Population genetics of the shrimp Artemesia longinaris (crustacea, penaeidae) on the south-west atlantic coast

Comp. Biochem. Physiol. Vol. 106B,No. 4, pp. 1015-1020, 1993 Printed in Great Britain

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

POPULATION GENETICS OF THE SHRIMP ARTEMESIA LONGINARIS (CRUSTACEA, PENAEIDAE) ON THE SOUTH-WEST ATLANTIC COAST L. I. WEBER, M. B. CONCEI~AO,M. L. RUFFINOand J. A. LEVY Fundaq~o Universidade do Rio Grande, Lab. Bioquimica Matin_ha, Dep. Quimica, C.P. 474, 96200, Rio Grande, RS, Brazil (Received 24 November 1992; accepted 15 January 1993) Abstract--1. In order to test the hypothesis of the existence of two subpopulations of the shrimp

Artemesia longinaris on the south-west Atlantic coast, as was suggested by Nascimento (Nascimento P. A. M., 1983; Naturalia, Sao Paulo, 8, 33-47), a gene frequency analysis of four A. longinaris subpopulations was done by starch-gel electrophoresis techniques. 2. Samples were collected along the Rio Grande (Brazil) and Mar del Plata (Argentine) coasts. 3. Three of the eight enzyme systems analyzed were polymorphic (MDH, PGI and IDH). 4. No significant allele frequency differences were found among samples from the putative subpopulations. 5. The contingency Chi-square test (P >0.112), the low values of Dm (0.0007), G,t (0.0039) and Fst (0.008), and the high genetic identity (I = 0.999) demonstrated the large homogeneity among A. longinaris samples. 6. It is concluded, thus, that there is no genetic evidence to support the existence of two subpopulations of A. longinaris on the south-west Atlantic coast.

INTRODUCTION

The shrimp, genus Artemesia (Bate, 1888) (Crustacea, Decapoda, Penaeidae) has only one species, A. longinaris Bate, op cit, which is endemic on the south-west Atlantic coast (Boschi, 1963, 1964, 1966, 1969a). Its distribution ranges along the coast from Cabo Silo Tome, Brazil (22°S) to Pto. Rawson, Argentina (43°S) (Iwai, 1973) (Fig. 1) and reaches depths of 30 m, showing higher concentrations of larvae and adults between 15 and 25 m depth. The spawning of this species occurs principally in autumn and spring, and seems to be restricted to the Rio Grande do Sul (Brazil) region (32°S) (Calazans, 1984). Eggs are demersal and not adhesive, which means that they can be transported by currents. Larval stages are meroplanktonic and migrate from deeper to coastal waters (Scelzo, 1971). The adults show a reproductive migration to deeper waters at the end of spring and the beginning of summer (Boschi, 1969a,b). Since 1988, A. longinaris has become commercially important in the State of Rio Grande do Sul (Brazil). Although of primary importance to the establishment of fisheries management plans, aspects of the population structure of A. longinaris within its range of distribution are very poorly known. Nascimento (1983) suggested the existence of two subpopulations of A. longinaris with different environmental preferences, which would migrate between Sta. Catarina State (Brazil) and Argentina, in response to the latitudinal displacement of the sub-tropical convergence during the year (Fig. 2). According to Nasci-

mento (1983), there is a more northern, warm-water sub-population, which migrates south (to higher latitudes) during the summer. This movement seems to follow the drift south of the warm waters of the Brazil Current, since, in early April, with the influx of sub-Antarctic waters to the Rio Grande do Sul, this subpopulation leaves the region, returning to the north. During the winter, the warm-water subpopulation is replaced on the coast of Rio Grande by a second, cold-adapted subpopulation, which comes from Argentina following the colder waters of the Falkland Current (Fig. 2). The aim of this work was to discover if two subpopulations of A. longinaris exist in the southwest Atlantic. For this, the technique of isozyme electrophoresis was utilized because of its proven reliability for the identification and discrimination of genetic stock (Selander et al., 1970; Ayala et al., 1974a,b; Ayala, 1975; Tracey et al., 1975; Fuller, 1977; Lester, 1979; Mulley and Later, 1980; Janson and Ward, 1984; Ward and Janson, 1985).

