Procambarus clarkii farming continues to expand, the model of “breeding and catching large and keeping small” has led to serious degradation of the fry. This has restricted the development of the P. clarkii industry. Therefore, the selection and breeding of optimal breeds of P. clarkii are very important. Investigating intraspecific patterns of genetic diversity may help unravel the species’ genetic structure, evaluate the quality of the fry, and design measures for breeding and conservation. In this study, the genetic diversity of P. clarkii from three regions of China was analysed using microsatellite markers. Populations of P. clarkii showed high genetic diversity and extensive gene flow. The Xuancheng population could be used as a selective breeding population because it possesses the highest diversity. The results of this study provide a scientific basis for the conservation of germplasm resources and the genetic selection and breeding of P. clarkii.High-quality seedlings are a prerequisite for healthy aquatic animal breeding. As the scale of Procambarus clarkii populations in Chongming, Shanghai; Gaoyou, Jiangsu; and Xuancheng, Anhui. The results indicated that the three populations were diverse and the number of alleles, observed heterozygosity, expected heterozygosity, Shannon information index, and polymorphic information content ranged from 4.8 to 6.2, 0.5567 to 0.6257, 0.6166 to 0.7086, 1.1292 to 1.3987, and 0.5446 to 0.6452, respectively. The Xuancheng population had the highest genetic diversity. The genetic differentiation coefficient and gene flow of the three populations were between 0.0553 and 0.1068 and 2.0908 and 4.2708, respectively, and there was extensive genetic exchange between the Chongming and Xuancheng populations. Analyses of molecular variance indicated that the genetic variation was mainly within the population (91.51%) and inter-population genetic variation accounted for 8.49%. The unweighted pair group method with an arithmetic mean clustering map was utilised based on the genetic distance between groups, and the results showed that the Gaoyou group was grouped alone, while the Chongming and Xuancheng groups were clustered together. The structural results indicated that the Chongming and Xuancheng groups had the same origin, although the Xuancheng group possessed a more complex genetic structure. This study indicated that all three populations had a high genetic diversity, with the Xuancheng population exhibiting the highest diversity. The results of the study provide a reference for the selection of base populations in breeding programs and confirm that the Xuancheng population in Anhui has a better genetic background. The selection of the Xuancheng population as one of the base populations for genetic breeding will be more efficient to accumulate superior traits.With the increasing scale of crayfish breeding, the self-propagation and “catch large and keep small” breeding patterns have led to serious degradation of the fry, so the selection and breeding of high-quality fry is very important. Selecting a population with a high genetic diversity as the base population for breeding can greatly improve the breeding efficiency. Fifteen microsatellite loci were used to understand the genetic structure and diversity of three  Procambarus clarkii, commonly known as crayfish and native to northern Mexico and the southern United States, was introduced to China in the 1930s and is now widely distributed there [P. clarkii industry in 2021 was CNY 422.195 billion, with a farming area of 26 million mu and an output of 2,633,600 tons, and it continues to grow rapidly [The red swamp crayfish ed there ,3,4. BasBy analysing the genetic diversity of organisms, it is possible to understand the genetic background and structure of a species and to also assess the quality of its seedlings in order to develop appropriate measures for breeding and conservation ,7,8.P. clarkii in China, indicating that the overall genetic diversity of P. clarkii in China is high and the high level of genetic differentiation in the population may be attributed to genetic drift and geographical isolation. Cui et al. [P. clarkii in Anhui with the wild and farmed populations in Jiangsu and Hubei. Their results showed that the level of genetic diversity of the farmed population of P. clarkii in Anhui was higher than that of the wild and farmed populations in Jiangsu and Hubei. Furthermore, it was considered necessary to strengthen the population introgression between different regions to improve the status of inbreeding. Using eight SSR molecular markers, Xing et al. [P. clarkii in eight main production areas in Jiangsu and identified extensive gene exchange and moderate genetic differentiation among the eight populations of P. clarkii, suggesting mutual exchange within Jiangsu Province due to anthropogenic factors. These studies demonstrate that SSRs are an appropriate molecular marker tool for analysing the genetic diversity among and within populations of P. clarkia. Related studies can reveal the genetic characteristics of different geographical populations of Chinese P. clarkii and also lay the foundation for molecularly assisted breeding. This allows scientists to select different breeding populations in a targeted manner according to diversity level and the genetic structure of different populations, helping to reduce effort regarding the selection of baseline breeding populations, improve breeding efficiency, and promote the progress of breeding work.Microsatellite markers, also known as simple sequence repeats (SSRs), have characteristics of large numbers, wide distribution, hypervariability, codominant inheritance, high conservativeness, and high reproducibility ,10,11. Si et al.  used 10 g et al.  analysedP. clarki is a hot research topic in China, and the study of genetic diversity is essential for breeding efforts. Current studies have focused on the genetic diversity of wild populations of crayfish, while relatively few studies have been conducted on cultured populations; thus, the main goal of this study was to compare the genetic diversity of cultured populations to determine if they could be used as a base population for breeding. Therefore, 90 muscle samples of P. clarkii from three cultured populations were used to analyse the genetic diversity of the P. clarkii germplasm using SSR molecular markers in order to compare patterns of genetic diversity among the three populations of P. clarkii in Xuancheng, Anhui; Chongming, Shanghai; and Gaoyou, Jiangsu. This study aimed to promote the effective use of germplasm resources and to provide a theoretical basis for quality improvement and molecularly assisted breeding of P. clarkii.Selective breeding of P. clarkii from Shanghai Chongming (CM), Jiangsu Gaoyou (GY), and Anhui Xuancheng (XC) were collected as the experimental samples. Shanghai samples were collected from the farms of Shanghai Daohong Aquaculture Technology Co., Ltd.; Shanghai, China, Jiangsu samples were collected from the farms of Jiangsu Wang Xianji Modern Agriculture Development Co., Ltd.; Gaoyou, China and Anhui samples were collected from the Anhui Niannian shrimp and rice cooperatives. In total, 30 samples from each location were collected for the experimental study. The tail muscles of all P. clarkii were sampled, fixed with anhydrous ethanol , and stored at −20 °C for later use.In June 2021, P. clarkii using an Animal Genome DNA Extraction Kit . The specific steps were performed according to the manufacturer’s instructions. The concentration and purity were determined using a micro-ultraviolet spectrophotometer  and DNA was stored at −20 °C.Genomic DNA was extracted from the muscle tissue of The 15 pairs of microsatellite primers were used following published studies with good amplification conditions and high polymorphism screening from Hu et al., Tan et al., and Huang et al. ,22,23. T2+, dNTPs) 12.5 μL and 1 µL of each forward and reverse primers, 0.5 μL of DNA template, and 10 μL of ddH2O. The PCR conditions were pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s; annealing at the appropriate annealing temperature for 30 s; extension at 72 °C for 1 min, 34 cycles; post-extension for 10 min at 72 °C; and storage at 4 °C. The PCR products underwent agarose gel electrophoresis to detect the quality of the products and were then sent to Sangon Biotech Co., Ltd. for capillary electrophoresis.Fifteen pairs of fluorescent primers were used to amplify the DNA of 90 samples by PCR. The reaction system comprised 25 μL 2 × Taq PCR Master Mix , effective allele number (eN), observed heterozygosity (oH), expected heterozygosity (eH), Shannon’s information index (I), gene flow (mN), Nei’s genetic distance (aD), and inbreeding coefficient for each microsatellite locus and population. The polymorphic information content (PIC) of each locus was calculated using PIC_CALC 0.6  [PIC was calculated for each population using Cervus 3.0.3  [stF) between populations was calculated using Arlequin 3.5  [aD values.CONVERT 1.31   softwareHungary) . The PICMT, USA) . The genzerland) , and thezerland)  to deterzerland) . The unwP. clarkii culture populations was performed using 15 primer pairs, and the amplified bands were genotyped by high-efficiency capillary electrophoresis. In all loci, no scoring errors were found due to stuttering, and no large allele dropout was detected. All three populations had null alleles in PclG37; in addition, the CM population had null alleles in PclG8 and PclG9, the GY population had null alleles in PclG4 and PclG29, and the XC population had null alleles in PclG4, PclG8, PclG9, and PclG28. Overall, a genetic diversity analysis of germplasm material from three aN, eN, oH, eH, I, and PIC values and mean values for 15 loci are summarised in eN values ranged from 1.9387 to 5.4619 with an average of 3.3147; the oH values ranged from 0.2111 to 0.9889 with an average of 0.5810; the eH values ranged from 0.4869 to 0.8215 with an