Heredity 61(1988)13—20 The Genetical Society of Great Britain Received 17 September 1987

Evolutionary divergence between sympatric species of southern African , capensis and M. paradoxus. I. Electrophoretic analysis of proteins

W. Stewart Grant,* Department of Microbiology, University of Cape Inga I. Becker and Town, Rondebosch 7700, South Africa. t Sea Fisheries Research Institute, Private Bag X2, Rob W. Lesliet Rogge Bay 8012, South Africa.

We estimated the amount of genetic divergence between two morphologically similar species of southern African , Merluccius capensis and M. paradoxus, with the electrophoretic analysis of proteins encoded by 31 loci. Nei's genetic distance between these taxa was 0•583 (±0.160) and is typical of evolutionary divergence between well differentiated congeneric species. We found no evidence of hybrid individuals. The mean heterozygosity over 13 samples of M. capensis was 0055 and over 10 samples of M. paradoxus was 0•067. The present sympatric distributions of these fish are most likely the result of secondary contact after speciation in allopatry or the result of repeated dispersals of ancestral populations of other Atlantic Ocean hakes to southern Africa, rather than the result of sympatric speciation. There were significant excesses of rare alleles in both species as compared with that expected for neutral alleles in species at drift-mutation equilibrium. Average heterozygosities, however, were not appreciably reduced in comparison with other marine fish. Using genetic distance and the assumptions of the molecular clock, we estimate that the lineages leading to these species diverged from one another between 7 and 13 million years ago.

