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Fine-scale morphological and genetic variability of geomys personatus subspecies in Southern Coastal Texas.

INTRODUCTION

The Texas pocket gopher (Geomys personatus) occurs in native coastal prairie and deep sandy soils throughout much of southern Texas (Fig. 1A; Williams and Genoways, 1981; Williams, 1982). Seven subspecies of G. personatus are recognized; three subspecies are endemic to portions of Kleberg and Nueces counties in the Gulf Coast region of south Texas: G. p. personatus, G. p. maritimus, and G. p. megapotamus (Williams and Genoways, 1981). Pocket gophers are poor dispersers, and areas of unsuitable soils often separate distinct maternal lineages. As a result, species-level differentiation may occur across a relatively small (several kilometers) geographic distance (Williams, 1982).

Morphological measurements often overlap among subspecies of pocket gophers, and the actual number of subspecies and species throughout the range of Geomys has been in question (Williams and Genoways, 1981; Williams, 1982). Early genetic data supported some of the morphology-based taxonomy of Geomys, but the phylogenetic relationships within the genus were hampered by complex patterns of chromosomal rearrangements that were difficult to resolve until modern DNA sequencing. Genetic data from mitochondrial DNA (mtDNA) 12S rRNA, cytochrome-6 (Oytfr, Jolley et al., 2000; Sudman et at., 2006), and nuclear interphotoreceptor retinoid-binding protein (Chambers et al, 2009) provided additional support for phylogenetic relationships within Geomys. Collectively, the combined mitochondrial and nuclear DNA data supported four species groups within Geomys across their distribution in North America and at least 12 species (Chambers et al., 2009).

The recent genetic data helped clarify the phylogenetic relationships among species of Geomys in the south Texas region. However, uncertainty remains as to how these lineages are geographically distributed, as well as the taxonomic status of G. personatus', some subspecies may warrant elevation to species status (Sudman et al., 2006; Chambers et al., 2009). Additional specimens from each taxon and wider geographic sampling of populations are needed to clarify the taxonomy and distribution of pocket gophers (Sudman et al., 2006; Chambers et al., 2009). Furthermore, the historical distribution of pocket gophers throughout south Texas is fragmented by agriculture and urban development. Pocket gophers are not considered threatened because the Geomys genus is widely distributed across the central plains and southeastern United States, but local subspecies or species with restricted distributions are vulnerable to further urban development and changes in land-use practices (Hafner et al., 2008).

The main goal of this study was to assess the population structure of G. p. personatus, G. p. maritimus, and G. p. megapotamus in the coastal region of southern Texas. We sampled pocket gophers in the geographic range of the three subspecies in Nueces and Kleberg Counties to evaluate the morphological differentiation among populations and the geographic distribution of genetic lineages. We also compared DNA sequence data from a portion of the mtDNA Cytb gene to published sequences of pocket gopher lineages to determine the relationship between our samples and previously established Geomys phylogenies in the region.

Methods

STUDY AREA AND SPECIMEN COLLECTION

We located gopher mounds using recent aerial photographs of Kleberg and Nueces Counties, Texas. We visited 52 mound sites and set traps at 44 that displayed signs of recent activity during 8 Jul. 2009 to 28 Feb. 2010. We captured an individual at each of the 44 sites using the Baker and Williams (1972) live trap, or kill-style traps (Gophinator, Peninsula Animal Trapping, Menlo Park, California; Gopher-getter, Wildlife Control Supplies, East Granby, Connecticut; Revenge, Roxide International, Larchmont, New York; and Black Hole traps, The Snare Shop, Liderdale, Iowa). Trap type depended on the potential subspecies of gopher and landowner preference. Collection and use of animals in this study were approved by the Texas A&M University-Kingsville Animal Care and Use Committee (ACUC 2009-04-29A) and were consistent with the American Society of Mammalogists (Sikes et al., 2011).

Trap locations were determined with a hand-held global positioning systems (GPS) unit (Garmin E-trex Legend, Garmin International, Inc., Olathe, Kansas). A 200 g soil sample was collected at each capture site. Gophers that were captured alive were anesthetized with ketamine hydrochloride and euthanized with C02 (University of California-Irvine, 2010). Gopher carcasses were washed with liquid soap, rinsed, and dried with a hand- held hair dryer to remove soil for pelage color analysis. All adult gophers were individually marked, sexed, and weighed to the nearest gram with a Pesola spring scale (Forestry Suppliers, Inc.). We measured body length, hind foot length, and tail length to the nearest 0.01 mm with a Digimatic caliper (Forestry Suppliers) and photographed each individual using a digital camera to document pelage coloration. We excised a pea-sized muscle biopsy for genetic analysis. Specimens and biopsies were stored on wet ice during transport and frozen at -40 C until analysis.

