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Assessment of self-recruitment in a pink abalone (Haliotis corrugata) aggregation by parentage analyses.

ABSTRACT The implementation of abalone aggregations as a tool for stock enhancement has been under discussion. For this purpose, understanding the self-recruitment capacity of managed reefs based on the amount of larval retention is an important task to evaluate potential success. Under the hypothesis that every reef is mostly self-restored within a short spatiotemporal scale because of a rather reduced planktonic period, the practice of aggregating mature adults of pink abalone in a reef would improve local recruitment. This study assessed local replenishment within an abalone reef of pink abalone Haliotis corrugata at Bahia Asuncion (El Riito reef), a location on the west coast of the Baja California Peninsula, where an aggregation of adult abalone was studied. A parentage analysis was carried out between adults transplanted to El Riito and the juveniles collected from the same site by comparing their genetic profiles at 8 microsatellite DNA loci. The parentage tests defined successfully the status of more than 97% of the juveniles, revealing that approximately 4% of them could have been produced within the aggregation area. Because not all the potential parental abalone were collected, this self-recruitment proportion in the aggregation experiment was possibly underestimated. The suitability of parentage analyses, based on genetic markers as a robust alternative for the assessment of future aggregations, is discussed.

KEY WORDS: abalone, local recruitment, local settlement, Haliotis corrugata, microsatellites, self-recruitment, parentage analysis

INTRODUCTION

The implementation of mature abalone aggregations to improve local recruitment of abalone (Haliotis spp. Linnaeus 1758) reefs has been discussed as an alternative for stock recovery. This approach was presented originally by Prince et al. (1987, 1988), who demonstrated that the abundance of breeding animals determines the abundance of local recruitment in Haliotis rubra (Leach 1814). However, in other abalone, such as Haliotis laevigata (Donovan 1808) Shepherd et al. (1992) rejected this hypothesis, concluding that immigration of larvae from over a large scale was sufficient to mask any local stock-recruitment relationship. In understanding fertilization biology of H. laevigata, Babcock and Keesing (1999) provided new insights, suggesting that, in populations where spawning individuals are separated by distances of 1.6 m or more, fertilization success may be expected to be 50% or less and decrease rapidly with increasing distance. Thus, stocks at higher densities are predicted to have higher fertilization rates (~90%) such that fertilization success is not a factor limiting recruitment.

The estimation of local recruitment in marine systems is undoubtedly a difficult proposition because most marine invertebrates produce large numbers of extremely small planktonic larvae that are spread into a vast volume and are subject to advection and diffusion, and extremely high mortality rates (Jones et al. 2005, Hedgecock 2010).

Difficulties associated with traditional mark-recapture studies, such as marking insufficient number of larvae and the low recovery rates, may be alleviated by the development of natural tags, such as genetic markers (Thorrold et al. 2002). Within these, microsatellites that are codominant DNA markers inherited in a Mendelian fashion and are hypervariable, have been used successfully to trace the pedigree of hatchery-reared organisms, such as the red sea bream (Pagrus major, Temminck & Schlegel 1843) (Perez-Enriquez et al. 1999), and to assess stock enhancement programs of species such as P. major and green abalone (Haliotis fulgens, Phillippi 1845) (Perez-Enriquez & Taniguchi 1999, Gutierrez-Gonzalez & Perez-Enriquez 2005).

More recently, genetic markers have been used in parentage analysis to estimate self-recruitment and connectivity in several marine species (Jones et al. 2005, Planes et al. 2009, Christie et al. 2010, Toonen & Grossberg 2011, Eble et al. 2011). Jones et al. (2005) showed, based on a genetic parentage analysis, that one third of juveniles of panda clown fish (Amphiprion polymnus, Linnaeus 1758) settled within 2 ha from the natal area, with many settling less than 100 m from their birth site. Planes et al. (2009) reported a higher level of local replenishment (~40%) than long-distance larval dispersal (up to 10%) in the orange clownish (Amphiprion percula, Lacepede 1802)using 16 polymorphic microsatellites. Parentage analysis in both A. polymnus and A. percula were compared with chemical and isotope labeling, confirming the DNA parentage identifications (Jones et al. 2005, Planes et al. 2009). Christie et al. (2010) demonstrated 2 parent-offspring pairs in the bicolor damselfish (Stegastes partitus, Poey 1868) at 2 sites in the Bahamas, indicating that larvae produced from this marine reserve settled within its boundaries. In contrast, Toonen and Grosberg (2011), in a study of the intertidal anomuran crab (Petrolisthes cinctipes, Randall 1840), found no consistent patterns of microsatellite parentage assignment to their natal population across years, indicating that the source of recruitment is unpredictable. Through indirect methods, Eble et al. (2011) found that the yellow tang (Zebrasoma flavescens, Bennett 1828) had an asymmetric gene flow between Hawaii and the western Pacific (16 times greater than the reciprocal), presumably because of larvae export.

