OCCASIONAL PAPERS THE MUSEUM TEXAS TECH UNIVERSITY NUMBER 75 22 JANUARY 1982 ZOOGEOGRAPHIC AND EVOLUTIONARY RELATIONSHIPS OF SELECTED POPULATIONS OF MICROTUS MEXICAN US Dallas E. Wilhelm, Jr. Upper elevations of the higher mountains of the southwestern United States are inhabited by several species of boreal mammals that have been isolated on these mountain refit,gia for varying periods of time. These restricted populations provide an oppor- tunii\ for investigating zoogeographic and evolutionary princi¬ ples. One such mammal, the Mexican vole {Microtus mexicanus), chosen as the basis of this study, presently inhabits the Transition Zone on scattered mountain ranges hom southwestern Colorado to the Mexican state of Veracruz. It typically is found in dry bum hgrass meadows and mesic grasslands scattered among yellow pine and fir at elevations generally above 2000 meters (m.). M. mexicanus is more tolerant of xeric conditions than are other mi- crotmes inhabiting this area and is often found considerable dis¬ tances from any source of permanent water (Findley and Jones, 1962). The isolated nature of mountains in the Southwest and rel¬ atively small geographic ranges of the species have resulted in a series of small, isolated Microtus mexicanus populations. This study concerns four populations of the Mexican \ole located in the San Mateo, Manzano, Sacramento, and Guadalupe mountain ranges of New Mexico and Texas. These mountain ranges are separated from one another by variable expanses of dry lowlands, generally consisting of unsuitable habitat for microtine rodents. Three of the four populations have been referred pre¬ viously to the subspecies M. m. guadalupcnsis (Manzano, Sacra- 2 OCCASIONAL PAPERS MCSKLM TEXAS 1 KCH t'NIVERSITY mento, and Guadalupe mountain populations); the remaining San Mateo Mountain population was referred to M, m. mogollo- nensts (Hall and Kelson, 1959), The primary objectives of this investigation were to reevaluate present subspecific assignments of populations of M. mexicanus in the region of study, to compare conclusions obtained by means of classical taxonomic methods with those obtained by using more recent systematic methods, to examine the zoogeographic history and evolutionary relationships of the populations con¬ cerned, and to attempt to place the evolutionary changes observed into a reasonable temporal framework. Additionally, this investi¬ gation was intended to explore aspects of the biology of M. mexi- canus that previously have not been covered in the literature. These four populations of the Mexican vole were examined using protein electrophoresis, karyotypic analysis, sperm morphology, bacular morphology, and c lassical morphometries. Methods and Materials Mexican voles were captured in Sherman live traps during the months indicated at the following localities: Texas: Culberson Co.: Upper Dog Canyon, Guadalupe Mountains National Park {April. August, December); The Bowl, Guadalupe Mountains National Park (August). New Mexico: Torrance Co.: 4th of July Campground, Cibola National Forest (September, October); Lin¬ coln Co.: South Fork Campground, Lincoln National Forest (May, October, November); Soccoro Co.: Beartrap Canyon, Cibola National Forest (October). The collecting localities and the extent of the mountain ranges involved are illustrated in Fig. L After collection, voles were transported from the field to the laboratory and were usually sacrificed within two weeks of cap¬ ture. A total of 379 specimens of M. mexicanus were examined, including 121 that were live trapped and maintained in the labor¬ atory for varying periods before being killed. All were prepared as standard museum skins and skulls and deposited in The Museum, Texas Tech University. Karyotypes were prepared following the in vivo technique of Baker (1970). A minimum of five metaphase spreads were counted lor each of 12 males and 49 females examined. Terminology des¬ cribing chromosomal morphology and fundamental number fol¬ lows that ol Patton (1907). Spermatozoa were obtained by removing the epididymis from freshly killed specimens, mincing it with scissors, anti suspending WILHELM—MICRO'I'LS MEXICANl N 3 Fig. 1.—Geographic relationships and approximate extent of the mountain ranges containing collecting localities for populations of Microtus tnexiranus examined. Localities are identified by numbers: ]) Beat trap Canyon (San Mateo Mountains, Soccoro County, New Mexico); 2) 1th of July Campground (Manzano Mountains, Torrance County, New Mexico); 3) South Fork Campground (Sacra¬ mento Mountains, Lincoln County, New Mexico); 4) Guadalupe Mountains (Cul¬ berson County, Texas). Circles indicate approximate tollerting localities within eac h mountain range. a small amount of fluid horn the minted tissue in an isotonic solution of sodium citrate. Several drops of this solution were placed on a microscope slide and allowed to air dry. Slides were then fixed in a solution of one part acetic acid and four parts methanol prior to staining with a 0.15 per cent solution of Giemsa in hot water. Photomicrographs of spermatozoa were OCCASIONAL PAPERS MI’SEI'M 1'EXAS LECH LNIVERsm 0_1 mm J' k.. 2.— Raculum of Microtus mexicanus guadaiupensis (III 27326) illustrating the dimensions described in the text: A-B, bacular length; D-E, width of base; F-G, width of shaft; B-C, length of median distal process; H-h width of median distal process. taken using a I.eitz Wetzlar microscope at a magnification of 950X, Measurements in millimeters were taken directly from the four by five-inch negatives using Helios dial calipers and a Saku- rai map measuring device. Measurements and terminology follow those of Linzey and Layne (1974). Ten spermatozoa were mea¬ sured for each of the 28 specimens examined, and average values were used in the subsequent analyses. Penes were removed from freshly killed specimens and stored in AFA; bacular preparation followed techniques outlined by Ander¬ son (1960). After staining and destaining, penes were dissected away from the bacula, and the bat ula were drawn in both dorsal and lateral views using a camera lucida attached to a Wild M5 microscope at a magnification ot 12X. Measurements (in millime¬ ters) of 64 specimens were taken directly from the drawings and included: bacular length, width at base, width of shaft, length of median distal process, and width of median distal process when an ossified distal process was present (Fig. 2). wil l — \ii (:ko n s \u-\k, \m s 1 he morphometric portion of the study was based on 379 spec¬ imens; only those specimens with a total skull length of 23 mil¬ limeters or more were included in the analysis. This corresponded to a minimum total body length of approximately 100 millime¬ ters. Four standard external measurements were recorded from specimen labels and 14 cranial measurements were taken. Cranial measurements follow Coin (1913), Anderson (1954), and Snyder (1954). Starch gel electrophoresis was used to assess allozymic variation for 80 Microtus mexicanus from the following localities: Tpper Dog Canyon (11 males, 9 females); South Fork Campground (12 males, 8 females); 4th of July Campground (8 males. 