Evolutionary associations between sand seatrout (Cynoscion arenarius) and silver seatrout (C. nothus) inferred from morphological characters, mitochondrial DNA, and microsatellite markers

The evolutionary associations between closely related fish species, both contemporary and historical, are frequently assessed by using molecular markers, such as microsatellites. Here, the presence and variability of microsatellite loci in two closely related species of marine fishes, sand seatrout (Cynoscion arenarius) and silver seatrout (C. nothus), are explored by using heterologous primers from red drum (Sciaenops ocellatus). Data from these loci are used in conjunction with morphological characters and mitochondrial DNA haplotypes to explore the extent of genetic exchange between species offshore of Galveston Bay, TX. Despite seasonal overlap in distribution, low genetic divergence at microsatellite loci, and similar life history parameters of C. arenarius and C. nothus, all three data sets indicated that hybridization between these species does not occur or occurs only rarely and that historical admixture in Galveston Bay after divergence between these species was unlikely. These results shed light upon the evolutionary history of these fishes and highlight the genetic properties of each species that are influenced by their life history and ecology.

Limited genetic structure of Gulf Menhaden (Brevoortia patronus), as revealed by microsatellite markers developed for the genus Brevoortia (Clupeidae)

Long-term sustainable management of wild populations should be based on management actions that account for the genetic structure among populations. Knowledge of genetic structure and of the degree of demographic exchange between discreet [sic] populations allows managers to better define management units. However, adequate gene loci for population assessments are not always available. In this study, variable co-dominant DNA loci in the heavily exploited marine genus Brevoortia were developed with a microsatellite-enriched DNA library for the Gulf Menhaden (Brevoortia patronus). Microsatellite marker discovery was followed by genetic characterization of 4 endemic North American Brevoortia species, by using 14 novel loci as well as 5 previously described loci. Power analysis of these loci for use in species identification and genetic stock structure was used to assess their potential to improve the stock definition in the menhaden fishery of the Gulf of Mexico. These loci could be used to reliably identify menhaden species in the Gulf of Mexico with an estimated error rate of α=0.0001. Similarly, a power analysis completed on the basis of observed allele frequencies in Gulf Menhaden indicated that these markers can be used to detect very small levels of genetic divergence (Fst≈0.004) among simulated populations, with sample sizes as small as n=50 individuals. A cursory analysis of genetic structure among Gulf Menhaden sampled throughout the Gulf of Mexico indicated limited genetic structure among sampling locations, although the available sampling did not reach the target number (n=50) necessary to detect minimal values of significant structure.

Genetic variation between outer-coastal and fjord populations of Pacific herring (Clupea pallasii) in the eastern Gulf of Alaska

Pacific herring (Clupea pallasii) from the Gulf of Alaska were screened for temporal and spatial genetic variation with 15 microsatellite loci. Thirteen collections were examined in this study: 11 from Southeast Alaska and 2 from Prince William Sound, Alaska. Although FST values were low, a neighbor-joining tree based on genetic distance, homogeneity, and FST values revealed that collectively, the Berners Bay and Lynn Canal (interior) collections were genetically distinct from Sitka Sound and Prince of Wales Island (outer-coastal) collections. Temporal genetic variation within regions (among three years of Berners Bay spawners and between the two Sitka Sound spawners) was zero, whereas 0.05% was attributable to genetic variation between Berners Bay and Sitka Sound. This divergence may be attributable to environmental differences between interior archipelago waters and outer-coast habitats, such as differences in temperature and salinity. Early spring collections of nonspawning Lynn Canal herring were nearly genetically identical to collections of spawning herring in Berners Bay two months later—an indication that Berners Bay spawners over-winter in Lynn Canal. Southeast Alaskan herring (collectively) were significantly different from those in Prince William Sound. This study illustrates that adequate sample size is needed to detect variation in pelagic fish species with a large effective population size, and microsatellite markers may be useful in detecting low-level genetic divergence in Pacific herring in the Gulf of Alaska.

