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1.
The response to chemical disturbance of an Italian river (Cecina River) characterised by high levels of trace metals was assessed in the sentinel species freshwater painter’s mussel (Unio pictorum). We exposed U. pictorum in the laboratory to metal, metalloid and other trace element polluted river sediments in order to evaluate genetic and cellular biomarkers. Bivalves exposed to sediments taken from all the impacted sites of the river basin exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. The lysosomal membrane stability of circulating haemocytes, assessed by the Neutral Red Retention Time, was found to be higher in specimens from Lake Maggiore and Berignone, while exhibited a significant decrease in specimens exposed to Ponteginori sediments. An increase of DNA migration in specimens exposed to the two contaminated sites in comparison with controls was found. Moreover, higher micronucleated haemocytes frequencies were observed in bivalves exposed to sediments collected at Possera 1 site. This study suggested that biomarkers evaluating cellular impairment, genetic and chromosomal damages, are highly sensitive and suitably applied to laboratory experiments. The painter’s mussel was confirmed to be an appropriate sentinel species for quality assessment in metal-contaminated freshwater environments.  相似文献   

2.
Two species of blue mussel, Mytilus galloprovincialis and M. trossulus, co-occur and hybridize along the Pacific coast of North America. Using a set of polymerase chain-reaction (PCR)-based genetic markers which diagnostically identify these species, we show that they are sympatric from the Cape Mendocino region to the Monterey Peninsula in northern and central California, USA. Mussels with hybrid genotypes were detected in all populations sampled in the region of sympatry, and the frequency of hybrid genotypes in individual hybrid populations ranged from 13 to 44%. Significant frequencies of first-generation backcross genotypes were detected in two individual hybrid zone populations (Berkeley and Monterey Marina) and in the hybrid zone as a whole, indicating that the potential exists for introgression between M. galloprovincialis and M. trossulus. Despite this potential, we found no evidence of advanced introgression beyond first-generation backcrosses, suggesting that gene flow between M. galloprovincialis and M. trossulus has been quite limited. The frequency of mussels with M. trossulus and hybrid genotypes declined abruptly south of Monterey Peninsula, while the frequency of mussels with M. galloprovincialis and hybrid genotypes declined precipitously north of Cape Mendocino. These abrupt genetic discontinuities indicate that this blue mussel hybrid zone is presently positioned between two prominent coastal features and there is little, if any, export of alleles from the hybrid zone into bordering parental populations. Received: 20 August 1997 / Accepted: 26 October 1998  相似文献   

3.
In Ireland, mussels on exposed rocky shores constitute an interbreeding mixture of two forms of mussels, the blue mussel, Mytilus edulis, and the Mediterranean mussel, M. galloprovincialis. Results from an Irish study in the 1980s, using partially diagnostic allozyme markers, indicated that mussels higher up the shore were more galloprovincialis-like than those lower down. In this study we set out to test two hypotheses: (a) recruits arriving on the shore are composed of genetically distinct cohorts that settle preferentially at different levels on the shore, and maintain genetic distinctiveness into adulthood; (b) recruits are genetically homogeneous, but once settled they diverge genetically over time, due to within-habitat site specific-selection. The diagnostic Me 15/16 DNA marker was used to analyse the genetic composition of newly-settled spat recruiting to artificial substrates, which were placed at two-week intervals from May–October 2002, on the mid- and low shore areas of two exposed sites in Galway Bay. Adult mussels were also collected on each sampling date. Results did not support the preferential settlement hypothesis, i.e., the genetic composition of primary settlers (≤ 500 μm) was similar between tidal heights and shores. Neither was there evidence of post settlement selective mortality, as adults were genetically similar to settling spat. In spat and adults the frequency of the M. galloprovincialis allele was high (0.56–0.80), due to high frequencies of M. galloprovincialis (> 37%) and hybrid (> 33%) genotypes, and correspondingly low frequencies of the M. edulis genotype (< 11%). Adult mussels from a nearby sheltered estuarine site, while significantly different to exposed shore mussels, still had low frequencies of the M. edulis genotype (< 17%), indicating no apparent advantage for the genotype in this environment. There are indications that the genetic composition of mussels may be changing on the Atlantic coasts of Ireland.  相似文献   

