A large number of species and taxa have been studied for genetic polymorphism. Microsatellites have been known as hypervariable neutral molecular markers with the highest resolution power in comparison with any other markers. However, the discovery of a new type of molecular marker—single nucleotide polymorphism (SNP) has put the existing applications of microsatellites to the test. To ensure good resolution power in studies of populations and individuals, a number of microsatellite loci from 14 to 20 was often used, which corresponds to about 200 independent alleles. Recently, these numbers have tended to be increased by the application of genomic sequencing of expressed sequence tags (ESTs) and the choice of the most informative loci for genotyping depends on the aims of research. Examples of successful applications of microsatellite molecular markers in aquaculture, fisheries, and conservation genetics in comparison to SNPs are summarized in this review. Microsatellites can be considered superior markers in such topics as kinship and parentage analysis in cultured and natural populations, the assessment of gynogenesis, androgenesis and ploidization. Microsatellites can be coupled with SNPs for mapping QTL. Microsatellites will continue to be used in research of genetic diversity in cultured stocks, and also in natural populations as an economically advantageous genotyping technique. The global production of aquatic animals for consumption was almost 178 million tonnes in 2020, including capture fisheries of 90.3 million, and aquaculture production of 87.5 million tonnes ,5,6,7. TProteins and allozymes have been continuously used in analyses of natural and hatchery populations since the 1960s and have provided a vast amount of information about their taxonomy, hybridization, genetic polymorphism and spatial differentiation in relation to aquatic environments . Allozymes were analyzed under assumptions of models based on neutrality, whereas more recently, they have been considered molecular markers operating under natural selection ,75,76. DMitochondrial DNA (mtDNA) as a molecular marker differs from nuclear DNA due to such properties as maternal inheritance in most animal species and a higher mutation rate due to relaxed selection . A few eThe marker MtDNA cytochrome c oxidase 1 (COI) has been commonly used for aquatic species identification—barcoding ,150,151.Nuclear DNA markers have increasingly been used since the 1990s. They were originally based on the detection of single changes in the genome: point mutations or rearrangements in a very limited number of genome sites recognized by cuts with restriction enzymes. DNA marker technologies and their applications in aquaculture genetics have been reviewed several times ,157,158.Microsatellites are highly polymorphic, neutral and co-dominant DNA markers based on a variable numbers of short, usually 2–4 bp, nucleotide repeats . MicrosaMicrosatellites have been most useful in terms of genetic characterisations and in the structure analysis of natural populations of many species. Examples of aquatic species for which population genetic structure has been successfully identified are presented in Loligo reynaudii and Doryteuthis (Amerigo) pealeii [Clupea pallasi) populations from the west coast of North America [Galeocerdo cuvier, with microsatellites revealed differentiation between populations from the Atlantic and Indo-Pacific including contemporary and archival samples [The list includes freshwater, diadromous and marine species. For the study of the genetic structure of the European hake, Pita et al.  distingu pealeii . Three d America . MtDNA r samples . O. keta, with 15 microsatellite loci exhibiting 349 independent alleles compared with 61 SNPs exhibiting 66 independent alleles, revealed that the SNP baseline performed considerably better than the microsatellite baseline [O. tshawytscha, in 60 populations from British Columbia obtained with 16 highly polymorphic microsatellite loci was projected to require 179 SNPs [S. salar, have been compared [A simulation study of the accuracy of assigning individuals to closely related populations of chum salmon, baseline . An equi179 SNPs . A popul179 SNPs . Microsa179 SNPs . The rescompared . Both SNcompared . Invasions of single or groups of aquatic species have been caused by natural events or human-mediated activities, such as shipping, aquaculture, recreational activities or attempts to enrich local ecosystems, including increasing fishing catches. Despite the understanding of the negative consequences of uncontrolled introductions of species into new areas, their numbers continue to increase, and the economic impact is increasing. There are examples of banning the entry of large ships with hulls contaminated by sessile exotic marine organisms to certain countries, e.g., New Zealand. The use of molecular markers makes it possible to identify invasive species, reconstruct their migration routes and source populations, reveal their hybridization with local congeners, estimate the economic and ecological impacts, predict their further spread and facilitate preventive actions. Micropterus dolomieu, introduced to the range of Guadalupe bass, M. treculii in central Texas and its introgressive hybridization in a few rivers [M. piceus, imported to U.S. aquaculture farms in the 1980 and present in the Mississippi River basin since the early 1990s [Hypophthalmichthys molitrix, and silver carp, H. nobilis, in Hungary, imported from China [Pacifastacus leniusculus, introduced to Europe from North American lakes for hatchery purposes [Population genetics of invasive species have been examined by studies of microsatellites in many species, including the evolution of brown trout populations originating from Poland and introduced to virgin rivers systems of the subantarctic Kerguelen Islands , smallmow rivers , Asian bly 1990s , bigheadom China , and sigpurposes . O. mykiss, native to the Pacific coast of North America and brown trout, S. trutta, from Europe, both introduced from Germany to South America over 100 years ago [O. nerka, into a new environment, Frazer Lake, Kodiak Island, Alaska [Platycephalus fuscus, and sand whiting, Sillago ciliata, objects of recreational and commercial fishers on the east coast of Australia [Artemia franciscana, in the Mediterranean Sea region [Ruditapes philippinarum, in the Mediterranean introduced for aquaculture purposes from the Indo-Pacific in 1983 [Perna perna, in the Gulf of Mexico [C. gigas, introduced for aquaculture and present as a wild populations in Ireland, France, The Netherlands [Microsatellites have extended the knowledge of invasive populations of estuarine and marine species, such as rainbow trout, ears ago . They ha, Alaska , estuariustralia , and thea region , Manila  in 1983 ,232, invf Mexico , and Ameherlands ,235 and Despite the great understanding of the need to protect entire aquatic ecosystems, actions to protect and restore individual species often remain difficult. One way to support these activities is to plan and manage endangered populations, taking into account their genetic characteristics. Based on the results of population genetics research, it is possible to define evolutionary conservation units . SignifiO. clarki seleniris, threatened by hybridization with introduced rainbow trout, O. mykiss [O. mykiss, tributary populations from the Bulkley-Morice River, British Columbia [O. mykiss, below and above natural barriers and man-made dams in rivers in California [O. clarki lewisi) on the northern periphery of its range [O. tshawytscha, in the North Fork Stillaguamish River, WA, USA [Salvelinus leucomaenis threatened by hybridization with non-native brook trout, S. fontinalis, in Japan [Thymallus thymallus, in Europe [Microsatellites have become a tool in the increasing the development and application of conservation genetics, and a few examples of its uses can be listed: analysis of populations of endemic California Paiute cutthroat trout, . mykiss , maintenColumbia , analysilifornia , descripts range , and the WA, USA . Signifi WA, USA . Microsain Japan  and analn Europe . Acipenser transmontanus) management units, two of which were listed as endangered populations under Canada’s Species at Risk Act, which were verified with microsatellite markers [Margaritifera margaritifera L.) in Europe, elaboration of the recommendation concerning conservation units [Crassostrea rhizophorae, in Brazil [The Fraser River system consisted of five white sturgeon  inhabited by theoretically separate stocks. The stocks correspond to spatial management units. The catch limits in Europe  are set annually for each management area. There is a growing understanding among fishery managers that the management units should be delineated using not only environmental but also biological criteria, including the genetic structure of stocks and populations. Mismatches between management units and population genetic structure have often been reported . Microsa America , Korean chlegeli , common ecimalis , geoduckColumbia , analysi Denmark ,256, par. trutta , and importh Sea . Studiesorth Sea . A. japonicus, in South Africa [O. nerka [O. tshawytscha, from the Yukon River, Alaska [Coregonus clupeaformis [Alosa sapidissima, in two Atlantic coast fisheries, Delaware Bay, USA, and Inner Bay of Fundy, Canada [S. trutta from the Gulf of Finland [Overfishing effects on dusky kob, h Africa , stock cO. nerka , mixed-s, Alaska , mixed seaformis , mixed-s, Canada , mixed-s Finland  have all Finland . Only ra Finland . Dissostichus mawsoni [Anguilla japonica, in the north of Taiwan from 1986 to 2007 [Esox lucius L., stocked population in Stege Nor, Denmark [C. harengus, in the Baltic Sea and Skagerrak waters over a 24-year period [Lates calcarifer, over 25 years [G. morhua, in the Western Bank over a few years [Sparus aurata, in wild samples from the Aegean and Ionian Seas [Stability in the genetic structure of populations throughout the age cohorts (juveniles and adults) has been related to a sufficiently large effective population size, which prevents genetic drift over generations, e.g., that detected with microsatellites and mtDNA in wild populations of the Antarctic toothfish,  mawsoni . Based o to 2007 , the tem Denmark , the star period , in the r period , and in 25 years , the genew years , and gilian Seas . Historiian Seas . Salmo trutta, before and after population decline and for its stocking with non-local strains of hatchery trout in rivers in Denmark over 60 years [Salvelinus namaycush, in the upper Laurentian Great Lakes of North America after their substantial decline in abundance and distribution during the mid-twentieth century and following their recovery with the partial contribution of enhancement from hatcheries [Esox lucius, population and the 8% loss of its heterozygosity over a 32-year period [On the other hand, temporal changes in the genetic