Coregonus albula) complex, to study the genetic similarity among two pairs of sympatric autumn- and spring-spawning populations from post-glacial German Lakes Stechlin and Breiter Luzin. For comparison, we included a similar pair of sympatric populations from the Swedish Lake Fegen. We wanted to explore potential genetic similarities between the three sympatric cisco population pairs in the three lakes, to evaluate whether the pairs may have emerged independently in the three lakes, or whether two different species may have colonized all three lakes independently. Furthermore, we considered allopatric C. albula populations from three Polish, three Finnish, and four Swedish locations, added one Siberian population of the sister species C. sardinella and a Swedish C. maraena (whitefish) population. By genotyping nine microsatellite markers in 655 individuals from these 18 populations, we wanted to elucidate how strongly the cisco populations differ across a larger geographical area within Europe. Finally, we compared the genetic differences between the spring- and autumn-spawning populations of ciscoes in the two German lakes to infer the potentially deteriorating effect of strong anthropogenic pressure on the lakes.Sympatric speciation along ecological gradients has been studied repeatedly, in particular in freshwater fishes. Rapid post-glacial ecological divergence has resulted in numerous endemic species or ecologically distinct populations in lakes of the temperate zones. Here, we focus on the Baltic cisco  by random genetic drift in isolated populations. However, earlier studies on the Swedish populations combining mitochondrial DNA and microsatellite data indicate that also post-glacial immigration from separate glacial refugia has shaped the present genetic population structure. The low neutral differentiation of the German sympatric pairs in contrast to the Swedish pair suggests that recent anthropogenic effects on the lakes in Germany may put the endemic spring-spawners at risk to extinction.The online version contains supplementary material available at 10.1186/s12862-021-01920-8. Traditionally, the process of speciation has been classified by using a geographical context. The allopatric mode is characterized by extrinsic barriers during divergence, the parapatric mode includes a partial extrinsic barrier, while the sympatric mode does not involve any extrinsic barriers . HoweverGasterosteus), Arctic charrs  and smelts (Osmerus) [Coregonus (whitefishes and ciscoes) is particularly diverse [Coregonus are considered typical examples for incipient and ongoing speciation events in post-glacial lakes, induced by segregation along ecological gradients, such as lake habitats  and feeding niches , by physiological differences along vertical temperature gradients, or by predator-induced morphological divergence [Coregonus, when sympatric species went extinct due to the deterioration of ecological gradients, for example following lake eutrophication, species invasion or fisheries [Coregonus is at risk with respect to the conservation of evolutionarily significant units in several lakes, and many extinction cases have been documented [Haplochromis) [Gasterosteus) [Alosa) [Freshwater fishes are good model systems for post-glacial ecological divergence in sympatry , 10, 11.Osmerus) –14. The  diverse , with nu diverse –18. Corevergence , 19, 20.vergence . Howeververgence . Accordiisheries , 23. Indcumented , 23. Simchromis) , sticklerosteus)  and shad [Alosa) .C. albula complex). Baltic ciscoes have smaller maximum size and age than whitefish and live predominantly in post-glacial lakes in Scandinavia, Finland, Poland and North Germany. Cisco species flocks are not known, but several sympatric population pairs have been described from lakes in Germany and Sweden [C. albula and a second form, which spawns in spring. The spring-spawning cisco in German Lake Breiter Luzin has been described as C. lucinensis [C. fontanae [C. trybomi [C. albula in the large Lakes Ladoga (C. ladogae) and Onega (C. kilez) [This risk seems also substantial for some populations of the Baltic ciscoes  .C. albula populations, including the only remaining sympatric populations from Lake Fegen, has comprehensively been studied [C. albula and circumpolar C. sardinella are present  in the two population assemblages and hence both species do not form two fully independent groups, but have experienced introgressive hybridization in their evolutionary past.Recently, the population differentiation based on microsatellite markers and mitochondrial DNA of several Swedish  studied , 33. Two studied . The sec studied , 34. BotPrevious analyses of genetic relationships between the German sympatric ciscoes have resulted in partly different evolutionary scenarios. By using six microsatellites and part of the mitochondrial DNA, Schulz et al.  found hiC. albula complex likely coexist not anywhere else than in the Lake Fegen. The situation in Finland remains to be studied further. In Germany, sympatric pairs seem to have evolved only in the two Lakes Stechlin and Breiter Luzin. The genetic differentiation between the autumn- and spring-spawners in both lakes was weak according to earlier studies [For the conservation of evolutionarily significant units, it is decisive to discriminate between a scenario that all spring-spawning populations have evolved independently and hence are endemic for each of the lakes, and a scenario by which two different ancestral genetic lineages have invaded all lakes with sympatric populations, suggesting that all spring-spawners would have a common evolutionary origin. However, three out of originally four Swedish sympatric population pairs have become extinct during the last decades , 33, pre studies . Further studies –38. TherC. albula [C. albula populations from Poland, Finland and Sweden. Our study had three aims. First, we wanted to elucidate