In the pink abalone (Haliotis corrugata, Wood 1828), Diaz-Viloria et al. (2009) found nonsignificant genetic differentiation among populations over a range of hundreds of kilometers along the Baja California Peninsula. Those gene flow estimates give information of long-term population connectivity, but do not necessarily provide information about local recruitment in an ecologically relevant timescale, suggesting the necessity of estimates of local replenishment. Taking into consideration the hypothesis that every reef is mostly self-restored within a short spatiotemporal scale, the practice of aggregating mature adults of pink abalone in a reef would improve local recruitment.

The goal of this study was to assess self-recruitment of the pink abalone Haliotis corrugata in an aggregation of adults within a limited area using microsatellite DNA markers. Self-recruitment was estimated by a direct approach of parentage analysis, using the individual genetic profiles from adults to match offspring to their parents.

MATERIALS AND METHODS

Sample Collection

Adult and juvenile pink abalone Haliotis corrugata were collected at El Riito reef, Bahia Asuncion, on the west coast of the Baja California Peninsula, Mexico (Fig. 1). An aggregation experiment was carried out from October 2001 through March 2003, when adult abalone (n = 267; shell length, 96-162 mm) were transplanted to El Riito from 5 neighboring reefs (Table 1, Fig. 1). Specimens were put into a circular nuclear area of ~50 [m.sup.2] at a depth of 11 m (Fig. 1). Four searches for juveniles and adults were carried out in June 2004 and November 2004, and September through November 2005. The searches included 4 transects oriented to the 4 cardinal points at 10, 20, 30, 40, 50, and 100 m from the aggregation center, searching by diving for 15 min at each distance at every cardinal point (Fig. 1). A total of 63 adults and 69 juveniles were collected for tissue analysis.

Tissue samples of mantle were cut from adults, and epipodial tentacles from juveniles were removed with clippers. Samples were preserved in 80% ethanol until genetic analysis. Juveniles were released back to the reef after sampling.

DNA Extraction and Microsatellite Analysis

The DNA of adult and juvenile specimens was extracted (Wizard Genomic DNA Purification Kit; Promega, Madison, WI) and purified using the protocol of Sweijd et al. (1998) or a kit (DNeasy Blood and Tissue Kit; Qiagen, Hilden, Germany).

After confirming Mendelian inheritance (Diaz-Viloria et al. unpubl, data), 8 microsatellite loci (Hco15, Hco16, Hco19, Hco22, Hco97, Hco194, Hka3, and Hka56) were analyzed in adults and juveniles from El Riito using the same reaction components and thermal conditions reported by Miller et al. (2001) and Diaz-Viloria et al. (2008). The polymerase chain reaction products from thermal cyclers (iCycler Bio-Rad, Hercules, CA; 2720, Applied Biosystems, Life Technologies, Carlsbad, CA) were separated on 5% polyacrylamide gel electrophoresis (at 1800 V, 100 Amp, 90 W); the fragments were visualized using Sybr-Gold (0.07x) within a 1.1% agarose matrix (Rodzen et al. 1998) and scanned (FMBIOIII-Plus; MiraiBio Group, Hitachi Solutions America, San Francisco, CA). Allelic size was determined by comparisons with samples of known allele sizes from previous studies (Diaz-Viloria et al. 2008, Diaz-Viloria et al. 2009).The error rate of scoring microsatellites was 0% at Hco15, Hco16, Hco22, Hco 194, and Hka56; approximately 4% at Hco19 and Hco97; and 10% at Hka3. Individuals with ambiguous genotypes were reprocessed entirely and scored 2 or 3 times, reducing the error rate to 0 at any locus.