average of 0.6973; the I values ranged from 0.8033 to 1.8259 with an average of 0.4243; and the PIC values ranged from 0.3670 to 0.7851 with an average of 0.6440. There were 12 loci with PIC values of >0.5, which were highly polymorphic and could be used for microsatellite genetic diversity analyses.The P. clarkii is listed in The genetic diversity of the three populations (90 samples) of P. clarkii populations are presented in p < 0.05) and five were extremely significantly deviated (p < 0.01). Among the fifteen GY population loci, the Hardy–Weinberg equilibrium indices of ten loci were less than 0, among which three deviated significantly (p < 0.05) and four deviated extremely significantly (p < 0.01). Among the fifteen loci of the XC population, the Hardy–Weinberg equilibrium indices of nine loci were less than 0, of which two deviated significantly (p < 0.05) and five deviated extremely significantly (p < 0.01).The Hardy–Weinberg equilibrium analysis results for the three is(F), total inbreeding coefficient (itF), inter-population genetic differentiation coefficient (stF), and gene flow (mN) of P. clarkii populations showed that isF was negative in five loci  and the rest were positive, with an average value of 0.1001. itF was negative for PcLG02, PcLG13, PcLG15, and PcLG33, and positive for the rest, with an average value of 0.1623. The stF values of the 15 sites ranged from 0.0079 to 0.1547, with an average value of 0.0691. The gene flow (mN) at each site ranged from 1.3662 to 31.3790, with an average of 3.3658 (The statistics of inbreeding coefficient f 3.3658 .D = 0.2560) and smallest between CM and XC populations (D = 0.1602) and, overall, the genetic distances were <0.3 (F-statistic was largest between CM and GY (stF = 0.1068) and smallest between CM and XC st(F = 0.0553) and, overall, the genetic differentiation index was higher than 0.05 and lower than 0.15, indicating that the degree of genetic differentiation among the populations was moderate. The ANOVA results (p < 0.01).The calculations of Nei’s genetic distance and Wright’s F-statistics for the three populations showed that the genetic distance was largest between CM and GY , while the Chongming and Xuancheng populations had one deviation from the equilibrium due to heterozygote excess  and the Gaoyou population had one genetic loci deviating from equilibrium due to other reasons  [aN, eH, and oH in oysters and significantly reduced isF. Hu et al. [The Hardy–Weinberg equilibrium index reflects the equilibrium relationship between Fis > 0) . The meaFis > 0) ,48,49. HFis > 0) . The preFis > 0)  have sugFis > 0)  suggesteu et al.  suggesteP. clarkii is a highly invasive species that reproduces rapidly, and coupled with the extensive farming activities and the negative effects of inbreeding, among others, of P. clarkia, their numbers will significantly expand in the absence of any measures. As shown in the previous results, there are certain differences in genetic diversity between different populations. Therefore, a more scientific breeding program should be developed to maintain the level of genetic diversity; however, such a breeding program is currently not existed. The establishment of a breeding program needs to be supported by a large number of studies, including more loci and as full a range of populations as possible, and, if possible, private alleles of different populations should also be found to establish a breeding program.mN level of each population was 3.3658, and the mN level among all populations was moderate, except for the Xuancheng and Chongming populations, which were slightly higher than 4.0. In general, there was a certain degree of genetic differentiation between the three populations, among which the Xuanxeng population and Chongming population had a higher gene flow and a lower genetic differentiation index, indicating that gene exchange occurred between the two populations. However, the distance between the two populations was relatively long, and Chongming was an island, so we inferred that the gene exchange between the two populations was caused by human factors. The results of the genetic diversity analysis also showed that the genetic diversity of the Chongming population was higher than that of the Gaoyou population but lower than that of the Xuancheng population. It is almost impossible for crayfish to reach the Chongming area through natural migration, indicating that crayfish in the Chongming area had been introduced. Rumishaa et al. found that the gene flow among sawn crab populations was large enough to offset population homogeneity [isF is an important parameter for evaluating the degree of inbreeding between individuals, and generally refers to the probability that two identical genes of an individual come from the same ancestor. A positive isF within a population indicates obvious inbreeding within that population [isF value of each locus was 0.1001, indicating that each population had a certain degree of inbreeding. Inbreeding is inevitable in aquaculture, and we should establish specific breeding programs based on the breeding characteristics in cultured species to try to avoid a large degree of inbreeding.The genetic differentiation index is an important parameter reflecting the degree of genetic differentiation between populations, which is measured by alleles at microsatellite loci . The resogeneity . Additioogeneity . Therefopulation ,56,57,58In this study, the minimum genetic distance (0.1602) was found between the Xuacheng population and the Chongming population, and the maximum genetic distance (0.2560) was identified between the Chongming and Gaoyou populations. The UPGMA phylogenetic tree showed that the Xuancheng and Gaoyou populations first clustered together, indicating that they were closely related, which is consistent with the results of genetic differentiation. There may be a possibility of mutual introduction between the Xuancheng and Chongming groups. Due to the interference of human factors, there was no absolute correlation between geographical distance and genetic distance. For example, in this study, the geographical distances between the three sites were close to each other, similar to an equilateral triangle; however, the genetic distance between the two differed.D values. The two populations of Chongming and Xuancheng were grouped together, indicating that they have some genetic homology. This may be because the initial populations of the two populations are closely related; however, they have been cultured in different environments and gradually localised to form different populations. When K = 3, the Chongming and Xuancheng populations also had a certain genetic similarity; however, when K = 4, the Xuancheng population was clearly different from the other two populations, which could be partly due to the increased mixing of other geographic groups.From the genetic structure diagram, when K = 2, the structure analysis results were consistent with the UPGMA cluster tree constructed based on Overall, all three crayfish cultured populations are potential genetic resources in China and should be protected. It is recommended that a genetic resource bank should be established to register the crayfish populations to more deeply understand genetic information, which will help in the development of breeding programs.P. clarkii breeding populations may be due to different regional introductions, with the Xuancheng population in Anhui having the highest genetic diversity. There was some genetic differentiation and germplasm mixing among various populations. The Shanghai Chongming and Anhui Xuancheng populations are closely related and may have mutual genetic introgression. Overall, the Anhui Xuancheng population had the highest genetic diversity and a relatively more diverse genetic structure, mixing individuals from multiple geographic groups. If the Anhui population is used as the base population for genetic breeding, there will be a higher chance of obtaining high-quality genes, and therefore the breeding efficiency can be improved. Although the population selected for this study was a high yielding area in China, the number was low and a wider group should be included in future studies. In this study, a genetic evaluation of three P. clarkii breeding populations was conducted to provide a reference for population selection in breeding efforts and to suggest a suitable breeding program to maintain genetic diversity and conserve genetic resources.Genetic diversity includes the level of variation and the distribution pattern of variation, namely the genetic structure of populations, and is an important theoretical basis for biodiversity conservation . The hig