how strongly the cisco populations differ across a larger geographical area including populations from Germany, Poland, Sweden and Finland, and by including a population of the geographically most distant Siberian C. sardinella. Second, we wanted to explore potential genetic similarities between the three sympatric cisco population pairs in the three lakes, to evaluate whether the pairs may have emerged independently in the three lakes, or whether two different species may have colonized the three lakes. Third, we wanted to compare the differences in genetic structure between the spring- and autumn-spawning populations of ciscoes within both German Lakes Stechlin and Breiter Luzin with similar data obtained about 20 years ago, to infer the potentially deteriorating effect of a strong anthropogenic pressure on these lakes.We used the microsatellite marker set developed for Swedish . albula  as a fraCoregonus population samples  and BWF1 (7.8%) and for the Finnish C. albula populations from Lakes Oulujärvi (19%) and Jerisjärvi (29%)  to 72 (BWF2) alleles per locus across all populations  compared to assemblage II . AR in all Finnish, German and Polish cisco samples was similar to the level in Swedish assemblage II (5.1–5.9), except for Lake Jerisjärvi (4.4), where AR was similar as in Swedish assemblage I , Stora Hålsjön, Oulujärvi and Jerisjärvi, C. maraena from Lake Bolmen and C. sardinella from Yenisei River did not show any deviation from HWE  and Cocl23 (N = 5).In nine of the 18 populations, the expected heterozygosity across all loci was significantly higher than the observed heterozygosity   , and  Fig. .Fig. 2DeC. albula from Lake Puruvesi, C. sardinella and C. maraena separated from the Baltic cisco populations, similar to the split of populations in the dendrogram . In the reduced space along PCoA-Axes 1 and 2, the major split between the populations of cluster I versus clusters II and III was confirmed, similar to the dendrogram  and the Finnish Lake Puruvesi. In contrast, the German sympatric populations from Lakes Stechlin and Breiter Luzin, the three Polish populations and the two Finnish populations from Lakes Jerisjärvi and Oulujärvi showed high levels of admixture , the cross-entropy criterion ure Fig. .Fig. 4MeThe AMOVA, splitting the variance among the six ancestral groups identified above, indicated that the among-populations variance 12.1%) was higher than the among-individuals within-populations variance (9.8%), whereas the variation within individuals represented the greatest share (78.1%)  are genetically most segregated from all other cisco populations included. Accordingly, the German and Swedish sympatric cisco pairs in Lakes Stechlin, Breiter Luzin and Fegen most likely do not share a common phylogeographic origin. Our data thus confirmed a stronger genetic similarity of sympatric cisco pairs within lakes than among the three autumn- or spring-spawners of the three lakes, suggesting independent evolution of the pairs in each of the lakes. Furthermore, we detected that the population differentiation of the sympatric cisco populations in the two German lakes, as estimated by microsatellite analyses, was very weak and seems to have decreased in recent years. Accordingly, the sympatric populations in German Lakes Stechlin and Breiter Luzin may be at risk of extinction. In contrast, the similar sympatric population pair in Lake Fegen showed strongly significant population differentiation, although the genetic difference between these sympatric populations may also be in decline [We aimed to analyze the genetic differentiation as based on neutral nuclear markers among 16 populations of Baltic ciscoes and one ribed in ), for wh decline .C. albula from the Finnish lakes, C. maraena and C. sardinella were separated from the populations forming the clusters I–III. This overall grouping of the populations was confirmed by the Principal Coordinate Analysis.The dendrogram Fig.  split thC. sardinella from Yenisei River has evolved (and is maintained) by random genetic drift in isolated populations. However, all lakes are relatively large (> 300 ha surface area) and hence the population sizes of ciscoes can be expected to be > 100,000 individuals, likely preventing strong effects of genetic drift. The documented shared ancestry of populations in the same geographic area can be explained by gene flow in connection to post-glacial immigrations.In line with the dendrogram and the PCoA biplots, the average admixture coefficients Figs.  and 5 geC. albula populations survived while the spring-spawners became extinct during the last decades  [C. sardinella populations, suggesting an immigration wave connecting these populations.The strong effect of post-glacial processes on the phylogeography of the Baltic cisco populations becomes obvious if the differentiation among the Swedish populations is taken into account. Lakes of the Swedish microsatellite assemblage I, in particular Lakes Fegen and Stora Hålsjön, were exceptions from a clear geography-based differentiation. Their admixture coefficients were very homogenous, with a single dominant putative ancestral genetic group that was only weakly shared by the other populations around the Baltic Sea. Populations of the assemblage I exist in southern Swedish higher-elevation lakes, including the sympatric populations from Lake Fegen and from the three other lakes, in which only the autumn-spawning n, Ören) . MitochoC. sardinella population from the Siberian Yenisei River in our study was more similar to Swedish assemblage II than to assemblage I , and by the fact that the Finnish, Polish and German populations together were more distant to the Swedish assemblage I than to assemblage II. It is further possible that the present populations in Germany and perhaps Poland are related to both colonization waves and are even connected to Siberian and North American C. sardinella