Anim. Biood Grps biochem. Genei. 2 (1971) 127-133

Isoenzyme polymorphism of the sorbitol dehydrogenase and the NADP-dependent isocitrate dehydrogenases in the fish family Cyprinidae

Wolfgang Engel, Joachim Faust and Ulrich Wolf

Institut fur Humangenetik und Anthropologie der Universitat Freiburg i.Br., West Germany

Received: 18 January 1971

Among members of the fish family Cyprinidae, the existence of a diploid-tetraploid relationship is well established. The analysis of individual gene loci, using isoenzyme polymorphism as genetic markers, does not always confirm the expected gene duplic- ation in the tetraploids. Of the markers used in this study, only the M-form of the NADP-dependent isocitrate dehydrogenase follows this expectation; the data suggest the existence of asingle gene locus for the enzyme in diploids, while the observations on tetraploids were consistent with control by two distinct loci. For two other enzymes, the S-form of the NADP-dependent isocitrate dehydrogenase and sorbitol dehydro- genase, no difference seems to exist in the number of gene loci between diploids and tetraploids. A comparison between Cyprinid fish (order Ostariophysi) and members of the order Isospondyli in which another diploid-tetraploid relationship was established, reveals that gene duplications are more frequently demonstrable within tetraploid Isospondyli than in tetraploid Cyprinidae. From this, it is concluded that polyploidiza- tion occurred earlier in evolution of Cyprinidae than of Isospondyli.

Introduction

Polyploidization has apparently operated as a powerful evolutionary force in verte- brate phylogeny (Ohno, 1970). In lower vertebrates, a more recent occurrence of poly- ploidization can be demonstrated in a few examples. These examples permit the conse- quences of polyploidization, i.e. the diploidization process starting after duplication of the genome, to be studied. In fishes of the orders Isospondyli and Ostariophysi analysis of the genome revealed a diploid-tetraploid relationship (Ohno et al., 1967; Klose et al., 1968; Wolf et al., 1969). The analysis of individual gene loci, using various genetically determined isoenzyme polymorphisms as genetic markers, does not how- ever confirm this diploid-tetraploid relationship in all markers examined. The follow- ing different conditions were found within the tetraploid group of species: a. No gene duplication is demonstrable.

128 WOLFGANG ENGEL, JOACHIM FAUST AND ULRICH WOLF

b. The genes are duplicated and have evolved divergently, following a disomic mode of inheritance. c. The genes are duplicated, behaving as alleles to eachother and following a tetrasa- mic mode of inheIitance.

These alternatives may represent different steps towards diploidization. Tetrasomic inheritance may be expected only in recent tetraploids. We discovered this mode of inheritance in some Salmonidae (Isospondyli) species for the gene loci of the sorbitol dehydrogenase and the S-form of NADP-isocitrate dehydrogenase (Wolf et al., 1970; Engel et al., 1970). When diploidization is completed, two different disomic loci arise from the former tetrasomic gene locus as in the case of the loci for lactate dehydrogen- ase in Salmonidae (Klose et al., 1968).

If a gene duplication is not demonstrable in the tetraploid group of species, the duplicated gene may have either been eliminated or it may have changed by mutation in such a way as to assume a new function. An example of this may be the S-form of NAD-dependent malate dehydrogenase in Cyprinus carpi0 (Aghasadeh and Ritter, 1971).

As mentioned, the genes for sorbitol dehydrogenase and S-form of NADP-isocitrate dehydrogenase follow a tetrasomic mode of inheritance in Salmonidae. Therefore, it was also of interest to study these enzymes in the tetraploid group of Cyprinidae species. It will be shown that in these species no gene duplication is demonstrable for these gene loci. These findings are interpreted as supporting the hypothesis that the respective Cyprinidae species are older tetraploids than the Salmonidae.

Materials and methods

The species of the fish family Cyprinidae and the number of specimens examined are summarized in Table 1.

Table 1. S w e y of the Cyprinidae species and number of specimens examined. The species within the fish family Cyprinidae; Barbus tetrazona, Rutilus, Tinca and Abramis represent the diploid level; Barbus barbus, Cyprinus, and Carassius have evolved from tetraploids (Wolf et al., 1969).

Species

Barbus tetrazona Rutilus rutilus Tinca tinca Abramis brama Barbus barbus Cyprinus carpia Carassius carassius

Diploid chromosome number

50 50 48 50 100 104 104

SDH

30 20 91 32 58 8

33

S-IDH

21 108 80 16 24 2 -

M-IDH

24 28 10 11 2 -

h i m . Blood Grps biochem. Genet. 2 (1971)

SDH AND IDH IN CYPRINID FISH 129

Of these, Barbus tetrazona was obtained from a local aquarium shop; all the other animals were caught from wild populations in the Rhine river.