Data Analysis

Genetic Diversity

The genetic diversity was assessed by the number of alleles ([N.sub.A]), the effective alleles per locus ([N.sub.EA]), and observed ([H.sub.O]) and expected ([H.sub.E]) heterozygosities. Deviations from Hardy-Weinberg equilibrium (HWE) were evaluated by Fisher's exact tests using the Markov chain (1,000,000 steps and 100,000 dememorization steps). A linkage disequilibrium test for all pairs of loci (10,000 permutations) was done, including individuals with genotype data from at least 6 loci. Hardy-Weinberg equilibrium and linkage disequilibrium analyses were performed with Arlequin 3.0 (Excoffier et al. 2005). The level of significance ([alpha] = 0.05) of multiple tests was then adjusted using the sequential Bonferroni approach (Rice 1989).

Larval Retention at El Riito

Larval retention was estimated for El Riito by a direct approach by means of parentage testing (single parent and sibship). To assess the validity of the microsatellites for these tests, Mendelian segregation, probability of identity, and polymorphic information content (PIC) were analyzed. The power of parentage analysis was calculated by the probability of identity. This is the probability of finding the same genotype in 2 individuals taken randomly from a population, which is given by I = [[summation].sub.i][p.sub.i.sup.4] + [[summation].sub.i] [[summation].sub.j>i] [(2[p.sub.i][p.sub.j]).sup.2], where [p.sub.i] and [p.sub.j] are the frequencies of the ith and jth alleles found in the population for every locus (Paetkau & Strobeck 1994). To calculate I, the allelic frequencies of adults and juveniles were used as total data. The total probability of identity was the product of each I value obtained at every locus (Paetkau & Strobeck 1994). The PIC, a measure of informativeness related to expected heterozygosity, was evaluated with CERVUS 3.0 (Slate et al. 2000).

The parentage analysis included adults collected within the nuclear area and the juveniles collected along the 4 transects to determine whether each juvenile could have been offspring of the adults within the nuclear area. The 8 loci showed unambiguous genotyping, HWE, and null allele frequencies less than 0.05 (Bernatchez & Duchesne 2000). The parentage analysis was processed with CERVUS 3.0 (Slate et al. 2000, Kalinowski et al. 2007), considering 55 candidate parents to a sampled parents ratio of 55/267 and parents' sex unknown. Nonexclusion probabilities for the most likely parents were obtained based on the likelihood of assignment obtained in CERVUS (LOD). The LOD is the natural logarithm of the likelihood ratio, which uses available data to test alternative hypotheses. The probability of obtaining data D under hypothesis H can be written P(D/H). The ratio of the probability of observing the data under one hypothesis ([H.sub.1]) compared with a second hypothesis ([H.sub.2]) is the likelihood ratio (Slate et al. 2000), and is written

L([H.sub.1], [H.sub.2]/D) = [P(D/[H.sub.1])/P(D/[H.sub.2])].

The LOD scores cannot be evaluated using a standard distribution, such as a chi-square distribution; therefore, CERVUS uses simulation of parentage analysis to evaluate the confidence in assignment of parentage to the most likely candidate parent (Slate et al. 2000). Critical LOD values were established with 2 levels of confidence--90% (relaxed) and 95% (strict)--based on simulations of 1,000,000 offspring.

For each putative parent-offspring pair, we calculated the probability of the pair being false, given the frequencies of shared alleles Pr([phi]|[lambda]) (Christie 2010, Christie et al. 2010). Simulations required for the calculations of Pr([phi]|[lambda]) were conducted with 10,000 false pairs generated over 100 null data sets, using methods that were implemented in R programs (http://sites. google.com/site/parentagemethods/).

Potential full-sibs and half-sibs from the groups of juveniles of the same age class were examined using 2 software systems: Colony 2.0.0.0 (Jones & Wang 2009) and Pedigree 2.2 (Herbinger 2005). Colony implements a likelihood method for inferring parentage and sibship from codominant marker data. Parameters were set as a polygamous mating system, long run, and the error rate was set at 0.0001. In the absence of parental information, Pedigree infers the kin and full-sib relationships of individuals in a sample by estimating the likelihood ratios and maximizing the overall score with a Markov chain Monte Carlo method. The parameters of the program (number of iterations, temperature, and weight) were optimized as described in Herbinger (2005). The accuracy of the kinship assignments were assessed using statistical inference in the same software, where 100 randomized sets are generated and the Markov chain Monte Carlo method algorithm is evaluated on each set with the same parameter that was used for the best full-sib partition and the best kin partition.

