JAP. JOUR. GENET. Vol. 38, No. 2: 89-96 (1963)

Hybridization Experiments in Cyprinid Fishes II. Reciprocal Crosses

between Pseudo esocinus and

elongatus elongatus

Ryo SUZUKI

Department of Biology, Aichi Gakugei University, Okazaki

Received December 24, 1962

Although both esocinus and elongatus belong to the same subfamily, , there are some wide morphological and habitual differ- ences between them. They are sympatric, viz., their habitats being confined to the same geographical area of river. The artificial hybridizations between the two are carried out to elucidate whether or not the success of hybrid development is correlated with supposed degree of taxonomic differences between them, which is based on morphological traits. If they were closely related phylogenetically we would expect that hybrids could be reared to reach the adult stage. Ripe unfertilized eggs are obtained artificially using the method reported previously (Suzuki 1957 a ; Suzuki and Kobayashi 1958). For the observation of both species, the trypsin-Ringer's solution of one per cent is employed to dissolve the egg membrane which has an opaque adhesive layer.

Observations

1) Viability About eighty-seven per cent of eggs from are successfully fertilized by the sperm of Gnathopogonelongatus elongatus and develop into embryos without any observable deformity. About ninety-three per cent of eggs from the reverse cross, viz., G. x P. ~, develop into embryos. These percentages are almost the same as in the cases of straight-fertilized eggs. Both the reciprocally crossed eggs hatch and resulted fry feed normally and reach the adult stage with the similar survival rate to the controls. 2) External characters The lower jaw of Pseudogobioesocinus is provided with a pair of long barbels and very thick lips are equipped with numerous nipplelike protuberances (Fig. 1). The eyes locate close together on the top of the head. The anus opens in the vicinity of the ventral fin insertion. The general color of the body is light yellow and blackish 90 R. SUZUKI brown spots are scattered over the head, body and fins. The breast is callous, where no scale is present. Large pectoral fins are extened to the ventral fin base. The mouth of Gnathopogon elongatus elongatus, however, is rather terminal and has fragile barbels (Fig. 4). The eyes are removed to sides. The anus opens just in front of the anal fin insertion. Silvery white sides shade to the dark gray above and none of the black spots is observed on entire body surface. There are many scales on the breast without callosity. The small pectoral fins reach to the midway between the base of the pectoral fin and the insertion of the ventral fin. External characters of three-year old hybrids are typically intermediate between those of the parental species (Figs. 2 and 3). That is, the mouth of hybrids from Pseudogobio esocinus x Gnathopogon elongatus elongatus is somewhat terminal, though more inferior than that of Gnathopogon control. Nipplelike protuberances covering the

Figs. 1-4. 1. An adult specimen of male Pseudogobio esocinus. 2. A hybrid male, Pseudogobio esocinus x Gnathopogon elongatus elongatu '. 3. A hybrid male, Gnathopogon elongatus elogatus x Pseudogobio esocinus'. 4. An adult specimen of male Gnathopogon elongatus elongatus. All of them are three-year old. HYBRIDIZATION EXPERIMENTS IN CYPRINID FISHES 91 lips are less conspicuous than those of Pseudogobio control. The eyes are closer to each other than those of Gnathopogon. The anus opens midway between the base of ventral fin and the insertion of anal fin. The ground color of the body is yellowish brown and faint spots are scattered over the head, body and fins. The breast with few scales is slightly callous. The pectoral fin is not so long to reach the ventral fin. These characters are almost similar to those hybrids of the reverse cross, except that no blackish brown spot is found on the caudal fin of the latter. 3) Behavior Pseudogobio esocinus always live on the sandy bed of the river and they actively dig out the bed strirring up sands and mud. Sometimes, they burrow into the bed exposing only their protruded eye balls outside. A number of funnel-like hollows, therefore, are formed on the bed, the diameter being about three times of the length of their head. Some typical hollows are shown in Fig. 5. Remarkably differing from such behavior, none of the hollow is observed in the bed of Gnathopogon elongatus elongatus as shown in Fig. 6, because they usually swim in the intermediate zcne between the surface and the bed. On the other hand, hybrids produced from Pseudo- gobio esocinus female and Gnathopogon elongatus elongatus male always creep on the bed and sometimes they swim few centimeters away from the bed. Although they do not burrow as Pseudogobio does, they sometimes dig out the bed. Thereby, a few hollows