12 females); and Beartrap Canyon (11 males, 9 females). Samples were pre- pared from liver, heart, and kidney extracts. Techniques of tissue preparation, electrophoresis, and biochemical staining were sim¬ ilar to those of Selander et al. (1971), hut staining procedures for sorbitol dehydrogenase (SDH) and glutamate dehydrogenase (GDI!) were modified after Shaw and Prasad (1970). A summary of electrophoretic methods used is given in Table 1. Designation of alleles follows that of Smith et al. (1973). The allele occurring in the highest frequency at each locus was assigned the value of 100 for anodally migrating systems, or 100 for those migrating cathodally. Remaining alleles at a locus were described as percentages of the 100 allele by comparing relative migration distances. When more than one locus was present in a system, the most anodal lot us in the system was designated “1” and more successively cathodal loci were assigned progressively higher numbers. Allozyme similaiity was assumed if side-by-side comparisons failed to establish differences (see Smith et al., 1973). Statistical and clustering analyses were carried out using SAS-76 (Ban et al ., 1976), IJNIVAR (Power, 1970), and NT-SYS pro¬ grams. In all analyses, specimens were grouped into four aggre¬ gate populations representing the primary collecting localities; these aggregates were considered as Operational Taxonomic Units (OTU’s). SAS programs were used m univariate analyses to provide standard descriptive statistics and to perform both single classifi¬ cation and two-way analyses of variance in order to test for signif¬ icant differences between or among means. When means were found to be significantly different, the Sums of Squares Simul¬ taneous Test Procedure (SS-STP) was conducted using a UNIVAR program to determine maximally nonsignificant subsets. Multi- OC C ASION AL PAPERS MI SKI M ! K\AS I EOH I 'MVER.SMA 6 variate analyses entailed calculation of Pearson product-moment correlation coefficients, again using SAS, to compare electropho¬ retic distance and correlation matrices with the corresponding morphometric and sperm morphology matrices, (duster analyses were conducted on the correlation and distance matrices and a two-dimensional phenogram was generated for each using the NT-SYS program. This program utilises the unweighted pair- group method using arithmetic averages (UPGMA). Pheriograms were compared with their respective matrices and a coefficient of cophenetic correlation was computed for each comparison to assess the reliability of the phenogram. A matrix of correlation among the characters was computed, and the first principal com¬ ponents were extracted. A three-dimensional plot of the OTI ’s onto the first three principal components was then prepared. The percentage of the total variation accounted for by each principal component was calculated, as w r as the contribution of each char¬ acter to each ot the principal components. Additional data con¬ cerning methods and materials may be found in Wilhelm f 1977>. S p i < a m en s E xA m i n f. n In addition to specimens collected for this study, other speci¬ mens were borrowed from the following institutions for examina¬ tion; Texas Cooperative Wildlife Collection. Texas A&M Univer¬ sity (TCWC); The Museum, Texas Tech University (TTU); University of New Mexico (UNM); and University of Texas at El Paso (UTEP). Localities are not plotted separately on Fig. 1, but ate grouped with the four primary collecting sites for purposes of analysis and discussion. The list below inc ludes all spec imens examined. Microtus mexicanus gwtdalupensis (300).— Ikxas: Culberson County: Guada¬ lupe Mountains National Park. The Bowl, 24 (6 TTU, 18 TCWC); Upper Dog Canyon, 33 (1 J I ■ Guadalupe Peak, 1 (Til i. New Mexico: Otero County: Tim- beron, 2 (T IT ■: 8.5 mi. E. 4.5 mi. N A!mo Peak. 2 (UNM 1 mi. N Cloudcroft, 2 (UNM); 7 mi. E Cloudcroft. 2 (UNM); 7 mi. E, 2.5 mi. N Cloudcroft, 7 (UNM); L mi. S Cloudcroft, 1 (UNM): 10 mi. S Cloudcroft, 15 (UNM); 2 mi. W Cloudcroft, L (UNM); 2.! mi. W Cloudcroft. 1 (UTEP); neat Cloudcroft, 19 (8 UTEP, 11 II' ; Russian Canyon. 5 mi. S. 2.5 mi. E Cloudcroft, 2 ft II P:, north of Ruidoso, 3 I 1 EPi. Lincoln County; Padilla Point, 3 (UTEP): South Fork Campground, 27 (TIT); 5 mi, N, 9 mi. E Capitan. 2 (UNM): 1.5 mi. W Capitan, 3 (UNM); Cajtitan Mountains, 20 (UNM); Monjeau Peak, 10,009 it., 6 (UNM); Lincoln County, 6 (TTU). Torrance County: 4th of July Campground, 36 (TTU); 0.5 mi. S Capillo Peak, 9000 ft., 1 (UNM): 5.5 mi. W Tajique, 8 (UNM); Red Canyon, 0.5 mi. S, 5 mi. W. Manzano. 31 (UNM); Red Canyon, 1 mi \V, 1 mi. S Manzano, 11 (UNM): 5 mi. W Manzano, 3 (UNM'. J 'orrance Connt>. 4 ■ ! I’Uj, Bernalillo ('ount\: Iree Spunks. 8600 ft., 21 (UNM). Table 1 . —Electrophoretic techniques utilized in this study. All gels were run for five hours. '.VIL1II LM —MICROTL X MKXICAN'l X i 2 _ W oo t 8 OCCASIONAL PAPERS MUSEUM TEX AS LECII UNIVERSITY Microtus mexicanus mogollonensis (79) — New Mexico: Socorro County: Beartrap Canyon, 78 (46 TIT, 28 UNM, 4 I T EP); 1.6 mi. from Water Canyon, 1 (UTEP). Results and Discussion Karyology The karyotype of Microtus mexicanus was described from a single female specimen by Matthey (1957), who established the diploid number as 44. Being unable to identify the sex chromo¬ somes, he was uncertain of the fundamental number and listed it as a minimum of 54 and perhaps 56. The karyotype of Microtus mexicanus given in Fig. 3 shows a diploid number of 44 and a fundamental number of 54. No chromosomal polymorphism was noted among specimens exam¬ ined. The autosomes consist of three 1 pairs of large submetacen- trics, one pair of medium submetacen tries, two pairs of small metacentrics, one pair of large acrocentrics, and 14 pairs of small acrocentrics. The X chromosome is a medium-sized submetacen- tric, and the Y is a small acrocentric. Although no chromosomal polymorphism was found in the karyotype of the Mexican vole in this study, two separate chromo¬ somal polymorphisms were described in Mexican voles from the states of Jalisco and Durango in Mexico (Lee and Elder, 1977), and involved differences in both the diploid and fundamental numbers. Thus, M. mexicanus is the only exclusively Nearctic species of microtine from which infraspecific differences in chromosome morphology or number have been reported, b In fra specific variation in chromosome number is more com¬ mon in Palearctic representatives of the genus, however. Microtus hyperboreus and M. middendorfi (Gileva, 1972) and M. juldaschi (Bol’shakov et ai, 1975) have been reported as polymorphic. Other genera of microtines that exhibit chromosomal polymor¬ phism include Dicrostonyx torquatus (Rausch and Rausch, 1972; Kozlovskii, 1974), Clethrionomys rutilus (Rausch and Rausch, 1975), and Pitimys subterraneus (Meylan, 1972). The relatively few number of karyotypic studies of Nearctic microtines and the apparent uniformity of infraspecific chromosome complements might reflect a lack of data rather than extreme chromosomal conservatism of the group. Sperm Morphology Spermatozoa of M. mexicanus from the four populations stud¬ ied wire similar in all respects. The sperm head, widest just above WILIIKLM—MICRO ITS MEXICANl’S Ifl 01 Al AA A A M »A Fu;. 