In recent decades, societies, states and local authorities have become increasingly aware that for effective long-term management and protection of aquatic ecosystems and populations, it is necessary to take into account the genetic changes occurring in these populations. One type of high-resolution molecular marker suitable for studying the neutral and adaptive genetic diversity of populations is single nucleotide polymorphism (SNP). This review is an attempt to show the benefits of using SNPs to recognize natural populations of aquatic animals and detect the threats to them from accidentally or intentionally released farm animals, fishery and global climate changes. It is postulated that conservation actions should protect not only pristine natural populations that are endangered or overfished, but also protect populations of non-threatened species from unnecessarily released semi-domesticated animals. The enhancement of natural populations with farmed material usually reduces their genetic diversity. Experimental size-selective catches of artificially created populations have caused evolutionary changes in the life cycles of fishes. However, fishery-induced evolution in natural populations is difficult to observe. The negative measurable effects on populations can be expected when the number of breeding individuals is reduced below 100, which occurs very rarely in the sea and more often in fragmented freshwater streams, ponds and seasonal rivers.An increasing number of aquatic species have been studied for genetic polymorphism, which extends the knowledge on their natural populations. One type of high-resolution molecular marker suitable for studying the genetic diversity of large numbers of individuals is single nucleotide polymorphism (SNP). This review is an attempt to show the range of applications of SNPs in studies of natural populations of aquatic animals. In recent years, SNPs have been used in the genetic analysis of wild and enhanced fish and invertebrate populations in natural habitats, exploited migratory species in the oceans, migratory anadromous and freshwater fish and demersal species. SNPs have been used for the identification of species and their hybrids in natural environments, to study the genetic consequences of restocking for conservation purposes and the negative effects on natural populations of fish accidentally escaping from culture. SNPs are very useful for identifying genomic regions correlated with phenotypic variants relevant for wildlife protection, management and aquaculture. Experimental size-selective catches of populations created in tanks have caused evolutionary changes in life cycles of fishes. The research results have been discussed to clarify whether the fish populations in natural conditions can undergo changes due to selective harvesting targeting the fastest-growing fishes. In recent decades, administrations and societies have become increasingly aware that for the successful long-term management and conservation of aquatic ecosystems and animal populations, it is essential to take into account the genetic changes that occur in these populations. As a consequence, the number of publications on the population genetics and genomics of aquatic species increases from year to year. The effectiveness of these studies has been significantly increased by the use of high-resolution molecular markers, such as microsatellites and, more recently, SNPs. The development of high-throughput genotyping techniques reduces the cost of population studies and increases their availability in countries around the world.The intensive exploitation of marine and freshwater resources requires the accompanying conservation of biodiversity at local and worldwide scales. Conservation is to maintain the threads of life across time and involves the ecosystem and a species flock or species perspectives . MoleculThe main concerns of conservation genetics are defining populations and closely related species, the characterization of their polymorphism and its changes over time, the identification of hybrids in wild populations, the estimation of effective population size, estimation of inbreeding, and understanding the local adaptations and genetic polymorphism at quantitative trait loci in populations. An understanding of the spatial structure of populations enables the delineation of evolutionary conservation units ,14. EvolThere have been many review papers concerning the application of molecular markers to aquaculture and the management of fisheries’ resource conservation ,21,22,23Barcoding studies, although incomplete, have demonstrated the existence of a much larger number of species than expected. Using SNPs and next-generation sequencing (NGS) reveals more species and more fine-scale genetic differentiation over large geographic areas at an unprecedented scale, but also contributes to the discovery of the extent of the expansion of cryptic species and the invasions of species transported by shipping, byproducts of aquaculture introductions, natural expansions and climate changes. The SNP loci have been discovered and characterized for assays in specimens and populations of an increasing number of aquatic species . The subThunnus thynnus and albacore, T. alalunga [Salvelinus in lakes in Idaho, USA [Oncorhynchus mykiss and Atlantic salmon, Salmo salar sequence data was evaluated to identify SNPs in three other species: O. tshawytscha, O. nerka and O. keta [O. mykiss and S. salar alignments were more successful than the primers designed based on Oncorhynchus-only alignments of sequences. Approximately half of the subsequently designed validation assays resulted in high-throughput SNP genotyping markers. Smith et al. [Oncorhynchus mykiss from the four major subspecies of cutthroat trout: westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus c.i bouvieri), coastal cutthroat trout (Oncorhynchus c. clarki), Lahontan cutthroat trout (Oncorhynchus c. henshawi), and their hybrids for stock assessments on the Clark Fork in Montana, USA [Salmo genus, Salmo marmoratus, S. obtusirostris and different evolutionary lineages of S. trutta was designed [Sebastes mentella and S. fasciatus in the North-West Atlantic (Canada), which will help to improve the assessment of fishery management units [Sola senegalensis and S. aegyptiaca [S. aegyptiaca. The identification of hybrids was more precise, with a large number of SNPs in comparison with earlier studies employing a low number of non-diagnostic markers. A large number of SNP markers useful for conservation purposes was discovered and characterized using RAD sequencing for four tetraploid sturgeon species, A. naccarii from the Adriatic Sea (hatchery), A. gueldenstaedtii and A. persicus from the Caspian Sea and A. baerii from Siberia [Acipenser gueldenstaedtii) from the Volga and Ural Rivers were essentially indistinguishable, but they differed from the Russian sturgeons in the Azov Sea, and from Persian (A. persicus) and Siberian river (A. baerii) sturgeons [A. sturio and American sea sturgeon A. oxyrinchus alleles [Oreochromis niloticus) and 3569 SNP were discovered [O. niloticus, O. aureus and O. mossambicus), different strains and different geographical locations. Native Nile tilapia populations have been studied in areas where there is or will be aquaculture developed in order to trace the escapees or introductions from aquaculture, including the Volta river in western Africa. The introduced populations of Nile tilapia were studied in the Congo Basin using over 27,000RAD-derived SNPs [Cyptinus carpio has been successfully used for genotyping eight other related species, including C. carassius and D. rerio [Ictalurus punctatus) and blue catfish (I. furcatus) [Management and conservation activities require the correct identification of species and their hybrids in natural and hatchery environments. In some cases, sets of diagnostic SNPs have been constructed that enabled the simultaneous identification of a few species. A panel of SNPs was developed by cross-species amplification for Atlantic bluefin tuna, alalunga . A set oaho, USA . A panelaho, USA . The use O. keta . The prih et al.  suggest ana, USA . A set odesigned . Forty-ont units . Restricgyptiaca . The gen Siberia . High-thturgeons . A speci alleles . The hybscovered . In totaved SNPs . The genD. rerio . Domestiurcatus) .Chionoecetes bairdi) from more commercial fishery targeted snow crabs (C. opilio) and their hybrids in the southeastern Bering Sea was developed [Bathymodiolus species , useful for both cross-species use and a fine-scale population differentiation assay [Chlamys farreri) SNP markers were also polymorphic in yesso scallop, Patinopecten yessoensis [A SNP-based assay for identifying tanner crabs  was found in the Pacific, with heterogeneity detected among tropical samples [Anguilla rostrata) is a panmictic species, despite it inhabiting different environments from the southern Sargasso Sea to Canada and Greenland. The polygenic basis of discrimination between highly differentiated ecotypes rearing in brackish, saltwater and freshwater and was demonstrated experimentally [SNPs have been useful in the analyses of the genetic structure of natural and wild populations of exploited species with high dispersal potential in ocean, anadromic, landlocked and sedentary freshwater populations and benthic species. Genetic differentiation between the pelagic populations of commercially important and over-fished albacore tuna, nean Sea ,114. A d samples , and bet samples . Alternamentally . Three hGadus morhua populations were found for the first time at the SNP allele frequencies in the transferrin gene [The differences between west and east Atlantic cod rin gene . Three hrin gene . A genomrin gene . In addirin gene . Signifirin gene . The genrin gene . The regrin gene . In totarin gene . The genrin gene ,128. Disrin gene . A set