A KARYOLOGICAL ANALYSIS OF TWO CYPRINID FISHES, NOTEMIGONUS CRYSOLEUCAS AND NOTROPIS LUTRENSIS*

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1 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 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 S1 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.

2 428 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.

430 LIEPPMAN AND HUBBS i t * 41 4 * ;.; a 411 " 4 4tric#74 4 4 tit 4,w't va. 4 4 icy c eci, i t * 4 4," % +i i* A, 4 * FIG. 2.

Representative karyotypes of the two species are presented in Figs. 3 and 4.

However, it is clear from these figures that the two species are quite different in respect to their karyological characteristics. N.

lutrensis has 5, 16 and 4 pairs respectively. Furthermore, N. crysoleucas has one pair of outstandingly long telocentrics lacking in N. lutrensis.

3 430 LIEPPMAN AND HUBBS i t * 41 4 * ;.; a 411 " 4 4tric# tit 4,w't va. 4 4 icy c eci, i t * 4 4," % +i i* A, 4 * 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 Sex No. No. cells specimens Notropis lutrensis female Notropis lutrensis male Notemigonus crysoleucas female Notemigonus crysoleucas male

KARYOLOGICAL ANALYSIS OF CYPRINID FISHES 429 8. A slide was placed over the coverslip and turned coverslip side up.

coverslip. This procedure was repeated, with increasing pressure. After the final press, the slide was examined microscopically for the quality of the preparation.

4 KARYOLOGICAL ANALYSIS OF CYPRINID FISHES 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 :1Ih % *I * et Ar V ( 44,,* P * 0 Tr i t ), dit *IA a 4 II t doe a e lb *Az* FIG. 1. Chromosome spread of a female Noternigonus crysoleucas.

Upper male, lower female. reflect sex chromosomes such as those found for Gambusia affinis by Chen and Ebling (1968).

5 KARYOLOGICAL ANALYSIS OF CYPRINID FISHES 431 m NW XI WI Alt Km st dtt Alt Jos ish sa XX AA 811 A.A t 10 Ad 40 4~A rs^ m AZ, 11, 7, XX XX sx Jut St AA /th aa AA 0 s t AP 41% AD ale. 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 I41 xx St TILL XI Ali LA?ix V, Ai AA 16 ita a* AA AA t A4 In IA roe 42 VA Ut St tit AI IN IL SI CC AN 41. 14 AA Ai õtt FIG. 4. Karyotype of Notropis lutrensis.

6 432 LIEPPMAN AND HUBBS m I41 xx St TILL XI Ali LA?ix V, Ai AA 16 ita a* AA AA t A4 In IA roe 42 VA Ut St tit AI IN IL SI CC AN AA Ai õtt 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)

7 434 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 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, 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, 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: , Brittan, M.R.: A revision of the Indo-Malayan freshwater fish genus Rasbora. Monographs Inst. Sci. and Tech. Manila, 3: 224, Chen, T. R., and A. W. Ebeling: Karyological evidence for female heteogamety in the mosquito fish, Garnbusia affinis. Copeia, 1968: 70-75, Darlington, P. J., Jr.: Zoogeography: The Geographical Distribution of Animals. John Wiley, New York, 675 pp., Gravell, M., and R. G. Malsberger: A permanent cell line from the fathead minnow (Pimephales promelas). Ann. N.Y. Acad. Sci., 126: , 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: , pls , 1966.

8 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

9 KARYOLOGICAL ANALYSIS OF CYPRINID FISHES Jordan, D. S., and B. W. Evermann: The fishes of North and Middle America. Bull. U.S. Nat. Mus., 47, pt. 1: , 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: , II. Maio, J. J., and C. L. Schildkraut: Isolated mammalian metaphase chromosomes. I. General characteristics of nucleic acids and proteins. 1. Molec. Biol., 24: 29-39, McPhail, J. D., and R. L. Jones: A simple technique for obtaining chromosomes from teleost fishes. J. Fish. Res. Bd., Canada 23: , pl. 1, Muramoto, J., S. Ohno, and N. B. Atkin: On the diploid state of the fish order Ostariophysi. Chromosoma, 24: 59-66, Ohno, S., J. Muramoto, and L. Christian: Diploid-tetraploid relationship among old-world members of the fish family Cyprinidae. Chromosoma, 23: 1-9, Post, A.: Vergleichende Untersuchungen der Chromosomenzahlen bei Susswasser-Teleosteern. Zeitschrift Zool. Syst. Evolut.-forsch., 3: 47-93, 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: , 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, Regan, C. T.: Fishes. In: Encyclopedia Brittanica, 14th ed. 9: , Roberts, F. L.: Chromosome cytology of the Osteichthyes. Progressive F ish- Culturist, 29: 75-83, Texas Rep Rio! Med 27:2, 1969

147. Triploidy appeared in the Back. Cross Offspring

702 Proc. Japan Acad., 51 (1975) [Vol. 51, 147. Triploidy appeared in the Back. Cross Offspring from Funa.Car p Crossing By Yoshio OJIMA, * > Makoto HAYASHI, *) and Koichi UENO* * (Comm. by Sajiro MAKING,