Determination of some dominant genetic characteristics and morpho-meristic features for selection of rainbow trout (Oncorhynchus mykiss) broodstocks

In this study, Iranian and French male and female Oncorhynchus mykiss broodstocks were divided into two groups 50 and 24 respectively in Research center of genetic and breeding of coldwater fishes, Yasouj, Iran and the genetic structure of them was investigated using 6 microsatellite markers. Then 19 morphometric and 5 meristic of broodstock were measured and compared in two populations. Along with broodstock maturation, fertilization 1:1(female:male) were randomly assigned and occurred in 25 of 12 Iranian and French treatment respectively. Reproductive parameters were recorded for the whole family. Average number of observed alleles in Iranian and French stocks was 6.68 and 6.83, respectively. Average number of effective alleles in Iranian and French stocks was 3.13 and 3.45 respectively. Fixation index Fst was calculated based on allelic frequency between two stocks was 0.058 with significant difference between 2 stocks. Morphometric analysis showed significant difference between two stocks in 8 characteristics. Meristic characters was without significant difference in broodstock groups. Eyed percentage for french broodstock calculated zero and deleted. Fertilization rate (100-0), the eyed percentage (98- 0), The hatch rate (98-0), the average fecundity 4114.708, the average eggs size 4.88 mm, Survival in the first three months 19-73% calculated for Iranian broodstocks. Considering the quality of eggs and larvae at different stages and selection between the different family and the within family remained 10 treatments and are kept as future broodstocks. The relationship between fecundity - egg size, fecundity - weight , fecundity - length, egg size- weight was performed using regression. The results showed that Fecundity was influenced more by weight and productive length. The research is beginning to ID the broodstock in our country.

Genetic variability in spotted seatrout (Cynoscion nebulosus), determined with microsatellite DNA markers

Variation in the allele frequencies of five microsatellite loci was surveyed in 1256 individual spotted seatrout (Cynoscion nebulosus) obtained from 12 bays and estuaries from Laguna Madre, Texas, to Charlotte Harbor, Florida, to St. John’s River on the Florida Atlantic Coast. Texas and Louisiana collection sites were resampled each year for two to four years (1998−2001). Genetic differentiation was observed. Spotted seatrout from Florida waters were strongly differentiated from spotted seatrout collected in Louisiana and Texas. The greatest genetic discontinuity was observed between Tampa Bay and Charlotte Harbor, and Charlotte Harbor seatrout were most similar to Atlantic Coast spotted seatrout. Texas and Louisiana samples were not strongly structured within the northwestern Gulf of Mexico and there was little evidence of temporal differentiation within bays. These findings are contrary to those of earlier analyses with allozymes and mitochondrial DNA (mtDNA) where evidence of spatial differentiation was found for spotted seatrout resident on the Texas coast. The differences in genetic structure observed among these markers may reflect differences in response to selective pressure, or may be due to differences in underlying genetic processes.

Microsatellite multiplex panels for genetic studies of gray snapper (Lutjanus griseus) and lane snapper (Lutjanus synagris)

Microsatellites are codominantly inherited nuclear-DNA markers (Wright and Bentzen, 1994) that are now commonly used to assess both stock structure and the effective population size of exploited fishes (Turner et al., 2002; Chistiakov et al., 2006; Saillant and Gold, 2006). Multiplexing is the combination of polymerase chain reaction (PCR) amplification products from multiple loci into a single lane of an electrophoretic gel (Olsen et al., 1996; Neff et al., 2000) and is accomplished either by coamplification of multiple loci in a single reaction (Chamberlain et al., 1988) or by combination of products from multiple single-locus PCR amplifications (Olsen et al., 1996). The advantage of multiplexing micro-satellites lies in the significant reduction in both personnel time (labor) and consumable supplies generally required for large genotyping projects (Neff et al., 2000; Renshaw et al., 2006).

Population genetic structure of Rutilus frisii kutum in Caspian Sea using biometry and microsatellite molecular technique