4.
 We investigated the utility of three polymerase chain-reaction (PCR)-based DNA molecular markers in analysing genetic structure of the populations of the blacklip abalone Haliotis rubra (Leach) of Victoria, Australia. The DNA markers included 84 randomly amplified polymorphic DNA (RAPD) bands amplified using six random primers, two minisatellites, GHR (putative growth-hormone-gene-repeat) and MIPR (putative mollusca-insulin-like peptide-gene-repeat), and three microsatellites, RUBGT1 [containing (GT)n repeats], RUBCA1 [containing (CA)n repeats] and RUBGACA1 [containing (GACA)n repeats]. All three types of DNA markers revealed significant subdivision in the H. rubra populations along the coastline. This is postulated as being related to the abalone's relatively short pelagic period and limited dispersion. Further analysis revealed that a Point Cook population sampled from within the semi-enclosed Port Phillip Bay was distinct from two other central zone populations (Apollo Bay and Cape Schanck). The genotypes of microsatellites indicated excessive homozygotes across all the populations at all three microsatellite loci, and possible causes such as larval recruitment pattern and asynchronous spawning are discussed. The excessive homozygotes recorded for the three microsatellite loci contrast with those observed in the minisatellite loci GHR and MIPR, the heterozygosities of which were at Hardy–Weinberg equilibrium. Received: 17 March 1999 / Accepted: 24 November 1999  相似文献   

5.
近几年来,菌根分子生物学得到了较快发展;尤其是一些用于首根真菌ITS区域的特异引物先后设计并合成成功,进一步推动了菌根学在分子水平上的研究。分子生物学技术在留根真菌分类鉴定及亲缘关系研究上应用较多。在菌根菌竟争性和持续性研究上.DNA分析技术已成为最重要的研究手段之一。在这些研究中,专为菌根真菌ITS区域分析而设计的ITS1-F和ITS4-B两种特异引物应用最为普遍。本文对菌根DNA分析技术、PCR-RFLP技术做了简单介绍;综述了PCR、ITh-RFLP等分析技术在菌根菌分类、亲缘关系及菌种持续性等方面的研究和应用情况。  相似文献   

6.
Geographic variation in mitochondrial large subunit (16S) ribosomal RNA haplotypes was examined for blue mussels, Mytilus trossulus Gould, 1850 and M. galloprovincialis Lamarck, 1819, sampled from ten sites along the Pacific coast of the USA in January of 1993. Using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) assays we determined haplotype frequencies for both the male and female mussel mitochondrial DNA (mtDNA) lineages. Populations from Morro Bay south to San Diego, California, contained only M. galloprovincialis male and female haplotypes, while those from Arcata Bay, California, north to Port Orford, Oregon, were fixed for M. trossulus haplotypes. Populations from Monterey Bay to Bodega Bay, California, contained a mixture of M. trossulus and M. galloprovincialis haplotypes. Overall only 2 of 97 heteroplasmic individuals had a mixed M. trossulus/M. galloprovincialis mitochondrial genotype indicating that hybridization is uncommon in the populations sampled. Further, there was no evidence of extensive introgression between these mussel taxa at the level of mtDNA. This is in contrast to previously published results which suggested the significant introgression of M. trossulus haplotypes into southern populations containing primarily M. galloprovincialis nuclear genotypes. We feel the discrepancy lies in the ability of our assays to detect haplotypes corresponding to both the male and female mtDNA lineages. Potential explanations for the lack of mtDNA introgression include, low levels of backcrossing between hybrids and parental taxa, epistatic interaction between nuclear and mitochondrial genes and the breakdown of a sex-specific inheritance pattern for mtDNA in hybrids.  相似文献   

7.
以栽培大豆和野生大豆的基因组DNA为材料,利用PCR技术扩增并克隆了豆类胰岛素基因,分析了两者的DNA序列.结果表明,测定的DNA序列分别为841bp和903bp,有4个核苷酸差异;在信号肽编码区域内各存在一个内含子,大小分别为368和370个核苷酸;在ORF范围内,与前人报导的cDNA序列分别有1个和2个位点的核苷酸差异,从而导致相应位点的氨基酸发生变化.Southern杂交表明,栽培大豆和野生大豆的豆类胰岛素基因分别以单拷贝和多拷贝形式存在于基因组中  相似文献   