MATERIALS AND METHODS

A total of 428 mature individuals of A. longinaris of both sexes were collected by trawl netting in the coastal waters of Rio Grande do Sul (Brazil, 32°S) and Mar del Plata (Argentina, 38°S) during the period of December 1989 to January 1991 (Fig. 1). All individuals are classified according to locality and season of collection: 1. Argentina (spring), 2.

1015

1016

L.I. WEBER et al.

Brazil (spring-summer), 3. Brazil (autumn), 4. Brazil (winter). Samples of muscle from each shrimp were homogenized in 0.02 M Tris-HCl, pH 7.5, and centrifugated at 10.000rpm for 5min in a refrigerated centrifuge. Horizontal starch-gel electrophoresis was carried out using 14% Penetrose-30. The buffer systems utilized were the following: (1) Tris-citrate, pH 8.0 (Siciliano and Shaw, 1976); (2) Tris-citric-boric LiOH, pH 8.26-8.31 (Redfield and Salini, 1980); (3) discontinuous Tris-citrate, pH 5.1-6.0 (Guinez and Gallegnillos, 1986); (4) Tris-citrate, pH 7.0 (Siciliano and Shaw, 1976); (5) discontinuous borate-Tris-citrate, pH 8.2-8.7 (Poulik, 1957); (6) Tris-boric EDTA, pH 9.0 (Ayala et al., 1974); (7) LiOH, pH 8.15-8.30 (Shaklee and Keenam, 1986). EC numbers, supports, buffers and resolutions of the enzymes assayed are shown in Table 1. The staining procedure followed the techniques of Brewer (1970), Shaw and Prasad (1970) and Harris and Hopkinson (1976). Allele frequencies for each sample were obtained for the enzymes successfully resolved. The genetic variability within subpopulations was estimated by the 99% criterion of polymorphic loci and by the unbiased mean heterozygosity (Snedecor and Irwin, 1933). Polymorphic loci were submitted to Chisquare tests for deviation from Hardy-Weinberg equilibrium, with correction for small samples (Snedccor and Irwin, 1933; Levene, 1949). F-statistics were calculated for the four subpopulations at each locus (Wright, 1965; Nei, 1977, 1987). The genetic

diversity and differentiation within and between subpopulations were estimated by the statistics of Nei (1973, 1987). The BIOSYS-I program (Swofford and Selander, 1981) was used for the Hardy-Weinberg equilibrium test, F-statistics and genetic differentiation indexes. Also calculated was the number of immigrants (Nm,t) according to Slatkin and Barton (1989), to estimate the levels of gene flow. RESULTS Three of the eight enzymatic loci successfully interpreted (Table 2) were polymorphic in at least one of the four subpopulations (Mdh, Pgi, Idh). No deviations from the Hardy-Weinberg equilibrium (HWE) were found in any of the samples. No significant (P > 0.05) deviations from HWE were found after the pooling of the isozyme data from all individual~ into one large "population" for any locus. The genetic diversity of the total population, assuming panmixis was 0.05 (including monomorphic loci). More than 99% of this diversity could be explained by within-sample differences (Gs = 0.996) (Table 3). In Table 3 can be found the inbreeding coefficients (Wright, 1965; Nei, 1977) for the loci in the samples. No significant (Chi-square test; Li, 1955; Snedecor and Irwin, 1933; P > 0.05) differences from zero were found, either for Fls (the mean deviation from random mating within subpopulations) or for Fst (the standardized variance across subpopulations) (Table 4). The contingency Chi-square test demonstrated that

B RAZI

L

Sta. Catarina

30", ;ao k~

sta 0 q"

Mar de Plata, 0

~

0

Krn 40"o

60"

50"

40"

Fig. 1. Geographic distribution of Artemesia longinaris. Arrows indicate the two sampling sites.

Artemesia longinaris o n the south-west Atlantic coast

1017

-,°..

..e, lAuT... |WINTER I SPRINal IJ.Ns .I~E. I.AmI.P.I.AvlJuNIJu- IAualsePIOCTINOVI[DEC~

: Brazi I

•.•. 0•

N. 8 t a . C a t a r i n a S. S t a . C a t a r i n a

i

:

i

**

.: "='.*O••o•, o..'•"

:

:

"!...

:

i "°e|l ii °,'e

.;.