structure of wild populations have been reported for brown trout, 60 years ,278,279,tcheries . Historir period , the temr period  and in Dr period , the intr period .Maccullochella ikei, has been found in the Clarence River system, eastern Australia, using microsatellite DNA markers [M. ikei population. This observation demonstrates the adverse effects of stocking programs and the necessity of support from genetic analysis in the design of the management of breeding and stocking strategies, particularly for threatened species [Hatchery populations may undergo genome-wide selective sweeps that can affect their fitness and linked neutral loci, such that individuals destined to be released to the wild, should be modified to minimize genetic adaptation to captivity . Tempora markers . Compari species . Microsa species . Salvelinus, populations have been found in two lakes in the Bavarian Alpine region [S. namaycush) from 72 unstocked and stocked lakes in Canada, in which an increase in genetic diversity and a twofold decrease in the extent of genetic differentiation among stocked populations compared to that among unstocked populations has been found [S. fontinalis, in Québec, Canada, related to long term stocking have been reported [Effects of stocking on the genetic integrity of Arctic charr, e region . A loss en found . Possiblreported . Salmo trutta in the Borne River in the Northern French Alps [S. t. m. trutta in Polish rivers, southern Baltic [Oncorhynchus masou ishikawae in Japan [O. keta [Morone saxatilis populations in the south eastern USA [Stocked populations were characterized by significant admixture at both population and individual levels, in populations of brown trout, nch Alps , stockednch Alps  and popunch Alps . Admixtunch Alps , in restnch Alps , and as nch Alps , sea tron Baltic ,296, amain Japan , Japanes[O. keta  and in stern USA . S. salar, on the genetic composition of populations within the small Ellidaar river system in SW Iceland [Enhancement of wild populations has generated changes such as the impact of straying of sea ranched hatchery-reared Atlantic salmon,  Iceland , differe Iceland ,302, dif Iceland ,304,305, Iceland . Alleles Iceland . Acanthopagrus schlegelii, in the South China Sea revealed the significant genetic differentiation of enhanced populations from native populations and the lower genetic diversity of the recaptured released groups of individuals [Pagrus major, in Sagami Bay and Tokyo Bay in the Kanagawa Prefecture [A. schlegelii, in Hiroshima Bay, Japan [Maccullochella peelii peelii) stocking in five river catchments in southern Australia [Paralichthys olivaceus [Piaractus mesopotamicus, because of declines in the number of wild populations in the Tiete and Grande rivers, Brazil, to be accompanied by the genetic monitoring of populations and broodstock to ensure the viability of such programs [Microsatellite DNA polymorphism evaluation of hatchery-based stock enhancement of black sea bream, ividuals . It has efecture , Kagoshiefecture , Shikokuefecture , and blay, Japan , and theustralia  have beelivaceus . Restockprograms . Acipenser oxyrinchus in North America (as opposed to the European A. sturio) as a native extinct species in the Baltic Sea and to the genetic control of its imported fry from Canada before the release into the wild in attempts to achieve the restoration of its population in drainages [A. naccarii) endemic to the North Adriatic region, in the Ticino River Park, Italy [Epinephelus marginatus, in the Mediterranean [Diplodus sargus, in a fishery reserve in Sicily, Italy [Epinephelus bruneus [Seriola lalandi, in Chile [Solea senegalensis [Microsatellite analysis has contributed to the identification of the sturgeon rainages ,317, resk, Italy , and theerranean , and of y, Italy . Microsa bruneus , yellowtin Chile  and a flgalensis . Sciaenops ocellatus, in some Texas bays and estuaries has been studied [Procypris rabaudi— an endemic fish in the upper Yangtze River, China, supplemented with hatchery-produced fish [H. discus [Haliotis kamtschatkana [Ruditapes decussatus, on Atlantic coasts of northern Spain using seeds produced in hatcheries [The recovery rate of hatchery-released red drum,  studied , as wellced fish . Assessm. discus , and usichatkana , and asstcheries  have beeOncorhynchus mykiss) crosses were released into two natural lakes. Parentage analysis was performed using microsatellite loci. The results indicated that domesticated fish can survive as well as wild fish. During the first summer, the fastest-growing crosses had the highest survival, but this trend was reversed after one winter and another summer. The experiment confirms the multigenerational risk of domesticated fish escaping or being released in the case of interbreeding with wild fish. Nile tilapia, Oreochromis niloticus, has been introduced throughout Africa, outside its native range for aquaculture purposes, and escapees hybridize with native populations of Oreochromis species, which result in negative effects on the conservation of fish biodiversity, aquaculture and capture fisheries in fresh water bodies [C. carpio var. color, in China [Orestias agassizii and O. luteus) mainly in the Lake Titicaca with implications for local fisheries, stocking and conservation [Pseudoplatystoma corruscans and P. reticulatum in the Upper Paraná River, in South America [To assess the impact of escapees from cultures and hatcheries, the fitness consequences of the introgression of fast-growing domesticated fish into a wild population were tested . Fry fror bodies ,330. Micin China , analysiervation , and ide America . However America . S. aurata, escapes was estimated to be from 14.1% to 30.2% using microsatellites [G. morhua, were identified in wild populations in Norway [S. salar, populations in Norway [S. salar, populations [Dicentrarchus labrax, on natural populations in waters around Cyprus [L. calcarifer, from a sea-cage facility in northern Australia [In the marine environments, net-cage aquaculture poses a risk of the escape of a large number of fish in the case of mechanical damage caused by natural factors  or related to human activities . In Japan, in the mariculture areas, the frequency of gilthead sea bream, tellites . Hybriditellites ,337,338.n Norway ,340. Farn Norway . Microsan Norway ,343 and n Norway , for theulations , in studulations , and ford Cyprus , and incustralia . Comparisons of feral populations and hatchery stocks using microsatellites have been published for many species . The lower genetic diversity of cultured populations in comparison with wild populations has been reported for many species, including fish, prawns, sea urchin, oysters, green mussels and abalones ,380,381.O. mykiss) are derived from imports from New Zealand, the latter being largely derived from Californian imports in 1883 and also having lower diversity in comparison with wild populations in the north Pacific [Phalacronotus bleekeri [L. crocea [Platichthys stellatus, in Korea [Micropterus salmoides floridanus [I. punctatus, in farms in Mexico [Acipenser baeri [A. gueldenstaedti and A. ruthens in a farm in Poland [Polyodon spathula, in Poland [L. calcarifer, hatcheries in Australia [Cherax quadricarinatus, introduced from Australia to culture in China [C. carpio, in the Czech Republic [D. labrax [Oreochromis, species cultured in Mexico [H. discus hannai [Panopea generosa) in Washington state, USA [A small loss of genetic variation in comparison to wild populations was found for Atlantic salmon in a hatchery in Canada , and for Pacific . Similar Pacific ,387. A lbleekeri , and a l. crocea , and stain Korea , Floridaoridanus , and chan Mexico , Siberiaer baeri , A. gueln Poland , American Poland , barramuustralia ,397, redin China , and comRepublic . A loss . labrax , three tn Mexico , in hatcs hannai , and in ate, USA . P. mesopotamicus, used in the stocking program of the Paranapanema River, Brazil, did not confirm reduction in genetic diversity [Pangasianodon gigas, in Thailand. Studies of the banana shrimp, Fenneropenaeus merguiensis, in Australia demonstrated the loss of alleles in the mass selection program carried out, even with over 1000 broodstocks being compared with similarly selected but outbred stocks [L. vannamei, in Pernambuco, Brazil, is sustained by the exchange of breeders between marine shrimp hatcheries [Cromileptes altivelis, captive breeding has been performed, and the recommendation has been made to increase the effective population size with wild fingerlings in order to avoid diversity reduction detected by microsatellites [Oncorhynchus [The genetic analysis of pacu broodstocks, iversity . Sriphaiiversity  concluded stocks . It is rtcheries . In an atellites . Sex-linrhynchus . S. salar) in Norway were estimated [C. gigas) populations with a set of 18 microsatellites -derived SSR) and 10 EST-derived SNP markers suggest that genomic SSRs and EST-SSRs are more suitable for population genetic analysis than are SNPs [Population parameters and the power of 16 microsatellites and 26 SNPs to assign single individuals to their sampling population in wild and farmed stocks of Atlantic salmon . A loss of genetic diversity decreases the fitness and adaptive potential of the progeny. If the process lasts for some generations, it results in increased relatedness (kinship) among individuals and the effect of inbreeding. Analysis of relatedness has been performed for different strains of rainbow trout, d stocks . Effectsof Fundy , and on of Fundy . Reprodulifornia . P. olivaceus, to be stocked in natural sea areas [S. maximus L. in hatcheries in Spain [S. salar, in Ireland [G. morhua, in Canada [L. calcarifer) broodstock in Thailand [S. ocellatus, in a marine fish stock-enhancement hatchery in the USA [E. encrasicolus, under a pilot project for aquaculture and enhancement of native populations in Spain [S. senegalensis, population [Epinephelus lanceolatus, broodstock management in Taiwan [Crassostrea gigas in Dabob Bay, Washington, USA [Megalobrama amblycephala, crosses for a freshwater polyculture system in China [S. dumerili [A. transmontanus [S. ocellatus) fingerlings used in stock enhancement [Argyrosomus regius to support the breeding program in the Mediterranean aquaculture [Microsatellite loci have been applied in pedigree tracing of a hatchery strain of Japanese flounder, ea areas , parentain Spain ,420, parin Spain , and Atl Ireland , parenta Ireland , parentan Canada , identifThailand , parenta the USA , and of in Spain , analysipulation , pedigren Taiwan , identifton, USA , parentaton, USA , parentain China , and gredumerili , estimatmontanus , and expancement . A microaculture .Litopenaeus vannamei, breeding program [Portunus trituberculatus [H. discus hannai abalone mixed family farming [H. midae abalone [H. asinina abalone larvae for parentage assignment in aquaculture in Australia in order to maintain the level of genetic diversity [Meretrix