RESULTS

Spatial Distribution of Juveniles

Collection sites of 51 juveniles were recorded within the study area, showing a higher abundance of pink abalone juveniles in the east and north transects (17 individuals and 14 individuals, respectively), followed by the west (n = 11) and south (n = 6) transects; 3 juveniles were found within the nuclear area (Table 2). The distribution of juveniles was rather homogeneous at each transect, although higher numbers were found 100 m along the north and east transects, and 10 m away along the west transect (Table 2).

Genetic Diversity Assessment

Genotypes at El Riito were obtained from 55 adults and 51 juveniles. The number of alleles per locus showed low (Hco22, Hco194, Hka56), moderate (Hco15, Hco16, Hco97), and high (Hco19, Hka3) polymorphism, but the number of effective alleles (~3) showed low polymorphism at most loci, with the exception of Hco19 and Hka3, which had 8 effective alleles and 15 effective alleles, respectively (Table 2, Figs. 2 and 3). All samples were in HWE at almost all loci after Bonferroni sequential adjustment, except at loci Hco19 and Hka3 of juveniles, which had heterozygote deficiencies (P < 0.008; Table 3). Linkage equilibrium was found in all samples from El Riito after the Bonferroni adjustment (P < 0.0029).

Direct Approach for Parentage Assignment at El Riito

The summary statistics from CERVUS showed that 8 loci were suitable for parentage analysis because all loci showed unambiguous genotypes, null allele frequencies less than 0.05 (including Hco97), and genotypic frequencies in HWE (Table 4). The exception was Hka3, for which the software was not able to proceed with the HWE test because there were many alleles. Five loci were less informative and discriminative (PIC values, 0.43-0.59; Pr(Z) values, 0.72-0.93), and 2 loci (Hco19 and Hka3) were highly informative and discriminative (PIC > 0.87; Pr(Z) < 0.35; Table 4). When multilocus genotypes were analyzed, the probability of identity was I = 6.73 x [10.sup.-9], meaning that one in about 148.5 million individuals would have exactly the same multilocus genotype by random selection.

The parentage analysis of 55 adults (potential sires/dams) and 51 juveniles resulted in the exclusion of 45 juveniles, with a potential assignment of 6 juveniles (Juv-1, Juv-32, Juv-36, Juv-46, Juv-55, and Juv-66) each to a single parent, but not to a parental couple (Table 5). This represents a potential offspring-single-parent assignation level of 11.7%. Three of these assignations showed high LOD values (>3.0), and 2 of them (Juv-32-Adult-11, Juv-66-Adult-29) were significant with a relaxed confidence level of 90% (LOD [greater than or equal to] 3.97), but none were significant under a strict confidence level of 95% (LOD [greater than or equal to] 4.95). Nevertheless, the 6 offspring-single-parent assignations showed considerably low values of average nonexclusion probabilities (0.0000106 [greater than or equal to] P [greater than or equal to] 0.000000122; Table 5). These values represent the average probability of not excluding a single randomly chosen, unrelated individual from parentage at all loci.

In contrast, after calculating the probability of a putative offspring pair being false using a Bayesian approach, the 6 putative parent-juvenile pairs were not supported (Pr ([phi]) = 1). With a set of loci that showed exclusion probabilities per locus that were not discriminative enough (except Hco19 and Hka3; Table 4), an expected 10 false pairs were obtained. After the simulation of 10,000 false parent-offspring pairs through a Bayesian approach, the probabilities of the 6 putative parent juvenile pairs being false were not rejected (Pr ([phi]|[lambda]) > 0.05). Despite the apparent lack of significant paternity assignments, the matching of rare alleles at Hka3 of juveniles and adults with high LOD values, suggests that at least juveniles 32, 55, and 66 show a possible parent assignment (Table 5).

To test for parentage among juveniles, the parentage analysis with Colony revealed 14 possible groups with half-sib and full-sib relationships of 2-4 related individuals each, with probabilities varying between 0.23 and 1.0. From Pedigree, 13 kinship groups with 2 or 3 related individuals each were partitioned, most of which coincided with the 14 kin groups found with the Colony software. The Pedigree and Colony software partitions agreed that there were at least half-sibs in 10 of the conformed groups. However, the statistical inference approach in Pedigree, with 100 randomized partitions in kin and full-sib analyses, did not produce any group with significant cohesion, indicating that the partition is not real and must be considered an artifact, meaning that it is highly unlikely that there are half-sib or full-sib families among El Riito juveniles.

