CHROMOSOMAL EVOLUTION IN THE POCKET GOPHER,

CRATOGEOMYS CASTANOPS (RODENTIA: GEOMYIDAE)

by

HAE KYUNG LEE. B.S.

A THESIS

IN

ZOOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

Approved

August, 1985

hl^» ^ ^ ACKNOWLEDGMENTS

I wish to express my deep appreciation to Robert J. Balder,

my major professor, for his guidance and support. I thank other

members of my committee. Dr. Raymond C. Jackson and Dr. Ronald K.

Chesser, for providing their time and assistance. I am also

grateful to the following for helpful comments and discussions on

various aspects of this paper: Mazln B. Qumslyeh. Craig S. Hood,

Robert H. Hollander, and Chuck Harris. For assistance in the

field In collecting specimens, I am Indebted to R. J. Baker. C. S.

Hood, R. H. Hollander, C. Harris, Cindy G. Dunn, Kala J. Haiduk.

and David A. McCullough. Special thanks go to my parents who

provided their moral support ajid encouragement. Finally, and not

least, I wish to thank my husband. Klhyung Song, for his constant

encouragement and support during this study. The collecting trip

to Mexico was partially supported by a research grant to Dr. R. J.

Baker.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS li

LIST OF FIGURES 1 v

I. INTRODUCTION 1

II. MATERIALS AND METHODS 4

III. RESULTS 6

IV. DISCUSSION 17

LITERATURE CITED 20

APPENDIX 22

111

LIST OF FIGURES

FIGURE PAGE 1. Geographic distribution of the two cytotypes of

Cratogeomys castanops 3

2. The G-banded karyotype of Cratogeomys castanops of 2N = 46 cytotype 9

3. The C-banded karyotypes of Cratogeomys castanops of 2N = 46 cytotype. demonstrating centromerlc distribution of heterochromatln 10

4. The G-banded karyotype of Cratogeomys castanops of 2N = 42 cytotype 11

5. The C-banded karyotype of Cratogeomys castanops of 2N = 42 cytotype 12

6. The G-banded karyotype of Cratogeomys gymnurus 13

7. The C-banded karyotype of Cratogeomys gymnurus 14

8. A composite comparison of haplold complement of Cratogeomys castanops of 2N = 42 and Cratogeomys gymnurus showing apparent G-bajided homologies 15

9. A composite comparison of haplold complements of the two cytotypes (2N = 46 and 2N = 42) of Cratogeomys castanops depicting proposed homologies.... 16

10. Phylogenetlc tree presenting the primitive elements of the castanops group 23

IV

I. INTRODUCTION

Cratogeomys castanops Is a highly fossorlal mammal that

lives virtually Its entire life below ground. The genus Crato­

geomys consists of seven species (Honeycutt and Williams, 1982),

most of which occur In the transverse volcanic chain of Mexico.

One species, Cratogeomys castanops, ranges northward as far as

Colorado (Figure 1). Russell (1968b) distinguished two subspecies

groups within C. castanops on the basis of cranial and body size.

Those with smaller crania were designated the subnubllus group and

those with larger skulls, the excelsus group. Distinctness of the

two groups and lack of evidence of Interbreeding between them sug­

gested to Russell (1968b) the possibility that they represent dis­

tinct species.

An alternative view of the Cratogeomys castanops group Is

based on chromosomal data. Two chromosomal races are recognized

within C. castanops based on non-dlfferentlally stained karyo­

types. Berry and Baker (1972) suggested that these probably re­

present two species, but the cytologlcal boundaries of the two

species was very different from that suggested by Russell (1968b)

based on skin and skull morphology. Berry and Baker (1972) re­

ported that all Cratogeomys castanops they examined north of 25

degree N. latitude had a diploid (2N) of 46 and a fundamental num­

ber (FN) of 86, whereas specimens of C. castanops taken south of

25 degree N. latitude possessed 2N = 42, FN = 78.

This study was designed to determine the nature and extent

of chromosomal evolution that distinguishes the two cytotypes. By

using another species of Cratogeomys (C. gymnurus) as an outgroup.

I hope to determine the direction of evolution in this group. The

ultimate goal Is to determine If the chromosomal data are best in­

terpreted as representing two species and to determine which of

the two cytotypes has the most derived karyotype.

Both G- and C-bandlng techniques were used in resolving the

above questions. G-band patterns have been used to demonstrate

types of chromosomal rearrangements and overall conservatism of

gene sequences for mammals, especially as related to the euchro-

matlc portion of the karyotype (Baker e;b al., 1983; Koop ejt al. .