MORPHOLOGY

We cleaned gopher skulls with the aid of a dermestid insect colony. Gopher skulls were measured to the nearest 0.01 mm with a Digimadc caliper (Forestry Suppliers) for total skull length, condylobasal length, basal length, palatal length, zygomatic breadth, mastoid breadth, mandible length, and upper and lower incisor width, following Elbroch (2006). Gopher carcasses and photographs were subjectively categorized into six color categories: dark hispid, light hispid, brown, reddish-brown, ash, and yellow. Soil particle size distribution was analyzed using the hydrometer method (Gee and Bauder, 1986).

We used a completely randomized design for statistical analyses. We tested for differences between collection locations within each sex on gopher body and skull measurements and soil particle size using analysis of variance (ANOVA). Multiple comparisons were made with Tukey's studentized range (HSD) test when significant main effects were found (Cochran and Cox, 1957). Homogeneity of variances among treatments was evaluated with the Bartlett's test (Steel and Torrie, 1980). Distributions of residual errors were tested for normality via the Shapiro-Wilk test. Non normal datasets were log-transformed (logio) and retested to ensure normality. We compared frequency of pelage color in gophers by Chi-square analysis. Statistical significance was inferred at P < 0.05. All means are reported [+ or -] 1 se. Finally, we performed a principal components analysis of the morphological measurements. Only females were considered in principal components analyses, as adult males display age-related characteristics that may complicate the analyses of cranial features (Honeycutt and Schmidly, 1979; Elrod et al., 2000). Statistical analyses were performed using the computer program SAS (SAS Institute, 1989).

DNA EXTRACTION AND AMPLIFICATION

We extracted DNA from tissue biopsies using a commercial kit (DNeasy, Qiagen Inc., Valencia, California). We sequenced a portion of the mtDNA Cytb gene using primers MVZ 04 (HI4542), 5'--GCAGCCCCTCAGAATGATATTTGTCCTC--3' and MVZ 05 (L14115), 5'--CGAAGCTTGATATGAAAAACCATCGTTG--3' (Smith and Patton, 1991). Sequences were amplified in 25 [micro]l reaction volumes containing 12.5 [micro]l Amplitaq Gold PCR Master Mix (Applied Biosystems, Carlsbad, California), 10 pmol of each primer, and 10-50 ng DNA. Reaction conditions consisted of an initial denaturation at 94 C for 10 min followed by 35 cycles of 94 C for 50 s, 60 C for 60 s, 72 C for 2 min, and a final extension at 72 C for 30 min. The PCR products were electrophoresed on 1% agarose gels containing ethidium bromide and viewed under UV light to verify successful amplification. Products from successful reactions were purified by an enzymatic method (ExoSAP-IT, USB Corporation, Wilmington, Maryland) and included as template for sequencing reactions using the BigDye Terminator Cycle Sequencing kit vl.1 (Applied Biosystems). Each sample was sequenced in both directions on an ABI 3130 (Applied Biosystems).

ANALYSIS OF SEQUENCE DATA

We assembled, aligned, and edited DNA sequences using the computer program sequencher 4.5 (Gene Codes, Ann Arbor, Michigan). We obtained Cytb sequences from a previous study of pocket gophers (Sudman et al., 2006; GenBank accession numbers AY393935--AY39371) to explore the phylogenetic relationships between our samples and published sequence data. The GenBank data included most currently recognized species (11/11) and subspecies (21/24) of pocket gophers (Fig. 1A): G. arenarius, G. attwaten, G. breviceps breviceps, G. breviceps sagittalis, G. bursarius bursarius, G. bursarius illinoensis, G. bursarius industrius, G. bursarius major, G. bursarius majuscules, G. bursarius missouriensis, G. jugossicularis halli, G. jugossicularis jugossicularis, G. knoxjonesi, G. lutescens, G. personatus davisi, G. personatus maritimus, G. personatus megapotamus, G. personatus personatus, G. pinetus mobilensis, G. pinetus pinetis, G. streckeri, G. texensis bakeri, G. texensis llanensis, G. texensis texensis, and G. tropicalis. Consistent with previous studies, we used closely related taxa (Pappogeomys bulleri, LI 1900; Cratogeomys castanops castanops, LI 1902) as outgroups (Sudman et al., 2006).

