2779 al8o1,

2779A1,89Al8o1,

BIOCHEMICAL GENETICS OF THE POCKET GOPHER

GENUS GEOMYS,AND ITS PHYLOGENETIC

IMPLICATIONS

DISSERTATION

Presented to the Graduate Council of the

North Texas State University in Partial

Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

By

Dan F. Penney, B. S., M. A.

Denton, Texas

December, 1974

Penney, Dan F., Biochemical Genetics of the Pocket Gopher

Genus GeoMys and its Phylogenetic Implications. Doctor of

Philosophy (Biology) December, 1974, 95 pp., 20 tables, 7

figures, literature cited, 55 titles.

Electrophoretic techniques were utilized for the demon-

stration of variation in 22 proteins from 24 natural populations

of four species ( G. bursarius, G. pinetis, G. arenarius and

G. personatus ) of the Geomys complex of pocket gophers.

Of the 24 structural loci , 19 were considered to be

polymorphic. Five of the six esterases contributed greatested

to the polymorphismwhile non-esterase proteins generally

showed low values. In the GeoMys complex of pocket gophers

in this study,selection appeared to be the most important

influence on genetic structure withsome evidence of random

drift in two of the four species.

Populations of G. arenarius and G. personatus had the

highest average interspecific genetic similarities to G.

bursarius and . pinetis was the most divergent. Biochemical

evidence supports the phylogeny of Geomys based on morphological

and fossil data.

LA PACIENCIA ES AMARGA,PERO SU FRUTO ES DULCE.

Rousseau

TABLE OF CONTENTS

PageLIST OF TABLES......................................... iv

LIST OF FIGURES..............* ....... **.*.*....* vi

Chapter

II. METHODS AND MATERIALS...................... 7

SamplesHorizontal Starch Gel Electrophoresis

Preparation of Tissue ExtractsElectrophoretic Apparatus and

TechniquesBuffer SystemsIdentification of Proteins

Treatment of Data

Electrophoretic ResultsScorable Proteins

Genetic VariabilityPolymorphic LociEffective Number of Alleles

IV. Ie IN...................76

Genetic VariationGenetic Similarity and Systematics

LITERATURE CITED....................................... 91

iii

LIST OF TABLES

Table Page

1 Collecting localities and sample sizes for24 populations of four species ofGeoms............. ............... 8

2 Allelic frequencies at the hemoglobin andesterase A loci for 24 populationsof Geomys......................... ... 27

3 Allelic frequencies at the albumin locusfor 24 populations of Geomys............ 31

4 Allelic frequencies at the transferrinlocus for 24 populations of Geomys...... 33

5 Allelic frequencies at the glutamateoxaloacetic transaminase locifor 24 populations of Geomys............ 36

6 Allelic frequencies at the oc-glycerophos-phate dehydrogenase locus for 24populations of Geomys................... 39

7 Allelic frequencies at the 6-phosphogluco-nate dehydrogenase locus for 24populations of Geo ys.................. 41

8 Allelic frequencies at the isocitratedehydrogenase locus for 24populations of Goy..........44

9 Allelic frequencies at the malate dehydro-genase loci for 24 populations of

_ . . .. . . . . .. ....... *.. 47

10 Allelic frequencies at the lactate dehydro-genase locus for 24 populations ofGeomy 50

11 Allelic frequencies at the lactate dehydro-genase locus for 24 populations of

G y............................... 54

iv

12 Allelic frequencies at the esterase-llocus for 24 populations ofGeomys..........*......****. ........... 56

13 Allelic frequencies at the esterase-3 and4 loci for 24 populations of Geos.... 59

14 Allelic frequencies at the esterase-5locus for 24 populations of Geomys..... 63

15 Allelic frequencies at the esterase-6 and8 locus for 24 populations of Geo ys... 65

16 Proportion of loci polymorphic per popula-tion (P), proportion of loci poly-morphic per individual (H ) in 24populations and four species ofGe . .67

17 Polymorphic proteins with proportion ofpopulation polymorphic for eachlocus and the effective number ofalleles at each locus for 24 popu-lations and four species of Geomys..... 68

18 Common or fixed allelic comparisons ofproteins among G. bursarius andG. pinetis. ....... ........ 72

19 Common or fixed allelic comparisons ofproteins among G. bursarius, G.arenarius, and G. personatus........... 72

20 Coefficients of genetic distance and simi-larity S below and I above for 24populations of Geomys.................. 84

v

PageTable

LIST OF FIGURES

Figure Page

1. Collecting localities for G. bursarius,G. personatus and G. arenarius ............. 11

2. Collecting localities for G. pinetis........ 12

3. Electrophoretic variation in albuminsand transferrins in pocket gophers ofthe genus Geo3ys............................ 50

4. Electrophoretic variation in 6-phospho-gluconate dehydrogenase and esterase-lin pocket gophers of the genus Geomys....... 53

5. Electrophoretic variation in esterases-A,3, 4, 5 and 8 in pocket gophers of thegenus Geomys .......................... 61

6. Dendrogram based on Roger's measure ofgenetic similarity for 24 populationsof Geomys----.................... *.. *...... 87

7. Dendrogram of genetic similarity basedon Nei's measure of genetic distancefor 24 populations of Geomys................ 88

vi

CHAPTER I

INTRODUCTION

Throughout its range, the pocket gopher genus GeoMys

exhibits a discontinuous distribution, associated with

edaphic conditions. The five extant species occur primar-

ily in sandy soils and are rarely if ever found in alluvial

silt, clay, or gravelly-slits. Therefore, numerous popula-

tions are more or less isolated on "islands" of sandy soils

surrounded by clay or silt deposits (Davis, 1940). This

frequently leads to an island-model type of distribution,

as defined by Wright (1943), that offers good potential

for study of intrapopulational variation. The fact that

seven genera and more than 300 kinds of pocket gophers of

the family Geomyidae have been formally named (Hall and

Kelson, 1959) reflects the great variety resulting from

this isolation.

At present, four species of the Geomys complex of

pocket gophers inhabit a large area of the Central and

Southeastern United States, while one species, G. tropi-

calis, is found only in a small area in southern Tamaulipas,

Mexico (Hall and Kelson, 1959). The ranges of the five

species reflect past distribution and isolation. Geomys

bursarius has the most extensive rangefrom the western

1

2

great plains, south to the Texas coast, and north into

Canada. Geomys personatus is restricted to the Texas Rio

Grande valley north of the Rio Grande River and is contig-

uous with G. bursarius from Corpus Christi to the San

Antonio River. Geomys arenarius occurs in extreme western

Texas and south central New Mexico, and G. pinetis is found

in Alabama, Georgia, and Florida. The last two species

occur as disjunct populations (Hall and Kelson, 1959).

The evolution of the genus Geomys is well-documented.

Remains of GeoMys are abundant from the Pleistocene soil

deposits of the Great Plains. Hibbard (1949) recorded the

genus from all five Pleistocene faunas studied in SW Kansas,

including two that were interpreted as having survived

under cold climatic conditions. Study of cave deposits by

Delquest and Kilpatrick (1973) indicated that the range of

G. bursarius extended over the Edwards Plateau of West

Central Texas during the period 10,000 years to 4,000 years

B.P., and probably later. The only remaining G. bursarius

populations of this region are in Mason and Llano counties.

Geomys personatus and G. arenarius probably diverged

from the southern and western portions of populations of

G. bursarius during Wisconsin or Recent time. Geomys

pinetis and G. bursarius were separated and diverged from

the ancestral stock during the mid-Illinoian glacial

period (Russell, 1968), with post-glacial habitats of the

3

Mississippi valley preventing reestablishment of the forms

in that region.

Morphological variation in pocket gophers is wide-

spreaddue to their island-model type distribution. It

was used in most of the early taxonomic studies of Geo s

(Davis, 1940; Merriam, 1895) and is still a useful tool for

species analysis (Anderson, 1966; Durrant, 1946; Russell,

1968). Russell (1968) used morphological characters of

fossil and extant forms to characterize the phylogenetic

evolution of the genusand regarded Geoys as one of the

most primitive of the living genera of gophers. However,

the genetic basis for most morphological features is poorly

understood, and it has become increasingly obvious in

recent years that newer techniques for studying genetic

variation should be utilized.

Cytologists have recently demonstrated the importance

of karyology as a tool for the study of mammalian taxonomy

and evolution (Matthey, 1964; Patton, 1967; Thaeler, 1968;

1972; Zimmerman, 1970). Valuable information regarding

trends within various taxa can be obtained when extensive

investigations are made. The genus Geomys has been studied

karyologically by Baker (1973), Davis et al. (1971), Hart

(1971),and Kim (1972), and Kim extended this investigation

to biochemical genetics of two of the five species of

Geomys.

4

Cytology and morphology offer some insight into

genetic variation in natural populations of mammals. The

latter utilizes gross morphological features controlled by

polygenic effects, while chromosome studies are limited to

numerical differences between taxa. The result has been

that the effect of individual genes could not be determined,

and that extent to which these genes vary in different popula-

tions has gone undetected.

Most of the inadequacies of morphological and cyto-

logical techniques can be overcome by ntiazingrebent

developments in the use of electrophoresis. The basis for

the use of electrophoresis in genetic studies is dependent

upon the net electrostatic charge of proteins and, to some

extent, their size and configuration. These properties

affect their mobility in an electric field. The charge

differences are the result of substitutions, deletions, or

additions in the amino acid sequences of the constituent

polypeptides of a protein, and these charge differences

represent mutations in the nucleotide sequence of the

structural genes.

The procedure utilizes small amounts of crude tissue

extracts applied to a slot in a starch gel, and an electric

potential is set up across the gel. After two or three

hours the gel is immersed in an assay solution of a general

protein stain or a specific enzyme substrate. The staining

5

procedure results in a band marking the location of the

migrated protein.

The advantage of the technique is that variation in

the banding pattern can be directly equated to the genetic

variation of the protein under investigation. These varia-

tions can be analyzed and utilized to explain observed

differences in populations. One explanation for observed

differences may be that the alleles are adaptively neutral,

and their variation is the product of random events

(genetic drift). Genetic drift has been suggested for the

lack of heterozygosity in at least two cases including cave

fish of the genus Astyanax (Avise and Selander e al.,

1971) and island populations of mice, genus Peromyscus

(Selander et al., 1971). A second explanation would be

that the variation is maintained by natural selection.

Selection for observed protein differences in mammals has

been documented by Jensen (1970) and Selander et al. (1971).

These studies represent pioneer investigations and should

further our knowledge of the phenomena responsible for the

genetic variability and evolutionary mechanisms of natural

populations.

The purposes of this study, through the use of starch -

gel electrophoresis were (1) to obtain estimates of the

degree of heterozygosity and variation in populations of

four species of Geoys, G. bursarius, G. pinetis,

6

oG. arenarius and G. personatus, (2) to assess the system-

atic relationships between the four species, (3) to compare

the genetic similarities and differences among and between

populations of the four species, and (4) to develop the

phylogenetic trends and relationships within the genus.

CHAPTER II

METHODS AND MATERIALS

Samples

Pocket gophers were collected from 24 naturally occur-

ring populations (Table 1) in Oklahoma, Texas, and Florida

(Figs. 1 and 2) using Victor-type jaw traps and live-traps

developed by Baker (1972). The animals were maintained in

the laboratory until blood samples could be taken and

prepared. Representative specimens were deposited in the

mammal collection of North Texas State University.