1985; Stangl and Baker, 1984). Analysis of C-bandlng Is used to

determine variation in the amount of constitutive heterochromatln.

By using both banding techniques, it should be possible to

determine which rearrangements are euchromatlc and which are

heterochromatlc.

I am following the taxonomy of Cratogeomys by Honeycutt ajid

Williams (1982).

FIGURE 1. Geographic distribution of the two cytotypes of Crato­geomys castanops. Specific localities where the specimens were collected are 1) U.S.A. Texas: Lubbock Co. 5 ml. N-. on Univ. Ave. (2N - 46 Cytotype). 2) Mexico: Zacatecas. 10.5 ml. S. of Concep-clon del Oro on Hwy 54. (2N = 42 Cytotype).

II. MATERIALS AND METHODS

Specimens used In this study were collected from natural

populations by using live traps for gophers (Baker and Williams,

1972). Specimens examined and collecting localities are described

in the appendix.

Specimens were subjected to two to four days of yeast stress

to Increase mitotic activity and karyotyped by the method of Lee

and Elder (1980). G-bands were obtained by trypsin digestion and

Glemsa staining (Seabrlght. 1971, as modified by Baker et al..

1982; Baker and Qumslyeh, 1985). and C-band procedures were those

described by Stefos and Arrlghl (1971. as modified by Baker and

(Qumslyeh, 1985).

A minimum of six G-banded cell spreads were examined for

each animal to document the degree of consistency of banding pat­

terns within each Individual. Three to ten mitotic spreads of

each Individual listed In the appendix were photographed and com­

pared In a side by side comparison with other individuals studied.

Banding results were photographed using 4 X 5 Kodak Plus-X film

and printed on Kodabromlde F-5 paper. Chromosomes were arranged

in order of decreasing size. Homologies were determined by side-

to-slde comparison of differential longitudinal staining patterns

of each chromosome. Figures representative for each species ,vere

prepared from one complete cell. Figures which are a comparison

5 between two cytotypes are a haplold complement from a single cell

from the respective individuals.

Photographic negatives and voucher-slides were deposited in

the laboratory of Dr. Robert J. Baker, Department of Biological

Sciences, Texas Tech University. Museum skins accompanied by par­

tial body skeletons were retained as voucher specimens and de­

posited In The Museum, Texas Tech University. Electrophoretlc

tissues (liver, heart, and kidney) were saved for all specimens

used In this study.

III. RESULTS

The 2N = 46 Cytotype of C. castanops—All autosomes in this

group are biarmed except two pairs (Figs. 2 and 3). One of the

two pairs of acrocentrics Is almost entirely heterochromatlc.

Heterochromatln Is restricted to or near the centromerlc regions

on all other autosomes. There is an Interstitial C-posltlve band

near the centromere on the long arm of the second largest chromo­

some. Also some interstitial C-bands were observed on several

small chromosomes. As noted in Berry and Baker (1972). the sex

chromosomes consist of a large metacentric or submetacentric X and

a medium-sized acrocentric Y, although the size of the sex chromo­

somes Is somewhat variable between individuals. The Y chromosome

is almost entirely heterochromatln (Figs. 2 and 3).

The 2N = 42 Cytotype of C. castanops—All autosomes are

biarmed except one small pair of acrocentrics which is entirely

heterochromatlc (Figs. 4 and 5). As In the 2N = 46 cytotype there

is interstitial C-band material on the second largest chromosome

and an addition of a large block of heterochromatln on the third

largest pair. The X chromosome is a large metacentric, and the Y

chromosome Is a medium-sized, entirely heterochromatlc acrocentric

element (Figs. 4 and 5).

Chromosomes are numbered according to the karyotype of the

2N = 42 cytotype of C. castanops.

7

The Outgroup—The G- and C-banded karyotypes of Cratogeomys

gymnurus are shown in figures 6 and 7. All chromosomes are

biarmed. including the sex chromosomes, and most heterochromatln

is restricted to or near the centromerlc regions (2N = 38). There

are several Interstitial C-posltlve bands on larger chromosomes

(Fig. 7). The Y chromosome is almost entirely heterochromatlc.

In figure 8. haplold complements of G-banded karyotypes of

C. castanops of 2N = 42 type and C. gymnurus (2N = 38) are com­

pared. The proposed homologies between the two species are shown

In box A. The remainder of the haplold complements for which

homology Is uncertain are shown In box B. The G-banded karyotype

of C. gymnurus. an outgroup of this study, shows homology of

chromosome numbers 1-13 when compared with the 2N = 42 cytotype

of C. castanops. An Inversion distinguishes the short arm of

chromosome number 3 of C. gymnurus and C. castajiops. Based on

cladlstlcal analysis. It is assumed that the homologies between C.

castanops (2N = 42) and C. gymnurus (2N = 38) are primitive ele­

ments for the castanops group.