We estimated haplotype and nucleotide diversity, number of polymorphic sites, and average number of nucleotide differences among sequences overall and within sampling locations (Nei, 1987) using the computer program DnaSP (Librado and Rozas, 2009). We estimated sequence divergence among our ingroup samples from south Texas and other Geomys GenBank sequences using the Kimura 2 parameter model of sequence evolution (Kimura, 1980) implemented in the computer program MEGA 4 (Tamura et al., 2007).

We determined the best-fit model of DNA sequence evolution based on PHYML likelihood calculations (Guindon and Gascuel, 2003) and Akaike's (1974) information criterion (AIC) using the computer program JMODELTEST (Posada, 2008). The GTR + I + [GAMMA] model of sequence evolution (general time-reversible plus invariant with a gamma correction; Lanave et al., 1984; Tavare, 1986; Rodriguez et al., 1990) was the best- supported model of sequence evolution. We conducted a nonclock Bayesian phylogenetic analysis using a Markov chain Monte Carlo (MCMC) approach implemented in the computer program MrBayes, version 3.2 (Ronquist et al., 2012). The Bayesian analysis consisted of two simultaneous, independent MCMC runs of four chains each for 20 million generations, sampled every 100th generation. Each set of four chains consisted one cold chain and three heated chains. A burn-in consisted of discarding the first 25% samples from each of the cold chains. A consensus tree is automatically constructed from the results of the two MCMC runs after the burn-in. Posterior probabilities were used to assess the strength of the inferred nodes of the Bayesian inference tree.

Results

We captured 44 pocket gophers (12 M, 32 F) at mound sites in the Flour Bluff district of Corpus Christi (on and near Naval Air Station-Corpus Christi), mainland Nueces County, Padre Island, and Kleberg County (Fig. 1, Appendix I). No males were captured in Kleberg County. All gophers were captured in areas with >80% sand (Appendix I). The proportion of clay varied from 6 to 7% for most sites, but Kleberg County sites contained twice the amount of clay (12.2%). The morphological measurements (Appendix II) suggested two general groupings among samples. In general, female gophers captured on mainland Nueces County were larger numerically for all body and skull measurements than females from Padre Island, followed by Flour Bluff and Kleberg County. We observed few size differences among locations for male gophers, with the exception of hind foot length which was 5 mm larger ([F.sub.2,8] = 9.18; P < 0.008), on average, for gophers from Padre Island than the other locations. We observed modest differences in body size between the sexes, except in the Flour Bluff region where males were larger than females in weight, lengths of total body, hind foot, skull, condylobasal, basal, palatal, and mandible, and zygomatic breadth (Appendix II).

The first two principal components accounted for 87% of the variation in physical and cranial measurements. The eigenvectors of the principal components suggested that the first principal component captured overall variation in size of the measured characters, with each character receiving similar weight. The second principal component was strongly influenced by mastoid breadth, palatal, and hind foot length while mandible, tail, and zygomatic breadth had lesser influence (Table 1). A plot of the first two principal component scores for all individuals revealed that specimens collected in mainland Nueces County and Padre Island clustered together (Fig. 2). Four of 5 individuals from Kleberg County clustered in the lower right quadrant, consistent with their smaller overall body size. In contrast, the Flour Bluff specimens displayed greater variation in principal component scores (Fig. 2).

We noted differences in frequency of pelage color (Chi-square = 43.7; 15 df; P < 0.005) among capture locations (Table 2). The most frequent pelage color differed by area, where 80% (n = 10) of the mainland Nueces County gophers were a dark hispid coloration, 72% (n = 18) of the gophers from Flour Bluff were brown to reddish-brown, 60% (n = 5) of the gophers from Kleberg County were a light hispid coloration, and 45% (n = 11) of the gophers from Padre Island were reddish-brown. The proportion of dark hispid, brown, and reddish brown differed significantly (Chi-square = 7.81; 3 df; P < 0.05) between the collection locations (Table 2).

We obtained mtDNA sequence data from 39 of the 44 specimens; sequences were deposited into GenBank, accession numbers KC567282-KC567290. We used 370 bp of sequence data for analyses. The 39 south Texas sequences contained 55 polymorphic sites and nine haplotypes; average number of nucleotide differences between sequences was 14.4 and nucleotide diversity was 0.041 (Table 3). Pocket gophers were highly structured in the region, with few shared haplotypes among sites (Table 3).

Haplotypes clustered in three distinct clades, concordant with the presumed number of subspecies in the region. One clade consisted of gophers collected in mainland Nueces County and Flour Bluff sites ("Maritimus clade") and a second clade was comprised of Padre Island samples. The third clade consisted of all Kleberg County samples. Unexpectedly, one sample from Padre Island clustered with samples collected from Kleberg County. The Kimura 2 parameter estimates of sequence divergence within the three south Texas clades were minimal (mainland Nueces County: d = 0.003, SE = 0.002; Padre Island: d = 0.003, se = 0.002; Kleberg County and the aberrant Padre Island sample consisted of a single haplotype).