3.— Karyotypf of ;i male (T 11 ‘ 27356) Microtus mexicanus guadalupensis. the base, is asymmetrical with one margin convex and the other nearly straight. The base is smoothly convex with a notch on one side. Almost one-half of the head is enveloped by the acrosome, which is elongated into a recurved hook that lies on the side of the head having the straighter margin. Attachment of the mid¬ piece is to the basal notch and is therefore eccentric. J he tailpiece tapers gradually toward the tip and is sometimes difficult to dis¬ tinguish from the midpiece. However, with the staining procedure used in this study, the tailpiece is generally of uniform appear¬ ance, whereas the midpiece is nearly always granular or mottled in appearance. Mean values, followed by range in parentheses and sample size, for selected sperm measurements of Microtus mexicanus follow {measurements are in microns and localities are given in the order Beartrap, 4th of July, South Fork, and Guadalupe}; length of head, 7.77(7.11-8.00) 7. 8.02(7.51-8.54) 7, 8.34(7,60-9.14} 10, 8.32(8.01-8.78) 8; width of head, 4,86(4.82-4.93) 5, 4.82(4.47-5.35) 5. 5.08(4.54-5.35) 10, 5.01(4.81-5.35) 8; length of mid piece , 18.45(17.12-20.32) 5, 18.10(16.80-18.80) 5, 18.32(16.72-19.36) 10. 18.46(15.84-20.72) 8; length of tailpiece , 66,02(60.16-71.36) 5, 70.21 (65.81-74.32) 5, 72.02(68.48-76.08) 10, 70.51(65.92-73.04) 8. Univariate analysis of sperm data showed a significant differ¬ ence in the head width of spermatozoa (T<0.05) between some localities and in the length of the tailpiece (7 J <0.01) between oth- 10 OCCASIONAL PAPERS MI SKl’M TEXAS 1KCII UNIVERSITY ers. The following nonsignificant subsets were generated by SS- STP tests: width of head —Guadalupe, 4th of July, Beartrap; length of tailpiece —South Fork, Guadalupe, 4th of July; Guada¬ lupe, 4th of July. Beartrap. In AT. m. mogollonensis, the sperm head is slightly shorter but just as broad as that of AT. m. guada- lupensis. The tailpiece of M. rn. mogollonensis is considerably shorter than that of the other subspecies. The four sperm measurements also were analyzed using the NT-SYS multivariate program. Both correlation and distance matrices were computed and phenograms representing the phe- netic relationships were plotted. A Pearson product-moment correlation matrix was computed comparing these two matrices with the corresponding electrophoretic matrices. Correlation coef¬ ficients for neither the distance matrices nor the correlation matri¬ ces were significant. The distance phenogram showed that the Beartrap and 4th of July specimens had smaller sperm than did those from the other two localities. Although on the basis of sperm data, the Beartrap {AT. m. mogollonensis) and the 4th of July (AT. m. guadalupensis) populations more closely resembled each other than either resembled the other two populations, their relationship to each other was not of the same magnitude as was that of populations from the South Fork and Guadalupe locali¬ ties. In the principal components analysis, the 1 amount of phenetic variation expressed in the first principal component was 65.58; in the second, 35.60; and in the third, 0.80. The percentage contribu¬ tion of each sperm character to each principal component, given in the order Component I, II, and III were length of head, 10.51, 0.71, 32.05; width of head, 5.87, 3.81, 22.89; length of tailpiece , 81.70, 59.85, 7.38; length of midpiece, 1.91, 35.62, 37.65. Length of tailpiece accounts for most of the phenetic variation. A three- dimensional perspective of the projection of the four OTU’s onto the first three components, based on a matrix of correlation among the four sperm measurements, is given in Fig. 4. Essen¬ tially the same pattern was seen in the distance phenogram. South Fork and Guadalupe OTU’s were clustered close to one another; Beartrap and 4 tli of July OTU’s were loosely grouped. This grouping of the populations sampled agreed in part with the results horn multivariate analyses of cranial morphometric data. Small sample sizes, however, render these results of limited value. \\ ILHKI.M — Ml( :R()1 I S MKXK AMs II Ill i n,. I ■ I'hree-itinifiisinnul pi<>jr< non of tin. 1 Tom Much populations of Mu rotus HWMrunui onto tlic Inst ihuv principal components based on a matrix of correla¬ tion among Four sperm measurements. Localities are coded as in Fig. I Bacular Morphology Anderson (1960) described the baculum of MU rotus mexicanus, and I noted no major deviations from his description. Of the 64 bacula examined, 20 possessed neither medial nor lateral pro¬ cesses; 32 possessed only the medial process; and 12 possessed both. Accordingly, of the five hat ular measurements taken, only three (bacular length, width of base, and width of shaft) were con¬ sistently available for all specimens. Mean values, followed by range in parentheses and sample sizes for selected bacular measurements were (measurements are in microns and localities arc given in the order Bcartrap, 4th of July, South Fork, and Guadalupe); bacular length, 2.81(2.37-3.76) 18; 2.98(2.07-3.53) 13; 2.69(2.05-3.21) 15; 2.90(2.52-3.23) 16; width of base , 1.35(0.98-1.85) 18; 1,36(0.55-1.87) 13; 1.25(0.71-1.77) 15; 1.39(0.97-1,71) 16; width of shaft, 0.27(0.16-0.37) 18; 0.28(0.17-0.38) 13; 0.27(0.17-0.38) 15; 0.30(0.22-0.40) 16. Univariate analysis of these three measurements revealed no significant differences among specimens from the four localities. The large amount of individual variation (CV's, 7.3-30.2) evident in bacular morphol¬ ogy, even among individuals of similar si/e (and presumably sim¬ ilar age), makes statistical treatment of bacular measurements dif- ficult. Bacula enlarge and c hange somewhat in shape throughout life. Although dividing the bacula into age classes would reduce variation due to age, estimating the age of individual microtines 12 OCCASIONAL PAPJ'RN MINKUM I EX AS TKCH UNIVERSITY is difficult, and the small number of bacula available rendered an attempt to age specimens unfeasabie at this time. Although there have been several surveys