Liver and heart was taken from each individual fish. The enzymes sorbitol dehydro- - se (SDH; E.C.: 1.1.1.14) and NADP-dependent isocitrate dehydrogenases (IDH; E.C. : 1.1.1.42) were examined using starch gel electrophoresis and histochem- ical staining. The enzymes SDH and S-form of NADP-IDH were extracted from liver, the M-form of NADP-IDH was derived from heal t.

Electrophoretic and staining techniques were performed for SDH according to op't Hof et a]. (1969), and for NADP-IDH according to Wolf et al. (1970).

Results

Sorbitol dehydrogenase The genetics and the quaternary structure of SDH may be considered as settled. According to studies on various vertebrates, including the Cyprinid fish Leuciscus cephalus, this enzyme represents a tetramer. If two different subunits exist as in hetero-

Fig. 1. Isoenzymes of sorbitol dehydrogenases in Cyprinid fish. a) Rvtilus rurilus. The homozygous wild type (AA) exhibits a single band; a faster (AA) and a slower (AA") variant occurred only in the heterozygote state. Of the five isoenzymes expected only 3 are visible . b) Tinca tincu. Two different homozygotes and the corresponding heterozygote are shown in the left block (phenotypes A'A, A A , and AA); another variant found in the heterozygote only (AA") is shown at right, compared with the homozygotes AA' and AA.

Anim. Blood Grps biuckem. Genet. 2 (1971)

130 WOLFGANG ENGEL, JOACHIM FAUST AND ULRICH WOLF

zygotes endowed with a single gene locus for this enzyme, random association of these subunits into tetramers results in 5 different isoenzymes (op't Hof, 1969; op't Hof et al., 1969).

Of the Cyprinidae species examined here, a single SDH-band was seen in starch gel either in all, or in the majority of cases. Variants were observed in Rutilus and Tinca only, giving rise to 5 different isoenzymes in the heterozygote.

In Rutilus, 3 different phenotypes occurred (Fig. la) which can be interpreted by assuming the existence of three different alleles at one gene locus. Of the 30 specimens examined, 23 exhibited a single band (wild type AA), in 5 a faster variant occurred (heterozygote AA'), and in 2 animals a slower variant was found (heterozygote AA").

In Tinca, 4 different phenotypes were identified (Fig. lb). Of these, 3 phenotypes belong to a polymorphism of 2 alleles (a and a'), the frequencies of which were AA : AA': A'A' = 21 : 35 : 27. The 4th phenotype represents a heterozygote with the variant A ' which is slower than A'; this type was observed in 8 specimens. In the sam- ples taken here, no other variants were observed.

The isoenzyme variation of SDH in the diploid species Rutifus and Tinca con- firms our previous observation that this enzyme behaves electrophoretically as a tetra- meric molecule, the heterozygotes exhibiting 5 different isoenzymes. Accordingly, the existence of a single gene locus for SDH in these species is suggested. Similar findings raised by Lin et al. (1969) in the tetraploid species Carassius lead to the conclusion that both, the species on the diploid as well as on the tetraploid evolutionary level are uni- formly endowed with a single gene locus for SDH.

S-form of the NADP-dependent isocitrate dehydrogenase The supernatant 6-) form of the NADP-IDH behaves electrophoreticaIly as a dimeric molecule (Henderson, 1968). Thus, in the presence of a single gene locus, homo- zygotes exhibit 1 band, heterozygotes 3. In the case of two different gene loci, the wild type is expected to show 3 bands, in heterozygotes for one of these loci 6 different iso- enzymes should be formed, if free association of subunits occurs. Depending on the electrophoretic mobility of the variant homomenc isoenzyme, however, intermediate isoenzymes may fall into the same position.

For this enzyme, all species examined in this study exhibited at least 3 bands after electrophoresis. The following variants were observed: Of the 108 specimens of Rutilus examined, 2 were heterozygous for a faster variant (Fig. 2b); in Barbus barbus, of the 24 specimens examined, 4 were heterozygous for a faster variant (Fig. 2e). .

These findings lead to the interpretation that the Cyprinidae species on the diploid and on the tetraploid level each possess two different gene loci for the S-form of NADP-IDH.