as indicated by the presence of both mtDNA clades in Breiter Luzin [C. sardinella, it has to be admitted that the results were most impacted by missing microsatellite alleles in our study, and hence the number of analyzed individuals was lower than for most of the other populations, likely reflected as low bootstrap supports in the dendrogram.The post-glacial colonization of central and north Europe from an eastern refugial area is also supported by the closer clustering of the Finnish er Luzin . HoweverC. albula and Siberian C. sardinella populations suggested that all contemporary populations in this area were formed by post-glacial hybridization between locally surviving ciscoes and invading West or East Siberian C. sardinella [Lota lota) for which populations from around the Baltic Sea formed a mitochondrial clade together with populations from Siberia, with single haplotypes even shared with burbot populations from Beringia and Alaska [Coregonus spp.) [Salmo trutta) [Perca fluviatilis) [Recent studies comparing Baltic and White Sea (Russia) rdinella , 40. Simd Alaska . In contus spp.) , brown t trutta)  and Euraiatilis)  indicateCoregonus cisco pairs in Swedish and German lakes evolved independently in each of the lakes, forming another example of parallel speciation, similar to the patterns found for example in other fish species [C. albula populations in the other lakes. The higher genetic similarity within than among the lakes supports earlier studies on these species pairs in German lakes based on microsatellites and mtDNA [C. artedi forms in the North-American Great Lakes area [The microsatellite analyses of this study support the hypothesis that the three sympatric  species –46, liza species  or snail species . If the nd mtDNA , and on nd mtDNA . A potennd mtDNA , and forkes area . In addikes area , 50, 51.C. albula and the spring-spawning C. fontanae in Lake Stechlin differ in their final thermal preferendum [More detailed investigations on ecological segregation have been performed on the sympatric pair in Lake Stechlin. A potential evolutionary split from a single ancestral population along the vertical temperature gradient has been demonstrated by an adaptive-dynamics model . As a coferendum , in the ferendum  and in tferendum . The sprferendum  or polycferendum . Howeverferendum  and feedferendum , suggestferendum , 16. We ferendum ) that woST estimates for samples taken in the years before 2005, but the differentiation was already weak. In the recent study, we could further follow up the genetic differentiation of these populations within lakes over time as based on repeated catches between 2011 and 2018. There was a tendency that genetic structure within the C. albula population in Lake Stechlin differed between 2011 and 2018, but we did not find evidence for a systematic differentiation of populations between all sampling years. Similar weak genetic differentiation has earlier been reported for the now extinct sympatric populations in Swedish Lake Ören [The statistically non-significant genetic differentiation between the sympatric pair in Lake Breiter Luzin and the very low differentiation in Lake Stechlin were not unexpected. Previous results on population differentiation of the German sympatric pairs , 35 had ake Ören , 61. In ake Ören , 33.Coregonus albula populations are well-known for abundance cycles with strongly varying recruitment [The spawning period (autumn: November to December vs. spring: April to June) of both sympatric pairs in the German lakes is still distinct, and hence reproductive isolation can be expected to be functionally maintained. Based on the available datasets of nine microsatellite markers, we have not evaluated the amount of potential hybridization between the species pairs. In the earlier study based on the numerically more abundant AFLP-markers, however, there were some signs of hybridization between the sympatric populations in both lakes, documented for fishes caught around the year 2000 . We cannruitment , 62 thatC. clupeaformis [Coregonus populations [There are three potential explanations why the genetic differentiation between the German sympatric pairs is much lower than that of the sympatric pair in Lake Fegen. First, it is possible that the population split between the sympatric populations from a common ancestor has occurred earlier in the Swedish than in the German populations, and hence the population differentiation is not yet strongly developed in Lakes Stechlin and Breiter Luzin. For Lake Fegen, time since divergence has been estimated to around 1900 years only (95% p.i.: 350 to 5900 years) assuming a generation interval of 4 years , a surpreaformis . In otheulations , 66. A rulations . AccordiAmphilophus spp.) demonstrated that such mono- or oligogenic traits may not suffice to efficiently reducing gene flow, such that genome-wide differentiation can remain weak and hence distinct species are ultimately not formed [Coregonus spp.) in lakes of Switzerland and Norway [Third, phenotypic traits may be affected by few genetic major effect loci only, but their ecological functions may induce strong additional effects on assortative mating and hence couple pre- and post-zygotic isolation. A recent study on Midas cichlids (t formed . If the d Norway . High-red Norway ), or whoCoregonus whitefishes, also sympatric Baltic ciscoes are considered distinct species in Central Europe, whereas they are considered the same species in Scandinavia [C. lucinensis was first described already in 1933), and also involve different taxonomical traditions in various parts of Europe. However, as discussed before, the level of genetic differentiation alone is not indicative to discriminate between complete and incomplete speciation. Non-overlapping spawning times, as found for the sympatric Baltic ciscoes, may be sufficient to limit gene flow between the co-occurring populations