Physiological performance of largemouth bass related to local adaptation and interstock hybridization: implications for conservation and management

Four genetically distinct stocks of age 2+ years largemouth bass Micropterus salmoides were produced using adults collected from two regions in the upper midwest (central Illinois, IL and south-eastern Wisconsin, WI, U.S.A.). Two pure stocks (IL × IL and WI × WI), as well as both of their reciprocal F1 interstock hybrids (IL × WI and WI × IL) were produced in research ponds in Champaign, IL. In general, swimming performance, routine oxygen consumption and activity were highest at 18 × C, intermediate at 12 × C, and lowest at 6. C for all stocks. However, performance indicators varied among stocks at each of the temperatures. The pure Illinois stock (IL × IL) had the lowest activity: cost ratio at 18 × C and the highest at 6_ C (based upon swimming strength, routine activity rates and routine metabolic rates). The opposite pattern was observed for the other pure stock (WI × WI). Although differences were less distinct at lower temperatures, the two pure stocks (IL × IL and WI × WI) outperformed both interstock hybrids. These results indicate that not only do non-native stocks appear to have reduced performance relative to locally adapted stocks, but also that interstock hybrids exhibit performance impairments, not hybrid vigour.     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.      

Hybrid Breakdown in Cichlid Fish

Studies from a wide diversity of taxa have shown a negative relationship between genetic compatibility and the divergence time of hybridizing genomes. Theory predicts the main breakdown of fitness to happen after the F1 hybrid generation, when heterosis subsides and recessive allelic (Dobzhansky-Muller) incompatibilities are increasingly unmasked. We measured the fitness of F2 hybrids of African haplochromine cichlid fish bred from species pairs spanning several thousand to several million years divergence time. F2 hybrids consistently showed the lowest viability compared to F1 hybrids and non-hybrid crosses (crosses within the grandparental species), in agreement with hybrid breakdown. Especially the short- and long-term survival (2 weeks to 6 months) of F2 hybrids was significantly reduced. Overall, F2 hybrids showed a fitness reduction of 21% compared to F1 hybrids, and a reduction of 43% compared to the grandparental, non-hybrid crosses. We further observed a decrease of F2 hybrid viability with the genetic distance between grandparental lineages, suggesting an important role for negative epistatic interactions in cichlid fish postzygotic isolation. The estimated time window for successful production of F2 hybrids resulting from our data is consistent with the estimated divergence time between the multiple ancestral lineages that presumably hybridized in three major adaptive radiations of African cichlids.     

Use of cellular oncogene probes to identify Morone hybrids.

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish.     

吉林爆玉米遗传距离与杂种优势关系的研究

根据吉林省80个爆玉米自交系数量性状的表现,估算了它们之间的遗传距离。又估算了这80个自交系间随机组配的67个杂交组合F1代小区产量、爆粒率和膨胀倍数的杂种优势。相关和回归分析表明,F1代产量的对照优势与其双亲之间的遗传距离呈显著的二次曲线关系,而F1代品质特性爆粒率和膨胀倍数的对照优势与其双亲之间的遗传距离无确定性关系。因此,可以根据亲本间数量性状的综合遗传差异——遗传距离来预测吉林省爆玉米杂种一代的产量优势。 An experiment was conducted in 1990 to 1991 at the university,to study the relationships between genetic distance,cluster analysis and heterosis in popcorn(Zea mays var.everta).Results showed that there is no sense in cludtering parental lines for prediction of yield heterosis,but blood pedigree can beidentifed from cluster analysis.The relation between genetic divergence or distance and yield heterosis of F1 hybrids can be predicted from the genetic distances of their parental lines according to this model,but no definite relation could be found between parental genetic divergence and the quality heterosis of F1 hybrids.     

黄海和东海小黄鱼遗传多样性的RAPD分析

小黄鱼（Pseudosciaena polyactis）是我国近海重要经济鱼类之一。本文分析了采自黄海和东海5个海区共计48个个体小黄鱼的随机扩增DNA多态性（RAPD）。从40个10 bp引物中选取20个用于群体遗传多样性分析,共检测出145个位点,其中132个（91.03%） 显多态性。用Shannon多样性指数量化的平均遗传多态度为1.93（1.50～2.44）,群体内和群体间的遗传变异比例分别为69%和31%;群体间的平均遗传相似度和遗传距离分别为0.9139和0.0861。用非加权配对算数平均法（UPGMA）聚类分析的结果表明,所分析的5个群体可分为3个地理群系,从分子水平上支持了过去有关学者把黄海和东海的小黄鱼划分为北、中、南3个地理群系的观点。     

