Cloning and characterization of novel microsatellite DNA markers for the grass rockfish,Sebastes rastrelliger,and cross‐species amplification in 10 related Sebastes spp

Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.     

Microsatellite markers for the heavily exploited canary (Sebastes pinniger) and other rockfish species

The isolation and characterization of nine polymorphic microsatellite loci (eight tetranucleotide and one dinucleotide) from the canary rockfish Sebastes pinniger are described. Polymorphism at these loci revealed from six to 28 alleles, with expected heterozygosities ranging between 0.42 and 0.88, enabling high‐resolution genetic population structure investigation for this overfished species in the northeastern Pacific. They also amplify in 13 other congeneric species, providing highly variable loci for research on other rockfishes.     

Multiplex PCR system applied for analysing microsatellite loci of Schlegel's black rockfish,Sebastes schlegeli

Two multiplex PCR amplifications were performed to analyse six microsatellite loci of Schlegel's black rockfish, Sebastes schlegeli, an important commercial fish in the northern part of Japan and an important species for the stock enhancement program in this area. We analysed 67 wild samples from Yamada Bay, Iwate Prefecture, Japan. The observed genotype frequencies agreed with the Hardy–Weinberg expectations at all loci, and the observed heterozygosities ranged from 0.072 to 0.897.     

Analysis of Microsatellite DNA Polymorphisms in Rockfish Sebastes thompsoni and Application to Population Genetics Studies

Population differentiation and relationships among 6 natural rockfish populations collected from northern coastal seas around Japan were assayed using microsatellite DNA loci. Seven loci examined were polymorphic in all populations. The number of alleles per locus ranged from 6.7 to 9.3, and the average of observed and expected heterozygosity ranged from 0.63 to 0.68, and from 0.66 to 0.69, respectively. The observed genotype frequencies at each locus were almost in agreement with Hardy-Weinberg expectations with two exceptions (P < .05). The allele frequencies of 16 population pairs were significantly different (P < .05). Genetic distance (D _A ) between 6 populations ranged from 0.03 to 0.08. According to a neighbor-joining tree generated from the D _A values, the 6 populations fell into 3 clusters. These clusters were correlated with the geographical positions of each population; larval dispersions due to water current were also found to have an effect on these results. Received May 2, 2000; accepted July 17, 2000.     

Cryptic speciation in the vermilion rockfish (Sebastes miniatus) and the role of bathymetry in the speciation process

A recent phylogenetic review of the genus Sebastes suggested the existence of a cryptic species of vermilion rockfish ( Sebastes miniatus ). To evaluate the geographical and bathymetric range of the Type 1 and Type 2 forms reported in that study, cytochrome b sequences were examined from 548 fish. Type 1 fish were found primarily south of Point Conception on reefs deeper than 100 m. Type 2 fish were common range-wide at sites shallower than 100 m. Reproductive isolation between the two types was tested using nine microsatellite loci. Estimates of genetic divergence were made using the fixation index ( F _ST) and correspondence between haplotype and genotype was tested by Bayesian population assignment and multivariate plotting of individual genotypes. Microsatellite analyses gave strong support for the presence of two distinct groups of genotypes. All fish with Type 1 haplotypes and fish with Type 2 haplotypes from < 100 m depth had genotypes unique to their haplotype group. However, most (68%) fish with Type 2 haplotypes from > 100 m depth assigned strongly to the Type 1 genotype group. Morphometric comparisons between the two genotypic groups revealed significant differences at three of the six examined measurements. Differences in both genetics, depth of occurrence, and morphology suggest these are separate species. This observation along with evidence of depth segregation in many recent species pairs led us to hypothesize a speciation model for Sebastes spp. by which the loss or truncation of a depth-related ontogenetic migration can lead to the creation of reproductively isolated populations.     

