Hatchery Samples Research Articles

Microsatellite DNA markers revealed genetic population structure among captive stocks and wild populations of mrigal, Cirrhinus cirrhosus in Myanmar

We investigated genetic diversity and population structure of mrigal in Myanmar using microsatellite DNA markers. A total of 211 individuals from five wild populations and 216 individuals from five hatcheries were analysed for six microsatellite loci ( Bgon22, Lr3, Lr12, Lr21, MFW1 and MFW17) which were developed for other cyprinids. For comparison, 43 individuals from a hatchery in northern Vietnam, of Indian origin and introduced in 1984, also were analysed. Tests for all loci revealed H–W equilibrium in only two hatchery samples. Allele richness ranged from 2.3 to 8.5. Overall, observed heterozygosity was high in all Myanmar samples (ranging from 0.654 to 0.756) but relatively low in the Vietnam hatchery sample (0.303). Pairwise F ST values among the Myanmar samples ranged from 0.000 to 0.096, and those between the Myanmar and the Vietnam samples from 0.353 to 0.506. Results of multidimensional scaling analysis (MDS) of pairwise F ST and Bayesian method revealed that one wild and two hatchery samples from Myanmar were differentiated from others, which appeared highly admixed. The study has important implications for genetic management of mrigal stocks in Myanmar, and possibly elsewhere in the region. For baseline stock for selective breeding, it would be best to include representation of samples from all groups we have identified to ensure a broad genetic base for genetic improvement programs. As for stock enhancement, seed produced from several hatcheries examined here should not be used for restocking in certain locations to avoid genetic contamination.

A longitudinal study of Campylobacter distribution in a turkey production chain

BackgroundCampylobacter is the most common cause of bacterial enteritis worldwide. Handling and eating of contaminated poultry meat has considered as one of the risk factors for human campylobacteriosis.Campylobacter contamination can occur at all stages of a poultry production cycle. The objective of this study was to determine the occurrence of Campylobacter during a complete turkey production cycle which lasts for 1,5 years of time. For detection of Campylobacter, a conventional culture method was compared with a PCR method. Campylobacter isolates from different types of samples have been identified to the species level by a multiplex PCR assay.MethodsSamples (N = 456) were regularly collected from one turkey parent flock, the hatchery, six different commercial turkey farms and from 11 different stages at the slaughterhouse. For the detection of Campylobacter, a conventional culture and a PCR method were used. Campylobacter isolates (n = 143) were identified to species level by a multiplex PCR assay.ResultsNo Campylobacter were detected in either the samples from the turkey parent flock or from hatchery samples using the culture method. PCR detected Campylobacter DNA in five faecal samples and one fluff and eggshell sample. Six flocks out of 12 commercial turkey flocks where found negative at the farm level but only two were negative at the slaughterhouse.ConclusionDuring the brooding period Campylobacter might have contact with the birds without spreading of the contamination within the flock. Contamination of working surfaces and equipment during slaughter of a Campylobacter positive turkey flock can persist and lead to possible contamination of negative flocks even after the end of the day's cleaning and desinfection. Reduction of contamination at farm by a high level of biosecurity control and hygiene may be one of the most efficient ways to reduce the amount of contaminated poultry meat in Finland. Due to the low numbers of Campylobacter in the Finnish turkey production chain, enrichment PCR seems to be the optimal detection method here.

Population genetics studies of half-smooth tongue sole Cynoglossus semilaevis using ISSR markers

Inter-simple sequence repeat (ISSR) analysis was performed in order to evaluate the genetic diversity of wild and hatchery samples of half-smooth tongue sole Cynoglossus semilaevis. A group of 200 genotypes belonging to four wild samples, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ) and one hatchery sample, Mingbo (MB) were screened using 15 different ISSR primers. A total of 137 loci were produced in the five studied samples. 41.80%, 45.26%, 44.27%, 42.86% and 41.59% of these loci were polymorphic over all the genotypes tested in LZ, WH, QD, RZ and MB samples, respectively. The number of polymorphic loci detected by single primer combination ranged from 2 to 7. The average heterozygosity of LZ, WH, QD, RZ and MB samples were 0.0710, 0.0814, 0.0793, 0.0727 and 0.0696, respectively. The WH sample showed a higher genetic diversity including total number of ISSR bands ( P < 0.05), total number of polymorphic bands ( P < 0.05), average heterozygosity ( P < 0.05) and total number of genotypes ( P < 0.05) than all the other samples. Among the five studied samples, the hatchery sample (MB) showed the lowest genetic viability.