Haplold complements of G-banded karyotypes of the two

chromosomal races of C. castanops are compared in figure 9. Box A

of figure 9 presents the chromosomes which appeared to be com­

pletely homologous between the two cytotypes. Chromosomes 10. 11

and 12 In box B appear partially homologous but some rearrange­

ments occurred in at least on one arm of one cytotype. It appears

probable that inversions occurred that distinguish these three

8 pairs of chromosomes. The remainder of the haplold complements

for which homology is uncertain are shown in box C. The G-banded

karyotypes of the two cytotypes of Cratogeomys castanops indicate

complete homology of chromosome numbers 1 - 8 , 14. 15, 16, 17. 18

and 19, and Incomplete homology of chromosomes 9, 10, 11, 12, 13

ana 20 m tne 2N = 42 cytotype ana 10, 11. 12. A, B. c, o and E m

the 2N = 46 cytotype.

?? ^ * Aa •'^ '̂ »*'

8

zz. y 5 6

^?B •?5 ^ J ^T- • • »6 5S ni - - •̂-

10 11 12 14

4f 15

B

^4 16

^ ^ ^

«r

C

* ^9

17

^

D

« •. ^

18

^ ^ ^

E

• "

19

C^ ^ F _

X

«« a

A

4 «

Y

FIGURE 2. The G-banded karyotype of Cratogeomys castanops of 2N = 46 Cytotype. X and Y chromosomes are illustrated at the lower right.

10

>

t

• • .

% " • •

r I ft

"

4\% * «

'< I X Y

FIGURE 3. The C-banded karyotype of Cratogeomys castanops of 2N = 46 Cytotype, demonstrating centromerlc distribution of heterochromatln. Interstitial C-bands are visible on several pairs of autosomes.

11

as 1

l{ 6

iii 11

16

2

7

12

17 18

a 3

If 8

1̂ 13

•

19

4 5

{if** 'it 9 10

14 15

20 ^ ^

FIGURE 4. The G-banded karyotype of Cratogeomys castanops of 2N = 42 Cytotype. X chromosomes are illustrated at the lov.er right.

12

)i il V ^ 0*

w 9r

II \\ C> '̂ -̂ I I ' • t t I t t l

X Y

FIGURE 5. The C-banded karyotype of Cratogeomys castanops of 2N = 42 Cytotype. X and Y chromosomes are illustrated at the lower right. The Y chromosome is almost entirely heterochroraouln

#4

a 1

m 6 7

10 11

c d

12

e

H *J 8

13

^ E

i

X Y

FIGURE 6. (2N = 38) r i g h t .

The G-banded karyotype of Cratpgeomyj gymnuras X and Y chromosomes are i l l u s t r a t e d at the lo.ver

14

FIGURE 7. The C-banded karyotype of Cratogeomys gymnurus. X and Y chromosomes are illustrated at the lower right.

15

14 15 16 17̂ 18 19 20

k a S H B

FIGURE 8. A composite comparison of haplold complements of Crato-geomys castanops of 2N = 42 and Cratogeomys gymnurus (2:: = 33) showing apparent G-banded homologies. Chromosome on left hand side of each comparison is from a representative of 2!: = 42 Crato­geomys castanops. whereas right hand chromosome is from a repre­sentative of Cratogeomys gymnurus. Box A presents the chromosomes which appeared to be completely homologous except pair 3. An In-verslon(*) distinguishes the upper arm of chromosome number 3 of these two species. The remainder of the haplold complements for which homology is uncertain are shown in box B.

iO

FIGURE 9. A composite comparison of haplold complements of the two cytotypes of C. castanops depicting proposed homologies. Chromosome on left hand side of each comparison is from a repre­sentative of the 2N = 46: FN = 86 ^^^f ̂ P^^^^^?;^^^^^!^^. p' - 78 Texas whereas right hand chromosome Is from the 2N - 42. K. - fii Stot^pe from Mexico, Zacatecas. Box A presents larger chromo­somes for which homologous segments are hypothesized^ c'lVnt. 11 ajid 12 m Box B appear partially homologous^ Box C presents the remainder of the haplold complements, for -.-hich no homology xs implied.

IV. DISCUSSION

The two chromosomal races of what are currently recognized

as a single species have fourteen pairs of autosomes (1, 2. 3. 4.