Comparative allometric growth of the gastrointestinal tract of two sympatric congeners, copper rockfish (Sebastes caurinus) and quillback rockfish (S. maliger)

Allometric growth of the stomach, intestine, and caeca was compared between two sympatric congeners, the copper rockfish ( Sebastes caurinus ) and the quillback rockfish ( S. maliger ), to examine the species-specific differences in their gut tract in relation to their general feeding habits. The intestine of copper rockfish was shorter and its mass less than the intestine of quillback rockfish. Copper rockfish also had a greater stomach mass and, on average, a greater number of pyloric caeca than quillback rockfish. The differences in the gut allometry between the species were consistent with differences in their general food habits as copper rockfish consume proportionally more fish and larger crustaceans than quillback rockfish.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.     

Estimation of genotyping error rate from repeat genotyping,unintentional recaptures and known parent–offspring comparisons in 16 microsatellite loci for brown rockfish (Sebastes auriculatus)

Genotyping errors are present in almost all genetic data and can affect biological conclusions of a study, particularly for studies based on individual identification and parentage. Many statistical approaches can incorporate genotyping errors, but usually need accurate estimates of error rates. Here, we used a new microsatellite data set developed for brown rockfish (Sebastes auriculatus) to estimate genotyping error using three approaches: (i) repeat genotyping 5% of samples, (ii) comparing unintentionally recaptured individuals and (iii) Mendelian inheritance error checking for known parent–offspring pairs. In each data set, we quantified genotyping error rate per allele due to allele drop‐out and false alleles. Genotyping error rate per locus revealed an average overall genotyping error rate by direct count of 0.3%, 1.5% and 1.7% (0.002, 0.007 and 0.008 per allele error rate) from replicate genotypes, known parent–offspring pairs and unintentionally recaptured individuals, respectively. By direct‐count error estimates, the recapture and known parent–offspring data sets revealed an error rate four times greater than estimated using repeat genotypes. There was no evidence of correlation between error rates and locus variability for all three data sets, and errors appeared to occur randomly over loci in the repeat genotypes, but not in recaptures and parent–offspring comparisons. Furthermore, there was no correlation in locus‐specific error rates between any two of the three data sets. Our data suggest that repeat genotyping may underestimate true error rates and may not estimate locus‐specific error rates accurately. We therefore suggest using methods for error estimation that correspond to the overall aim of the study (e.g. known parent–offspring comparisons in parentage studies).     

Characterization of 11 new microsatellite loci in taro (Colocasia esculenta)

Eleven new microsatellite markers were isolated from taro, Colocasia esculenta (L.) Schott, a root crop widely distributed all over the world. Forty-eight primer pairs were designed from a microsatellite-enriched genomic library, of which 11 primer pairs have polymorphisms in 30 individuals tested from a population in China, which revealed two to six alleles per locus with the observed and expected heterozygosity levels ranging from 0 to 0.733 and from 0.381 to 0.731, respectively. These new genetic markers will be useful for the study of taro germplasm management and population evolution in the future.     

Detection of highly polymorphic microsatellite loci in a species with little allozyme polymorphism

Microsatellite loci are regions of DNA containing tandem repeats of a short sequence motif; they occur abundantly in all eukaryotic genomes and have been shown to be a rich source of highly polymorphic genetic markers in humans and other mammals. These loci are particularly suitable for population studies because they can be relatively easily scored using a combination of polymerase chain reaction (PCR) amplification of each locus followed by electrophoresis to separate alleles. This paper details a method for finding these loci in any species. This method demonstrates that trinucleotide microsatellite loci are abundant and highly polymorphic in the social wasp Polistes annularis , whereas allozyme electrophoresis reveals very little polymorphism. The first six loci examined were all polymorphic with a mean observed heterozygosity of 0.62; in comparison average heterozygosity of 33 allozymes was 0.035. We suggest that this method can be used to detect variation where other methods have failed, making it an ideal tool for population and conservation geneticists who must deal with populations lacking other types of genetic variability.     