Distribution of luminescent Vibrio harveyi and their bacteriophages in a commercial shrimp hatchery in South India

Luminescent Vibrio harveyi is a natural microflora of marine and coastal water bodies and is associated with mortality of larval shrimp in penaeid shrimp hatcheries. It is also known that the bacteriophages occur virtually in all places where their hosts exist. In this study, distribution of luminescent V. harveyi and the bacteriophages affecting these hosts was examined in a commercial Penaeus monodon hatchery during three shrimp larval production cycles, including a cycle affected by luminescent bacterial (LB) disease outbreak.Out of a total of 1195 samples drawn from seawater source, sand-filtered water, nauplius, zoea, mysis and post larval rearing tanks, maturation and spawning tanks, Artemia hatching tank and algal culture tanks processed using conventional microbiological techniques, 21.4% of the samples harboured luminescent bacteria. During the larval production cycle affected by LB disease (LBD), luminescent V. harveyi could be recovered from 52% of the hatchery samples, whereas during luminescent bacterial disease-free larval production cycle (LBDF), these bacteria could be recovered from only about 9% of the samples. The predominant source of luminescent bacteria was the brood shrimp and their rearing tanks in maturation and spawning facilities. 73% of the maturation and 80% of the spawning tank water samples harbored LB during LBD, whereas, only 20% and 32% of the maturation and spawning tanks respectively harbored LB during LBDF. LB could be isolated from 17% of the water samples in tanks from nauplius stage onwards with increasing counts that subsequently lead to LB disease.Bacteriophages affecting the luminescent V. harveyi could be isolated from as many as 36% (21% and 43% of the samples analysed during LBDF and LBD respectively) of a total of 181 water samples drawn from various sources in the hatchery, using 27 luminescent V. harveyi hosts by agar overlay technique. The maturation tank water samples were found to be the predominant source of bacteriophages, followed by spawning tank water samples as observed with the LB. Sixty five bacteriophages, 13 during LBDF and 52 during LBD were isolated, which were grouped in to seven types based on their plaque morphology.The study has indicated that the brooders, maturation and spawning facilities in the shrimp hatchery are the main source of luminescent V. harveyi and their bacteriophages and that occurrence of LB even in low counts during early larval stages can possibly lead to development of LB disease despite presence of bacteriophages in the larval rearing tanks.

An investigation of sources of Campylobacter in a poultry production and packing operation in Barbados

Chicken meat is frequently contaminated with Campylobacter jejuni and is thought to be the major source of organisms causing human Campylobacter enteritis. Genotypic similarities between Campylobacter isolates from chicken meat at retail outlets and patients with gastroenteritis in Barbados suggested that it is a vehicle for infection of humans on the island and prompted this investigation of transmission of Campylobacter in a local poultry operation. Campylobacter testing was conducted at the hatchery, on the broiler farm and in the processing plant for two consecutive production cycles. The genetic relatedness of Campylobacter isolates was determined by RAPD typing with primer OPA 11. Hatchery samples and week-old chicks were negative for Campylobacter. Flocks became colonized as early as three weeks after introduction to the farm. Ten distinct RAPD genotypes were identified among isolates. Some genotypes were similar and may be of clonal origin. There was no evidence of vertical transmission of Campylobacter. The results suggest that the broiler flock was infected from more than one source in the farm environment.

Loss of genetic variation in hatchery‐reared Indian major carp, Catla catla

The hypothesis that effective population sizes are low in hatchery‐reared catla (Catla catla) from Bangladesh, possibly leading to inbreeding and loss of variation, was tested. The study was based on analysis of seven microsatellite loci in three samples of hatchery‐reared catla and four samples representing wild populations. Pair‐wise estimates of genetic differentiation between samples were low between wild samples (θ ranging from 0·012 to 0·034), but high between hatchery samples (θ ranging from 0·153 to 0·185), suggesting strong genetic drift in hatcheries. Genetic variation, both in terms of expected heterozygosity and allelic richness, was significantly lower in hatchery samples than in samples of wild catla. Application of a method for reconstructing families among offspring without parental genetic data showed that the hatchery samples consisted of very few half‐ and full‐sib families, whereas the wild samples consisted of a high number of families, suggesting that most individuals were unrelated. Finally, estimation of the effective number of parents (Nb) in the largest sample of hatchery fish confirmed that effective population size was low (Nb= 14·9 for multiallelic loci and Nb= 10·6 if alleles were pooled into two composite alleles). The results show that low effective population sizes leading to loss of variation and possibly inbreeding depression should be a matter of serious concern in aquaculture production of catla.