8.
Interference competition for limited habitat or refuges is known to produce density-dependent mortality and generate patterns of micro-habitat distribution. While in mobile species the outcome of interference at a local scale can usually be determined from differences in body size and behavior, the population-level consequences of such interactions vary depending on rates of settlement and recruitment at a site, which are not directly correlated to local reproductive success. Previous experimental studies in central Chile demonstrated that interference competition for refuges is the primary factor driving microhabitat segregation between the predatory crabs Acanthocyclus gayi and Acanthocyclus hassleri, with the latter species monopolizing galleries inside mussel beds and excluding A. gayi to rock crevices. Between April 2001 and March 2006 we quantified monthly recruitment rates in artificial collectors at 17 sites over 900 km of the central coast of Chile. Results show that recruitment rates of A. hassleri are almost two orders of magnitude lower than those of A. gayi, and that they are tightly and positively correlated among sites across the region, suggesting that at scales of kilometers larval stages of these species are affected by similar oceanographic processes. Total crab densities per site were also positively correlated between species and strongly associated to mussel cover, with overall low crab densities at all sites where mussel cover was lower than about 60%. At all sites with mussel cover >60%, the ratio of A. gayi to A. hassleri density progressively decreased from recruits (2.6) to juveniles (0.5) to adults (0.04), overcoming initial differences in recruitment rates. The relative success of the inferior competitor at sites with low mussel cover does not appear to provide a potential mechanism favoring regional coexistence through dispersal to other sites (“mass effects”), because their densities were lower than at sites of high mussel cover. Yet, at many sites of low mussel cover the dominant competitor is virtually absent, allowing A. gayi to attain larger population sizes at the scale of the region. Thus, the factors limiting the dominant competitor from successfully utilizing other microhabitats seem to be the most critical factor in promoting both local and regional coexistence between these species.  相似文献   

9.
In this paper we describe a cryopreservation protocol followed by the culture of Symbiodinium sp. isolated from the Caribbean gorgonian Pseudopterogorgia elisabethae as a potential renewable source of the dinoflagellate symbiont. Four different freezing protocols were designed: a controlled cooling device designed to cool at 1°C/min, a three-step protocol (−20°C for 2 h, −70°C for 2 h, liquid nitrogen-LN2), a two-step protocol (−70°C for 2 h, LN2), and a one-step protocol (LN2). All cells were stored in LN2 after cryopreservation. The cryoprotective agents (CPA) used were ethanol (EtOH) and methanol (MeOH) at 10 and 20%, and seawater (FSW) was used as a control. Viability measurements using cell counts showed that all cryopreservation protocols were relatively successful, and no trends were observed regarding freezing protocol or CPA used. After 19 weeks in culture the viability of samples which had high biomass was determined by the fluorescent assay CellTiter Blue™. The most viable cultures were those cryopreserved by a two-step protocol using 20% MeOH or 20% EtOH as a CPA. A genetic examination of the DNA of these samples using Symbiodinium-specific PCR primers confirmed that the composition of the culture had not changed. For the first time, we report that Symbiodinium sp. isolated from a gorgonian can be cryopreserved and subsequently cultured successfully. Lory Z. Santiago-Vázquez and Nealie C. Newberger contributed equally to this publication.  相似文献   

10.
Scleractinian coral species harbour communities of photosynthetic taxa of the genus Symbiodinium. As many as eight genetic clades (A, B, C, D, E, F, G and H) of Symbiodinium have been discovered using molecular biology. These clades may differ from each other in their physiology, and thus influence the ecological distribution and resilience of their host corals to environmental stresses. Corals of the Persian Gulf are normally subject to extreme environmental conditions including high salinity and seasonal variation in temperature. This study is the first to use molecular techniques to identify the Symbiodinium of the Iranian coral reefs to the level of phylogenetic clades. Samples of eight coral species were collected at two different depths from the eastern part of Kish Island in the northern Persian Gulf, and Larak Island in the Strait of Hormuz. Partial 28S nuclear ribosomal (nr) DNA of Symbiodinium (D1/D2 domains) were amplified by polymerase chain reaction (PCR). PCR products were analyzed using single stranded conformational polymorphism and phylogenetic analyses of the LSU DNA sequences from a subset of the samples. The results showed that Symbiodinium populations were generally uniform among and within the populations of eight coral species studied, and there are at least two clades of Symbiodinium from Kish and Larak islands. Clade D was detected from eight of the coral species while clade C was found in two of species only (one species hosted two clades simultaneously). The dominance of clade D might be explained by high temperatures or the extreme temperature variation, typical of the Persian Gulf. Publication of this article was held up owing to technical problems. The publisher apologizes sincerely for this lengthy delay.  相似文献   

