2015. Translocation tactics: a
391 framework to support the IUCN Guidelines for wildlife translocations and improve the quality
392 of applied methods. Journal of Applied Ecology 52:15981607. Page 19 of 34 Journal of Wildlife Management and Wildlife Monographs
18 Vaughn et al.
393 Brichieri‐Colombi, T.A., and A. Moehrenschlager. 2016. Alignment of threat, effort, and
394 perceived success in North American conservation translocations. Conservation Biology
395 30:11591172.
396 Brown, D. E., J. Cancino, K. B. Clark, M. Smith, and J. Yoakum. 2006. An annotated
397 bibliography of references to historical distributions of pronghorn in southern and Baja
398 California. Bulletin, Southern California Academy of Sciences 105:116.
399 Cancino, J. 2005. Factores ecológicos y antropogénicos que influyen en el estatus del Berrendo
400 peninsular (Antilocapra americana peninsularis): La estrategia para su recuperación. PhD
401 Dissertation, CIBNOR, La Paz, Baja California Sur, México.
402 Cancino, J., A. Ortega-Rubio, and R. Rodriguez-Estrella. 1998. Population size of the peninsular
403 pronghorn in Baja California Sur, Mexico. California Fish and Game 84:2530.
404 Cancino, J., R. Rodríguez-Estrella, and P. Miller. 2010. Using a population viability analysis for
405 management recommendations of the endangered endemic peninsular pronghorn. Acta
406 Zoológica Mexicana 26:173189.
407 Cancino, J.,V. Sánchez-Sotomayor, and R. Castellanos. 2002. Alternative capture technique for
408 the peninsular pronghorn. Wildlife Society Bulletin 30:256–258.
409 Cancino, J., V. Sanchez-Sotomayor, and R. Castellanos. 2005. From the Field: Capture, hand-
410 raising, and captive management of peninsular pronghorn. Wildlife Society Bulletin 33:6165.
411 Carling, M. D., C.W. Passavant, and J.A. Byers. 2003. DNA microsatellites of pronghorn
412 (Antilocapra americana). Molecular Ecology Notes 3:1011.
413 Cavalli-Sforza, L. L., and A. W. F Edwards. 1967. Phylogenetic analysis: models and estimation
414 procedures. American Journal of Human Genetics 19:233–257. Journal of Wildlife Management and Wildlife Monographs Page 20 of 34
19 Vaughn et al.
415 Dunn, S. J., K. K. Barnowe-Meyer, K. J. Gebhardt, N. Balkenhol, L. P. Waits, and J. A. Byers.
416 2010. Ten polymorphic microsatellite markers for pronghorn (Antilocapra americana).
417 Conservation Genetics Resources 2:8184.
418 Earl, D. A., and B. M. vonHoldt. 2012. Structure Harvester: a website and program for
419 visualizing Structure output and implementing the Evanno method. Conservation Genetics
420 Resources 4:359361.
421 Evanno, G., S. Regnaut, and J. Goudet. 2005. Detecting the number of clusters of individuals
422 using the software Structure: a simulation study. Molecular Ecology 14:26112620.
423 Excoffier, L., and H. E. L. Lischer. 2010. Arlequin suite ver 3.5: A new series of programs to
424 perform population genetics analyses under Linux and Windows. Molecular Ecology
425 Resources 10:564567.
426 Fischer, J., and D. B. Lindenmayer. 2000. An assessment of the published results of animal
427 relocations. Biological Conservation 96:111.
428 Gedir, J. V., J. W. Cain, G. Harris, and T. T. Turnbull. 2015. Effects of climate change on
429 long‐term population growth of pronghorn in an arid environment. Ecosphere 6:120.
430 Guindon, S., J. Dufayard, V. Lefort, M. Anisimova, W. Hordijk, and O. Gascuel. 2010. New
431 algorithms and methods to estimate maximum-likelihood phylogenies: assessing the
432 performance of PhyML 3.0. Systematic Biology 59:307321.
433 Hall, E. R., and K. R. Kelson. 1959. The Mammals of North America. The Ronal Press
434 Company, New York, USA.
435 Hasegawa, M., H. Kishino, and T. Yano. 1985. Dating of the human-ape splitting by a molecular
436 clock of mitochondrial DNA. Journal of Molecular Evolution 22:160174. Page 21 of 34 Journal of Wildlife Management and Wildlife Monographs
20 Vaughn et al.
437 Hassanin, A. et al. 2012. Pattern and timing of diversification of Cetartiodactyla (Mammalia,
438 Laurasiatheria), as revealed by a comprehensive analysis of mitochondrial genomes. Comptes
439 Rendus Biologies 335:3250.
440 Hervert, J. J., J. L. Bright, R. S. Henry, L. A. Priest, and M. T. Brown. 2005. Home-range and
441 habitat-use patterns of Sonoran pronghorn in Arizona. Wildlife Society Bulletin 33:815.