Wild and Aquaculture Populations of the Eastern Oyster Compared Using Microsatellites

Five new microsatellite markers were developed for the eastern oyster (Crassostrea virginica), and allelic variability was compared between a wild Chesapeake Bay population (James River) and a hatchery strain (DEBY). All loci amplified readily and demonstrated allelic variability with the number of alleles ranging from 16 to 36 in the wild population and from 11 to 19 in the DEBY strain. Average observed and expected heterozygosities were estimated at 0.66 and 0.80 in the hatchery sample. The corresponding estimates were 0.91 and 0.75 in the wild sample. Results indicated lower genetic variability in the DEBY strain and significant genetic differentiation between the wild population and hatchery strain. These microsatellite loci will prove valuable for future population genetic studies and in tracking of hatchery strains used in restoration.

Widespread hybridization among species of Indian major carps in hatcheries, but not in the wild

Twenty‐one allozyme loci in samples of wild‐caught and hatchery‐reared Indian major carps from Bangladesh were analysed. Bayesian model‐based clustering analysis revealed the presence of four taxa, corresponding to the three known species along with a fourth unknown taxon present in two hatchery samples. Individual admixture coefficients showed that 24% of all hatchery‐reared fishes were hybrids, whereas a single hybrid was observed in the wild‐caught samples. Only catla Catla catla× rohu Labeo rohita and mrigal Cirrhinus cirrhosus× rohu hybrids were observed, the vast majority of which were F1 hybrids, though five individuals represented putative backcrosses. Mitochondrial DNA analysis revealed that catla × rohu hybridization primarily involved catla males and rohu females, whereas mrigal × rohu hybrids primarily resulted from rohu males and mrigal females. Despite the high percentage of F1‐hybrids in hatchery samples, reproductive barriers among species have so far precluded widespread introgression. Continued hybridization may eventually lead to a breakdown of species barriers, thereby compromising the genetic integrity of the species in the wild, and leading to production losses in aquaculture.

Migration barriers protect indigenous brown trout (Salmo trutta) populations from introgression with stocked hatchery fish

Brown trout populations in the Belgian rivers Scheldt and Meuse have been intensively stocked in the past decades, often with material of uncertain origin. Moreover, the species habitat has become increasingly fragmented, preventing gene flow between neighboring populations. We assessed how this impacted genetic diversity and population structure by analyzing 12 wild populations (total n=309) and seven hatchery stocks (n=200) at the mitochondrial control region with SSCP and at 27 RAPD loci. Historical records indicate that brown trout from distant locations have been used to supplement hatchery stocks; nevertheless we detected non-Atlantic mitochondrial genomes in only one population of the Scheldt basin and in one hatchery. In general, the hatchery samples displayed a higher genetic diversity and differentiated less among each other (global F-ST(mtDNA)=0.311/F-ST(RAPD)=0.029) compared to the wild populations (global F-ST(mtDNA)=0.477/F-ST(RAPD)=0.204). This is due to frequent exchanges between hatcheries and regular supplementation from several indigenous populations. Gene pools present in most downstream sections from tributaries of the Meuse were similar to each other and to the hatchery samples, despite the presence of migration barriers. Assignment analyses indicated that the contribution of hatchery material to the upstream parts was limited or even completely absent in populations separated by a physical barrier. Intensive stocking and exchange between hatcheries has homogenized the downstream sections of the Meuse River, whereas the migration barriers preserved the indigenous upstream populations. As such, uncontrolled removal of barriers might result in an irreversible loss of the remnant indigenous gene pools.