Characterization of microsatellite loci in fulvous fruit bat Rousettus leschenaulti

Rousettus leschenaulti is an abundant species in many countries of South‐East Asia, including south China. We isolated seven microsatellite loci in R. leschenaulti from genomic DNA enriched for CA repeats with the enriched library method. A total of 56 samples from a population in the Guangxi Province of China were tested with these microsatellite markers. The polymorphism ranged from seven to 16 alleles, and the observed heterozygosity was 84–94%. It is the first time microsatellite markers were characterized from R. leschenaulti, and these markers can be an important tool for analysing population structure and genotypic diversity.     

Temporal variation and feeding ecology of juvenile Sebastes in rocky intertidal tidepools of northern California, with emphasis on Sebastes melanops Girard

This study examined the seasonal use of rocky intertidal areas by young-of-the-year (YOY) Sebastes spp. at three locations in northern California, 2003 to 2005. Six species of Sebastes were noted during the study with black rockfish Sebastes melanops comprising >99% of the total number sampled. Sebastes melanops were noted in rocky intertidal areas from May to August with peak abundances occurring in May or June. Variation in recruitment and feeding was evident among years. Diet analysis, using the index of relative importance (% I _RI) and the modified index (% I _M), showed that YOY S. melanops and Sebastes mystinus consumed a variety of prey items with harpacticoid copepods and gammaridean amphipods being the dominant prey categories. The rocky intertidal appears to serve as an important habitat for YOY S. melanops in northern California.     

Ovarian aging in two species of long-lived rockfish, Sebastes aleutianus and S. alutus

Little is known about the ovary during aging in long-lived fish with respect to follicular stages and de novo oogenesis. We examined two species of rockfish, Sebastes aleutianus (rougheye rockfish) and Sebastes alutus (Pacific ocean perch). Fish were sampled offshore of British Columbia, age was estimated by otolith annuli, and the ovaries were examined histologically. In S. aleutianus, age up to 80 yr did not markedly alter the frequency distribution of oocytes, follicles, or their total numbers. Similarly, in a larger sample of S. alutus, the abundance of oocytes and follicles showed little age trend up through 77 yr. However, fish older than 50 yr lacked the largest and smallest oocyte size classes (40-60, >80 microm) and the smallest follicle size class (200-350 microm), which results from the later seasonal developmental state of these older fish. These data provide evidence that oogenesis continues at advanced ages in these two species, in contrast with long-held assumptions about mammals. These species represent an iteroparous extreme in the spectrum of life history strategies and merit investigation to determine the mechanisms for such an extended reproductive life span.     

Development of microsatellite loci in Pacific herring (Clupea pallasi)

Fifteen polymorphic di‐, tri‐ and tetranucleotide microsatellite markers were developed in Pacific herring (Clupea pallasi) to aid in the delineation of population structure of herring in British Columbia. Twelve of the microsatellite loci were analysed in over 4000 wild herring. Expected heterozygosities ranged 0.73–0.95, and only two loci significantly deviated from Hardy–Weinberg equilibrium.     

Development of microsatellite loci in pinto abalone (Haliotis kamtschatkana)

Twelve novel di‐, tri‐ and tetranucleotide microsatellite loci to the pinto abalone (Haliotis kamtschatkana) are described. Over 400 individuals were analysed at each microsatellite locus. Observed heterozygosities ranged from 0.44 to 0.93, and numbers of alleles from 20 to 63. Six of the loci contained excesses in homozygosity indicative of inbreeding, nonrandom mating, population admixture, or null alleles.     

