Texas Reports on Biology and Medicine,
Volume 27, Number 2, Summer, 1969
A KARYOLOGICAL ANALYSIS OF TWO CYPRINID
FISHES, NOTEMIGONUS CRYSOLEUCAS AND
NOTROPIS LUTRENSIS*
MICHAEL LIEPPMAN AND CLARK HUBBS
Department of Zoology, University of Texas at Austin,
Austin, Texas 78712
Chromosome number and morphology; karyotypes; cytological
preparation of fish chromosomes; cyprinid minnows
ABSTRACT
A cytological study of two species of Texas cyprinid minnows, Notemigonus
crysoleucas and Notropis lutrensis, found a diploid chromosome number of 50 for
both species. A modification of McPhail's method for karyological preparation provided a reliable source of good mitotic figures.
INTRODUCTION
Karyological characteristics have been used as a valuable aid to
taxonomic and evolutionary studies in many groups of plants and
animals. Few ichthyologists have made use of fish cytology because
the chromosomes are small and the available techniques have often
yielded questionable counts and minimal morphological detail.
The intrafamilial supergeneric classification of cyprinid fishes,
which includes the largest number of fish species, is controversial or
nonexistent. Greenwood, et al (1966) criticized the systems proposed
by Regan (1911), Brittan (1954), and Ramaswami (1955), but did
not propose an alternate system. Cytotaxonomic studies of cyprinids
could, therefore, provide data that might resolve controversy based on
osteological studies. This report presents karyological data on two
cyprinids, Notemigonus crysoleueas and Notropis lutrensis, and an
improved cytological preparation technique that may permit additional studies on cyprinids.
MATERIAL
Specimens of both species were seined from the Colorado River below Tom Miller
* Supported by Clinical Cancer Training Grant CA 8000-02S1 from National
Cancer Institute to the University of Texas M. D. Anderson Hospital and Tumor
Institute at Houston. Rec'd for publication Dec. 9, 1968.
Texas Rep Biol Med 27:2, 1969
428
LIEPPMAN AND HUBBS
Dam at Austin, Texas, from Shoal Creek in Austin, Texas, and from Braes Bayou
in southwest Houston, Texas. Subsequent specimens of Notemigonus were obtained
through the courtesy of the Riverside Tackle Shop in Austin in order to check possible chromosomal variation between native and hatchery-reared specimens. No
variation was found, so these hatchery specimens were included in the data presented
in Table 1.
METHODS
Karyological methods involving the use of squash preparations of gill arch epithelial cells gave satisfactory results. McPhail and Jones' description of gill arch
technique (1966) was used with modifications which improved the results. Freshly
netted fish were brought into the laboratory in their native water in styrofoam boxes.
This water was aerated, the fish identified, and those in poor condition discarded.
The cytological procedure follows:
1. Average specimens of both species, weighing from 1 to 2 g, received intramuscular injections of 0.03 to 0.04 ml of 0.025% colchicine solution, the amount
varying with individual weight. Injection was made in the right epaxial muscle
mass posterior to the right gill arch and operculum. The specimen was maintained
in the native water for 3 hours at room temperature before sacrifice. Failure to
aerate water would inhibit the mitotic activity of the gill arch epithelial cells, resulting in a lowered mitotic index.
2. With jeweler's forceps and fine-tip scissors, the forward 3 gill arches on the
right side of the animal were removed and were then individually incubated in a
hypotonic solution. For freshwater specimens taken from regions where little or
no saltwater intrudes into freshwater streams or rivers, the ideal treatment was incubation in double-distilled water for 30 minutes at room temperature. For freshwater fish native to slightly brackish water, such as Braes Bayou in Houston, a 1%
solution of TM (Maio and Schildkraut, 1966) for a duration of 20 minutes provided
ideal hypotonicity—the cell integrity was maintained, and the cells squashed successfully with minimum breakage of the cell membranes. Distilled water treatment
of a brackish-water fish gill arch would cause the cells to explode.
3. The right gill arches having been removed and the left side of the fish left unmarked for identification purposes, the sex was determined by microscopic examination. All specimens were sexually mature. Midventral incision disclosed roe-filled
coe!oms and the displaced Wolfian tubules of females. Males were examined for
presance of testes. Notropis males were easily identified by their blue bodies and red
fins. Specimens were preserved in 10% formalin solution for one week, then transferred to 50% ethyl alcohol to preserve their coloration.