DISCUSSION

An important issue in marine ecology of invertebrates with sessile or sedentary adults, high fecundity, tiny planktonic propagules, few obvious barriers to dispersal, and broad range is the dispersal potential of a species as a function of the breeding site. The very small size of the larval stages of marine organisms, such as abalone, and the complexity of the environment (advection and diffusion jets, retention zones, seasonal shifts in currents, and episodic events of upwelling) make tracking the flow and fate of propagules difficult (Levin 2006, Cowen & Sponaugle 2009, Weersing & Toonen 2009).

Indirect Inference of Aggregation Success

The presence of juveniles in the area surrounding the center of the aggregation could indicate the success of this strategy for stock enhancement. Juveniles derived from the aggregation do not have dominant directional dispersal from the center, although movement to the south seems to be the least probable. For the other directions, there is a tendency toward larvae settling farther from the center.

The significant departures of HWE of juvenile allelic frequencies at loci Hcol9 and Hka3 indicate that the transplanted adult abalone come from neighboring reefs at El Riito. Toonen and Grosberg (2011) suggested that departures from HWE from mixing of individuals from breeding groups with different allelic frequencies could account for heterozygote deficiencies on a small scale.

Direct Inference of Success from Aggregation

The level of identity and parentage indicate that some juveniles probably came from the adults in the aggregation. Genetic matching with the markers supports high certainty because the probability of 2 abalones having exactly the same multilocus genotype at random is only one in ~148.5 million individuals, as abalone are scarce in this area. The average catch at El Riito and neighboring reefs between 2008 and 2010 was 7,500-10,000 individuals (Guzman-del Proo, unpubl, data).

The ability to assign progeny to particular parents is contingent on the number of loci available and the method's ability to exclude nonparents, which is highly correlated to the allelic diversity of the loci (Bernatchez & Duchesne 2000, Selvamani et al. 2001). According to Bernatchez and Duchesne (2000), the minimum number of loci for assignment of parentage is 5, with 11 alleles per locus if 50 potential parents are tested. In our study, 8 loci and a mean 13-15 alleles per locus were used; however, the mean number of alleles per locus are overestimated because only a low number of alleles were equally frequent in most loci, leading only to 2 loci with higher numbers of effective alleles and discriminative probabilities of exclusion. Within this set, locus Hka3 was the most discriminative because it showed the largest number of effective alleles and the lowest probability of exclusion.

Although our results indicate the lack of match between the genetic profiles of most juveniles with any of the sampled adults, one important consideration here is that not all the potential parents were sampled. The possibility remains that some of the adults that were not sampled sired some juveniles.

The parentage analyses meant to reconstruct full- or half-sib relationships among juveniles did not show any such relationships. This result occurs when most families are extremely small (e.g., 1 offspring per sibship). It is difficult to have replicate runs in Colony (using different random numbers seeds) converging to the same best configuration (Jones & Wang 2009). For this purpose, more microsatellites would have to have been analyzed or more juveniles in the experimental area would provide more reliable results.

At least 2 juveniles were progeny of transplanted adults, which represents a local recruitment of 3.9%. Even though this appears to be low, it is likely an underestimate of the real local recruitment because only 20.6% of the adult abalone that were transplanted (putative parents) were retrieved and used for the DNA analysis. According to Christie et al. (2010), for species with a large population size and low sampling, the finding of a few parent offspring pairs suggests that there are high rates of self-recruitment.

Factors Influencing Retention of Larvae

Larval retention, as much as dispersal, is influenced by oceanographic and biological forces, such as larval and post-larval behavior, including the duration of the planktonic stage (Leighton 2000, Takami et al. 2006, Weersing & Toonen 2009), the direction and intensity of currents (Guzman-del Proo et al. 2000), and the habitat and feed available for settled juveniles (Pineda 1994).

Veliger larvae of abalone are roughly spherical and have difficulty orientating in flowing water; consequently, swimming behavior might be expected to confer little directional movement. However, vertical movement (buoyancy movements) may confer an ability to use or avoid surface currents (McShane 1992). Even though the duration of the pelagic larval stage is 4-17 d in Haliotis corrugata (Leighton 1974, Leighton 2000), it could be an important element in understanding dispersion. There are findings that refute that the pelagic larval stage is a good predictor of the magnitude of dispersal and gene flow (Shanks 2009, Weersing & Toonen 2009, Selkoe et al. 2010). Temby et al. (2007) found that recruitment of the blacklip abalone (Haliotis rubra) is primarily local, and suggest that limited dispersal of larvae is explained by the negatively buoyant eggs and the early stages of development occurring near the seabed. According to Shanks (2009), null models of passively dispersed propagules are poor predictors of larval dispersal because they do not include the behavior of the larvae. In addition to swimming behavior, high water temperatures in the inshore countercurrent zone could promote recruitment over short distances because it decreases the length of the planktonic larval stage (Takami et al. 2006), which in H. corrugata (Leighton 1974, Leighton 2000) occurs frequently within the first 4 d.