of microtine bat ula, few have treated bacular measurements statistically. Dearden (1958) performed an analysis of variance and reported standard errors for some bacular characters in several microtines. HL results indicated that there were subspecific differences in the length of the* bacular shaft in several subspecies of Lagurus curta tus, although the standard errors were nearly twice those that I found in the bacular shaft length of Microtus mexicanus. Coeffi¬ cients of variation for bacular characters in the Mexican vole were large, compared to the- corresponding statistic for any cranial measurement. This high degree of variation within a population, coupled with small sample sizes, makes interpopulational com¬ parisons difficult. As a result, bacular morphology offered no information concerning relationships among the populations examined in this study. Morphometric Analysis Fourteen cranial and four external measurements were- analyzed using the SAS univariate program. Descriptive statistics and results of the SS-STP tests for these measurements are given in Table 2. Coefficients of variation obtained for the c ranial charac¬ ters (3.26-8.95) agree with those values reported by Long (1968) for rodents. A two-way analysis of variance was conducted to detect possible sexual dimorphism and to determine if differences existed among localities for cranial and external characters. Differences were found between sexes for depth of skull, total length, and length of hind foot befow the- probability level of 0.05; for interorbital breadth, 0.01. Total length of females was consistently greater than that of males, and the interorbital breadth was always larger in males than it was in females. Within the Beartrap population, females had deeper skulls and longer hind feet than did males; in the' other three populations, males were larger than females in these two characters. For diastema length, condylozygomatic length, lambdoidal breadth, and le ngth of hind foot, there was no significant differ¬ ence 1 among localities. Nonoverlapping subsets were found in only two characters: the inc isive foramen in specimens from the Cuadalupe Mountains was significantly longer than in those lrom the othei three populations; interorbital breadth was larger IVII.HKI M MICROTI'S Ml XICAM S 1 i 1 abi.k 2 .—Descriptive statistics derived from two-way analysis of variance for external amt cranial measurements of Micro! us mexicanus. Groups of means found to be significantly different at V>OJ'n were tested with the sums of squares simultaneous testing procedure to find the nonovertapping subsets. Groups of means that were found to be not significantly different at P >0.05 are marked ns. Localities are coded as in b ig, !. Kcsulu Locality SS-S I I 1 Mcarr Range SE cv Total length of skull 2 25.5 23.0-27.6 .10 1.29 t 25.4 23.1-27.6 .13 1.02 4 25.4 23,0-27.2 .16 1.51 3 25.1 22.8-27.4 .00 3.68 Diastema length 1 7.* 6.7-9.1 .06 5.72 2 7.7 6.7-8.7 .04 5.41 3 ir- 7.7 6.7-8.8 .04 5.36 ! 7.6 6.0-8.6 .05 5.26 l ength of incisive foramen 4 | 4.7 4.1-5.3 .05 7.27 1 1.5 3,5-5.4 .03 6.22 3 4.5 3.8-5.3 .03 6 38 2 i. [ 3.4-5.3 .04 8.59 Pa bit ilar length 1 13.0 11.5-14.3 .it? 1.28 2 13.0 11.7-14.2 .05 4.25 3 12.8 11.3-14.1 .05 3.92 4 12.8 11.5-14.2 .08 4.82 Candy lazy gomatit length I 10.2 17.5-20.7 .03 3.49 2 10.2 17.6-20.8 .07 3.87 3 ! s 10.2 17.6-12.1 .06 3.26 4 10 1 17.7-20.8 .in 3.95 I ength of nasals 2 7.7 6.1-9.0 .04 5.71 1 7.6 6.0-8.4 .07 6.07 3 i 7.5 59-8.6 .01 6.10 1 1 7.3 6.2-8.1 .06 6.24 Rostral breadth 1 1.1 3.7-4.5 .03 1.80 2 4.1 3.7*4.5 .02 1.44 1 1.1 3,7-4.6 .02 1.78 3 4.0 3,6-4.8 .02 4.32 ] 1 OCCASIONAL PAPERS MI SI. CM i LX A .3 I K( II I'NIVERSITV L a b j i. 2. — Co tit mu ed. Interorbital breadth (males', 2 U 2.9-3.9 .0.4 7.26 1 3. 1 3.0-3.0 .04 4.76 1 3.2 2,8-8.6 .01 6.07 3 3.2 2.7-3.9 .04 6.66 Interorbital breadth (females) I 3.4 2.9-3.8 .01 7.58 2 3.3 2.9-8.8 .04 6.87 1 3.2 2.7-5.7 .01 6.56 3 3.1 2.4-3.6 .05 0.76 Zygomatic breadth 1 15.0 13.3-10.7 .Id 5.00 1 14.7 13.1-16.0 .08 1.57 3 14.7 13.4-16.4 .06 1.01 9 14.7 12.8-16.1 .06 1.60 1‘relamboidal hr cad t h 9 10.5 9.5-11.7 .04 3.51 i 10.1 9.4-11.2 .04 3.54 i 10.3 9.4-11.0 .01 3.41 3 10.3 9.5-11.8 .0.5 3.56 I . a m b d o idal b readt h 1 11.7 10.6-12.6 .06 4.05 9 11.7 10.5-12.6 .01 3.90 i ns 11.5 10.1-12.4 .01 3.85 1 11.5 10.2-12.4 .0" 1.70 Height of skull 2 10.0 9.2-11.0 .03 4.29 1 9.9 9.0-11.2 .04 4.10 1 9.9 9.2-10.9 .06 1.10 3 9.8 9.0-10.6 .04 3.16 Depth of braitua.se (males > 2 7.8 7.3-9.2 .0-1 1.21 3 7.7 7.0-8.8 .05 4.75 1 1 7.0 7.1-8.2 .07 1.08 1 7.4 6.3-8.1 .08 5.12 Depth of brairn ase (females >, 9 7.8 7.1-8.5 .01 3-68 1 7.7 6.9-8.5 .06 1.87 3 7.6 6.9-8.6 .04 1.64 1 7.1 6.7-8.3 .0? 5.13 WII.HI-I.M-MICROTI'S Mi \i(,\M S 13 Ta.bi k 2— Continued. Rostral U ngth 2 ■1.9-7.3 .05 8.23 I 6,2 4.9-7.5 .07 8.91 i 1 6.0 4.7-7.3 .07 8.95 3 1 5.9 4.7-7.1 .04 7.68 / trial lengtf (males} l 127.1 111.0-148.0 2.01 8.23 2 131.2 107.0-150.0 1.18 6.87 3 11-. 131,2 119.0-148.0 1 05 5.80 1 127.4 114.0-140.G 1.75 6.30 Total length (females} 1 130,4 95.0-151.0 1.91 8.90 2 134.3 109.0-175.0 1.63 9.17 3 ns i:u.i 110.0-158.0 1 88 7.M i 129.2 111.0-152.0 1.82 8.09 / nigth o / tail 4 30,9 22.0-42.0 .18 11.34 2 29.5 22.0-58.0 .12 15.13 i 28,7 15.0-42." .55 15.30 3 28.3 18,0-39.0 .37 18.58 Length of hind foot (males. ] 17.3 15.0-20.0 .21 6.35 2 17.8 15.0-22.0 19 8.15 3 11 s 17.5 16.0-19.0 .1 1 5.79 1 17.7 15.0-20.0 .37 9.53 / ength of hmd foot ( female vy i 17.5 15.0-20.0 19 6.70 > 17.2 12.0-23.0 90 9 85 .3 ns 17.2 16.0-20.0 .12 5.36 l 175 15.0-21.0 .29 9.37 Length of ear ■1 12.7 10 0 18.0 .25 11.19 2 12.4 9.0-19.0 .1 1 12.28 1 LM 10.0-19.0 .17 1 1.55 3 12.0 9.0-15.0 .10 8.32 in individuals from Beartrap and 4th of July populations than those from South Fork and Guadalupe localities. The remaining characters showed from two to three overlapping subsets but pro¬ vided no distinct groupings. Characters with high coefficients of variation (length of tail and length of ear) and the four characters which displayed sexual dimorphism (depth of skull, total length, length of hind foot, and 1<> OCCASIONAL PAPl-.RS Ml’SEl'M I'EXAS H-t II ! MVKRM iA interorbital breadth) were eliminated from the subsequent multi¬ variate analyses. The remaining 12 cranial measurements were then analyzed using the NT-SYS- multivariate analysis program. Correlation and distance matrices were computed and pheno- grams representing the phenetic relationships of the four Oi l s were plotted. In addition, a Pearson product-moment correlation was computed comparing these two morphometric matrices w ith (he corresponding electrophoretic matrices. The correlation coeffi¬ cients for the two distance matrices and for the two correlation matrices were not significant. The distance phenogram for mor¬ phometric characters differed from that produced for sperm