M-form of the NADP-dependent isocitrafe dehydrogenase The M-form of NADP-IDH also manifests a dimeric structure under electrophoretic study (Wolf et al., 1970). This enzyme did not show any variant in ail samples taken

Anim. Blood Grps biochem. Genet. 2 (1971)

131

Fig. 2. Isoenzymes of the NADP-dependent isocitrate dehydrogenases. a) Barbus tefrazona; the 3 wild type bands of the S-form. b) Rurilus rutilus. From left to right: the single band of the wild type M-form, 3 bands of the wild type S-form, and a faster variant of S-form in a heterozygote. c) Tinca tinca; M- and S-forms of the wild type. d) Abramis brama; M- and S-forms of the wild type. e) Barbus barbus. The wild type exhibits 3 bands in the M-form (left); in the S-form, the pattern corresponds to Rutilus (b).

here. In Rutilus, Tinca and Abraniis a single band was stained (Fig. 2 b-d); in Barbus barbus (Fig. 2e) and Cyprinus carpio 3 bands occurred in each invididual fish.

According to these findings, in the diploid group the existence of a single gene locus is presumed; in the species on the tetraploid level two different gene. loci should be responsible for the 3-banded pattern.

Discussion

Of the species within the fish family Cyprinidae examined here, Barbus tetrazona, Rutilus, Tinca, and Abramis represent the diploid level; Barbus barbus, Cyprinus, and Carassfus have evolved from tetraploids (Wolf et al., 1969, and Table 1).

The study of three independent isoenzyme systems suggests a gene duplication in the tetraploids for one system only, the M-form of NADP-IDH, while in both groups of species, the enzyme SDH may be encoded by a single gene, and the S-form of NADP-IDH by two different genes.

As shown previously, in the diploid Cyprinid fish Leuciscus cephalus also a single SDH gene locus is to be assumed (op’t Hof et al., 1969). The tetraploid goldfish

Anim. Blood Grps biochem. Genet. 2 (1971)

132 WOLFGANG ENGEL, JOACHIM FAUST AND ULRICH WOLF

Table 2. The number of gene loci for various isozymes in the fish orders Isospondyli and Ostariophysi. Comparison between species on the diploid and tetraploid level.

Gene loci for: Isospondy li Ostariophysi

genome: 2n 4n 2n 4n

LDH 2n 4n 2n 4n &PGD 2n 2n 2n; 4n 4n S-fOm NAD-MDH 2n 4n 2n 2n; 4n S-fom NADP-IDH 2n 4n* 2n 2n M-form NADP-IDH 2n 4n 2n 4n SDH 2n 4n+ 2n 2n

~

* Tetrasomic inheritance in some species.

(Curassius uurutus) was shown to maintain an SDH polymorphism on the basis of one gene locus and a number of alleles (Lin et al., 1969).

The S-form of NADP-IDH was already examined in Cyprinus curpio and Curassius uurutus by Quiroz-Gutierrez and Ohno (1970) who conclude that two gene loci exist for this enzyme.

Thus, while no duplication is demonstrable for the genes coding for SDH and S- form of NADP-IDH within the family Cyprinidue, in the order Isospondyli the genes for these two enzymes are duplicated in the tetraploids [Wolf et al., 1970; Engel et al., 1970). A comparison of the number of gene loci for various isozyme systems between

diploid and tetraploid members of the fish orders IsospondyZi and Ostariophysi is given in Table 2. As shown in the table, in tetraploid Isospondyli the number of duplicated gene loci

exceeds that of the tetraploid Ostariophysi. Furthermore, in some Isospondyli species a tetrasomic mode of inheritance is found which represents a transient stage towards diploidization, while in Ostariophysi this stage has not been observed. This difference may be due to the vaiiation in the time periods which have elapsed since polyploidiza- tion took place. While in recent tetraploids each single gene locus is duplicated, during the course of evolution some duplicated genes may have disappeared due to lack of selective advantage (Ohno, 1970). Our findings thus suggest that the tetraploid Zso- spondyli represent a more recent tetraploid group than the tetraploid species of Cyprinidue.

References

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Anrm. Blood Grps bioehem. Genet. 2 (1971)

SDH AND IDH IN CYPRlNlD FISH 133

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Lin, C.-C., G. Schipmann, W. A. Kittrell & S. Ohno, 1969. The predominance of heterozygotes found in wild goldfish of Lake Erie at the gene locus for sorbitol dehydrogenase. Biochem. Genet. 3: 603-607.

Ohno, S., 1970. Evolution by gene duplication. Springer, Berlin-Heidelberg-New York. Ohno, S., J.-I. Muramoto, L. Christian & N. B. Atkin, 1967. Diploid-tetraploid relationship among

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Anim. Blood Grps biochem. Genet. 2 (1971)