Food habits and relative abundances of native piscivores: implications for controlling common carp

Common carp (Cyprinus carpio, carp) are a widespread and ecologically destructive invasive fish species. Carp management is critical for maintaining healthy aquatic ecosystems, and many control options are available, but most have proven to be ineffective. Carp abundances have increased at The Nature Conservancy’s Emiquon Preserve, Illinois, since its restoration in 2007 despite management efforts to suppress this species. We conducted a comparative diet study in Illinois, Tennessee, and Wisconsin to test whether bowfin (Amia calva), spotted gar (Lepisosteus oculatus, gar), and largemouth bass (Micropterus salmoides) commonly preyed upon carp. We focused on bowfin and gar because they are hypoxia-tolerant, similar to carp. We also assessed whether specific fish community characteristics were correlated with carp relative abundances. We found no evidence that bowfin, gar, and bass consumed large numbers of carp. However, carp may be limited in some ecosystems (e.g., Reelfoot Lake, Tennessee) through alternative mechanisms associated with bowfin, gar, bass, and bluegill (Lepomis macrochirus) included in a diverse native fish community.     

Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus

The genetic basis of species differences provides insight into the mode and tempo of phenotypic divergence. We investigate the genetic basis of floral differences between two closely related plant taxa with highly divergent mating systems, Mimulus guttatus (large-flowered outcrosser) and M. nasutus (small-flowered selfer). We had previously constructed a framework genetic linkage map of the hybrid genome containing 174 markers spanning approximately 1800 cM on 14 linkage groups. In this study, we analyze the genetics of 16 floral, reproductive, and vegetative characters measured in a large segregating M. nasutus x M. guttatus F2 population (N = 526) and in replicates of the parental lines and F1 hybrids. Phenotypic analyses reveal strong genetic correlations among floral traits and epistatic breakdown of male and female fertility traits in the F2 hybrids. We use multitrait composite interval mapping to jointly locate and characterize quantitative trait loci (QTLs) underlying interspecific differences in seven floral traits. We identified 24 floral QTLs, most of which affected multiple traits. The large number of QTLs affecting each trait (mean = 13, range = 11-15) indicates a strikingly polygenic basis for floral divergence in this system. In general, QTL effects are small relative to both interspecific differences and environmental variation within genotypes, ruling out QTLs of major effect as contributors to floral divergence between M. guttatus and M. nasutus. QTLs show no pattern of directional dominance. Floral characters associated with pollinator attraction (corolla width) and self-pollen deposition (stigma-anther distance) share several pleiotropic or linked QTLs, but unshared QTLs may have allowed selfing to evolve independently from flower size. We discuss the polygenic nature of divergence between M. nasutus and M. guttatus in light of theoretical work on the evolution of selfing, genetics of adaptation, and maintenance of variation within populations.     

Inbreeding and outbreeding depression in Stylidium hispidum: implications for mixing seed sources for ecological restoration

The benefits of composite rather than local seed provenances for ecological restoration have recently been argued, largely on the basis of maximizing evolutionary potential. However, these arguments have downplayed the potentially negative consequences of outbreeding depression once mixed provenances interbreed. In this study, we compared intraspecific F1 hybrid performance and molecular marker differentiation among four populations of Stylidium hispidum, a species endemic to Southwestern Australia. Multivariate ordination of 134 AFLP markers analyzed genetic structure and detected two clusters of paired sites that diverged significantly for marker variation along a latitudinal boundary. To test for outbreeding depression and to determine the consequences of molecular population divergence for hybrid fitness, we conducted controlled pollinations and studied germination and survival for three cross categories (within‐population crosses, short‐ and long‐distance F1 hybrids) for paired sites distributed within and between the two genetically differentiated regions. We found evidence of outbreeding depression in long‐distance hybrids (111–124 km), and inbreeding depression among progeny of within‐population crosses, relative to short‐distance (3–10 km) hybrids, suggesting an intermediate optimal outcrossing distance in this species. These results are discussed in light of the evolutionary consequences of mixing seed sources for biodiversity restoration.     