Two Geomys bursarius from Aubrey, Denton Co., Texas

were used as control animals. These animals had identical

blood protein banding patterns and were alternately bled

and run on gels with each population sample. The standard

banding pattern was then compared with the population

sample to assess the fast and slow migrating bands. By

placing a standard on both ends of the samples, variation

due to distortion in the gels could be detected. The 22

proteins utilized in this study allowed comparisons with

polymorphic proteins found in other fossorial rodents

(Kim, 1972; Nevo et al., 1974; Patton et al., 1972;

Selander et al., 1971).

7

8

Table 1, Collecting localities and sample sizesfor 24 populations of four species ofGeomy

Population Locality Number ofnumber individuals

Geomys bursarius

1 4 mi. S. Huntsville, Hwy. 75,Walker Co., Texas

2 8 mi. E. Denton, Y mi. W. Aubrey,Denton Co., Texas 15

6 mi. E. Arthur City, on farm 7road, Lamar Co., Texas

4 35 mii N. Cushing, Hwy. 225, Rusk 12Co., Texas

3 mi. S. Centerville, IH 45, Leon5 Co., Texas. 2 mi. N. Buffalo, IH 10

45, Freestone Co., Texas 2

3 mi. S.E. Thackerville, on farm6 road, and 5 mi. S. Thackerville, 8IH 35, Love Co., Oklahoma 7

7 8 mi. N. Stratford, Hwy. 177,Garvin Co., Oklahoma

8 2-18 mi. N. Mason, Hwy. 87, Mason 8Co., Texas

9 6 mi. N.E. Bastrop, Hwy. 21, 10Bastrop Co., Texas

10 4 mi. S.W. Weatherford, IH 20, 4Parker Co., Texas

11 4 mi. N. Burkburnett, on farm road,Wichita Co., Texas

12 3 mi. N. Decatur, Hwy. 81, Wise Co.,Texas

9

Table 1 --Continued


Geomys pinetis

Ya mi. N. Old Town, Hwy. 349, Levy 11Co., Florida. 3 mi. E. Homasassa,

13 Hwy. 490, Citrus Co., Florida. 4 1mi. W. Lake City, Hwy. 90,Columbia Co., Florida 1

14 4 mi. N. Haines City, Hwy. 27, PolkCo., Florida

3 mi. E. Mossyhead, Hwy. 90, Walton 1Co., Florida. 3$ mi. N. Washington

15 and Bay County line, Washington Co., 3Florida. Hwy 79, between Warsauand Greenhead, Hwy. 77, WashingtonCo., Florida 4

16 5 mi. N., 1-5 mi. E. Youngstown, 4Hwy. 20, Bay Co., Florida

Geomys arenarius

2 mi. W.N.W. Ft. Handcock, Hudspeth17 Co., Texas. 2 mi. S.S.W. Tornillo, 3

El Paso Co., Texas 10

10-12 mi. N.W. El Paso, W. bank18 Rio Grande River, El Paso Co., 13

Texas,

2 mi. E. Sunland Park, W. bank Rio19 Grande River, Dona Ana Co., New 5

Mexico

Geomys personatus

20 6 mi. N. Beeville, Hwy. 181, Bee Co.,Texas

10

Table 1 -- Continued


21 Park Road 53, Mustang Island, 7Nueces Co., Texas

22 East end Padre Island, Nueces 7Co., Texas

10 mi. N. Raymondville, Hwy. 77,23 Willacy Co., Texas. 11 mi. S. 2

Sarita, Why. 77, Kenedy Co.,Texas

24 2-6 mi. W. Falfurrias, Hwy. 285, 10Brooks Co., Texas

11

Fig. 1, Collecting localities for G. bursarius,G. personatus and G. arenarius-

70

120 02

010 0

0801

09

020

2224o

230

12

Tig. 2, Collecting localities for G. pinetis

0150 o 16 3

0 1

o13

o 14

13

Horizontal Starch Gel Electrophoresis

Preparation of Tissue Extracts

Blood was obtained by inserting a 1.0 x 100-mm capil-

lary tube into the suborbital canthal sinus. For serum

samples, approximately two capillary tubes of blood were

collected in a dry 6 x 50-mm culture tube and allowed to

clot for approximately 20 minutes. The samples were then

centrifuged at 1000 g for 10 minutes. Serum samples were

placed in a clean culture tube and were either used immedi-

ately or stored at - 2 0 O0 until used.

Transferrin samples were prepared by removing one

drop of the serum and placing it in a clean 10 x 75-mm

culture tube containing one drop of a 0.15% ferric

chloride solution. Four drops of a 0.6% rivanol solution

(2-ethoxy-6,9-diaminoacridine lactate) were added, the

solution was thoroughly mixed by shaking, and the precip-

itate was centrifuged at 1000 g for 3 minutes (Chen and

Sutton, 1967). This process removed most of the proteins

other than transferrin (Sutton and Karp, 1965). Electro-

phoresis of the clear yellow supernatant solution was

completed within 6 hours of sample preparation.

The remaining serum was diluted with an equal volume

of buffered saline solution (0.08 M dibasic sodium phos-

phate, 0.04 M manobasic potassium phosphate, pH 4.4,

14.45 g sodium chloride per 1700 ml of deionized water,

Jensen, 1970). Albumins and plasma esterases were run

from dilute serum samples.

For hemoglobin and erythrocyte protein samples,

approximately one capillary tube of blood was collected in

a 10 x 75-mm test tube containing 0.5 ml of 4% sodium

citrate solution. Samples were centrifuged at 1000 g for

three minutes. Erythrocytes were washed two times in

buffered saline solution and hemolysed by the addition of

three to four drops of deionized water. Hemolysed

erythrocyte preparations were then centrifuged at 1000 g

for 10 minutes. Hemoglobin samples were run within 5hours of bleeding. Storage, even at low temperature, has

been reported to result in denaturing of hemoglobin

(Jensen, 1970; Selander et al., 1969).

Tissue extracts were prepared by hemogenizing samples

of liver, kidney and testis in two volumes of buffer

(0.1 M tris pH 7.0, 0.001 M EDTA) for approximately 1

minute in a cooled 7-ml glass tissue grinder (Selander

et a., 1971). Extracts were centrifuged at 10,000 rpm at

0 C for 30 minutes. The supernatant solution was removed

and used immediately, or stored at -20 C for as long as a

month.

Organs not hemogenized immediately after the death of

the animal were dissected and frozen in two volumes of

15

homogenizing buffer. Selander et al. (1971) reported that

most proteins ramain undenatured in intact organs frozen

for weeks or months at -20P , but in solution the activity

of many enzymes was soon lost at this temperature.

Electrophoretic Apparatus and Techniques

Horizontal starch gel electrophoresis (Smithies, 1955)

was used to fractionate all samples. Gel molds were modi-

fied from those described by Kristjansson (1963). The

mold consisted of a glass plate (152 mm x 220 mm x 6 mm)

and four plexiglass strips, two 6 mm x 19mm x 196 mm and

two 6 mm x 19mm x 114 mm. The plexiglass strips were held

in place with petroleum jelly. When the liquid gel was

poured into the mold, a plexiglass plate (152 mm x 220 mm x

6mm) was used to cover the mold.

All gels were prepared from a 12% suspension of hydro-

lysed starch (Connaught National Laboratories, University

of Toronto, Toronto, Canada). Suspended starch was poured

into the buffer, heated to boiling in a 1,000 ml round-

bottom flask, and shaken vigorously for 1 minute. The

mixture was then degassed with an aspirator for approxi-

mately 1 minute. After degassing, the clear liquid gel

was poured into the mold, covered with a plexiglass plate,

and allowed to cool at room temperature for a minimum of

90 minutes.

16

After the gels had cooled the plexiglass plate and

long plexiglass strips were removed. Gels were cut parallel

to and 2.0 cm from one of the short sides of the gel to form

an insertion line, and the smaller portion of the gel was

gently pushed back. All samples were absorbed into no. 3filter paper (4 mm x 5 mm),with the exception of albumin

and plasma esterase samples,which were absorbed into no. 1

filter paper. Filter paper inserts were blotted and placed

approximately 3 mm apart against the exposed cut surface of

the smaller portion of the cut gel. After the samples were

placed on the gel, the smaller portion was carefully pushed

back in contact with the larger portion of the gel.

Saran Wrap (Dow Chemical Company) was used to cover

the surface of the gel during electrophoresis (Kristjansson,

1963). The edges of the Saran Wrap were folded back to

expose approximately 1.7 cm of the gel, to allow contact

with the bridge from the electrode chambers of the electro-

phoresis apparatus.

The electrophoresis chamber consisted of a plexiglass

tray (405 mm x 360 mm x 88 mm) which was divided into three

compartments. The central compartment (157 mm x 405 mm x

57 mm) was filled with ice to cool the gel during electro-phoresis. The two outer electrode chambers (405 mm x 101 mm)

each contained a 304-mm no. 22 platinum wire. The gel was

supported on a glass plate placed across the central com-

partment and sponge cloths (203 mm x 139 mm x 6 mm) were

17

used as bridges between the gel and the electrode buffer.

A glass plate was placed on top of sponge cloth bridges to

hold them flat in contact with the gel. Power was supplied

by either a Gelman Electrophoresis Power Supply Model 38206

or a Heathkit lPl7 H.V. Power Supply. All electrophoresis

was completed in a controlled temperature chamber between

00 and LC.

Buffer Systems

Seven buffer systems were used to separate the various

proteins examined in this study. Hemoglobins and ester-

ase-1 (erythrocyte esterase) were separated in a

discontinuous buffer system consisting of a 0.01 M tris-

hydrochloric acid gel buffer, pH 8.5, and an electrode

buffer of 0.3 M sodium borate pH 8.2 (Selander et al.,

1971). Most efficient separation was obtained at 25 ma

with voltage not exceeding 250 v. Two other erythrocyte

esterases and 6-phosphogluconate dehydrogenase were

separated from the hemolysed in a continuous buffer system

consisting of an electrode buffer of 0.1 M tris- 0.1 M

maleic acid- 0.01 M EDTA -0.01 M magnesium chloride, pH 7.4

and a gel of a 1:9 dilution of the electrode buffer of the

same pH (Selander et al., 1971). A potential of 100 v was

applied for 5 hours to provide sufficient separation.

Most serum proteins (transferrins and esterases) and

liver and kidney esterases were separated in a system

18

consisting of a gel of a 1:9 mixture of stock solutions A

and B as follows: Stock solution A was a 0.03 M lithium

hydroxide- 0.19 M boric acid, pH 8.1, and stock solution B

was a 0.05 M tris- 8 mM citric acid, pH 8.4. The electrode

buffer consisted of stock solution A (Selander et al.,

1971). Optimum separation was obtained at 25 ma per gel

with the voltage not exceeding 350 v for a period of 2.5

hours. Albumins were separated from the sera in a dis-

continuous system consisting of a tris-citrate gel (0.004 M

citric acid), pH 6.0. The electrode buffer was a 0.3 M

sodium borate solution, pH 6.5 (Jensen and Rasmussen, 1971).