In order to carry out Biometric studies, 75 samples were caught from 3 locations ( Tajan river, Sefidrud and Shirud) using Salic and the length (±1 mm) and weights (± 5 gr) of samples were determined. Using One-way ANOVA by SPPSS software, there wasn’t significant difference between locations in length and fecondity (P ≥0.01(, but there was significant difference between Shirud and tajan samples with sefidrud in weight ) P≤0.01(. In order to carry out genetic variation studies, 210 fish were caught from 3 different regions of the Iranian coastline (Khoshkrud, Tonekabon, Gorganrud) and 1 region in Azerbaijan (Waters of the Caspian Sea close to Kura River mouth) during 2008-2009 . Genomic DNA was extracted of fin using the phenol-chloroform. The quantity and quality of DNA from samples were assessed by spectrophptometer and 1% agarose gel electro-phoresis. PCR was carried out using 15 paired microsatellite primers. PCR products were separated on 8% polyacrylamide gels that were stained using silver nitrate. Molecular weight calculate using UVTech software. The recorded microsatellite genotypes were used as input data for the GENALEX software version 6 package in order to calculate allele and genotype frequencies, observed (Ho) and (He) expected heterozygosities and to test for deviations from Hardy-Weinberg equilibrium. Genetic distance between two populations was estimated from Nei standard genetic distance and genetic similarity index (Nei, 1972). Genetic differentiation between populations was also evaluated by the calculation of pairwise estimates of Fst and Rst values. From 15 SSR markers were used in this investigation, 9 of them were polymorph. Average of expected and observed heterozygosity was 0.54 and 0.49 respectively. Significant deviations from Hardy-Weinberg expectations were observed in all of location except Anzali lagoon- autumn in AF277576 and EF144125, Khoshkrud in EF144125 and Gorganrud and Kura in AF277576. Using Fst and Rst there was significant difference between locations ) P≤0.01(. According to Fst , the highest population differentiation (Fst= 0.217) was between Gorganrud and Khoshkrud that have the lowest Nm and the lowest (Fst= 0.086) was between Gorganrud and Tonekabon that have the highest Nm. Using Rst the highest population differentiation (Rst= 0.271) was between Tonekabon and spring Anzali lagoon and the lowest (Rst= 0.026) was between Tonekabon and Autumn Anzali 159 lagoon. Also the difference between Spring Anzali lagoon and Autumn Anzali lagoon was noticeable (Fst=0.15). AMOVA analysis with consideration of 2 sampling regions (Iran and Azerbaijan) and 7 sampling locations (Iran: Khoshkrud, Tonekabon, Gorganrud, Spring Anzali lagoon and Autumn Anzali lagoon ; Azerbaijan: the Kura mouth) revealed that almost all of the variance in data namely 83% )P≤0.01( was within locations, Genetic variances among locations was 14% )P≤0.01( and among regions was 3% )P≤0.01(. The genetic distance was the highest (0.646) between Gorganrud and Autumn Anzali lagoon populations, whereas the lowest distance (0.237) was between Gorganrud and Tonekabon River. Result obtained from the present study show that at least 2 different population of Rutilus frissi kutum are found in the Caspian sea,which are including the kura river population and the southern Caspian sea samples and it appears that there is more than one population in southern Caspian sea that should be attantioned in artifical reproduction Center and stoke rebilding.

The genetic diversity of Acipenser stellatus populations in the Caspian Sea was studied using microsatellite technique

A total of 361 caudal fin samples were collected from adult A. stellatus specimens caught in the north Caspian Sea, including specimens from Kazakhstan (Ural River), Russia (Volga River), Azerbaijan (Kura River), specimens caught in the south Caspian Sea including specimens from Fishery Zone 1 (from Astara to Anzali), Fishery Zone 2 (from Anzali to Ramsar), Fishery Zone 3 (from Nowshahr to Babolsar), Fishery Zone 4 (from Miyankaleh to Gomishan) as well as from specimens caught in Turkmenistan (all specimens were collected during the sturgeon stock assessment survey). About 2 g of fin tissue was removed from each caudal fin sample, stored in 96% ethyl alcohol and transferred to the genetic laboratory of the International Sturgeon Research Institute. Genomic DNA was extracted using phenol-chloroform method. The quality and quantity of DNA was assessed using 1% Agarose gel electrophoresis and Polymerase Chain Reaction (PCR) was conducted on the target DNA using 15 paired microsatellite primer. PCR products were electrophoresed on polyacrylamide gels (6%) that were stained using silver nitrate. Electrophoretic patterns and DNA bands were analyzed with BioCapt software. Allele count and frequency, genetic diversity, expected heterozygosity and observed heterozygosity allele number, and the effective allele number, genetic similarity and genetic distance, FST and RST were calculated. The Hardy Wienberg Equilibrium based on X2 and Analysis of Molecular Variance (AMOVA) at 10% confidence level was calculated using the Gene Alex software. Dendrogram for genetic distances and identities were calculated using TFPGA program for any level of the hierarchy. It is evident from the results obtained that the 15 paired primers studied, polymorphism was observed in 10 pairs in 12 loci, while one locus did not produce DNA bands. Mean allele number was 13.6. Mean observed and expected heterozygosity was 0.86 and 0.642, respectively. It was also seen that specimens from all regions were not in Hardy Wienberg Equilibrium in most of the loci (P≤0.001). Highest Fst (0.063) was observed when comparing specimens from Fishery Zone 2 and Fishery Zone 4 (Nm=3.7) and lowest FST (0.028) was observed when comparing specimens from the Volga River and those from the Ural River (8.7). Significant differences (P<0.01) were observed between RST recorded in the specimens studied. Highest genetic distance (0.604) and lowest genetic resemblance (0.547) were observed between specimens from Fishery zones 2 and 4. Lowest genetic distance (0.311) and highest genetic resemblance (0.733) was observed between specimens from Turkmenistan and specimens from Fishery zone 1. Based on the genetic dendrogeram tree derived by applying UPGMA algorithm, A. stellatus specimens from Fishery zone 2 or in other words specimens from the Sepidrud River belong to one cluster which divides into two clusters, one of which includes specimens from Fishery zones 1, 3 and 4 and specimens from Turkmenistan while the other cluster includes specimens from Ural, Volga and Kura Rivers. It is thus evident that the main population of this species belongs to the Sepidrud River. Results obtained from the present study show that at least eight different populations of A. stellatus are found in the north and south Caspian Sea, four of which are known populations including the Ural River population, the Volga River population, the Kura River population and the Sepidrud River populations. The four other populations identified belonging to Fishery zones 1, 3, and 4 and to Turkmenistan are most probably late or early spawners of the spring run and autumn run of each of the major rivers mentioned. Specific markers were also identified for each of the populations identified. The Ural River population can be identified using primers Spl-68, 54b and Spl-104, 163 170, 173, the Volga River population can be identified using primers LS-54b and Spl-104, 170, 173 113a and similarly the population from the Kura River can be identified using primers LS-34, 54b and Spl-163, 173 and that from the Sepidrud River can be identified using primers LS-19, 34, 54b and Spl-105, 113b. This study gives evidence of the presence of different populations of this species and calls for serious measures to be taken to protect the genetic stocks of these populations. Considering that the population of A. stellatus in Fishery zone 2 is an independent population of the Sepidrud River in the Gilan Province, the catch of these fishes in the region needs to be controlled and regulated in order to restore the declining stocks of this species.