°~°°°°°=o.°.o.°°•o°°°°•°°~

. / ~ Argentina

Fig. 2• Migration associated with water bodies o f t w o Artemesia longinaris subpopulations. Modified from Nascimento (1983). ( . . . ) Brazil current (tropical waters); (- --) Falkland current (sub-Antarctic waters).

the four samples were not heterogeneous in allele frequencies for the loci studied (P=0.112, Table 5). This was ratified by the high values of unbiased Nei's Genetic Identities (Nei, 1978) obtained between all pairwise comparisons, that was 0.999 (Brazil, autumn-Brazil, winter) or higher. The high average number of migrants exchanged between local populations (Nm,t = 32.98, calculated from rare alleles with the adjusting for sample size) is further evidence of high levels of gene flow (Slatkin, 1985; Slatkin and Barton, 1989)•

DISCUSSION

The single most significant result of this work was the very high genetic homogeneity of the samples of Artemesia longinaris both in space and time. As was shown in Results, all samples of A. longinaris were in Hardy-Weinberg equilibrium. The lack of deviances from equilibrium, even when the whole data were pooled into one single "population", further indicates the high homogeneity of the samples. If there were differences among samples, the pooling would produce a heterozygote deficiency (Wahlund,

Table 1. Enzyme systems assayed in

Artemesia longinaris Buffer

Enzyme

EC Number

Support

system

Resolution

Acid phosphatase Alcohol dehydrogenase Alfa-glycerophosphate

3.1.3.2 I.l.l.8

I 1

2 1

NA NA

dehydrogenase Alkaline phosphatase

l.l. 1.81 3.1.3.1

1

2

I

1

2,3,4

Aspartate Aminotransferase Esterase Glutamate dehydrogenase

NA

2.6.I.1 3.1. I. 1 1.4. i.3

1,2

2 2,7 1,3,4

Hexokinase

Isocitrate dehydrogenase Lactate dehydrogenase Malate dehydrogenase Malic enzyme Octanol dehydrogenase Phosphoglucose isomerase Phosphoglucomutase 6-Phosphogluconate dehydrogenase

Sorbitol dehydrogenase Superoxide dismutase

2 1

i.1.1.42 1.1.1.27 1.1.1.37 1.1.1.40 1.1.1.73 5.3.1.9 2.7.5.1 1.1.1.44 1.1.1.14 I. 15. I. 1

Support: 1, Starch; 2, Polyacrylamide. Buffer systems: see text. Resolution: I, interpreted. NA: not analyzed due to poor resolution.

I

NA NA NA

2

I I I I

l

NA

2

I I

l l

I l

l

1 l

1,2,5,6 1,3,4,6

NA NA NA

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L . I . WEBER et al.

Table 2. Allele frequencies in Artemesia longinaris subpopulations Subpopulation Locus Mdh (N) I 13 100 84 Pgi (N) 120 109 100 90 81 72 ldh (N) 108 100 90 82

Argentina

Brazil (spring--summer)

Brazil (autumn)

Brazil (winter)

(115) 0.004 0.904 0.091

(166) 0.000 0.931 0.069

(60) 0.000 0.867 0.133

(65) 0.000 0.931 0.069

(115) 0.004 0.013 0.861 0.017 0.096 0.009

(164) 0.000 0.012 0.875 0.015 0.095 0.003

(64) 0.000 0.039 0.836 0.039 0.086 0.000

(83) 0.000 0.012 0.928 0.012 0.048 0.000

(115) 0.000 0.991 0.009 0.000

(166) 0.003 0.991 0.000 0.006

(60) 0.000 1.000 0.000 0.000

(65) 0.015 0.985 0.000 0.000

(90) 1.000

(42) 1.000

(47) 1.000

(54) 1.000

(ll5) 1.000

(ll8) 1.000

(47) 1.000

(54) 1.000

(90) 1.000

(66) 1.000

(25) 1.000

(54) 1.000

(70) 1.000

(18) 1.000

(47) 1.000

(30) 1.000 (54) 1.000 0.375 0.039 0.037

Locus

(70)

(66)

(64)

1.000

1.000

1.000

0.250 0.058 0.055

0.250 0.050 0.047

0.025 0.068 0.066

F~t

FiL

Fs~

Mdh Pgi Idh

- 0.109 -0.022 - 0.011

- 0.100 -0.014 - 0.006

0.008 0.008 0.006

Mean

- 0.057

- 0.049

0.008

Fis: mean deviation from random mating within subpopulations; Fit: mean deviation from random mating over all subpopulations; Fst: standardized variance across subpopulations; measure of the degree of genetic differentiation among subpopulations.