Optimum separation was obtained at 25 ma per gel with a

maximum potential of 300 v until the borate boundary had

migrated 8 cm from the origin (approximately 3 hours).

Glutamate oxaloacetic transaminase was separated from

liver or kidney extracts in a continuous buffer system

consisting of a gel buffer of 22.89 mM tris- 5.22 mM citric

acid, pH 8.0, and an electrode buffer of 0.687 M tris-

0.157 M citric acid, pH 8.0 (Selander et al., 1971). A

potential of 100 v was applied for 4 hours for sufficient

separation.

Lactate dehydrogenase was separated from kidney and

testicular extracts in a discontinuous buffer system con-

sisting of a gel buffer of 0.076 M tris- 0.005 M citric

acid, pH 8.7 and an electrode buffer of 0.3 sodium borate,

19

pH 8.2 (Selander et al., 1971). Sufficient separation was

obtained at 25 ma per gel with a maximum potential of

250 v for 3 hours.

Malate dehydrogenase and Isocitrate dehydrogenase

were separated from kidney extracts in a continuous buffer

system consisting of a gel buffer of 8 mM tris - 3 mM

citric acid, pH 6.7 and an electrode buffer of 0.223 M

tris - 0.086 M citric acid, pH 6.3 (Selander et al., 1971).

A potential of 170 v was applied for 3 hours to produce

desired separation.

After electrophoresis, gels were allowed to cool for

a few minutes and sliced in 2-mm horizontal sheets with a

0.2-mm wire stretched tightly across a frame. The two

halves were then separated onto two glass plates.

Identification of Proteins

Hemoglobins, albumins and transferrins were stained

with a general protein stain of 2% solution of buffalo

black NBR (naphthol blue black) for 20 minutes in a 5:5:1

mixture of methanol, deionized water, and glacial acetic

acid. The gels were destained and fixed with repeated

washes in 5:5:1 mixture of methanol, deionized water, and

glacial acetic acid.

Enzymes were identified by using specific biochemical

staining techniques. Glutamate oxaloacetic transaminase

was identified by the staining technique of DeLorenzo and

20

Ruddle (1970),consisting of a stain of 50 ml 0.2 M tris

hydrochloric acid buffer, pH 8.0; 0.5 mg pyridoxal- 5'-

phosphate; 200 mg o( -aspartic acid; 100 mg o(-ketoglutaric

acid; and 150 mg Fast Blue RR Salt. Stain was prepared

prior to use each time and gels were stained in the dark for

30 minutes at 3700.

Isocitrate dehydrogenase produced two forms, IDH-1

(supernatant) and IDH-2 (mitochondrial). Staining tech-

nique by Selander et al. (1971) was used; 50 ml 0.2M tris-

hydrochloric acid buffer, pH 8.0, 0.2 ml 0.25M Manganese

chloride, 3 ml 0.1 trisodium DL-isocitric acid, 10 mg NADP,

5 mg NBT and 7mg PMS. Incubate for 30-60 minutes, 3700.

LDH was inhibited on these gels by Isobutyramide (4.08 g

isobutyramide, 250 ml deionized water, adjust to pH 7.5

with NaOH solution. Add I ml isobutyramide solution per

10 ml stain).

Lactate dehydrogenase was detected by a technique

modified from Markert and Massaro (1966). The stain con-

sisted of 30 ml of deionized water, 20 ml 0.2 M tris-

hydrochloric acid buffer (pH 8.0), 9 Ml 0.5 M sodium

DL-lactate, 20 mg P -nicotinamide adenine dinucleotide,10 mg MTT tetrazolium, and 8 mg phenazine methosulfate.

Gels were stained in the dark at 37 0 for 1 to 2 hours.

Malate dehydrogenase activity was detected by a

technique modified from Shows and Ruddle (1968),using a

21

stain consisting of 30 ml 0.2 M tris-hydrochloric acid

buffer, pH 8.0, 5 ml 2.0 M malate solution (pH adjusted to

7.0 with 1.0 M sodium hydroxide), 10 mg . -nicotinamide

adenine dinucleotide, 20 mg MTT tetrazolium, and 5 mg

phenazine methosulfate. The gels were stained in the dark

at 37 C for 1 hour. LDH was inhibited on these gels with

isobutyramide.

c(.- Glycerophosphate dehydrogenase was detected with

stain technique by Selander et al. (1971). Fifty ml 0.2M

tris-bydrochloric acid buffer pH 8.0, 1 ml 0.IM magnesium

chloride, 50 mg disodium c(-DL-glycerophosphate, 20 mg NAD,

13 mg NBT, and 4 mg PMS. Incubate in dark for 1 to 2 hours.

LDH was inhibited on these gels by isobutyramide.

The enzyme 6-phosphogluconate dehydrogenase was

detected by staining technique of Carter et a. (1968).

The stain consisted of 7 ml 0.2 M tris-hydrochloric acid

buffer, pH 8.0, 7 ml 0.1 M magnesium chloride, 3 ml

demonized water, 20 mg barium-6-phosphogluconic acid, 1 mg

nicotinamide adenine dinucleotide phosphate, 4 mg MTT

tetrozolium, and I mg phenazine methosulfate. Gels were

stained in the dark at 200C for 1 hour.

Esterases were detected by and coded according to the

methods outlined by Selander et al. (1971). Esterases in

the sera and kidney extracts were stained with a mixture

of 1 ml 0.2 M monobasic sodium phosphate, pH 4.4, 1 ml 0.2 M

22

dibasic sodium phosphate, pH 8.7, 47 ml deionized water,

25 mg Fast Blue RR Salt, 1 ml of a solution of 0.1 g

CC-naphthyl propionate (kidney extracts) or OC-naphthyl

butyrate (serum) in 10 ml of acetone. Gels were stained

at 3/C in the dark for 10 to 30 minutes. Some esterases

of kidney and liver extracts were inhibited by the use of

0.001 M eserinewhich allowed identification of the non-

inhibited esterases. Gels were preincubated at room

temperature for 20 minutes prior to staining at 3700 in

substrate solution.

Erythrocyte esterases were stained with 24 ml 0.2 M

monobasic sodium phosphate, pH 4.4, 6 ml 0.2 M dibasic

sodium phosphate, pH 8.7, 20 ml deionized water, 25 mg

Fast Garnet GBO Salt, and 1 ml of a solution of 0.1 g

oC-naphthyl propionate in 10. ml of acetone. Staining was

accomplished at 37C in the dark for a period of 1 to 2

hours. All gels were fixed in the 5:5:1 methanol, deionized

water, glacial acetic acid solution for 24 hours, scored or

photographed, and wrapped in a clear plastic film for

storage.

Treatment of Data

Since heterozygous individuals are identifiable,

electrophoretic data allowed direct calculation of allelic

frequencies. Gene frequencies were calculated by summing

the occurrence of a given allele for all loci at which

25

it occurs and dividing by the number of loci at which the

allele could occur, times two.

The systematic relationships of populations of G.

bursarius, G. personatus, G. arenarius and G. pinetis were

analyzed on the basis of genetic similarity and differences

of the 24 loci examined. Roger's (1972) coefficient of

genetic similarity (S) was utilized for comparison of popu-

lations on the basis of gene frequencies of the populations.

Genetic similarity (I), distance (D), and time (T) were

calculated for paired combinations of all populations,

using Nei's coefficient (1971). From these analyses, genetic

similarity dendrograms for 24 populations were drawn and

correlated with time.

The genetic similarity (S) between two populations is

calculated by summing the probabilities of drawing identi-

cal genotypes from the two populations for each genotype

of the locus, divided by the sum of the probabilities of

drawing identical genotypes from the same population on

two successive independent draws from each genotype of the

locus, as shown below:

L A

S( = 1 -1(P PL ijx ~Pijy

i=1 j=1

24

where L is the number of loci, A is the number of alleles

at the ith locusand P.. and P.. are the frequencies of

the jth allele at the ith locus in populations x and y,

respectively. From a matrix of coefficients of genetic

similarity for populations of Geomys, cluster analysis was

performed by the weighted pair group method of Sokal and

Sneath (1963).

Genetic similarity is also calculated for paired com-

binations for all populations sampled,using the coefficients

of Nei (1971). The normalized identity of genes between

two populations designated as x and y with respect to each

locus is defined as:

I. =

where 0. is the probability of identity of two randomly

chosen genes in population x and jy is the probability of

identity of randomly chosen genes in population y. Nei's

similarity measure has the added advantage of permitting

the calculation of an expected divergence time for popula-

tions based on biochemical data,as defined below:

D

t =

(2cnt a)

where D is the geometric mean of genetic similarity

(- loge I), c is the proportion of amino acid substitutions

25

which can be detected by electrophoresis% is the rate

of amino acid substitutions per polypeptide per site per

year, and nt is the total number of amino acids (codons)

concerned with synthesis of a protein.

Levels of heterzygosity provide estimates of genetic

variability within populations. Since proportion of poly-

morphic loci is strongly dependent upon sample size and is

therefore a poor index of the degree of genetic variation

within populations, Lewontin and Hubby (1966) proposed an

index utilizing the proportion of loci heterozygous per

individual. This estimate is calculated by summing the

observed frequencies of heterozygotes at each locus and

then averaging over all loci for each population separate-

Effective number of alleles was calculated as the

reciprocal of the sum of squares of allelic frequencies

for a given locus for each population.

CHAPTER III

RESULTS

Electrophoretic Results

Twenty two proteins were identified from 200 animals

representing 24 populations of the GeoMys complex of pocket

gophers. These samples were represented by 105 G. bursar-

ius, 30 G. pinetis, 31 G. arenarius, and 34 G. personatus,

from which 19 proteins were considered polymorphic. Hemo-

globin (Table 2) and plasma esterase A (Table 2) were

monomorphic, but were not fixed for the same allele in all

species. Esterase-6 was monomorphic in all individuals

except one from Love Co., Oklahoma, where a mutant or rare

allele was found and was not considered indicative of

polymorphism. Indole phenol oxidase was also monomorphic

for the same allele for all specimens examined. Of the 19

proteins considered polymorphic in this study (Table 17),

18 were polymorphic in G. bursarius, 12 in G. pinetis, 11

in G. arenarius, and 12 in G. personatus.

No evidence was found to suggest the linkage of the

24 structural loci encoding the 22 proteins examined with

genetic material of the X chromosome. Both males and

females were found to be heterozygous for all loci for

which heterozygous types were observed.