Tuvans are one of the most compactly living peoples of Southern Siberia, settled mainly in the territory of Tuva. The gene pool of the Tuvans is quite isolated, due to endogamy and a very low frequency of interethnic marriages. The structure of the gene pool of the Tuvans and other Siberian populations was studied using a genome-wide panel of autosomal single nucleotide polymorphic markers and Y-chromosome markers. The results of the analysis of the frequencies of autosomal SNPs by various methods, the similarities in the composition of the Y-chromosome haplogroups and YSTR haplotypes show that the gene pool of the Tuvans is very heterogeneous in terms of the composition of genetic components. It includes the ancient autochthonous Yeniseian component, which dominates among the Chulym Turks and Kets, the East Siberian component, which prevails among the Yakuts and Evenks, and the Far Eastern component, the frequency of which is maximum among the Nivkhs and Udeges. Analysis of the composition of IBD-blocks on autosomes shows the maximum genetic relationship of the Tuvans with the Southern Altaians, Khakas and Shors, who were formed during the settlement of the Turkic groups of populations on the territory of the Altai-Sayan region. A very diverse composition of the Tuvan gene pool is shown for various sublines of Y-chromosomal haplogroups, most of which show strong ethnic specificity. Phylogenetic analysis of individual Y-chromosome haplogroups demonstrates the maximum proximity of the gene pool of the Tuvans with the Altaians, Khakas and Shors. Differences in frequencies of Y-chromosome haplogroups between the Todzhans and Tuvans and a change in the frequencies of haplogroups from south to north associated with the East Asian component were found. The majority of the most frequent Y-chromosome haplogroups in the Tuvans demonstrate the founder effect, the formation age of which is fully consistent with the data on their ethnogenesis. From the point of view of studying population and evolutionarygenetic processes, analyzing genetic diversity, andreconstructing the genetic history of populations, the genepool of the indigenous population of Southern Siberia is aunique system. The problems related to the analysis of thecomposition and ratio of various substrate components amongthe Siberian peoples, despite the high level of study, have anumber of unanswered questions. In this regard, geneticsprovides the richest opportunities for studying these problems,since the development of new approaches to the analysis of thepopulation gene pool makes it possible to bring ethnogeneticstudies to a completely new level. Modern methods used inmolecular genetic research and new bioinformatic developmentsmake it possible to reliably identify various ancestralgenetic components in the gene pool of various peoples andindividuals.One of the most important problems of ethnology and anthropologyof the population of Southern Siberia is the issueof the formation of indigenous ethnic groups, in the solutionof which, at present, methods of analyzing genomic data playan important role. The gene pool of the indigenous populationof this region was formed due to the long-term and multi-stagemixing of a large number of local gene pools of various tribesof Caucasoid and Mongoloid origin. The indigenous ethnicgroups of Southern Siberia are characterized by variousanthropological types, complex ethnic and demographic history.The mixture of numerous Turkic, Mongolian, Yeniseian,Samoyed and Ugric groups based on the genetic substrateof the ancient Indo-European tribes and taiga Mongoloidsformed as a result a motley picture of the genetic diversityof the population of this region .The processes of merging and assimilation with the participationof various migration flows played an importantrole in the formation of modern Turkic-speaking populationsof Southern Siberia, especially the Tuvans. In the era of theEneolithic, Bronze and Early Iron Ages, the territory of Tuvawas part of the habitat of the ancient Caucasoid population,which later developed the cultures of the Scythian-Siberianworld . The penetration of the Central AsianMongoloid component into the territory of Southern Siberiadates back to the VII–VI centuries BC. The appearance ofthe forest, taiga Mongoloid component also dates back toapproximatelythe same time . Gradually,there was an increase in the Mongoloid component, from thepredominance of the Caucasoid in the Scythian time to theformation in the XIII–XIV centuries AD of the modern CentralAsian anthropological type of the Tuvans .Tuvans are one of the most compactly living peoples ofRussia, settled mainly in the territory of Tuva. In Russia,according to the All-Russian Population Census of 2010, thenumber of the Tuvans is 263,934 people. At the same time, thegene pool of the Tuvans is relatively isolated, due to endogamyand a very low frequency of interethnic marriages . The heterogeneity of the tribalcomposition of the Tuvans was shown . Forsome groups of Tuvans, isolation of local populations wasnoted, caused both by geographical factors and historically,which is especially pronounced for Tuvans-Todzhans fromthe northeastern mountainous part of Tuva. In recent years,a number of scientific publications have been devoted to thestudy of the Tuvinian gene pool, which were focused on thestudy of the general spectrum of mtDNA lines, Y-chromosomehaplogroups, and the detailing of individual clades .The purpose of this study is a comprehensive analysis ofthe structure of the gene pool of Tuvans and the reconstructionof their origin in comparison with other populations ofthe indigenous population of Siberia. To address the issues ofgenetic proximity of Tuvans with other indigenous peoples,genotyping of a wide genomic set of autosomal markers usinghigh-density biochips, as well as an expanded set of SNP andSTR-markers of the Y-chromosome was performed in variousindigenous peoples of Siberia.The material of the study was DNA samples of men with atotal number of 419 samples, representing the indigenouspopulation of the Republic of Tuva. Samples were collectedin the village Teeli (west of Tuva) (N = 44), village Kungurtug(south-east of the Republic) (N = 48), village Toora-Khem(north-eastern part of Tuva) (N = 23), and the city of Kyzyl(N = 304). Samples from Kyzyl were assigned to the correspondingterritorial group according to the birthplaces of thedonors. The samples were divided into five territorially distantgroups: west (N = 169), center  (N = 179), east (N = 71), includingthe northeast (Todzhinsky district) (N = 23) and southeast(Tere-Kholsky district) (N = 48).The sampling of primary biological material (venous blood)from donors was carried out in compliance with the procedureof written informed consent for the study. For each donor, a questionnaire was compiled with a brief pedigree, indicatingethnicity and places of birth of ancestors. An individual wasassigned to a given ethnic group based on his own ethnicidentity, his parents and place of birth.For the analysis of Y-chromosome haplogroups and haplotypesof Tuvans, all 419 male DNA samples were used.For genotyping on chips, unrelated accessions from the villageof Teeli of Bai-Taiga kozhuun (N = 28) were selected.Other populations of the indigenous population of Siberia arerepresented by: Chulyms, Khakas-Sagays, Khakas-Kachins,Southern Altaians, Kets, Khanty, Tomsk Tatars, Buryats,Yakuts,Evenks, Nivkhs, Udeges, as well as Kalmyks, Dungansand Kirghiz.Genome-wide genotyping data were obtained using InfiniumMulti-Ethnic Global-8 (Illumina) microarrays for SNPgenotyping, including over 1.7 million markers. The materialwas deposited in the bioresource collection “Biobank of thePopulation of Northern Eurasia”. For comparative analysis,we used genotype data for 1677114 autosomal SNPs  of 917 samples and genotypingdata for more than 3000 Y-chromosomal SNPs and36 YSTRs from more than 1600 male samples representingthe indigenous population of Siberia and neighboring regions.More than 30 population samples have been characterized,which are described in detail in our previous works . The NGSadmix method and the ADMIXTURE program were used to analyze the component composition and amountof impurities in individuals and populations, and a comparativeanalysis of autosomal SNP data and haplogroups andhaplotypes of Y-chromosomes.Autosomal SNP genotype array clustering and qualitycontrol were performed using a protocol developed by  using GenomeStudio , a software package that Illuminahas developed for various genomic analyses. For filtering,normalizing and calculating standard genomic statistics andindicators, the standard set of programs, including vcftools,bcftools, and plink, proved to be optimal. To analyze linkageblocks identical in origin, the Refined IBD algorithm  was used, which shows moreaccurate results compared to the algorithms built into plink.The genotypes were preliminarily phased using the Beagle 5.1software . To comparethe populations, the sums of the average lengths of blocksidentical in origin  wereobtained between pairs of individuals.To study the composition and structure of Y-chromosomehaplogroups, two systems of genetic markers were includedin the study: diallelic loci represented by SNPs and polyallelichighly variable microsatellites (YSTRs). With the helpof 156 SNP markers, the belonging of the samples to differenthaplogroups was determined. The classification ofhaplogroups is given in accordance with the data of theInternational Society for Genetic Genealogy (website www.isogg.org). Analysis of STR haplotypes within haplogroupswas performed using 44 STR markers of the non-recombiningpart of the Y-chromosome .STR markers were genotyped using capillary electrophoresison an ABI Prism 3730 genetic analyzer. Genotypingof SNP markers was performed using PCR and subsequentanalysis of DNA fragments using RFLP analysis. Experimentalstudieswere carried out on the basis of the Centerfor Collective Use of Research Equipment “Medical Genomics”. The construction ofmedian networks of Y-chromosome haplotypes was carriedout using Network v.10.2.0.0  using the Bandelt median networkmethod . The generation age of the observeddiversity of haplotypes in haplogroups was estimated using theASD method , based on the mean squaredifferences in the number of repeats between all markers.Genotyping of a large array of SNPs makes it possible to studyin great detail the patterns of haplotype diversity that markvarious substrate and superstrate layers of the population genepool, the degree of miscegenation with the alien populationat various levels – from individual to generic and ethnic, toconduct a detailed analysis of the demographic history ofvarious populations and analyze the molecular phylogeneticand phylogeographic structure of Y-chromosome haplogroups.This makes it possible to more accurately reconstruct the geneticand demographic events that occurred in the past. Theuse of modern bioinformatic approaches on a wide array ofSNPs and a detailed phylogeny of uniparental lines makes itpossible to more accurately reconstruct the formation of theTuvan gene pool.After processing the data on the results of a microchip studyto filter the progenotyped samples and carry out further calculations,a search was first made among the