Relative to the additional GenBank sequence data, the mainland Nueces County and Flour Bluff samples clustered with G. p. maritimus, while the Padre Island samples formed a well-defined subcluster similar to but distinct from G. p. personatus and G. p. megapotamus sequences (Fig. 3). The Kleberg County and aberrant Padre Island sample formed a separate clade, with variable placement near other Geomys groups, but outside of other G. personatus sequences. Estimates of sequence divergence based on the Kimura 2 parameter model between the Kleberg County clade and G. personatus were >0.12 (Table 4). The Kleberg County clade was most similar to G. texensis, where sequence divergence ranged from 0.06 to 0.08 (Table 4).

DISCUSSION

Physical measurements were an inconsistent means of differentiating among presumed subspecies, as evidenced by the large degree of overlap in physical measurements among and between the sexes. Morphological differentiation within presumed Geomys subspecies may occur at fine geographic scales, such as on opposite sides of creeks or intrusions of unsuitable soils (Wilkins and Swearingen, 1990). Specimens from Kleberg County were consistently smaller for most physical measurements. Previous studies have associated smallei body size in inland populations with the presence of indurate soils (summarized in Williams, 1982), and the Kleberg County specimens occurred in soils with greater clay content than we encountered elsewhere in the region. Nevertheless, physical measurements appeared to provide weak discriminatory power for the identification of unique lineages in the coastal areas of southern Texas.

Previous descriptions of the G. personatus complex described individuals as 'Broccoli brown' in color (Williams, 1982). However, Williams (1982) noted that Kennedy (1954)

proposed that pelage coloration was adaptive to soil coloration, though abnormal coloration and albino specimens were occasionally observed. The proportion of color morphs varied among our collection sites, but >1 morph was present at each site. The complex distribution of pelage color morphs was unexpected and warrants further attention.

The association of mainland Nueces County and Flour Bluff samples with G. p. maritimus supports previous range maps that depict G. p. maritimus to occur on suitable soils in the vicinity of Corpus Christi and southward to Baffin Bay until interrupted by clay soils between Nueces and Kleberg Counties (Williams et al., 1982; Wilkins and Swearingen, 1990). The phylogenetic association of the Kleberg County samples was unexpected and inconsistent with G. p. megapotamus; this conclusion was supported by the Kimura 2 parameter distances and Bayesian analyses. Our phylogenetic analyses produced topologies similar to previously published associations among Geomys taxa (Sudman et al., 2006), with minor differences in placement of taxa that may be due to the shorter length of the sequence fragments we used; previous studies used the entire Cytb gene. Baker and Bradley (2006) proposed that genetic distances based on Cytb sequences in the range of 5 to 11 % as worthy of further examination for taxonomic status. The extent of sequence divergence (d > 12%) between Kleberg County samples and members of G. personatus suggests that the Kleberg County samples may lie outside the G. personatus species group entirely. Compared to other Geomys in central and southern Texas, the Kleberg County samples were most similar to the G. texensis group (d = 6 to 8%), rather than G. streckeri or G. attwateri (d > 10%). Therefore, the Kleberg County samples appear to represent a previously undetected, cryptic lineage warranting further taxonomic consideration.

Pocket gophers exhibit restricted dispersal across water barriers and unsuitable soils, and morphologically cryptic species are known to exist in close geographic proximity (Cramer and Cameron, 2001). Previous studies of pocket gopher taxonomy used samples of G. p. megapotamus taken in the sand plain between Los Olmos Creek and the Rio Grande to the south and southwest (Sudman et al, 2006; Chambers et al, 2009). Wilkins and Swearingen (1990) identified a narrow band of unsuitable soil flanking Los Olmos Creek, where erosion from the creek exposed an underlying layer of clay (Fig. 1). Los Olmos Creek bisects the region along an east-west line and may serve as a barrier between the Kleberg County and G. personatus lineages to the south. If so, Kleberg County lineages may reside in the small geographic area from Los Olmos Creek northward to clay soils near the Kleberg- Nueces County line. The aberrant haplotype found on Padre Island may reflect simple human-mediated transportation {i.e., through translocation of topsoil or fill dirt for construction) or a more complicated history of colonization and isolation events.