26

27

Table 2, Allelic frequencies at the hemoglobinand Osterase-A loci for 24 populationsof Geomys

Population Number of Hemoglobin Esterase-wAindividuals a b a b

Geomys bursdrius

1 Walker Co., Texas

2 Denton Co., Texas

3 Lamar Co., Texas

4 Rusk Co., Texas

5 Leon Co., Texas

6 Love Co., Oklahoma

7 Garvin Co., Oklahoma

8 Mason Co., Texas

9 Bastrop Co., Texas

10 Parker Co., Texas

11 Wichita Co., Texas

12 Wise Co., Texas

Geoys inetis

13 Levy Co., Florida

14 Polk Co., Florida

15 Washington Co., Florida

16 Bay Co., Florida

10

15

7

12

12

15

4

8

10

4

3

5

13

5

8

4

-- 1.00

-- 1.00

-o 1.00

am o1.00

--o 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

--m 1.00

-- 1.00

1.00

1.00

1.00

1*00

-- 1.00

--f 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

--o 1.00

-- n 1.00

-_ 1.00

-o 1,00

-- 1.00

-- 1.00

-- 1.00

am O 1.00

-- 1.00

-- 1.00

28

Table 2 --Continued

Population.Number of Hemoglobin Esterase-Aindividuals a b a b

Geom arenarius

17 south El Paso Co., Texas 13

18 north El Paso Co., Texas 13

19 Dona Ana Co., New Mexico 5

-- 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

Geo ys personatus

20 Bee Co., Texas

21 Mustang Island, Texas

22 Padre Island, Texas

23 Willacy-Kenedy Co., Texas

24 Brooks Co., Texas

5

7

7

5

10

-- 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

1.00

1.00

1.00

1.00

1.00

29

Scorable Proteins

Albumin (plasma). Four albumin alleles were demon-

strated segregating at a single locus on tris-citrate gels

(Fig. 3). Kim (1972) found only two alleles in Geomys

with polymorphism only in G. bursarius. Geomys bursarius

had the b allele fixed in most populations, with poly

morphism for the a and b alleles in populations 1 (Walker

Co., Texas) and 12 (Wise Co., Texas). Most populations of

_G. pinetis were fixed for the C allele, and only population

14 (Polk Co., Florida) demonstrated polymorphism for the

c and d alleles (Table 3). Geonys arenarius was monomorphic

for the a allele, whereas populations of G. personatus were

generally fixed for the b allele, with the exception of

populations 22 (Padre Island, Texas) and 23 (Willacy and

Kenedy Co., Texas),which were polymorphic for the b and c

alleles.

Transferrin (plasma). Three transferrin alleles,

segregating at a single locuswere demonstrated on lithium

hydroxide gels (Fig. 3). Polymorphism was found in all

four species of Geomys in this study (Table 4). Five popu-

lations of G. bursarius demonstrated polymorphis, whereas

G. pinetis was polymorphic for the b and c alleles, while

in G. personatus, only populations 22 (Padre Island, Texas)

and 24 (Brooks Co., Texas) were polymorphic.

30

Fig. 3, Electrophoretic variation in albumins andtransferrins in pocket gophers of thegenus Geonys

+

0

ALB AA BB CC DD AB BC CD

+

ft

AA BB CC AB BCTRF

31

Table 3, Allelic frequencies at the albuminlocus for 24 populations of Geomys

Population .Number of Albuminindividuals a b c d

Geom s bursarius

1 Walker Co., Texas

2 Denton Co., Texas

3 Lamar Co., Texas

4 Rusk Co., Texas

5 Leon Co., Texas



8 Mason Co., Texas




12 Wise Co., Texas

Geomys pinetis




16 Bay Co., Florida

10

15

7

12

12

15

4

8

10

4

3

5

0*05

-- o

--om

--

-- f

--om

--

--

am-a

--

--

0.30

13

5

8

4

0.95

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1. 00

1.00

1.00

0.70

1OO --

0.60 0.40

1.00 --

1.00 --

--w

--40

-- w

-- ow

--M

-- m

--

-- vi

-- m

--u

--mo

--m~

--q

--af

--o

32

Table 3 --Continued

Population Number of Albuminindividuals a b c d

Geomys arenarius

17 south El Paso Co., Texas

18 north El Paso Co., Texas

19 Dona Ana Co., New Mexico

Geomys personatus

20 Bee Co., Texas





13

13

5

5

7

7

5

10

1.00

1.00

1.00

-- 1.00 --

-- 1.00 --

-- 0.71 0.29

-- 0.40 0.60

-- 1.00 --

--at --o

33

Table 4, Allelic frequencies at the transferrinlocus for 24 populations of Geomys

Population Number of Transferrinindividuals a b c

Geomys bursarius

1 Walker Co., Texas

2 Denton Co., Texas

3 Lamar Co., Texas

4 Rusk Co., Texas

5 Leon Co., Texas

6 Love Co., Texas

7 Garvin Co., Texas

8 Mason Co., Texas




12 Wise Co., Texas

10

15

7

12

12

15

4

8

10

4

3

5

Geomys pjnetis




16 Bay Co., Florida

Geo arenarius



13

5

8

4

13

13

-- 0.75

-- 1.00

-- 1.00

0.54 0.33

--w 1.00

0.30 0.70

1.00 --

-- 1.00

-- 1.00

-- 0.50

-- 0.83

-- 1.00

0.50

0.50

1.00

1.00

0.45

0.50

-- e

-- 0.65

-- 0.58

0.25

0.13

0.55

0.42

0.3

0.42

34

Table 4 --Continued

Population. Number of Transferrinindividuals a b c


Geomys prsonatus

20 Bee Co., Texas





5

7

75

10

0.70 0.30

-- .00

-- 1.00

-- 0.72

-- 1.00

-- 0.85

0.28

0.15

35

Glutamate Oxaloacetic Transaminase (kidney). Tris-

citrate gels demonstrate two forms of glutamate oxalate

transminase, GOT-1 (supernatent form) migrating anodally

and GOT-2 (mitochondrial form) migrating cathodally.

Three-banded heterozygotes in GOT-1 were reported in Mus

(DeLorenzo and Ruddle, 1970), Peromyscus (Selander_ et al.,

1972) and Geomys (Kim, 1972) demonstrating that the protein

occurs as a dimer. Polymorphism in Geomys was shown at the

GOT-1 locus, with three alleles occurring in three of the

four species (Table 5). Geonxs arenarius was monomorphic

and fixed for the c allele. Most G. pinetis were poly-

morphic, with only population 16 (Bay Co., Florida) being

monomorphic for the b allele. Populations of G. personatus

were generally fixed for the c allele, but population 21

(Mustang Island, Texas) was polymorphic for the b and c

alleles, while population 24 (Brooks Co., Texas) was

polymorphic for the a and c alleles. In G. bursarius

four populations were found to be polymorphic, two of which

were polymorphic for three alleles.

GOT-2, having two alleles segregating at a single

locus, was polymorphic in G. bursarius and G. pinetis

but was monomorphic in G. arenarius and fixed for the a

allele. In G. personatus GOT-2 was monomorphic and fixed

for the b allele.

o( -Glycerophosphate dehydrogenase (kidney). dO-GPD,demonstrated on tris-citrate gels, produces three bands in

OH 00

HO H H O

0 1 C\J IO 0* I * IH O0

HO 0

C D0I 5 0 0

000

0

0SN1 5

00r- 0

OH

. . .0

H HO

'0 0

00S.H

010O

S .

0N I I 0 1

. I I " IO H

ON0%

000000H H H

01 10I I

H

0000 1

S SH- H

I I I I I II I I I I I

0

4') CO

co 0o

001

0o

14 *

o A

00

4)~

C4-')

430

04-4

43

00

r402()00

6-44

56

^ 4

N'JI

100

0

oe

0(0

OH

00

C>

4

0p-'E

0 f\C1M ( N N L\ co o 4 K\ \H H- H H HH

rn o co 0 e eF40 o c 0 ) o

S O cO 4E1 02 e 0o O 0,4) 4) x o 0 0c X E-4() E-4 0E-1 E-1 OX X H 04) xE-4 4 M) 4 E-i 4

.P 4)SE-4 E- 0 E4

0* 05d0 0 0 0 0000 0 00

0 0 4) 0 0

4-O)VCO00CO0 0 0o 0 0 0 0 oHc *H

0 r-4 kj 0 Z P1 Pco 0

4)H H H

I

.N I*

00-O * 0 *OH. H

0 I I II I I

0 0

. .0

H H

Q0N

.0

00000O00000O

I I I I I

00

.H

0 tN

0 0

0 0 0.000.

00

0

0 0

. 0

H H

00H

I II I

t(\ UrN0H-

0

H

I I II I I

I I I1 1 1

0 'o 000000 0

* 0 0 0H OHO HO

I H II " I

0

r\ r< -UN

02 02 00O 0 m00

rdXX r'H 4) 4) --ri oeHFA E4 E-q 4)0 2:H-4

rd 0o 0 0 4)'H -H ' 00 ZP4 P4 . -rH0 0 0 p4 0 0 VH HO q 0 0 w2 0P4 P4 H o o 0

.~ 0 P4, P4

-P 0

* *Om 0

00

-0bo r A rA

0 o m 0 4o o o

02X 0coa

E- X4)

C)) rd0 a :>40 'ri

w q fb" e

o H H ? 0

0 P En H 0P- 4)02 vo H

0 4 y 0 .,I

00:

e

.

toco

0 020 0o

E-4d pt

37

00

co

rd4)

*r4

00

4c)

co

EH

0)

ea

o

02

rd4) -r4

,a t

I II I

i i ii ei i i i0

UN\ 0HA

0o'

0P-4

38

the heterozygous conditionindicating the protein occurs

as a dimer (Selander et al., 1971). Three alleles were

found, although Kim (1972) demonstrated five alleles in

Geomys at the OC-GPD locus. Kim (1972) found variation at

the O(-GPD locus in four populations of G. bursarius and

three populations of G. personatus. This study demonstrated

polymorphism in three of the four species (Table 6). All

populations of G. pinetis demonstrated monomorphic alleles:

two populations were fixed for the b allele, while two were

fixed for the a allele. GeoMys arenarius had the c allele

fixed in all three populations studied and showed polymorph-

ism in only one. Geomys personatus demonstrated the fixa-

tion of the c allele in most populations, with two populations

being polymorphic. In G. bursarius the b allele was fixed

in the monomorphic populations, and polymorphism was

detected in four populations, three of which were poly-

morphic for all three alleles.