Tuvans of mestizosusing the NGSadmix program. The NGSadmix method, whenlaunched on the data array that we formed, showed that allprogenotyped samples of the Tuvans do not have crossbreeding,which is fully consistent with the data of the DNA donorquestionnaire. The obtained data on the frequencies of SNPsin the studied population samples were used to elucidate thegenetic relationships between different ethnic groups. TheADMIXTURE algorithm was used to reduce the dimensionand identify the genetic components.Component composition of the gene pool of the TuvansTo identify individual genetic components in the gene pool ofthe studied populations, the ADMIXTURE program was used,which makes it possible to identify the mixed composition ofa set of individuals based on genotype data and, thereby, tomake assumptions about the origin of the population. Modelingusing ADMIXTURE has recently become one of the mainmethods of analysis in the study of the gene pools of modernand ancient human populations, allowing you to analyze thesame data at different hierarchical levels.Tuvans, in comparison with most Siberian populations,show a very diverse composition of genetic components Their distribution is most clearly manifested at K = 12. Foralmost all Siberian populations, the complete dominance ofone genetic component, characteristic of individual samplesor closely related indigenous peoples, is shown. In addition tothe Tuvans, a rather heterogeneous component compositionwas also found among the Khakas-Kachians. The spectrumof genetic components of the Kachins almost completelycoincides with the Tuvans, but differs in their proportionsAltai component. With the maximum frequency in the Tuvans(53 %), the genetic component that dominates in theSouthern Altaians (up to 90 %) is represented. Taking intoaccount the fact that the analyzed sample of the Tuvans representsthe westernmost region bordering the Republic ofGorny Altai, this is quite natural. It is presented with sufficientfrequency among the Kyrgyz (9.8 %) and Khakas-Kachins(7.6 %), related to the Southern Altaians. Probably, this geneticcomponent is associated with the influence of Turkic speakersin the formation of modern South Siberian peoples. Previously,the proximity of the Altaians and Tuvans was shown by analyzingthe allele frequencies of the ZFX gene , X-linked STR markers ,enzymes and blood proteins , frequenciesblood groups of the ABO system and according to theiranthropological parameters .East Siberian component. The second most commonamong the Tuvans is the East Siberian genetic component(21 %), which is dominant among the Yakuts (94 %), Evenksfrom Yakutia (93 %) and Transbaikalia (62 %). This correspondsto the linguistic data on the South Siberian origin ofthe ancestors of modern Yakuts. It is 30 % among the Buryats,12 % among the Kachins, and 4 % among the Southern Altaians.The distribution of this genetic component is consistentwith the classification of racial types. Tuvans, Tofalars, Yakutsand Dolgans are carriers of the traits of the North Asian minorrace – one of the subdivisions of the continental branch of thegreat Mongoloid race. Two moderately different types aredistinguished in the composition of the North Asian Mongoloids– Baikal and Central Asian. The first type is typicalprimarily for the Tungus-Manchurian peoples, the second – forthe Turkic and Mongolian peoples .East Asian component. In third place in the Tuvans (11 %)is the dominant component of the Dungans (91 %), Buryats(63 %) and Kalmyks (54 %). It manifests itself most clearlyat K = 12. It makes up a larger proportion among the Kirghiz(49 %), Kazakhs (46 %), Uzbeks (43 %), Khakas-Kachins(41 %) and Tomsk Tatars (24 %) and has a small shareamong the Kachins (4 %) and Southern Altaians (4 %). It isthis genetic component that reflects the contribution of thelatest groups of immigrants from the territory of Mongoliato the gene pool of the population of Southern Siberia.Almost all other studied populations of Siberia and the FarEast – the Yakuts, Shors, Khakas-Sagays and Chulyms demonstratethe almost complete absence of this component.It was not found among the Evenks, Khanty, Kets, Chulyms,Chukchi, Koryaks and Nivkhs. The general picture of thedistribution of this genetic component is in good agreementwith anthropological and ethnographic data on the influenceof the Mongol expansion on the ethnogenesis of the studiedethnic groupsYeniseian component. The largest share of this componentis characteristic of the Chulym Turks (94 %) and Kets (65 %).In the Kets, its proportion is lower due to miscegenation andthe detection by the NGSAdmix method of a recent Caucasoidadmixture in many samples. Among Tuvans, its frequency is6.9 %, and among Kachins, 20 %. The results obtained are ingood agreement with the data of ethnology, anthropology andlinguistics on the contribution of the Yeniseian component tothe formation of various peoples of the Altai-Sayan region andthe historical areas of the Yeniseian languagesFar Eastern component. The last genetic component in theTuvans present with a significant frequency (4.9 %) prevails inthe Nivkhs (96 %) and Udege (56 %). It is present with a lowfrequency among the Trans-Baikal Evenks (11 %), Buryats(10 %), Kalmyks (8 %) and Dungans (6 %). Probably, itspresence reflects the contribution of the taiga Mongoloids,who in ancient times settled westward from Primorye andTransbaikalia.It can be assumed that the Samoyed component can also bepresent in the Tuvinian gene pool, however, its determinationrequires an analysis of the population groups in which it isdominant .Identical in origin clutch blocks. As a result of bioinformaticsprocessing of genotyping data from high-density biochipsof various Siberian populations, an analysis was madeof the coincidence of DNA fragments common in originbetween populations and individuals. A segment with identicalnucleotide sequences is IBD in two or more individualsif they inherit it from a common ancestor without recombination,that is, in these individuals the segment has a commonorigin. The expected length of an IBD segment depends onthe number of generations since the last common ancestor.One of the applications of the analysis of genome regions ofcommon origin is the quantitative assessment of the degreeof relationship between individuals, which can also supplementinformation on the genetic relationships of populations.Samples from the sample of the Tuvans showed the maximummatch in IBD blocks among themselves (10.07 %), thenwith a sample of the Southern Altaians (1.62 %), Evenks(0.81 %), Yakuts (0.77 %), Chulyms ( 0.70 %), Khakas-Sagays(0.66 %), Khakas-Kachins (0.64 %), Buryats (0.58 %),Kalmyks (0.57 %), Udeges (0.39 %) and Khanty (0.38 %).The degree of overlap of IBD blocks between the Tuvansand other population samples is consistent with the resultsof ADMIXTURE on the distribution of allele frequenciesand common genetic components in these populations. TheFROH inbreeding coefficient was also calculated for all individualsby homozygosity blocks (ROH). For the Tuvans, itsvalue (0.0151) is much lower than for the Chulyms (0.0292),Kazyms (0.0280) and Russkinskaya Khanty (0.0266), Kets(0.0259) and Khakas-Sagays from the foothill Tashtyp region.Almost equal to the Tuvans in terms of FROH value are thesamples of the Southern Altaians (0.0168) and the Khakas-Kachins of the Shirinsky district (0.0146). This indicates theabsence of a significant role of inbreeding in the formation ofthe gene pool of modern Tuvan populations.Haplogroups of the Y-chromosomeFor the most frequent Asian haplogroups of the Y-chromosomein the Tuvans, additional terminal SNPs were genotyped,which made it possible to more accurately separate thesamples into individual specific sublines. The frequencies ofoccurrence are indicated only for them (see the Table). Thefrequencies of other rather rare haplogroups represented byseparate samples, indicated in an earlier article , are not given here, since additional SNPs were notselected for themThe most frequent Y-chromosome haplogroup in the Tuvansis N1a2b1-B169, which makes up 24 % of the total array ofmale samples. It is divided into three sublines that differ interminal SNPs and haplotype clusters. Its variant N1a2b1b2b1 is present with themaximum frequency among the Tuvans. In addition to theTuvans, two samples of the Southern Altaians belong to it.According to the YFull website, this line was also found inone man from Kyrgyzstan and two from China. The haplotypesof this lineage have a stellar phylogeny, indicating a strongfounder effect .The age of this line among the Tuvans according to YSTRis 1442 years (SD = 368 years). Its presence among the Altaians,Kirghiz and Chinese in the form of single samples ispossibly associated with the inclusion of individual men ofTuvan origin in their composition. This line among the Tuvansrepresents a common genetic substrate for them, whichis unequivocally connected with the heritage of the Samoyedpopulation of the territory of Southern Siberia. The presenceof different ethnospecific variants of the N1a2b1 haplogroupamong the Tuvans, Khakas, and Shors indicates a significantgenetic differentiation between them. This confirms the absenceof migrations of carriers of this haplogroup and geneexchange over the past few hundred years. The main factorin its spread on the territory of Tuva was the genetic isolationof local Samoyedic groups and the intensive increase intheir population. Four samples of Tuvans belong to a veryrare parallel line N1a2b1b2a1~ . It was previously found in Mongols . The third Tuvan subline includes 10 samples.The second most frequent among the Tuvans is the haplogroupN1a1 (19 %), which is divided into three branches.In the total sample, its frequency is inferior to N1a2b1 by only5 %, covering slightly less than 30 % of samples in the westof Tuva. The first line N1a1a2~ (B187 xB449) in the totalsample of the Tuvans has a frequency of 6.4 %. In the easternregions – Todzhiinsky and Tere-Kholsky, this haplogroup