Allozyme variation in Synodontis leopardinus (Pisces, Siluriformes)

Comp. Biochem. Physiol. Vol. 105B,No. 2, pp. 333-336, 1993 Printed in Great Britain

0305-0491/93$6.00+ 0.00 © 1993PergamonPress Ltd

ALLOZYME VARIATION IN S Y N O D O N T I S LEOPARDINUS (PISCES, SILURIFORMES) F. H. VAN DER B A N K

Research Unit for Aquatic and Terrestrial Ecosystems, Rand Afrikaans University, P.O. Box 524, Auckland Park, 2006, Republic of South Africa (Fax 011 489241 I)

(Received 23 October 1992; accepted 27 November 1992) Abstract--1. Gene products of 51 protein coding loci in Synodontis leopardinus were examined by horizontal starch-gel electrophoresis. 2. Electrophoretic analysis of the loci, using blood, liver and white muscle samples, revealed genetic variation at 15 loci. 3. An average heterozygosity value of 0.0854 (+0.0228) was calculated, which is more than previous estimates based on fewer loci for other species. 4. The results correspond with recently published karyological, morphometrical and meristical data.

INTRODUCTION Some 2400 siluriform species are distributed in all continents, chiefly Africa, Asia and America (Agn~se et al., 1990) and the genus Synodontis accounts for a quarter of all the African siluriformes. Members of this genus are commonly known as "squeakers", due to the sound they make when taken from the water (Jubb, 1967). The specific name of the Leopard Squeaker (S. leopardinus) refers to the leopard-like markings on the body (Bell-Cross and Minshull, 1988). This species has recently become a very popular aquarium fish, due to its peculiar shape and distinct markings. It is a moderately large species with a maximum recorded standard length of 196 mm (White, 1987). The success of conservation efforts can be enhanced by knowing the genetic structure of the species in question and since the artificial production of squeakers is being executed, the apparent importance of such fundamental and applied data became evident. This study therefore aims to provide information on the allozyme diversity of wild S. leopardinus since genetic variation was, until now, not studied in any of the 112 recognised Syndontis species in all of Africa (Skelton and White, 1990) or elsewhere. MATERIALS AND METHODS

White muscle samples were obtained from 35 individuals and liver and blood samples were taken from 11 Leopard Squeakers from the Upper Zambezi River System (34°25'S, 10°11'E). Whole (heparinized) blood samples together with the other tissue samples were stored in liquid nitrogen and transported to the laboratory. These tissue samples were analysed by starch-gel electrophoresis (13% gels), using the electrophoretic procedures, buffers, method of interpretation of gel banding patterns, locus nomenclature

and statistical analysis as described and referred to by van der Bank et al. (1992). RESULTS

Fifty-one protein coding loci provided interpretable results, of which 29.4% displayed polymorphism. Locus abbreviations, enzyme commission numbers, tissues and buffers giving the best results are listed in Table 1. Thirty-six of the 51 loci displayed monomorphic gel banding patterns (Table 1). Products of the following loci migrated anodally: CK-4, GAPD-3, GPI-3, SORD-3, and MPI-I. In addition to these loci, PEP-D was stained for using phenylalanylproline as substrate which, together with GAPD-1 and 2, IDH-1 and ME-2, did not show sufficient activity or resolution to score it satisfactorily. Allele frequencies for polymorphic loci are presented in Table 2 as well as G-test values for loci deviating significantly (P >0.05) from expected Hardy-Weinberg proportions. Monomeric gel banding patterns were obtained at the CK, EST, SORD, PGM and PROT loci whereas dimeric patterns were obtained for GPI, MDH and MPI. The enzyme subunit structure for the HK locus could not be determined since no heterozygotes were detected (only alternate homozygotes). The number of alleles for the MDH-3 locus was four, whereas two alleles were present at all of the other polymorphic protein coding loci studied. Average heterozygosity was 0.0854 with a standard error of 0.0228. DISCUSSION Two-banded variant phenotypes were observed for CK (Table 2), reflecting the monomeric nature of this enzyme. This result is in agreement with the subunit

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Table 1. Locus abbreviations, EC No., tissues and buffers giving the best results for each protein Protein Alcohol dehydrogenase Adenylate kinase Creatine kinase

Esterase

Glyceraldehyde-3-phosphate dehydrogenase Guanine deaminase Glycerol-3-phosphate dehydrogenase Glucose-6-phosphate isomerase Hemoglobin Hexokinase L-lditol dehydrogenase Isocitrate dehydrogenase L-Lactate dehydrogenase Malate dehydrogenase Malic enzyme Mannose-6-phosphate isomerase Peptidase: Substrate: Glycyl-L-leucine L-Lecylglycylglycine Leucyl-tyrosine 6-Phosphogluconate dehydrogenase Phosphoglucomutase Purine-nucleoside phosphorylase General protein

Superoxide dismutase

Locus

EC No.

Tissue

Buffer

*(ADH-I) *(ADH-2) *(ADH-3) *(AK) *(CK-1) (CK-2) (CK-3) (CK-4) *(EST- 1) *(EST-2) (EST-3) *(EST-4) (EST-5) *(GAPD-3)

1.1.1.1

B,L B,L,M L B,L,M L,M B L,M M B,L,M B B M L,M M

TC TC TC TC RW RW RW RW RW RW RW RW RW TC

B,L,M L,M B,L,M B,L,M B,L B,M B L B L B,L,M B,L,M L B,L,M B,L,M M B,L L B,L,M L B,L,M

MF TC TC RW RW RW RW MF TC TC TC TC RW RW RW RW RW RW TC MF MF

*(PEP-A)

B,L,M

*(PEP-B)

L

*(PEP-C,I) *(PEP-C,2) *(PGD-I) *(PGD-2) *(PGD-3) (PGM-I) (PGM-2) *(PGM-3) *(NP)

2.4.2. I

B,L,M B,L,M B,L L,M B,L,M L B,L,M B,L B,L,M

MF MF MF MF TC TC TC RW RW RW MF

1.15.1.1.