4. Individual gill arches were carefully transferred to 50% acetic acid and left
for 20 minutes. Care in transfer assured minimal loss of the swollen epithelial cells.
5. Arches were air-dried for one minute after removal from the acid. This technique dissociated the cells while leaving the cellular membranes intact.
6. Individual gill arches were transferred to individual coverslips. One drop of
1 % acetic orecin was introduced to each arch, then the entire coverslip was covered
with a watch-glass to prevent precipitation of the stain during the 10-minute staining period.
7. With jeweler's forceps, each gill arch was then gently agitated over the coverslip, allowing the gill arch to shed the epithelial cells. Clumps of cells were removed.
Texas Rep Biol Med 27:2, 1969
430
LIEPPMAN AND HUBBS
i *
t
4
41
411
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.;
a
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tit 44,w't vA.4 4
,
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FIG. 2. Chromosome spread of a female Notropis lutrensis.
lutrensis was found to be 50 (Table 1). There was no polymorphism
among the epithelial cells of gill arches.
Representative karyotypes of the two species are presented in Figs.
3 and 4. Matching of homologous pairs was rather subjective since
many elements were morphologically similar. However, it is clear
from these figures that the two species are quite different in respect to
their karyological characteristics. N. crysoleucas has 8 pairs of metacentric or near metacentric choromosomes, 12 pairs of submetacentric
or subtelocentric, and 5 pairs of telocentric, whereas N. lutrensis has
5, 16 and 4 pairs respectively. Furthermore, N. crysoleucas has one
pair of outstandingly long telocentrics lacking in N. lutrensis.
We find no karyotypic sexual dimorphism in either species that may
TABLE 1
Chromosomal data for Notropis and Notemigonus
Species
Notropis lutrensis
Notropis lutrensis
Notemigonus crysoleucas
Notemigonus crysoleucas
Sex
female
male
female
male
47
0
2
0
0
48
2
4
2
1
49
50
8 126
0 122
3 84
4 89
51
6
2
0
2
52
No.
No.
cells specimens
0 142
1 131
0 89
0 96
41
64
34
27
Texas Rep Biol Med 27:2, 1969
429
KARYOLOGICAL ANALYSIS OF CYPRINID FISHES
8. A slide was placed over the coverslip and turned coverslip side up. A piece of
filter paper was placed over the coverslip area and the air bubbles were gently
squeezed out by light thumb pressure applied from the center of the coverslip until
the thumb was off the coverslip. This procedure was repeated, with increasing pressure. After the final press, the slide was examined microscopically for the quality
of the preparation. If the metaphase chromosomes were not sufficiently spread, the
slide was heated by passing it over an alcohol-lamp flame, protected by filter paper,
and repressed with the thumb.
9. The slides were sealed with Kronig cement and kept in a refrigerator. Microscopic observations were made with the aid of a Zeiss phase contrast microscope.
RESULTS
Two metaphases, one from each species (Figs. 1 and 2), show the
type of chromosome spreads which we analyzed for karyotypes. Proper
concentration of colchicine and duration of colchicine treatment were
most important factors in procuring good metaphase figures.
Invariably the forward-most gill arch contained a higher mitotic
index than others. If this conclusion also applies to other fishes, it
should be feasible to study the chromosomal characteristics of any individual by removing one gill arch without necessarily sacrificing the
fish. This procedure would be advantageous in cases of hybrids or of
rare species.
The diploid number of both Notemigonus crysoleucas and Notropis
4141
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),
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et
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FIG. 1. Chromosome spread of a female Noternigonus crysoleucas.
Texas Rep Biol Med 27:2, 1969
431
KARYOLOGICAL ANALYSIS OF CYPRINID FISHES
NW XI WI
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st
Alt Km
dtt Alt Jos iSh
sa XX AA
811 A.A
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FIG. 3.
Karyotype of Noterrzigonus crysoleucas. Upper male, lower female.
reflect sex chromosomes such as those found for Gambusia affinis by
Chen and Ebling (1968).