wasnot found. This variant is very ethnospecific and is not foundin other populations. The sister line parallel to it (N1a1a2 ~B499) with a relatively recent divergence from the Tuvanline is also characteristic of the Khakas-Sagais and Shors. Itdominates in frequency in the Khakas seoks Khyi and Khobyi.Among the Shors, this haplogroup includes all men of the seokKyi and Kobyi . On the median networks, thehaplotypes of these lines in the Tuvans, Khakas, and Shorsform three clusters that do not intersect with the Tuvans, exceptfor one sample .At the same time, the haplotypes of the Khakas-Sagays ofthe Tashtyp district, bordering Shoria, are very close to theShors and demonstrate a strong recent founder effect. The totalage of the Tuvan haplotype cluster was 1863 years (SD = 294years). This shows the long-standing division of these lineagesbetween the Tuvans, Khakas, and Shors, and rather strongfounder effects for individual seoks of Khakas and Shors.This subline has a very limited geographic range. Most likely, the initial place of its distribution was the territory of Tuva,from where it spread to Gornaya Shoria and then to Khakassia.It separated from the main stem of N1a1 very early and,like many other rare Y-chromosomal lineages, was preservedwith a sufficiently high frequency only in relatively isolatedmountain populations. Its separation from the main stem ofthe N1a1a haplogroup occurred approximately 10,700 yearsago (YFull). Due to its population specificity and isolation,the relationship of this variant with the Samoyedic, Ugric, orother genetic components is ambiguous.The second subline N1a1a among the Tuvans isN1a1a1a1a3a2 (B219 xB199). It is represented in all districtsand has a frequency of 11.9 %. It also includes threesamples of the Altaians. It was not found among the Khakas,Shors and Chulyms. The line N1a1a1a1a3a2c2-B199, whichis closely related to it, dominates in the Eastern Buryats and isrepresented by a rather high frequency in the Western Buryats.The appearance of these lines is unambiguously connectedwith the settlement of Mongolian ethnic groups in Tuva, Buryatiaand Altai. The age of this line according to haplotypesamong the Tuvans was 1500 years (SD = 304 years). Thespread of this Y-chromosome lineage occurred a little laterthan the carriers of the Tuva-Shor-Khakas branch N1a1a2~.Only two specimens of Tuvan-Todzhans belong to a veryrare lineage N1a1a1a . In terms of haplotypes,it is very close to the Yakut-Evenki haplogroupN1a1a1a1a4a1a1, but is not mutated in its terminal SNPs. The presence of thisY-chromosomevariant in the Todzhans is consistent with thedistribution of the East Siberian genetic and overlap in IBDblocks with the Yakuts and Evenks. This lineage also includesfour samples of Khakas-Sagay men from the Askizsky districtwith haplotypes close to those of Tuva.Haplogroup Q1b1a3b1a~ occupies 13 % of the total sample of the Tuvans. Its maximumfrequency falls on the eastern samples of the Todzhans andTuvans of the Tere-Kholsky kozhuun (25 %). Four samples ofSouthern Altaians also belong to this lineage .The descending gradient of this haplogroup from east towest was shown on the territory of Tuva earlier . Thehighest frequency of the haplogroup Q1b1a3b1a~ for Tuvans in Todzha is apparently a consequence of their relative geneticisolation and the preservation of a greater proportion of thelocal autochthonous Yeniseian genetic component. The ageof this line according to haplotypes among the Tuvans was2187 years (SD = 446 years). The distribution frequency ofthe haplogroup Q1b1a3b1a~ and its related lines Q1b1a3b1a2-B33 and Q1b1a3b4-B31 in the populations of the indigenouspeoples of Southern and Western Siberia reflects the contributionto their gene pools of local aboriginal population groupsbelonging to the Yeniseian language family, which are quiteancient in origin. Analysis of the Y-chromosomal sublinesQ1b1a3b shows that the original center of origin and settlementof its carriers is the territory of modern Tuva.Different populations with a share of the Mongolian geneticcomponent have different haplogroups and sublines, the originof which is associated with the settlement of various ethnicgroups and migration events of different times. Among theTuvans, the result of the Mongolian contribution, in additionto N1a1a1a1a3a2, is the haplogroups of the clades C2b1,O2 and O3. All of them are very close to the variants presentedwith a high frequency among the Mongols, Buryatsand Kalmyks. The share of C2b1c (M504) and C2b1a1a1a1(F3850) is the highest in the southeastern sample (15 %).Lineage C2b1a1a1a1 (F3850) was found only in the southernand southeastern regions. The more frequent line C2b1b1(M77) shows a clinal decrease in frequency from southeastto west. The same is true for haplogroups O2 and O3. In thegene pool of almost all the populations studied, in which theMongolian genetic component is not detected by autosomalSNP, the indicated Y-chromosome haplogroups are also absent.Phylogenetic analysis of Y-chromosomal sublines andhaplotypes shows that the center of origin and distribution ofthe carriers of the Mongolian component is the territory ofCentral AsiaThese haplogroups among the Tuvans are a legacy of thegenetic contribution of late Mongoloid migrants, reflectingthe contribution of the Xiongnu and Mongolian settlers tothe territory of Tuva. Thus, genetic data confirm that thepenetration of Mongolian nomads into the territory of Tuvacame from the south, gradually spreading to the northernregions, and, accordingly, the mongolization of the populationof Tuva was most pronounced precisely in the southernregions. This coincides with the data of paleoanthropology and anthropology of the modern population. The data of linguistics characterizing thesoutheastern dialect as formed as a result of the significantinfluence of the Mongolian language also completely coincidewith the distribution of this component and the frequencies ofhaplogroups that we obtained.The haplogroup R1a1a (12 %) among the Tuvans includesseven different lines. Six Tuvan men belong to theR1a1a1b2a2a(Z2123) lineage. Three Altaians and two Kirghizalso belong to it. Five more Tuvans belong to the lineR1a1a1b2a2a3b1a1~ close to it. It dominatesin frequency among the southern Altaians and Teleuts.Twenty-seven Tuvans had the R1a1a1b2 (Y43109) line, whichwas divided into three variants differing in haplotypes. SixteenTuvans and five Southern Altaians belong to one variant. Inthe second, there are four Tuvans, Khakas from the Turan andKhyzyl Khaya seoks, and almost all Shors from the Tartkyn,Shor-Kyzai and Kara-Shor seoks. In the third, there are sevenTuvans, Khakas from various seoks of the Beltir and Biryusin,and Shors of the seoks of the Cheley and Chediber. Thisconfirms the data that some groups of Tuvans who roamedin the Minusinsk Basin and were later called the “Beltyr”were completely assimilated by local tribes, constituting oneof the components of the formation of the ethnos of modernKhakas.The haplogroup R1a1a1b2e1~ (YP1509) among the Tuvansis also divided by haplotypes into two lines. Nine samples ofTuvans of the first variant are very similar in haplotypes tothis variant among the Khakas of the Kharga seoks and theShors of the Karga and Cheli seoks and one Teleut. Eightsamples belong to another specific variant common amongthe Telengits and Northern Altaians.A very large diversity of the R1a1a haplogroup was shownamong the indigenous population of the Altai-Sayan region.Its various sublines split long ago and show no star-like haplotypephylogeny, reduced diversity, or traces of the foundereffect. This indicates a significant size of the effective sizeof the populations of the ancient Caucasoids and Turks, whointroduced these components into the gene pool of modernTuvans, Khakas and Shors. Founder effects with significantdemographic growth were found only in the Southern Altaians,Kirghiz and Teleuts in the haplogroup R1a1a1b2a2a3b1a1~.The distribution of various discovered sublines of the haplogroupR1a1a in the territory of Tuva, Altai, Khakassiaand Shoria is most likely associated with the Turks and theYeniseian Kyrgyz.Of the other haplogroups among the Tuvans, eight moreare single samples . Mostlikely, their presence is partly due to the recent miscegenationand earlier dispersal of the Central Asian populations. Theresults of the study of the detailed phylogeny of Y-chromosomehaplogroups made it possible to more accurately analyzethe component composition of the Tuvan gene pool. This is amore accurate addition to the analysis of autosomal markers,which makes it possible to reconstruct in detail the formationof their gene pool. This information is also important fordescribing the similarities and differences between the comparedgroups, as well as the processes of their ethnogenesis.Various Y-chromosome haplogroups in the Tuvan gene pooldemonstrate their genetic affinity with the Altaians, Khakas,Shors, Buryats, Mongols, Evenks, Kets, Chulym Turks, andTeleuts. This allows us to characterize in more detail thegene pool of the indigenous South Siberian population andthe genetic relationships and continuity of populations livingin this territory.Thus, in the present study, a detailed study of the gene poolof Tuvans was carried out based on the data of high-densitybiochips and a wide range of SNPs of the non-recombiningpart of the Y-chromosome. A very heterogeneous compositionof the gene pool of the Tuvans and Khakas was found, both inautosomal SNPs and in various sublines of Y-chromosomalhaplogroups. The maximum closeness of the gene pool of theTuvans with the Altaians, Khakas and Shors is shown. Analysis of IBD blocks and individual rare variants of male linesdemonstrates traces of more ancient connections with the ancientaboriginal population of this region and the populationsof Eastern Siberia and the Far East. Within the Tuvan ethnos,significant differences were found between samples from thewestern, southern, and eastern regions of Tuva in terms of theproportion of the Mongolian and Yeniseian geneticcomponent.The genetic diversity of the Tuvans in Y-chromosomalhaplogroups and the most heterogeneous composition of geneticcomponents indicate the highest diversity of the Tuvangene pool, compared to all other indigenous peoples of SiberiaIn the future, we plan to analyze in more detail the structureof the gene pools of the South and West Siberian populationsby adding population samples of the Samoyedic peoples – theNenets and Selkups.The authors declare no conflict of interest.Agdzhoyan A.T., Damba L.D., Gurianov V.M., Zaporozhchenko V.V.,Balanovsky O.P. Phylogenetic analysis of the South SiberianQ-YP1102 haplogroup based on the data on Y-SNP and Y-STRmarkers in Tuvans and surrounding populations. Russ. J. Genet.2021;57:1398-1407. DOI 10.1134/S1022795421120024.Alekseev V.P. 