with differentiation accumulating over longer time spans, and hence may be seen as a central element to consider spring-spawning ciscoes as separate species. However, we do not know enough about the differences in genomic architecture of the sympatric species to evaluate their degree of post-zygotic incompatibility. As long as this ambiguity remains, the spring-spawning ciscoes in the two German lakes are considered separate evolutionary significant units, and their population status is monitored continuously.The complex phylogeographic history and the low differentiation at neutral markers in the German sympatric populations challenge the current taxonomy of sympatric Baltic ciscoes. Similar to the taxonomy of ndinavia . The difC. albula populations in other Swedish lakes of the microsatellite assemblage I are genetically most segregated from all other cisco populations included here. In contrast, the two sympatric pairs in the German Lakes Stechlin and Breiter Luzin are only weakly differentiated and most closely related to populations in neighboring Poland. The statistically non-significant neutral genetic differentiation of both German sympatric pairs in comparison with the strong genetic differentiation of the sympatric pair in Lake Fegen makes both pairs more prone to the loss of the locally endemic spring-spawners. Recent eutrophication in both lakes may add to the disturbance of strong ecological gradients that may have been decisive during the ecological split between autumn- and spring-spawners. In the complex of Baltic ciscoes, disappearance of endemic spring-spawners from several locations has already been documented, presumably caused by anthropogenic effects on the lakes. These observations support the general notion that biodiversity is at risk from anthropogenic changes, such as global warming. Species such as the endemic spring-spawning ciscoes that only recently evolved may be particularly vulnerable.Our data suggest that for the three sympatric pairs of autumn- and spring-spawning ciscoes, a common phylogeographic history is unlikely. The sympatric ciscoes in Lake Fegen and the allopatric Coregonus populations from 15 localities into the analyses  or assemblage II  [C. albula populations from western Polish Lakes Ińsko, Siecino and Miedwie, and three populations from the Finnish Lakes Jerisjärvi, Oulujärvi and Puruvesi were also added. Finally, we obtained individuals from a population of the Russian Yenisei River near Turukhansk, which is preliminarly identified as C. sardinella. For comparison, we also considered a population of C. maraena (whitefish) from the Swedish Lake Bolmen as a more distantly related taxon.We included 18 f Kalix)   , becauseC. albula, spring: C. fontanae and C. lucinensis) in the years between 2011 and 2018. We only included individuals with almost running gonads, to guarantee that we do not mix specimens from the sympatric populations. C. albula from Lake Stechlin was sampled in 2011, 2012, 2014 and 2018. C. fontanae (Lake Stechlin) was sampled in 2011, 2012 and 2018. Lake Breiter Luzin ciscoes were sampled in 2011 (both species) and 2012 . Samples from the other seven populations were obtained by gillnetting in 2002 (Finland), 2012 (Poland) and 2013 (Russia). One pectoral or caudal fin per fish was clipped and instantaneously fixed in ethanol for genetic analyses. Material from Swedish populations wasalso obtained by gillnetting and sampled for tissue in 2005, 2007, 2008, 2009 and 2010. Sympatric populations in Lake Fegen were sampled during spawning seasons, including only specimens with running or almost running gonads. See [Individuals from the German sympatric populations were sampled by gillnetting during the spawning times of the respective populations , Sfo23 (Cyanine 5), Sfo8 (Dy-751) and Cocl23 (BMN-6), set 2 was composed of Cisco157 (Dy-751), Cisco126 (BMN-6) and BWF1 (Cyanine 5), and set 3 consisted of Str73 (BMN-6) and BWF2 (Cyanine 5).We conducted the laboratory analyses for the 11 populations from Germany, Poland, Finland and Russia, whereas already existing microsatellite data from the 7 Swedish populations were added later to the dataset (see below). Total DNA was extracted from fin tissue using the Tissue DNA Mini Kit peqGOLD  following the protocol of the supplier. DNA concentration was determined spectrographically and aliquots diluted to a final concentration of ~ 20 ng/µl. All individuals were genotyped at nine polymorphic microsatellites following the protocol developed by , namely F1, BWF2 , Cisco90Cisco157 , Cocl23  [Cocl23 , Sfo8, S8, Sfo23 , and Str® Multiplex PCR mastermix, 1 µl DNA (~ 20 ng/µl), 0.44 to 1.91 µl primer mix and 2.1 to 3.6 µl H2O (PCR grade). The thermocycling profile started with an initial denaturation step at 95 °C for 5 min, followed by 28 cycles of 30 s at 95 °C, 90 s at 53 °C, 45 s at 72 °C and ended with a final extension step of 20 min at 60 °C. A quantity of 0.85 μl of the PCR product were added to a mix of 30 μl formamide  and 1 μl size standard-400 (Sciex), and denatured fragments were resolved on an automated DNA sequencer (Beckmann Coulter CEQ 8000). Genotypes were determined with the fragment analysis module of the GenomeLab™ GeXP Genetic Analysis System, version 10.2 (Beckman Coulter). Analyses of individuals with missing genotypes  were repeated once or twice, but were left incomplete after the second repetition.The Qiagen Multiplex PCR kit was used for PCR amplification according to the recommendations of the manufacturer. PCR amplification was carried out in 10 µl reaction volumes, containing 5 µl QIAGENC. albula populations from Swedish lakes [C. albula individuals from Lakes Fegen (autumn- and spring-spawners), Stora Hålsjön, Åsnen, Vättern, from the Baltic Sea  population of C. albula, and for C. maraena from Lake Bolmen. In this way, the data from Swedish populations were expressed with similar read lengths as if