11.
Previous studies of a hybrid zone between the mussels Mytilus edulis Linnaeus and M. galloprovincialis Lamarck have not resolved the relative importance of the genetic composition of settling larval cohorts versus post-settlement selection in determining the distribution of the parental species and their hybrids. In the present study, recently settled mussels (spat) were collected from 20 sites in southwest England throughout the summer and fall (May–October) in 1998 and 1999. This study investigated the spatio-temporal patterns of settlement and genetics of mussel spat by genetically identifying M. edulis, M. galloprovincialis and their hybrids using the diagnostic PCR marker Glu-5. Settlement was observed earlier in populations of M. edulis than in populations of M. galloprovincialis. Settlement occurred in hybrid populations at times intermediate to and overlapping with both of the parental populations. Temporal genetic variation within years was rare at most sites, while there was some variation between the two years. Spatial genetic variation, however, was common among spat settling within the hybrid populations and matched that observed in small, sub-adults at the same sites. No consistent directional changes in allele frequency were observed over the course of several weeks after settlement. These data suggest that the observed spatial variation in the adult populations is the result of spatial variation in settling larval cohorts and not of either temporal genetic variation or of selection soon after settlement.Communicated by J.P. Grassle, New Brunswick  相似文献   

12.
选用高分子量麦谷蛋白亚基(HMW-GS)在Glu-D1位点已知的 26个普通小麦品种,包括 15个 (TriticumaestivumL. )川育系列品种及其 11个亲本,根据其亚基Dx5和Dy10基因的特异序列设计引物.通过对Dx5基因的PCR扩增及对Dy10基因的AS-PCR扩增,结果表明,所设计的引物对Dx5和Dy10都能扩增出特异条带,而携有Dx5基因的材料同时也携有Dy10基因.说明用所设计的引物可快速有效的鉴定出普通小麦中是否带有优质的 5 10亚基.图 4参 15  相似文献   

13.
Individuals of Mytilus edulis were collected from two populations in easterm Long Island Sound, USA, at 6 wk intervals from January 1982 to February 1983, and the hypothesis that nutrient availability controls the timing of gametogenic events in this mussel was tested using genetic variants at the Lap locus. In eastern Long Island Sound, Lap genotypes differ in the net rate of nitrogen accumulation. A highly significant difference in reproductive cycle among genotypes occurred in a population of M. edulis where Lap genotypes differ in nitrogen budget. Genotypes with lower rates of nutrient accumulation delayed the reproductive cycle by approximately six weeks relative to genotypes with high rates of nutrient accumulation. No significant difference in reproductive cycle occurred at a locality where Lap genotypes do not differ in nitrogen budget. These data indicate that nutritional status can exert significant control over the timing of gametogenesis in M. edulis. Differences in reproductive cycle among Lap genotypes provide a mechanism of non-random mating among genotypes which may result in heterozygote deficiencies at this locus.  相似文献   

14.
环境DNA(eDNA)宏条形码(Metabarcoding)技术越来越多地被应用于环境中物种定性识别,但如何定量监测物种在环境中的丰度尚未得到解决。本研究以太湖流域常见的5种浮游动物拟同形溞、大型溞、蚤状溞、多刺裸腹溞、老年低额溞为研究对象,建立了一种基于eDNA宏条形码技术的物种定量方法,并与实时荧光定量PCR(qPCR)相比较,研究了eDNA宏条形码技术多物种定量的准确性。结果表明,PCR引物对eDNA宏条形码的物种检测和定量影响显著。313 bp COI313引物对浮游动物物种覆盖度高,但是物种间DNA扩增的偏好性大,不适用于eDNA宏条形码定量检测。基于COI序列重新设计的短COI116引物能够同时检测出所有5个物种。荧光定量PCR(qPCR)物种拷贝数与物种相对占比呈正相关。eDNA宏条形码所检出每个物种的序列数与qPCR定量拷贝数高度一致。综上,eDNA宏条形码技术可实现对浮游动物物种的半定量检测,在生物多样性监测和生物完整性评价有显著的应用价值。  相似文献   