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Cruzia tentaculata is a helminth parasite of marsupials and has a wide geographic distribution from Mexico to Argentina. The aim of this study was to analyse the genetic population structure of this nematode along the Atlantic Forest biome. Cruzia tentaculata specimens were recovered from Didelphis aurita, Didelphis albiventris and Philander quica in 9 localities. Morphological and morphometric data were investigated for phenotypic diversity among localities and hosts using multivariate discriminant analysis of principal components. Phylogenetic relationships of C. tentaculata were determined using maximum likelihood and Bayesian inference. The population structure was analysed by fixation indices, molecular variance analysis, Tajima's D and Fu's Fs neutrality tests, Mantel tests and Bayesian clustering analysis. A higher significant morphometric difference for males was observed between localities. In the haplogroup networks, 2 groups were recovered, separating locations from the north and from the south/southeast. The morphometric variation in C. tentaculata between different localities was compatible with this north and southeast/south pattern, suggesting adaptation to different ecological conditions. Population genetic analyses suggested a pattern of evolutionary processes driven by Pleistocene glacial refugia in the northeast and southeast of the Atlantic Forest based on the distribution of genetic diversity. Cruzia Travassos, 1917 comprises 10 species that parasitize the caecum and large intestine mainly of mammals but also amphibians and reptiles , Didelphis albiventris Lund, 1841, Didelphis aurita Wied-Neuwied, 1826, D. marsupialis, Marmosa murina , Metachirus myosuros , Metachirus nudicaudatus , Monodelphis domestica, Wagner, 1842 and Philander opossum Linnaeus, 1758, with the larger geographic distribution, occurring in Argentina, Bolivia, Brazil, Colombia, Paraguay, Peru, French Guiana and Mexico , suggested by the fact that D. aurita and D. albiventris have large vagility compared to other small mammals, with the ability to cross degraded areas, exploring anthropized rural and urban environments; and (ii) C. tentaculata specimens present morphological variation related to the host localities.We hypothesized that (i) C. tentaculata were recovered from 3 species of marsupials of the subfamily Didelphinae . These 3 species are the most commonly found and easily captured along the Atlantic Forest on the east coast of South America. Nine different localities were sampled along the Atlantic Forest biome: the municipality of Santa Rita (SRT-PB) in the state of Paraiba, the municipalities of São Cristovão (SCR-SE) and Capela (CAP-SE) in the state of Sergipe, the municipalities of Petrópolis (PET-RJ) and Rio de Janeiro (RIO-RJ) in the state of Rio de Janeiro, the municipality of Conceição dos Ouros (CDO-MG) in the state of Minas Gerais, the municipalities of Curitiba (CUR-PR) and Ponta Grossa (PGR-PR) in the state of Paraná and the municipality of Porto Alegre (POA-RS) in the state of Rio Grande do Sul. The number of helminths studied for each locality was based on the number of hosts collected in each locality and on the parasitic load of each animal to obtain representative samples from each local population  model 201 traps (16″ × 5″ × 5″). Each trap was baited with a generalist mixture of peanut butter, banana, oats and bacon , preferring odorous items for mammals' attraction. After capture, the marsupials were conducted to a field laboratory, where they were anaesthetized, killed and had their bionomic data and samples taken for different studies.C. tentaculata were recovered through examination of the host caecum and large intestine. The helminths were washed in saline solution and stored in 70% ethanol for further morphological, morphometric and molecular studies.The marsupials were necropsied, and their organs were removed and placed separately in Petri dishes, washed in saline solution (0.9% NaCl) and dissected with the aid of a stereoscopic microscope. Specimens of Cruzia and taxonomic keys according to Vieira et al.  and among host species . In this analysis, the same morphometric characteristics described above were used. The DAPC describes variations between defined groups, selecting principal components (PCs) that explain the greatest variation between groups while minimizing the variation within each group   according to the manufacturer's instructions and stored at −20°C until use. Partial cytochrome c oxidase subunit 1 (MT-CO1) mitochondrial gene fragments were amplified by polymerase chain reaction (PCR) using the primer cocktail described by Prosser et al.  ; 10.5 μL of ultrapure water for molecular biology, 0.5 μL of sense  and of anti-sense  oligonucleotide cocktail (10 μm); and 1.0 μL of genomic DNA (~1–4 ng μL−1). We used a Veriti Thermal Cycler (Life Technologies) thermocycler under the following thermal cycling conditions: initial denaturation at 94°C for 1 min; 5 cycles of denaturation at 94°C for 40 s, hybridization at 45°C for 40 s, and extension at 72°C for 1 min; followed by 35 cycles of denaturation at 94°C for 40 s, hybridization at 51°C for 40 s, and extension at 72°C for 1 min; and a final extension at 72°C for 5 min .The resulting amplicons were visualized using 1.5% agarose gel electrophoresis with GelRed Nucleic Acid Stain  on an ultraviolet light transilluminator. Successfully amplified PCR products were purified using the Illustra GFX PCR DNA and Gel Band kit  according to the manufacturer's instructions and sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit . The reactions were prepared at a final volume of 10 r et al. . Sequencet al., Ascaris lumbricoides ; Baylisascaris schroederi ; Parascaris equorum ; Pseudoterranova krabbei Paggi, Mattiucci, Gibson, Berland, Nascetti, Cianchi and Bullini, 2000; Toxascaris leonina ; and Toxocara cati  based on phylogenetic proximity to the Kathlaniidae family  and Bayesian inference (BI) methods. Phylogenetic reconstructions by ML were conducted using the online web server PhyML 3.0  method  in relation to host species  (P < 0.01) and among hosts , although to a lesser extent in the latter  . Males we latter .Table 3In the DAPC for female parasites, 4 principal components (PCs) were retained to discriminate host species groups, and 2 PCs were retained to discriminate locality groups. For males, 2 PCs were retained to discriminate both host species and locality groups. In each of these DAPCs, the principal components retained explained approximately > 98% of the total variance. The proportion of female specimens correctly classified to their original groups was approximately 50% for localities and 86% for host species. For males, the proportion of specimens correctly classified to their original groups was approximately 57% for localities and 75% for host species.C. tentaculata occurring in different host species than among some localities.When analysing the differences between localities, DAPC identified 2 sets of localities for females, 1 from the southeast region  and the other formed by the localities of the northeast region (CAP-SE and SCR-SE) and (CUR-PR) southern region . For malThe variables that best discriminated parasites between the localities for female specimens were the length of the intestinal diverticulum and the body length . For malC. tentaculata studied, 144 haplotypes with 161 polymorphic sites were found, and the mean nucleotide diversity and haplotypic diversity were 0.03480 and 0.998, respectively. To estimate the genetic diversity indices, the C. tentaculata sequences were organized into groups according to the host species and localities studied  used for phylogenetic inferences.Three matrices were constructed. The first was generated to calculate genetic diversity indices, AMOVA, Fst, Tajima's C. tentaculata formed a monophyletic group in both phylogenies . In the ML  and BI , Sergipe (CAP and SCR), Paraná (CUR), Rio Grande do Sul (POA), by parasites from D. aurita in the states of Rio de Janeiro (RIO and PET), Paraná (CUR) and Minas Gerais (CDO) and from P. quica in the state of Paraná (PGR).In the ML analysis, the nucleotide replacement model selected by the AIC in PhyML SMS was GTR + R, with an estimated invariable site proportion of 0.000, estimated gamma parameter of 1.000 and variation of rates with 4 categories. The most likely tree presented an LnL of −4970.750524. The estimated base frequencies were A = 0.15645, C = 0.06752, G = 0.26508, and T = 0.51095. For BI, the estimated mean marginal probability was −4982.4043, and the median was −4982.079. The ESSs for all parameters were above 100 samples and were effectively independent, indicating the robustness of our sampling. Phylogenetic reconstructions by ML and BI resulted in equal topologies. Specimens of ) and BI , the groD. albiventris and D. aurita. Interestingly, in the host haplotype network, there were 3 groups of haplotypes for D. albiventris at the periphery of the haplotypes of D. aurita (The haplotype network suggested separation of 2 groups: SRT-PB and SCR-SE (group I) and PET-RJ, RIO-RJ, POA-RS, CUR-PR, PGR-PR, SCR-SE, CAP-SE and CDO-MG (group II), separated by a genetic distance of 32 mutational steps. The sharing of haplotypes between localities and host species was observed. Haplotype H-5 was shared between the POA-RS and PET-RJ localities . Haploty. aurita .Fig. 9.D. aurita and D. albiventrtis (0. 