they would have been analyzed entirely on our sequencer.To facilitate the comparison of population structure as obtained for the 11 analysed populations with that determined earlier for sh lakes , we re-aR, i.e. the allele number corrected for the smallest sample size (N = 10) [IS) and population-wide deviations from Hardy–Weinberg equilibrium (HWE) were tested by the exact (probability) test using genepop_in_R based on Genepop 4.7.0 [Most analysis and graphical output were performed in R version 3.6.2 . Genetic(N = 10) , by PopG(N = 10) . Allele op 4.7.0 , 78. Theop 4.7.0  per popuop 4.7.0 , completop 4.7.0  as impleop 4.7.0 .Coregonus populations, a neighbor-joining tree [Population differentiation was estimated by F-statistics  between ing tree  based oning tree  chord diing tree . This tring tree . In addiing tree , and theing tree .K for N = 1 to 15 ancestral genetic groups with 10 repetitions per K, the maximum number being similar to the number of sampled locations. We then ran the ancestry estimates for the 655 individuals with the optimum K, and displayed the coefficients as barplot. We re-run an Analysis of Molecular Variance with the optimum number of ancestry groups determined by LEA, and compared the results with those from the AMOVA on the 18 contemporary populations. Finally, we calculated the population means of the ancestry coefficients, and displayed them as pie charts on geographical maps, to illustrate the geographical distribution of the ancestry genetic groups.To estimate and visualize population genetic structure, Bayesian computer algorithms as implemented in the stand-alone programs STRUCTURE  or ADMIXAdditional file 1: Table S1. Proportion of missing genotypes among the nine microsatellites (columns) in the 18 Coregonus populations (rows). Individuals with more than a single missing locus were omitted from subsequent statistical analyses. AS = autumn-spawner, SS = spring-Spawner. Table S2. Matrix of pairwise θ between sympatric populations of Lake Stechlin , sampled in different years , and their lower 95% confidence intervals . Weak structure between (sub)populations is indicated by the lower CI including zero. Table S3. Matrix of pairwise θ between sympatric populations of Lake Breiter Luzin , sampled in different years , and their lower 95% confidence intervals . Weak structure between (sub)populations is indicated by the lower CI including zero. Table S4. P-values of deviations from Hardy–Weinberg equilibrium per locus and population, with P < 0.05 considered a significant deviation. The original alpha = 0.05 was controlled by the false discovery rate for multiple tests. Table S5. Matrix of pairwise θ between 18 sympatric and allopatric Coregonus populations from 15 lakes or rivers in Germany, Sweden, Poland, Finland and Russia , and their P-values as obtained by G-tests . Strong structure between populations is indicated in bold. AS = autumn-spawner, SS = spring-spawner. Table S6. Overview of systematic differences in repeat length of nine bi-allelic microsatellite markers for eight cisco individuals from six Swedish populations, analysed either on an ABI3130xl sequencer (Applied Biosystems) (SWE) or an Beckmann Coulter CEQ 8000 sequencer (GER). The differences in read length were added to the original data for the Swedish populations included in the study. Figure S1. Overview on number of alleles per nine microsatellite loci, for 655 individuals of Baltic ciscoes from 18 lake and river populations in Germany, Sweden, Poland, Finland and Russia. Figure S2. Separate biplots of 18 Coregonus populations in reduced space along axes 1 and 2 of the Principal Coordinate Analysis. The axes dimensions are identically scaled to facilitate comparison of location and extent of population diversification. Figure S3. Plot of cross-entropy vs. a range of 1 to 15 ancestral genetic groups, as estimated by cross-validation by the R-package LEA.


Panthera leo). When planning translocations, the genetic background of populations needs to be taken into account; failure to do so risks disrupting existing patterns of genetic variation, ultimately leading to genetic homogenization, and thereby reducing resilience and adaptability of the species. We urge wildlife managers to include knowledge of the genetic background of source/target populations, as well as species‐wide patterns, in any management intervention. We present a hierarchical decision‐making tool in which we list 132 lion populations/lion conservation units and provide information on genetic assignment, uncertainty and suitability for translocation for each source/target combination. By including four levels of suitability, from ‘first choice’ to ‘no option’, we provide managers with a range of options. To illustrate the extent of international trade of lions, and the potential disruption of natural patterns of intraspecific diversity, we mined the CITES Trade Database for estimated trade quantities of live individuals imported into lion range states during the past 4 decades. We identified 1056 recorded individuals with a potential risk of interbreeding with wild lions, 772 being captive‐sourced. Scoring each of the records with our decision‐making tool illustrates that only 7% of the translocated individuals were ‘first choice’ and 73% were ‘no option’. We acknowledge that other, nongenetic factors are important in the decision‐making process, and hence a pragmatic approach is needed. A framework in which source/target populations are scored based on suitability is not only relevant to lion, but also to other species of wildlife that are frequently translocated. We hope that the presented overview supports managers to include genetics in future management decisions and contributes towards conservation of the lion in its full diversity.Conservation translocations have become an important management tool, particularly for large wildlife species such as