519
Preservation of genetic diversity in restocking of the sea cucumber Holothuria scabra investigated by allozyme electrophoresis Sven Uthicke and Steven Purcell
Abstract: Population genetics analyses should be considered when releasing hatchery-produced juveniles of the sea cucumber Holothuria scabra when spawners from nonlocal populations are used. In New Caledonia, within-region genetic heterogeneity of H. scabra populations (examined through allozyme electrophoresis of 258 animals) indicated high gene flow between nine sites and FST values did not deviate significantly from zero. However, exact tests indicated that populations at two sites with limited water exchange in the southern location were significantly different from populations at three other locations on the west coast. Inclusion of H. scabra sampled in Bali (n = 90) and Knocker Bay, Australia (n = 47), and comparisons with existing data from the west Pacific (Torres Strait, Solomon Islands, Upstart Bay, Hervey Bay) showed that populations were significantly different (using exact tests) and samples partitioned distinctly using unweighted pair group method with arithmetic mean clustering. Rogers’ genetic distance values between populations were significantly related to geographic distances, showing a pattern of isolation by distance. The rapid increase in genetic distance over the first few hundred kilometres supports the view that the spatial extent of any translocation needs to be carefully considered on the basis of knowledge of variation in allele frequencies within the target area. Résumé : Lorsqu’on ensemence en nature des jeunes Holothuria scabra élevés en pisciculture et issus de parents qui n’appartiennent pas aux populations locales, il est indiqué de penser faire des analyses génétiques de population. En Nouvelle-Calédonie, l’hétérogénéité génétique des populations d’H. scabra au sein d’un même région (basée sur l’examen par électrophorèse des allozyme de 258 individus) démontre l’existence d’un important flux génétique entre neuf sites; de plus, les valeurs de FST ne diffèrent pas significativement de zéro. Cependant, des tests précis montrent que les populations à deux sites du sud, qui font peu d’échange d’eau, diffèrent significativement de trois autres populations de la côte occidentale. L’inclusion de spécimens d’H. scabra prélevés à Bali (n = 90) et à la baie Knocker en Australie (n = 47), de même que la comparaison avec les données existantes pour le Pacificique ouest (détroit de Torres, îles Solomon, baie Upstart et baie Hervey), indiquent que les populations sont significativement distinctes (à l’aide de tests précis) et que les échantillons se séparent de façon nette dans une analyse de groupement de type UPGMA (méthode de groupement par associations moyennes UPGMA). Les valeurs de distance génétique de Rogers entre les populations sont en corrélation significative avec les distances géographique, ce qui indique un système d’isolement par la distance. L’accroissement rapide de la distance génétique sur les premières centaines de kilomètres appuie l’opinion que la répartition spatiale de toute translocation doive être prise en compte sérieusement à partir de ce qu’on connaît de la variation dans les fréquences d’allèles dans la région ciblée. [Traduit par la Rédaction]
Uthicke et al.
528
Introduction About 20 species of sea cucumbers of the order Aspidochirotida are fished commercially in the tropical IndoPacific. The processed body wall, known as “bêche-de-mer” or “trepang”, is exported to Asia, where it is consumed as a delicacy, an aphrodisiac, or a natural medicine. Fueled by an increase in trade with China, widespread overfishing of sea cucumbers in most countries over the past decades has de-
pleted stocks of commercially important species (Conand 1997, 2001; Uthicke and Benzie 2000a). The species of highest value, the sandfish Holothuria scabra, is overfished in many regions because it often occurs in easily accessible, shallow coastal waters (Conand 1989; Hamel et al. 2001). Although countries such as Australia, Fiji, and the Solomon Islands have imposed long-term fishing closures for this species, there is some evidence that natural recovery of sea cucumber stocks may take several
Received 7 May 2003. Accepted 23 December 2003. Published on the NRC Research Press Web site at http://cjfas.nrc.ca on 12 May 2004. J17511 S. Uthicke.1 Australian Institute of Marine Science, PMB No. 3, Townsville, Queensland 4810, Australia. S. Purcell. WorldFish Center, c/o Secretariat of the Pacific Community, B.P. D5, 98848 Noumea CEDEX, New Caledonia. 1
Corresponding author (e-mail:
[email protected]).