INTRODUCTION tal waters of southern Africa. Merluccius capensis is distributed between 12°S on the west coast and Isolationin allopatry followed by genetic drift or 31°E on the south coast. , in adaptive divergence appears to be the most com- contrast, has a more restricted range between 18°S mon mode of speciation in sexually reproducing and 27°E (Inada, 1981). The ranges of these hakes (Futuyma and Mayer, 1980). Two kinds coincide with the cool temperate waters of the of population events can lead to allopatric isolation Benguela Upwelling System and are bounded by in marine fish. One is the subdivision of an ances- the warm southward flowing Southern Equatorial tral population into two or more large populations Current on the west coast and the southwestward by geologic events or by changes in oceanic cur- flowing Mocambique Current on the south coast. rents or temperatures, and the other is the estab- There is a broad area of bathymetric overlap; Mer- lishment of small founder populations outside of luccius capensis inhabits the continental shelf to a species' usual geographic range. The sympatric about 400 m, and M. paradoxus inhabits the con- occurrence of closely related species is therefore tinental shelf and slope from about 140 m to 850 m. assumed to reflect secondary contact after the for- The taxonomies of species in the genus Merluc- mation of reproductive barriers has prevented cius are in considerable disarray because many reassimilation into a single gene pool. species are morphologically similar to one another. In this paper we describe genetic divergence The two nominal species of southern African hake between two sympatric species of hake, Merluccius were considered to be a single species, M capensis, capensis and M. paradoxus, which inhabit the coas- until Franca (1954) demonstrated the existence of a second sympatric taxon, M. paradoxus, from a *Presentaddress: Department of Genetics, University of the study of vertebral numbers. Subsequent morpho- Witwatersrand, Johannesburg 2050, South Africa. logical studies (van Eck, 1969; Bentz, 1976) and 14 W. S. GRANT, I. I. BECKER AND R. W. LESLIE an electrophoretic study of non-specific proteins Whitt, 1970) to examine glyceraldehyde phosphate (Jones and Mackie, 1970) have confirmed Franca's dehydrogenase (Gap-i and Gap-2, EC 1.2.1.12), (1954) hypothesis of two distinct taxa. The actual glycerol-3-phosphate dehydrogenase (Gpd-A, amount of genetic divergence between these Gpd-B and Gpd-C, EC 1.1.1.8), isocitrate dehy- species and their phylogenetic relationships to drogenase (Idh-A, EC 1.1.1.42), malate dehy- other species of hake, however, are still matters of drogenase (Mdh-A and Mdh-B, EC 1.1.1.37), much speculation (Szidat, 1961; Franca, 1971; malate dehydrogenase-NADP (=°malic enzyme, Meyer-Rochow, 1972; Kabata and Ho, 1981). Me, EC 1.1.1.40), and a tris-borate-EDTA buffer The goal of this study is to document geneti- (pH 87; Markert and Faulhaber, 1965) to examine cally-based electrophoretic variation in these taxa guanine deaminase (Gda, EC 3.5.4.3), man- and to estimate the amount of genetic divergence nosephosphate isomerase (Mpi, EC 5.3.1.8), between them. The following paper in this issue nucleoside phosphorylase (Np, EC 2.4.2.1) and reports genetic divergence between these taxa as peptidase (Pep-D, EC 3.4.13.9 and Pep-X, EC measured by the restriction-endonuclease analysis 3.4.?.?). of mitochondrial DNA (Becker et a!., 1988). Sub- sequent papers in this series will examine the phy- logenetic relationships of these taxa with other RESULTS Atlantic and Pacific Ocean hakes. Weidentified the gene products of 31 protein- coding loci by the criteria of Allendorf and Utter MATERIALSAND METHODS (1979) and by comparison with other related teleosts. Proteins encoded by Ck-B, Gap-2, Gpd-A, Weexamined genetic variation in a total of 1135 Gpd-B, Ldh-C, Pep-X, Pt-i, Pt-2, Pt-3, Pt-4 and individuals of Merluccius capensis from 13 loca- Sdh appeared as invariant bands on the gels with tions (extending from 18°42'S, 1i°48'E to 34°20'S, the same mobilities for both species. The products 25°40'E), and a total of 882 individuals of M of Np and P1-5 were invariant within species but paradoxus from 10 locations (23°02'S, 13°05'E to were fixed for different alleles between species. 34°40'S, 25°40'E) (fig. 1). Samples of eye, liver, Three enzymes encoded by Mpi, Pgm-i and Pgm-2 skeletal and cardiac muscles were held at —25°C (fig. 3) showed double-banded heterozygous for up to 3 months until laboratory analysis. phenotypes in one or both species. Ten proteins Individuals were identified to species by gill arch encoded by Gda, Gpd-C, Gpi-A, Gpi-B (fig. 2), tubercle morphology (van Eck, 1969). Starch-gel Idh-A, Mdh-A, Mdh-B, Pep-D, Pgd and Sod-i had electrophoretic methods followed May et a!. triple-banded heterozygotes typical for dimeric (1979), and staining protocols followed Shaw and enzymes in at least one species. Although a dimer, Prasad (1970) and Harris and Hopkinson (1976). Ck-A exhibited double-banded heterozygous To facilitate comparison between species, alleles phenotypes typical for teleosts (Ferris and Whitt, for each locus were designated by their elec- 1978). The products of Gap-i, Ldh-A, Ldh-B and trophoretic mobilities relative to the most common Me showed broad-banded or five-banded allele in M. capensis for that locus. Alleles with heterozygous phenotypes typical for tetrameric products migrating cathodally were prefixed with enzymes in at least one species. a minus sign. The amount of genetic variation present in both The gene-products of the following 31 species was very similar. In M capensis, 13 of 31 homologous loci were examined in each species. loci (41.9 per cent) showed at least some poly- We used a discontinuous lithium-borate tris-citric morphism and 7 loci (22.6 per cent) had common- acid buffer (pH 8'!; Ridgway et al., 1970) to allele frequencies less than 095 in at least one examine variation for creatine kinase (Ck-A and sample, In M. paradoxus, 13 loci (41.9 per cent) Ck-B, EC 2.7.3.2), glucosephosphate isomerase were polymorphic, and 8 loci (25.8 per cent) were (Gpi-A and Gpi-B, EC 5.3.1.9), lactate dehy- polymorphic using the 095 criterion of poly- drogenase (Ldh-A, Ldh-B and Ldh-C, EC 1.1.1.27), morphism. The proportion of expected heterozy- phosphoglucomutase (Pgm-1 and Pgm-2, EC gotes at a locus may be defined by 2.7.5.1), phosphogluconate dehydrogenase (Pgd, h =1 EC 1.1.1.44), non-specific protein (Pt-i, Pt-2, Pt-3, —x P1-4, P1-5), superoxide dismutase (Sod-i, EC where x, denotes the frequency of the ith allele. 1.15.1.1) and sorbitol dehydrogenase (Sdh, EC Average heterozygosity (H) is the arithmetic mean 1.1.1.14). We used a tris-citric acid buffer (pH 69; of h over all loci including monomorphic loci. In SYMPATRIC FISH SPECIES: PROTEIN ANALYSIS 15

Figure 1Geographic distributions of southern African hakes, Merlucciuscapensisand M. paradoxus.