meretrix, by crosses and parentage assignment [C. gigas, in a hatchery in France [Patinopecten yessoensis [Microsatellites have been employed in shellfish aquaculture for kinship analysis and genetic variation monitoring in a whiteleg shrimp,  program , identifrculatus , parenta farming  and in a abalone , genotypiversity , geneticsignment ,443, parn France ,445, andssoensis . Dicentrarchus labrax, with microsatellies, in aquaculture in the larval stage, when families of progeny had been mixed to start the production cycle, has been conducted to help attain balanced parental contribution [Cynoscion nebulosus, dams and sires in two restoration enhancement facilities in Texas were assessed throughout a spawning year by using parentage analysis based on 12 variable microsatellite loci. That and other studies indicate that reductions in Ne of hatchery- or farm-raised progeny stem primarily from non-contributing dams, suggesting that periodic identification and removal of low-contributing dams from broodfish stocks constitute a critical step toward maximizing the Ne levels of hatchery offspring used in restoration enhancement [Genetic analysis of broodstock and progeny of the European sea bass, ribution . The resancement . The disancement . Based oancement  reportedancement . In the ancement ,452.O. tshawytscha, from the Wenatchee River, Washington [Thymallus arcticus, from the Lubbock River, Yukon, were sampled for adults and young-of-the-year independently in order to enable the identification of parent–offspring pairs [Statistical analysis of parentage assignment has been conducted for artificially propagated hatchery fish (removed adipose fin) with parents originating from wild salmon (preset adipose fin) in a population of Chinook salmon, shington . Simulatng pairs . The genO. nerka) population in Alaska demonstrated that the assignment success of 80 SNPs  was higher than that of 11 microsatellites  but the identification of full-sib groups without parental information from relatedness measures was possible using both marker systems [Raja clavate, and the Pacific white shrimp, L. vannamei, with high potential for further applications [A comparison of SNP and microsatellite applicability in parentage and kinship assignment of a wild sockeye salmon  in cultured species can be useful when disease control and prevention is a goal [S. solea) in an intensive recirculation aquaculture system (RAS) and in a semi-natural outdoor pond (POND) has shown low genetic correlations for growth between environments, which implies that the best genotypes in an intensive aquaculture environment are not necessarily the best genotypes in more natural environments such as ponds [Selection programs have been executed with the aim to improve the quality of cultured fish and shellfish for releases to the wild or for consumption. Microsatellites were employed in selection in the larval stage for the faster growth of adults of Asian sea bass (barramundi), lcarifer , selecti. labrax , geneticresponse , commercresponse , confirmresponse , and a sviatilis . Selectiormities . Immune-s a goal . Assessmas ponds . D. labrax) affecting fish weight and size at age [S. ocellatus, in southern USA [S. alpinus, eggs were obtained for research from two North American sources, an eastern  and western  stock and fishes were mixed and grown for 2 years in tanks [Spares aurata, has been shown to be significant [Microsatellites were used for parentage identification in studies of heritability and genotype by diet interactions of European sea bass  exposed to the cold or chemical shock followed by pressure treatment ,487. Inhriepinus , in the cephalus , large ya crocea  , e.g., in the loach, caudatus , amago sshikawae , and in . crocea . Microsay 12 MYA , functioturgeons , naturalycephala ,509, pard, Japan , analysid larvae  and testneration . G. morhua [S. senegalensis, for aquaculture [D. labrax [C. gigas, for improved selection for fast growth in aquaculture [Siniperca scherzeri, and X  mandarin fish, S. chuatsi (sic), F1 and F2 hybrids [Haliotis rufescens with H. discus hannai produced in Chile [H. discus hannai Ino with Haliotis gigantea in China [P. trituberculatus, distributed in the coastal waters of Asia-Pacific with the aim to increase its performance in hatchery [S. maximus [Microsatellites have been found to have applications in research concerning the induction of triploids in cod, . morhua , inductiaculture , pressur. labrax , parentaaculture , productaculture , charact hybrids   using two backcrosses between genetically divergent strains [C. carpio [D. labrax [S. aurata [H. discus hannai [Tolerance to environmental stress factors such as temperature, salinity, low oxygen (hypoxia), changes in pH, food availability and biological factors, resistance to viral and bacterial pathogens and parasites determine survival under natural conditions and productivity under aquaculture aconditions. Genomic regions associated with the trait of interest can be mapped in order to identify quantitative trait loci (QTL). Microsatellites have been commonly used successfully for finding the linkage of molecular markers with QTL. Subsequently, such markers can be used in marker-assisted selection (MAS) in aquaculture. Analysis of genomic sequences and genetic differentiation of associated tandem repeat markers (microsatellites) in the growth hormone somatolactin and insulin-like growth factor-1 genes of the sea bass, ntiation . Geneticntiation . MicrosaS. salar ,527, for strains , a commo. carpio ,530, Eur. labrax , gilthea. aurata , and Pacs hannai . S. ocellatus [Verasper moseri, spotted halibut, Verasper variegatus [S. maximus, and one possible QTL associated with body length was detected [S. rhombus, for a preliminary study on growth-related QTL for body weight, length and Fulton’s condition factor [O. kisutch [O. mykiss [A genetic map was developed for a population of European sea bass with the use of ove 90 microsatellites and enabled the finding of two QTL for body weight, six QTL for morphometric traits and three suggestive QTL for stress response . Geneticcellatus ,536, barriegatus , Atlantiriegatus , and fordetected ; howeverdetected . Microsan factor  and for n factor . Locatedn factor , QTL for kisutch , QTL inf kisutch  and uppe. mykiss . Thirtee. mykiss . for a h. mykiss . Faster S. aurata [C. carpio) based on LDH Activity [Eriocheir sinensis, and used for QTL detection, and nine growth-related QTL for body length, width and weight were mapped to seven linkage groups [Nodipecten subnodosus, and putative QTL were identified for morphometric traits [Hyriopsis cumingii in China [H. rubra from Australia [Argopecten irradians [Penaeus (Fenneropaeneus) chinensis [P. olivaceus, and F-1 progeny of crosses between disease-resistant and disease-susceptible parents were used for the detection of QTL associated with resistance to Streptococcal disease (streptococcosis) caused by Streptococcus iniae [S. salar, using a genome scan [O. mykiss [S. salar [Flavobacterium psychrophilum, the causative agent of BCWD in rainbow trout, O. mykiss [QTL for morphometric traits in gilthead seabream, . aurata ,550,551,Activity  have beee groups . Additioc traits , QTL forin China , and forustralia  were fourradians , was fouhinensis  was consus iniae . These come scan , and in . mykiss   were used to construct a linkage map and to identify associations between molecular markers, QTL determining resistance to infectious hematopoietic necrosis virus (IHNV) and associations were observed for four of the markers [Photobacterium damselae, subsp. Piscicida [R. philippinarum [C. gigas [O. edulis, for resistance to a parasite, Bonamia ostreae [C. gigas) opens new possibilities of selection for resistance to oyster herpesvirus, OsHV-1 [O. edulis, in France and around the world, with worldwide mortality being observed since 2008 [Pinctada martensii pearl oyster, the primary cultured species of marine pearls in southern China [Backcrosses of rainbow trout and steelhead  on Atlantic and Pacific salmonids from wild and farmed fish, by analyses of microsatellite DNA [Microsatellites have been used also in studies of phenotypic changes in transgenetic ar. Jian . Microsa latipes . Populatlite DNA . Microsalite DNA ,579, andlite DNA . NeverthSimple sequence repeats (SSR—microsatellites) have been found to have a variety of applications in genetic research related to natural populations and cultured stocks over the past 30 years. Microsatellites have been known as hypervariable neutral molecular markers with the highest resolution power in comparison with any other markers. The largest number of publications concerned the detection of the genetic structure of pristine populations in natural conditions, identification of invasive species, detection of evolutionary management units for conservation genetics, identification of fishery management units and mixed stock analysis, effects of stocking or escapes of farmed fish and their interactions with natural populations, analysis of hatchery stocks including kinship and parentage analysis, assessment of gynogenesis, selection for characteristics of choice and heritability in the wild and identification of quantitative trait loci (QTL). However, the discovery of a new type of molecular marker—single nucleotide polymorphism (SNP) has put the existing applications of microsatellites to the test. Both types of markers have become more available with the development of genomics. In population related studies, several (14–20) microsatellite loci correspond to about 200 independent alleles, which can ensure good accuracy when distinguishing populations. Recently, these numbers tend to be increased by the application of genomic sequencing of expressed sequence tags (ESTs), and using only the most informative loci for genotyping, depending on the aims of research. However, the number of SNP loci can be increased to thousands, which makes it possible to obtain more detailed and precise information on populations. Because SNPs are neutral or outliers , they are able to uncover more effectively both differentiation among populations and the various processes taking place in them, such as introgression, hybridization or the reconstruction of demographic changes. It is noticeable that the number of recent papers related to population level processes associated with microsatellites has decreased, whereas of those using SNPs has increased. Nevertheless, microsatellites can be considered to be superior markers in such topics as kinship and parentage analysis in cultured and natural populations, assessment of gynogenesis, androgenesis and ploidization. For other purposes, microsatellites coupled with SNPs for mapping QTL will remain feasible. Microsatellites will continue to be used in studies of genetic diversity in cultured stocks, and also in the research of natural populations as an economically advantageous research technique.