5, 6, 7. 8. 14, 15, 16. 17, 18. and 19), that appear to be un­

changed between the two. Additionally, the X chromosome has a

homologous portion that appears unchanged. Pairs 10, 11, and 12

appear to be altered by an inversion, and, because the condition

for these three chromosomal pairs is the same in the outgroup (C.

gymnurus) and the 2N = 42 cytotype, it is concluded that the

primitive condition is found in the 2N = 42 cytotype, and all

three chromosomes are derived in the 2N = 46 cytotype. Moreover,

the two cytotypes are distinguished by rearrangements that change

pairs 9, 13, and 20 of the 2N = 42 cytotype into pairs A. 3. C. D,

and E of the 2N = 46 cytotype (or vice versa) . Kov.ever because 9

and 13 both appear in the outgroup in the same condition as found

In the 2N = 42 cytotype, the rearranged condition is likely found

In the A, B, C, D, and E chromosomes of the 2N = 46 cytotype. Al­

though there are few bands on some of these smaller elements, it

is a matter of conjecture how 9. 13, and 20 were rearranged into

A. B. C, D, and E. But probably three or more rearrangements v.ere

required to produce the observed differences. Therefore, the 2\\ =

42 cytotype and the 2N = 46 cytotype are probably distinguished by

six or more rearrangements, and most if not all were established

in the 2N = 46 cytotype.

17

18 Are these chromosomal differences sufficient to result In

genetic Isolation? In the absence of breeding data the matter is

conjectural, but this Is certainly more chromosomal rearrangement

than usually distinguishes two closely related species of mammals.

At this time, probably the best course of action is to recognize

two species wnicn empnasizea tnat tne two cytotypes represent dis­

crete genetic entitles. If two species are recognized then the

two species would probably be named C. castanops (Balrd, 1852) (2N

= 46 cytotype) and C. goldmanl Merrlam (1895) (2N = 42 cytotype).

There Is a problem in assigning a name to the 2N = 42 cytotype

(Berry and Baker, 1972). The type locality of C. goldmanl is well

withm the range of the 2N = 42 cytotype. but, although Indivi­

duals collected from near the type locality, Canltas, Zacatecas,

of goldmanl had a 2N = 42. they differed morphologically from the

type specimen and topotypes of goldmanl. If it should prove that

goldmanl is from the 2N = 46 cytotype, then C. subnubllus (l.'elson

and Goldmaji, 1934) would be the oldest available name for that

species.

Although it is attractive to propose an adaptive role for

the karyotype, data documenting such are few and have been

reviewed by Baker et al. (1983a). Robblns et al. (1983) suggested

that in Peromyscus and Onychomys there was a positive correlation

between number of derived rearrangements In a species and the

total geographic range occupied by that species. A similar

correlation may also be found in Cratogeomys .vhere C. cai.'tanops Is

19 the most widely distributed of the species of Cratogeomys. C.

castanops is the only species of the genus to occur outside the

Mexican central core region. Although it Is tempting to suggest

that the rearrangements found In the 2N = 46 castanops may have

been critical to the species Invading this new region, such is

only an hypothesis.

LITERATURE CITED

Baker, R.J . R. K. Chesser. B. F. Koop. and R. A. Hoyt. 1983a. Adaptive nature of a chromosomal rearrangement: dif­ferential fitness In pocket gophers. Genetlca, 61:161-164.

Baker, R.

Baker. R,

J., M. W. Haiduk. L. W. Robblns. A. Cadena. and B. F. Koop. 1982. Chromosomal studies of South American bats and their systematic Implications. Pp. 303-327. In Mammalian biology in South America (M H. H. Genoways. eds.). Spec. Publ. Ser. Lab. Ecol.. Univ. Pittsburgh, 6:1-539.

A. Mares and Pymatunlng

J., B. F. Koop, and M. W. Haiduk. 1983. Resolving sys­tematic relationships with G-bands; A study of five genera of South American crlcetlne rodents. Syst. Zool.. 32:403-416.

Baker, R. J., and M. B. Qumslyeh. 1985. Methods In Chlropteran mitotic chromosomal studies. (In press) Methods for behavioral and ecological studies of bats. (Thomas E. Kunz. ed.), Smithsonian Inst. Press.

Baker, R. J., and S. L. Williams. 1972. A live trap for pocket gophers. J. Wlldl. Manage.. 36;1320-1322.

Berry, D. L. and R. J. Baker. 1972. Chromosomes of pocket gophers of the genus Cratogeomys. subgenus Cratogeomys. J. Mamm., 53:303-309.