Genetic and morphometric divergence in threespine stickleback in the Chignik catchment,Alaska

Divergent selection pressures induced by different environmental conditions typically lead to variation in life history, behavior, and morphology. When populations are locally adapted to their current environment, selection may limit movement into novel sites, leading to neutral and adaptive genetic divergence in allopatric populations. Subsequently, divergence can be reinforced by development of pre‐ or postzygotic barriers to gene flow. The threespine stickleback, Gasterosteus aculeatus, is a primarily marine fish that has invaded freshwater repeatedly in postglacial times. After invasion, the established freshwater populations typically show rapid diversification of several traits as they become reproductively isolated from their ancestral marine population. In this study, we examine the genetic and morphometric differentiation between sticklebacks living in an open system comprising a brackish water lagoon, two freshwater lakes, and connecting rivers. By applying a set of microsatellite markers, we disentangled the genetic relationship of the individuals across the diverse environments and identified two genetic populations: one associated with brackish and the other with the freshwater environments. The “brackish” sticklebacks were larger and had a different body shape than those in freshwater. However, we found evidence for upstream migration from the brackish lagoon into the freshwater environments, as fish that were genetically and morphometrically similar to the lagoon fish were found in all freshwater sampling sites. Regardless, few F1‐hybrids were identified, and it therefore appears that some pre‐ and/or postzygotic barriers to gene flow rather than geographic distance are causing the divergence in this system.     

Tree hybridization and genotypic variation drive cryptic speciation of a specialist mite herbivore

Few studies have investigated the roles that plant hybridization and individual plant genotype play in promoting population divergence within arthropod species. Using nrDNA sequence information and reciprocal transfer experiments, we examined how tree cross type (i.e., pure Populus angustifolia and P. angustifolia x P. fremontii F(1) type hybrids) and individual tree genotype influence host race formation in the bud-galling mite Aceria parapopuli. Three main findings emerged: (1) Strong genetic differentiation of mite populations found on pure P. angustifolia and F(1) type hybrids indicates that these mites represent morphologically cryptic species. (2) Within the F(1) type hybrids, population genetic analyses indicate migration among individual trees; however, (3) transfer experiments show that the mites found on heavily infested F(1) type trees perform best on their natal host genotype, suggesting that genetic interactions between mites and their host trees drive population structure, local adaptation, and host race formation. These findings argue that hybridization and genotypic differences in foundation tree species may drive herbivore population structure, and have evolutionary consequences for dependent arthropod species.     

Individual variation in whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance in a marine teleost

Hypoxia is a pervasive problem in coastal environments and is predicted to have enduring impacts on aquatic ecosystems. Intraspecific variation in hypoxia tolerance is well documented in fish; however, the factors underlying this variation remain unknown. Here, we investigate the role of the heart in individual hypoxia tolerance of the European sea bass (Dicentrarchus labrax). We found individual whole-animal hypoxia tolerance is a stable trait in sea bass for more than 18 months (duration of study). We next examined in vitro cardiac performance and found myocardial muscle from hypoxia-tolerant individuals generated greater force, with higher rates of contraction and relaxation, than hypoxic-sensitive individuals during hypoxic exposure. Thus, whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance. As the occurrence of aquatic hypoxia is expected to increase in marine ecosystems, our experimental data suggest that cardiac performance may influence fish survival and distribution.     

Genetics of esterases in Drosophila of the virilis group. I. Characteristics of esterase patterns in D. virilis,D. texana,D. littoralis,and their hybrids

Starch and polyacrylamide gel electrophoreses have detected six esterase fractions in Drosophila of the virilis group. These esterases have been characterized in detail using a series of substrates and inhibitors and also thermal treatment. Differences in esterase patterns have been found between D. virilis, D. texana, and D. litoralis as well as between D. virilis stocks. An interstock polymorphism for different esterase patterns has been established with respect to the electrophoretic mobilities of a number of esterase fractions. In rare instances, it has been observed within some D. virilis stocks, too. There is specificity in organ distribution of esterase fractions in Drosophila. Monogenic control of the electrophoretic mobilities of esterase-2 and esterase-4 has been demonstrated in D. virilis, and a dimer structure has been found in esterase-2. Genes controlling esterase-2 and esterase-4 are located on the second chromosome (209.3 for esterase-2 and 192.0 for esterase-4). In interstock and interspecific hybrids, esterases usually manifest codominance. In interstock hybrids, esterase-2 forms a hybrid band not observed in interspecific hybrids. In third instar larvae of interspecific hybrids, differential expression of certain esterase isozymes has been noted. These observations are in agreement with data from histochemical studies of organs of different hybrids.     