6-Phosphogluconate dehydrogenase (erythrocyte). Four

different 6-PGD alleles segregating at a single locus were

demonstrated on tris-maleic acid-EDTA gels (Fig. 4). Poly-

morphism was demonstrated in three of the four species, with

G. arenarius being monomorphic and fixed for b allele (Table

7). The b allele was fixed in all populations of G. bursar-

ius except population 3 (Lamar Co., Texas), which was polymor-

phic for the c and d alleles, with polymorphism for the b and c

39

Table 6, Allelic frequencies at the OC-glycerophosphatedehydrogenase locus for 24 populations ofGeomys

Population Number of D(-GFDindividuals a b c

Geomys bursarius

1 Walker Co., Texas 10 -- 1.00 --

2 Denton Co., Texas 15 0.09 0.73 0.18

3 Lamar Co., Texas 7 1.00

4 Rusk Co., Texas 12 1.00

5 Leon Co., Texas 12 -- 1.00 --

6 Love Co., Oklahoma 15 0.44 0.53 0.03

7 Garvin Co., Oklahoma 4 -- 1.00 --

8 Mason Co., Texas 8 -- 1.00 --

9 Bastrop Co., Texas 10 0.10 0.90 --

10 Parker Co., Texas 4 -- 1.00

11 Wichita Co., Texas 3 -- 1.00

12 Wise Co., Texas 5 0.10 0.40 0.50

Geomys pinetis

13 Levy Co., Florida 13 -- 1.00 --

14 Polk Co., Florida 5 1.00 -- ..

15 Washington Co., Florida 8 1.00 -- ..

16 Bay Co., Florida 4 -- 1.00 --

40

Table 6 --Continued

Population Number of OC-GPDindividuals a b c

Geomys arenarius




Geomys personatus

20 Bee Co., Texas





13

13

5

5

7

7

5

10

-- 0.12

-- 0.20

-- 0.10

0.88

1.00

1.00

1*00

1.00

1.00

0.80

0.90

41

Table 7, Allelic frequencies at the 6-phospho-gluconate dehydrogenase locus for 24populations of Geomys

Population Number of 6-PDGindividuals a b c d

Geomys bursarius

1 Walker Co., Texas

2 Denton Co., Texas

3 Lamar Co., Texas

4 Rusk Co., Texas

5 Leon Co., Texas

6 Love Co., Texas


8 Mason Co., Texas




12 Wise Co., Texas

Geomys pinetis




16 Bay Co., Florida

10

15

7

12

12

15

4

8

10

4

3

5

13

5

8

4

-- 0.40 0.60 --

-- 0.97 0.03 --

-- -- 0.86 0.14

-- 1.00 -- M -

-- 1.00 -- --

O 0.435 0.53 0.04

-- 1.00 -- --

-- 0.44 0.56 -

0.15 0.85 -- --

-- 1.00 -- --

-- 1.00 -- --

-- 1.00 --

-- 1.00 --

1.00 -- --

-- 0.69 0.31

-- 0.63 0.37

42

Table 7 --Continued

Population Number of 6-PDGindividuals a b C d

Geomys arenarius




Geomys personatus

20 Bee Co., Texas





13

13

5

5

7

5

10

-- 1.00 --

-- 1.00 --

-- 1.00 --

-- 1.00 --

-- 0.29 0.71

-- 0.29 0.71

-- 1.00 --

-- 1.00 --

43

alleles in two populations. Populations of G. personatus

on Mustang and Padre Islands were polymorphic for the b and

c alleles. Kim (1972) also found polymorphism for 6-PGD on

Mustang Island, but he had no specimens from Padre Island.

Isocitrate dehydrogenase (kidney). Two forms of iso-

citrate dehydrogenase, )IDH- (supernatant) and IDH-2

(mitrochondrial), were shown on tris-citrate gels, and both

are NADP dependent. The three--banded pattern of hetero-

zygotes in IDH-I is interpreted as a dimer configuration

for IDH molecules. This pattern has been shown in Mus

(Henderson, 1968), Dipodomys (Johnson and Selander, 1971),

and Peromyscus (Selander et al., 1971). Kim (1972) found

polymorphism in IDH in five populations of G. bursarius and

none in G. personatus. He also found a cathodally migrat-

ing band he called IDH-2 negative. This band was detected

by this author on several gels but could not be coded

consistently.

Three alleles were detected at the IDH-1 locus

(Table 8), with polymorphism in all four species at this

locus. Seven of the twelve populations of G. bursarius

were polymorphic. Polymorphism was found in all popula-

tions of G. pinetis, population 17 (south El Paso Co.,

Texas), of G. arenarius, and population 24 (Brooks Co.,

Texas),of G. personatus.

I I I I I .f- II I I "*

0

10 00 1I . 0 S 1

O Hr- H-

- 0 -H H H HH OH H

0

I SH-

00

S 0K C IN 0 0- H- OH 0 H HO

O 00

0I C

0

0L0 K\0 0- M \C

co

U00C)

P4

rd

co

-P

00P 0-H N

to

0 0co

4) o

0 P44) 0

-P0 0

*H

0

r-i

co-r

E-4

00-H 4

a)

Ho

0 a

P r-d

H

0200o

P4FdC) -HirO>

-1-4

0

-r

r-f

ec 0 A r-co (a

0

0 9 X 0 E-0 E-2 c0 r- 0 0 E-4

N ~~ 00 0 N 0 0)E-E-4 C) C) M E-i .- ' C)E-1 E-4 0 * -0' 0 5 000 0 0 * 0 0 0 0

0 0000-0P 00r0 0 0 p- -r0

co M 0 0 -,P 020 w 0 > 0E0w HtH

co (1) 0 co co co c -ri -rl

K\ t LE\ C- CN O Cr- MH r-I H

,0

CMj

H1

02 02m m0 )0

El E-4

* e

02 0 0ri

P4

P4 H -'e 0 C)PD M

4 rCMd

0 LE\ C C CM L\ r 000 4 \ LE\Sr- r-I

45

0 0 0

- H H H H

1 1 I I

I I-I e

S 0 0 o 00000 .

00

1

0o

p-i9

0 T

H

H

o >

r d

0

00

ord

co0-I

CMco

0

uo0

00

00

I

0 0. .

H- H

0 0. .S

H H

I I II I I

r-i r-i U

U)

E-0

0

0

coa4000U)

m 0o 0x0

E-4 0

* *0 0)O z0 *

0o 0a r*1

0 U N KIN1O CM 'e H

0 0 0 0

00 00H

0'H

-4i

rd rd o94 *e ri0 0 0 p

H H 00N - I

0 0 04H Hol 0

-fe f e Q

* 0 0 4 #.

0 0H 0

I-0h

8H H

I I I I II I I I I

0000000000

. . 0 .

H rI H H H

i

'd

0

OD

0

-Ilraco

0

F4Ioco-ri 0 0 0

p o

coo O O

00 O N

ii

i 1 1 1 I I

0000 o000 0 0 0 w

H H H HO

HI I I I HI II I

0

UN > C>r r\ 0r--I

H

to E -.o

m w

0 cl 0

E-4 0 W

to rd

co 9 0% PX) r-4 0 0

H H 0

r 14

0

rI 0o * o UP

i p-4 H 0

O H H 0

46

At the IDH-2 locus, two alleles were detected.. Poly-

morphism was shown only in G. bursarius and G. arenarius.

Geoays pinetis and G. personatus were monomorphic and fixed

for the a allele. The a allele was fixed in G. arenarius

and all but populations 10 (Parker Co., Texas) and 11

(Wichita Co., Texas) of G. bursarius. Populations 6 (Love

Co., Oklahoma) and 9 (Bastrop Co., Texas) were polymorphic

in G. bursarius,and population 17 (south El Paso Co., Texas)

in G. arenarius was polymorphic.

Malate dehydrogenases (kidney). Two NAD-dependent

malate dehydrogenase forms were shown on tris-citrate gels.

The MDH-NADP form was not scorable- on some gels; therefore,

it was not included in the results. The supernatant MDH-l

migrates anodally and was monomorphic in three of the four

species, G. bursarius, G. pinetis, and G. personatus. All

were fixed for the c allele (Table 9). Populations 17

(south El Paso Co., Texas) and 18 (north El Paso Co., Texas)

of G. arenarius were polymorphic for the a and b alleles.

Kim (1972) showed no polymorphism in MDH-l from 17 popula-

tions of G. bursarius and G. personatus.

The mitochrondrial MDH-2 migrates cathodally and was

polymorphic in all four species. Four alleles were shown

in G. bursarius,with the d allele (slowest migrating band)

being detected in populations 5 (Leon Co., Texas), 6 (LoveCo., Oklahoma), and 7 (Garvin Co., Oklahoma). Polymorphism

47

I I I I 0 KN 0 1 I 1 1 I'd 1 0 O i\ 0 1 1 1 1

SOr-

0 0008 HO 0NV 00 0 0 1~- I co C* I I \

r 4 1 * I * * I I0H H H H 0 00 0

ON 00 00I I I I I I I H \ 0 0 N

00O H H-iO

to 0o I I I I I I I I 1 I 1 I

0 101010I0I0I0I0I0I01010

0r 0S 0 5 5 0 0 0 S 0 0

H H H H H H H H H H H H H

0d P

(1) I I I I I I I I I I I I4 J

co

H 01

co 4)'ca I I I I I I I I I 1 1 1

0 1 1111

04

- 4 4-1 rHH0 0 0

ri :j043 0 Lf ll- C\J N j L\ 4 cao) 0 4 Kl\ In

(1) a (1 -r- -I r-i - H -

'0AA rd

(1)4 0

4-4 0 (1co44 (

H 0 0 ( 0o 0 ( E 0 Xdi X w 0 (0 (0 H 0 0 o 0w4HI co co w 0 co W x c

(1 -r m 0 (1) a 4 r i o 0 xo O

H0() ( 0 X 0 0 0 O X E- 0.E-4 0HO &4 E 0 X HOM 0 E-4 x4 4I E-4 0 0 >M E-4 0Selk E 2- 4 0 E

S G . .-40 0 w-5 0* 0

ON 0 I0 0 * * 0.0 0 0r4 do0 0 0 0 0 R 0o0) 4)p 4 p 0 0 4 0 F4pH 0 0o0 0 P P -- 0 r4I.M H E(0,1143 0 co4 0 4-) 10f42-o z 0P 0 El0 0 0 ca 0 pc0- A 0 o 4)a co 4) 0 co C5 ca c - r

0 p-V4 IC(r1 )L \ ~ ~ C ' H C

H0H1

48

I I I II I I I

H

0

O

0

0

I II I

00H-

0000

S 0

H H

1 1 1I 1 1

I I II I I

N

0

S

0

I I I II I I I

CMH

Zrd

o

r4

P4

co

Co

0 co-

0rd

H

0

p-,

00OH

0 0

* 0 I

0O

0

t<\L(\CO

K- a

Hco 05

000- 0

CocCo 0d o 0

o0 0 0H HO0 0 co

r= I4 P-10 0 0 tot

0 pd0 CoI

4) 0 o coq P4 -r co

r H O r-4% N HtoH A

O~

. .0H H

I II I

1 1I I

0 0

OHL'\

0

,...4; .H

mtoCo -s

c o0 0 C* e-i x o to

o 0rd 0Ct o 'E-

So *o F e gto * X mC o li 0co 0 O H4 0Ht.

<- 0 to 0C oH CO 4) alp,0

0 00 0

0 OC c '

H H 4 () m r

0 00I4) coC C

0 ON 0 0(1J N Y Lr0 \ ' CM0 0 0 0 0

O H N00CO CM 0 'ACO0 0 0 0 0

0 888800 0 0 0H H H H

I 1 1 1 I

Lr\Q

coco

0C::

4)

co

000

H

was shown in all five populations of G. personatusias well

as population 13 (Levy Co., Florida) of G. pinetis and

population 17 (south El Paso Co., Texas) of G. arenarius.

The c allele was the common allele in G. bursarius, appear-

ing in all except four populations. Allele b was fixed in

certain populations of G. pinetis and G. arenarius, and

alleles a and b appeared in all populations of G. personatus.

Lactate dehydrogenases (kidney and testis). Two

lactate dehydrogenase systems (LDH-1 and LDH-2) were found

in kidney extract. Genetic control of the protein is con-

sidered to involve two loci (Appella and Markert, 1961).

LDH-1 and LDH-2 migrate anodally and combine to form a five-

banded pattern explained by Appella and Markert (1961) as a

tetramer. The faster migrating band is controlled by the

LDH-l locus and the slower band by the LDH-2 locus. Inter-

mediate bands represent tetrameric combinations of alleles

from the two loci. Two alleles (a and b) were detected in

bbth LDH-1 and LDH-2, and polymorphism was found only in

G. bursarius (Table 10). The a allele (fastest migrating

allele) in G. bursarius was fixed in all populations except

2 (Denton Co., Texas) and 11 (Wichita Co., Texas) for both

LDH-l and LDH-2. Polymorphism was found in four populations

of G. bursarius at both LDH loci. A cathodally migrating

band was found by Kim (1972), but the allele was not

detected in this study.