orin gene ,132,133.Salmo salar with the application of genome-wide SNP arrays [Salmo salar) in Scotland and northeast England [S. salar SNP loci and loci determining sex and sea age at maturity SNP modules developed for the analysis of long-term genetic changes in the Teno River, Finland, were postulated to also be sufficient for the stock identification for other populations in Europe [FST enabled the improvement of genetic stock identification of Chinook salmon (Oncorhynchus tshawytscha) from western Alaska in a study of twenty-eight populations [Oncorhynchus keta populations found with SNPs had a regional structure in the northeastern Pacific Ocean, similar to that detected with microsatellites and allozymes [Oncorhynchus tshawytscha allowed for the grouping of populations into major lineages and mixed-stock analysis [Oncorhynchus mykiss from the northwestern United States and British Columbia were divided into two major lineages by genotyping with a panel of 276 SNPs [Oncorhynchus nerka), occur as two reproductive ecotypes with no morphological differences in British Columbia, Canada. Eighteen outlier SNP loci were identified as putative under the divergent selection between the ecotypes in the absence of a neutral SNP structure, indicating the early stages of ecological divergence [Salmo trutta native and supplemented populations has been reported in Europe [A much stronger differentiation has been revealed for migrating Atlantic salmon P arrays . Two maiP arrays . A panel England . The basn Europe . Ninety-ulations . The genllozymes . The genllozymes . The anaanalysis . Eleven 276 SNPs . The evivergence . Mixed-svergence . The difvergence . A diffen Europe ,146,147.Merluccius merluccius [Engraulis encrasicolus populations in the Atlantic and Mediterranean areas was observed and lower differentiation among Mediterranean populations [Clupea harengus) populations are genetically structured at a fine scale within the Baltic Sea, which is explainable by oceanographic connectivity [Sparus aurata) as well as between western and eastern Mediterranean European sea bass (Dicentrarchus labrax) [Lates calcarifer populations has been observed in Australia and Papua New Guinea, whereas lower differentiation has been seen in the Indonesian archipelago to Southeast Asia, with gene-associated SNP markers [Scatophagus argus wild populations in China [Thaleichthys pacificus, populations ranging from the Cook Inlet, Alaska, and along the west coast of North America to the Columbia River were analyzed [Genetic differentiation, on European and local scales in the Atlantic, North Sea, northern Portugal, western, central and eastern Mediterranean have been found for populations of European hake, rluccius ,149, in ulations . Atlantiectivity . Whole-gectivity . Howeverectivity . The app labrax) . A signi markers  and genein China . Eulachoanalyzed . The popMacrobrachium rosenbergii) in India were studied using high-throughput sequencing [Haliotis discus hannai) from China and Japan have been developed and can be used for the assessment of genetic structure changes in both wild and cultured populations [Haliotis midae [Chlamys farreri, populations in China: Changdao, Rongcheng, Rizhao and Qingdao [Pinctada maxima) in the ecologically and economically important Indo-Pacific region: Aru, Bali and West Papua [Pinctada margaritifera was studied across its ~18,000 km Indo-Pacific distribution, using 580 specimens [Four wild stocks of the giant freshwater prawn  was also delineated using neutral and outlier SNPs [Northern and southern populations of blue mussels ybridize . Novel Sincialis ,166,167.ltic Sea . The coereported . Invasivrn Ocean ,171,172.rn Ocean ,174. A sier SNPs .Salvelinus fontinalis), either not stocked or stocked with a different intensity since 1971, were sampled in the Portneuf Wildlife Reserve in Quebec, Canada and genotyped at 280 SNPs [Salmonid populations have been strongly affected by introgressive hybridization caused by stocking with hatchery-reared fish over a long time. Nine populations of brook charr  in the southern Baltic rivers in Poland in 1996–2009 related to enhancement practices were revealed with SNP analysis [S. t. marmoratus) individuals, a lineage of brown trout S. trutta [Oncorhynchus mykiss, called redband trout, in the upper McCloud River watershed in California [O. mykiss with native cutthroat trout O. clarkii populations in western North America due to widespread stocking and invasion into historical cutthroat trout habitats has been reported [O. clarkii seleniris) population, endemic to Silver King Creek, California, USA, revealed that it was weakly structured and introgressed from the long-time stocking by California golden trout (O. mykiss aguabonita) and hatchery rainbow trout O. mykiss [Salmo salar) in hatcheries along the North Atlantic coasts has resulted in frequent escapes to the wild. It has been known that domestic salmon interbreed with wild populations and lower their fitness. The application of a 220,000-SNP array to studies of wild European and North American populations, farmed salmon and escapees confirmed and quantified the levels of European alleles and their introgression in Canada [SNP genotyping of historical fish scales, e.g., in sockeye salmon, us nerka , and bon pallasi , has beeNP array ,181. Attanalysis . Two Soč. trutta . Non-purlifornia . Populatreported . Genetic. mykiss . The cutn Canada . Subsequn Canada .Haliotis laevigata) is an endemic Australian shellfish species targeted by fisheries and stock enhancement [Greenlip abalone  in Canada [Sciaenops ocellatus, were grown in three ponds and genotyped using a panel of 2000 SNPs [Oncorhynchus nerka, which allowed for their economic survey [The first condition for the protection of natural populations and their proper management is the identification of species’ genetic characteristics of their populations. Molecular markers such as mtDNA, microsatellites and MHC turned out to be sensitive enough to find a population differentiation generally congruent with the genomic methods based on the genotyping of a large number of SNPs in the conservation units of coho salmon (n Canada . Howevern Canada . Neverthn Canada . Broodfi000 SNPs . It has c survey .Sphyrna lewini) is a critically endangered shark species characterized by a circum-global distribution in tropical and subtropical ocean zones. Genotyping with over 5000 SNPs confirmed the differentiation of Gulf of California samples from other regions of the Indo-Pacific, a lack of gene flow between populations from the Seychelles (Indian Ocean), Hawaii and Gulf of California and central Indo-Pacific locations and significant STF among distant geographic regions [Clupea harengus, in the North and Baltic Seas [The scalloped hammerhead  was more diagnostic than the 18 microsatellites. This diagnostic panel of SNPs can be optimized further for identification of all 37 Oreochromis species and their hybrids. Such a panel of SNPs can be very useful in conservation activities directed at protection and restitution of native populations from hybridization and introgression of the introduced alien species originating from aquaculture.Recent research encompassed 12 aracters . Whole-gLates japonica. This freshwater species is distributed only in Kochi and Miyazaki areas in Japan, where two populations were identified using a set of 780 SNPs obtained from ddRAD-seq (restriction site—associated sequencing) [Brachionichthys hirsutus), which are recommended to be considered as separate conservation management units by the authors [Patella candei distributed only in the northeast Atlantic Macaronesian islands  have been identified with over 3000 SNP [Raja clavata samples including 159 individuals from the Bay of Biscay [SNPs are also useful in studies of the genetic structure of endemic species with local distribution only, such as Japanese lates (akame), uencing) . In Tasm authors . The thr3000 SNP . SNPs caf Biscay  revealedThe use of a large number of genotyped SNPs makes it possible to identify the outlier loci and determine their correlation with environmental conditions, such as salinity, temperature, precipitation, food accessibility or migration distance and timing, and thus indicate possible adaptations . HoweverOncorhynchus tshawytscha [Salvelinus alpinus in Gander Lake in Newfoundland, Canada [Hypophthalmichthys nobilis) from the Yangtze River [Understanding the interactions between organisms and the environment requires the construction of genetic maps and the identification of quantitative trait loci (QTLs) related to candidate genes associated with the phenotypic traits influencing adaptations . Most trawytscha . The app, Canada . An outlze River ,201.Gadus morhua) populations, 40 outlier SNPs have been associated with the temperature in populations on both sides of the North Atlantic, which indicates the existence of discrete regions of genomes (linkage groups) under directional selection and may represent local adaptations [O. mykiss) also have been reported [Clupea harengus stocks differing in spawning time in North Atlantic, the genetic bases for adaptation were related to mutations in the TSHR gene and retrotransposon insertion [S. salar), a genome-wide association study (GWAS) was performed using a pool-sequencing approach on fish from rivers and cultures in Norway [Diplodus sargus and striped red mullet Mullus surmuletus revealed outlier SNPs correlated with temperature in the latter only [Prionace glauca was thought to have a panmictic world-wide population. The genome scan (RAD-DArT sequencing) enabled the discovery of over 37,000 SNPs [Of over 1600 SNPs genotyped in cod  [citlr7) gene [C7N1 [Ctenopharyngodon idella. In golden pompano (Trachinotus ovatus) from China, out of over 700,000 SNPs surveyed, four were significantly associated with hypoxia tolerance [Nemadactylus macropterus) from around New