BiochemicalGenetics, Vol.30, Nos. 1/2, 1992
Allozyme and Mitochondrial DNA Analysis of a Hybrid Zone Between White-Tailed Deer and Mule Deer (Odocoileus) in West Texas Scott W. Ballinger, 1'3 Lytle H. Blankenship, 1'4J o h n W. Bickham, 1 and Steven M. Carl" 2
Received21 July1988--Final30 Oct. 1991
Thirty allozyme loci and 35 mitochondrial DNA (mtDNA) restriction sites were examined in 24 white-tailed deer and 46 mule deer from a hybrid zone in West Texas. A common mtDNA genotype is shared by all of the mule deer with 67% of the white-tailed deer. At the albumin locus, 13% of the white-tailed deer and 24% of the mule deer are heterozygous, sharing alleles that are otherwise species-specific in allopatric populations; 7% of the mule deer are homozygous for the allele that is characteristic of allopatric white-tailed deer. Gene flow appears to have been bidirectional, with greater genetic introgression into mule deer. The mtDNA data suggest that matings between white-tailed and mule deer have occurred in the past. Despite evidence of genetic introgression, analysis of multilocus genotypes indicates that none of the deer examined is an F 1 hybrid. Production of such hybrids appears to be generally uncommon in North American deer; management plans that assume otherwise should be reconsidered. KEYWORDS: mitochondrial DNA;
Odocoileus;allozymes; hybrid zones.
This work was supported by an NIH Biomedical Research Support Grant, Texas Agricultural Experiment Station Program Development and Expanded Research Awards, the Caesar Kleberg Research Program in Wildlife Ecology, and a Natural Sciences and Engineering Research Council Operating Grant. J Wildlife Genetics Laboratory, Department of Wildlife & Fisheries Sciences, Texas A&M University, College Station, Texas 77840. 2 Genetics, Evolution, and Molecular Systematics Laboratory, Department of Biology, Memorial University of Newfoundland, St. John's NF A1B 3X9, Canada. 3 To whom correspondence should be addressed at Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322. 4 Present address: Safari Adventures, P.O. Box 5220, Uvalde, Texas 78802.
1 0006-2928/92/0200-0001506.50/0 © 1992 Plenum Publishing Corporation
2
Ballinger, Blankenship, Bickham, and Carr
INTRODUCTION White-tailed deer (Odocoileus virginianus) and mule deer (O. hemionus) differ in a number of qualitative and quantitative morphological and ecological attributes that are maintained in both allopatry and sympatry (Baker, 1984; Marchinton and Hirth, 1984). Although their distributions overlap in several areas of the southwestern United States, white-tailed deer thrive in pine/oak montane forests, whereas mule deer favor more open, arid terrain at lower elevations. The range of white-tailed deer has expanded westward at the expense of mule deer in the Trans-Pecos region of West Texas within historic times (Wiggers and Beasom, 1986), however, leading to speculation that interspecies hybridization might have occurred during this displacement. Interspecies hybrids have been obtained in captivity (Gray, 1972; Derr et al., 1991), and animals of intermediate appearance have been reported in the wild from the Southwest and elsewhere for many years (reviewed by Ballinger, 1987). As a case in point, Bailey (1931) concluded that the type specimen of the desert mule deer (0. h. crooki taken in 1892 from New Mexico) was probably a hybrid between the two species. Sympatric populations of these two species in West Texas share a common mtDNA restriction map genotype, a result which was hypothesized to be the product of introgressive hybridization (Carr et al., 1986). This study is a test of that hypothesis based on biparentally-inherited nuclear gene products (allozymes) and maternally inherited cytoplasmic genes (mtDNA) from 70 deer taken from an area of sympatry.