Of the currently recognized subspecies of Geomys, only G. p. maritimus is listed as a Species of Concern in Texas (Texas Parks and Wildlife Department, 2011). The distribution of G. p. maritimus includes the Flour Bluff region of Corpus Christi and the adjacent United States Navy Naval Air Station-Corpus Christi. Our data confirm G. p. maritimus is found throughout sandy soils of southern Nueces County and may be less vulnerable to development than previously feared. Additional sequence data (complete Cytb and perhaps other nuclear and mtDNA genes) and sampling in the region are warranted to fully understand the distribution and degree of uniqueness of the Kleberg County gophers.

Appendix I.--Capture location and soil characteristics for
pocket gophers (Geomys) trapped in southern coastal Texas
during Jul. 2009 to Feb. 2010. Soil composition is given as
average % and se of soil samples collected at trap sites

ID      Sex        Location

1       [female]   Mainland       Nueces Co.
2       [female]
3       [male]
4       [male]
5       [female]
6       [female]
7       [female]
8       [female]
9       [female]
10      [female]
11      [male]     Padre Island   Nueces Co.
12      [female]
13      [female]
14      [female]
15      [male]     Padre Island   Nueces Co.
16      [male]
17      [female]
18      [male]
19      [female]
20      [female]
35      [male]
21      [female]   Flour Bluff    Nueces Co.
22      [male]
23      [female]
24      [male]
25      [female]
26      [female]
27      [female]
28      [male]
29      [male]     Flour Bluff    Nueces Co.
36      [female]
37      [female]
38      [female]
39      [female]
40      [female]
41      [female]
42      [male]
43      [female]
44      [female]
30      [female]   Kingsville     Kleberg Co.
31      [female]
32      [female]
33      [female]
34      [female]

ID      Coordinates

1       27[degrees]36'08.5"N, 97[degrees]27'04.1"W
2       27[degrees]34'33.6"N, 97[degrees]23'24.7"W
3       27[degrees]36'08.1"N, 97[degrees]22'05.0"W
4       27[degrees]34'33.4"N, 97[degrees]23'27.7"W
5       27[degrees]34'18.0"N, 97[degrees]23'30.4"W
6       27[degrees]36'06.5"N, 97[degrees]22'06.1"W
7       27[degrees]34'17.3"N, 97[degrees]23'30.4"W
8       27[degrees]36'08.1"N, 97[degrees]21'57.8"W
9       27[degrees]36'10.2"N, 97[degrees]22'03.9"W
10      27[degrees]34'34.0"N, 97[degrees]23'25.0"W
11      27[degrees]37'53.2"N, 97[degrees]13'55.3"W
12      27[degrees]36'26.8"N, 97[degrees]12'45.3"W
13      27[degrees]36'1.8"N, 97[degrees]12'57.3"W
14      27[degrees]35'57.4"N, 97[degrees]13'1.0"W
15      27[degrees]36'3.6"N, 97[degrees]12'57.3"W
16      27[degrees]37'15.2"N, 97[degrees]13'57.1"W
17      27[degrees]37'15.2"N, 97[degrees]13'57.1"W
18      27[degrees]36'03.4"N, 97[degrees]13'2.0"W
19      27[degrees]36'03.1"N, 97[degrees]13'4.4"W
20      27[degrees]36'01.1"N, 97[degrees]12'56.4"W
35      27[degrees]37'29.0"N, 97[degrees]13'20.5"W
21      27[degrees]39'15.4"N, 97[degrees]18'48.9"W
22      27[degrees]39'10.1"N, 97[degrees]18'29.5"W
23      27[degrees]39'08.8"N, 97[degrees]18'31.1"W
24      27[degrees]40'59.9"N, 97[degrees]17'35.1"W
25      27[degrees]39'15.4"N, 97[degrees]18'49.4"W
26      27[degrees]39'17.3"N, 97[degrees]18.5'04"W
27      27[degrees]41'26.1"N, 97[degrees]15'31.9"W
28      27[degrees]39'15.4"N, 97[degrees]18'48.9"W
29      27[degrees]39'59.9"N, 97[degrees]18'49.2"W
36      27[degrees]41'26.5"N, 97[degrees]16'06.1"W
37      27[degrees]41'27.3"N, 97[degrees]15'34.7"W
38      27[degrees]41'25.6"N, 97[degrees]15'31.6"W
39      27[degrees]41'02.3"N, 97[degrees]16'06.1"W
40      27[degrees]41'21.9"N, 97[degrees]15'40.9"W
41      27[degrees]41'26.6"N, 97[degrees]15'34.8"W
42      27[degrees]41'41.4"N, 97[degrees]15'33.3"W
43      27[degrees]41'22.4"N, 97[degrees]15'41.1"W
44      27[degrees]41'26.8"N, 97[degrees]15'30.6"W
30      27[degrees]32'40.1"N, 97[degrees]51'45.1"W
31      27[degrees]24'10.8"N, 97[degrees]54'47.6"W
32      27[degrees]23'21.5"N, 97[degrees]51'37.7"W
33      27[degrees]23'51.0"N, 97Q46'05.7"W
34      27[degrees]31'21.0"N, 97[degrees]53'44.2"W