Hart. E. B. 1978. Karyology and evolution of the plains pocket gopher Geomys bursarlus. Occas. Papers Mus. Nat. Hist., Univ. Kansas. 71:1-20.

Honeycutt, R. L., and S. L. Williams. 1982. Genie differen­tiation In pocket gophers of the genus Cratogeomys. with comments on Intergeneric relationships in the subfamily Geomylnae. J. Mamm., 63(2):208-217.

Honeycutt, R. L., and D. J. Schmldly. 1979. Chromosomal and morphological variation In the plains pocket gopher, Geomys bursarlus. in Texas and adjacent states. Occas. Papers Mus., Texas Tech Univ., 58:1-54.

Koop, B. F., R. J. Baker. M. W. Haiduk. and M. D. Engstrom. 1935. Cladlstlcal analysis of the primitive G-band sequences for the karyotype of the ancestor of the Crlcetldae complex of rodents. Genetlca. 64:199-203.

20

21 Lee. M. R.. and F. F. B. Elder. 1980. Yeast stimulation of bone

marrow mitosis for cytogenetic Investigations. Cytogenet. Cell Genet.. 26:36-40.

Merrlam. C. H. 1895. Monographic revision of the pocket gophers, family Geomyldae (exclusion of the species of Thomomys). N. Amer. Fauna.. 8;1-258.

Nelson. E. W., and E. A. Goldman. 1934. Revision of the pocket gophers of the genus Cratogeomys. Pro. Biol. Soc. Washington., 47;135-153.

Robblns. L. W., M. P. Moulton and R. J. Baker. 1983. Extent of geographic range and magnitude of chromosomal evolu­tion J. Blogeography. 10;533-541.

Russell. R. S. 1968a. Evolution and classification of pocket gophers of the subfamily Geomylnae. Univ. Kansas Publ. Mus. Nat. Hist.. 16:473-579.

Russell. R. S. 1968b. Revision of pocket gophers of the genus Cratogeomys. Univ. Kansas Publ., Mus. Nat. Hist., 16:581-776.

Seabrlght, M. 1971. A rapid banding technique for human chromo­somes. Lancet 2:971-972.

Stangl, F. B., Jr.. and R. J. Baker. 1984. A chromosomal sub­division In Peromyscus leucopus: implications for the subspecies concept as applied to mammals. Pp. 139-145, In Festschrift for Walter W. Dalquest in honor of his sixty-sixth birthday (N. V. Horner, ed.). Dept. Biol., Midwestern State Univ.. Texas, 163 pp.

Stefos. K., and F. E. Arrlghl. 1971. Heterochromatlc nature of the W chromosome in birds. Exp. Cell Res.. 68:228-231.

APPENDIX : SPECIMENS EXA:'II:;ED

Specimens examined were as follows:

TK numbers are karyotype numbers used to reference tissues and voucher specimens.

Cratogeomys castanops. 2N = 46 Cytotype.

TK 20395 (F). TK 20397 CM). U.S.A. Texas; Lubbock Co., 2 ml N. on University Ave.

TK 26238 (F), TK 27143 (F). TK 27144 (M). U.S.A. Texas; Lubbock Co., 2 ml. W. of Loop 289 on 4th St.

TK 27145 (.M) . TK 27146 (F) . TK 27147 (M) , TK 27149 CF) . U.S.A. Texas; Lubbock Co. 5 ml. W. on Univ. Ave.

Cratogeomys castanops. 2N = 42 Cytotype.

TK 27105 (F) . Mexico; San Luis Potosl, 12 mi. W. of CD. del Mlaz.

TK 27647 (F). TK 27650 (F). TK 27651 (M). TK 27652 (F), TK 27655 <M). TK 27656 CM). TK 27567 (M). TK 27659 (M), TK 27661 (M). TK 27663 (F), TK 27664 (M).

Mexico; Zacatecas. 10.5 ml. S. of Concepcion del Oro on Hwy 54.

Cratogeomys gymnurus (2N = 38).

TK 28550 (M). TK 28551 (M). TK 28552 (F), TK 28554 <M). Mexico; Michoacan. 9.4 ml SE of Patzcuaro.

22

23

OUTGROUP C. castanops

C. gymnurus (2N = 38) 2N = 42 2M = 46

i::v. 10

:::v. ii

INV. 12

?Fissio::s

Primitive chromosomes for C. castanops: 1-8 as In the 2N = 46 cytotype and 1-13 as in the 2N = 42 cytotype.

FIGURE 10. Phylogenetlc tree presenting the primitive elements of the castanops group.