Background: Capitulum mitella is a widely distributed and ecologically important stalked barnacle that settles extensively on rocky shores. This species contributes to the structural complexity of intertidal habitats and plays a critical role in the marine ecosystem. This study aimed to reveal the genetic diversity and population structure ofC. mitella by analyzing the mitochondrial cytochrome oxidase I (COI) gene.Methods: A 683bp fragment of the COI gene was sequenced from 390 individuals sampled from six localities in Fujian, China.Results: A total of 84 distinct haplotypes were identified through the analysis of 82 polymorphic sites, resulting in an average haplotype diversity (h) of 0.660 and nucleotide diversity (π) of 0.00182. Analysis of molecular variance (AMOVA) and pairwise FST statistics showed no significant population structure. Neutrality tests and mismatch distributions provided evidence of recent population expansion for the species.Conclusions: We suggest that the species' high dispersal ability, and ocean currents coupled with limited physical barriers in the region, contribute to its current phylogeographic structure. These findings enhance our comprehension of the genetic diversity and population structure ofC. mitella, providing valuable insights for future conservation efforts. Capitulum mitella , the single species within the genusCapitulum Gray , is an ecolgically significant stalked barnacle that aggregates and settles extensively on rocky shores  has emerged as a valuable tool for studying genetic diversity and population structure in various organisms  was employed to extract DNA from tissue samples preserved in ethanol, following the standard protocol for animal tissues. The quality and quantity of the extracted DNA are measured using BioDrop . The amplification of the mitochondrial COI gene was carried out using the Lco1490/Hco2198 primers . We evaluated the mismatch distribution and neutrality statistics to study demographic history, such as Tajima'sD  segment of the COI gene was obtained from 390 individuals sampled from six populations. The average composition of the four nucleotides  was found to be 18.19%, 42.93%, 14.65%, and 24.22%, respectively. It was determined that none of the sequences contained premature stop codons, insertions, or deletions. A nucleotide pair frequency analysis of the entire dataset revealed the presence of 82 variable sites (12.00%) among 683 sites, including 38 parsimony informative sites and 44 singleton sites.C. mitella specimens. Two haplotypes (H5 and H9) were shared by populations from five localities, while 59 haplotypes (accounting for 70.24%) were private. The ND population exhibited the highest number of unique haplotypes (32), followed by PT (27), FZ (20), QZ (19), ZZ (17), and XM (12), according toA total of 84 haplotypes were identified among 390 individuals, with 59 of them being private and 25 being shared . The mosC. mitella populations, molecular variation analysis (AMOVA) and pairwiseFST values were employed. Results from the AMOVA analysis indicated that 99.77% of the genetic variation was found within populations, however, 0.23% have corresponded to among-population variation , indicating a lack of significant genetic variation among these populations. The pairwise populationFST estimates obtained through an exact test were generally low, ranging from 0.00574 to 0.01144 among the six populations  of 0.660 and a nucleotide diversity (π) of 0.00182, with 84 haplotypes identified and a star-like haplotype network   plays a significant role in determining population genetic structure of marine invertebrates. Although the authors stated in the introduction that I advise utilizing MEGA software that is more recent, such as MEGA 11, rather than the old version MEGA 7.FST test, I advise doing additional statistical tests to support the findings of population genetic structure. As a result, I advise using hierarchical AMOVA. To investigate every clustering possibility, SAMOVA would be the best choice. In order to determine whether isolation by distance (IBD) may be influencing the genetic structure of the population in this species, the author may also need to undertake testing for IBD.Although the authors performed a pairwise FST estimates were carried out; instead, the description is shown in the section of results.I recommend performing post hoc analysis, such as sequential Bonferroni correction for pairwise analysis, because it is advised to perform a multiple-comparison correction when several dependent or independent statistical tests are being performed simultaneously. The authors indicated how molecular variation analysis (AMOVA) was performed in the methodology part, but did not describe how pairwise populationCapitulum mitella, the authors can estimate this population size using software like n-MIGRATES.In order to compare the results of the genetic diversity and demographic history with the effective population size ofFST  orΦST for subpopulation comparisons.Additionally, authors must confirm whether they used pairwise population However, the manuscript is suitable for publication following some minor revisionsIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Aquatic resource managementI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 1. Although the authors stated in the introduction that the Capitulum mitella species is a stalked barnacle that aggregates and settles extensively on rocky shores, the authors should clearly at least describe the developmental mode of the study species because it is well-known that developmental type  plays a significant role in determining population genetic structure of marine invertebrates.  Thank you very much for your valuable suggestion. We have supplemented the content accordingly. C. mitella is a dominant organism in intertidal coastal ecosystems with a widespread distribution throughout warmer regions of the Indo-Pacific, from Korea through India to the West Pacific Ocean. It like other barnacles, has a biphasic life history: sessile adults and planktonic larvae. They have six naupliar stages and one cyprid stage, when it fixes itself in place, undergoes metamorphosis, and becomes a sessile juvenile. It is commonly found attached to rocks in the lower part of the intertidal zone, particularly in areas with strong currents. It tends to occur in dense populations, often crowded together in cracks and grooves on otherwise smooth rocky surfaces. Its attachment to rocks provides shelter and refuge for various organisms, influencing their distribution and interactions. Additionally, the population density of C. mitella in cracks and grooves can shape the physical structure of the intertidal zone. C. mitella also plays a vital role in intertidal ecosystems by filtering food particles from the water. This feeding behavior contributes significantly to nutrient cycling and energy flow within the ecosystem. By consuming organic detritus, algae, and small invertebrates, C. mitella helps maintain the overall productivity and balance of the intertidal community. 2. I advise utilizing MEGA software that is more recent, such as MEGA 11, rather than the old version MEGA 7. Thank you very much for your suggestion， we have made the changes as per your request. 3. Although the authors performed a pairwise FST test, I advise doing additional statistical tests to support the findings of population genetic structure. As a result, I advise using hierarchical AMOVA. To investigate every clustering possibility, SAMOVA would be the best choice. In order to determine whether isolation by distance (IBD) may be influencing the genetic structure of the population in this species, the author may also need to undertake testing for IBD. Thank you very much for your feedback. This suggestion is excellent and addresses the question of whether isolation by distance (IBD) is influencing the genetic structure of the population. In our study, we focused only on resources within a single province, where the geographic distances were relatively small, and no genetic differentiation was observed among populations. Therefore, we did not employ this method. However, moving forward, we will adopt your recommended approach to investigate the distribution, dispersal, genetic structure, and variations of this species along the Chinese coast. By doing so, we hope to obtain more comprehensive and in-depth results. 4. I recommend performing post hoc analysis, such as sequential Bonferroni correction for pairwise analysis, because it is advised to perform a multiple-comparison correction when several dependent or independent statistical tests are being performed simultaneously. The authors indicated how molecular variation analysis (AMOVA) was performed in the methodology part, but did not describe how pairwise population FST estimates were carried out; instead, the description is shown in the section of results. Thank you for your suggestion. We have already supplemented that section. 5. In order to compare the results of the genetic diversity and demographic history with the effective population size of Capitulum mitella, the authors can estimate this population size using software like n-MIGRATES. Thank you for your suggestion. We highly appreciate your proposed approach, and similar to the third point, we will adopt this method to study the distribution, dispersal, genetic structure, and variations of this species along the Chinese coast, to achieve more comprehensive and in-depth results through this approach. 6. Additionally, authors must confirm whether they used pairwise population FST  or ΦST  for subpopulation comparisons. Thank you for your suggestion. We have already confirmed and supplemented this section. Thank you once again for your valuable suggestions. “ C. mitella is a dominant organism in intertidal coastal ecosystems with a widespread distribution throughout warmer regions of the Indo-Pacific, from Madagascar to southern Japan.” Please rephase the sentence. Ecosystem services of the species can be mentioned. More context is needed, alone this is subjective.“Mitochondrial DNA (mtDNA) is widely used as an ideal marker for investigating genetic diversity and population structure due to its rapid evolutionary rate, non-recombining nature, and simple amplification procedure .” The authors should explain the importance of using different genetic markers, including COI gene, to better understand the current population structure.Please could you include the populations to be studied in the main text? They are only detailed in the caption of figure. Please include the full name in addition to the abbreviation. Some information describing the study area would be great.In the section "DNA extraction and PCR amplification", how the DNA was extracted is missing. Please indicate the methodology used. Also indicate the commercial name of the reagents used. In the sentence, 'MEGA 7.0 was used to edit and align the sequences,' the word 'aligned' should be changed to 'align'. Please specify what you mean by SSD before the acronym (sum of squared deviation) and include the acronym after raggedness index analysis (Rg). The authors present an interesting study on Genetic diversity and population structure of Capitulum mitella  in Fujian (China) by mtDNA COI sequences. The bright side of the manuscript is that to provide the current genetic structure of the species in Fujian (China)  using mtDNA COI sequences. However, some points are missing (mentioned below) in the manuscript. Therefore, I would like to make some suggestions to improve the quality of the paper as below:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Molecular biological oceanographyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 1.“ C. mitella is a dominant organism in intertidal coastal ecosystems with a widespread distribution throughout warmer regions of the Indo-Pacific, from Madagascar to southern Japan.” Please rephase the sentence. Ecosystem services of the species can be mentioned. More context is needed, alone this is subjective. Thank you. We have already rewritten that section. 2.“Mitochondrial DNA (mtDNA) is widely used as an ideal marker for investigating genetic diversity and population structure due to its rapid evolutionary rate, non-recombining nature, and simple amplification procedure .” The authors should explain the importance of using different genetic markers, including COI gene, to better understand the current population structure. Thank you for your suggestion. We appreciate the importance of incorporating different genetic markers, including the COI gene, to enhance our understanding of the current population structure. We have already rewritten that section and made some additions. 3. Please could you include the populations to be studied in the main text? They are only detailed in the caption of figure. Please include the full name in addition to the abbreviation. Some information describing the study area would be great. Thank you for your suggestion. We have made the necessary revisions. 4. In the section "DNA extraction and PCR amplification", how the DNA was extracted is missing. Please indicate the methodology used. Also indicate the commercial name of the reagents used.  Thank you. We have already added that section as per your request. 5. In the sentence, 'MEGA 7.0 was used to edit and align the sequences,' the word 'aligned' should be changed to 'align'.  Thank you. The word 'aligned' has been changed to 'align'.  6. Please specify what you mean by SSD before the acronym (sum of squared deviation) and include the acronym after raggedness index analysis (Rg).  Thank you. We have made the necessary revisions as this suggestion.