MATERIALS AND M E T H O D S
Tissue samples for allozyme and mtDNA analyses were collected from 24 white-tailed deer and 46 mule deer between 1983 and 1985 at the Longfellow Ranch, Pecos County, Texas. Tissue samples (heart, liver, kidney, and skeletal muscle) were taken during field dressing of the deer and were stored in liquid nitrogen. Individuals were assigned to species based on the general morphology of tails, ears, and antlers (when present) and further verified by metatarsal gland measurements. White-tailed deer have significantly shorter metatarsal glands than mule deer at the Longfellow Ranch (Carr et al., 1986) and elsewhere (Wishart, 1980). The ranges of metatarsal gland measurements from: the samples of both species in this study were significantly different (t = 25.4, P < 0.0005) and there were no individuals with intermediate metatarsal gland lengths (Cart et al., 1986). Preparation of extracts and procedures for horizontal starch gel electrophoresis were essentially as described by Manlove et al. (1975) and Selander et al. (1971). Procedures for vertical starch gel electrophoresis followed
Allozyme and Mitochondriai DNA in Hybrid-Zone Deer
3
Siciliano and Shaw (1976). Staining and buffer systems were those described by Harris and Hopkinson (1976). The more common allele at each locus was designated 1.00 or - 1.00, corresponding to an anodal or cathodal migration product, respectively. The designation of the alternate allele at each locus corresponds to the relative mobility of its product compared to the common allele product. Allele frequencies, heterozygosities, and Nei (1972, 1978) and Rogers (1972) indices were calculated with the BIOSYS-1 program of Swofford and Selander (1981). Probabilities of the observed heterozygote distributions at each locus were calculated by Haldane's (1954) exact method. This method is superior to a chi-square test when the sample sizes of some classes are fewer than 10 (Haldane, 1954). The mtDNA analysis was performed as described by Carr et al. (1986). MtDNA data from 19 of the white-tailed deer and 44 of the mule deer were given in that report. Additional mtDNA samples were purified as described by Carr and Griffith (1987). Thirty-five restriction sites were identified with the restriction endonucleases EcoRI, HindIII, HpaI, XbaI, BamHI, PvulI, SstI, XhoI, BclI, SstlI, and ClaI.
RESULTS AND D I S C U S S I O N
Allelic and genotypic frequencies were determined for the seven polymorphic protein coding loci (Table I): Ab (serum albumin), Gd (glucose-6phosphate dehydrogenase; EC 1.1.1.49), GPD-1 (alpha glycerophosphate dehydrogenase 1; EC 1.1.1.8), MPI-1 (mannose phosphate isomerase 1; EC 5.3.1.8), PGD (phosphogluconate dehydrogenase; EC 1.1.1.44), Pep-B (peptidase B, substrate leucyl-glycl-glycine; EC 3.4.11), and DH-2 (an unidentified dehydrogenase that appeared when staining for ADH). The remaining 23 loci (Table I) were monomorphic. Although none of the loci showed fixed species differences, alternate alleles were more common in each of the two species at three loci: GPD-1, Ab, and DH-2 (Table I). TheAb (1.00) allele, GPD-1 (1.00) allele, and DH-2 ( - 1.00) allele were the common alleles in white-tailed deer, whereas theAb (1.03) allele, GPD-1 (1.45) allele, and DH-2 ( - 1.39) allele were the common alleles in mule deer. Allele frequencies were most distinctive at the albumin locus. Statistical analyses revealed intrapopulation heterozygote deficiencies and genetic similarity between species. Haldane's (1954) exact test for deviations from Hardy-Weinberg expectations at each polymorphic locus (Table I) showed no significant heterozygote excesses. Significant deficiencies of heterozygotes were found at three of seven (43%) white-tailed deer loci (GPD, Pep-B, and MPI) and at five of seven (71%) mule deer loci (all
Ballinger, Blankenship, Bickham, and Carr Table I. Allele and Genotype Frequencies of Polymorphie Loci Among Deer from the Longfellow Ranch; Exact Probabilities of Heterozygote Distributions°
Allele Locus Ab DH-2 Gd GPD-1 MPI-1 PGD Pep-B
Genotype
Allele
Species
F
S
FF
FS
SS
P (FS)
1.03 (F) 1.00 (S) -1.39 (F) -1.00 (S) 1.00 (F) 0.82 (S) 1.45 (F) 1.00 (S) 1.00 (F) 0.82 (S) 1.00 (F) 0.73 (S) 1.17 (F) 1.00 (S)
WT MD WT MD WT MD WT MD WT MD WT MD WT MD