The Settlement pattern can also be shaped by the direction and intensity of currents, especially during the spawning season. The breeding period of pink abalone starts at the end of summer, with the highest intensity in autumn and early winter (Ortiz-Quintanilla et al. 1990). This season coincides with the declining northwestern winds that induce upwelling and the start of a nearshore countercurrent with poleward flow (Lynn & Simpson 1987, Zaitsev et al. 2007). Kelp cover reduces current speed (Rosman et al. 2007), an additional factor favoring retention of larvae (Selkoe et al. 2010).

Self-recruitment on an extremely local scale has important implications for conservation and management (Planes et al. 2009). In Haliotis cracherodii, Chambers et al. (2006) found significant genetic differences among populations, and genetic similarity between adults and juveniles, implying that recruitment may come primarily from local sources. Miller et al. (2009) found significant genetic subdivision among Haliotis rubra populations, with most of the subdivision occurring at the smallest sampling scale, inferring local recruitment. However, they found significantly higher levels of genetic variability in the collapsed populations compared with the healthy populations, because in depleted populations the numbers of adults diminished and local recruitment was reduced, then the changed ratio of migrant to local larvae may have resulted in an apparently higher diversity in recovery populations. Thus, resilience of abalone fisheries not only relies on the local scale, where most recruits originate, but also on the mesoscale (tens of kilometers), where more distant populations provide sporadic recruits. Miller et al. (2009) suggested that ongoing larval dispersal from nearby healthy reefs should eventually result in the natural recovery of these populations.

Other examples of self-recruitment or dispersion have been reported in marine larvae of panda clownfish (Amphiprion polymnus), with 16 juveniles as self-recruiters and 43 juveniles as immigrants that traveled more than 10 km (Jones et al. 2005). In the orange clownfish (Amphiprion percula), 40% of larvae were derived from resident parents and 10% from parents that resided up to 35 km away (Planes et al. 2009). In bicolor damselfish (Stegastes partitus), 2 parent-offspring pairs were identified as self-recruiters; homogeneity in allelic frequencies among sites suggested that connectivity among populations occurs less frequently (Christie et al. 2011). Local sustainability seems demographically significant and probably contributes most to persistence of discrete populations within a larger metapopulation (Planes et al. 2009); larval dispersal maintains genetic homogeneity among locations and can also contribute to local recruitment of neighboring or distant reefs (Miller et al. 2009, Planes et al. 2009).

Based on our results, self-recruitment within short distances of abalone aggregations appears to be low. Larger sample sizes should be tested to evaluate the suitability of parentage analysis as a tool for larval retention assessment.

ACKNOWLEDGMENTS

We thank fishermen cooperative personnel F. Lopez-Salas (SCPP "Leyes de Reforma") for providing the aggregation samples. Thanks to R. Burton (Scripps), M. R. Christie (Oregon State University), and C. Herbinger (Dalhousie) for their suggestions during design and data analysis. S. Avila-Alvarez provided laboratory assistance. I. Fogel of CIBNOR provided editorial services. This project was funded by Consejo Nacional de Ciencia y Tecnologia (CONACYT grant 79482) to R. P. E. and by Instituto Politecnico Nacional to S.A.G.D.P. (grant SIP-782009). N.D.V. was a recipient of a CONACYT doctoral fellowship (162710).

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NOE DIAZ-VILORIA, (1 [dagger]) SERGIO A. GUZMAN-DEL PROO, (2 [dagger]) PEDRO CRUZ (1) AND RICARDO PEREZ-ENRIQUEZI (1) *

(1) Aquaculture Genetics Laboratory, Centro de Investigaciones Biologicas de/Noroeste, Instituto Politocnico Nacional 195, Col. Playa Palo de Santa Rita Sur, La Paz, B.C.S. 23096, Mexico; (2) Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prol. Carpio y Plan de Ayala s/n, Mexico City 11340, Mexico

* Corresponding author. E-mail: rperez@cibnor.mx

([dagger]) Current address: Centro Interdisciplinario de Ciencias Marinas--IPN, Av. Instituto Politecnico Nacional s/n, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico

DOI: 10.2983/035.032.0116

TABLE 1.
Number of abalone adults transplanted to the aggregation
center at El Riito reef from source reefs.