Gene expression associated with indicated faster growth of crossbreeding in Sinocyclocheilus grahami

In this study, pure‐breeding and crossbreeding of Sinocyclocheilus grahami were established to test heterosis and expression of growth‐related genes which were examined in the first‐generation (F1). Genetic distances results showed that Muyang river (M) and Sanbaimu reservoir (S) populations had smaller interspecific distances (genetic distance: 0.000) while larger distances were shown to other two populations (0.024 of Jiuzhai vauclusian spring [J] and 0.044 of Chenglong vauclusian spring [C]). Full‐sib families were created by using the Muyang river population as females: MM, MS, MJ and MC. Growth performance had significant differences between each group of F1 hybrids; the fastest growth group was shown in MJ while the slowest was in MS. For growth‐related genes, the fast growth group of MJ hybrids showed significantly higher levels of ghrelin expression in strong expression tissues of the intestine and liver. Among the four combinations, mRNA levels of insulin‐like growth factor I (IGF‐I) were the highest expression in MJ hybrids than others in all tested tissues. Myostatin b and somatostatin1 (SST1) were strongly expressed in the brain and MJ hybrids showed relatively lower expression level. In the muscle, slow growth group of MS hybrids showed extremely higher expression levels of these two genes, especially SST1 gene. In conclusion, associating growth with growth‐related genes expression indicated faster growth of crossbreeding in S. grahami and can make a contribution to commercial fish production in the future.     

Nuclear and cytoplasmic contributions to intraspecific divergence in an annual legume

The genetic architecture of trait differentiation was evaluated between two ecologically distinct populations of Chamaecrista fasciculata. Individuals from Maryland and Illinois populations were crossed to create 10 types of seed: Maryland and Illinois parents, reciprocal F1 and F2 hybrids, and backcrosses to Maryland and to Illinois on reciprocal F1 hybrids. Reciprocal crosses created hybrid generation seeds with both Maryland and Illinois cytoplasmic backgrounds. Experimental individuals were grown in a common garden near the site of the Maryland population. In the garden, plants from the Illinois population flowered, set fruit, and died earlier than those from Maryland, likely reflecting adaptations to differences in growing season length between the two populations. Although reproductive components at the flower and whole plant level differed between the two populations, reproductive output as measured by fruit and seed production was similar. Cytoplasmic genes had a subtle but pervasive effect on population differentiation; hybrids with Maryland cytoplasm were significantly differentiated from those with Illinois cytoplasm when all characters were evaluated jointly. The nuclear genetic architecture of population differentiation was evaluated with joint scaling tests. Depending on the trait, both additive and nonadditive genetic effects contributed to population differentiation. Intraspecific genetic differentiation in this wild plant species appears to reflect a complex genetic architecture that includes the contribution of additive, dominance, and epistatic components in addition to subtle cytoplasmic effects.     

Low temperature cardiac response to exhaustive exercise in fish with different levels of winter quiescence

We examined the cardiac responses of different fish species to anaerobic exercise at low temperatures (3 degrees C). Three species of sympatric warmwater fish with perceived differences in winter activity were used for this comparative study: the winter-quiescent largemouth bass (Micropterus salmoides); the winter-active white bass (Morone chrysops); and the intermediately winter-active black crappie (Pomoxis nigromaculatus). Perceived differences in winter activity were reflected in cardiac responses; e.g. basal cardiac values were lowest for largemouth bass, highest for white bass, and intermediate for black crappie. In addition, cardiac recovery was most rapid for white bass, slowest for largemouth bass and intermediate for black crappie. When disturbed at low temperatures, largemouth bass and black crappie elevated cardiac output principally through increases in heart rate despite substantial decreases in stroke volume. Conversely, white bass principally used stroke volume modulation to change cardiac output. The results of this study indicate that different species respond differently to exercise at low temperatures. Management strategies should recognize that such variation exists and ensure that management decisions are based upon an understanding of the low temperature exercise physiology and winter biology of the species of interest.     

Early life traits of farm and wild Atlantic salmon Salmo salar and first generation hybrids in the south coast of Newfoundland

This study examined fertilization rates, survival and early life‐trait differences of pure farm, wild and first generation (F1) hybrid origin embryos after crossing farm and wild Atlantic salmon Salmo salar. Results show that despite a trend towards higher in vitro fertilization success for wild females, differences in fertilization success in river water are not significantly different among crosses. In a hatchery environment, wild females' progeny (pure wild and hybrids with wild maternal parent) hatched 7–11 days earlier than pure farm crosses and hybrids with farm maternal parents. In addition, pure wild progeny had higher total lengths (L_T) at hatch than pure farm crosses and hybrids. Directions in trait differences need to be tested in a river environment, but results clearly show the maternal influence on early stages beyond egg‐size differences. Differences in L_T were no longer significant at 70 days post hatch (shortly after the onset of exogenous feeding) showing the need to investigate later developmental stages to better assess somatic growth disparities due to genetic differences. Higher mortality rates of the most likely hybrids (farm female × wild male hybrids) at egg and fry stages and their delayed hatch suggest that these F1 hybrids might be less likely to survive the early larval stages than wild stocks.     