Aquaculture Asia, Vol.14, No.2, pp.1-40, April-June 2009

Sustainable aquaculture GLOBALG.A.P. standard in Thai shrimp farms: Mission (im)possible? p.4 Leepaisomboon, T., Chuchird, N., Limsuwan, C., Steenbruggen, E.R., and Mungkung, R. The Victorian trout industry & the bushfires p.6 Mosig, J. Small-scale aquaculture in the Ayeyarwady Delta, Myanmar p.10 Edwards, P. The history, status, and future prospects of monosex tilapia culture in Thailand p.18 Belton, B., Turongruang, D., Bhujel, R. and Little, D.C. Mangroves of Nakhon Si Thammarat Province in southern Thailand: Species diversity, community structure and current status p.20 By Amarasinghe, M.D., Dulyapurk, V., Taparhudee, W., Yoonpundh, R. and Jumnongsong, S. Research and farming techniques Induced breeding of pacu (Piaractus brachypomus) in captivity with pituitary extract p.23 Chattarjee, N.R. and Mazumdar, B. Aquatic animal health Fumonisins - mycotoxins of increasing importance in fish! p. 24 Griessler, K. and Encarnação, P. Genetics and biodiversity Microsatellite DNA markers, a fisheries perspective. Part 1: The nature of microsatellites p.27 Sekar, M., Suresh, E., Kumar, N.S., Nayak, S.K., Balakrishna, C. Asia-Pacific Marine Finfi sh Aquaculture Magazine Formulated feed for tiger grouper grow-out p.30 Rachmansyah, Usman, Palinggi, N.N. and Williams, K. NACA Newsletter 36

Population connectivity among Dry Tortugas, Florida, and Caribbean populations of mutton snapper (Lutjanus analis), inferred from multiple microsatellite loci

Determining patterns of population connectivity is critical to the evaluation of marine reserves as recruitment sources for harvested populations. Mutton snapper (Lutjanus analis) is a good test case because the last known major spawning aggregation in U.S. waters was granted no-take status in the Tortugas South Ecological Reserve (TSER) in 2001. To evaluate the TSER population as a recruitment source, we genotyped mutton snapper from the Dry Tortugas, southeast Florida, and from three locations across the Caribbean at eight microsatellite loci. Both Fstatistics and individual-based Bayesian analyses indicated that genetic substructure was absent across the five populations. Genetic homogeneity of mutton snapper populations is consistent with its pelagic larval duration of 27 to 37 days and adult behavior of annual migrations to large spawning aggregations. Statistical power of future genetic assessments of mutton snapper population connectivity may benefit from more comprehensive geographic sampling, and perhaps from the development of less polymorphic DNA microsatellite loci. Research where alternative methods are used, such as the transgenerational marking of embryonic otoliths with barium stable isotopes, is also needed on this and other species with diverse life history characteristics to further evaluate the TSER as a recruitment source and to define corridors of population connectivity across the Caribbean and Florida.