Source Reef                Date of Transplant   Transplanted Adults

El Bajito                  10/26/2001                   29
                           10/9/2002                    55
La Barra de Punta Prieta   10/28/2001                   11
                           3/20/2002                    39
Los Duritos                10/28/2001                   15
El Lopon                   3/21/2002                    29
                           5/19/2002                    24
La Rama Verde              6/11/2002                    28
El Riito                   1/19/2003                    20
                           03/7//2003                   17
Total                                                  267

TABLE 2.
Distance of pink abalone juveniles from the
aggregation center.

                       Distance (m)

Site       0    10   20   30   40   50   100   Total

Center      3   --   --   --   --   --    --       3
Transect
North      --    1    2    1    3    3     4      14
East       --    1    3   --    2    4     7      17
West       --    6    1    3   --   --     1      11
South      --   --    1    3   --    2    --       6
Total       3    8    7    7    5    9    12      51

TABLE 3.
Genetic diversity of Haliotis corrugata adults and juveniles
at El Riito.

Locus          Juveniles    Adults

Hco15
  n            44          55
  [N.sub.A]    13          13
  [N.sub.EA]    2.198       2.392
  [H.sub.E]     0.592       0.590
  [H.sub.O]     0.545       0.582
  P             0.147       0.717
Hco16
  n            48          58
  [N.sub.A]     4           7
  [N.sub.EA]    2.398       2.519
  [H.sub.E]     0.532       0.555
  [H.sub.O]     0.583       0.603
  P             0.886       0.889
Hco19
  n            53          56
  [N.sub.A]    18          20
  [N.sub.EA]    8.850       3.731
  [H.sub.E]     0.880       0.896
  [H.sub.O]     0.887       0.732
  P             0.649       0.000 *
Hco22
  n            51          59
  [N.sub.A]     4           4
  [N.sub.EA]    3.003       2.950
  [H.sub.E]     0.677       0.651
  [H.sub.O]     0.667       0.661
  P             0.445       0.942
Hco97
  n            49          56
  [N.sub.A]     9           6
  [N.sub.EA]    1.815       1.751
  [H.sub.E]     0.493       0.440
  [H.sub.O]     0.449       0.429
  P             0.503       0.427
Hco194
  n            46          61
  [N.sub.A]     2           3
  [N.sub.EA]    1.395       1.524
  [H.sub.E]     0.276       0.378
  [H.sub.O]     0.283       0.344
  P             1.000       0.288
Hka56
  n            49          58
  [N.sub.A]     5           6
  [N.sub.EA]    2.041       1.757
  [H.sub.E]     0.562       0.491
  [H.sub.O]     0.510       0.431
  P             0.507       0.581
Hka3
  n            48          57
  [N.sub.A]    49          60
  [N.sub.EA]   15.873      11.364
  [H.sub.E]     0.982       0.988
  [H.sub.O]     0.937       0.912
  P             0.299       0.001
Mean
  n            48.5        57.5
  [N.sub.A]    13          14.875
  [N.sub.EA]    4.696       3.499
  [H.sub.E]     0.624       0.624
  [H.sub.O]     0.608       0.587

* P < 0.008. HE, expected heterozygosities; [H.sub.O], observed
heterozygosities; n, size sample, [N.sub.A], number of alleles;
[NE.sub.A], number of expected alleles; P, Fisher's exact test
probabilities of Hardy-Weinberg equilibrium.

TABLE 4.
Summary statistics of 8 loci suitable for parentage analysis.