Can. J. Fish. Aquat. Sci. 61: 519–528 (2004)
doi: 10.1139/F04-013
© 2004 NRC Canada
520
decades (Battaglene and Bell 1999; Lincoln-Smith et al. 2000; Uthicke et al. 2004). Following the development of culture methods for H. scabra (Battaglene 1999; James 1999; Pitt 2001), many countries are now considering restocking with hatcheryproduced juveniles as a way to accelerate the recovery of sizeable breeding populations (Battaglene and Bell 1999). This strategy raises concerns about altering the genetic diversity of remnant stocks (see Hindar et al. 1991; Utter 1998) because it may be desirable to translocate the hatchery-produced juveniles to sites other than where the adult broodstock were collected. Release of hatchery-produced animals can cause reduced genetic diversity in wild stocks in two ways. Firstly, if broodstock used for hatchery production are from different genetic stock, the native alleles may be reduced in frequency through introgression of exogenous alleles. This introgression may be caused either by interbreeding of animals from the two stocks or by the introduced alleles outcompeting native alleles because of higher fitness of individuals carrying these introduced alleles (Hindar et al. 1991; Utter 1998). Thus, introgressions may reduce genetic differences among stocks (Utter 1998). Secondly, interbreeding of introduced stock with the native stock may disturb potentially complex adaptations to the local environment that have evolved over time, causing “outbreeding depression” (Templeton 1986; Waples 1995). Such hybridizations tend to have long-lasting effects that are disadvantageous to native stocks rather than beneficial through added genetic variation (Hindar et al. 1991). Therefore, unless the genetic structures of stocks at release and source sites are known, juveniles should be released only at native sites to preserve the genetic diversity of stocks (Shaklee and Bentzen 1998). These cautions arise mostly from translocations of temperate fishes but could be more pertinent for sedentary invertebrates with limited postsettlement dispersal such as H. scabra, which moves at most 1–2 m·day–1 (Hamel et al. 2001) and probably only a couple of kilometres in a lifetime. However, even when native populations are used, the number of spawners must be carefully chosen to avoid a reduction in overall effective population sizes (Ryman et al. 1995), especially in highly fecund species with large variations in reproductive success (Tringali and Bert 1998), such as holothurians. A reduction in effective population size can lead to losses of genetic variation, for example through decreased heterozygosity or the loss of rare alleles (Utter 1998). Another potential problem when releasing animals bred in captivity into the environment is domestication selection, which can lead to a high supplementation load of deleterious alleles in wild populations (Lynch and O’Hely 2001). Hardly any work on these issues has been conducted for marine invertebrates. However, studies on abalone (Haliotis spp.) have provided some indication for reduced diversity in hatchery-produced animals (Mgaya et al. 1995; Gaffney et al. 1996). In general, long-lived marine invertebrate larvae provide an efficient means of gene flow between populations, leading to small population differentiation even over large geographic scales (Avise 1994; Palumbi 1997). Within the Pacific, Holothuria nobilis (Uthicke and Benzie 2000b, 2003) and a variety of other echinoderms show very little
Can. J. Fish. Aquat. Sci. Vol. 61, 2004
population differentiation over distances larger than 1000 km (e.g., Williams and Benzie 1998; Benzie 1999; McCartney et al. 2000). Holothuria scabra is a broadcast spawner with a relatively long planktotrophic larval duration of 10–14 days in hatchery culture (Battaglene et al. 1999; Mercier et al. 1999), although larvae could survive longer in the wild in low concentrations of algae. Unlike the examples above, allozyme genetic studies on H. scabra (Uthicke and Benzie 1999, 2001) indicated that gene flow between populations greater than ~800 km apart was more restricted than expected from estimates of larval duration. In one instance, significant genetic differentiation occurred between sites separated by only 16 km, possibly because of the geomorphology at one site that reduced larval connectivity and severe depletion of one stock through overfishing (Uthicke and Benzie 2001). This study used allozyme electrophoresis to determine genetic differentiation of H. scabra among multiple sites along New Caledonia, which are under consideration as release sites of cultured H. scabra in a project designed to determine optimal restocking methods (Purcell et al. 2002). We use these data to examine patterns of within-region genetic heterogeneity of H. scabra with a view to determining whether hatchery-produced juveniles from spawners collected at a single site could be released at other sites 60–400 km away. In addition, we investigated larval connectivity on a larger geographic scale by comparing data obtained in New Caledonia with previously published data from other Pacific regions and two additional populations in the Indo-Pacific. With this approach, we aim to assist restocking programs through an improved understanding of the genetic differentiation between stocks of H. scabra at different spatial scales so that the release of cultured juveniles can be planned in a responsible way to preserve the genetic diversity of existing stocks.