M. capensis, H per sample ranged from 0046 to by the gene diversity statistics of Nei (1973) with 0055 and averaged 0051 (±0.023) over 13 the computing algorithm of Chakraborty et al. samples. H for M. paradoxus ranged between (1982). Total gene diversity (HT; heterozygosity of 0059 and 0070 and averaged 0063 (±0.026)over pooled allele frequencies) was partitioned into two 10 samples. The difference between these two components average values of H was not significant (t-test on arcsin transforms of locus heterozygosities; Archie, HT= Hs+DST 1985). where II is the average subpopulation heterozy- The relative amounts of subdivision at different gosity and DST is the proportion of HT that is due levels of population organisation was summarised to differences between subpopulations. Therefore, 16 W. S. GRANT, I. I. BECKER AND R. W. LESLIE

PGM G Pt

—100 — 80Gp/-B — 70

—400 — 50 6p1-A —-100 —-200

000 N) ON- 00ON-N-N-N-N- N- OriqIn 00000 000 00000 00oct 0)0)0)0)0)0) 22222200 N N:;: NNN N NNN N NNN N N Pgrn-2 NNN NN N Gp/-B 20 ON-O 000) 0010 00 0001) toN-N-N-L)O 00002 222N- 2' 0000 00 000000000020)0)0)0) (000 cu c'Jc'J 000 toc CJOJC'J IL)00000000000to 000OOIO10Q10 0010000N N N N N Of) N N0N- 0000100Pgm-/ N Gp/-A tO 010(0 2 2 N-2 ° co 1000000000o 41.copensis M.paradoxus CC C C C CC P P P P Species

Figure 2 Electrophoretic variability for phosphoglucomutase Figure3 Electrophoretic variability for glucosephosphate in muscle tissues of Merluccius capensis (C) and Merluecius isomerase in muscle tissues of Merluccius capensis (C) and paradoxus (P). Merluccius paradoxus (P).

H5/ HT is the relative proportion of the total gene was 00007 and that between pairs of samples of diversity that is contained within subpopulations M. paradoxus was 00006. I between the two and was 0983 and 0987 for M capensis and M. species was 0558 (±0.160) and D between species paradoxus, respectively. GST =DST/HT is the rela- was 0583 (±0. 160). tive proportion of the total diversity that is due to differences between subpopulations and was 0017 and 0013 for M. capensis and M. paradoxus, DISCUSSION respectively. Since these values represent very low Severalloci were fixed or nearly fixed for different levels of population differentiation, allele frequen- alleles between these species and we interpret this cies were pooled by species for further analysis as convincing evidence that M capensis and M. (table 1). paradoxus do not share a common gene pooi. We Genetic identities between taxa for a locus may be computed from allele frequency data by also did not observe any individuals with hybrid genotypes. Several studies show that there is gen- I =J/(JJ)1R erally good agreement between traditional and molecular distance. In some in- where is the probability of identity of two alleles stances, however, where there is little morphologi- drawn at random, one each from populations x cal differentiation, as with the hakes in the present and y (Nei, 1972). This probability is standardised study, molecular distance may then be used as a by the geometric mean of the probabilities of iden- guide to taxonomy. Thorpe (1982) summarised tity within populations x and y. An estimate of I genetic distance data for 2664 pairs of taxa of over all loci may be computed by averaging J,, invertebrates and vertebrates and found that an J, and J, over loci. Genetic distance (D) is defined average D between congeneric species was 062. as In a review of genetic distances specifically between fish taxa, Shaklee et al. (1982) found that D=—logel. for pairs of taxa classified as related species, D Nei and Roychoudhury (1974) present a formula ranged from 0025 to 065 and averaged 030. Thus for computing the standard error of D. The average our finding of D =0583between these two hakes D (31 loci) between pairs of samples of M. capensis indicates that they should be treated as full species. SYMPATRIC FISH SPECIES: PROTEIN ANALYSIS 17

TableI Summary of alleles frequencies for southern African hakes, Merluccius capensis and M. paradoxus. Alleles are arranged according to their electrophoretic mobility beginning from the cathodal end of the gel

Allele

Locus' Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 h

Ck-A C <0001 0-198 0-004 0-797 0-001 0-327 p — — — — 1-0 0•0 Gap-I C 1-0 — 0-0 P 00020998 0-004 Gda C 0999 0001 0-002 P 10 — .0 Gpd-C C 10 0-0 P 09980002 0004 Gpi-A C 0052 07530-003 0-190 0002 — — — — 0394 P 00010001— 0005— 0967— 00050020 0001 0001 0-059 Gpi-B C 0•00100150001 00270001 0956 0001 0-081 P 0002 — 0•9930005 — — 0-016 Idh-B C 0•001 0.999 0001 p 00210816— 0163 0308 Ldh-A C 0•0010•999 — 0-002 p 0•00l000209850012 0030 Ldh-B C 0005 0995 0009 P 0-00200030•995 0059 Mdh-A C 00020997000 1 0-0 p 10 0-0 Mdh-B C 10 0-0 p 0002 09950-003 0-010 Me C 0998 0-002 — — 0-040 p 0005 00180931— 00430-003 0131 Mpi C — — 1-0 — — — 0-0 p 0011 017300100656 0-001 0143 0003 0003 0-520 Np C 1-0 — 0-0 P 10 0-0 — Pep-D C 0•0050-358 0010 0057 — 0-5690001 — 0-551 p 0-001— — 02320005 0548 0209 0-005 0602 Pgd C — — 10 0-0 P 001 097200020-0230-001 0-054 — Pgm-1 C 0004 00090026— 0-003— 0-8930004 0-054 — 0007 — — 0204 p — 0-006— 00380002— — 0906— 0-0030-042 0-0030169 Pgm-2 C 0002 — 0006— 0984— 0008 0028 p — 0-00200650921— 0-012 0-147 Pt-S C — 1-0 0-0 P 1-0 0-0 Sod-i C — — 1-0 0-0 p 0-0010008 0991 — 0-019