the lion ( Although these patterns are largely congruent, in certain parts of the lion range, there are differences in patterns derived from these two types of genetic markers, which can be explained by local demographic histories and migration patterns Bertola,  Figure . Since nP. l. melanochaita) as a pragmatic and hierarchical approach for finding suitable source populations for conservation translocations. We define the ranking roughly as follows: (1) first choice: source and target populations fall within the same nuDNA and same mtDNA clade; (2) second choice: source and target populations fall within the same nuDNA clade but are from differentiated mtDNA clades; (3) third choice: source and target populations are from differentiated nuDNA clades, but from the same subspecies. We also include ‘no option’, which represents the introduction of the alternative subspecies or individuals of unknown genetic origin, which we strongly advise avoiding. We also categorize a translocation from the same subspecies from West or Central Africa into India (or vice versa) as ‘no option’ because of the strongly differentiated character of the Indian population. This categorization is in line with widely accepted recommendations, often summarized as keeping populations separated if there is no evidence of recent gene flow . To review the magnitude and direction of past international transport of lions, statistics available for legal live lion exports/imports were mined from the online Trade Database for the period 1983—2019 . We stress that the numbers retrieved from the database merely reflect the intention of trade and will thus deviate from the number of actual translocated individuals. Details of our approach and the definitions of source and purpose codes, as captured in the CITES Trade Database, can be found in Appendix CITES  is a multilateral legally binding treaty covering the transboundary trade of >35,000 species of plants and animals, including lions. Parties to CITES are obliged to compile and submit annual reports on their international trade in species listed on Appendix The available CITES trade data were amalgamated into four categories for analysis, namely the numbers of lions listed on consolidated permits that were (i) wild lions , and ranched), intended for (re)introduction into the wild (purpose code ‘N’); (ii) captive lions (source code ‘C’), intended for (re)introduction into the wild (purpose code ‘N’); (iii) wild lions , to be exported with the intention of commercial trade, breeding in captivity, and trophy hunting purposes  and (iv) captive lions (source code ‘C’), to be exported with the intention of commercial trade, breeding in captivity and trophy hunting purposes  Table .Based on scientific and grey literature and expert knowledge of authors, it became clear that, following our strict rules as outlined in Appendix 33.1Based on the phylogenetic assignment, associated certainty Table  and suggIt must be noted that around natural suture zones and in the case of potential hybrid populations, the matrix is not symmetrical: movement into suture zones (of lions from one of the overlapping clades) was scored as more favourable than movement from suture zones . We scored the admixed populations resulting from past translocations  of the individuals included in the CITES Trade Database would still receive a ‘no option’ recommendation. The vast majority of the individuals in this category receive this recommendation due to their captive origin. We summarize these results, both for CITES data only and for CITES data adjusted in Table Focussing on the origin of the individuals for which CITES records were obtained, and using Table 4Here we present an overview of LCUs and their association to genetic clades, based on the best available evidence. We connect this to recommendations for future conservation translocations, by providing a hierarchical approach, which includes a ‘first choice’, ‘second choice’, ‘third choice’ and ‘no option’ Figure  and a de4.1Despite people being involved in moving wildlife, including lions, throughout history  , to intensively managed free‐roaming populations (‘managed metapopulation’), to traditional free‐roaming (listed LCUs)  can be consulted for the most recent information on these populations and combined with genetic testing to determine their suitability for translocation.The managed metapopulation in RSA has been stocked with individuals from Kruger NP, Kgalagadi Transfrontier Park and Etosha NP (Namibia), and it mostly represents a mix of two mitochondrial haplogroups (East/Southern and South West)  alleviating the negative fitness effects of inbreeding depression  genetic clades, measured between populations, and (ii) heterozygosity, measured within populations or individuals. Our recommendations in this study focus mainly on the first component, expressed by the association of each LCU to a specific genetic clade. Therefore, we largely disregard within‐country translocations, even though these conservation interventions are fairly frequent, mostly as a response to human‐wildlife conflict. However, we do highlight those countries where within‐country translocations risk crossing boundaries between distinct genetic clades (see below). As mixing of genetic clades may have adverse effects, such as range‐wide homogenization and loss of adaptive potential, we urge managers to acknowledge differentiation between populations in their considerations of suitable source and target populations. Simultaneously, we acknowledge that, especially in the context of a reintroduction, i.e., a small initial population, within‐population diversity should be monitored and consecutive augmentation may be necessary to ensure that heterozygosity levels do not drop in subsequent generations.As translocations may be approached as mimicking natural gene flow, in general, translocations with source/target