Materials and methods Study region Sea cucumbers are diverse and ubiquitous in New Caledonia (Conand 1989). The expansive shallow habitats of the lagoon around the main island (La Grande Terre, hereafter referred to as New Caledonia) covering approximately 24 000 km2 are enclosed by the second largest barrier reef in the world (Fig. 1). Mangrove systems and shallow fringing coral reefs with sandy or muddy sea grass beds are abundant along the west coast of the island. A number of the inshore holothurian species have been fished periodically since the 1840s, especially the sandfish H. scabra (Conand 1989). This species is common mostly on inshore fringing reef flats and bays < 5 km from the mangrove systems, but there is evidence of stock depletion at some sites because of intense fishing. Sampling Five locations, each separated by 60–130 km, were selected along the coast of New Caledonia (Fig. 1). Two replicate sites with H. scabra, 8–23 km apart, were then chosen within four of these locations on the west coast of the island, whereas only one site was selected at the single northeast lo© 2004 NRC Canada
Uthicke et al.
521
Fig. 1. Sampling sites (solid circles) for H. scabra in New Caledonia, Knocker Bay (Australia), and Bali (Indonesia). The barrier reef around New Caledonia is indicated with thick lines, letters denote sampling sites, and numbers denote locations. Sites: A, Baie de Sainte-Marie; B, Ilot Maître; C, Ouano; D, Ilots Kundogi; E, Plateau de Béco; F, Pointe Pindaï; G, Pouangué; H, Ouaco; I, Récif Thaavaam (Ilot Cocotier). Locations of previous samples from other regions (Uthicke and Benzie 2001) are indicated with open circles.
cation. Adult H. scabra (n = 14–34) were collected by hand from the nine sites (labelled A to I; see Fig. 1) in the five locations within search areas of 2.0. Because data points were not independent, a Mantel (1967) test was used to test for matrix correlation. These tests used genetic distances between all populations thus far analysed, with one exception: the population from Solomon Islands site A was omitted because it represented a sample from an isolated lagoon (Uthicke and Benzie 2001). The significance of Mantel’s normalized Z was tested by 999 random permutations using TFPGA.
Results Most allozyme loci investigated were highly variable and two to five alleles could be reliably scored (Table 1). The only exception was MDH*, which displayed fixed or nearfixed alleles in each population, similar to previous studies on H. scabra (Uthicke and Benzie 2001). Genetic variability within sites (e.g., allelic richness, number of polymorphic loci, heterozygosity) did not show any distinct differences between populations sampled in New Caledonia, Knocker Bay, and Bali (Table 2). The observed heterozygosity is, in each case, very close to that expected under Hardy–Weinberg equilibrium. Exact tests for deviations from Hardy–Weinberg equilibrium for each locus at each site indicated no significant deviations (P > 0.05) after corrections for multiple tests, indicating that all H. scabra populations investigated in New Caledonia, Knocker Bay, and Bali were in Hardy–Weinberg equilibrium. Genotypic diversity (expressed as number of genotypes observed, Ngo) was high in each population, but some genotypes occurred more than once (Table 2). Exact tests for population differentiation indicated no significant difference (P > 0.05) between any sites within locations along the coast of New Caledonia. Therefore, data from sites within locations were pooled for all subsequent analyses to increase statistical power of the analyses. The FST statistics for data from New Caledonia (Table 3) show low FST values for each locus, and the confidence intervals
Can. J. Fish. Aquat. Sci. Vol. 61, 2004
of the averages include zero. Therefore, this analysis did not indicate restrictions in gene flow within New Caledonia. Rogers’ (1972) genetic distance was low between all pairs of locations (Table 4). However, the distances between location 1 and locations 2, 3 and 4 were higher than other comparisons. This was confirmed by exact tests of population differentiation between locations: after Bonferroni corrections, the pooled population from location 1 was significantly different from those at locations 2, 3, and 4. No significant difference existed for other between-location comparisons in New Caledonia. Within New Caledonia, a cluster analysis illustrated the larger difference between populations at location 1 and those at other locations (Fig. 2). Populations from each of the large geographic regions (New Caledonia, Hervey Bay, Upstart Bay, Torres Strait, Solomon Islands, Northern Territory, Bali) form separate clusters, which are in most cases supported by high bootstrap values obtained from 1000 permutations. The two populations west of the Torres Strait (i.e., Bali and Knocker Bay) show the largest genetic differences from the other populations. Differences between all populations from the main geographic regions were highly significant (exact tests, all P values