'The following loci were fixed for the same allele in both species Ch-B, Gap-2, Gpd-A, Gpd-B, Ldh-C, Pep-X, Pt-I, P1-2, P1-3, Pt-4 and Sdh.

Average heterozygosity for M. capensis was for equilibrium populations having a specific 0.051 and that for M. paradoxus was 0063 and average heterozygosity. A comparison of the both values were similar to those found in other observed distribution of locus heterozygosities in fishes. Winans (1980) summarised average the two hakes with that expected under the infinite- heterozygosity in 82 fish and found an overall alleles model for H =005is shown in fig. 4. This average of 0048 0•033. In the inflite alleles model agreement suggests that these populations are in of neutral mutations the expected heterozygosity drift-mutation equilibrium. in an equilibrium population is equal to 4NeV, An examination of the distributions of expected where Ne is the effective population size and v is and observed allele frequencies, however, gives a the mutation rate per locus per generation (Kimura different picture. When a bottleneck in population and Crow, 1964). Fuerst eta. (1977) have generated size occurs, as for instance in a small founder theoretical distributions of locus heterozygosities population, average heterozygosity declines by 18 W.S. GRANT, I. I. BECKER AND R. W. LESLIE

80- M. copensis M. poradoxus TheoreticalH 0.05

40 :3 r;3. -

TheoreticolLeoreticalH0.05 0 0:5 1.0 0 0.5 .0 Locus heterozygosity Locus heterozygosity

Figure 4 Comparisons of observed (broken line) locus heterozygosities with theoretical (solid line) distributions of heterozygosities expected at equilibrium in the infinite-alleles model of mutation.

1/(2Ne) for a one time bottleneck (Nei eta!., 1975; with the expected distributions under the infinite Chakraborty and Nei, 1977). If population growth allele model of neutral mutations. The expected is slow after the bottleneck, heterozygosity con- number of alleles with frequencies between p and tinues to decline. With time, new alleles appear q is through mutation and accumulate in the popula- tion at a rate that depends upon 4NeV. But there Nq is a delay in the return to original levels of heterozy- n(p,q)= 0N1/i(O+N—1) gosity as rare alleles drift to intermediate frequen- NpI cies (Maruyama and Fuerst, 1984). Thus during the transit to equilibrium, there is an excess of whereN is the total number of genes sampled and low-frequency alleles compared with that expected O4NeV=H/(1H) (p. 400, Waterson, 1984). for a given value of H in populations at drift- This theoretical distribution for 0-05 frequency mutation equilibrium. intervals is compared with the observed distribu- We compared the observed distributions of the tions of the two hakes in fig. 5. It is apparent that number of alleles at different allele frequencies there is a large excess of alleles with frequencies

50 50 M capensis M poradoxus 4°. H 0.055 40 H= 0.067 11)

3Q. 30

20 E :3 lO' 10

. It.I I I Ji 0 0.5 1.0 0 0.5 1.0 Frequency Frequency Figure 5 Comparisons of observed (broken line) numbers of alleles at 005 frequency intervals with the theoretical (solid line) distribution expected at equilibrium in the infinite-alleles model. SYMPATRIC FISH SPECIES: PROTEIN ANALYSIS 19 less than 005 for both species. The Kolmogorov- Acknowledgments Wethank Dr E. MacPherson and the crew Smirnov test (Sokal and Rohlf, 1981) for fit to the of the M.F.V. Chicha Touza, Institutode Investigaciones Pesqueras, Barcelona, Spain for collection of samples in theoretical distribution indicates that this deviation Namibia and C. Hart and the crew of the R.V. Africana for is highly significant for both M. capensis (D67 = samples in South Africa. Dr Wessels of the Fishing Research 0337,P<0'Ol) and M. paradoxus (D54=0424, Institute, University of Cape Town provided laboratory P<001). facilities. 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