Zealand and Tasmania [A GWAS was used to find loci associated with resistance to rotten body disease caused by scuticociliates in large yellow croakers ( crocea) . A larger7) gene  and one ne [C7N1  in grassolerance . A correTasmania .Parastichopus californicus) [Homarus americanus) populations are under fishery pressure and have weak genetic structure on the Atlantic coast of North America as shown with neutral markers. In an extensive survey of over 14,000 SNPs, putatively adaptive SNPs located in such genes as a thermal stress response, salinity tolerance and growth demonstrated a higher sensitivity for finding a fine-scale population genetic structure and the capabilities of the identification of management units [Carcinus maenas is invasive on the Pacific coasts of North America and has been found to have some SNPs linked to a chromosomal inversion associated with latitude and winter temperature [MYH in Pinctada fucata martensii had 18 out of 95 SNPs discovered in the exons region, were significantly associated with growth, of which 2 were confirmed in 2 populations [Nerita yoldii north of the Yangtze River estuary, neutral SNPs confirmed that it consisted of founders and was distinct from the southern source population [SNP alleles associated with the warm temperature of the sea floor were identified in the giant California sea cucumber (ornicus) . Americant units . Europeaperature . In the ulations . In the pulation . The newMicropterus salmoides), in natural conditions such as small lakes (trout Oncorhynchus mykiss), or fish (guppy Poecilia reticulata) transferred in creeks between environments with different pressures of predators isolated by waterfalls. It has been shown that fishing selection can have similar effects to the pressure of natural predators. The effects observed were changes in the time to reaching maturity for reproduction, size and growth rate, rapid changes in phenotypic features and life cycles [Danio rerio originated from natural conditions and caused evolutionary changes in life cycles in the 5th generation: increased energy allocation for reproduction, reduced adult size (breeding maturity) and less exploratory behavior and boldness [Danio rerio caused a phenotypic selection in growth, metabolism and social behavior [Since the late 1990s, there have been publications indicating that fishing can be a selection factor inducing genetic changes and evolution in the exploited populations. Experimental studies of the impact of fishing on genetic changes in fish populations were carried out on several species of fish . Common e cycles . The expboldness . The obsboldness . Also unbehavior . A genomMenidia menidia, were created in tanks of 1100 individuals each [Six captive populations of the marine Atlantic silverside fish, als each . Two popals each . A reducSander vitreus [Coregonus clupeaformis, the Lesser Slave Lake population experienced a collapse due to overfishing and in 1965–1972 a ban on fishing was imposed. Out of 61 SNPs located in the genes, one was identified as a candidate SNP under divergent selection, which may be explained as an effect of overfishing [O. mykiss was more intensive in comparison with slow-growing wild genotypes in two small lakes stocked for research purposes in southwestern British Columbia, Canada. However, no changes in the genetic composition as analyzed with 349 neutral SNPs have been observed, despite the collapse of the population of the European anchovy, Engraulis encrasicolus in the Biscay Bay in the period 2002–2010 [Gadus morhua archival samples collected in 1907  and 1940  and modern samples in 2013 (Canada) and 2011 and 2014 (Norway) from these same locations using 346,290 SNPs obtained by whole-genome sequencing did not reveal differences in the genetic diversity in populations from before and after periods of overfishing and a rapid decline of the age of maturation [Thunnus alalunga, populations over time [Using approximately 9000 SNPs, changes in the structure of harvested populations, neutral diversity and signatures of selection have been reported in Mistassini Lake, Quebec for 15 years, whereas no such changes have occurred in the reference, weakly exploited population of walleyes,  vitreus . Populat vitreus , and therfishing , the gil002–2010 . A compaturation . No overver time . Hutchinver time  in theirThere is growing awareness that local populations as well as genetic structures on a large scale in marine and freshwater environments should be protected, along with the ecosystems to which they belong. An increasing number of aquatic species has been studied for genetic polymorphisms, which extends the knowledge on their natural populations. In previous years, population polymorphism studies were carried out using various genetic markers, some of which turned out to be particularly useful and enabled obtaining results on a local and macro-geographical scale comparable in different laboratories, e.g., allozymes, mtDNA, single nuclear DNA loci, restriction fragment length polymorphism (RFLP) and microsatellites. In the last decade, thanks to the progress of genomics and next-generation sequencing (NGS) methods, the use of single nucleotide polymorphism (SNP) markers has become particularly popular in population genetics studies. Although the reduction in the cost of sequencing whole genomes makes it possible to use it in population studies, it does not yet apply to non-model species. It allows the discovery of millions of SNPs, from which a smaller or larger number  can be selected for population genetics research. However, with such a large number of SNPs, a subset of diagnostic SNPs can be used for cost-effective genotyping in individual laboratories. It should be noted that the use of these revolutionary markers in population genetic studies confirmed the results obtained earlier with other markers, e.g., allozymes or microsatellites, and substantially extended them. The advantage of using SNPs lies not only in the quantity, but also in the possibility of identifying neutral loci showing differentiation as well as loci under the action of selection. Moreover, the use of genetic mapping enables the discovery of SNPs associated with candidate genes localized in quantitative trait loci (QTLs) of interest and finding favorable genotypes in natural populations. This could be helpful in the application of gene-editing technology for aquaculture purposes.The most successful application of SNPs in population genetics lies in their ability to identify populations on a local and intercontinental scale, which is of fundamental importance for conservation and management. SNPs can be used for the precise identification of the origin of introduced populations, finding interactions between natural and introduced or invasive populations and species, identification of hybrids, levels of inbreeding and estimating effective population sizes with the linkage disequilibrium method. Enhancement leads to a reduction in the diversity of the population compared to other wild populations because the number of broodfish contributing to stocking material production is usually too low. Domesticated broodfish should be replaced with individuals originating from wild populations.SNP genetic data can be used for finding the adaptations of populations to changing environmental factors. Neutral and outlier SNP alleles can be correlated with the environmental variables and show usually weak linkages. The genotyping of a larger number of SNPs located in genes with known function can reveal adaptations to the specific environmental drivers, such as temperature, salinity, oxygen, pH, precipitation, etc. Such knowledge will enhance the power to predict the ability of populations to adapt to expected climate changes and help to implement more efficient conservation prioritization. Studies conducted on natural populations and stocks in aquaculture enabled the identification of QTLs, including SNPs located in genes, which regulate quantitative characters such as growth, age and size of fish at maturity, resistance to pathogens, stress and tolerance to varied environmental conditions. Fish stocks subjected to strong size-selective fishery pressure under experimental or semi-natural culture conditions change their performance and experience alteration in genetic composition  over several generations depending on the individual size. The negative measurable effects on populations affecting genetic polymorphism can be expected from a reduction in the effective population size below 100 individuals, which is very rarely the case in marine environments and is more frequent in fragmented freshwater creeks, ponds and periodic rivers. Fishery-induced selection against fast-growing and bold fish could be observed in wild populations if QTLs and SNPs located in the candidate genes are identified. In the case of polygenic traits, the fishery-induced evolution may not be detectable in wild populations.It is postulated that protection measures should encompass not only pristine endangered or overfished natural populations, but also non-endangered species populations from unnecessarily released hatchery animals. In the case of the foreseen extinction of local populations, supportive breeding and enhancement of populations can be considered as an ultimate measure for rescuing declining populations. For the purposes of population analyses, it is imperative that the most appropriate set of baseline and reference populations or groups of individuals be chosen. Otherwise, the low number of loci used in an economical survey can determine inappropriate and misleading results, as an occurrence or lack of temporal changes in a population, or the rejection or confirmation of the presence of a taxon in a geographic region under study. The full potential of SNPs in studies of populations has yet to be explored in population genomic research.