DISCUSSION
A comparison of karyotypes among related fishes may emphasize
chromosome number, arm number, or DNA volume. One treated
alone can cause misleading conclusions. Centromeric fusion can reduce
Texas Rep 13iol Med 27:2, 1969
432
LIEPPMAN AND HUBBS
m
Ali
LA
V, Ai AA 16 ita
a*
TILL
St
t
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42 VA Ut
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AN 41. 14
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FIG. 4. Karyotype of Notropis lutrensis. Upper male, lower female.
chromosome number without an equivalent fundamental change in
chromatin content. Similarly unequal reciprocal translocations can
alter the arm numbers but not alter chormatin significantly (Booke,
1968). Polyploidy can cause marked changes that imply greater phylogenetic effects than have occurred (Ohno, et al. 1967).
Very little published evidence is available about cyprinid karyotypes. We find no reports on any North American species in the reviews by Post (1965) and Roberts (1967). McPhail and Jones (1966)
Texas Rep Biol Med 27:2, 1969
434
LIEPPMAN AND HUBBS
fasciatus are remote on gross morphological grounds. The other two,
Scardinus erythrophthalmus and Abramis branza (Post, 1965), are
leuciscine cyprinids to which the American genera are related (Regan,
1911, [929). Furthermore, the close relationship of Abramis and Notemigonus is extensively recorded in the literature, (Jordan and Evermann, 1898; Berg, 1949) . It is unfortunate that Post did not publish a
plate of his Abramis slide because the morphology might provide additional support for the assumed close relationship.
Close relationship between a monotypic genus, Notemigonus, from
eastern North American, and a western Eurasian genus, Abramis
(Berg, 1949; Banarescu, 1964), presents some biogeographic complications. Darlington (1957) and Regan (1929) assumed that North
American cyprinids entered the New World via the Bering Strait. The
absence of either form in the intervening connecting area suggests a
migration across the Atlantic.
ACKNOWLEDGMENTS
The authors wish to express their sincere thanks to Dr. T. C. Hsu
for his advice and assistance. This research was supported in part by
National Science Foundation grant GB 6657 awarded to Dr. Hsu and
GB 6429. We also thank Craig L. Campbell for assistance in acquiring
the specimens.
REFERENCES
1. Banarescu, P.: Fauna republicii populare Romine. Pisces-Osteichthyes. Academia
Republicii Populare Romine, 13: 959, 1964.
2. Berg, L. S.: Freshwater fishes of the U.S.S.R. and adjacent countries. Akademiya
Nauk SSSR Zoologicheskii Institut (Israel Program for Scientific Translations,
1964) 2: 496, 1949.
3. Booke, H. E.: Cytotaxonomic studies of the coregonine fishes of the Great Lakes,
U.S.A.: DNA and karyotype analysis. Fisheries Research Board, Canada, 25:
1667-1687, 1968.
4. Brittan, M.R.: A revision of the Indo-Malayan freshwater fish genus Rasbora.
Monographs Inst. Sci. and Tech. Manila, 3: 224, 1954.
5. Chen, T. R., and A. W. Ebeling: Karyological evidence for female heteogamety
in the mosquito fish, Garnbusia affinis. Copeia, 1968: 70-75, 1968.
6. Darlington, P. J., Jr.: Zoogeography: The Geographical Distribution of Animals.
John Wiley, New York, 675 pp., 1967.
7. Gravell, M., and R. G. Malsberger: A permanent cell line from the fathead
minnow (Pimephales promelas). Ann. N.Y. Acad. Sci., 126: 555-565, 1965.
8. Greenwood, P. H., D. E. Rosen, S. H. Weitzman, and G. S. Myers: Phyletic
studies of teleostean fishes, with a provisional classification of living forms. Bull.
American Museum Natural History, 131: 339-456, pls. 21-23, 1966.
Texas Rep Biol Med 27:2, 1969
KARYOLOGICAL ANALYSIS OF CYPRINID FISHES
433
used Rhinichthys evermanni to illustrate their gill arch technique.
They showed that it had 2n = 50 chromosomes and their figure indicates most chromosomes are metacentric. In addition, we can find only
the reports or stem line chromosomes in Pimephales promelas in
Grave11 and Malsberger (1965) and Levan, et al. (1966). The former
had modal numbers of 2n = 49, 50 or 51 based on cultured cells. It is
apparent that karyotype changes may occur in culture so that use of
this technique for precise taxonomic comparisons must be done with
caution. For example, cell 7 of Levan, et al., has 1 less chromosome
than the other 9 in the same culture, and additional variations occurred in arm length. Therefore, intrahemispheric valid comparisons
are limited to R. evermanni and our two species, which clearly are different as is each from that of the P. promelas karyotype published by
Levan et al.