15.
This study examined the effects of two selection treatments (elevated water temperature and air exposure) on the genetic and physiological characteristics of the juvenile marine mussel, Mytilus edulis (<10 mm). Genetic effects were measured on five allozymes and fitness assessed using physiological tests to estimate energy balance (scope for growth) as well as size, growth and survival. The in vitro treatments resulted in 48% mortality from an air exposure of 11 h at 27°C and 76% mortality from a 6-h exposure to 33°C water. Survivors (n = 1,152) of each treatment along with controls (n = 2,304) were measured and randomly placed in compartmentalized cages. Mussels were deployed to three bays in Prince Edward Island, Canada and monitored over a 10-month period. Initially, both of the treatments had an effect on mussel size and increased the heterozygosity of the surviving mussels. Physiological analyses after 3 months in the field showed that the two treated mussels showed lower metabolic rate that the control group. After 10 months in the field, the treated mussels were larger and had lower mortality than the untreated control mussels. Unexplained environmental interaction in each of the bays had an effect on allelic frequencies and heterozygosity. Overall, the results demonstrate that simple husbandry techniques can be used to increase the productivity of mussel seed and heterozygosity measures can be used to assess fitness. However, more field data is needed to determine the consistency of the increased productivity and if the increased productivity justifies the costs of a selective treatment. Furthermore, because the level of heterozygosity in juvenile mussel populations can vary considerably, both spatially and temporally, it may be effective as a warning of future natural mass mortality when overall heterozygosity levels are found to be low.  相似文献   

16.
The Atlantic surfclam, Spisula solidissima (Dillwyn), is broadly distributed in sandy sediments of the western North Atlantic between the Gulf of St. Lawrence and the Gulf of Mexico. In the United States, a substantial commercial fishery between Long Island and Cape Hatteras harvests offshore populations of one subspecies, S. s. solidissima. A smaller coastal form, S. s. similis Say (also known as S. s. raveneli Conrad), has a partially sympatric geographic distribution, but differs in several life-history characteristics. DNA sequence variation in mitochondrial cytochrome oxidase I (COI) and in introns at two nuclear calmodulin loci was examined to measure genetic divergence between the two subspecies and to test for population structure among populations of S. s. solidissima. Surfclams were collected from seven localities between 1994 and 2001. Based on both mitochondrial and nuclear DNA variation, the two subspecies of S. solidissima are reciprocally monophyletic, with a net COI divergence of 13.9%, indicating long-term reproductive isolation. The only significant differentiation among populations of S. s. solidissima (based on an AMOVA analysis of COI sequences) was between the Gulf of St. Lawrence and more southerly populations. A long internal branch in the S. s. solidissima genealogy coupled with low haplotype diversity in the northern-most population suggests that populations north and south of Nova Scotia have been isolated from each other in the past, with gene exchange more recently. Populations of S. s. similis from Atlantic and Gulf of Mexico coasts had a net COI divergence of 9.2%. Thus, diversification of Spisula spp. clams in the western North Atlantic involved an early adaptive divergence between coastal and offshore forms, with later barriers to dispersal emerging in the offshore form from north to south and in the coastal form between Atlantic and Gulf of Mexico populations.  相似文献   