Population differentiation in the beetleTribolium castaneum. I. Genetic architecture

We used joint-scaling analyses in conjunction with rearing temperature variation to investigate the contributions of additive, non-additive, and environmental effects to genetic divergence and incipient speciation among 12 populations of the red flour beetle, Tribolium castaneum, with small levels of pairwise nuclear genetic divergence (0.033 < Nei's D < 0.125). For 15 population pairs we created a full spectrum of line crosses (two parental, two reciprocal F1's, four F2's, and eight backcrosses), reared them at multiple temperatures, and analyzed the numbers and developmental defects of offspring. We assayed a total of 219,388 offspring from 5147 families. Failed crosses occurred predominately in F2's, giving evidence of F2 breakdown within this species. In all cases where a significant model could be fit to the data on offspring number, we observed at least one type of digenic epistasis. We also found maternal and cytoplasmic effects to be common components of divergence among T. castaneum populations. In some cases, the most complex model tested (additive, dominance, epistatic, maternal, and cytoplasmic effects) did not provide a significant fit to the data, suggesting that linkage or higher order epistasis is involved in differentiation between some populations. For the limb deformity data, we observed significant genotype-by-environment interaction in most crosses and pure parent crosses tended to have fewer deformities than hybrid crosses. Complexity of genetic architecture was not correlated with either geographic distance or genetic distance. Our results support the view that genetic incompatibilities responsible for postzygotic isolation, an important component of speciation, may be a natural but serendipitous consequence of nonadditive genetic effects and structured populations.     

Growth and mortality of black bass, Micropterus salmoides (Pisces, Centrachidae; Lacapède, 1802) in a reservoir in southern Brazil.

The black bass, Micropterus salmoides, was introduced to Brazil from North America in 1922. Since then the species has been reared in aquaculture facilities intended to stock reservoirs as additions to native stocks available for angling. At present no scientific information on the biology of black bass in Brazilian waters is available. Since black bass dispersion may cause severe impacts on native Brazilian fish fauna, information on the basic biological parameters of this species is necessary. The objective of the present study is to provide information on the growth, age structure, and mortality of the species in a small reservoir in southern Brazil, where the species reproduces regularly. Based on scale readings, the von Bertalanffy growth curve was calculated and compared to the length-frequency distribution of the population. Both methods showed similar results. Maximum length was about 44 cm. The mean length at the end of the first year was 23.1 cm (s.d. = 13.88); at that of the second, 37.3 cm (s.d. = 12.52); and 41.4 cm (s.d. = 9.92) at the third. Oldest fish were three years old. The growth performance index phi' was 3.28 cm year(-1). Mortality increased from 0.16 year(-1) between the first and the second cohort, to 0.8 year(-1) between the second and third. The results show that black bass in Brazil grows faster than in its area of origin, but longevity is shorter and body shape, stouter. The cause of high mortality at a relatively early age may be connected with the loss of genetic diversity due to inbreeding of the Brazilian stocks, which originated from few introduced individuals a long time ago. The fact that black bass reproduces in reservoirs and grows rapidly may be considered a threat to conserving fish diversity in Brazilian ecosystems.     

Comparison of maturation timing,egg size and fecundity between hatchery lines of chinook salmon and their wild donor stocks

Increasing concern has been expressed about the genetic effects of cultured salmonid fishes on natural populations. Avoidance of extreme negative outcomes was one reason for the establishment of a genetic management policy for the State of Alaska. However, domestication within the hatchery may still cause divergence from the wild donor population. This divergence could potentially lead to adverse impacts on wild stocks through straying and introgression. This study examines potential domestication in two Alaskan chinook salmon stocks. The Little Port Walter (LPW) Hatchery Chickamin River stock resulted from a small collection of wild broodstock in 1976. The LPW Unuk stock was founded with a larger number of individuals in 1976 and has had subsequent infusion of wild gametes. These lines have been maintained at LPW through ocean ranching of tagged smolts. Comparisons are made between the hatchery lines, progeny of wild chinook collected from the Chickamin and Unuk Rivers, and hybrids between the hatchery and wild groups. Mature ocean‐ranched female chinook salmon returning to the facility were periodically graded for ripeness and spawned. Body size and meristic measurements were collected from these mature spawners. Maturation timing, fecundity, and individual egg size of these fourth generation hatchery fish are compared with that of offspring of wild fish from the same donor stock. Stock of origin is confirmed for all spawners and offspring using microsatellite DNA analysis.