Characteristics of Sexual Maturation of Wild and Hatchery-Reared Baltic Salmon (Salmo Salar L.) Parr

Considering conservation issues, qualitative features of fish intended for release are becoming the most important factor. We compared some morphological and physiological parameters of hatcheryreared (Žeimena Hatchery) and wild Baltic salmon juveniles originating from the same wild population of the Žeimena River (Svencionys district, Lithuania). Hatchery salmon parr were characterised by the accelerated growth rate. Their body mass and length, as well as the mass-length ratio and condition factors, were higher than those in wild salmon. Female gonads in both groups were at the stage of protoplasmic growth. However, differences in the development of hatchery-reared female gonads were observed. Histological analysis revealed that substage V oocytes were present in hatchery salmon females. No oocytes of this stage were found in wild females. The share of precocious parr males in hatchery samples was significantly higher than in the samples of wild population. The constituents of hatchery conditions – tempe...

Genetic population structure of central Oregon Coast coho salmon (Oncorhynchus kisutch)

We surveyed microsatellite variation from 22 spawning populations of coho salmon (Oncorhynchus kisutch) from the Oregon Coast to help identify populations for conservation planning. All of our samples were temporally replicated, with most samples obtained in 2000 and 2001. We had three goals: (1) to confirm the status of populations identified on the basis of spawning location and life history; (2) to estimate effective population sizes and migration rates in order to determine demographic independence at different spatial scales; and (3) to determine if releases of Washington hatchery coho salmon in the 1980's into Oregon Coast streams resulted in measurable introgression into nearby wild Oregon Coast coho populations. For the last question, our study included a hatchery broodstock sample from 1985, after the Puget Sound introduction, and a 1975 sample taken from the same area prior to the introduction. Our results generally supported previously hypothesized population structure. Most importantly, we found unique lake-rearing groups identified on the basis of a common life-history type were genetically related. Estimates of immigrant fraction using several different methods also generally supported previously identified populations. Estimates of effective population size were highly correlated with estimates of spawning abundance. The 1985 hatchery sample was genetically similar to contemporary Washington samples, and the contemporary Oregon Coast samples were similar to the 1975 Oregon Coast sample, suggesting that introductions of Washington coho salmon did not result in large scale introgression into Oregon populations.

Detection by PCR of hepatopancreatic parvovirus (HPV) and other viruses in hatchery-reared Penaeus monodon postlarvae.

The prevalence of hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in samples of Penaeus monodon postlarvae (PL10 to PL20, 10 to 20 d old postlarvae) in India was studied by PCR. Samples collected from different hatcheries, and also samples submitted by farmers from different coastal states, were analyzed. HPV was detected in 34%) of the hatchery samples and 31% of the samples submitted by farmers, using a primer set designed for detection of HPV from P. monodon in Thailand. However, none of these samples were positive using primers designed for detection of HPV from P. chinensis in Korea. This indicated that HPV from India was more closely related to HPV from P. monodon in Thailand. MBV was detected in 64% of the samples submitted by the farmers and 71% of the hatchery samples. A total of 84 % of the samples submitted by farmers, and 91% of the hatchery samples, were found positive for WSSV. Prevalence of concurrent infections by HPV, MBV and WSSV was 27% in hatchery samples and 29%, in samples submitted by farmers. Only 8% of the hatchery samples and 16% of the samples submitted by farmers were negative for all 3 viruses. This is the first report on the prevalence of HPV in P. monodon postlarvae from India.

Abundance of potentially pathogenic micro-organisms in Penaeus monodon larvae rearing systems in India

Monodon baculovirls (MBV), external fouling organisms (EFO) and bacteria (especially Vibrio species) were monitored during 1996-1997 at nine different Penaeus monodon rearing hatcheries in India. Total cultivable heterotrophic bacteria, Vibrio-like-bacteria, presumptive Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus counts were determined from shrimp eggs, post larvae, rearing tank water, source sea water, feed (Artemia nauplii and microencapsulated feed). The MBV infected post larvae and their environment showed higher Vibrio-like-bacteria than uninfected post larvae. An overwhelming predominance of presumptive Vibrio harveyi and Vibrio anguillarum was observed in post larval rearing tank water, MBV infected and uninfected post larvae. Vibrio-like-bacteria in Artemia nauplii clearly showed the possible source of these pathogenic bacteria in the hatchery environments. Quantitative analysis of Vibrio-like-bacteria in hatcheries revealed that when the Vibrio-like-bacteria increases to 2 x 10(2) CFU mortality of the post larvae occurs. Abundance of these micro-organisms in hatchery samples indicated that they are opportunistic pathogens which can invade the shrimp tissue, subsequently cause disease when the post larvae were under stressful conditions.