mea¬ surements presented earlier, with tw r o clusters: Beartrap, 4th of July, and South Folk forming the first, and Guadalupe forming the second. The first three principal components were extracted and plot¬ ted, yielding the relationships illustrated in Fig. 5. Phenetic varia¬ tion expressed by the first principal component was 54.78 per cent; second, 37.75; third, 7.47. The major contributing characters for each component and their percentage contribution were: first principal component; total length of skull (14.48), palatilar length {13.27;, condylozygomatic length (11.34), zygomatic breadth (12.95), and lumbdoidal breadth (12.40); second component: total length of skull (27.42) and condylozygomatic length (20.62); third component: total length of skull (15.55) and prelambdoidal breadth (18.74). Thus, characterization of the OTU’s by principal components is, to a large degree, dependent on total length of skull. The three-dimensional plot arranged the OTU’s into two groups that corresponded to those of ihe distance phenogram. I he Beartrap and 4th of July samples w ere again grouped much closer to each other than to the South Fork and Guadalupe sam¬ ples. The latter two populations are separated somewhat along axis II. Thus, morphometric analyses yielded no conclusive evidence concerning relationships of the four populations studied. The four populations examined generally fell into two groups, corres¬ ponding to tlu same two groups produced by analysis of sperm morphology. There is some evidence from the phenogram based on morphometric characters and the results of the SS-STP test that the Guadalupe population is more distinct than the other three populations. The Guadalupe population has a significantly longer incisive foramen. However, for interorbital breadth, the Beartrap and 4th of July populations have relatively greater inter- orbital breadth than the South Pork and Guadalupe samples. W!I IILLM —MICROTI'S Ml XTCAM S 17 nu'xttanu\ onto the first three principal components based on a matrix ot ton ela¬ tion among 12 cranial measurements. Localities are coded as in Pig. 1. Principal component analyses separated ihe four populations into the same two groups identified in the sperm analysis and in the 11 u a phometric phenogram. Microtine subspecies generally have been delimited by size dif¬ ferences as well as qualitative characteristics, such as pelage color. Microtus mexicanus guadalupensis was distinguished by Bailey (1902) from M. m. mogollonensis primarily on (he basis of cranial characters. Bailey (1902) reported measurements for the male holotype of M. rn. guadalupensis, six of which may be com¬ pared with those taken in this study: total length, 152; length of tail, 34; length of hind foot, 20; basal length of skull. 24.5; length of nasals, 7.5; and zygomatic breadth, 16.0. A comparison of these measurements with those in Table 2 reveals that the total length <>f the holotype exceeds that of any Guadalupe male examined, and equals that of the largest Guadalupe female examined. I he tail of the holotype was longer than the average length of tail found in this study, and the length of hind foot equals the largest value reported herein. The three skull measurements of the holo¬ type all are near the average for those measurements in the Guad- alupe specimens examined. Electro p h oret ic A n a lysis Nineteen protein systems were investigated, but only 16 were scored with confidence. .These systems contained 24 scorable loci, which are listed in Table 3 along with the frequencies of each in the population. In the total sample, 14 loci (58 per cent) were polymorphic, and five (L.DH-2, ADH, G-6-P, MDH-1, and ES I -7) OCCASIONAL PAPERS MLSELM l'EXAS ITCH LNIVERSITV 18 J abie 3. — Alleles and frequencies (in parentheses) at 24 loci in Microtus mexiea- uus and the mean proportion of individuals heterozygous at each locus (h); h is averaged over all four populations (N=80). 1 1 HUS Hi .utiap 4th of July South Fork Guadalupe h LDH 1 100 (1.00) 100 (1.00) 100 (1.00) 100 (1.00) 0.00 LDH-2 100(,975) 72(.025> 100 (1.00) 100 (1.00) 100 (1.00) 0.01 MDH-1 100(.975> 70(.025) 100 (1.00) 100 (1.00) 100 (1.00) 0.01 MDH-2 -100(1.00) -100(1.00) -100(1.00) -100(1.00) 0.00 ME 100 (1.00) 100 (1.00) 100 (1.00) 100 (1.00) 0.00 mu 100 (1.00) 100 (1.00) 100(.925) 1000650) 0.19 85(.075) 85(.350) son 100 (1.00) 100 (1.00) 100(.930) 1160050} 100 (1.00) 0.03 ADH -100(1.00)) 100(.825) -1000950) -100(1.00) 0.11 —86(. 175) -86(.050) LAP 100 (1.00; 100 (1.00) 100 (1.00] 100 (1.00) 0.00 PCI 100(.750) 100(.800) 100 (.H00) 1000250) 0.40 54(.250) 33(.200) 54(.200) 54(.750) PCM 100G825) 160(. 175) 100 (1.00) 100 (1.00) 100 (1.00) 0.06 AB 100 (1.00; 100 (1.00) 100 (1.00) 00 (1.00) 0.00 IPO 100 (1.00) 100 (1.00) 100 (1.00) 100 (1.00) 0.00 GOT-1 100 (1.00) 100 (1.00) 100 (1.00) 00 (1.00) 0.00 GOT-2 -100(1.00) —100(.900) -1000 700) ■ 100(1.00) 0.15 -41 (TOO) —41 (.300) G-6-P 100 (1.00) 100 (1.00) 100 (1.00) 00(.975) 88(.025) 0.01 GDH-I 100 (1.00) 100 (1.00) 100 (1.00) 100 (1.00) 0.00 (.1)11-2 100 (1.00] 100 (1.00) 100 (1.00) 100 (1.00) 0.00 GDH-3 100 { 1.00) 100 (1.00) 100 (1.00) 100 (1.00) 0.00 CrGPDH-1 100 (1.00) 100(.950) 115(.050) 100 (1.00; 100 (1.00) 0.03 aGPDH-2 100(.925) 100(.975) 1000950) 1000975) 0.09 129(.050) 90(.025) 129(.025) 129(.050) 129(025) aGPDH-3 10U(.925) 100 (1.00) 1000950) 100 (1.00) 0.04 112(.075) 142(.050) EST-1 1000475) 100(.800) 1000850) 1000925) 0.43 93(.525) 93 (.200) 930150) 93(.075) EST-7 1000975) 100(.975) 100 (1.00) 100 (1.00) 0.03 65(.025) 65(.025) were present in a frequency of only five per cent. Acid phospha¬ tase (AGP), alkaline phosphatase (AKP), and xanthine dehydroge¬ nase (XI)H) were incompletely scored, but did exhibit some W1I HI'.LM MICRO n s MKMCAM'S 19 polymorphism. 1'he major features of the allozyme variations observed in Microtus mexicanus are listed below. Malate dehydrogenase (MDH).— I’wo MDH loci were observed; the cathodally migrating MDH-2 was monomorphic for all popu¬ lations. MDH-1 exhibited two alleles; the MDll-1 70 allele was present in a single specimen from Beartrap. Isocitrate dehydrogenase (IDH).—This system was represented by two alleles. Hie IDH 85 allele was found in three individuals from South Fork and in la individuals from the Guadalupe sam¬ ple. Sorbitol dehydrogenase (SDH).— Two SDH alleles were identi¬ fied; the SDH 116 allele was found in two individuals from South Fork. Alcohol dehydrogenase (ADH).—Two ADH alleles were observed in this cathodally migrating system. The ADH 516 allele was present in seven individuals from 4th of July and in two individuals from South Fork. Phosphoglucose isomerase (PGI).—Three alleles were repre¬ sented m this system. The PGI 100 allele occurred in all samples. PGI 54 was identified in eight individuals from Beartrap, eight from South Fork, and in all individuals from Guadalupe. PGI^ was present in eight specimens from the 4th of July Campground. Phosfihoglucomutase (PGM).