Agave potatorum Zucc. locally known as Tobalá, is an important species for mezcal production. It is a perennial species that takes 10 to 15 years to reach reproductive age. Because of high demand of Tobalá mezcal and the slow maturation of the plants, its wild populations have been under intense anthropogenic pressure. The main objective of this study was to estimate the genome-wide diversity in A. potatorum and determine if the type of management has had any effect on its diversity, inbreeding and structure. We analyzed 174 individuals  from 34 sites with a reduced representation genomic method (ddRADseq), using 14,875 SNPs. The diversity measured as expected heterozygosity was higher in the nursery and wild plants than in cultivated samples. We did not find private alleles in the cultivated and nursery plants, which indicates that the individuals under management recently derived from wild populations, which was supported by higher gene flow estimated from wild populations to the managed plants. We found low but positive levels of inbreeding (FIS = 0.082), probably related to isolation of the populations. We detected low genetic differentiation among populations (FST = 0.0796), with positive and significant isolation by distance. The population genetic structure in the species seems to be related to elevation and ecology, with higher gene flow among populations in less fragmented areas. We detected an outlier locus related to the recognition of pollen, which is also relevant to self-incompatibility protein (SI). Due to seed harvest and long generation time, the loss of diversity in A. potatorum has been gradual and artificial selection and incipient management have not yet caused drastic differences between cultivated and wild plants. Also, we described an agroecological alternative to the uncontrolled extraction of wild individuals. Agave L., is a species-rich genus, with over 200 described species native to America’s arid lands . Although the agroecological management at the SJR nursery can be considered a success, other A. potatorum producers should be aware of the importance of reducing the relatedness among cultivated individuals. Seeds should be collected from different mother plants and localities. These practices will decrease inbreeding and maintain the genetic diversity of the cultivated individuals. Nevertheless, it is also essential to maintain local adaptation and avoid outcrossing depression; therefore, the ecological characteristics of the habitat should also be considered. We believe that a global breeding program can also be implemented for A. potatorum where the wild samples’ origin, initial genetic diversity and differentiation are registered. These practices will reduce inbreeding, inbreeding depression and serve as a gene bank for future breeding necessities and for adapting to the global changes.The uncontrolled extraction of wild individuals and seeds increases the extinction risk of the populations  and, evein situ conservation efforts hinge on a careful consideration of the demography patterns of the wild populations, which can effectively serve as germplasm banks [On the other hand, successful sm banks . Additioin situ, [Given the absence of significant genetic differences between wild and cultivated populations, we propose the reintroduction of a percentage of nursery-raised plants into the wild populations (as it has been done in SJR and SLA). Also, where appropriate, implementing agroecological management for this species in situ,  is recomin situ, , as wellin situ, .We found higher genetic diversity in the wild and nursery than in cultivated samples. Moreover, no private alleles related to management type (under cultivation and nursery) were found, meaning that most of the managed samples were recently derived from natural populations. Inbreeding levels varied widely among studied samples, apparently associated with isolation and fragmentation, but unrelated to human selection and management. Genetic differentiation at the species level was low. However, we found three genetic groups. Also, we identified higher gene flow in populations growing in less fragmented habitats. Furthermore, elevated gene flow from wild populations to the other two types of management was detected. Finally, we found loci with possible selection signals mainly related to biological processes and apparently to self-incompatibility.A. potatorum.As a conservation proposal, we suggest following the examples of SJR and SLA, with the production of plants in nurseries to avoid over-exploitation of wild plants and with the use of agroecological management, together with analysis of breeding and genetics, in order to trace the lineage of the plants and prevent inbreeding processes in populations of S1 File(DOCX)Click here for additional data file.


Ramulus mikado across Japan, based on a multifaceted molecular approach . Subsequently, we identified unique phylogeographic patterns, including the discovery of identical COI genotypes spanning considerable distances, which substantiates the notion of passive long-distance genotypic dispersal. Overall, all the molecular data revealed the low and mostly non-significant genetic differentiation among populations, with identical or very similar genotypes across distant populations. We propose that long-distance dispersal facilitated by birds is the plausible explanation for the unique phylogeographic pattern observed in this flightless stick insect.Exploring how organisms overcome geographical barriers to dispersal is a fundamental question in biology. Passive long-distance dispersal events, although infrequent and unpredictable, have a considerable impact on species range expansions. Despite limited active dispersal capabilities, many stick insect species have vast geographical ranges, indicating that passive long-distance dispersal is vital for their distribution. A potential mode of passive dispersal in stick insects is via the egg stage within avian digestive tracts, as suggested by experimental evidence. However, detecting such events under natural conditions is challenging due to their rarity. Therefore, to indirectly assess the potential of historical avian-mediated dispersal, we examined the population genetic structure of the flightless stick insect  The devConsequently, flightless insects offer intriguing models for examining population genetic structure, with numerous species demonstrating substantial genetic differentiation between populations within relatively small geographical distances ,6. PhasmTimema) is regarded as a crucial innovation in phasmid evolution [Stick insects display remarkable masquerade crypsis as a defensive mechanism against visually hunting avian predators, by morphologically and behaviourally imitating twigs, bark, lichen, moss and leaves ,12. Addivolution . This alvolution ,14.The sturdiness of phasmid eggs might allow them to remain viable even when contained within gravid female stick insects consumed by avian predators ,15. ThisDue to their infrequent occurrences, it is likely challenging to demonstrate that avian predation serves as a factor promoting dispersion in natural settings . One metRamulus mikado, a predominantly parthenogenetic stick insect species found commonly in Japan, with only a few documented instances of males [R. mikado (= R. irregulariterdentatum) eggs in experimental conditions  markers and genome-wide single nucleotide polymorphisms (SNPs) to determine the phylogenetic relationships among individuals collected throughout the species distribution range. Mitochondrial sequence analysis with maternally inherited markers is anticipated to reveal a genetic structure similar to that of sexually reproducing species, even in predominately parthenogenetic species. SSR markers, which have high mutation rates, are probably appropriate for detecting limited intraspecific genetic variation in parthenogenetic species. Additionally, genome-wide SNP markers can offer more reliable data for estimating kinship relationships among individuals sharing a common parthenogenetic ancestor.By integrating the results from these three genetic markers, each with distinct advantages, we have examined the intraspecific phylogeographic pattern of parthenogenetic and flightless stick insect species with unparalleled resolution. Consequently, we discovered unique phylogeographic patterns in the flightless stick insect, such as the identification of identical mitochondrial DNA cytochrome oxidase subunit I (COI) haplotypes across significant distances, which do not contradict the hypothesis of passive long-distance genotypic dispersion.. 2 (a)Ramulus mikado is a predominantly parthenogenetic and flightless stick insect widely distributed throughout Japan  was employed to extract total genomic DNA, following the manufacturer's instructions. This extracted DNA was used for the subsequent molecular analysis.For data analysis, three populations were defined based on their respective regions: SI, WH and EH. To obtain genomic DNA,  (b)http://www.geospiza.com/finchtv/). The assembled sequences were aligned with all analysed samples using CLUSTAL W [For the analysis of genetic diversity, phylogenetic relationships among haplotypes, and their geographical distribution among three populations, we determined the partial sequences of the mitochondrial DNA cytochrome oxidase subunit I (COI) gene. The COI gene fragment was amplified and sequenced using primers LCO_Phas3 (5′– AAC TCA GCC ATT TTA CTA ATG AAA CG –3′) and HCO_Phas1 (5′– TAT ACT TCT GGA TGA CCA AAA AAT CA –3′), which were designed based on the sequences of related taxon found in the DNA Data Bank of Japan (DDBJ). PCR amplification was conducted in a GeneAmp PCR System 2700 thermal cycler . The PCR products were purified by using a High Pure PCR product purification kit , and purified products were sequenced directly using an ABI BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems) on the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Both strands of the amplified PCR products were sequenced, and electropherograms were assembled using Finch TV R. mikado and designed corresponding SSR primers . We employed these newly designed primers to determine genotypes