Thryonomys swinderianus) is a large-body old world rodent found in sub-Saharan Africa. The body size and the unique taste of the meat of this major crop pest have made it a target of intense hunting and a potential consideration as a micro-livestock. However, there is insufficient knowledge on the genetic diversity of its populations across African Guinean forests. Herein, we investigated the genetic diversity, population structures and evolutionary history of seven Nigerian wild grasscutter populations together with individuals from Cameroon, Republic of Benin, and Ghana, using five mitochondrial fragments, including D-loop and cytochrome b (CYTB). D-loop haplotype diversity ranged from 0.571 (± 0.149) in Republic of Benin to 0.921 (± 0.013) in Ghana. Within Nigeria, the haplotype diversity ranged from 0.659 (± 0.059) in Cross River to 0.837 (± 0.075) in Ondo subpopulation. The fixation index (FST), haplotype frequency distribution and analysis of molecular variance revealed varying levels of population structures across populations. No significant signature of population contraction was detected in the grasscutter populations. Evolutionary analyses of CYTB suggests that South African population might have diverged from other populations about 6.1  MYA. Taken together, this study reveals the population status and evolutionary history of grasscutter populations in the region.Grasscutter ( Thryonomys swinderianus) is one of the two known extant cane rats found exclusively in sub-Saharan Africa (T. gregorianus) are the only known extant members of the genus Thryonomys and the family Thryonomidae . Samples from Republic of Benin (586/DGEFC/DCPRN/SCPRN/SA) and Cameroon were collected from bush meat markets. We have complied with ARRIVE at submission.n = 36) and Republic of Benin (n = 17). All samples were collected from January to March 2019 , Osun, Ogun, Ondo and Edo (rain forest) and Cross River (humid rainforest) states . Five ofrch 2019 . Hair ro™ NanoDrop 2000 spectrophotometer to assess purity. Furthermore, the DNA extracts were checked for molecular quality by running them through a 2% agarose gel together with a 2 kb DNA ladder marker. The five mitochondrial fragments were sequenced in 175 grasscutters samples using primer pairs amplifying 384–658 bp fragments of D-loop, cytochrome b (CYTB), cytochrome c oxidase I (COI), ribosomal subunits 12 S and 16 S , and approximately 30 ng genomic DNA. PCR cycling conditions included an initial denaturation of 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, annealing . The nucleotide sequences of all the newly sequenced samples were deposited in GENBANK under accession numbers MZ418538 - MZ418687; MZ418390 - MZ418537; MZ418252 - MZ418389; MZ418839 - MZ418996; MZ418688 - MZ418838 for D-loop, CYTB, COI, ribosomal subunits 12 S and 16 S respectively. In addition, previously published D-loop (n = 86), CYTB (n = 25), COI (n = 26), 12 S ribosomal subunit (n = 22) and 16 S (n = 27) sequences of T. swinderianus from Ghana, Nigeria, Equatorial Guinea, and South Africa (http://www.ncbi.nlm.nih.gov) (http://www.ncbi.nlm.nih.gov). In total, 236 D-loop, 173 CYTB, 164 COI, 180 ribosomal subunits 12 S and 178 16 S nucleotide sequences of T. swinderianus were analysed in this study. The CYTB sequences of dassie rat , naked mole rat  and guinea pig  were also included in the estimation of divergence times.The identities of the newly generated sequences were confirmed by BLAST searches  in the Nh Africa  were downih.gov) . FurtherCYTB and COI were independently translated into amino acids using the vertebrate mitochondrial code. No premature stop codons were observed, demonstrating that the open reading frame was maintained in the protein-coding loci. In all the loci, no unexpected gap was found within the alignments.First, the sequences of each region were aligned in MEGA7  using CLSequence comparison and identification of haplotypes were performed with DNASP 5.10.1 . GeneticCYTB, COI, 12 S and 16 S), which constituted the second partition, were concatenated and further analysed as a unit.Because of the limited number of variable sites, we explored the possibility of merging all the five regions. We first evaluated the molecular evolution models for the five mitochondrial regions using PartitionFinder2 . The beshttp://www.fluxus-engineering.com). When needed, the networks were cleaned using maximum parsimony (MP) options.The phylogenetic analyses were conducted using both maximum likelihood  and BayeD  computed from ARLEQUIN, were then investigated and presented in terms of correlation coefficients and scatter plots. This analysis was done separately for both the four concatenated mitochondrial regions, and the D-loop region.Because some of the samples were collected from meat markets, it was difficult to obtain the exact locations of all the samples. Hence, only the approximate locations were used, assuming that the animals sampled at a market were hunted from a location close to the market. We followed the procedure used in a Muscovy duck study . For eacCOI, CYTB, 12 S and 16 S) and D-loop.The analyses of the genetic ancestry were first computed in STRUCTURE version 2.3.4 . We ran df2genind in adegenet package. The data were first converted into principal components (PCs). The top 30 pCs were used for the analysis. The PC-transformed data were then used as inputs for DAPC. The results were presented using compoplot in adegenet package.We further investigated the genetic components with the discriminant analysis of principal components (DAPC)  implemenST values were used for the computation of distances while NJ method (The population phylogenetic trees were made for the four concatenated and D-loop regions using POPTREE2 . CorrectJ method  was usedprcomp in R base (https://www.R-project.org/) was then used to compute the PCA from the pairwise distances.The pairwise nucleotide distances were computed with MEGA7 using maximum composite likelihood method with 5 Gamma parameters. Bootstrap method with 1,000 replicates was used for the estimation of variance. The principal component analysis (PCA) was computed according to the procedure reported by CYTB haplotypes of grasscutter samples were used for the estimation of the divergence times. In addition, the CYTB sequences of dassie rats , naked mole rat  and guinea pig  were also retrieved. The best evolutionary model for the aligned sequences was checked. The divergence time estimates were then computed using *BEAST . The overall exact test of differentiation based on the haplotypes frequencies . Pairwise comparisons of the FST values further showed the existence of population structures across various Nigerian population pairs  were obtained in 83 Nigerian samples spread across the investigated locations . On the contrary, the analyses of the Tajima D and Fu’s Fs tests on CYTB revealed significant negative values for most populations  was found in Cross River (n = 14), Edo (n = 8), and to a less extent in Ondo (n = 1). The second most abundant haplotype (n = 22 or 21%), which seemed to be more central, and more likely to have contributed to the radiation of several other haplotypes is found in all populations except Edo and Cross River populations. Although, low-frequency haplotypes were exclusively restricted to certain populations, several haplotypes occurred in multiple populations. Similar analyses with CYTB, 16 S, 12 S and COI mitochondrial regions, which were known to contain sites under various levels of purifying selections, revealed different patterns   was within-population  (ST) computed from D-loop sequences showed consistency with the geographical locations; Cross River population seemed to be isolated from other populations  of D-loop for the Nigerian wild grasscutter populations revealed that 86.14% of the population variance were within-population variance (< 0.001) . AMOVA o< 0.001) . The paiulations . Indeed,e manner .COI, CYTB, 16 S and 12 S). Note that the D-loop region was analyzed separately. The aligned sequences of the four concatenated regions were 1,974 bp long and included 131 grasscutter samples from Nigeria, Cameroon, Republic of Benin, Ghana, and South Africa. These aligned sequences with 276 variant sites were assigned to 45 haplotypes , and Republic of Benin is very close to Ogun and Oyo states (Nigeria) . We alsoplotypes . The happlotypes . The nucpulation . Howeverpulation .n = 31) was shared between Nigerian and Republic of Benin populations, while the other (n = 3) was shared between Ghana and Republic of Benin samples. This suggested the existence of population structures across countries. The phylogenetic analyses using Bayesian inference  with the genetic distance between the populations  for models with or without admixture, and model involving migration . At all In any case, we proceeded to investigate the “best” k value for the grasscutter populations. For all the k values, the STRUCTURE’s choice criterion was higher for models without migration . The modBecause of the limited number of loci and STRUCTURE’s assumptions, we used DAPC to further investigate the genetic components in the populations studied. Consistent with the cluster results for models without migration, South African samples were separated from other populations at all k values tested . The resn = 104), Republic of Benin (n = 15), Cameroon (n = 31) and Ghana (n = 84) were assigned to 60 haplotypes  was found in the four countries under investigation. However, many of the haplotypes in the D-loop regions were specific to each country. The analyses of CYTB, COI, 12 S and 16 S regions revealed patterns expected under purifying selections or selective sweep with fewer haplotypes dominated by numerous low-frequency haplotypes (CYTB haplotype with the highest frequency in Nigeria populations (68%), Republic of Benin population (93%) and Cameroon population (55%) are not found in Ghana  was found between Ghana and Cameroon populations, while the lowest FST value (0.042) was found between Nigerian and Republic of Benin populations. Indeed, the correlation between the geographical distance and the genetic distance was higher in D-loop region  than in the merged regions  and Equatorial Guinea (n = 2) populations, the signatures of populations structures were still revealed.Principal component analyses were then computed using pairwise genetic distances of the D-loop haplotype sequences. PC1, which accounted for about 52% of the variance separate a cluster of Ghanian haplotypes from others . Also, Cing CYTB  revealeding CYTB . DespiteWe next checked the ancestral genetic components for the populations based on the D-loop sequences. We checked the components for k = 2 to k = 11 using three different models in STRUCTURE software . ConsistWe next repeated the analyses of the D-loop region using DAPC. Like for the STRUCTURE results, the South African samples were consistently shown to be genetically different at all k values investigated . The resP. typicus). However, there are no nucleotide sequences of any of the five studied mitochondrial regions for lesser cane rats, and there was paucity of the sequences of mitochondrial regions for dassie rats. Thus, we restricted the divergence time estimate to CYTB region with dassie rat sequence  MYA. Although only three samples from South African were analysed, the fact that the haplotype was observed in none of the 234 West African and Cameroonian individuals  MYA. The phylogenetic relationship of the CYTB, 16 S, 12 S and CO1 are likely to show incomplete lineage sorting, D-loop tends to reveal more recent evolutionary history. Consistent with functional importance, the D-loop region reveals more population dynamics and history, potentially because of the relaxed purifying selection. The overall D-loop haplotype diversity for Nigerian grasscutter population (0.912 ± 0.015) is slightly lower than 1.000 (± 0.016) reported for Nigerian cattle (This study investigates the population dynamics of Nigerian wild grasscutter population with populations from other African Guinea forest countries using the nucleotide sequences from five mitochondrial regions. While the n cattle , but slin cattle , higher n cattle  and 0.67n cattle , suggestn cattle . ImportaFs and Tajima’s D could reveal recent population size change, they are originally tests for neutrality which could reflect signatures of selections and/or selective sweeps (The analyses of demographic histories reveals the recent population dynamics of the wild grasscutter populations. Whereas significant values of Fu’s e sweeps . This sue sweeps  and Bayee sweeps  assumes e sweeps . NotwithThe determination of the exact number of genetic components in a population or group of populations remains a Herculean task . We use Although there is high within-population variance and Nigerian populations tend to have the same genetic component, our analyses reveal some level of genetic structures among the Nigerian populations. This is more pronounced when populations are compared across countries. Shared haplotypes are few, especially at the mostly neutrally evolving D-loop locus. The strength of genetic structure relates to geographical distance. The high within-population variance could reflect recent population divergence or admixture, and thus geographical distance. Indeed, a significant positive relationship occurs between the genetic and geographical distances of the populations. However, our data cannot delineate between the recent divergence or admixture hypotheses. Moreover, the genetic distinctness of Cross River subpopulations among the Nigerian grasscutters may be attributable to the elevation of Cross River state, thereby limiting migration. However, the population structure might not be entirely due to migratory limitations as grasscutter have relatively good migratory ability as they can run on land and swim across rivers. One factor that might contribute to the population structure is the colony system . Each grCYTB haplotypes diverged at least 6.1 MYA. It is possible that the haplotype divergence may predate population divergence (CYTB sequence suggest that Ghana population diverged before the Cameroon population diverged from the Nigerian and Republic of Benin populations. However, the population divergence tree based on D-loop suggests that the Cameroon population diverged first before the split of Ghana and Nigerian/Benin populations. This suggests that CYTB might reflect more of gene tree than the population tree. Further, the impacts of purifying selection on the divergence time estimation cannot be fully ascertained. The analyses of nuclear data of different mammalian orders reveal that the use of sites under purifying selection gave more consistent results (Although grasscutter is believed to have evolved in Africa , the exavergence . Indeed,vergence . The inc results . HoweverGrasscutter has been described as a potential livestock for the future . SeveralAlthough this study reveals some important status of the wild grasscutter populations from the Guinea Forests of West Africa, there are certain limitations of the study. First, the number of individuals that could be analysed is few in some populations. Indeed, the description of the wild grasscutter populations from Equatorial Guinea and South African cannot be extensively investigated because of the extremely low sample sizes. How representative these sampled individuals are for their respective populations cannot be ascertained. Second, the number of informative sites was small. Although the effects of these two factors could be minimized by the number of replications or samplings, the results are still not completely free from stochasticity. Therefore, further studies involving larger sample sizes are recommended for these populations. Finally, this study uses maternally inherited mitochondrial regions with limited number of informative sites, and some analyses that assume infinite site models might not be reliable. Future studies to further confirm the results should be based on nuclear regions sampled from sufficiently large number of individuals from across the locations.