— Phis locus was represented by two alleles. All populations exhibited the PGM 100 allele; PGM 160 was identified in six indiv iduals from Beartrap. Glutamate oxalate transaminase (GOT)- — Two loci were observed; GOT-1 was monomorphic for all populations, and the polymorphic GOT-2 allele migrated cathodally. Four individuals from the 4th of July sample and 10 from South Fork exhibited the GOT-2* 41 allele. Glucose-6-phosphate dehydrogenase (G-6-P).— Two alleles were observed; the G-6-P 100 allele was represented in all populations; the G-6-P 88 allele w as found in one individual from the Guada¬ lupe sample. cxGlycerophosphate dehydrogenase (aGPDH).— Fhree loci were identified and all exhibited polymorphism. The ctGPDH l 113 allele was found in two individuals from 4th of July. nGPDH -2 was represented by three alleles; all populations exhibited the a GPDH-2 100 and aGPDFI-2 129 alleles, and the aGPDH—2 90 allele was present in onlv one specimen from Beartrap. oGPDH-3 142 was detected in two specimens from Beartrap and in two from South Fork. 20 OCCASIONAL P\PI RS Ml SI l M EEXAS TEC H I MVI RSI LA Tahi.e 4. —Proportion of individuals heterozygous per locus per population f hP is the proportion of 24 loci polymorphic in each population (loci with commonest allele at a frequency of > 0.95 were considered monomorphic), II, mean proportion of loci heterozygous per individual, N = 20. 1j(K us Populations sampled lit mi i;. )ih ot JuK South Fork (.-uatkihipt I.DH-2 .0f» MDII-1 .05 JDH .15 .60 SDII .10 ADH .35 .10 PCI >< i .10 . 10 .50 PGM .25 G<) 1 -2 .20 .10 G-6-P .05 ctGPDH-1 .10 aGPDH-2 .15 .05 .10 .05 crGPDH-3 05 .10 EST-1 . 1.5 .10 .20 .15 ESI-7 .05 .05 P .160 .208 .35:1 .125 H .05 1 .065 .065 .056 Esterases (ESI).—A total of seven esterase systems were identi¬ fied, but only two could be scored with confidence. EST-1 con¬ tained two alleles found in all four populations. EST-7 also was composed of two alleles; EST-7 65 was found in one specimen from Beartrap and in one from 1th of July Campground. Of the five remaining esterases identified., EST-2 and EST-3 exhibited polymorphism in all populations. EST-4 and ESI-5 were poly¬ morphic in all populations except that from the Guadalupe Mountains, and EST-6 was monomorphic in all populations. Monomorphic proteins.— The following proteins were scored as monomorphic in all populations sampled: lactate dehydrogenase (LDH); malate dehydrogenase-2 (MDH-2); malate enzyme (ME); leucine arninopeptidase (LAP); albumin (AB); indolphenol oxi¬ dase (IPO); glutamate oxalate transaminase-1 (GOT-1); and glu¬ tamate dehydrogenase-1, -2, -3 (GDII-1, GDI 1-2, GDII-3). Genic variability .-^Table 1 gives the proportion of polymor¬ phic loci in each population, the proportion of heterozygous loci per individual, and the proportion of heterozygous individuals per locus per population. Genic heterozygosity (II) is based on the analysis of 24 loci (11 polymoprhic, 10 monomorphic). WIl HI ! M MICROTI'S MKXICANTS 21 I abt.k 5.— Coefficients of genic similarity (Rogers’ S), genic identity (Nei*s h, and genetii distance, (\'ei’.s D), respectively, for four populations of Mu \ ■ m iiy tm-xicanus. Bi-iiiliaiJ Prh of Juh Smith Hut; (•u adaiupi' l-4f .t r r r ij I 0000 0,9535 0.9401 0.9279 0.0000 0-9883 0,9847 0.9526 0.0000 0 0117 0,0155 0.0-186 1th of !ul\ ] .000(1 0.9572 0.91 12 0.0000 0.9933 0,9543 0.0000 0,0067 0.0468 N< mi h I ork i .0000 0.9119 0.0000 n 9566 0,0000 0.0444 GtKukuupr- 1.0000 0,0000 0.0000 1 he loci that contributed most to the heterozygosity values var ied among populations. Phosphoglucose isomerase {PGI} was a major contributor in all populations, whereas EST-1 was respon¬ sible for most of the individual heterozygosity in the Beartrap and 4th of July populations. Other loci contributing significant amounts of heterozygosity were PGM in Beartrap, ADH in 4th of July, GOT-2 in South Fork, and IDH m the Guadalupe sample. The Beartrap population exhibited four unique alleles (LDH- 2 7 \ MDH l ( \ PGM 160 , and aGPDII-f), whereas the 4th ol July {population possessed only two unique alleles (PGI’ and aGPDH-l 115 ). The remaining two populations had one unique allele each; South Fork (SDH 116 ) and Guadalupe (G-6-P 88 ). Genic similarity ,—Coefficients of genic similarity between {pop¬ ulations were calculated, using Rodgers’ similarity, S (Rodgers, l'J72) and Nei’s identity, / (Nei, 1972). Values for / are generally slightly higher than those for S lint both measures give compara¬ ble results. Both are reported in 'Fable 5. Electrophoretic studies generally show high levels of polymor¬ phism in natural populations, with reduced levels of heterozygos¬ ity in small, isolated populations. Selander et al. (1971) demon¬ strated low levels of genic variability (IT = 0.018) in insular {populations of Peromyscus polionotus compared to the larger mainland populations (H 0.054-0.088), Similar results were obtained for several species of Peromyscus (Avise et al., 19746), in w hich insular subspecies had an average ol less than one per cent heterozygous loci. Reduced levels of heterozygosity in small, iso¬ lated {populations arc thought t<> be due to genetii drift. 22 OCC ASIONAL PAPERS MUSEUM TEXAS LECH UNIVERSITY The level of genetic variability is relatively uniform for the four populations of Mexican voles studied, ranging from 0.054 to 0.065 (mean, 0.060). These values are consistent with heterozygosity values for other mainland vertebrate populations (II — 0.0T0.09) as reported by Selander and Johnson (1973). The proportion of the 24 loci examined that were polymorphic (P) ranged from 0.125 to 0.353 (mean. 0.213). This figure also agrees with the value of 0.202 that Selander (1976) listed as an average value for rodents. Rodgers’ coefficient of genic similarity (S) between the Guada¬ lupe sample and the other three populations studied is less than 0.93, whereas those between the other three all are more than 0.94. This relationship is more pronounced in Nei’s identity coefficient, where the values separating the Guadalupe sample and the remaining populations are less than 0.96; those between the other three are greater than 0.98. Coefficients of similarity generally range from 0.90 to 1.00 for conspecific populations of rodents, although in strongly divergent populations of Peromyscus polio- notus on Florida’s barrier islands, the average similarity value drops to 0.84 (Selander and Johnson, 1973). Other biochemical studies comparing subspecies have yielded Rodgers’ similarity values of 0.769 in Mus musculus (Selander et al., 1969), 0.89 to 0.95 in Peromyscus boylii, and 0.75 to 0.79 in P. polionotus (Avise et al., 1971a), and 0.86 in P. erernicus (Arise et al., 19745). The low values for