for analysing genetic diversity among the three populations, phylogenetic relationships among multilocus SSR genotypes and their geographical distribution. We performed PCR amplification of the 13 SSR loci using 5 µl reactions with the QIAGEN Multiplex PCR Kit and a fluorescent dye-label protocol [We successfully isolated 13 SSR loci from the nuclear genome of protocol . Each reA), allelic richness (RS), observed heterozygosity (HO), expected heterozygosity (HE) and the fixation index (FIS). These parameters were calculated using GenAlEx 6.5, except for allelic richness, which was calculated using FSTAT 2.9.3 [DA [DA was evaluated for (1) all populations, (2) SI and WH populations, (3) WH and EH populations, and (4) each of the three populations through Mantel tests. Mantel tests were performed using GenAlEx.We established multilocus genotypes based on 13 SSR loci genotypes. The probability of identical genotypes resulting from sexual reproduction was calculated using GenAlEx 6.5 . For eacAT 2.9.3 . To evalAT 2.9.3  and perf.9.3 [DA  among mu.9.3 [DA  was used (d)MIG-seq is a recently developed genome-wide genotyping methodology employing a high-throughput sequencing platform . This tem) = 3, maximum distance permitted between stacks (M) = 2 and the number of mismatches allowed between sample loci during catalogue construction (n) = 2. SNP sites containing fewer than three minor alleles were filtered, and only SNPs retained by 33 or more samples were extracted. We restrict data analysis to only the first SNP per locus to avoid linkage between SNPs. The SNP filtering for excess heterozygosity was not performed because of the predominantly parthenogenetic reproduction of R. mikado [. Ultimately, 980 SNPs were procured for subsequent analyses. During these processes, one sample (EH09) was excluded due to its high missing rate. The nucleotide diversity (ND), observed and expected heterozygosity was assessed by the program populations of Stacks. The number of allelic differences between two individuals was calculated using R package poppr 2.9.4 [FST values were calculated, and the probability of each pairwise FST value being different from zero was tested based on 999 permutations using GenAlEx 6.5 [p-distance matrix and disregarding ambiguous sites, with the use of SplitsTree version 4.15.1 [Upon eliminating primer sequences and low-quality reads , 9 558 5. mikado . Ultimatpr 2.9.4 . OverallAlEx 6.5 . A neighn 4.15.1 . Moreoven 4.15.1 . The cor. 3 (a)R. mikado due to parthenogenetic reproduction. The observed number of alleles at the 13 newly developed loci ranged from 1 to 20 . The observed heterozygosity for eight loci was either 0 or nearly 0, while the remaining five loci displayed high values ranging from 0.64 to 1.00. From the 67 samples, 55 multi-locus genotypes were identified. The combined random match probability for the 13 loci was 5.53 × 10−6, with a high power for discriminating among individuals. The MIG-seq-based SNP heterozygosity (the highly heterozygous and homozygous loci) corresponds to these SSR heterozygosity patterns .Genetic analysis based on mitochondrial COI sequences and nuclear SSR markers revealed a notable accumulation of mutations in HD and the nucleotide diversity of COI sequences, as well as the allelic richness RS of SSR markers, which are crucial indicators for assessing genetic diversity, were relatively high in the SI and WH populations and low in the EH population . Each FST value calculated from mitochondrial sequence data and genome-wide SNP data was significant for every pair of populations, while the pairwise FST value between the EH and WH populations did not exhibit a significant difference when assessed using SSR genotype data a,c and 4a,c). However, aside from these few instances, the correspondence between phylogenetic relationships and distribution was not evident. In all analyses of genetic markers, individuals with close genetic relationships were found to be dispersed across different populations. This pattern remained consistent across distinct methods employed in phylogenetic analysis and measures of genetic distance, such as the construction of a most parsimonious network based on COI sequence data, as well as the use of neighbour-joining trees based on Nei's genetic distance [Our molecular analyses, which employed COI haplotypes, SSR markers and MIG-seq analysis, revealed limited associations between lineage and geographical distribution –5, with 3distance  and Cavadistance .Figure a,c). Although a weak yet significant correlation was detected in the SSR data , the R2 value was low (R2 = 0.041). Notably, genetically very similar individuals are found up to approximately 700 km in the mitochondrial sequence data and up to about 600 km in the SSR data. The combinations of individuals with the highest level of genetic distance between individuals were observed even at a distance of about 100 km in all three genetic markers.　Within the samples merging Western Honshu (WH) and SI, ‘isolation by distance' was not observed based on COI haplotypes, SSR and SNP data . By contrast, within Eastern Honshu (EH), genetic distance exhibited a significant positive correlation with geographical distance based on all molecular methods. This correlation was particularly pronounced in SSR markers .The Mantel test results, examining the correlation between genetic distance and geographical distance within specific regions, yielded different outcomes among regions . The Manfigure 5. 4R. mikado . This pattern, also observed in genome-wide SNP data , suggests the predominance of asexual reproduction, though historical cryptic gene flow cannot be excluded [R. mikado [Our SSR analysis shows high heterozygosity in five loci and zero or near-zero in the remaining eight loci in excluded . This as. mikado  but also. mikado . The pat. mikado ,64. Auto. mikado . The mixR. mikado individuals  exhibit significant genetic differentiation even on a small spatial scale [R. mikado (0.7–1.0 Ma versus 0.34–0.51 Ma) [Although al scale –30. The al scale ,69. This0.51 Ma) ,25, outpR. mikado, did not expand until after the last glacial period [Distinct patterns in the EH population, which is probably a recently formed population following the last glacial period, may also support the infrequency of long-range dispersal. Pollen analysis indicates that coniferous forests predominantly covered this region during the last glacial period, while temperate broadleaf forests, which serve as suitable habitats for l period . The lowR. mikado populations is probably facilitated by long-distance dispersal. Internal dispersal through the digestive tract of stick insect predators is a plausible mechanism for dispersal, particularly considering the demonstrated resilience of their eggs in withstanding passage through avian digestive tracts [Megacrania, possess sponge-like eggs that can float for long periods without needing to be attached to vegetation [R. mikado since their eggs do not possess either a sponge-like structure or the ability to stick to the branches.As discussed earlier, the low and mostly non-significant genetic differentiation among e tracts . Oceanice tracts . Additiogetation . HoweverR. mikado and humans. Thus, the somewhat plausible human-mediated dispersal of R. mikado is accidental long-distance transportation with plant seedlings in the past few centuries, coinciding with the advent of steam engines. In such recent human-mediated transportation, genetic variation typically does not accumulate after dispersal, and specific genotypes associated with human transport can be identified [R. mikado specimens indicates the presence of certain levels of genetic variations, with several identical or very similar genotypes widely but disjunctly intermixed across long distances. This pattern deviates from the typical patterns observed in recent human-mediated dispersal.While anthropogenic translocation is another possible explanation ,72,73, ientified ,75. HoweClitarchus stick insects in New Zealand show distinct genetic structuring in populations with sexual reproduction, whereas populations with parthenogenetic reproduction, which enables internal dispersal, demonstrate a much weaker structure [Therefore, internal dispersal via predation provides a plausible explanation for the unexpected phylogeographic pattern. In fact, similar non-significant geographical differentiation can be observed in certain plants dispersed by birds in Japan, where the majority of haplotypes are widespread throughout the country ,77. Howetructure . The contructure ,81, the Corvus macrorhynchos), Eurasian jay (Garrulus glandarius), brown-eared bulbul (Hypsipetes amaurotis), bull-headed shrike  and eastern buzzard (Buteo japonicus), have the potential to aid in the passive dispersal of R. mikado due to their consumption of stick insects in Japan [Hypsipetes amaurotis migrate southward during autumn and winter [Macaca fuscata and the marten Martes melampus feed on stick insects in Japan, and stick insect eggs are frequently excreted in the faeces of the latter [R. mikado.Various bird species, including the large-billed crow (in Japan ,82. For d winter . Consided winter . While cd winter . As largd winter . Omnivore latter ,87. ThesR. mikado eggs can potentially survive avian gut passage in the wild, enabling occasional long-distance dispersal. Our finding presumably provides new perspectives by challenging the longstanding notion that insects invariably perish when consumed by predators.Overall, we have identified distinct phylogeographic patterns, including instances where identical COI genotypes are found at sites that are geographically distant and disconnected. This supports the idea of passive long-distance dispersal of the genotypes. The question of how organisms with limited active dispersal capabilities achieve extensive distribution has captured curiosity since the time of Darwin . Based o