M0 (N) 100 Aat (N) 100 Ldh (N) 100 a-Gpdh (N) 100 Pgm (N) 100 Po.99 Ho Hc

Table 4. Summary of F-statistics at all Artemesia longinaris subpopulations

P0.99: proportion of polymorphic loci (99% criterion); Ho: observed mean heterzogysity; He: Hardy-Weinberg expected mean beterozygosity.

1928). Less than 1% (Gst ~- 0.0039) of the total genetic variation of A. longinaris is explained by the differences between subpopulations. Fst values were similarly low (Fst = 0.008). The absolute degree of genetic differentiation (Dm = 0.0003) obtained for A. longinaris was very low compared with values from other crustacean species including those obtained for subpopulations without genetic differentiations (Selander et al., 1970; Tracey et al., 1975; Fuller, 1977; Lester, 1979). The minimum Nei's Genetic Identity (I) obtained for the subpopulations of A. longinaris

was 0.999. Values of I equal or higher than 0.997 have been considered in Penaeus species as indicative of random mating (Lester, 1979). Characteristic alleles were observed for some of the subpopulations (Mdh) ll3, (Pgi) J2°, (Idh) 9° for Argentina and (Idh) 82 for Brazil (spring-summer). These alleles could be used to infer the level of gene flow between the samples (Slatkin, 1985) and the low frequencies of these alleles gave a very high value of migrants (Nm = 32.98), indicating that the population is effectively panmictic (Slatkin, 1985; Slatkin and Barton, 1989). This contradicts the results found in the only other work on population dynamics of this species (Nasciemento, 1983). Naturally, because of the very nature of science, only differences can be positively stated, whereas similarities always must remain provisory (things can only be disproved, never proved). It is possible, thus, that some structuring does exist in A. longinaris, and that we simply failed to reveal it with loci sampled. However, the work by Nascimento (1983) was based on few morphological characters, which have been proven to be much more influenced by the environment than the isozymes (Lindenfeiser, 1984). Isozymes have, thus, a higher heuristic value for population genetics than does morphology. We therefore conclude that, since for the data set used no structuring was found among samples, then it is likely that they belong to a single, large; panmictic population. High fecundities, two dispersal phases (planktonic larvae and vagile adults) and the migration patterns observed in penaeid shrimps have been used to explain the high level of genetic similarity between

Table 3. Genetic diversity analysis of four subpopulations of Artemesia Ionginaris Locus

Mdh Pgi Idh Mean * Mean t

Dst

Ht

Hs

Dm

Gst

Gs

0.00069 0.00089 0.00004 0.00054 0.00020

0.16620 0.22608 0.01636 0.13621 0.05108

0.16552 0.22519 0.01631 0.13567 0.05088

0.00092 0.00119 0.00006 0.00072 0.00027

0.09413 0.60393 0.00272 0.00397 0.00397

0.99586 0.99606 0.99728 0.99603 0.99603

*Mean of three polymorphic loci. tMean for all eight loci. Dst: diversity between subpopulations; Ht: total diversity (heterozygosity); Hs: diversity within subpopulations; Dm: absolute degree of gene differentiation between subpopulations; Gst: genetic differentiation coefficient between subpopulations; Gs: genetic differentiation coefficient within subpopulations.

Artemesia longinaris on the south-west Atlantic coast Table 5. Contingency Chi-square analysis at all polymorphic loci of Artemesia longinaris Locus Alleles Chi-squares d[f P Mdh Pgi Idh Totals

3 6 4

7.742 17.781 14.111 39.634

6 15 9 30

0.257 0.274 0.118 0. I 12

penaeid populations (Lester, 1979) and also may be the factors determining the genetic homogeneity found in the A. longinaris population within the region studied. Acknowledgements--The authors are very grateful to A. Sole-Cava for revision and additions to the manuscript and to E. Tellechea for invaluable help during the collection of samples. This study was supported by grant No. 931/90 from Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and FURG.

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