0.063 0.815 0.167 0.511 0.958 0.620 0.271 0.967 0.833 0.913 0.958 0.609 0.083 0.011
0.938 0.185 0.833 0.489 0.042 0.380 0.729 0.033 0.167 0.087 0.042 0.391 0.917 0.989
0.000 0.696 0.083 0.370 0.917 0.522 0.208 0.956 0.792 0.891 0.917 0.500 0.083 0.000
0.125 0.239 0.167 0.283 0.083 0.196 0.125 0.022 0.083 0.044 0.083 0.217 0.000 0.022
0.875 0.065 0.750 0.348 0.000 0.283 0.667 0.022 0.125 0.065 0.000 0.283 0.917 0:978
0.936 (+) 0.127 ( - ) 0.090 ( - ) 0.002 (-)* 0.979 (+)
Allozyme and Mitochondrial DNA Analysis of a Hybrid Zone Between White-Tailed Deer and Mule Deer (Odocoileus) in West Texas Scott W. Ballinger, 1'3 Lytle H. Blankenship, 1'4J o h n W. Bickham, 1 and Steven M. Carl" 2
Received21 July1988--Final30 Oct. 1991
Thirty allozyme loci and 35 mitochondrial DNA (mtDNA) restriction sites were examined in 24 white-tailed deer and 46 mule deer from a hybrid zone in West Texas. A common mtDNA genotype is shared by all of the mule deer with 67% of the white-tailed deer. At the albumin locus, 13% of the white-tailed deer and 24% of the mule deer are heterozygous, sharing alleles that are otherwise species-specific in allopatric populations; 7% of the mule deer are homozygous for the allele that is characteristic of allopatric white-tailed deer. Gene flow appears to have been bidirectional, with greater genetic introgression into mule deer. The mtDNA data suggest that matings between white-tailed and mule deer have occurred in the past. Despite evidence of genetic introgression, analysis of multilocus genotypes indicates that none of the deer examined is an F 1 hybrid. Production of such hybrids appears to be generally uncommon in North American deer; management plans that assume otherwise should be reconsidered. KEYWORDS: mitochondrial DNA;
Odocoileus;allozymes; hybrid zones.
This work was supported by an NIH Biomedical Research Support Grant, Texas Agricultural Experiment Station Program Development and Expanded Research Awards, the Caesar Kleberg Research Program in Wildlife Ecology, and a Natural Sciences and Engineering Research Council Operating Grant. J Wildlife Genetics Laboratory, Department of Wildlife & Fisheries Sciences, Texas A&M University, College Station, Texas 77840. 2 Genetics, Evolution, and Molecular Systematics Laboratory, Department of Biology, Memorial University of Newfoundland, St. John's NF A1B 3X9, Canada. 3 To whom correspondence should be addressed at Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322. 4 Present address: Safari Adventures, P.O. Box 5220, Uvalde, Texas 78802.
1 0006-2928/92/0200-0001506.50/0 © 1992 Plenum Publishing Corporation
2
Ballinger, Blankenship, Bickham, and Carr
INTRODUCTION White-tailed deer (Odocoileus virginianus) and mule deer (O. hemionus) differ in a number of qualitative and quantitative morphological and ecological attributes that are maintained in both allopatry and sympatry (Baker, 1984; Marchinton and Hirth, 1984). Although their distributions overlap in several areas of the southwestern United States, white-tailed deer thrive in pine/oak montane forests, whereas mule deer favor more open, arid terrain at lower elevations. The range of white-tailed deer has expanded westward at the expense of mule deer in the Trans-Pecos region of West Texas within historic times (Wiggers and Beasom, 1986), however, leading to speculation that interspecies hybridization might have occurred during this displacement. Interspecies hybrids have been obtained in captivity (Gray, 1972; Derr et al., 1991), and animals of intermediate appearance have been reported in the wild from the Southwest and elsewhere for many years (reviewed by Ballinger, 1987). As a case in point, Bailey (1931) concluded that the type specimen of the desert mule deer (0. h. crooki taken in 1892 from New Mexico) was probably a hybrid between the two species. Sympatric populations of these two species in West Texas share a common mtDNA restriction map genotype, a result which was hypothesized to be the product of introgressive hybridization (Carr et al., 1986). This study is a test of that hypothesis based on biparentally-inherited nuclear gene products (allozymes) and maternally inherited cytoplasmic genes (mtDNA) from 70 deer taken from an area of sympatry.