Post (1965) lists the chromosome number of 15 cyprinids and presents figures of spermatocyte metaphase plates for 3. Thirteen (including the 3 illustrated) have a haploid number of 24. The 3 figures
indicate that some (notably in Barbus oligolepis) chromosomes are
metacentric so that the arm numbers often exceed n=24. It is obvious
that chromosome number varies among closely related cyprinids because two Asian species, Barbus tetrazona (n=25) and B. fasciatus
(n=26) (Ohno, et al., 1967), have more chromosomes than the 6 species of Barbus studied by Post. Unfortunately, the only karyotype
studies and DNA analyses on cyprinid fishes are by Ohno and his
colleagues on 3 "diploid" and 2 "tetraploid" species, so that phylogenetic speculation must be restricted to number alone.
Most teleost fishes have n=24 chromosomes and the majority of
those that do not, have fewer (Post, 1965 and Roberts, 1967). Therefore, reduction in chromosome number occurs more often than an
increase. Moreover, most of the high numbers belong to the primitive
order Isospondyli. If one assumes that the primitive cypriniform fish
had n=24, an increase from that number would be of evolutionary
significance. The chromosome number of all 3 (or even 4) North
American cyprinid fishes for which a count is available is n=25. The
number of Old World species available for comparison is minimal and
only Barbus tetrazona and Labeo chrysophekadion (Muramoto et al.,
1968) have the same number. Both are quite different from N otemiionus and Notropis on gross morphology, and Labeo has many more
"subterminal" chromosomes than do the North American minnows
studied. In contrast, if one assumes that the North American cyprinids
may have fused two pairs of chromosomes into one, the 3 known n=26
species are available for comparison. The relationships with Barbus
Texas Rep Biol Med 27:2, 1969
KARYOLOGICAL ANALYSIS OF CYPRINID FISHES
435
9. Jordan, D. S., and B. W. Evermann: The fishes of North and Middle America.
Bull. U.S. Nat. Mus., 47, pt. 1: 1-1240, 1898.
10. Levan, A., W. W. Nichols, M. Peluse, and L. L. Coriell: The stem line chromosomes of three cell lines representing different vertebrate classes. Chromosoma,
18: 343-358, 1966.
J. J., and C. L. Schildkraut: Isolated mammalian metaphase chromosomes. I. General characteristics of nucleic acids and proteins. 1. Molec. Biol.,
II. Maio,
24: 29-39, 1967.
12. McPhail, J. D., and R. L. Jones: A simple technique for obtaining chromosomes
from teleost fishes. J. Fish. Res. Bd., Canada 23: 767-768, pl. 1, 1966.
13. Muramoto, J., S. Ohno, and N. B. Atkin: On the diploid state of the fish order
Ostariophysi. Chromosoma, 24: 59-66, 1968.
14. Ohno, S., J. Muramoto, and L. Christian: Diploid-tetraploid relationship among
old-world members of the fish family Cyprinidae. Chromosoma, 23: 1-9, 1967.
15. Post, A.: Vergleichende Untersuchungen der Chromosomenzahlen bei Susswasser-Teleosteern. Zeitschrift Zool. Syst. Evolut.-forsch., 3: 47-93, 1965.
16. Ramaswami, L. S.: Skeleton of cyprinoid fishes in relation to phylogenetic
studies. 7. The skull and Weberian apparatus of Cyprininae (Cyprinidae) Acta
Zool. Stockholm, 36: 127-158, 1955.
17. Regan, C. T.: The classification of the teleostean fishes of the order Ostariophysi
1. Cyprinoidea. Ann. Mag. Nat. Hist., ser. 8, vol. 8: 13-32, 1911.
18. Regan, C. T.: Fishes. In: Encyclopedia Brittanica, 14th ed. 9: 305-329, 1929.
19. Roberts, F. L.: Chromosome cytology of the Osteichthyes. Progressive F ishCulturist, 29: 75-83, 1967.
Texas Rep Rio! Med 27:2, 1969