China is located in East Asia. With a high genetic and cultural diversity, human migration in China has always been a hot topic of genetics research. To explore the origins and migration routes of Chinese males, 3333 Chinese individuals  with 27 Y-STRs and 143 Y-SNPs from published literature were analysed. Our data showed that there are five dominant haplogroups  in China. Combining analysis of haplogroup frequencies, geographical positions and time with the most recent common ancestor (TMRCA), we found that haplogroups C-M130, N-M231 and R1-M173 and O1a-M175 probably migrated into China via the northern route. Interestingly, we found that haplogroup C*-M130 in China may originate in South Asia, whereas the major subbranches C2a-L1373 and C2b-F1067 migrated from northern China. The results of BATWING showed that the common ancestry of Y haplogroup in China can be traced back to 17 000 years ago, which was concurrent with global temperature increases after the Last Glacial Maximum. As a consequence, China has long been one of the best places to study human evolution, civilization, and genetics.Covering a land area of about 9.6 million square kilometres, China ranks as the third largest country in the world, following Russia and Canada. It is a multi-ethnic country with a long history of civilization. Archaeologists have discovered that Chinese civilization originated 5800 years ago along the Yellow River, the middle and lower reaches of the Yangtze River and the Western Liaohe River . About 1−4 −1.0 × 10−2 mutations/generation) can generate unique haplotypes which have been used in sexual assault cases and familial searching [et al. explored genetic variation among geographical regions and among ethnic groups through information on 17 commonly used Y-STRs in the YHRD database [The male specific region of the Y chromosome (MSY) with paternal inheritance and lack of recombination is a powerful tool for inferring paternal ancestry and identifying paternal genealogy . As one earching ,4. In 20database . Y-chromdatabase . To the database . Y-SNPs database .et al. proposed a model of human expansion of East African origin by studying the demographic history of human males with Y-SNP and STR markers from 51 populations [Combining analysis of Y-SNP and STR has been proved to be a good strategy to predict population sub-structure and to explore human origins/migrations . In 2010ulations . SeveralIn this study, we conducted a detailed analysis of five ethnic groups , which account for 93.51% of the total population of China. We focused on the following questions: Does geography/ethnicity play an essential role in genetic affinity within China? What is the genetic relationship between Chinese populations and its surrounding populations? Can we find any evidence of early human activity among Chinese ancestors? If so, is it possible to characterize them?. 2. 2.1We searched PubMed with the keywords ‘Y-STR’, (Y-SNP’ or ‘Y haplogroup), and (Chinese’ or ‘China') from 2019 to 2021. Inclusion criteria were as follows: (1) containing at least the 27 Y-STR data information as the Y Filer Plus kit; (2) containing high-resolution Y-SNP information, not just under one of the major haplogroups; (3) data can be obtained from the attached table. A total of 3333 individuals were collected from five high-resolution Y-SNP-STR studies as shown in electronic supplementary material, table S1 –16. To f. 2.2https://isogg.org/tree/index.html). We analysed Y-STRs strictly following the recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) [All these five studies were performed in a laboratory accredited by the China National Accreditation Service for Conformity Assessment (CNAS). The Y haplogroup was named according to Y-DNA Haplogroup Tree 2019–2020 (s (ISFG) .. 2.3. 2.3.12)/(n-1) . Principal component analysis (PCA) was conducted based on the haplogroup frequencies in a previous study [Haplogroup frequency of each population was calculated by direct counting. Haplogroup diversity was calculated as: n/(n − 1) . Discrimination capacity (DC) was calculated as: DC = the number of observed haplotypes/the number of total samples. Match probability (MP) was computed as: MP = Σpi2. The pairwise genetic distances of FST were calculated using Arlequin 3.5.2 with 23 Y-STRs .Allele and haplotype frequencies were direct counted by Office Excel. The allele of DYS389b was obtained as: DYS389b = DYS389II-DYS389I. DYS385 and DYF387S1 were treated as allelic combinations. The gene diversity (GD) and haplotype diversity were calculated as: http://www.maths.abdn.ac.uk/~ijw). In this study, minor modifications were applied to the genetic and population parameters according to a previous study [Time to the most recent common ancestor (TMRCA) was estimated based on Y-STR using BATWING  [To present median-joining networks of these five populations, 23 single-locus STRs (excluding DYS385 and DYF387S1) and 143 Y-SNPs were imported into Network 5.0 . The hig. 3. 3.1The highest haplogroup diversity was found in the Hui population (0.979), followed by Han (0.966), Mongolia (0.956), Yi (0.910) and Kyrgyz (0.791). As shown in a). Intensive analysis of haplogroup C showed that the frequency of sub-haplogroup C2a-L1373 decreased from north to south (b). However, a higher frequency of sub-haplogroup C2b-F1067 was found in Henan Han (c). Haplogroup D showed higher haplogroup frequency in the Yi population, followed by the northwestern Hui group, which may be related to the geographical proximity of Tibet (d). The frequency of haplogroup J showed a decreasing trend from southwestern China to northern China (e). A higher frequency of haplogroup N exists in northern China and decreases southwards along the coast (f). The highest frequency of haplogroup O1-F265 was found in the southeast Han (greater than 25%) and it decreases gradually toward the northwest (g). Haplogroup O2-M122 is widely distributed in the eastern coastal region of China, and its frequency gradually decreased from the east to the west (h). Haplogroup R-M207 possessed a high frequency in western China and gradually declined toward eastern China (i).In order to further explore the haplogroup distribution trend, the frequency contour plots with haplogroup were conducted. The results showed that haplogroup C mainly distributed in northern China, with decreasing haplogroup frequency to the south a. Intensto south b. Howeveenan Han c. Haplogof Tibet d. The frrn China e. A highhe coast f. The hiorthwest g. Haplogthe west h. Haplogrn China i.Figurea, electronic supplementary material figure, S3b). The frequency of haplogroup O2a1 showed a decline from eastern to western China . The frequency of haplogroup O2a2 showed a decreasing trend from eastern to northwestern China .Further analysis of sub-haplogroups C and O revealed that different sub-haplogroups existed in these five ethnic groups. In haplogroup C, most of the Kyrgyz distributed in sub-haplogroup C2a-L1373, while most of the Han and Yi distributed in sub-haplogroup C2b-F1067 . In haplogroup O, the highest haplogroup diversity was Hui (0.956), followed by Han (0.951), Mongolia (0.925), Yi (0.887) and Kyrgyz (0.793). Interestingly, no haplogroup O1-F265 was found in Kyrgyz . We found that the frequency of haplogroup O1a-M119 decreased from southeastern to northwestern China, while haplogroup O1b spread from southern to northern China according to the results of haplogroup frequency contour analysis , followed by Hui (0.99987), Yi (0.99977), Mongolia (0.99950) and Kyrgyz (0.99803). The highest DC was found in the Han population (0.998), followed by Hui (0.960), Yi (0.953), Mongolia (0.878), and Kyrgyz (0.793). The MPs of these five ethnic populations were 0.0008, 0.0017, 0.0020, 0.0026 and 0.0052, respectively.Gene diversities for the 27 Y-STR loci were shown in electronic supplementary material, table S3 and electronic supplementary material, table S4. DYS385 was the highest polymorphic marker overall (GD = 0.967), while DYS391 showed the least diversity (GD = 0.435) overall. Interestingly, the GD values of DYS385 ranked first in Han, Hui, and Yi, while DYF387S1 ranked first in Kyrgyz and Mongolia. The GD values of DYS391 ranked bottom in Han and Hui, while DYS437 ranked bottom in Kyrgyz, Mongolia and Yi.. 3.3. 