Pieris rapae), a major agricultural pest, has become one of the most abundant and destructive butterflies in the world. It is widely distributed in a large variety of climates and terrains of China due to its strong adaptability. To gain insight into the population genetic characteristics of P. rapae in China, we resequenced the genome of 51 individuals from 19 areas throughout China. Using population genomics approaches, a dense variant map of P. rapae was observed, indicating a high level of polymorphism that could result in adaptation to a changing environment. The feature of the genetic structure suggested considerable genetic admixture in different geographical groups. Additionally, our analyses suggest that physical barriers may have played a more important role than geographic distance in driving genetic differentiation. Population history showed the effective population size of P. rapae was greatly affected by global temperature changes, with mild periods  leading to an increase in population size. Furthermore, by comparing populations from south and north China, we have identified selected genes related to sensing temperature, growth, neuromodulation and immune response, which may reveal the genetic basis of adaptation to different environments. Our study is the first to illustrate the genetic signatures of P. rapae in China at the population genomic level, providing fundamental knowledge of the genetic diversity and adaptation of P. rapae.The cabbage white butterfly ( Exploring the effects of changeable environments on genetic diversity of a species can elucidate its evolutionary history and underlying adaptations . Pest poPieris rapae, is a lepidopteran pest whose larvae feed on plenty of cash crops such as Brassica juncea, Cleome spinosa and Tropaeolum majus [P. rapae has been dispersed from eastern Europe to inhabit every continent except South America and Antarctica [P. rapae, this butterfly still causes considerable economic losses in China annually. P. rapae has adapted to an abundant and predictable agricultural resource through a narrowing of niche breadth and loss of genetic variants [P. rapae is not only essential for understanding the distribution pattern of this butterfly, but also has great implications for uncovering the genetic basis of adaptation to the local environment.The notorious cabbage white butterfly, um majus . Its lartarctica , 9. In mtarctica , 11, thavariants . ExploriP. rapae have mainly focused on morphology, control methods, immune mechanisms and transcriptomics [P. rapae is limited due to the lack of studies at the genomic level. Population genomics of P. rapae has been hampered by limitations in obtaining sufficient samples and high-resolution molecular markers. Spurred by rapid developments in sequencing technology, increased data production is accompanied by a drive to develop efficient computational approaches to interpret patterns of genetic variation at the genomic scale [P. rapae has been sequenced and annotated successively [At present, studies on iptomics –16. Howeic scale . Genome-ic scale . In receessively , providiP. rapae from different locations across China were collected and the whole genomes of a total of 51 individuals were resequenced. We investigated genomic polymorphisms, population structure, and demographic history in these butterfly populations. Moreover, the rapidly evolving genes related to adaptation to local environments in P. rapae were also identified. This study provides insights into the genetic diversity and evolutionary history of P. rapae, as well as theoretical support for the population genome study of the cabbage white butterfly.In this study, 19 feral colonies of 51 adult butterflies were collected from 19 areas in China, covering multiple climates from temperate to tropical and various terrains such as plains, mountains, basins, and islands. These butterflies have been preliminarily divided into four major populations based on their geographical location, with 18 individuals in the north population, 10 in the coastal population, 11 in the southeast population and 12 in the southwest population . We employed fastp  to filteP. rapae reference genome  using Burrows-Wheeler Alignment (BWA) aligner [95.8 Gb of clean reads were retained and mapped to the latest version of the  aligner  with the aligner . Polymer aligner  with the aligner  was used7 raw SNPs were detected. To obtain reliable SNPs, we set the following filtering criteria using vcfutils and vcftools [Single nucleotide polymorphisms (SNPs) and insertion-deletion sites (INDELs) were detected using sentieon  with thevcftools : (1) SNPAfter applying the above filters, high-quality clean data were retained for downstream analyses. Subsequently, the table_annovar.pl script provided by the ANNOVAR  was usedIn addition, we utilized BreakDancer  to detec2) ≤ 0.2 was used as the input file. The optimal K was determined by the lowest cross-validation error. Principal component analysis (PCA) with biallelic SNPs was performed using the PLINK tool [https://itol.embl.de/) was then utilized to visualize the phylogenetic tree.To further estimate individual admixture, we used ADMIXTURE v1.23 software  to invesINK tool , and theINK tool  with 100−9 per site per generation [2) ≤ 0.2 as the input file. A maximum likelihood tree was inferred according to the hypothetical number of gene flows, and the direction of gene flow was calibrated on the tree.We applied the pairwise sequentially Markovian coalescent (PSMC) model analysis  to traceneration , and theneration  was applST) between populations, nucleotide diversity (θπ), and Tajima’s D was calculated using vcftools software [Genetic differentiation index  between any two loci in each population, with the parameter of MaxDist set to 2, i.e. the maximum distance calculated by r2 was 2 Kb. The LD decay curve was then plotted using a Plot_MultiPop.pl script (provided with the PopLDdecay software).To assess the Linkage Disequilibrium (LD) pattern in cabbage white butterflies, PopLDdecay software  was usedP. rapae individuals from southwestern and southeastern areas into south populations to compare with north populations. In general, positive selection results in higher genetic differentiation of loci between populations and lower genetic diversity within populations [ST values and θπ ratios with a sliding-window method (100-kb windows with 10-kb step). Z transformation for FST values and log2 transformation for θπ ratios were applied. The windows with the top 5% Z(FST) and log2 (θπ ratio) values were simultaneously considered as the candidate outliers under strong selection, and the genes in the outlier windows were screened as selected genes.To further investigate the effects of different latitudes or climates on this butterfly, we combined the ulations . In ordeP. rapae, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The reference coding sequence of P. rapae was proactively annotated on the eggNOG-MAPPER online website (http://eggnog-mapper.embl.de/) to obtain GO and KEGG background annotation information. An R package, clusterProfiler [In an effort to further explore the role of these selected genes in the adaptive evolution of Profiler , was utiP. rapae indicated an average depth of 9.5× and coverage of 93% relative to the reference genome . The cross-entropy error rates were lowest for K-values of 2–4 . When K Next, a maximum likelihood tree was constructed to infer the phylogenetic relationship between different geographical groups . We foun6. Then, a dramatic population decline of the four populations occurred during Xixiabangma Glaciation  and Naynayyxungla Glaciation (780 to 500 thousand years ago) [P. rapae. Unlike other populations, the southwest population continued to decline even in warmer climates. Notably, over the last 10 000 years, the effective population sizes of the north and southeast populations have reached nearly 700 000 and 750 000, respectively, which is 3–4 times that of the southwest and coastal populations.We inferred the historical effective population size to gain a better understanding of the demographic history in this butterfly . The popars ago) . Subsequars ago) , the lasP. rapae, the development of the Silk Road of maritime trade likely facilitated the introduction of this butterfly into Asia [P. rapae in China.Populations of a species often have complex histories that contribute to their current genetic makeup. For further insight into population history, we also investigated gene flow between populations. Significant gene flow was detected from the coastal population to the southwest population . During nto Asia . It is sST to quantify population genetic differentiation  using whole genome-wide SNP data. The heterozygosity of the four geographical groups did not differ much, but the level of heterozygosity was high, with an average heterozygosity of 0.231. This value is much higher than that of Lepidopteran model insect silkworms (0.0032) and wild silkworms (0.0080) [−3), whereas the isolated coastal population showed the lowest π value (π = 4.08 × 10−3). The two southern populations, namely southwest and southeast, displayed similar θπ, which ranged between north and coastal groups (To explore the genetic diversity among (0.0080)  Fig 6A)P. rapae (0.0080) . The norl groups .2 decreasing to 0.15 at only 62bp and tending to stabilize after falling to 0.08. In contrast, coastal populations had the slowest LD decay, with r2 decreasing to 0.15 at 441 bp. The two southern populations exhibited similar LD decay patterns, with r2 decreasing to 0.15 at 139 and 148bp, respectively , we mainly focused on the identification of genomic signatures of positive selection between the two populations. Using Fpulation .P. rapae, including the lipid and creatine metabolism , synaptic structure , the Notch signaling pathway , the ErbB signaling pathway , and the TGF-beta signaling pathway    . Further × 10−7) .