01 0 1 I I - II " I I I I

-10

C000C 0 LF\Q CO00e HO

0 0 00 C \QQCO Q 0

- H- H- HO H- H 060co

0

rd

4)

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00

0 0

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-rH r-4

001

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SA

'H

00

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r-e

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'H C0 H

ro

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p

ca

40H,p4

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oc

0 \ O CM C \ O CO C0 i\ r'C LCH H H H H H

to c aE A c 0 CD 0co co Ea 0 co w

0 0 C 0 0 - CD E 0 N-E- N 0 CD X CX ri H0 0( ) E 0-C0 0 E 0 0 0 )E E-I 0 E eE e- 0 X H-O 0 NE

& 0 0 , . . 00-I0E0I 9 0 * oE9 0 0 90 . .0

0 00 b V0 *c0 0000 * * 0 0 0-

0 0 0-ri0 4 0 0M 000 >0 4 0 4H 0 0 0 00 0 0 0 wEas ( o :j 4 0 o o m -ri -i

H- CMN ( PC\ '0 E*%- co O'Y\ O Hf CMH-4 Hi H

50

C' 0 nO001 (J f 0 c

00 HO

01 0 1 1 1 1 1I " I I I " I

r- 0

51

to

co'

0000

0000

000

00 0

O( LE O OH OO

rd X -ri

E-4 E-4 4)o0zH

0 a :P4 0rd 0o 0 0 4)-ri - 0dP P *H*o o 0 4 00 0

r-I r-I O 0 U) U)OP4 P4 H- 0 0 0

0 -r4 P-4 .4 0

-r 4 04 0 o 40 0 4U)

OH 04-' - 0 co '0 A

0) q p-, Lp q -o m 0

Lr C I I - 00 ON0

0-ri o ea 4 o H

O~

* 0 0

- H- H

I I II I I

000 O000 O* 0 0

H- H- H-i

r-( r-r\

rij

4-)

-i

0

0

-)Hto

1 1 I 1 I

* 0 0 0 0

1- 1- -1 -I -I

Lf"NN NL\ 0H

E)

E-1 C, . o4) 0-r -

E--

) r4o $.

X 0o rd 4)E-) H

co b o p z

U2 d r e

:j c p,pq ;E: r-i 4 p

0 e 0 - --I N 4004))I0I91 00

COH

0 -ro00

-ri

4--)ci

P40

P4

52

Geomys pinetis, G. arenarius, and G. personatus showed

no polymorphism at the LDH-l and LDH-2 lociwith the a

allele being fixed. However, Kim (1972) found three alleles

in G. personatuswith polymorphism for the allelesin popu-

lations from Kenedy and Kleberg Counties, Texas.

LDH-3 was found in testis extracts (Goldberg and

Hawthrey, 1967) and demonstrated four alleles (Table 11).

Geomys bursarius was the only species showing polymorphism.

The b allele was fixed in all four species except three

populations of G. bursariuswhich were fixed for the e

allele. Population 6 (Love Co., Oklahoma) of G. bursarius

was polymorphic for three allelesi b, c, and d.

Esterase 1 (erythrocyte). Es-1 demonstrated 4 alleles

on tris-bydrochloric gels in the Geomys complex of pocket

gophers (Fig. 4). Geomys bursarius was the only species

that had all four alleles present (Table 12). Only four

populations of G. bursarius demonstrated polymorphism. All

other species studied showed polymorphism except one popula-

tion of G. pinetis. Considerable variation was shown in

populations of G. pinetis and G. arenarius. Alleles b and c

were found in all populations of G. pinetis. Geom

arenarius was polymorphic for alleles a and b in two popula-

tions, and population 17 (south El Paso Co., Texas)

demonstrated only the b alleles. Most G. personatus popula-

tions were polymorphic for the b and c alleles.

53

Fig. 4, Electrophoretic variation in 6-phospho-gluconate dehydrogenase and esterase-Lin pocket gophers of the genus Geo

0

6PGD AA BB CC DD AB BC CD

0Es-1 AA BB CC DD AB BC

54

Table 11, Allelic frequencies at the lactate dehydro-genase locus for 24 populations of Geoys

Population Number ofindividuals aindividuals

Geonys bursarius

1 Walker Co., Texas

2 Denton Co., Texas

3 Lamar Co., Texas

4 Rusk Co., Texas

5 Leon Co., Texas



8 Mason Co., Texas




12 Wise Co., Texas

Geomys pinetis




16 Bay Co., Florida

10

15

7

12

12

15

4

8

10

4

3

5

13

5

8

4

-- 1.00

-- 0.80

-- 1.00

( ).25 0.75

-- 1.00

-- 0.62

-- 1.00

-- 1.00

-- I r "mo

a" 100

"- -- 1.00

-- -- 1.00

100 a

- 1.00 --

-- 1.00 -

-- 1.00 --

0.20

0.15 0..2

0.13 0.25

LDH-3

Flb c d-

i

55

Table 11 --Continued

Population .Number of LDH-3individuals a b c d

Geors arenarius




Geo ys personatus

20 Bee Co., Texas





13

13

5

5

7

7

5

10

-- 1.00

-- 1.00

-- 1.00

--m 1.00

-- 1.00

-- 1.00

-- 1.00

-- 1.00

00 0

H H

I - WIN1

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H

co*0

OHH

0

H

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00

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si o

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l A 00 0 U X00o H 0 ) X CCO M M - Ei 0 -i10 (1X) H C)EH4

m E-1 4P%

. 0 0 0 000-0 0

SF4r4 *C 0 04-) ~ CC0 co *Hx> U) 4 0

o e m P .rI .

56

I HK*

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I D1 0

o ('1

0

0 r\JL\4 000C 4 K\ /\Hr- H H H

0 00 0 UXX0 0) 0

c) ) X 0 0

co 0 o 0:: 0F0 0

000

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rd 'ri

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w 00 0

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I I I I II I I I I

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caN o0 0c

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:3 EA CO c

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0 0 4

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021

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C10co

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58

Esterase 3 (kidney). This esterase was detected on

lithium hydroxide gels and demonstrated three alleles

(Fig. 5). Polymorphism was found in most G. bursarius

populations (Table 1). inetis was polymorphic in

all populations for the a and b alleles, except population

16 (Bay Co., Florida),which was monomorphic for the b allele.

Geomys arenarius was polymorphic for alleles a and b, and

G. personatus generally demonstrated polymorphism for the

a and b alleles, also. Three alleles, a, b, and c were

detected in populations 21 (Mustang Island, Texas) and 22

(Padre Island, Texas) of G. personatus.

Esterase 4 (kidney). Lithium hydroxide gels demon-

strated three alleles for esterase 4 (Fig. 5). The a

allele was the most common in G. bursarius, and was found

in all populations except 1 (Walker Co., Texas) and 2

(Denton Co., Texas). Polymorphism was demonstrated in sev-

eral populations of G. bursarius (Table 13). Geoys pinetis

was polymorphic for the a and b alleles except in population

14 (Polk Co., Florida), which was monomorphic for the a

allele. Geomys arenarius was polymorphic for alleles a and

b,while G. personatus was polymorphic for the a and b

alleles in all populations except 20 (Bee Co., Texas).

Esterase 2 (plasma). Four Es-5 alleles were demon-

strated in the Geomys complex on the lithium hydroxide gels

(Fig. 5). Allele b was found in 6 of the 12 populations

tr\

0

0 IN IHO

I I I I I I I I II I I I I I I I I

i\ D tLr\

I 0 . I eI 0 0

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00

.

H

02

4),-P

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co

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59

I 1 I I I I I I I I I I

0 t\ "\ N (NJ N \ 4 00 \ 0N Ur\r-I - q -4 -I -

0 0 020 02

XoX 0 02 02 ) o 0 4) ox 0 )0a) co cX 0 0 o0 x X H-4 () EH1 0E-4 HE-4 I) x x H O a) E-4H (1) C) HEa)

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N Lr\ to C11- o ON 0 -r - r i

60

I I 1 II I 1 I

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00

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CMO

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0 0

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KN K\H H

4-4

o0

r d

rd

000 d

-rq

r--440H-

co

co00

0

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0

CCH

000O

0

00

00

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00

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0 .0 0

tr\ tr\ t\0UN UN C- ~ UN

00-ri

x

0

0

co9i

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0O

00

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X C0 0cE-1

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CO E4 a CLI4) OH H0 cae0o CC o * bC

A 4o 0 0o 0

mCo

o to

coo 0r4 00

*4 0I 0

o CoH 0

0

co r

4

co0

00 0 UNH CM I

S0 0

rd

rc U o :J

0

r--4 oa NH V0 % r

0 0 0M e-ri r 04

o 0 0 40ri)

0 0 c

q PH H

co

co0

0

0o c

o

02I 0-)i r4

0

P44

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ii

61

Fig. 5, Electrophoretic variation in esterases-A,3, 4, 5 and 8 in pocket gophers of thegenus Geomys

Es-8

Es-3

Es-4

Es-8

Es-3

Es-4

m-m

- - - --

==~==

AA

AA

AA

BB CC AB BC

BB CC AB BC

BB CC AB BC

+

- -

Es-5

=

BB CC DD AB BC CD

Es-A BB

0

Es-A0

Es-5 AA

AA

62

of G. bursarius, and nearly all polymorphic populations

were highly variable (Table 14). pinetis showed

polymorphism for alleles a and b, and G. arenarius was poly-

morphic for b and c alleles. Allele b was found in G.

personatus populations, and allele a was in two populations.

Esterase 8 (kidney). Three Es-8 alleles were demon-

strated from lithium hydroxide gels (Fig. 5). In G.

bursarius, polymorphism for the a and b alleleswas found

in five populations (Table 15). Three populations of G.

bursarius demonstrated monomorphism for the a allele, and

four were monomorphic for the b allele. Geonjs pinetis was

polymorphic for the a and b alleles in two populations and

two were monomorphic for the a allele. Geomys arenarius was

polymorphic in all populations for alleles a and b. Three

populations of G. personatus demonstrated polymorphism for

all three alleles whereas two were polymorphic for a and b.