17.
 Sedimentary DNA, RNA and ATP concentrations were measured at six stations on a tidal flat in the German Wadden Sea from April to December 1995 to determine factors controlling microbial abundance and activity. Two stations (1 and 2) were located in a mussel bed (Mytilus edulis) in the middle of the flat, and the other four were arranged in a line following ebb tide direction. DNA and ATP concentrations were converted into carbon equivalents using literature coefficients and considered to represent total microbial biomass and viable microbial biomass, respectively. The difference between CDNA and CATP was considered as detrital biomass. CDNA and detrital CDNA biomass both increased from spring to summer and decreased in autumn to reach lowest values in December. The most prominent seasonal increase was observed at the mussel bed stations and two adjacent stations. CATP biomass peaked in early spring, especially at the mussel bed stations, and showed a secondary peak in summer. Regression analysis yielded a highly significant relation between the silt content of the sediment and CDNA and CATP. However, CDNA and CATP biomass are not correlated if silt is taken into account. The relation between silt and microbial biomass in combination with the decrease of the silt content with distance from the mussel bed indicate the importance of mussel beds for the microbial activity on the tidal flat, most likely through deposition of feces and pseudofeces. The low silt content and microbial biomass in winter are attributed to resuspension events. The zero intercepts of the regressions between DNA and ATP or RNA suggest that extracellular DNA is absent in our samples. Hence, detrital CDNA biomass in our samples probably consists of intact but dormant cells. Compared to other marine habitats the percentage of detrital CDNA biomass on the tidal flat is quite high, i.e. up to >95% of the total CDNA. However some doubt is expressed about the validity of factors used to convert ATP and DNA to carbon. The proportion of active biomass, expressed by the ratio CATP/CDNA being the complement of the detrital CDNA percentage shows a steady decrease with time and with diminishing distance towards the mussel beds. The role of a mussel bed as a biosedimentary system influencing spatial and temporal trends in specific activity is discussed. Received: 26 January 2000 / Accepted: 3 July 2000  相似文献   

18.
We examined the inheritance of theMytilus edulis CaM-1 Intron 3 locus, a non-coding DNA locus with two potentially neutral length-variants. The polymerase chain reaction (PCR) was used to determine theCaM-1 genotype for 799 larvae obtained from 11 laboratory crosses. Larvae were typed singly only 8 to 24 h after fertilization. We find evidence that each allele can be inherited from either sex, that there are no barriers to fertilization between gametes of different genotypes, and that most larvae have genotypes compatible with Mendelian inheritance of the locus. Deviations from expected genotype frequencies were found in some crosses; we suggest that a contributing factor is aneuploidy in early larvae, but a major cause is more likely to be selection at a locus linked toCaM-1, occurring under the artificial laboratory culture conditions. This study demonstrates the feasibility of applying molecular genetic techniques to early larval stages of marine bivalves, and presents a new non-destructive biopsy method for DNA analysis from living adult mussels.  相似文献   

19.
The degree of population subdivision among collections of young-of-the-year (YOY) Spanish mackerel Scomberomorus maculatus sampled from along the Atlantic coast of the United States (Atlantic) and Gulf of Mexico (Gulf) was examined. Young-of-the-year were collected from spawning areas to minimize possible mixing among putative spawning stocks that may occur at later life history stages along migratory pathways or at over-wintering grounds. Restriction fragment length polymorphism (RFLP) analysis of the mitochondrial DNA (mtDNA) ND4 region and a nuclear actin intron was conducted. Collections in each of two years were analyzed to minimize bias associated with sampling at a single point in space or time. Substantial variation was detected at ND4 (haplotype diversity=0.81) and at the actin intron locus (gene diversity=0.21). Significant differences in gene diversity or allele frequencies were not detected among temporal samples at any of three locations. A homogeneous distribution of genetic variance among samples from widely spaced geographic regions was consistent with the hypothesis that Spanish mackerel comprise a single intermingling genetic stock. Power analysis showed that the mitochondrial analysis from this study was much more likely to detect population subdivision than analysis of the nuclear actin locus, despite lower mtDNA sample size. Received: 3 January 2000 / Accepted: 28 August 2000  相似文献   

20.
The genetic structure of populations of the Mytilus edulis (L.)-M. galloprovincialis (Lmk.) complex has been characterized by studying polymorphism by means of isoenzyme electrophoresis. Three known loci (MPI, EST-D, GPI) have been used to characterize samples taken from two hybridization sites on the French Atlantic coast. The purpose of this study was to investigate the genotypical composition of a mussel brood during two consecutive years (1990 and 1991) in relation to the genetic structure of parental populations analyzed in 1989. The 1989 parental samples showed a strong hybridization at the two sites, and there was a correspondingly high degree of evolution in the 1990 and 1991 broods towards M. edulis. This indicates that genetic structure is not stable in the syntopic zones over time, and that there can be a marked evolution of genetic components, as demonstrated by the results from Groix Island between 1990 and 1991, where brood structure did not conform to that of the parental population. Our study takes into account environmental characteristics, gene flow, and the selection of dependent genotypes responsible for the evolution of the observed biodiversity at hybridization sites where bivalve culture is a contributory factor.  相似文献   

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