3.3.1Based on the GD of Y-STR, we found that DYS389b, DYS460, DYS390, DYS481, DYS533, DYS389I and DYS449 were significantly associated with haplogroup F , while DYS439 and DYS518 were significantly associated with haplogroup H. In an attempt to further explore the correlation of STRs within each haplogroup, alleles of STRs were analysed by different haplogroups . Using the frequency of alleles, we achieved a significant correlation between Y-STRs and haplogroups . The above results suggested that the specific Y-STR haplotype can be used to predict haplogroups, which can improve the precision of existing Y haplogroup prediction software, especially in East Asian populations.. 3.3.2a). Interestingly, we found that haplogroup C* only distributed in southern China. Further analysis of sub-haplogroup C2a1a3-M504, the result showed that the Mongolian and Kyrgyz individuals divided into two clusters, indicating that the sub-haplogroup C2a1a3-M504 in the Kyrgyz was different from in Mongolians. Star-like networks of haplogroup D-M174 in electronic supplementary material, figure S6 showed that the sub-haplogroup D presented in the Han, Hui, Mongolia and Kyrgyz populations might derive from Yi-related ancestral populations. Further analysis of sub-haplogroup D showed that the Yi population expanded in haplogroup D1a1a1a1b-SK541, while the Kyrgyz population only distributed in D1a1b1a-M533. No haplogroup J-M304 was found in the Yi population . High polymorphism of haplogroup J was found in the Hui population. High polymorphism of haplogroup N-M231 was found in the Han population . Most Yi individuals clustered in haplogroup-N1b, while Mongolians distributed in N1a. In sub-haplogroup N1a1a1a1a3-B197, Mongolians was clustered and expansion. However, in haplogroup N1a2-F1008, Han individuals and Mongolians distributed in different branches which indicated that the two populations have different origins. No Kyrgyz individual was found in haplogroup O1-F265 (b). Yi, Hui and Mongolia populations distributed in the downstream of haplogroup O1- F265, except sub-haplogroup O1b1a1a1a-F1252. In sub-haplogroup O2a1-L467, these five populations distributed alternately. But in the O2a1*, only Hui and Mongolian individuals appeared (c). In haplogroup O2a2-P201, most Hui, Mongolian, Yi and Kyrgyz individuals distributed the downstream of the central reference Han people, such as sub-haplogroup O2a2a1-F2588, O2a2b1a1a1-F438 and O2a2b1a2a1a-F46. It suggested that haplogroups O2a1-L467 and O2a2-P201 have different origins in Chinese populations. Only Hui and Mongolians distributed in R2-M479 . Star-like networks of haplogroup R1a1a1b2a2-Z2124 were observed in Kyrgyz individuals, which reflected likely population expansion happening in these people. No Yi population was found in haplogroup R-M207. In the Chinese Han population, haplogroup R was mainly distributed in the northern Han.We applied Network analysis to further infer the possible patriarchal activities within China. Mongolia, Kyrgyz and some Northwest Hui were clustered in C2a1a1b1-F1756, C2a1a2-M48 and C2a1a3-M504, while Han populations distributed in C2b1-Z1338 and C2b1b-F845 a. Intere O1-F265 b. Yi, Huappeared c. In hap. 3.3.3The TMRCA for the Chinese populations can be traced to 17 kya . The TMRCA of haplogroup C with Mongolia, Kyrgyz and Northwest Hui (approx. 13.6–17.5 kya) predated other groups. Further analysis of sub-haplogroups C2a and C2b showed TMRCA can be dated back to 7.6 and 14.5 kya in the Mongolian group. TMRCA of haplogroup N in the northern Chinese populations was earlier than that of the southern populations, which was similar to the results of the frequency distribution contour heat map. It indicated that haplogroup N is spreading from north to south. Although higher frequency of haplogroup O1a was found in southern China, the TMRCA of Northwest Hui groups can trace to 12.2–16.7 kya.. 3.3.4ST values were calculated by using 23 Y-STRs . The results showed that Kyrgyz was the most distantly related to other populations (p < 0.05). The Hui populations were divided into three groups: Cluster 1 , Cluster 2 (Sichuan Hui and Shandong Hui) and Yunnan Hui. The small genetic distance was shown in Cluster 1 (FST < 0.0027). Cluster 2 Hui populations were closer to Northern Han populations than other Hui populations. Yunnan Hui were closer to Yi populations than other Hui populations. Chinese Han populations were separated into two clusters: Northern Han and Southern Han.To reveal the substructure within Chinese populations, pairwise FST . Han, Kyrgyz and Hui were separated with PC1. Northern Han and Southern Han were separated with PC2. From the quadrant perspective, Cluster 1 Hui populations were clustered with Mongolians, while Cluster 2 Hui populations were clustered with Chinese Han.In a principal component analysis (PCA) of these five ethnic groups, similar genetic profiles were observed as F. 3.3.5a). We found that Cluster 1 Hui populations had closer genetic distance with Mongolia and Dongxiang populations. Han populations were clustered with the other Hui groups, Yi and Lingao populations. To further illustrate the genetic relatedness of the studied populations and worldwide populations, PCA analyses were conducted by 34 Y-SNP markers and 91 populations (b). The results showed that Han, Hui, Mongolia, Yi, Southeast Asian and other East Asians clustered in the left quadrant. South Asians and central Asians were located in the upper right quadrant. West Asians, Europeans and Africans were located in lower right quadrant. However, Kyrgyz located at the centre, which indicated that Kyrgyz population exhibited a genetic admixture of Asian and European populations.To better understand the Chinese genetic flow and mixture, the relationships of populations in this study and 15 reference populations were explored via 23 single locus Y-STR a. We fouulations b. The re. 4Historically, China has experienced a number of population migrations, such as the migration of nomads from the north to the interior during the late Eastern Han Dynasty, and the mass migration of northerners to the south to escape the war during the An-Shi Rebellion. Therefore, population genetic structure and population migration have been a hot topic in Chinese genetic research. In this study, we explored the genetic affinities of the Han, Hui, Mongolia, Yi, and Kyrgyz populations to each other and to neighbouring populations using a variety of analytic methods, and investigated the values of Y chromosome markers (both Y-SNP and Y-STR) in biogeographical ancestry inference.et al.'s study [The population comparisons by PCA showed three intercontinental clusters: Cluster 1: East and Southeast Asian populations; Cluster 2: South and Central Asian populations; Cluster 3: African, American and European populations. In this study, we found that Han and Yi populations were clustered with East and Southeast Asian populations. It was consistent with Cheng 's study . Hui and's study .et al. study [Haplogroup O-M175 is the most common paternal lineage in East Asian and Southeast Asian populations . It was l. study . Interreet al. found that O2-M122 haplotypes in southern East Asia were more diverse than that in northern East Asia by typing 2,332 East Asian individuals, suggesting that haplogroup O2 originated in the south and migrated northward [Ding orthward . The freHaplogroup C-M130 is the strong supporting evidence for the migration and settlement of modern humans from the Middle East to South and East Asia . In thisA previous study showed that haplogroup D mainly distributed in East Asia, especially in Tibetan and Japanese (30%–40%) . In thisHaplogroup R occurs at high frequency in modern Europeans, especially in Western Europe . AlthougThe Y haplogroup is a valuable tool for studying the origin of humans and inferring the admixture among populations ,38. Due . 5Human migration has always been a hot topic in genetics research. In this study, 3333 Chinese individuals  were analysed by Y-STR and Y haplogroup. We found that differences of Y haplogroup and Y-STR not only existed among ethnic groups, but also in geographical locations. Furthermore, combining analysis of haplogroup frequencies, geographical positions and TMRCA, it was shown that Chinese populations have undergone a complex process of population migration. The population migration was concurrent with global temperature increases after the Last Glacial Maximum.