Temporal recruitment patterns and gene flow in kelp rockfish (Sebastes atrovirens)

Pelagic dispersal of marine organisms provides abundant opportunity for gene flow and presumably inhibits population genetic divergence. However, ephemeral, fine-scale, temporal and spatial genetic heterogeneity is frequently observed in settled propagules of marine species that otherwise exhibit broad-scale genetic homogeneity. A large variance in reproductive success is one explanation for this phenomenon. Here, genetic analyses of 16 microsatellite loci are used to examine temporal patterns of variation in young-of-year kelp rockfish (Sebastes atrovirens) recruiting to nearshore habitat in Monterey Bay, California, USA. Population structure of adults from central California is also evaluated to determine if spatial structure exists and might potentially contribute to recruitment patterns. Genetic homogeneity was found among 414 young-of-year sampled throughout the entire 1998 recruitment season. No substantial adult population structure was found among seven populations spanning 800 km of coastline that includes the Point Conception marine biogeographic boundary. Comparison of young-of-year and adult samples revealed no genetic differentiation and no measurable reduction in genetic variation of offspring, indicating little variance in reproductive success and no reduction in effective population size for this year class. Simulation analyses determined that the data set was sufficiently powerful to detect both slight population structure among adults and a small reduction in effective number of breeders contributing to this year class. The findings of high gene flow and low genetic drift have important implications for fisheries management and conservation efforts.     

Dietary inclusion of mealworm (Tenebrio molitor) meal as an alternative protein source in practical diets for juvenile rockfish (Sebastes schlegeli)

An 8‐week feeding trial was designed to evaluate the potential of yellow mealworm (Tenebrio molitor) as a locally available nutrient‐rich feedstuff for juvenile rockfish (Sebastes schlegeli). Experimental diets containing elevated levels of mealworm meal (WM) supplemented with synthetic methionine were formulated to be isonitrogenous, isolipidic and isoenergetic to a WM‐free fishmeal (FM) based control diet (designated as WM0, WM8, WM16, WM24 and WM32, respectively). To determine the necessity of dietary methionine supplementation at the highest inclusion of WM, a diet was prepared to contain 32% WM without methionine supplementation (WM32‐AA). Triplicate groups of rockfish juveniles (Mean ± S.E.; 3.11 ± 0.01 g) were fed one of the experimental diets to apparent satiation twice daily for 8 weeks. Fish growth performance in terms of weight gain and specific growth rate increased with increasing dietary inclusion of WM from 0 to 16% and then tended to decrease with further increase in dietary WM levels to 32%. Protein retention (PR) values followed the same trend as growth rates with the highest values found in fish offered WM16 diet. Although fish fed WM32‐AA diet showed significantly lower growth rate and PR values compared to those fed WM16 diet, their performance was still comparable to that of the WM‐free control group. Plasma triglyceride level was negatively affected by dietary WM inclusion and the lowest values were observed in the WM32‐AA group. Whole‐body and fillet proximate and essential amino acid compositions were not altered by dietary treatment and these values were comparable to those of the WM0 group. These findings suggested that WM might prove to be a promising alternative to FM in practical diets for juvenile rockfish and could be used at an inclusion level of up to 32% without having any adverse consequences for the health and performance of the fish. Although the diet containing 32% WM seemed to support a performance similar to that of the control diet, the recommended dietary inclusion level was no more than 16% of the diet dry matter.     

Characterization of microsatellite loci in the lesser dawn bat (Eonycteris spelaea)

We constructed a genomic DNA library enriched for CA repeat motifs in Eonycteris spelaea. Nine microsatellite loci were isolated and tested on a population of 39 samples from Yunnan Province, China. These nine loci had three to 22 alleles per locus. Observed and expected heterozygosity values ranged from 0.079 to 0.963 and from 0.078 to 0.959. Two loci revealed significant departure from Hardy-Weinberg equilibrium and no linkage disequilibrium was found between loci pairs. These microsatellites can be a powerful molecular tool for population-level studies of E. spelaea.     

Characterization of microsatellite loci in the Kaka,Nestor meridionalis

Eight microsatellite loci were characterized from the Kaka (Nestor meridionalis), a New Zealand parrot, using a polymerase chain reaction‐based isolation technique. Locus‐specific primers were used to genotype nine Kaka populations and tested on 25 other species of parrot. The number of alleles observed within Kaka ranged from one to 16 in the 12–126 individuals screened and two loci exhibited greater than 60% heterozygosity. Furthermore, these primers are likely to be useful in population‐level studies of two other New Zealand parrots.