−16) and “defense response to fungus”  being particularly significant, suggesting the importance of resistance to pathogenic microorganisms in the adaptation of P. rapae to northern environments.In the northern cabbage white butterfly, the biological process of “defense responses”  is highl−11), MAPK signaling pathway , Toll and Imd signaling pathway  and melanogenesis , which may indicate that the immune response may be involved in the positive selection during evolution of this butterfly.Moreover, a number of immune-related pathways were found to be significantly enriched in both the south and north populations , includiST analyses suggest that physical barriers may have contributed more to genetic differentiation than geographic distance, although we acknowledge that distance may also have played a role. Demographic history suggests that historical global temperature has profoundly affected the effective population size, with a colder climate  inhibiting population growth, while warmer periods  have been associated with increased population size. Finally, we identified genes associated with positive selection.Studying pests at the genomic level not only provides insights into their structure, history and adaptation, but also presents novel approaches to pest control. We have found a dense map of variation in the butterfly genomes, with large genetic admixture between different geographical groups. This is a dense variation compared to published data on model insect variation, fruit flies , and humP. rapae is not easily distinguished based on geographical location, with terrain features being more likely to influence the population’s genetic structure than geographical distance. In particular, populations inhabiting areas with closed terrain, such as Chongming (CM) with island terrain, Shandong (SD) with peninsular terrain, and southwest areas  dominated by mountains and basins, show significant divergence from other populations. Like other lepidopteran pest populations [P. rapae exhibits considerable genetic admixture among geographically connected groups, based on ADMIXTURE results and PCA analyses. This may be attributed to the rapid dispersal of P. rapae, allowing for gene flow among populations in the absence of physical barriers. The global invasion of the cabbage white butterfly is a prime example of this potential for rapid dispersal, with its geographic spread and divergence being driven by host plant diversification and human trade [The data from our study suggest that the genetic structure of ulations , 44, P. an trade . In ChinST and lower θπ. In contrast, populations that are less isolated  displayed reduced differentiation, higher heterozygosity, fast LD decay, and greater genetic diversity, which is another potential evidence of high gene flow. Genetic diversity is an important factor in the survival and adaptive potential of populations and species [P. rapae may be a key genetic factor for adaptation to different environments and resistance to various insecticides in China.Our analysis also revealed that isolated populations  exhibit higher genetic differentiation and lower genetic diversity, as evidenced by higher pairwise F species . For but species , which m species . Heteroz species , and a h species . Moreove species . Thus, wP. rapae, we found that it fluctuates almost in tandem with global temperature changes, with cold glacial periods leading to significant population declines and warm interglacial periods allowing recovery from population bottlenecks. This mirrored relationship between historical effective population size and temperature has been observed in many other insects [P. rapae from northern and southeastern China are likely to exhibit a more rapid recovery from population bottlenecks and reach a higher effective population size level. Acknowledging the potential impact of population structure on our PSMC model-based inferences is crucial. Although the PSMC method operates under the assumption of panmixia, real populations frequently demonstrate structuring. Conventional population genetics methods typically overlook these structural intricacies, possibly resulting in misinterpretations or inaccuracies when inferring historical population sizes, as in these instances the inverse instantaneous coalescent rate (IICR) corresponds to the effective population size [In exploring the historical effective population size of  insects –52. Popuion size . This ovP. rapae, especially concerning their responses to environmental changes.The abundance of a species is thought to depend on its adaptation to environmental temperature and food resources , 55. StuDrosophila [P. rapae and warming-induced key biochemical pathways have changed significantly in warming environments [Drosophila, those living in the tropics were found to lack genetic variation in tolerance to extreme environments such as dry and cold [P. rapae in south population.The selected genes are identified with an outlier approach, using Fst and θπ, with the 5% right tail determining the threshold. It should be noted that if selection is rare will lead to an overestimate, and the effects of demographic history, purifying and background selection, and variation in recombination and mutation rates were not considered in this research , 58. Theosophila , 61. Morosophila , and theosophila . Evolutiosophila , 65. Preronments . In a stand cold . CompareP. rapae is a variety of selected genes involved in defense responses in northern China. The climate disparity between North and South China results in differing pathogenic microorganisms. The enrichment of genes involved in defense responses is a possible indication of adaptation to the local environment. Delving deeper into these genes may reveal the underlying mechanisms of the cabbage white butterfly’s response to external stimuli. Furthermore, we identified several pathways related to immune response in both the south and north population, including the JAK-STAT signaling pathway, MAPK signaling pathway, and melanogenesis. Unlike mammals, insects rely on their innate immune system to protect themselves from foreign substances and microorganisms [Another important characteristic of positive selection during evolution in rganisms . The JAKrganisms , while trganisms . Melaninrganisms , 72. TheP. rapae in China, utilizing population genomics approaches to provide insights into the population differentiation, structure, demographic history and adaptive evolution of this species. We reveal significant genomic variation and heterozygosity across the entire genome of P. rapae. In addition, we characterized the population genetic structure of P. rapae and uncovered the intrinsic relationships among different geographical groups in China. Geographical distance did not prevent genetic communication between P. rapae populations, while terrain barriers may have a more profound effect on the population divergence of this butterfly. The effective size of the population is strongly influenced by global temperatures. Additionally, we identified the selected genes under different climates to address the potential mechanisms of local adaptation, which will promote a better understanding of how the environment has shaped patterns of genetic variation and rapid evolution that are triggered by selective pressures.Our research has focused on the widespread populations of S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig−8), and the thin red line illustrates the PSMC estimates for 20 rounds of bootstrap.The clear red line indicates the effective population size measured by the scaled mutation rate per generation (0.2×10(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.


Semaprochilodus insignis is a migratory fish of commercial and subsistence importance to communities in the Amazon. Despite the high intensity of exploitation, recent studies have not been carried out to assess the genetic status of its stocks.S. insignis through sequencing of the mtDNA control region (n = 241) and eight microsatellite loci (n = 180) of individuals sampled at 11 sites distributed in the Brazilian Amazon basin.This study is the first to estimate genetic diversity and to test the existence of spatial and temporal structuring of Results for both markers were congruent, revealing a homogeneous genetic diversity in all the sampled locations, in addition to the absence of spatial and temporal genetic structure, indicating that the species forms a large panmictic population in the Brazilian Amazon.S. insignis, signals of reduction of the effective population size and a bottleneck provide an early alert to the effects of overfishing. Thus, the ever-decreasing populations may threaten S. insignis in the future. Therefore, it is hoped that the results of this study may contribute to the elaboration of management plans or any other measures that aim at the management and conservation of this species of great importance for the Amazon basin.Although overfishing does not yet appear to have affected the levels of genetic variability of  Semaprochilodus insignis . Due to its comparatively low commercial value, S. insignis is the main fish consumed by low-income urban population  list of threatened species as overexploited or threatened with overexploitation. The species is a flagship species that plays an important role as an ecosystem engineer  classified the engineer , and itsengineer  makes itS. insignis by sequencing the control region of the mitochondrial DNA and genotyping eight microsatellite loci from individuals collected from 11 localities along the main channel of the Amazon River and its tributaries, which include rivers with different water types, such as black water (Negro River), clear water (Tapajós River) and white water (Solimões-Amazon Rivers). Considering the migratory patterns of this r-strategist species, we expect it to form a single large panmictic population despite being found in a diversity of water types and forming different migratory groups throughout the Amazon basin. In addition, considering the historical situation of overfishing of the species, we test whether populations already show signs of population size reduction in their genetic stocks.The present study examines the population genetics of Semaprochilodus insignis were sampled from small artisanal fishing boats at 11 localities in the Amazonian basin . Sampling collection was undertaken in accordance with the ethical recommendations of the Conselho Federal de Biologia , Resolution 301 .In total, 241 specimens of the an basin , Table 1For the spatial analysis, 241 individuals were sequenced for mitochondrial DNA control region and 180 individuals were genotyped for eight microsatellite loci . The samvia the polymerase chain reaction (PCR) using the primers F-TTF (5′-GCCTAAGAGCATCGGTCTTGTAA-3′) and 