Genetic Variability

Genetic variability within a population can be

expressed in several ways: the proportion of loci which

are polymorphic for each population, the total number of

alleles detected at each locus, and the mean number of

alleles segregating for each polymorphic population. These

indices of genetic variability for the species of the

Geomys complex are presented on Tables 16 and 17. Eighteen

0

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0 0 0 0OHO OHO

0 0 0 0

H H H H

d0

r-el

co mr r c'o

H H 0 o0

64 0

0

0 0.

rN4 'H q.r\

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0 0

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02

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0

00

00

0 0 NOON ( L U O\0 0 0 0

o 0

0 0 0 0

00000o

0 0 S0 0

I I I I II I I I I

o 00 oN 'H

E-

0~* A

O Z

02N 0

coeE-i N

Sa

o o (12 C d0 0 dm m . m rd0 0 0 0 OH 0

P4 e PE (120H H H

o co coco H H O 02

0004) 4z M 0 $4-.)4 rd

o O 0 H m

-

UO

020 c

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0

o 1O 0

4)

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67

Table 16, Proportion of loci polymorphic perpopulation (P), Proportion of locipolymorphic per individual (H) in24 populations and four species ofGeomys

Population (P) All Nonesteraseproteins proteins

G. bursarius

Walker Co., TexasDenton Co., TexasLamar Co., TexasRusk Co., TexasLeon Co., TexasLove Co., OklahomaGarvin Co., OklahomaMason Co., TexasBastrop Co., TexasParker Co., TexasWichita Co., TexasWise Co., Texas

0.1360.1360.0450.1360.0910.1820.0910.3180.2270.0910.0910.136

X 0.140

0.0310.0180.0250.0220.0190.0330.0230.0800.0500.0340.0300.045

S0.034

0.0250.0210.0000.0150.0110.0280.0000.0630.0560.0310.0200.037

X 0.025

-. pinetis

Levy Co., FloridaPolk Co., FloridaWashington Co., FloridaBay Co., Florida

0.1820.1360.1820.182

Y 0.171

0.0280.0550'0610.045

X 0.047

0.0090.0250.0440.031

X 0.027G. arenarius

south El Paso Co., Texasnorth El Paso Co., TexasDona Ana Co., New Mexico

0.3640.182

_ 0.136X 0.227

0.0800.0450.045

Y 0.057

0.0630.0380.0380.046

G. personatus

Bee Co., TexasMustang Island, TexasPadre Island, TexasWillacy-Kenedy Co., TexasBrooks Co., Texas

0.1360.0910.2270.1820.182

X 0.164! 0.161

0.0450.0390.0710.0550.0550.0570.049

0.0250.0270.0080.0220.0190.0200.027

123456789

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of the 24 structural loci were observed to be polymorphic

in the populations of G. bursarius (Table 17),while 12 loci

were observed to be polymorphic in populations of G.

pinetis. In G. arenarius,11 of the loci were observed to

be polymorphic (Table 17), and 12 loci were polymorphic in

G. personatus (Table 17).

Of the 24 loci, 19 were considered to be polymorphic

in one or more populations. Eight of the 24 loci were

polymorphic in all four species of Geoniys,while 12 loci

were polymorphic in three of the four species.

Two loci were observed to be polymorphic in only two

species. Malate dehydrogenase -l was polymorphic only in

G. arenariuswhile a single variant was observed in G.

bursariusat the esterase-6 locus.

Significant variation was demonstrated in the propor-

tion of polymorphic loci among major regions inhabited by

Geomys. However, within each species variation was local-

ized and could not be contributed to geographic distribution.

Variation in the loci of eight proteins provided the

major differences between G. pinetis and G. bursarius

(Table 18). Four loci had distinguishing alleles for G.

pinetis: hemoglobin, fixed for allele a; albumin, fixedfor allele c; esterase-5, polymorphic for the a and b

alleles, and plasma esterase A, fixed for allele a. Four

71

polymorphic proteins demonstrated interspecific variation,

but their loci contributed only weakly to the variation.

Variation in the loci of seven proteins contributed

the greatest differences between G. bursarius and G.

arenarius (Table 19). Esterase-l was highly variable, demon-

strating polymorphism for four alleles in G. arenarius but

only three alleles in G. bursarius. Distinguishing loci for

G. arenarius were albumin, (a allele), GOT-2 (a allele),

OC-GPD, (c allele) and MDH-1 (b allele).

Two loci distinguishd G. personatus from G. bursarius

(Table 19). Plasma esterase A was fixed for the a allele

in G. personatusas was OC-GPD locus for the c allele.

Polymorphic Loci

The mean number of polymorphic loci per population

was 0.161 (Table 16). The lowest proportion of polymor-

phic loci was found in G. bursarius (range = 0.045 to 0.318;

X = 0.140). The greatest proportion of loci polymorphic

per population was observed in G. arenarius (range = 0.136

to 0.364; X = 0.227). Polymorphic loci per population in

G. pinetis ranged from 0.136 to 0.182, with a mean of 0.171,

while that of G. personatus ranged from 0.091 to 0.227,with

a mean of 0.164. The highest polymorphic value per popula-

tion was 0.364 observed in G. arenarius from south El Paso

Co., Texas. The second highest value of polymorphic loci

was 0.318, detected in the population of G. bursarius from

Table 18, Common or fixed allelic comparisons ofproteins among G. bursarius and G. pinetis

Species Hb Alb Trans Got-2 MDH-2 Est-1 Est-5 plasmaEst-A

G.bursarius b b b b c b-d b-c-d b

Gopinetis a c a a b b-c a-b a

Table 19, Common or fixed allelic comparisons ofproteins among G. bursarius, G. arenariusand G. personatus

Species Alb Trans Got-2 @(..GPD MDH-l Est-1 Est.-4 plasmaEst-A

G.Tursarius b b b b c b-d a b

arenarius a b-c a c b a-b a-b b

G.Personatus b b b c c b-c a-b a

72

73

Mason Co., Texas. The lowest value of 0.045 was detected

in the population of G. bursarius from Rusk Co., Texas.

The mean number of polymorphic loci per individual

for all species was 0.049 (Table 16). The lowest mean of

polymorphic loci per individual was found in G. bursarius

(range 0.018 to 0.080; X = 0.034). The greatest proportion

of polymorphic loci per individual was observed in G.

arenarius (range 0.045 to 0.080; YX= 0.057) and G. personatus

(range 0.039 to 0.071; X = 0.057). The highest polymorphic

value per individual was 0.080, observed in G. arenarius from

south El Paso Co., Texas and G. bursarius from Mason Co.,

Texas. The lowest values of 0.018 and 0.019 were detected in

the individuals of G. bursarius from Denton Co., Texas, and

Leon Co., Texas, respectively.

Effective Number of Alleles

The effective number of alleles at each locus (Table

17) is a measure of the amount of variation contributed by

the various alleles at any polymorphic locus. Any mono-

morphic locus would have only one allele. Therefore, the

effective number would be one. Five of the six esterases

showed high values of effective number of alleles (Table 17).Esterase-1 and Es-5 had the highest values of 1.755 and

1.789, respectively. These values are indicative of the

esterases in general, as these were the most polymorphic

proteins studied. Non-esterase proteins generally

74

showed low values, except transferrin, with a value of

1.411 at this locus. In transferrin, the b allele was

detected in all but three of the 24 populations. Allele

a was found at high frequencies in all four populations of

G. pinetis and in three populations of G. bursarius.

Moderate frequencies of allele c were found in the popula-

tions of G. arenarius, while low frequencies of c were

found in 7 populations of the other three species. This

indicates allele b contributes greatest to the variation

at this locus, whereas allele a contributes the next

greatest and the c allele only a small portion. In

esterase-1, the b allele was found at high frequencies in

most all populations studied except in G. pinetiswhere

allele c showed high values. Alleles a and d were detected

in four populations each and contributed less. Allele b

was the most common, while allele c was the next greatest

contributor.

Esterase-5 had the highest effective number of alleles

(1.789). Polymorphism was rather high for this protein,

and alleles b and c contributed about evenly to the varia-

tion. Allele a was high in most populations of G. pinetis,

while allele d was detected generally at low frequencies in

some G. bursarius populations.

Esterase-6 had the lowest effective number of alleles

of the 19 polymorphic proteins. Allele b was detected in

75

all individuals. However, one specimen was heterozygousfor alleles a and b. The effective number at this locuswas 1.003, indicating that allele a contributed only 0.003to polymorphism at this locus.

Esterase-8, with the effective number of alleles ateach locus of 1.457, was highly polymorphic. Alleles a andb contributed about equally in effectiveness, and allelec was detected in three populations of G. bursarius.

CHAPTER IV

DISCUSSION

Genetic Variation

The estimates of heterozygosity for the 24 populations

representing four species of Geomys is based on 22 blood

and tissue proteins controlled by 24 genetic loci. The

average value for heterozygosity (H) for the four species

of Geomys is 0.0487, indicating that only 4.87% of the 24

loci are heterozygous in the average individual. Hetero-

zygosity varies from a low of 0.018 in G. bursarius to a

high of 0.0800 in the same species. The lowest average

heterozygosity was found in G. bursariuswith a mean value

for H of 0.0342, while the highest mean value of ff was

0.0556 in G. arenarius and G. personatus.

Among non-fossorial rodents, comparable but still

higher values of heterozygosity have been found in

Peromyscus attwateri, and probably reflects random fixation

(Sewell Wright effect) of low heterozygosity during

Pleistocene isolation (Kilpatrick, 1973). Smith et al.

(1973) reported a range of heterozygosity in P. floridanus,

a Pleistocene relict, of from 0.046 to 0.064, with a mean

of 0.053, and suggested P. floridanus to be one of the

least variable species of the genus Peromyscus. In

76

77

mainland populations of P. polionotus, Selander et al.

(1971) reported heterozygosity ranging from 0.0496 in

South Carolina and Georgia to 0.086 in peninsular Florida.

In isolated populations of P. polionotus inhabiting small

barrier islands and peninsulas of the Gulf Coast of

Florida, lower estimates of heterozygosity were obtained,

ranging from 0.018 on islands to 0.033 on peninsulas, with

a mean of 0.028. Other rodents which are reported to have

low levels of heterozygosity include kangaroo rats of the

genus Dipodomys (Johnson and Selander, 1971). Johnson and

Selander (1971) examined several possible relationships

affecting degree of genetic variability in kangaroo rats

and observed that the major contribution of heterozygosity

to a population came from one or two loci. They concluded

that sampling error might significantly affect estimates of

genetic variability and assumed that much of the variation

reflected some significant degree of interspecific variation

in levels of heterozygosity. They observed that no rela-

tionship existed between degree of genetic variability and

the extent of geographic range. For example, of the seven

species of kangaroo rats exhibiting low levels of heterozy-

gosity, three have small ranges, and four have extensive

ranges, including D. ordii, the most widely distributed

member of the genus. Likewise, G. bursarius, the most

widely distributed species of the genus Geomys is no more

78o

variable than the other three species examined with

restricted ranges.

Factors relating to the maintenance of protein poly-

morphism are unclear at this time. Recent discussion hasbrought about the consideration that most, if not all, genicvariation is neutral (Kimura and Crow, 1964). Thus, it hasbeen maintained that alleles which demonstrate differentpatterns of electrophoretic mobility are functionally

identical and selectively neutral. In addition, Kimuraand Crow (1964) have suggested that on a theoretical basis,heterozygosity, even of the magnitude observed in popula-tions of Geomys, should create an excessive genetic load.Nevertheless, the level of polymorphism in vertebratepopulations examined to present is far higher than couldexist under conditions of excessive genetic load (Selanderand Johnson, 1973). If one accepts the hypothesis thatprotein polymorphism is influenced, for the most part, byselection, protein variation correlates with physiologicalvariation and, to some degree, with environmental condi-tions. For instance, Johnson (1973) has demonstrated from13 species of Drosophila that enzymes whose substrates areenvironmentally influenced are more variable than thoseenzymes of narrow specificity. Indeed, the proteins of_e~oj;s which have the most variable substrates are theesterases, and these were found to be the most polymorphic.