combinations from close proximity are favoured. As a rough guideline for acceptable translocation distance from a genetics perspective, we propose to use a distance, which can reasonably be covered by natural, possibly multigenerational, dispersal. Natural dispersal of lions depends on a range of factors, including geography, landscape use, prey density and climate, males especially being capable dispersers, covering up to 200 km , we advise against translocations from African lions to India or vice versa. The only situation in which a translocation of African P. l. leo individuals into India should be considered as if the Indian population has entered the extinction vortex , and genetic rescue is needed to safeguard survival of the population. Despite the Indian population being very low in diversity as a result of multiple bottlenecks , assuming sustainable off‐take from a source population is possible. Although we focus our assessment on continent‐wide patterns of diversity, population structure can also be determined on a more local scale. Few countries have had a country‐wide or regional assessment of lion population structure so far, but if this information is available, e.g., for Tanzania . However, the presence of hybrid individuals resulting from translocations may introduce doubt into the genetic origin of a lion or lion body part being used as evidence, thereby hindering the ability to utilize geographic assignment testing for forensic purposes. Documentation of management interventions will be helpful for understanding future patterns of diversity and possible forensic applications.Beyond the impact on lion populations, undocumented translocations can impede legal actions against the trade in both live lions and lion body parts. Successful prosecution for such illegal trade may require evidence of their likely origin, which is commonly determined using genetic methods to assign an evidence item to a particular population while excluding it from others. This requires the population of origin to be sufficiently genetically distinct from other populations (Ogden & Linacre, As we move towards the future, with ongoing land conversion and possible future local extinctions, we need to maintain a flexible attitude and be willing to reconsider our recommendations as new data become available and the situation on the ground may change. Making decisions on a case‐by‐case basis, including a wider context of both source and target populations, will contribute towards maintaining diversity. We emphasize that this paper is purely on the genetic aspect of translocations, and we acknowledge that other considerations must be taken into account. Several resources are available for dealing with translocations in general, such as the IUCN Translocation Guidelines IUCN, , or spec4.5The genetic variation present across the natural range of a species serves as its evolutionary potential to adapt to changes in its environment, such as climatic shifts, epidemics etc., thereby making it more resilient. If this variation declines, e.g., by local extinctions or by uncontrolled moving of individuals during translocations, the adaptability and the chances for long‐term survival of the species in the wild are impacted. We therefore urge managers to take genetic information, such as assignment to a genetic clade, into account when selecting source/target populations for conservation translocations. We present the current knowledge on the distribution of genetic variation in the lion in a comprehensive overview, hoping that this will support managers to integrate these insights when making decisions regarding source/target populations for translocations. This paper is not meant to fuel a debate on the feasibility of conservation interventions; rather, our hierarchical approach reflects our aim to deliver a tool, which includes the idealism‐pragmatism spectrum. Although this paper uses the lion as a case study, this approach is more broadly applicable. Co‐distributed species often show similarity in their phylogeographic patterns, depending on their ecology and demographic history, and hence may benefit from similar recommendations as are represented here for the lion.The authors declare that there is no conflict of interest.Appendix S1Click here for additional data file.Table S1‐S6Click here for additional data file.


LIMS1 for hair morphology, and COL24A1 for bone formation), and they are enriched in neurological functions either at an identical locus , or different loci in an identical gene . The bASN ancestry could also have formed the substrate of the genetic architecture of the dark pigmentation observed in the TIA peoples. We hypothesize that phenotypic convergence of the dark pigmentation in TIAs could have resulted from parallel  or genetic convergence driven by admixture , new mutations , or notably purifying selection . Our results provide new insights into the initial peopling of Asia and an advanced understanding of the phenotypic convergence of the TIA peoples.Tropical indigenous peoples in Asia (TIA) attract much attention for their unique appearance, whereas their genetic history and adaptive evolution remain mysteries. We conducted a comprehensive study to characterize the genetic distinction and connection of broad geographical TIAs. Despite the diverse genetic makeup and large interarea genetic differentiation between the TIA groups, we identified a basal Asian ancestry (bASN) specifically shared by these populations. The bASN ancestry was relatively enriched in ancient Asian human genomes dated as early as ∼50,000 years before the present and diminished in more recent history. Notably, the bASN ancestry is unlikely to be derived from archaic hominins. Instead, we suggest it may be better modeled as a survived lineage of the initial peopling of Asia. Shared adaptations inherited from the ancient Asian ancestry were detected