Human salmonellosis associated with young poultry from a contaminated hatchery in Michigan and the resulting public health interventions, 1999 and 2000.

Although approximately 95% of disease caused by nontyphoidal salmonella is transmitted by foodborne vehicles, four documented salmonella outbreaks in the 1990s have been traced to contact with young poultry. No environmental studies of source hatcheries were completed. This case-control study was performed by comparing culture-confirmed Salmonella Infantis in Michigan residents, identified between May and July 1999, with two age- and neighbourhood-matched controls. Eighty environmental and bird tissue samples were collected from an implicated hatchery; all salmonella isolates underwent pulsed-field gel electrophoresis (PFGE) analysis. The study included 19 case-patients sharing the same PFGE subtype and 37 matched controls. Within 5 days before illness onset, 74% of case-patients resided in households raising young poultry compared with 16% of controls (matched OR 19.5; 95% CI 2.9, 378.1). Eight hatchery samples yielded Salmonella Infantis with PFGE subtypes matching the patients' isolates. This investigation identified birds from a single hatchery as the source of human illness and confirmed the link by matching PFGE patterns from humans, birds and the hatchery environment. Subsequent public health interventions reduced, but did not eliminate, transmission of poultry-associated salmonellosis. Five additional PFGE-linked cases were identified in Spring 2000, necessitating quarantine of the hatchery for depopulation, cleaning and disinfection.

Genetic Characterization of Naturally Spawned Snake River Fall-Run Chinook Salmon

We sampled juvenile Snake River chinook salmon Oncorhynchus tshawytscha to genetically characterize the endangered Snake River fall-run population. Juveniles from fall and spring–summer lineages coexisted in our sampling areas but were differentiated by large allozyme allele frequency differences. We sorted juveniles by multilocus genotypes into putative fall and spring lineage subsamples and determined lineage composition using maximum likelihood estimation methods. Paired sMEP-1* and PGK-2* genotypes—encoding malic enzyme (NADP+) and phosphoglycerate kinase, respectively—were very effective for sorting juveniles by lineage, and subsamples estimated to be 100% fall lineage were obtained in four annual samples. We examined genetic relationships of these fall lineage juveniles with adjacent populations from the Columbia River and from Lyons Ferry Hatchery, which was established to perpetuate the Snake River fall-run population. Our samples of naturally produced Snake River fall lineage juveniles were most closely aligned with Lyons Ferry Hatchery samples. Although fall-run strays of Columbia River hatchery origin found on spawning grounds threaten the genetic integrity of the Snake River population, juvenile samples (a) showed distinctive patterns of allelic diversity, (b) were differentiated from Columbia River populations, and (c) substantiate earlier conclusions that this population is an important genetic resource. This first characterization of naturally produced Snake River fall chinook salmon provides a baseline for monitoring and recovery planning.

Microsatellite polymorphism and genetic impact of restocking in mediterranean brown trout (Salmo trutta L.)

The genetic impact of restocking Mediterranean brown trout populations with hatchery stocks was investigated in the Orb River drainage (France), using genetic data from three microsatellite loci. We sampled two wild populations, the main river which is restocked each year and one of its tributaries which has not been restocked for 6 years. Each sample was divided into two age groups (juveniles/adults). Introgression of each native population by hatchery stocks was previously estimated using allele frequencies from two diagnostic protein-coding loci and one mtDNA haplotype. The genetic structure and allelic frequency at three microsatellite loci in native populations were compared with two hatchery samples belonging to stocks usually used for restocking this drainage. High levels of polymorphism (23-27 alleles per locus) were detected for two loci, whereas the third was less polymorphic. Polymorphism was significantly higher in the restocked population than in the now undisturbed population. Significant differences between age groups were observed in the main river, but not in its tributary. The introgression estimates using microsatellites were compared to those obtained from proteins and mtDNA. The different possible origins of alleles common to hatcheries and wild populations (homoplasy, ancestral polymorphism or introgression) are discussed.

Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae.