M B,L,M M M L,M

RW RW RW RW RW

*(GDA) *(GPD- 1) *(GPD-2) *(GPI-I) (GPI-2) (GPI-3) *(HB) (HK) *(SORD-I) (SORD-2) *(SORD-3) *(IDH-2) *(LDH-I) *(LDH-2) *(LDH-3) (MDH-I) (MDH-2) *(MDH-3) *(ME-I) *(MPI-I) (MPI-2)

2.7.4.3 2.7.3.2

3.1.1 .--

1.2.1.12 3.5.4.3 1.1.1.8 3.5.1.9

2.7.1.1 1.1.1.14 1.1.1.42 1.1.1.27 1.1.1.37 1.1.1.38 5.3.1.8 3.4.- .

*(PROT-1) *(PROT-2) (PROT-3) *(PROT-4) *(SOD)

1.1.1.44 5.4.2.2

*Monomorphic loci, B = blood, L = liver, M = muscle, MF = a continuous Tris, boric acid, EDTA buffer (pH 8.6) described by Markert and Faulhaber (1965), RW = a discontinuous Tris, citric acid, (gel pH 8.7), lithium hydroxide, boric acid (electrode pH 8.0) buffer system (Ridgway et al., 1970), TC = a continuous Tris, citric acid (pH 6.9) buffer system (Whitt, 1970).

structure for this enzyme as described by Scopes and Hamoir (1971). Harris and Hopkinson (1976) found it to be dimeric in humans. Double-banded heterozygotes were also observed for the EST, PGM and PROT loci, and three-banded heterozygotes were observed for the GPI, and M D H loci to conform with the results obtained by other authors for freshwater fish in general (as discussed by van der Bank et al., 1989). The enzyme subunit structure of SORD was monomeric in S. leopardinus and MPI was dimeric. McAndrew and Majumdar (1983) found the former locus to be dimeric in cichlid species and Harris and

Hopkinson (1976) described it as being tetrameric and MPI as monomeric in humans. Deviations from expected Hardy-Weinberg proportions occurred at the CK-4, GPI-3, HK, MPI-2 an PGM-1 loci (Table 2). Various factors can shift the equilibrium and disrupt the stability of a population, giving rise to change in the genetic structure, as ideal Hardy-Weinberg populations do not actually occur in nature (Altukhov, 1981). Although small sample sizes could account for the deviations at the last three loci, recurrent bottlenecks involving founder populations with little variability could account for the

Allozyme variation in Synodontis leopardinus

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Table 2. Allelefrequenciesof polymorphicloci and G-test values with degrees of freedom (DF) for loci which deviate significantlyfrom expected Hardy-Weinbergproportions(P > 0.05) Relative mobilitiesof alleles Locus 2N -95 - 100 85 90 95 100 105 G-test DF 0.136 0.864 CK-2 22 0.614 0.386 CK-3 70 0.152 0.848 28.072 1 CK-4 66 0.773 0.227 EST-3 22 0.014 0.986 EST-5 70 GPI-2 22 0.045 0.955 0.114 0.886 11.543 1 GPI-3 70 0.222 0.778 9.535 1 HK 18 SORD-2 20 0.150 0.850 MDH-1 70 0.214 0.300 0.100 0.386 MDH-2 22 0.864 0.136 MPI-2 20 0.700 0.300 12.217 1 PGM-I 22 0.227 0.773 5.486 1 PGM-2 70 0.014 0.986 PROT-3 70 0.743 0.257

deviations at the other loci. The latter hypothesis is supported by the incidence of recurrent droughts over the past 20 years in southern Africa, which undoubtedly produced numerous population bottlenecks. It is also true that factors (e.g. natural selection) can result in the production of a larger number of progeny by some genotypes than by others (Altukhov, 1981; Kirpichnikov, 1981). Other factors include selfsorting crossings and linking (Allendorf and Utter, 1979), and when a sample include individuals from two or more populations, it can lead to the Wahlund (1928) effect which would also influence expected Hardy-Weinberg equilibrium. The extent that such factors could have had on S. leopardinus is unknown since very little is known about their biology. Average heterozygosity (/7/) was 8.5% (Table 2), which is slightly higher than values obtained for other species of bonefishes. For instance, Powell (1975), Nevo (1979), Avise and Aquadro (1982), Mork et al. (1982) and van der Bank et al. (1992) recorded B levels of 5.4, 5.1, 5.4, 8.2 and 4.7%, respectively, for various bonefish species. The relatively high/7 value obtained for S. leopardinus is probably reflected by its morphological features which were found to be very variable (White, 1987). This hypothesis is also supported by the results obtained by Agnrse et al. (1990) who found that a fundamental number of the chromosomes varied in other Synodontis species and that all the studied females revealed the existence of heteromorphic chromosomes. The observed higher value of heterozygosity found in the present study, compared to the levels found for other species, can best be accounted for by the analysis of different species, sample sizes, increased number of loci now studied, and the choice of loci which may have differences in heterozygosities (Lewontin, 1974). For example, Harris and Hopkinson's (1976) original estimate of 9.9% f o r / 7 in man, based on the first 10 gene loci examined, turned out to be 50% too small. In conclusion, this is the first account of electrophoretic variants of this species. The results of the present study indicates that the species examined possesses a healthy amount of genetic variation (i.e.

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