442 Hughes, K. S. 1991. Sonoran pronghorn use of habitat in southwest Arizona. M.S. Thesis, The
443 University of Arizona, Tucson, Arizona, USA.
444 Huson, D. H., and D. Bryant. 2006. Application of phylogenetic networks in evolutionary
445 studies. Molecular Biology and Evolution 23:254267.
446 Jakobsson, M., and N. A. Rosenberg. 2007. CLUMPP: a cluster matching and permutation
447 program for dealing with label switching and multimodality in analysis of population
448 structure. Bioinformatics. 23:18011806.
449 Jombart, T. 2008. adegenet: a R package for the multivariate analysis of genetic markers.
450 Bioinformatics 24:14031405
451 Klimova, A., A. Munguia-Vega, J. I. Hoffman, and M. Culver. 2014. Genetic diversity and
452 demography of two endangered captive pronghorn subspecies from the Sonoran Desert.
453 Journal of Mammalogy 95:12631277.
454 Koressaar, T., and M. Remm. 2007. Enhancements and modifications of primer design program
455 Primer3. Bioinformatics 23:12891291.
456 Laliberte, A. S., and W. J. Ripple. 2004. Range contractions of North American carnivores and
457 ungulates. Bioscience 54:123138. Journal of Wildlife Management and Wildlife Monographs Page 22 of 34
21 Vaughn et al.
458 Lamb, T., J. C. Avise, and J. W. Gibbons. 1989. Phylogeographic patterns in mitochondrial DNA
459 of the desert tortoise (Xerobates agassizi), and evolutionary relationships among the North
460 American gopher tortoises. Evolution 43:7687.
461 Lee Jr, T. E., J. W. Bickham, and M. D. Scott. 1994. Mitochondrial DNA and allozyme analysis
462 of North American pronghorn populations. The Journal of Wildlife Management 58:307318.
463 Leigh, J. W., and D. Bryant. 2015. popart: full‐feature software for haplotype network
464 construction. Methods in Ecology and Evolution 6:11101116.
465 Lou, Y. 1998. Genetic variation of pronghorn (Antilocapra americana) populations in North
466 America. PhD Dissertation, Texas A&M University, College Station, Texas, USA.
467 McKenna, M. C., and S. K. Bell. 1997. Classification of mammals: above the species level.
468 Journal of Vertebrate Paleontology 19:191195.
469 McRae, B. H., P. Beier, L. E. Dewald, L. Y. Huynh, and P. Keim. 2005. Habitat barriers limit
470 gene flow and illuminate historical events in a wide‐ranging carnivore, the American puma.
471 Molecular Ecology 14:19651977.
472 Medellin, R. A., C. Manterola, M. Valdéz, D. G. Hewitt, D. Doan-Crider, and T. E. Fulbright.
473 2005. History, ecology, and conservation of the pronghorn antelope, bighorn sheep, and black
474 bear in Mexico. Pages 387404 in J. L.E. Cartron, G. Ceballos, and R. S. Felger, editors.
475 Biodiversity, Ecosystems, and Conservation in Northern Mexico. Oxford University Press,
476 New York, USA.
477 Meirmans, P. G., and P.H. Van Tienderen. 2004. Genotype and Genodive: two programs for the
478 analysis of genetic diversity of asexual organisms. Molecular Ecology Notes 4:792794. Page 23 of 34 Journal of Wildlife Management and Wildlife Monographs
22 Vaughn et al.
479 Mercure, A., K. Ralls, K. P. Koepfli, and R. K. Wayne. 1993. Genetic subdivisions among small
480 canids: mitochondrial DNA differentiation of swift, kit, and arctic foxes. Evolution
481 47:13131328.
482 Morgart, J. R., J. J. Hervert, P. R. Krausman, J. L. Bright, and R. S. Henry. 2005. Sonoran
483 pronghorn use of anthropogenic and natural water sources. Wildlife Society Bulletin
484 33:5160.
485 Munguia-Vega, A., A. Klimova, and M. Culver. 2013. New microsatellite loci isolated via next-
486 generation sequencing for two endangered pronghorn from the Sonoran Desert. Conservation
487 Genetics Resources 5:125127.
488 Nelson, E. W. 1925. Status of the pronghorned antelope, 1922-1924. United States Department of
489 Agriculture Department Bulletin 1346:164.
490 O’Gara, B., C.J. Knowles, P. R. Knowles, and J. Yoakum. 2004. Capture, translocation and
491 handling. Pages 705761 in B. O'Gara, and J. Yoaku, editors. Pronghorn: ecology and
492 management. University of Colorado Press, Boulder, Colorado, USA.
493 Otte, A. 2006. Partners save the Sonoran pronghorn. Endangered Species Bulletin 31:2223.
494 Paetkau, D., R. Slade, M. Burdens, and A. Estoup. 2004. Genetic assignment methods for the
495 direct, real-time estimation of migration rate: a simulation-based exploration of accuracy and
496 power. Molecular Ecology 13:5565.
497 Paetkau, D., W. Calvert, I. Stirling, and C. Strobeck. 1995. Microsatellite analysis of population
498 structure in Canadian polar bears. Molecular Ecology 4:347354.