Mus in reality could be due to the fact that the two subspecies studied are incipient species, whereas those reported for P. polionotus result from comparison of a sub¬ species occurring on Florida’s barrier islands to a mainland popu¬ lation. The values obtained in this study ranged from 0.9119 to 0.9572, and, because these are well within the range of values reported for other rodent subspecies, would seem to indicate that the populations examined are no more differentiated than would be expected on the basis of their current taxonomic rank. Unfor¬ tunately, no data from other microtines are available for compari- son. Electrophoretic analysis revealed a close association among all four populations studied, hut also indicated that there could he some slight differences between the Guadalupe population and the remaining three. Inconsistencies in the morphometric and electrophoretic anal¬ ysts result from two different approaches to the same problem and are to be expected. Electrophoretic analysis frequently fails to dis¬ tinguish between or among subspecies that were described WILHELM —MIt.ROTUS MEXICAN! S 23 initially on the basis of classical systematic criteria. Whether many subspecies are arbitrary units that do not reflect major gene differences or whether the resolution resulting from electropho¬ retic techniques is insufficient to detect the differences is not known at present, but both elements probably are involved (Avise, 1975). Contrasts between genic similarity and organismic similar¬ ity are not unknown, and it might be a general rule that orga¬ nismic evolution and stuctural gene evolution proceed at virtually independent rates, as suggested by Wilson (1976). Therefore, in this study, electrophoretic evidence was accorded less considera¬ tion than were other data in determining systematic placement of the foui populations. < 'Jironolo# \ Findley (1969) explained many of the present mammalian dis¬ tributional patterns in the southwestern United States on the basis of a series of boreal expansions and contractions in the late Pleis¬ tocene, the contractions leaving isolated boreal habitats and popu¬ lations of certain mammals on scattered mountain ranges. The Mexican vole was probably widespread over the Mexican and Colorado plateaus during the cooler, more mesic pluvial periods of the late Pleistocene. The aridity ot interpluvial periods caused fragmentation of the distribution of that vole, and with increasing aridity, those fragments became restricted to relatively mesic mountaintops (see also Smartt, 1977). The chronology of late Pleistocene events in the southwestern United States vvas estimated by Wendorf (.1975), based on pollen profiles from the Llano Estacado of West Texas. Evidence of a continuous boreal forest on the Llano as low as 1000 meters dur¬ ing the Early Tahoka Pluvial (17,000 BP) corresponds tn a depres¬ sion of vegetative zones of from 1300 to 1500 meters be low their current levels. Because intermontane altitudes in the area under study range from about 1250 to 2050 meters, the presence ot more or less continuous boreal forest there in the Early Tahoka seems probable. The Late Tahoka Pluvial, dated approximately 11.500 BP, evi¬ dently was the most recent period of extensive pine-spruce forest in this area (Wendorf, 1975). Ibis rather open forest covered much of the Southwest, as evidenced by the fact that the Llano Estacado was at least 50 per cent covered by spruce-pine forest (Wendorf, 1961). During this pluvial period, then, the Mexican vole was probably still widely distributed over Arizona, New Mex- Of CANIOXAI. PAPKRS MlSKl M i 1 \ AS Il( il l XU I-RSI ] \ ico, and western Texas. AI. mexicanus was present on the Llano Estacado at this time in extreme eastern New Mexico, at an ele\a- tion of 1350 meters in what is now Roosevelt County (Slaughter, 1975). Fragmentation in the distribution of Minot us mexicanus prob¬ ably began with the Scharbauer Interval (10,500 BP), which marked the end of the Late 1’ahoka Pluvial. This was a time of increasing aridity and was marked h\ a decline of the pine-spruce forest in the Southwest, although subsequent periods of resur¬ gence of cooler and more moist conditions could have allowed temporary expansion of boreal habitats. The boreal extensions, both in time and geographic scope, since the late Tahokan are not presently known, hut in all likelihood they were not of suffi¬ cient magnitude to connect previously isolated montane habitats. Fhe climate of the Southwest has been one ol increasing aridity since the Lubbock Subpluvial (9500 BP) and has undoubtedly resulted in the progressive geographic isolation of boreal elements on mountain tops. The subspecies of Microtus mexicanus in this study almost cer¬ tainly result from 9000 to 10,000 years of isolation (Slaughter placed the isolation of M. mexicanus at post-10,000 BP). The degree of differentiation observed between the two nominal taxa provides, therefore, some idea of the gross rate of evolution in this species, and, in a general way, underscores the relationship between geographic isolation and evolution in a small mammal. Relative divergence times (T) for paired combinations of all populations were calculated following Nei’s (1971a) technique using tlie expected number of amino acid differences (D) per pro¬ tein that can be detected by electrophoresis. Nei (19715) renamed the function D, calling it “genetic distance,” and later presented a detailed description (Nei, 1972). Genetic distance is defined as D - logj, where I is Nei’s identity value. The values of Nei’s genetic distance are given in Table 5. Relative divergence times were then estimated using the formula T - Di IT, where Di and IL are the genetic distance values for the population pairs in question, and are presented in Table 6. FJsing this table, it is possible to postu¬ late the order of divergence of the lour populations by listing the population pairs from one column in order of increasing diver¬ gence times. This suggests the following sequence of events. Isola¬ tion of the Guadalupe population (4) occurred first. It initially was separated from the Beartrap population (1), followed quickly by separation from the 4th of July (2) and South Fork (3) popula- YVII.HEI.M—MICRO i l S MEXICANI 'S 25 Tabi.e 6. —Matrix of relative divergence times (T). Parentheses indicate exceptions from the general order discussed in the text. Pairs of populations compared on both the ordinate and abassa are the same as coded in Pig. /. n 1,2 1,3 1.1 2.3 2,4 3,1 n 1,2 l.IJO 1,32 1.15 o V, l (Hi 3.79 1. i (C75 1 .