12SR5 (5′-GGCGGATACTTGCATGT-3′) obtained from From each sample, the total DNA was extracted following a standard proteinase K, phenol–chloroform extraction protocol . The mtDEight microsatellite loci characterized by ® version (Life Technologies).PCR products generated with labeled primers were visualized on an ABI-3500 automatic sequencer (Life Technologies). Allele sizes were scored against a ROX size standard  and the S. insignis a haplotype network based on a maximum likelihood phylogenetic tree topology was used in the Haploviewer software (FIS) for each locality. Furthermore the number of alleles observed and expected heterozygosities, and linkage disequilibrium (LD) between pairs of loci, and Hardy-Weinberg equilibrium (HWE) were calculated for each locus within the localities. All these measures of genetic variability were estimated in the software ARLEQUIN version 3.5 (For the mtDNA control region we estimated genetic diversity as gene diversity (Ĥ), nucleotide diversity (Π), number of haplotypes and number of segregating sites (S). For the reconstruction of genealogical relationships among individuals of software . For micsion 3.5 . Statistsion 3.5 . Considesion 3.5 , a rarefsion 3.5  that solsion 3.5 . The sofsion 3.5  was usedST (control region) and FST (microsatellite) metrics, we tested the hypothesis of overall population structure , temporal structure , and structure associated with differences in water chemistry. The statistical significance for all analyzes was assessed using 20.000 permutations after adjusting for multiple comparisons   and pairparisons .v. 2.3.4 software  and discarded the first 100,000 as burn in. The most likely K value was estimated using Bayes Factors  , using aclusters .ST/(1 − ΦST) for the control region and FST /(1 − FST) for the microsatellites, and the distances in kilometers representing the shortest river path between each pair of populations  compared to allelic spread (r) is below the critical value 0.68, which indicate that the population has experienced a recent reduction in size, featuring a bottleneck event.Considering the situation of overfishing of the species, we estimated the effective population size (Ne) for each population in the software NeEstimator v 2.0 , using tThe control region data were composed of 241 nucleotide sequences with a total of 1,145 base pairs (bp) of which 1,031 sites were monomorphic and 114 variables. The sequences are available at GenBank: ON972834–ON973074. In the 241 individuals collected along the main channel and tributaries, 116 haplotypes were found. The lowest number of haplotypes was observed in the locality of Careiro (seven) and the highest in Purus and Santarém (27) . The H4 The results of the genetic diversity parameters show that gene diversity was 1 in all locations . The lowS. insignis showed 191 alleles in the eight analyzed loci. Matrix of genotypes is available at https://github.com/legalLab/datasets and https://doi.org/10.5281/zenodo.7641680. The number of alleles per locus ranged from 12 (SinC10 locus) to 31 (SinF05 and SinE08 loci) . The average expected heterozygosity(HE) ranged from 0.79 (locus SinB04) to 0.94 (locus SinE08), with an average of 0.88 per locus (FIS) ranged from 0.021 in Tefé to 0.234 in Manaus, showed statistical significance for eight out of 11 sampled locations ; it also was significant when the analysis was conducted considering a single and large population. Proportion of private alleles ranged from 0.40 in Tefé and Coari to 0.85 in Jacareacanga. Private alleles values above 0.5 were observed in the locations with the greatest geographical distance   .ST values of AMOVA results (p < 0.010). For this locality 22.19% of variance is found between samples from different years, indicatating temporal structure. The values of FST of AMOVA for the temporal analysis based in microsatellite data ranged from −0.00183 for Santarém (2006 × 2008) to 0.01429 for Itaituba (2006 × 2008), however, none of the values were significant  comparison . For the control region the values of pairwise comparisons of ΦST for spatial analysis ranged from −0.008 for Novo Airão and Manaus to 0.148 for Tabatinga and Careiro, however, no value was statistically significant after Bonferroni correction (p < 0.0009)  and Tefé and Jacareacanga  comparisons were significant .The AMOVA based on the spatial analysis revealed that the higher genetic variation was observed within sampled populations (99.65% for control region and 99.19% for microsatellites), and no genetic differentiation was observed among populations (Φ 0.0009) . For thenificant . All Nm S. insignis  nor for microsatellites .The Mantel test results suggest no significant correlation between genetic divergence and geographical distance of sampled localities neither for control region (Ne) ranged from 61.1 in Itaituba to 491.2 in Novo Airão, with nine locations tending to infinity  across populations; all values were lower than the critical value of 0.68 proposed by Garza and Williamson  was significant for all localities, a characteristic that has been observed in studies carried out with populations of other Amazonian migratory fish ) and the piramutaba ), but the schools of jaraquis are seen migrating over hundreds of kilometers. Although there has been no previous genetic study to confirm the hypothesis of S. insignis forming a single panmictic population in the Amazon basin, this characteristic was expected due to the life style of the species, which migrates extensively through a wide-ranging habitat during its life cycle. Semaprochilodus insignis is an r-strategist species which has high reproductive rates, complex schooling behavior, and a migratory and reproductive pattern associated with flooding pulses, typical of the Amazon basin  and the Bottleneck software (heterozygosity) showed that the reduction of the effective population size has been ongoing for some time (Arapaima gigas), a sedentary species and of great economic value for the Amazon region, and also considered overfished in the rivers of the Amazon basin  decrease of census population size indicated by lower fishing efficiency, (2) its adaptive response of reduction in size at first reproduction (S. insignis must be a high conservation priority. Loss of genetic diversity (effective population size) lags behind the loss of census size, and thus provides a window of opportunity for implementing conservation and management strategies that are able capitalize on the still available genetic diversity.Given that oduction , and (3)S. insignis fishing regulations have been implemented in a very irregular way, if at all, and independently by each region of the state of Amazonas . Rather, the entire fishery focuses on harvesting reproductive migrations. Considering the results presented here, S. insignis is clearly showing signs of rapid demographic population collapse and loss of genetic diversity. Compounded on the overfishing, loss of genetic diversity and lack of fishing regulations, a study by S. insignis. The varzea and igapo forests are floodplain forests and integral part of the riverine ecosystem, but also are the main area of human settlement and agricultural conversion and unsustainable resource extraction.In 2007, IBAMA established a closed fishery period to protect selected overfished species . The closed period aims to close fishing during the reproductive season, in order to guarantee the reproduction of the selected species and help in the maintenance of fish stocks. However, despite clear evidence of overexplitation detected as early as 40 years ago , S. insiS. insignis appears to be analogous to that of the north Atlantic cod (Gadus morhua). The north Atlantic cod, a historically abundant r-strategist, has been subject to sustained but sustainable fishery since the Viking period . This will not only decelerate observed negative population trends, but also provide time during which managers and scientists can devise and implement an effective conservation and management plan.The scenario of S. insignis, one of the most important fish species in the Amazon. Here, populations distributed in 11 localities along of Amazon River and their tributaries were analyzed from mitochondrial and microsatellites markers providing information about genetic diversity, temporal and spatial structure and effective population sizes. The results reveal that S. insignis forms a single panmitic population, with high levels of genetic variability and gene flow in the Amazon basin. Despite high levels of genetic variability, S. insignis shows signs of bottlenecks indicating that the species is suffering the effects of overfishing. Therefore, we hope that the results presented in this study will contribute to the elaboration and implementation of future management plans and fishing regulations. Last but not least, we strongly recommend that the species enters on the list of species protected by fishing closure during its reproductive season.This study is the first to explore the population genetics of 10.7717/peerj.15503/supp-1Figure S1Click here for additional data file.10.7717/peerj.15503/supp-2Table S1Click here for additional data file.10.7717/peerj.15503/supp-3Table S2Tab: Tabatinga, Eir: Eirunepé, Tef: Tefé, Coa: Coari, Ppu: Piagaçu-Purus, Mao: Manaus, NAi: Novo Airão, Car: Careiro, San: Santarém, Itai: Itaituba, Jac: Jacareacanga.Click here for additional data file.10.7717/peerj.15503/supp-4Table S3O: observed heterozygosity, HE: expected heterozygosity, P: significance level for Hardy Weinberg equilibrium. * Values in bold correspond to significant P values after Bonferroni correction (<0.000625).Na: Number of alleles, HClick here for additional data file.10.7717/peerj.15503/supp-5Table S4Click here for additional data file.10.7717/peerj.15503/supp-6Table S6Note: Tab: Tabatinga, Eir: Eirunepé, Tef: Tefé, Coa: Coari, Ppu: Piagaçu-Purus, Mao: Manaus, NAi: Novo Airão, Car: Careiro, San: Santarém, Itai: Itaituba, Jac: Jacareacanga. In bold loci with evidence for a null allele.Click here for additional data file.10.7717/peerj.15503/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.15503/supp-8Supplemental Information 8Click here for additional data file.