002). For localities, the Fst values were significant between CDO-MG and SCR-SE, CDO-MG and CAP-SE, SCR-SE and POA-RS, SCR-SE and CUR-PR, SCR-SE and RIO-RJ, SCR-SE and CAP-SE, SCR-SE and SRT-PB, POA-RS and CUR-PR, POA-RS and CAP-SE, POA-RS and SRT-PB, CUR-PR and CAP-SE, CUR-PR and SRT-PB, RIO-RJ and CAP-SE and CAP-SE and SRT-PB  species than between hosts. There was a higher genetic variation between localities (52.82%) than within localities . The vald SRT-PB . The neud POA-RS . Accordiclusters .Fig. 11r: 0.6764 P = 0.001)  , Fig. 12C. tentaculata, agreeing with the taxonomic key  . This fact is corroborated by the low and non-significant Fst values between localities, indicating low genetic differentiation, low geographic structure and high gene flow of the parasite populations among the studied areas. A mixture was found between the clusters , corroborating the haplotype network, the phylogenetic analyses and the low Fst values. In addition, cluster 2 recovered in BAPS was congruent with group II found in the haplotype network. However, the heterogeneity observed between populations can be attributed to geographic distance, corroborated by the Mantel test, which indicated a significant and high (68%) correlation between geographic and genetic distance, suggesting isolation by distance. Most of the genetic divergence, shown by haplotype networks, is between the northern populations and the southern ones, separated by 1200 km.C. tentaculata were not correlated with the host species. In addition, the host species were also observed to share haplotypes. The pattern found in phylogenies was corroborated in the haplotype network, and no structure was observed associated with the host species of C. tentaculata, with AMOVA indicating greater genetic variation within each host species (88.85%). In addition, the Fst value was very low and significant and was more evident between D. aurita and D. albiventris. Didelphine marsupials are phylogenetically close and can occur in sympatry in regions of ecotone between the Atlantic Forest and the Cerrado, showing overlap in their diets, which allows parasite sharing and therefore sharing of their haplotypes   in Didelphis marsupialis, D. virginiana and Philander opossum in central and southeastern Mexico. Phylogenetic analyses demonstrated a similar pattern to that of C. tentaculata, in which the specimens of O. microcephalus were not correlated with host species or geographical locality.López-Caballero et al.  conducteC. tentaculata was also identified in Haemonchus contortus , a parasite of sheep and goats  and another from the northeast . The haplotype network showed the separation of the 2 groups. We believe that the centre of origin was in D. aurita (southeast) and that secondary infections infected D. albiventris (northeast), as shown in the haplotype network.Our results showed that there is a possibility of having 2 lineages, 1 from the southeast , giving rise to group I. The second lineage may have differentiated and expanded by the northeast region (Sergipe state), southeast (Rio de Janeiro and Minas Gerais states) and dispersed to the south (Paraná and Rio Grande do Sul states), giving rise to group II.The ‘Hypothesis of refuges’ predicts that during the glacial periods, the flora and fauna of forested areas became isolated in different forest fragments (refuges), differentiating and leading to speciation. When the glacial period ended, the forests expanded, and the species met again. The Atlantic Forest has 5 areas of great genetic diversity, which correspond to forest refuges (Carnaval and Moritz, C. tentaculata may represent a panmictic population along the Atlantic Forest. However, we cannot affirm that the analysed individuals of C. tentaculata form a panmictic population, which is supported by the neutrality tests, morphometric differences found, and the haplotype networks, which showed the existence of 2 groups. Thus, despite the low genetic differentiation observed, the first hypothesis cannot be confirmed with the data of the present study, unlike the second hypothesis, which was corroborated. We suggest that the morphological differences and the haplotypes grouping into 2 groups observed are attributed to ecological differences, which can be explained by the hypothesis of refuges. We suggest a more comprehensive study including other molecular markers to corroborate the observed patterns and the inclusion of parasite samples from the Cerrado and Amazon biomes to test the hypothesis of refuges.Some analyses, such as the Mantel test, AMOVA, the low values of Fst and the sharing of haplotypes, indicate that there is a high gene flow between the subpopulations of the Atlantic Forest, corroborating our initial hypothesis that the specimens of


This article discusses genetic traits  of the Amazon sailfin catfish. Domesticated fish of this species escaped from aquariums to establish invasive populations in large areas of North America and Asia. In Brazil, this species is an important fishery resource and a potential for fish farm management and production. Its karyotype showed 52 chromosomes with a similar conservative gross morphology to that of related species. The Cytochrome Oxidase I (COI) gene revealed a low molecular divergence between native and exotic populations.Pterygoplichthys pardalis is an armored catfish native to South America and an important resource for the ornamental fish industry. Recently, several exotic populations have been introduced into rivers on five continents. Despite its commercial and environmental importance, P. pardalis is poorly studied from a genetic perspective. In this study, we analyzed the karyotype of P. pardalis from the Amazon River and molecular variations in the mitochondrial gene Cytochrome oxidase I (COI) between native and exotic populations. The karyotype presented diploid number 2n = 52 and NF = 100 without cytogenetic variation between males and females. Nucleolus organizer regions (Ag-NOR) in the distal region of the long arm of pair 12 coincided with the 18S hybridization signal, whereas 5S was syntenic to this chromosome but localized in the short arm. The constitutive heterochromatin was restricted in the distal regions of pairs 4, 12, 25, and 26. Telomeric probes showed only distal hybridization signals. The karyotype of P. pardalis diverged from that of its congeners, and COI molecular variation revealed four haplotypes. The Philippine population revealed the greatest diversity with three haplotypes, while haplotype H1 was the most abundant and observed in both native and exotic populations. This new genetic data contributes to species management and provides useful information from an aquaculture perspective. The Amazon basin is the largest hydrographical province in South America and supports considerable ichthyofauna with thousands of species. The armored catfish  is one of the main fish groups in this region and currently encompasses 1041 valid species [Pterygoplichthys  encompass 15 species [Pterygoplichthys is closely related to Hypostomus and part of Hemiancistrus, which are nested in the clade–Tribe Hypostomini [Hemiancistrus”, which was demonstrated to be a paraphyletic group [Hypostomus and Pterygoplichthys. For example, a recent integrative taxonomic study demonstrated that Hypostomus chrysostictus should be properly reclassified as P. chrysostictus [The Loricariidae family is considered a monophyletic group; however, the systematic relationships at the subfamily level are unclear ,3,4,5. Tys Gill, 858 encomostomini . While tostictus .Pterygoplichthys have been recorded in the Amazon basin: P. disjunctivus (Weber 1991), P. gibbiceps (Kner 1854), P. joselimaianus (Weber 1991), P. lituratus (Kner 1854), P. pardalis (Castelnau 1855), P. punctatus (Kner 1854), P. weberi (Armbruster & Page 2006), and P. xinguensis (Weber 1991). Pterygoplichthys pardalis inhabits marginal lagoons and floodplains along the Amazon River and the confluence zones of its main tributaries [Pterygoplichthys pardalis was possibly introduced into Asia and North America through the release or escape of animals sold in the aquarium market, that subsequently bioinvaded various river systems [P. pardalis reportedly caused severe ecological disturbances to native fish communities in Mexico and Southeast Asia [Eight species of butaries . Pterygops Kner 154, P. jos Weber 191, P. giast Asia ,11.P. pardalis is an important fishery resource that was considered one of the ten most exploited species in the lower Amazon region [P. pardalis for domestication and aquaculture programs. Those authors highlighted that the limited genetic data on P. pardalis was a constraint for its use in aquaculture production. Genetic information is necessary to achieve the management and conservation of commercial fish stocks, either in the wild or in fish farms [In Brazil, n region . Moroni n region  evaluatesh farms ,15,16.Pterygoplichthys have been karyotyped . The voucher specimens were fixed with 10% formalin followed by preservation in 70% ethanol for deposit in the Fish Collection of the Federal University of Western Pará, collection numbers UFOPA-I 1393–UFOPA-I 1415. The collections were authorized by the Brazilian government under SISBIO permission No. 69419-1.v/v).The preparation of mitotic chromosomes from the cephalic kidney involved 24 h of mitosis stimulation with yeast , followeAir-dried slides were processed with 5% Giemsa conventional staining for karyotyping and the nucleolar organizing regions were detected using silver staining (Ag-NOR) . C-bandiP. pardalis specimens  was extracted following the salting-out protocol [2, 2.8 μL of 1.25 mM DNTPs, 0.5 μL of 5 μM for each primer, 0.2 μL of Taq