(H) 3.14 0 Cl 3.02 2So 1.1 0 21 0,32 1 00 0 i 17 (0 HO) ( 0 'M 2,3 1.75 2,31 7.25 1.00 0.0S) (6,t>3| 2 A 0.25 (1.33 1.0 ; 0 l n 1 00 0.95 3,4 0.26 0-35 1.00 0.1 , 1 03 1.00 tions. The next population to be isolated was that at Bear nap. It was first separated from South Fork, then from 1th of Jul). The last separation isolated the 1th of July and the South Fork popu¬ lations. It can be seen from the geographic relationships of the four study areas (Fig. 1), that the isolation of Guadalupe from South Fork probably occurred at about the >aine time as separa¬ tion of the Guadalupe- 1th of July and of the Guadalupe-Beartrap populations. Inasmuch as the calculation of relative lime of divergence is intimately related to Nei’s identity value (I), it is not surprising that these values correlate perfectly with the hypothetical sequence (derived from observed divergence of characters) of isolation of the four populations studied. Ihe highest identity values are found between those populations theoretically in contact for the longest period of time. Rodgers’ similarity values (S) indicate the same situation, except that they suggest that the Guadalupe population was in contact with the Beartrap population for a slightly longer period than it was with either the South Fork or the 4th of July populations. Although the calculation of relative divergence times can offer only gross estimates of temporal isolation, the sequence of events indicated by these data agrees with what would be expected from examination of the geologic evidence. Other recent studies on mammals have shown that Nei’s evolutionary divergence time does correlate well with both fossil and morphological evidence (Nevo et al., 1974; Zimmerman et al., 1975; Kilpatrick and Zim¬ merman, 1976). As the Pleistocene glaciers retreated northward and the climate of the Southwest became warme r and drier, boreal taxa such as the Mexican vole were forced into isolation on mon¬ tane refugia. Such restrictions evidently first took place on the southernmost Guadalupe Mountains, was followed by isolation of OCCASIONAL IWPKR.N Ml'SKl'M I LX AS TECH I'NIVERSI'I'Y 2f> the Beartrap population west of the Rio Grande on the San Mateo Mountains, and ended with isolation of the 4th of July and South Fork populations on the Manzano and Sacramento mountains, respectively. It is significant to note that the population thought to have been isolated for the longest period of time is the one showing the greatest degree of genic and morphological differen¬ tiation. Results of this study indicate that Microtus mexicanus is an evolutionarily conservative microtlne. Only slight change evi¬ dently has taken place in isolated populations of the species over a time span in which several European microtines have developed well-marked karyotypic and morphologic differences. No other North American microtine has been the subject of a detailed sys¬ tematic investigation, however, and the Mexican vole could prove to be an exceptionally conservative species. 7 'axonomic Conclusions Subspecific differentiation of Microtus mexicanus in the region of study undoubtedly has occurred since the isolation of popula¬ tions on mountain ranges in the late Pleistocene. The current subspecific boundries were established b', Bailey (1932), with the Rio Grande designated as the line of demarcation between M. m. mogollonensis (occurring in the mountains to the west of the river) and M. m. guadalupensis (inhabiting montane areas to the east). This arrangement seems logical in a geographic sense, and places the Beartrap sample (San Mateo Mountains) in the subspe¬ cies mogollonensis and the remaining three samples (4th of July. Manzano Mountains; South Fork, Sacramento Mountains; and Guadalupe, Guadalupe Mountains) in the suhspet ies guadalupen- sis. Results of the sperm and morphometric analyses demonstrate, however, that the 4th of July sample bears a closer relationship to the Beartrap sample than it does to either of the other two popu¬ lations. Therefore, the 4th of July population is here transferred to the subspecies M. m. mogollonensis. The South Fork and Guadalupe populations remain referrable toM. m. guadalupensis. There exists, however, a certain degree of divergence between the Guadalupe and South Fork samples, as evidenced by both cranial characters and values of electrophoretic similarity. Consid¬ ering the isolated nature of these two populations and the degree of divergence exhibited, I conclude that they constitute an exam¬ ple of incipient subspeciation. The Guadalupe Mountains are W 11,1 i11 \J - MICRO 1 l s me \ K M s 27 lower in altitude mul considerably drier than are the other three mountain masses. This results in extremely restricted areas of hab¬ itat for Mexican voles and severely limits the population size. The Guadalupe sample thus represents a population with several of the attributes typically thought to contribute to evolutionary div¬ ergence. Geographic isolation, small population size, occupancy of a heterogenous habitat dividing this population into several smaller denie s, and a harsh or stringent environment relative to other conspecific populations, might be reflected in the degree of divergence found in the Guadalupe population of Microtus mexi- can us. , VCK N ()WLF !)f, M ENTS A portion of this research was carried out under National Park Service contract CX70004Q145 awarded to Dr. II. II. Genoways and Dr, R. J. Baker. Computer time, some expenses, and research costs were furnished by 1 he Museum and the Graduate Sc hool. Texas Tech University. I wish to thank the following individuals for the loan of, or opportunity to examine, specimens under their care: Dr. J. S. Findley, Museum of Southwestern Biology, Univei- sity of New Mexico: Dr. A. H. Harris, Laboratory for Environ¬ mental Biology. University of Texas at FI Paso; and Dr. 1). J. Selim idly, 1’exas Cooperative Wildlife Collection, Texas A&M University. Dr. R. J. Baker generously made laboratory facilities available and Dr. 1. Greenbaum provided technical advice on the electrophoretic portion of the study. I am particularly grateful to Dr. J. Knox Jones, Jr., for his advice and assistance throughout the course of this study. I would also like to express my apprecia¬ tion to my wife, Moira, who prepared the figures and aided in collecting, processing, and examining the specimens. Literature Cited Andkksos, S. 1954. Subsperiaiion in the meadow mouse, Microtus montanus, in Wvfmiing and Colorado, ['em. Kama* Pul>U Mils. Nat. liist.. 7:489- 506. -, I960. The bacutum in mitroiiric rodents. Inn. Kansas Pub)., Mas. Nat. Hist., 12:181-216 A vise, J. C. 1975. Systematic value of electrophoretic data. Syst. Zool., 23:465- 481 Avise, J. C.. M. II. .Smith, akd R. K. Selandek. J974. Biochemical polymor¬ phism and systematic* in the genus Peromy-scies. VI. I he boylii species group. ] Mamm., 55:751-763. 28 OCCASIONAL PAPERS Ml'SECM TEXAS I Till IMVERSI1 V Avi.sk, J. C., M. H. Smith, R. K. Selander, T. E. Lawlor, and P. R. Ram- sf.y. 19746. Biochemical polymorphism and systematic® in the genus PeTomyscu