Recurrent outbreaks of a disease leading to mass mortalities in an oyster (Crassostrea gigas) hatchery located in western Japan were investigated. The disease occurred regularly in 2- to 8-d-old larvae and has been experimentally controlled in the hatchery by treating the larval rearing water with streptomycin, without ascertaining the etiological agent. The signs of the disease and the course of infection resembled bacillary necrosis reported in oysters and other bivalve molluscs in the USA and Europe. Quantitative and qualitative examinations of the bacterial flora of hatchery samples including source water, broodstock, larval feed and larvae revealed a very high total bacterial load and presumptive vibrios in diseased larvae. Further, the bacterial profile revealed that Vibrio spp. constituted approximately 60 to 95% of the bacteria isolated from infected larvae and most isolates were identified as V. splendidus biovar II and V. harveyi, suggesting their possible role in the disease. However, experimental challenges proved the pathogenicity of V. splendidus II. Several isolates of V. splendidus II from infected larvae were highly pathogenic, producing 100% mortality at levels of 10(5) cfu ml-1 in 24 h, while isolates from other sources demonstrated a low degree of virulence. Detection of V. splendidus II from broodstock, especially in the gonad of a few breeders, suggests the probability that broodstock could be the source and route of transmission of this pathogen.

Localized Genetic Effects of a Long-Term Hatchery Stocking Program on Resident Rainbow Trout in the Metolius River, Oregon

Abstract Hatchery rainbow trout Oncorhynchus mykiss have been stocked in the Metolius River in central Oregon since 1938, and legal-sized (≥160 g) yearling trout were stocked annually from 1947 until 1995. In 1996, management objectives shifted to emphasize wild trout, and hatchery stocking ceased. We examined allozyme and mitochondrial DNA (mtDNA) variation among three naturally occurring populations of rainbow trout in the Metolius River to investigate possible hybridization with hatchery-produced rainbow trout. We also examined two commonly used hatchery strains of rainbow trout, one of which has supplied nearly all of the catchable hatchery trout in the Metolius. Both allozyme and mtDNA data showed the two hatchery samples to have genetic characteristics typical of hatchery populations derived from coastal rainbow trout O. mykiss irideus. Rainbow trout sampled from the lower Metolius River, approximately 30 km downstream of the headwaters, had allozyme and mtDNA characteristics typical of interior rai...

Genetic heterozygosity and meristic character variance in a wild Atlantic salmon population and a hatchery strain derived from it

Heterozygosity at seven enzyme loci was compared with the degree of fluctuating asymmetry of three bilateral traits in a wild population of Atlantic salmon (Salmo salar L.) and a hatchery strain derived from it. In the wild population, average heterozygosity per fish was higher (p < 0.01) and average asymmetric value per fish was lower (p < 0.01), compared with the hatchery strain. Analyses at the intrapopulation level did not provide unequivocal support for the relationship between heterozygosity and developmental stability, with correlation between the number of heterozygous loci per fish and the asymmetric value per fish being non-significant. However, comparisons of asymmetric value per fish between homozygous and heterozygous individuals at separate loci indicated that heterozygotes had significantly lower asymmetry values per fish at IDDH-1* in the hatchery sample. These results are discussed in relation to the potential utility of fluctuating character asymmetry as an indicator of loss of heterozygosity in cultured Atlantic salmon stains.

Heritage Brook Trout in Northeastern USA: Genetic Variability within and among Populations

Abstract Brook trout Salvelinus fontinalis from 21 unstocked waters, 3 naturalized lakes, and 4 hatcheries in New York and Pennsylvania were analyzed electrophoretically for allozyme expression. Thirty-two of the 68 loci examined were polymorphic. Average heterozygosity of samples from populations classified as wild–unstocked was 0.050 (range, 0.026–0.076). Differences (P < 0.05) occurred among the 21 wild–unstocked samples at 25 of 31 possible locus comparisons. All wild–unstocked samples were significantly different from each other and from hatchery samples (P < 0.01). A high fixation index (F ST = 0.375) indicated that the wild–unstocked samples represented highly differentiated populations. A considerable portion of the gene diversity was found among major river basins (22.5%); the remainder was due to differences among minor river drainages within basins (10.0%) and among samples within minor drainages (5.0%). Cluster analysis of genetic distances organized samples into three main groups that were al...