Population structure of chum salmon (Oncorhynchus keta) across the Pacific Rim, determined from microsatellite analysis

The Pacific Rim population structure of chum salmon (Oncorhynchus keta) was examined with a survey of microsatellite variation to describe the distribution of genetic variation and to evaluate whether chum salmon may have originated from two or more glacial refuges following dispersal to newly available habitat after glacial retreat. Variation at 14 microsatellite loci was surveyed for over 53,000 chum salmon sampled from over 380 localities ranging from Korea through Washington State. An index of genetic differentiation, FST, over all populations and loci was 0.033, with individual locus values ranging from 0.009 to 0.104. The most genetically diverse chum salmon were observed from Asia, particularly Japan, whereas chum salmon from the Skeena River and Queen Charlotte Islands in northern British Columbia and those from Washington State displayed the fewest number of alleles compared with chum salmon in other regions. Differentiation in chum salmon allele frequencies among regions and populations within regions was approximately 18 times greater than that of annual variation within populations. A regional structuring of populations was the general pattern observed, with chum salmon spawning in different tributaries within a major river drainage or spawning in smaller rivers in a geographic area generally more similar to each other than to populations in different major river drainages or geographic areas. Population structure of chum salmon on a Pacific Rim basis supports the concept of a minimum of two refuges, northern and southern, during the last glaciation, but four possible refuges fit better the observed distribution of genetic variation. The distribution of microsatellite variation of chum salmon on a Pacific Rim basis likely reflects the origins of salmon radiating from refuges after the last glaciation period.

Microsatellite primers for red drum (Sciaenops ocellatus)

In this note, we document polymerase-chain-reaction (PCR) primer pairs for 101 nuclear-encoded microsatellites designed and developed from a genomic library for red drum (Sciaenops ocellatus). Details of the genomic library construction, the sequencing of positive clones, primer design, and PCR protocols may be found in Karlsson et al. (2008). The 101 microsatellites (GENBA NK Accession Numbers EU015882-EU015982) were amplified successfully and used to genotype 24 red drum obtained from Galveston Bay, Texas (Table 1). A total of 69 of the microsatellites had an uninterrupted (perfect) dinucleotide motif, and 30 had an imperfect dinucleotide motif; one microsatellite had an imperfect tetranucleotide motif, and one had an imperfect and compound motif (Table 1 ). Sizes of the cloned alleles ranged from 84 to 252 base pairs. A ‘blast’ search of the GENBANK database indicated that all of the primers and the cloned alleles were unique (i.e., not duplicated).

Determination of population structure and stock composition of chum salmon (Oncorhynchus keta) in Russia determined with microsatellites

Variation at 14 microsatellite loci was examined in 34 chum salmon (Oncorhynchus keta) populations from Russia and evaluated for its use in the determination of population structure and stock composition in simulated mixed-stock fishery samples. The genetic differentiation index (Fst) over all populations and loci was 0.017, and individual locus values ranged from 0.003 to 0.054. Regional population structure was observed, and populations from Primorye, Sakhalin Island, and northeast Russia were the most distinct. Microsatellite variation provided evidence of a more fine-scale population structure than those that had previously been demonstrated with other genetic-based markers. Analysis of simulated mixed-stock samples indicated that accurate and precise regional estimates of stock composition were produced when the microsatellites were used to estimate stock compositions. Microsatellites can be used to determine stock composition in geographically separate Russian coastal chum salmon fisheries and provide a greater resolution of stock composition and population structure than that previously provided with other techniques.

Mitochondrial DNA markers to identify commercial spiny lobster species (Panulirus spp.) from the Pacific coast of Mexico: an application on phyllosoma larvae

Molecular markers based on mitochondrial DNA (mtDNA) are extensively used to study genetic relationships. mtDNA has been used in phylogenetic studies to understand the evolutionary history of species because it is maternally inherited and is not subject to genetic recombination (Gyllensten et al., 1991). The high mutation rate of mtDNA makes it a useful tool for differentiating between closely related species (Brown et al., 1979)—a tool that is especially important when significant variations occur between species, but not within species (Hill et al., 2001; Blair et al., 2006; Chow et al., 2006a).