Locus     N    [N.sub.A]    PIC    [H.sub.O]   [H.sub.E]

Hco15     98      17       0.558     0.561     0.587
Hco16    106       7       0.481     0.594     0.543
Hc019    108      22       0.873     0.806     0.887
Hco22    110       4       0.594     0.664     0.662
Hco97    104       9       0.434     0.442     0.466
Hco194   107       3       0.281     0.318     0.336
Hka3     104      80       0.982     0.923     0.987
Hka56    105       6       0.463     0.476     0.529

Locus    f null     HWE      I      Pr(Z)

Hco15    0.0103     NS     0.1991   0.749
Hco16    -0.0480    NS     0.2706   0.831
Hc019    0.0448     NS     0.0239   0.350
Hco22    -0.0003    NS     0.1814   0.723
Hco97    0.0278     NS     0.3177   0.856
Hco194   0.0315     NS     0.4962   0.935
Hka3     0.0311     ND     0.0006   0.039
Hka56    0.0449     NS     0.2874   0.811

f null, null alleles frequencies; [H.sub.E], expected
heterozygosities; [H.sub.O], observed heterozygosities; HWE,
Hardy-Weinberg equilibrium test; I, probability of identity; n,
size sample, NA, number of alleles; ND, not determined; NS, not
significant; PIC, polymorphic information content; Pr(Z),
probability of exclusion per locus.

TABLE 5.
Multilocus genotypes of juveniles assigned to collected
adults at El Riito, with shared alleles in gray.

Parentage      Hco15         Hco16         Hco19         Hco22

Juv-1 (I)      218    222#   209#   240    166#   172#   217#   223#
  Adult-13     222#   226    209#   212    166#   196    217#   217
  Adult-52     222#   222    209#   212    172#   178    212    223#
Juv-32 (III)   222#   222    209#   209    166#   166    212#   223
  Adult-11     222#   222    209#   209    166#   166    912#   223
Juv-36 (I)     208    222#   209#   209#   172#   174#   221    223#
  Adult-77     222    222#   209#   209    174#   184    217    223#
  Adult-29     222    222#   209#   212    162    172#   217    223#
Juv-46 (I)     222#   222    209#   234    166#   168    212#   217
  Adult-1      222#   224    209#   212    166#   166    212#   223
Juv-55 (I)     222    222#   209#   212    166#   200    212    217#
  Adult-33     196#   222#   209#   209    160    166#   217#   223
Juv-66 (III)   222    234    209#   212#   168#   172#   212#   223#
  Adult-55     218    222#   209#   237    168#   168    212#   223
  Adult-29     222    222#   209#   212#   162    172#   217    223#

Parentage      Hco97         Hco194        Hka56         Hka3

Juv-1 (I)      201#   201#   198#   198#   242#   244    250#   260#
  Adult-13     201#   204    196    198#   242#   244    250#   252
  Adult-52     201#   201    196    198#   242    244#   253    260#
Juv-32 (III)   201#   212    198#   198    140    244#   250    295#
  Adult-11     201#   225    198#   198    242    244#   249    295#
Juv-36 (I)     201#   201#   198#   198#   242    244#   257#   280
  Adult-77     201    201#   198    198#   239    244#   253    257#
  Adult-29     201    201#   198    198#   244    244#   245    257#
Juv-46 (I)     201#   201    196    198#   242    244#   248#   305
  Adult-1      201#   201    198    198#   244    244#   248#   259
Juv-55 (I)     201    201#   198    198#   244    244#   241#   266
  Adult-33     201    201#   198    198#   242    244#   241#   249
Juv-66 (III)   201#   201#   198#   198#   244#   244#   236#   245#
  Adult-55     201#   207    198#   198    242    244#   236#   310
  Adult-29     201    201#   198    198#   244#   244    245#   257

Parentage      LOD      P value

Juv-1 (I)
  Adult-13     0.60     1.06 x [10.sup.-5]
  Adult-52     1.56     1.06 x [10.sup.-5]
Juv-32 (III)
  Adult-11     4.29 *   1.29 x [10.sup.-6]
Juv-36 (I)
  Adult-77     3.12     1.22 x [10.sup.-7]
  Adult-29     2.76     1.22 x [10.sup.-7]
Juv-46 (I)
  Adult-1      0.912    1.36 x [10.sup.-6]
Juv-55 (I)
  Adult-33     2.97     3.54 x [10.sup.-6]
Juv-66 (III)
  Adult-55     1.86     5.39 x [10.sup.-6]
  Adult-29     4.44 *   5.39 x [10.sup.-6]

* Significant (P < 0.1). Juv, juvenile; LOD, likelihood of
assignment obtained in CERVUS; P value, average nonexclusion
probabilities. The roman numerals in parentheses correspond
to the juvenile's age class.

Note: Shared alleles indicated with #.
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Author:Diaz-Viloria, Noe; Guzman-del Proo, Sergio A.; Cruz, Pedro; Perez-Enriquezi, Ricardo
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:1MEX
Date:Apr 1, 2013
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