Ruditapes philippinarum in the world. Most of the clam seeds for aquaculture are mainly derived from artificial breeding in southern China, likely resulting in the loss of genetic variation and inbreeding depression. In this paper, we investigate the genetic diversity and differentiation of R. philippinarum populations. The present results not only provide the scientific basis for the phylogenetic relationship of clam resources, but also lay the foundation for natural population conservation and genetic improvement of R. philippinarum.China has the largest production yield of Manila clam Ruditapes philippinarum in the world. Most of the clam seeds for aquaculture are mainly derived from artificial breeding in southern China, likely resulting in the loss of genetic variation and inbreeding depression. To understand the genetic and haplotype diversity of R. philippinarum, 14 clam populations sampled from different regions of China were analyzed by three molecular markers, including COI, 16SrRNA and ITS. Based on the results of the COI and ITS genes, the 14 populations showed a moderate to high level of genetic diversity, with an average haplotype diversity of 0.9242 and nucleotide diversity of 0.05248. AMOVA showed that there was significant genetic differentiation among all populations . Pairwise FST analysis showed that genetic differentiation reached significant levels between Laizhou and other populations. Two Laizhou populations showed great divergence from other populations, forming an independent branch in the phylogenetic tree. The shared haplotypes Hap_2 and Hap_4 of COI appeared most frequently in most clam populations. In contrast, 16SrRNA analysis of the clam populations revealed the dominated haplotype Hap_2, accounting for 70% of the total number of individuals. The haplotype diversity of the Laizhou population (Laizhou shell-wide (KK) and Laizhou dock (LZMT)) was relatively higher than other populations, showing multiple unique haplotypes . These findings of genetic and haplotype diversity of clam populations provide guiding information for genetic resource conservation and genetic improvement of the commercially important R. philippinarum.China has the largest production yield of Manila clam  Genetic diversity is an essential component of biodiversity, with positive significance for the evolution and conservation of a species ,2,3,4. GRuditapes philippinarum, native to the Pacific and Indian Ocean coast, has been introduced to North America and Europe, becoming a worldwide cultured bivalve in mudflats [ITS richness in nutrition, R. philippinarum are very much enjoyed by worldwide consumers [R. philippinarum becomes one of the most commercially important bivalves from the northern to southern coast, with a high annual production rate (~4 million tons) [R. philippinarum have been reported using mitochondrial markers, such as COI and 16S rRNA [The Manila clam mudflats ,21. Due onsumers . In Chinon tons) . For manon tons) ,24,25. C16S rRNA ,27,28,29R. philippinarum, 14 clam populations along the coast of China were collected and analyzed by two mitochondrial genes (COI and 16SrRNA) and one nuclear gene (ITS). The present results not only provide the scientific basis for the phylogenetic relationship of clam resources, but also lay the foundation for natural population conservation and genetic improvement of R. philippinarum.Most of the clam seeds for aquaculture are mainly derived from artificial breeding in southern China, likely resulting in the loss of genetic variation. To explore the genetic and haplotype diversity of R. philippinarum samples representing 14 populations were taken from the north and south coast of China, including Beihai (BH), Donggang (DG), Dongying (DY), Guangdong (GD), Haiyang (HY), Hongdao (HD), Laizhou shell-wide (KK), Laizhou dock (LZMT), Lianjiang (LJ), Ningbo (NB), Rizhao (RZ), Sanya (SY), Zhangzhou (ZZ) and Zhuanghe (ZH) (Manila clam ghe (ZH) . Two popghe (ZH) .R. philippinarum using the standard phenol chloroform method. After DNA extraction, the products were tested by 1% agarose gel electrophoresis for DNA quality. The concentration and purity of DNA was measured using a NanoDrop Lite ultra-micro spectrophotometer . The obtained DNA was diluted to 50 ng/μL and stored in an ultra-low-temperature refrigerator at −80 °C.The DNA was extracted from the foot muscle of COI primers of LCO1490 and HCO2198 were used for PCR amplification, while 16SrRNAar and 16SrRNAbr were used for PCR amplification of 16SrRNA [COI and 16S, the internal transcribed spacer (ITS) of nuclear DNA also served as one of the most extensively sequenced molecular markers for investigating genetic diversity among populations. In this study, the primers of ITS-F and ITS-R were used to amplify the ITS2 region [The universal  16SrRNA ,31. In a2 region . The aboCOI, 16SrRNA and ITS gene sequences were submitted to the NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 30 August 2023)) database under accession numbers OR486714—OR486966, OR499223—OR499487 and OR499488—OR499723, respectively.The polymerase chain reaction (PCR) system (50 µL) consisted of 5 µL of DNA template solution at 50 ng/µL, 25 µL of MIX enzyme , 2 µL of each forward and reverse primers and a fixed volume of 50 µL of ultrapure water. The amplification reaction conditions were as follows: pre-denaturation at 94 °C for 5 min; cyclic denaturation at 94 °C for 30 s; annealing and denaturation at 56 °C for 30 s; extension at 72 °C for 1 min for total 35 cycles; and finally, extension at 72 °C for 10 min, ending at 4 °C. Each PCR product was examined on 1% agarose gel to verify the amplified fragment length. Finally, they were sent to Sangon Biotech (Shanghai) Co., Ltd.  for Sanger sequencing. In this study, R. philippinarum populations and to calculate the coefficient of genetic differentiation (FST) within and among populations [The obtained DNA sequences were visualized and checked using SeqMan 7.1.0.44 software . The splulations . A haploulations .COI, 16SrRNA and ITS genes were used for multiple sequence alignment of the homologous sequences. The primers and partial end sequences were removed. Gene fragments of 559 bp, 431 bp and 349 bp in length were obtained. A total of 253 COI sequences, 265 16SrRNA sequences and 242 ITS sequences derived from 14 populations were compared using MEGA11 software. For COI, the average contents of AT and GC were calculated as 66.1% and 33.9%, respectively. The mean contents of 16SrRNA genes A + T and G + C were estimated to be 67.3% and 32.7%, respectively. In contrast to COI and 16S, ITS showed the different pattern of AT and GC contents, with AT content (31.6%) significantly lower than GC content (68.4%).The sequences of COI genes are shown in ITS own haplotype. Among them, the largest number of individuals was 14 in the HY and ZH populations, and the smallest number was 6 in the HD population. There were 49 unique haplotypes in the population, accounting for 70% of the total number of haplotypes. There were 21 shared haplotypes, accounting for 30% of the total number of haplotypes. The maximum number of individuals of shared haplotype Hap_2 was 58, accounting for 22.9% of the total number of individuals. The shared haplotype Hap_2 was distributed in 12 populations, except for KK and LZMT. In contrast, some unique haplotypes  were only distributed in the KK and LZMT populations. A total of 68 nucleotide polymorphic loci were detected in the COI gene, including 36 single-mutant loci and 32 parsimony informative loci. The average number of nucleotide differences across all populations was 4.156, and the nucleotide diversity index was 0.00746.The results of genetic diversity based on 16SrRNA gene sequences were summarized in R. philippinarum. For 16S, haplotype diversity index was calculated to be 0.488 for all populations. The ZZ population had the most haplotypes (six), and the GD population had the least haplotypes (one). There were 19 population-specific haplotypes, accounting for 79.2% of the total number of haplotypes. There were five shared haplotypes, accounting for 20.8% of the total number of haplotypes. Among them, the maximum number of individuals with shared haplotype Hap_2 was 187, accounting for 70.6% of the total number of individuals. As the dominant haplotype, haplotype Hap_2 was distributed and shared in all populations, except for the DG population. A total of 30 nucleotide polymorphic loci were detected in the 16SrRNA gene, including 23 single-mutant loci and 7 parsimony informative loci. The average nucleotide difference number of the total population was 0.872, and the nucleotide diversity index was 0.00203. Based on 16S analysis, the two highest numbers of haplotype diversity were found in the LZMT population (0.59) and the ZZ population (0.44), showing relatively higher values than those of the other populations. In contrast, the extremely low level of haplotype diversity (≤0.10) was detected in some populations, including the DG, GD and HD populations.Genetic diversity parameters based on ITS gene sequences showed that a total of 115 haplotypes were detected in 242 individuals  and three southern populations  were grouped into one branch, indicating the close relationship between the northern and southern populations (ITS-based NJ tree (The phylogenetic tree of 14 populations constructed using NJ (neighbor-joining method) is shown in branches a,b. KK aulations a. The cl NJ tree b,c.COI, 16S and ITS are shown in The maximum likelihood (ML) trees for COI genes ranged from 0.04753 to 0.61486  to 0.96041 (between BH and GD). As indicated by ITS analysis, significant differentiation was detected between GD and other populations. The highly significant differentiation was also found between LZMT and BH, HD and RZ, and BH and HY. Furthermore, the significant differentiation appeared among HY and DG and RZ groups, as well as LZ and DG groups.The genetic differentiation coefficients among the 14 populations and their significance levels are shown in  0.61486 . The sma 0.89497 . The lowCOI, 16S and ITS in COI, the results show that the contribution of genetic variation within the population was 83.73%, while the contribution of genetic variation among populations was 16.27%  is comparable with the previous results of other clam populations, indicating large stable populations over time in most clam populations [Mytilopsis sallei and mussel Musculista senhousia [dH < 0.5 and iP < 0.05) in most studied populations. In particular, the extremely low level of haplotype diversity in some populations suggests that the founder effects may drive the genetic structure of passively dispersed aquatic invertebrates [For animals, a high level of genetic diversity is essential for the long-term survival of populations, having great impacts on their ability to adapt to changing environments ,39. DNA ulations ,27,28,40enhousia ,41,42. Aenhousia . In conttebrates .dH < 0.5, iP < 0.005) suggested that a population bottleneck effect or an establishment effect by a single, small population has recently occurred [16SrRNA was more conserved mitochondrial DNA across species compared to the COI and ITS genes [16SrRNA gene contains highly conserved regions and highly variable regions, it serves as an effective marker for studying the phylogenetic relationship among species below the family level [According to the previous study, the low level of haplotype diversity and nucleotide diversity (occurred . In the occurred . For thoTS genes ,45,46. Bly level ,27,29. TCOI appeared most frequently in most clam populations. Moreover, 16SrRNA analysis of the clam populations revealed the dominate haplotype Hap_2, accounting for 70% of the total number of individuals. The haplotype Hap_2 was distributed in all populations, except for the DG population. One possible reason for this is that the pattern of southern breeding and northern culture in the clam industry may cause high genetic homogeneity [In the present study, the shared haplotypes Hap_2 and Hap_4 of ogeneity . Notablyogeneity ,25.COI was highly significant between Laizhou and other populations, ranging from 0.35 to 0.61 (R. philippinarum, the mode of southern breeding and northern culture may increase the gene flow of clams, resulting in low genetic differentiation between northern and southern populations [COI, 16S and ITS support the conclusion that artificial breeding and culture may serve as one of the major factors influencing the population genetic structure of clams in China.Population genetic structure and differentiation are affected by many factors, such as habitat adaptation, natural selection, random genetic drift, and gene flow ,28,39. I to 0.61 . Consist to 0.61 . This isulations ,25,40,47R. philippinarum will provide some guiding information for the conservation, management and genetic improvement of R. philippinarum in China.In the clam aquaculture industry, frequent translocation of artificial clam seeds to natural habitats may result in altering genetic composition in wild populations, even displacing the natural clam populations. The genetic changes affected by clam seed translocation were evidenced by microsatellite marker analysis for western Korean populations . The freR. philippinarum Manila clam from different regions of China were analyzed by three molecular markers, including COI, 16SrRNA and ITS. Based on the results of the COI and ITS genes, the 14 populations showed a moderate to high level of genetic and haplotype diversity. The shared haplotypes of COI and 16SrRNA were detected in most studied populations, while the LZ populations had some unique haplotypes, showing great divergence from other populations. The present findings support the conclusion that the genetic population structure of clams may be influenced by translocation of artificial clam seeds to different regions for culture in China. The present findings of genetic and haplotype diversity of R. philippinarum based on three molecular markers will improve our understanding of the genetic status of clam populations and provide some guiding information for the conservation, management and genetic improvement of R. philippinarum in China.In this study, 14 populations of