DNA polymerase (5U/μL), and 1 μL of genomic DNA (50–100 ng/μL). The cycling profile was 95 °C/2′, 35 cycles of 94 °C/30″, 54 °C/30″, and 72 °C/60″ followed by a final extension of 72 °C/10′. The PCR products were visualized in 1% agarose gel stained with GelRedTM .Genomic DNA of six protocol . The 5′ protocol . The reaPositive reactions were cleaned with 20% PEG 8000  and prochttps://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 2 March 2022) and an ID engine tool . Supplementary sequences of P. pardalis and other Hypostomini members were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/) , including specimens collected from natural populations (Brazil and Colombia) and exotic populations from Asia, representative of six countries. In addition, P. edentaculatus (n = 5), P. zuliaensis (n = 1), and Hypostomus cochliodon (n = 1) were used as outgroups. The best model for the data was that of T92+G (Tamura 3-parameter) and the gamma shape parameter was 0.63. A distance matrix and neighbor-joining clustering were generated with MEGA X [The COI sequences were visually inspected and trimmed with BioEdit v.7.2.5 . We checih.gov/) . For molh MEGA X . Statisth MEGA X .P. pardalis populations, we created a haplotype network using the median joining algorithm [To explore the molecular variation and microevolutionary relationships within natural and exotic lgorithm . Haplotylgorithm  and the P. pardalis from the lower Amazon River has 2n = 52 chromosomes and an FN = 100, a karyotypic formula of 20m + 20sm + 8st + 4a, and no distinction of differentiated sex chromosomes  in the P. pardalis reference mitogenome (Access Number NC_060468.1). G+C content was 0.421, polymorphic sites were S = 5, and nucleotide diversity was Pi = 0.00071. The genetic distance within P. pardalis populations was zero  and P. etentaculatus (0.030 to 0.031). The neighbor-joining distance-clustering tree is shown in P. pardalis (n = 80) that were representative of indigenous and exotic populations, and the haplotypic diversity was Hd = 0.121. All COI variants were observed in the Asian populations, whereas the native populations from Brazil and Colombia had only a single haplotype (H1). This haplotype was the most frequent and shared by both native (Brazil and Colombia) and exotic populations (Asia). The haplotype H2 was a singleton recorded from a specimen collected in Thailand, while H3 and H4 were recorded in specimens from the Philippines. Both H2 and H4 haplotypes diverged from H1 by one mutation, whereas H3 diverged by four mutational steps  showed extensive conservative patterns of karyotype features and chromosome morphologies, as previously observed in the literature [P. pardalis from the Manaus section of the Amazon River [P. pardalis from Manaus [terature ,27,28,29P. pardalis populations throughout the main channel of the Amazon River without topographic barriers that promote isolation, this species is a non-migratory fish that is limited to a local distribution [P. pardalis has a karyotypic polymorphism along its natural geographic range. This mechanism of chromosomal evolution in not uncommon in the genetic divergence of Amazon fishes. In addition, populations of Peckoltia vittata  from the Xingu River diverged in terms of a variation in chromosome arms, which was explained by pericentric inversions [Karyotypic evolution in freshwater fish is linked to species biological traits  and environmental structures that could restrain gene flow between populations, thereby favoring the fixation of chromosomal rearrangements . Despiteribution . Moreoveversions .P. pardalis diverged from P. multiradiatus, P. anisitsi, and P. joselimaianus  to 16 (P. anisitsi from the Miranda River) [P. pardalis (pair 26) is acrocentric and shows a positive C-band subterminal, that is characterized as GC-rich heterochromatin  . The lithromatin b,e. ThisP. pardalis specimens from Santarém, which contrasted with the large number observed in the population from Manaus [P. pardalis shows heterochromatic magnification when exposed to a polluted environment. The collection site of P. pardalis from the lower Amazon River in the present study was assumed to be an unpolluted environment, because it was distant from urban zones and other potential sources of water pollution, such as large-scale agriculture, mining, and industry. The heterochromatic C-bands in P. pardalis from Catalão Lake, an unpolluted site from the Manaus sector, showed marks in the centromeric regions of all chromosomes [P. pardalis has an intrinsic C-banding polymorphism that is not exclusively associated with genomic adaptation to environmental disturbances. The mechanisms involved in the heterochromatin organization of fish genomes are not completely understood; however, it is clear that heterochromatin accumulation can play a role in sex chromosome evolution [Heterochromatin (C-bands) was rare in the m Manaus . Araújo omosomes . Therefovolution  and specvolution .Pterygoplichthys species investigated to date and is considered to exist in the ancestral Loricariidae karyotype [Pterygoplichthys karyotypes showed a size heteromorphism and a preferential subterminal position in submetacentric (4 sp), metacentric (2 sp), and subtelocentric 1 sp; P. chrysostictus) pairs.Karyotypes with a single Ag-NOR-bearing chromosome are considered plesiomorphic in many fish groups, including the primitive Actinopterygii . This paaryotype ,55,56. DPterygoplichthys  showed coincidental Ag-NOR and 18S FISH signals  and P. joselimaianus (metacentric), although the subterminal location of Ag-NOR was preserved. Similarly, 5S rDNA cistrons were detected in synteny with 18S in P. ambrosettii and P. pardalis  that are shared with Hemiancistrus [Aphanotorulus emarginatus (Hypostomus emarginatus) [Hypostomus is cytogenetically diverse with karyotypes having diploid numbers ranging from 2n = 64 to 2n = 84 [Pterygoplichthys in the Hypostomini phylogeny is well supported cytogenically.The Ag-NOR labeling detected by silver nitrate staining was highly associated with hybridization signals of the 18S rDNA probe . Three snals see . Ag-NOR alis see . In thiscariidae . Pterygoncistrus  and Aphaginatus) . However 2n = 84 . TherefoP. pardalis and revealing its considerable divergence (3–8.5%) from its congeners (P. etentaculatus and P. zuliaensis). Molecular species delimitation by DNA barcoding has been successfully used for monitoring biodiversity and addressing taxonomic ambiguities, especially in commercially exploited species [Pterygoplichthys is controversial and that the delimitation of some species of the genus is based on morphological characters that are not very robust, such as the pattern of ventral spots [P. pardalis and P. disjunctivus, molecular identification by DNA barcoding and morphological patterns is limited due to possible interspecific hybridization [DNA barcoding (COI) was effective for identifying  species . Consideal spots , the COIdization ,59,60,61P. pardalis have established themselves in Mexico [P. pardalis COI sequences within the native range (Brazil), with only a single mitochondrial haplotype (COI) detected in P. pardalis from the Amazon River (Manaus sector) [P. pardalis exhibit high morphological and genetic diversity, which has been suggested to result from interspecific hybridization between P. pardalis and P. disjunctivus [P. pardalis in the Philippines remains unclear, but it is suspected that the founder individuals were imported from the United States through international trade in ornamental fish for domestic aquariums [Populations of introduced species commonly show distinct losses of genetic diversity associated with the colonization process, due to the founder effect and genetic drift . Howevern Mexico  and seven Mexico . We foun sector) ,57. This sector) , was rec sector) ,59,60,61quariums .P. pardalis extends over a wide region of northern South America, including the Amazon and Orinoco River basins. To date, DNA barcoding has been used in only two locations in this region, the middle (Manaus) and lower Amazon River (Santarém), which limits or prevents a reliable assessment of the genetic diversity of the native population. Despite this scarcity of genetic data, P. pardalis is a heavily exploited fishing resource in the Amazon basin, both for subsistence fishing for traditional populations and commercial fishing to supply regional urban centers [P. pardalis is considered the most important fishing resource for traditional riverside communities in the floodplain region of Santarém. In communities where this species is often captured, a decrease in average body size of the fish has been observed [P. pardalis from the Amazon basin are recommended to evaluate the possible effects of overfishing and loss of intraspecific genetic diversity.The natural geographic distribution of  centers ,64. Sant centers . P. pardobserved ,67. FurtP. pardalis) is a recognized aquaculture candidate species; however, the lack of genetic data is a main limiting factor and a high priority for research aimed at the domestication of this species for cultivation in aquaculture systems [P. pardalis from the wild population contribute to advances in the debate on Loricariidae taxonomy, the management of invasive species, and considerations regarding fishery pressures, stock conservation, and food security in the context of the sustainability of traditional Amazon riverside communities.Construction of genetic databases are mandatory to subsidize technological packages for domestication and aquaculture programs. The sailfin catfish ( systems . This nePterygoplichthys pardalis from the lower Amazon region retain the diploid number 2n = 52 but differ from congeners due to variations in the karyotypic formula. Pericentric inversion-type rearrangements may explain the changes in chromosomal morphology observed in the group. COI gene variation revealed that native and exotic populations of P. pardalis are highly homogeneous and linked by a single shared haplotype.The