ID      Sand         Silt        Clay

1       91.0 (3.0)   3.3 (3.6)   5.8 (1.1)
2
3
4
5
6
7
8
9
10
11      88.5 (4.6)   4.8 (3.5)   6.7 (2.0)
12
13
14
15
16
17
18
19
20
35
21      88.8 (3.8)   4.3 (1.8)   6.9 (2.6)
22
23
24
25
26
27
28
29
36
37
38
39
40
41
42
43
44
30      80.5 (2.9)   7.3 (0.7)   12.2 (2.5)
31
32
33
34

Appendix II.--Body, skull, and habitat measurements (mean
and SE) of 12 male (cr) and 32 female (9) pocket gophers
(Geomys spp.) collected in Kleberg and Nueces counties,
Texas, during Jul. 2009 to Feb. 2010

               Mainland       Padre island
               nueces CO.

Variable          [male]        [female]        [male]
(1,2)            (N = 2)        (N = 8)        (N = 5)

Weight (g)     355.4 (67.3)   309.4 (14.9)   316.6 (63.8)
                    Aa             Ba             Aa
Total bodv     212.5 (12.5)   203.2 (2.8)    173.6 (30.2)
                    Aa             Ba             Aa
Tail            85.8 (4.6)     80.4 (2.0)    78.0 (13.3)
                    Aa            ABa             Aa
Hindfoot        34.0 (1.0)     33.9 (1.2)     40.2 (1.6)
                    Aa             Ba             Ba
Skull           49.1 (5.1)     51.2 (1.0)     50.4 (3.2)
                    Aa             Ba             Aa
Condylobasal    48.2 (5.4)     50.3 (1.1)     49.2 (3.2)
                    Aa             Ba             Aa
Basal           46.6 (4.8)     48.2 (1.0)     43.6 (5.6)
                    Aa             Ba             Aa
Palatal         31.5 (2.8)     32.1 (0.6)     32.1 (2.6)
                    Aa             Ba             Aa
Zygomatic       33.2 (1.4)     32.1 (0.6)     30.8 (2.9)
  breadth           Aa             Ba             Aa
Mastoid         28.0 (2.4)     28.9 (0.6)     27.3 (1.4)
  breadth           Aa             Ca             Aa
Mandible        34.8 (1.3)     35.1 (0.9)     27.9 (7.5)
                    Aa             Ba             Aa

               Flour bluff

Variable         [female]        [male]
(1,2)            (N = 6)        (N = 5)

Weight (g)     282.4 (24.9)   408.7 (19.2)
                   ABa             Aa
Total bodv     190.8 (4.4)    224.0 (4.8)
                   ABa             Aa
Tail            85.7 (3.5)     81.6 (3.7)
                    Ba             Aa
Hindfoot        32.9 (0.6)     34.9 (0.4)
                    Bb             Aa
Skull           49.4 (1.7)     53.4 (1.0)
                    Ba             Aa
Condylobasal    48.4 (1.9)     52.6 (0.8)
                    Ba             Aa
Basal           46.2 (1.8)     50.0 (1.3)
                    Ba             Aa
Palatal         31.1 (1.1)     35.3 (1.3)
                    Ba             Aa
Zygomatic       29.4 (1.2)     32.7 (1.1)
  breadth          ABa             Aa
Mastoid         27.8 (1.0)     27.9 (1.6)
  breadth          BCa             Aa
Mandible        32.2 (1.8)     35.9 (0.8)
                   ABa             Aa

               Kleberg        CO.

Variable         [female]        [male]
(1,2)            (N = 13)       (N = 5)

Weight (g)     255.7 (18.8)   191.3 (22.5)
                   ABb             A
Total bodv     182.5 (5.0)    171.0 (9.1)
                    Ab             A
Tail            75.0 (2.5)     69.0 (2.0)
                    Aa             A
Hindfoot        31.7 (0.4)     27.7 (1.3)
                    Bb             A
Skull           47.9 (1.1)     42.8 (2.2)
                   ABb             A
Condylobasal    46.5 (1.2)     40.8 (2.2)
                   ABb             A
Basal           44.6 (1.1)     38.8 (1.7)
                    Bb             A
Palatal         30.7 (1.0)     25.6 (1.2)
                    Bb             A
Zygomatic       28.4 (0.8)     25.6 (1.5)
  breadth           Ab             A
Mastoid         24.8 (0.9)     23.3 (0.9)
  breadth          ABa             A
Mandible        31.7 (0.9)     27.7 (1.5)
                   ABb             A