Figs. 5-8. 5. Funnel-like hollows formed on the bed where Pseudogobio esocinus controls are reared. 6. No. hollow on the bed where Gnathopogon elongatus elonga- tus are reared. 7. Hollows formed on the bed where the hybrids, Pseudo- gobio esocinus x Gnathopogon elongatus elongatus 13 are reared. 8. Hollows formed on the bed where the hybrids, Gnathopogon elongatus elongatus X Pseudogobio esocinus are reared. 92 R. SUZUKI

are formed on the bed though they are smaller than those of Pseudogobio (Fig. 7). Such intermediate behavior is also observed in the reciprocal hybrids (Fig. 8). When food is dropped from one foot above the surface as shown in Fig. 9, Gnathopogon controls come up immediately the surface, swarm around the food and feed before they sink to the bed. Neither such rising nor swarming, however, take place by Pseudogobio control. Also they feed solitarily creeping on the bed after they sank. Both hybrids always swarm around fallen foods creeping on the bed and feed at few centimeter

Fig. 9. A semi-diagrammatic figure of the feeding distance from the bed. behavior of hybrids and controls, when 4) Sex and male sterility foods are dropped from one foot above the water surface. a; Gnathopogon elongates Of the cross between Pseudogobio elongatus. b ; Pseudogobio esocinus x Gnatho- esocinus female and Gnathopogon elonga- pogon elongatus elongatus 3. c; Gnathopogon tus elongatus male, eleven hybrids reach elongatus elongatus .2 x Pseudogobio esocinus ~. d ; Pseudogobioesocinus. the three-year old. Ten of them are male and the remaining one is ap- parently neuter which the gonads are not observed. Ten hybrids of three-year old are obtained from the reverse cross. Eight of them are male, one is female and another hermaphrodite. In the control, the ratio of male to female is approximately 1:1. In order to observe whether or not the hybrids display the spawning behavior, one female and two males are kept together in a cement aquarium during the spawn- ing season. But no sexual display is observed during one month rearing. Anatomical investigation during the spawning season reveals that the testes of most hybrids are nearly normal in external form, while those of the others are reduced. To observe the spermatozoa of hybrids, their testes are fixed with formalin and then a piece of them is crushed with a cover glass. None of the spermatozoa is observed in any hybrids, while there are numerous spermatocytes (Fig. 10). This fact may indicate that spermatogenesis is impaired. On the other hand, many sperma- tozoa are observed in control testes (Fig. 11). The ovary of one hybrid male is filled with abundance of semi-ripe ovarian eggs. In the gonad of a hermaphroditic hybrid, the anterior part is prevailingly testicular and the posterior region is chiefly ovarian. HYBRIDIZATION EXPERIMENTS IN CYPRINID FISHES 93

rigs. lu-li . 10. No spermatozoon in hybrid testis, Pseudogob zo esocinus X Gnat hopogon elongatus elongatus ~. 11. Spermatozoa stripped from the control testis of Pseudogobio esocinus.