MATERIALS AND M E T H O D S
Tissue samples for allozyme and mtDNA analyses were collected from 24 white-tailed deer and 46 mule deer between 1983 and 1985 at the Longfellow Ranch, Pecos County, Texas. Tissue samples (heart, liver, kidney, and skeletal muscle) were taken during field dressing of the deer and were stored in liquid nitrogen. Individuals were assigned to species based on the general morphology of tails, ears, and antlers (when present) and further verified by metatarsal gland measurements. White-tailed deer have significantly shorter metatarsal glands than mule deer at the Longfellow Ranch (Carr et al., 1986) and elsewhere (Wishart, 1980). The ranges of metatarsal gland measurements from: the samples of both species in this study were significantly different (t = 25.4, P < 0.0005) and there were no individuals with intermediate metatarsal gland lengths (Cart et al., 1986). Preparation of extracts and procedures for horizontal starch gel electrophoresis were essentially as described by Manlove et al. (1975) and Selander et al. (1971). Procedures for vertical starch gel electrophoresis followed
Allozyme and Mitochondriai DNA in Hybrid-Zone Deer
3
Siciliano and Shaw (1976). Staining and buffer systems were those described by Harris and Hopkinson (1976). The more common allele at each locus was designated 1.00 or - 1.00, corresponding to an anodal or cathodal migration product, respectively. The designation of the alternate allele at each locus corresponds to the relative mobility of its product compared to the common allele product. Allele frequencies, heterozygosities, and Nei (1972, 1978) and Rogers (1972) indices were calculated with the BIOSYS-1 program of Swofford and Selander (1981). Probabilities of the observed heterozygote distributions at each locus were calculated by Haldane's (1954) exact method. This method is superior to a chi-square test when the sample sizes of some classes are fewer than 10 (Haldane, 1954). The mtDNA analysis was performed as described by Carr et al. (1986). MtDNA data from 19 of the white-tailed deer and 44 of the mule deer were given in that report. Additional mtDNA samples were purified as described by Carr and Griffith (1987). Thirty-five restriction sites were identified with the restriction endonucleases EcoRI, HindIII, HpaI, XbaI, BamHI, PvulI, SstI, XhoI, BclI, SstlI, and ClaI.
RESULTS AND D I S C U S S I O N
Allelic and genotypic frequencies were determined for the seven polymorphic protein coding loci (Table I): Ab (serum albumin), Gd (glucose-6phosphate dehydrogenase; EC 1.1.1.49), GPD-1 (alpha glycerophosphate dehydrogenase 1; EC 1.1.1.8), MPI-1 (mannose phosphate isomerase 1; EC 5.3.1.8), PGD (phosphogluconate dehydrogenase; EC 1.1.1.44), Pep-B (peptidase B, substrate leucyl-glycl-glycine; EC 3.4.11), and DH-2 (an unidentified dehydrogenase that appeared when staining for ADH). The remaining 23 loci (Table I) were monomorphic. Although none of the loci showed fixed species differences, alternate alleles were more common in each of the two species at three loci: GPD-1, Ab, and DH-2 (Table I). TheAb (1.00) allele, GPD-1 (1.00) allele, and DH-2 ( - 1.00) allele were the common alleles in white-tailed deer, whereas theAb (1.03) allele, GPD-1 (1.45) allele, and DH-2 ( - 1.39) allele were the common alleles in mule deer. Allele frequencies were most distinctive at the albumin locus. Statistical analyses revealed intrapopulation heterozygote deficiencies and genetic similarity between species. Haldane's (1954) exact test for deviations from Hardy-Weinberg expectations at each polymorphic locus (Table I) showed no significant heterozygote excesses. Significant deficiencies of heterozygotes were found at three of seven (43%) white-tailed deer loci (GPD, Pep-B, and MPI) and at five of seven (71%) mule deer loci (all
Ballinger, Blankenship, Bickham, and Carr Table I. Allele and Genotype Frequencies of Polymorphie Loci Among Deer from the Longfellow Ranch; Exact Probabilities of Heterozygote Distributions°
Allele Locus Ab DH-2 Gd GPD-1 MPI-1 PGD Pep-B
Genotype
Allele
Species
F
S
FF
FS
SS
P (FS)
1.03 (F) 1.00 (S) -1.39 (F) -1.00 (S) 1.00 (F) 0.82 (S) 1.45 (F) 1.00 (S) 1.00 (F) 0.82 (S) 1.00 (F) 0.73 (S) 1.17 (F) 1.00 (S)
WT MD WT MD WT MD WT MD WT MD WT MD WT MD
0.063 0.815 0.167 0.511 0.958 0.620 0.271 0.967 0.833 0.913 0.958 0.609 0.083 0.011
0.938 0.185 0.833 0.489 0.042 0.380 0.729 0.033 0.167 0.087 0.042 0.391 0.917 0.989
0.000 0.696 0.083 0.370 0.917 0.522 0.208 0.956 0.792 0.891 0.917 0.500 0.083 0.000
0.125 0.239 0.167 0.283 0.083 0.196 0.125 0.022 0.083 0.044 0.083 0.217 0.000 0.022
0.875 0.065 0.750 0.348 0.000 0.283 0.667 0.022 0.125 0.065 0.000 0.283 0.917 0:978
0.936 (+) 0.127 ( - ) 0.090 ( - ) 0.002 (-)* 0.979 (+)