(1) All body and skull measurements are in mm except where
designated otherwise

(2) Means with different upper-case letters are
statistically different (P < 0.05) between areas within the
same sex; means with different lower-case letters are
statistically different between sexes within the same area


Acknowledgments.--We thank the United States Navy for funding this project. R. L. Honeycutt provided helpful comments on an earlier version of the paper. Informal discussions with P. Sudman, R. Dowler, J. Light, and D. Reed helped clarify our interpretation of the results. This is contribution 13-136 of the Caesar Kleberg Wildlife Research Institute.

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SUBMITTED 28 SEPTEMBER 2012

ACCEPTED 25 NOVEMBER 2013

JAMES R. JUSTICE (1) and RANDALL J. BERNOT

Ball State University, Department of Biology, Muncie, Indiana 48306

Table 1.--Eigenvectors derived from principal components
analysis of physical measurements and cranial morphology for
32 female pocket gophers captured in southern Texas during
Jul. 2009 to Feb. 2010

                               Eigenvectors

Measurement             Principal     Principal
                        component 1   component 2

Total body length       0.307716      -0.057815
Tail                    0.280934      -0.085887
Hind foot               0.292530      -0.297143
Skull                   0.338193      -0.050460
Condylobasal            0.341009      0.038829
Basal                   0.342565      -0.012861
Palatal                 0.322147      -0.354071
Zygomatic breadth       0.329134      -0.071022
Mastoid breadth         0.266298       0.865241
Mandible                0.331328       0.133098
Proportion of           0.813          0.053
  variation explained

Table 2.--Frequency and distribution of pelage coloration
in 44 pocket gophers (Geomys spp.) captured in
southern Texas during Jul. 2009 to Feb. 2010

                                              Location

Color     Chi-square              Mainland    Padre    Flour
(1)                                nueces     island   bluff

Dark      Observed                    8         0        1
Hispid    Expected                   2.3       2.5      4.1
          [chi square] value        14.1       2.5      2.3
          % total [chi square]      32.2       5.7      5.2
Light     Observed                    1         3        1
Hispid    Expected                   1.8       2.0      3.3
          [chi square] value        0.36       0.5      1.6
          % total [chi square]       0.8       1.1      3.7
Brown     Observed                    1         0        6
          Expected                   1.8       2.0      3.3
          [chi square] value        0.36       2.0      2.2
          % total [chi square]       0.8       4.6      5.0
Reddish   Observed                    0         5        7
Brown     Expected                   2.7       3.0      4.9
          [chi square] value         2.7       1.3      0.9
          % total [chi square]       6.2       3.0      2.1
Ash       Observed                    0         2        3
          Expected                   1.1       1.2      2.0

          [chi square] value         1.1       0.5      0.5
          % total [chi square]       2.5       1.1      1.1
Yellow    Observed                    0         1        0
          Expected                   0.2       0.2      0.4
          [chi square] value         0.2       3.2      0.4
          % total [chi square]       0.5       7.3      0.9
Total                                10         11       18

                                         Location

Color     Chi-square              Kleberg     Total
(1)                                  Co.

Dark      Observed                    i         10
Hispid    Expected                   it
          [chi square] value        0.009
          % total [chi square]      0.02
Light     Observed                    3         8
Hispid    Expected                   0.9
          [chi square] value         4.9
          % total [chi square]      11.2
Brown     Observed                    1         8
          Expected                   0.9
          [chi square] value        0.01
          % total [chi square]      0.02
Reddish   Observed                    0         12
Brown     Expected                   1.4
          [chi square] value         1.4
          % total [chi square]       3.2
Ash       Observed                    0         5
          Expected                   0.6
          [chi square] value         0.6
          % total [chi square]       1.4
Yellow    Observed                    0         1
          Expected                   0.1
          [chi square] value         0.1
          % total [chi square]       0.2
Total                                 5         44

(1) Chi-square value = 43.74 DF = (r-l)(c-l) = 15
Tabular Chi-square value = 32.8 (P = 0.005)

Table 3.--Number of haplotypes (h), haplotype diversity (H),
average number of nucleotide differences (k), and
distribution of nine Cytb haplotypes for pocket gophers at
five sampling sites in southern coastal Texas