499 Paradis, E., and A. Potts. 2010. pegas: an R package for population genetics with an integrated-
500 modular approach. Bioinformatics 26:419420. Journal of Wildlife Management and Wildlife Monographs Page 24 of 34
23 Vaughn et al.
501 Peakall, R., and P. E. Smouse. 2006. GenAlEx 6: genetic analysis in Excel. Population genetic
502 software for teaching and research. Molecular Ecology Notes 6:288295.
503 Peakall, R., and P.E. Smouse. 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic
504 software for teaching and research—an update. Bioinformatics 28:25372539.
505 Philibosian, B., T. Fumal, and R. Weldon. 2011. San Andreas fault earthquake chronology and
506 Lake Cahuilla history at Coachella, California. Bulletin of the Seismological Society of
507 America 101:13–38.
508 Pritchard, J. K., M. Stephen, and P. Donnelly. 2000. Inference of population structure using
509 multilocus genotype data. Genetics 155:945959.
510 Riddle, B. R., and D. J. Hafner. 2006. A step-wise approach to integrating phylogeographic and
511 phylogenetic biogeographic perspectives on the history of a core North American warm
512 deserts biota. Journal of Arid Environments 66:435461.
513 Seddon, P. J., C. J. Griffiths, P. S. Soorae, and D. P. Armstrong. 2014. Reversing defaunation:
514 restoring species in a changing world. Science 345:406412.
515 Seddon, P. J., D. P. Armstrong, and R. F. Maloney. 2007. Developing the science of
516 reintroduction biology. Conservation Biology 21:303312.
517 Secretaría de Medio Ambiente y Recursos Naturales 2009
518 [SEMARNAT]. 2009. Programa de acción para la conservación de la especie: berrendo
519 (Antilocapra americana). México D.F., México: Comisión Nacional de Áreas Naturales
520 Protegidas. [In Spanish]
521 Smith, M. F., and J. L. Patton. 1980. Relationships of pocket gopher (Thomomys bottae)
522 populations of the lower Colorado River. Journal of Mammalogy 61:681696. Page 25 of 34 Journal of Wildlife Management and Wildlife Monographs
24 Vaughn et al.
523 Stephen, C. L., J. C. Devos, T. E. Lee, J. W. Bickham, J. R. Heffelfinger, and O. E. Rhodes.
524 2005. Population genetic analysis of Sonoran pronghorn (Antilocapra americana sonoriensis).
525 Journal of Mammalogy 86:782792.
526 Takezaki, N., and M. Nei. 1996. Genetic distances and reconstruction of phylogenetic trees from
527 microsatellite DNA. Genetics 144:389–399.
528 U.S. Fish and Wildlife Service [USFWS]. 1967. Native Fish and Wildlife Endangered Species.
529 Federal Register 32:4001.
530 U.S. Fish and Wildlife Service [USFWS]. 2010. Final environmental assessment for
531 reestablishment of Sonoran pronghorn. Southwestern Region. Arizona, USA.
532 U.S. Fish and Wildlife Service [USFWS]. 2015. Endangered and threatened wildlife and plants;
533 Sonoran pronghorn draft recovery plan. Federal Register 80:3822638228.
534 Untergasser, A., I. Cutcutache, T. Koressaar, J. Ye, B. C. Faircloth, M. Remm, and S. G. Rozen.
535 2012. Primer3 - new capabilities and interfaces. Nucleic Acids Research 40:e115.
536 van Oosterhout, C., W. F. Hutchinson, D. P. Wills, and P. Shipley. 2004. Micro-Checker:
537 software for identifying and correcting genotyping errors in microsatellite data. Molecular
538 Ecology Notes 4:535538.
539 Vaughn, E. E. 2016. Conservation genetics and epigenetics of pronghorn, Antilocapra
540 americana. PhD Dissertation, University of Arizona, Tucson, Arizona, USA.
541 Webber, B. L., J. K. Scott, and R. K. Didham. 2011. Translocation or bust! A new
542 acclimatization agenda for the 21st century? Trends in Ecology and Evolution 26:495496.
543 Wilson, D. E., and D. M. Reeder. 2005. Mammal species of the world: a taxonomic and
544 geographic reference. Third edition. Johns Hopkins University Press, Baltimore, Maryland,
545 USA. Journal of Wildlife Management and Wildlife Monographs Page 26 of 34
25 Vaughn et al.
546 Woodruff, S. P., P. M. Lukacs, D. Christianson, and L. P. Waits. 2016. Estimating Sonoran
547 pronghorn abundance and survival using fecal DNA and capture‐recapture methods.
548 Conservation Biology 5:11021111.
549 Yoakum, J.D. 1972. Antelope-vegetation relationships. Antelope States Workshop Proceedings
550 5:171177.
551
552 Associate Editor: Page 27 of 34 Journal of Wildlife Management and Wildlife Monographs
26 Vaughn et al.
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.