79

Those enzymes with narrow specificities are the least

variable. If selection were not a determining factor, all

proteins should show the same magnitude of polymorphism, a

condition not found in Geomys or other vertebrates. Like-

wise, in a constant environment, selection for the optimal

form of a protein is the best strategy, while in a variable

environment, heterozygous individuals have an advantage

(Johnson, 1974). Therefore, a narrow, subterranean niche

like that of G probably necessitates a low level of

heterozygosity in all proteins of narrow specificity.

The low levels of heterozygosity in G are best

correlated to the restricted subterranean habitat of a fos-

sorial rodent. Indeed, evidence from several species of

fossorial rodents has accumulated and indicates that

relatively low levels of heterozygosity are shared by most

subterranean mammals. The lowest value of H in rodents has

been found in mole rats of the p ehrenbergi complex of

Israel (Nevo and Shaw, 1972; Nevo and Cleve, 1974) with a

mean heterozygosity of 0.034. Similar values of H have

been demonstrated in other North American pocket gophers

such as Thomomys talpoides (Nevo et al., 1974), T. bottae,

and T. umbrinus (Patton et al., 1972) with average values

of heterozygosity of 0.047, 0.070, and 0.033, respectively.

The mean value of heterozygosity for four species of Geomys

of 0.0487 is comparable to that found for two species of

80

Geomys by Kim (1972), who reported an average R of 0.047

for G. bursarius and 0.053 for G. personatus.

Evidence substantiating the uniformity of the subter-

ranean niche is now accumulating, most studies indicating

seasonal and daily uniformity in temperature and relative

humidity independent of environmental vicissitudes. Daily

temperature variation in burrows of Geomys is inversely

related to depth, and McNab (1966) has shown the temperature

variation to be 4.80C at 11 cm, 1,00C at 30 cm, and 1.00 C

at 50 cm. The atmospheres of burrows of Geomzs and the

African fossorial rodent Heterocephalus are nearly sat-

urated with moisture, and humidity appears to be indepen-

dent of burrow temperature (McNab, 1966). Saturation of

Geomys burrows has also been demonstrated by Kennerly (1964),

even with soil moisture content of only 1.0%. Compounding

this is the apparent restriction of Geomys to sandy, loose

soils (Davis, 1940), a factor that further narrows the

niche of this fossorial rodent.

The effect of a constant subterranean environment in

no way precludes the role of isolation and random proces-

ses. However, the pattern of genetic variation in Geo ys

does not correspond to that expected if genetic drift were

operating, with the exception of certain loci in G. pinetic

and G. arenarius. The random drift hypothesis would predict

alternative fixation of genes and the same average degree

81

of heterozygosity in different populations. Such is the

case in isolated troglobitic populations of the Mexican

fish, Astyanax mexicanus, where low genetic variability

was attributed to random drift rather than to selection in

a uniform cave environment (Avis and Selander, 1972).

Certain species of lizards of the genus Anolis inhabiting a

small island in the Bahamas have an absence or reduction in

heterozygosity attributed to random drift by Webster et al.

(1973). Similarly, in island populations of Peromyscus

polionotis, lower estimates of heterozygosity were obtained

than those found on the mainland (Selander et al., 1971).

Thus, the island-model type distribution of populations of

G. bursarius and the restricted ranges and localization of

populations of G. arenarius, G. ersonatus and G. pinetis

would appear to fit a prerequisite for the random drift

hypothesis. However, examination of genetic data for

Geomys reveals uniformity of monomorphic or weakly poly-

morphic loci in populations of all species.

The major contributors to heterozygosity in all popula-

tions of Georys were the esterases, Es-1, Es-3, Es-4, Es-5,

and Es-8 and, to a lesser degree, transferrin and gluta-

mate oxaloacetic transaminase. Non-esterase proteins con-

tributed no heterozygosity in some populations to a high

of 0.063 with an overall mean of 0.027 (Table 16). This is

true in reproductively as well as geographically isolated

populations. Although the pattern of geographic variation

82

in heterozygosity is remarkably uniform in all populations,

except one sample each of G. bursarius, G. pinetis, and G.

personatus, it seems highly improbable that fixation or

near-fixation of the same alleles would be so uniform

among all species of Geomys. Ewens (1963) has shown that,

at the most, only 2.8N generations, in an effective popula-

tion size of N, are necessary for a polymorphism whose most

common allele has a frequency between 0.50 and 0.99 to

become fixed under the influence of random drift. If

selection were not acting, the five esterases mentioned

above should have the same fate as other alleles.

Although it would appear to be obvious that selection

has been most important in influencing the genetic struc-

ture of Geomys, contradicting data are evident from this

study. The two species most geographically isolated from

the obvious parental stock, G. bursarius, have unique

alleles or fixed alleles that occur only rarely in G.

bursarius. In G. pinetis, the unique hemoglobin pattern

and albumin alleles c and d that occur only rarely in G.

personatus, may well have reached fixation or near fixa-

tion under the influence of random drift and continued

lack of gene flow. Likewise, fixation of the unique a

albumin allele as well as the monomorphism of several pro-

teins in G. arenarius would tend to support the hypothesis

that random drift has played at least a minor role in

structuring the genome of this isolated species. Thus,

it would appear that selection for homozygosity in a uniform

subterranean niche has been the adaptive strategy for

genomic structuring in , with evidence for random

events having occurred in isolated cases. In addition, the

fixation or near fixation of genes within Gom populations

in no way reflects the loss of adaptability of the species.

Thus, low levels of heterozygosity appear to be an adaptive

strategy in this fossorial genus.

Genetic Similarity and Systematics

Relatively low coefficients of genetic similarity (S)

were obtained from comparisons of paired populations of

conspecifics (Table 20). Furthermore, inspection of Table

20 indicates that S values obtained for paired combinations

of conspecifics are often lower than those obtained for

interspecifics. This is especially evident in G. bursarius,

and a similar pattern of genetic similarities among species

of the Thomomys talpides complex of pocket gophers was

found by Nevo et al. (1974).

Avise and Selander (1972) have pointed out that the

values of S for members of the same species are generally

high, usually in the high 0.80's to the 0.90's. The degree

of interspecific genetic heterogeneity in Geomys probably

reflects extensive genetic modification under isolation.

Indeed, collection of samples taken during this study

indicated that pocket gophers occur disjunctly, even when

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85

suitable habitat exists between populations. In addition,

genically divergent populations, such as those of G.

bursarius from Parker Co., Wichita Co., and Wise Co., Texas,

populations 10, 11, and 12, respectively (Fig. 1), are

isolated from other G. bursarius sampled by the Western

Cross Timbers and the Red River in Northern Texas. There

is no evidence to suggest that potential gene flow between

conspecifics of G. bursarius has been interrupted, and

Davis (1940) obtained morphological data that appear to

parallel the genetic heterogeneity in G. bursarius. Davis'

work demonstrated a high degree of variation in size among

local, but disjunct, populations.

The low levels of genetic similarity between inter-

specifics is indicative of the extensive genetic change

with speciation and is the result of genetic divergence

during a period of at least 300,000 years. Fossil records

indicate the Great Plains to be the center of differentia-

tion of Geomys (Russell, 1968), with expansion of the range

of the genus to the Pacific Coast during the mid-Pleisto-

cene. Utilizing morphological features and fossil evidence,

Russell (1968) placed the divergence of G. bursarius and G.

pinetis from a common ancestor (probably G. bisiculatus)

during the Illinoian glacier about 300,000 years BP, with

complete differentiation by the Sangamon interglacial,

200,000 to 150,000 years BP. Illinoian glacier resulted

in separation of two geographic units in the central and

86

southeastern United States by the flood plain of the

Mississippi River. Isolation of certain populations of

gophers in suitable habitats along the Rio Grande River

in the arid southwest contributed to the speciation of

G. arenarius during the Wisconsin or post-Wisconsin

(100,000 to 15,000 years BP). According to Russell (1969),

G. personatus differentiated from G. bursarius during the

mid-Wisconsin, due to isolation on sandy soils in south-

western Texas with retreat of G. bursarius eastward.

Biochemical evidence would appear to support the phy-

logeny of Geomys based on morphological and fossil data,

although certain discrepancies exist, with divergence times

estimated from the electrophoretic analysis. Populations

of G. arenarius and G. personatus had the highest average

interspecific genetic similarities to G. bursarius, 0.559

and 0.630, respectively. This reflects their recent

evolution from G. bursarius (Fig. 6). As suggested by

Russell (1968), G. pinetis is the most divergent species

studied,with an average genetic similarity to G. bursarius

of only 0.508, and this is also born out from chromosomal

data. Kim (1972) has shown that the diploid number in G.

bursarius ranges from 70 to 74, while the diploid number inpersonatus is 68 to 74. The karyotypes of both species are

comprised of mostly acrocentric chromosomes. Geomys pinetishas been shown to have a diploid number of only 42, with

predominantly biarmed chromosomes (Penney, 1974). It would

87

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89

appear that the dendrogram constructed from biochemical

data in Fig. 6 corresponds quite well to the phylogeny of

the four species of Geomys based on the study by Russell

(1968).

Nei (1971) has demonstrated that evolutionary diver-

gence time can be ascertained from electrophoretic data.

Although the method yields only crude estimates, data from

fossils of pocket gophers of the genus Thomomys and deer

mice, genus Perognyscus, have been shown to correspond

remarkably well with estimates of divergence time from

biochemical evidence (Nevo et al., 1974; Zimmerman et al.,

1974).

Using Nei's index for divergence time, G. pinetis and

G. bursarius diverged about 320,000 years BP during the

Illinoian glaciation (Fig. 7), the same period suggested

by Russell (1968). Geomys arenarius and G. personatus

diverged from G. bursarius about 275,000 years BP and

218,000 years BP, respectively. These times correspond

rougly to the late Illinoian glaciation and the Sangamon

interglacial, but Russell (1968) placed the divergence of

these two species from G. bursarius within the last

100,000 years during the Pleistocene. It would appear thatin the case of GeoMys, fossil evidence may be more reliable

than use of Nei's distance measure for determining

90

divergence times, and the extensive genetic modification

seen has resulted from the effect of extreme selection

in a uniform subterranean habitat.

LITERATURE CITED

Anderson, Sydney. 1966. Taxonomy of gophers, especiallyThomomys in Chihuahua, Mexico. Syst. Zool. 15:189-197.

Appella, E., and C. L. Markert. 1961. Disassociation oflactate dehydrogenase into subunits with guanidinehydrochloride. Biochem. Biophys. Res. Commun. 6:171-176.

Avise, J. C., and R. K. Selander. 1972. Evolutionarygenetics of cave-dwelling fishes of the genus 'Atynax.Evolution 26:1-19.

Baker, R. J., and S. L. Williams. 1972. A live trap forpocket gophers. J. Wildly. Manag. 36:1320-1322.

Baker, R. J., S. L. Williams, and J. C. Patton. 1973.Chromosomal variation in the Plains Pocket Gopher,Geomis bursarius major. J. Mammal. 54:765-769.

Carter, N. D., R. A. Fildes, L. I. Fitch, and C. W. Parr.1968. Genetically determined electrophoretic variationof human phosphogluconate dehydrogenase. Acta Genet.18:109-122.

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