                                   Haplotype

Site           h   H(SD)              k    1    2   3   4

Mainland
  Nueces Co.   4   0.644 (0.152)   1.33    6
Flour Bluff    3   0.522 (0.101)   1.04    11       5
Padre Island   3   0.679 (0.122)   11.85        1       3
Kleberg Co.    1   --                --         4
Total          9   0.776 (0.056)   14.41   17   5   5   3

               Haplotype

Site            5     6     7     8     9

Mainland
  Nueces Co.           1    2            1
Flour Bluff                       1
Padre Island    4
Kleberg Co.
Total           4     1     2     1      1

Table 4.--Average genetic distances between selected taxa of
Geomys based on the Kimura 2 parameter model (Kimura 1980) of
sequence evolution (lower diagonal) and se derived from 1000
bootstrap reps (upper diagonal)

                                         Taxon

     Taxon                               1       2       3       4

1    Mainland Nueces, Flour Bluff                0.011   0.021   0.02
2    Padre Island1                       0.060           0.02    0.017
3    Kleberg Co.                         0.127   0.122           0.018
4    G. attwateri (n = 3)                0.132   0.111   0.110
5    G. personatus davisi (n = 1)        0.068   0.064   0.135   0.110
6    G. personatus maritimus (n = 3)     0.004   0.060   0.126   0.131
7    G. personatus megapotamus (n = 4)   0.046   0.026   0.130   0.111
8    G. personatus personatus (n = 2)    0.049   0.033   0.130   0.114
9    G. texensis bakeri (n = 1)          0.127   0.134   0.079   0.140
10   G. texensis llanensis (n = 1)       0.127   0.130   0.069   0.131
11   G. texensis texensis (n = I)        0.123   0.129   0.063   0.131
12   G. streckeri (n = 3)                0.139   0.139   0.103   0.133
13   G. tropicalis (n = 2)               0.058   0.063   0.150   0.134

     Taxon                               5       6       7       8

1    Mainland Nueces, Flour Bluff        0.015   0.002   0.011   0.011
2    Padre Island1                       0.012   0.011   0.005   0.006
3    Kleberg Co.                         0.022   0.021   0.021   0.021
4    G. attwateri (n = 3)                0.017   0.02    0.018   0.018
5    G. personatus davisi (n = 1)                0.015   0.012   0.012
6    G. personatus maritimus (n = 3)     0.068           0.011   0.011
7    G. personatus megapotamus (n = 4)   0.049   0.046           0.004
8    G. personatus personatus (n = 2)    0.056   0.048   0.014
9    G. texensis bakeri (n = 1)          0.131   0.125   0.133   0.133
10   G. texensis llanensis (n = 1)       0.127   0.125   0.131   0.129
11   G. texensis texensis (n = I)        0.134   0.121   0.129   0.129
12   G. streckeri (n = 3)                0.143   0.138   0.133   0.142
13   G. tropicalis (n = 2)               0.063   0.059   0.050   0.054
                                         Taxon

     Taxon                               9       10        11      12

1    Mainland Nueces, Flour Bluff        0.021   0.021   0.021   0.022
2    Padre Island1                       0.021   0.02    0.021   0.021
3    Kleberg Co.                         0.016   0.015   0.014   0.018
4    G. attwateri (n = 3)                0.022   0.02    0.021   0.02
5    G. personatus davisi (n = 1)        0.021   0.021   0.022   0.023
6    G. personatus maritimus (n = 3)     0.021   0.021   0.021   0.022
7    G. personatus megapotamus (n = 4)   0.022   0.021   0.022   0.022
8    G. personatus personatus (n = 2)    0.022   0.021   0.021   0.022
9    G. texensis bakeri (n = 1)                  0.01    0.008   0.02
10   G. texensis llanensis (n = 1)       0.035           0.008   0.02
11   G. texensis texensis (n = I)        0.023   0.023           0.02
12   G. streckeri (n = 3)                0.122   0.126   0.126
13   G. tropicalis (n = 2)               0.131   0.134   0.134   0.147
                                         Taxon

     Taxon                                 13

1    Mainland Nueces, Flour Bluff        0.013
2    Padre Island1                       0.012
3    Kleberg Co.                         0.023
4    G. attwateri (n = 3)                0.02
5    G. personatus davisi (n = 1)        0.014
6    G. personatus maritimus (n = 3)     0.013
7    G. personatus megapotamus (n = 4)   0.012
8    G. personatus personatus (n = 2)    0.012
9    G. texensis bakeri (n = 1)          0.021
10   G. texensis llanensis (n = 1)       0.021
11   G. texensis texensis (n = I)        0.021
12   G. streckeri (n = 3)                0.022
13   G. tropicalis (n = 2)

(1) aberrant sample collected at Padre Island not included
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