Discussion

A bulk of researches have revealed that the success of hybrid development in fishes is correlated with the closeness of taxonomic position of the crossed fishes (cf. Moenkhaus 1910 ; Hubbs, C. L. 1955 ; Suzuki 1957 b, 1957 c, 1957 d ; Hubbs, C. and Strawn 1957; Hubbs, C. 1959; Hubbs, C. and Drewry 1959), though the results cited here are not completely agree with this situation. Hubbs, C. and Drewry suggested that the following four hypothetical levels as to the relation between the success of hybrid development and the taxonomic position of the crossed fishes are distin- guishable: "Hatching -intraordinal; interfamilial . Feeding-intrafamililial; intertribal. Rearing- intratribal ; intergeneric. Fertility-intrageneric ; interspecific." Although much more studies may be necessary to justify this generalization, many experimental results among the cyprinid and cobitid fishes have almost substantiated it. For instances, according to Suzuki (1953, 1956, 1957 c, 1957 d) and Kobayashi and Yamabayashi (1957), all interfamilial hybrids between Cobitidae and die before of shortly after hatching, apparently without feeding. Intersubfamilial hybrids in Cyprinidae, studied by Suzuki (1957 c, 1957 d), Kobayashi, S. and Mizumoto (1950) and Balon, Duyvene de Wit and Hoh ik (1962), also died as larvae. Whereas, many intergeneric hybrids produced in the same subfamily belonging Cyprinidae have been reared to adult stage by Matsui (1931), Matsui and Makino (1935), Duyvene de Wit (1960a, l961 a to 1961d) and Suzuki (1961 a, 1961 b, 1962). Present minnow hybrids are also the cases. Furthermore, natural intergeneric hybrids in Cyprinidae are found by Hubbs, C. L. and Miller (1943), Miller (1945), Hubbs, C. L. and Bailey (1952), Weisel (1954, 1955) and Holcik (1962). Interspecific hybrids among subfamily, Acheilognathinae, are produced by Duyvene de Wit (1960 b, 1962a, 1962 b). Some of these hybrids are normally fertilizable and Fz progeny could be artificially obtained. From the above mentioned facts the author suggests here that cyprinid hybrids which 94 R. SUZUKI are able to be reared until the adult stage are confined to the intergeneric or interspecific hybrids among the same subfamily. Newman (1915) reported that success in hybrid development is not correlated with the taxonomical neighborhood of the crossed fishes. The similar non-correlation is detected in Suzuki's results. That is, some of his interfamilial hybrids between cobitids and cyprinids live longer than the intersubfamilial hybrids among cyprinids. These discrepancy may be due either to some technical factor in the laboratory or to some unsuitable based on morphological studies of parental fishes. Be that as it may, much more studies would be necessary to solve this doubtful point. The present hybrids are not only morphologically intermediate between the parental species but also have typically intermediate behavior. Thus characters of hybrids are similar to those of the other hybrids reported by many investigators. As described in the preceding paper of this series (Suzuki 1962), both hybrid males and females from reciprocal crosses of Gnathopogon elongatus elongatusx parva are fertilizable and F2 offspring can be produced. Present hybrids, however, are apparently male sterile. These results may show that the phylogenetical relation between Gnathopogon elongatus elongatus and Pseudogobio esociuns is far more apart than that between Gnathopogon elongatus elongatus and Pseudorasboraparva. From the investigations of Matsui, Duyvene de Wit and the present author, it seems to be a general phenomenon that some intergeneric hybrids in Cyprinidae become neuter or hermaphroditic. These facts may indicate that the hybrids are liable to be subjected to the outbalance of genetic sex-determining mechanism.

Summary

The reciprocal crosses between Pseudogobio esocinusxGnathopogon elongatus elongatus are successful and hybrids are reared until they reach adult stage with the similar survival rate to the controls. The hybrids are not only intermediate in morphology but also have typically intermediate behavior between their parental forms. Of twenty one hybrids, reared to adult hood, eighteen are sterile males, while one is a female with abundance of semi-riped ovarian eggs, one a neuter and the remain- ing one a hermaphrodite.

Acknowledgement

The author wishes to express his sincere gratitude to Prof. Dr. T. Yamamoto of Nagoya University and Prof. Dr. H. Kobayashi for their valuable advice and criticism, and to Prof. Dr. H. Gamo for his help in preparing the manuscript. HYBRIDIZATION EXPERIMENTS IN CYPRINID FISHES 95

Literature Cited

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