among the TIA groups (e.g.,  Asia is home to approximately 370 million self-identified indigenous peoples, comprising two-thirds of the total indigenous population in the world. These populations are culturally and physiologically diverse and have been largely shaped by their local environment and subsistence. These unique characteristics offer an incredible opportunity to study human evolution and have long been a topic of interest and speculation. Archaeological records document a long history of modern human occupation in Asia, which probably occurred 50–70 ka along the coast of the Indian subcontinent to the Sundaland, subsequently reaching the Sahul Shelf . SeveralOne question that has long been debated is whether the distinctive physical features  shared by the tropical hunter-gatherers are ancestral or derived as a result of genetic convergence. Efforts have been made to investigate the full landscape of human genomic diversity in Asia , and theFST) in Asia was among the NL-TIA populations , and the Papuans (PNG)  A, and waDespite the fact that most of the global populations have multiple sources of genetic components, ADM, MLS, and the Oceanic populations contributed three additional ancestry components and showed minimal evidence of recent admixture B. This rConsistent with the autosomal genetic pattern, mitochondrial DNA (mtDNA) haplogroups were generally restricted in local regions. Populations with shared genetic ancestry may share the same mtDNA haplogroups . For insK = 5, where we found one basal component specifically prevailing in Asia, with leading proportions in the Oceanic populations, followed by some South and Southeast Asian populations , suggesting that the peopling of Asia might have occurred prior to the Altai Neanderthal introgression (directly or indirectly) to the modern human populations in this region , but this was not observed in West Eurasian samples (P = 0.89)   ancestry to be an ancient lineage, as evident by the following. First, the bASN ancestry is negatively correlated with the Altai Neanderthal introgression in Asian populations  . The bAS = 0.89)  fig. 2C = 0.89) . The sigC = 0.89  × 10−5) .MixMapper  fit all LIMS1. It consists of 40 bASN alleles spanning the entire gene (∼150 kb), all of which are derived alleles. The frequency of this haplotype is 0.97 in PNG, 0.8 in ADM, <0.3 in MLS and PHI, and is very rare in other worldwide populations. The high frequency and long stretch of this haplotype suggested that it could be adaptive in PNG and ADM , but did not show signals of adaptation in any of these populations. We also identified a group of candidate haplotypes (∼51 kb) in COL24A1, the frequency of which is higher in PNG (0.47), MLS (0.31), Vellalar , CheWong , Tibetan (0.20), and African populations (0.25–0.42) than the rest of populations studied. COL24A1 encodes for collagen XXIV predominantly functioning in osteoblast differentiation and bone formation and is associated with some morphological traits in humans, such as waist–hip ratio and height (DDB1/DAK was identified in ADM (0.95), PHI (0.5), and African populations (0.52–0.83), and it included a key missense variant  associated with the melanin index of Africans   .FCS based on the integrated haplotype score (iHS) and locus-specific branch length (LSBL) . Remarkably, we identified two genomic regions that exhibited sweep signals in all four populations  and is associated with certain neurological diseases. Notably, it has been reported to be height-associated in the Europeans , we compiled a comprehensive list of 1,057 pigmentation genes in human from previous genetic studies or biological pathways , the risr2=0.23 and P = 2.25 × 10−5 across individuals; r2=0.77 and P = 0.006 across populations) and South Asia   over the other Eurasians (AF=0.20–0.45), and two DPAs (rs6510760-A and rs112332856-C) in MFSD12 were enriched in the TIA populations in each geographic region. However, in most of the cases, the DPAs were lost in several of the TIA populations during evolution. For instance, we observed the accumulation of three DDB1-DAK DPAs  in ADM and PHI , which are in strong linkage disequilibrium (LD) with the aforementioned missense mutation at rs2260655 . In the DPAs reported in Europeans, we also found such examples like the ancestral allele (T) at an intronic variant TRPS1-rs2737212 . As for other pigmentary variants without determined effect alleles associated with pigmentation or depigmentation, we assumed that the alleles showing higher frequencies in the representative TIAs than in the nonindigenous Eurasians are more likely to be DPAs (hereafter denoted as putative DPAs). Interestingly, the population-specific putative DPAs, presenting in at least one representative TIA population but missing in all the nonindigenous Eurasians , tend to show in the Africans =0.36, Fisher’s exact test), whereas those population-specific non-pigmentary alleles are more likely to be novel mutations in the representative TIAs   and other 13 putative DPAs at RP11-6N13.1. The allele age at rs113432620-G was estimated to be older than 400 ka. This result, again, suggests that dark skin in the TIAs could be one of the most important phenotypes inherited from African ancestry. However, the adaptation of inherited DPAs mingled with that of LPAs in the skin-color determination of TIAs. We identified adaptation at the OCA2 loci in the TIA populations and found that the adaptive alleles matched the Asian prevalent alleles, which could be a consequence of recent admixture —number of DPAs in each individual—is positively correlated with the bASN ancestry in mainland Southeast Asia (lations) . We furts2260655 . Althougct test) . For exadmixture .MTHFD1-rs138662720-T, is related to different haplotypes between Africans and TIAs. The haplotypes in TIAs are similar to the Altai Neanderthal sequences and were inferred to be introgressed haplotypes by ArchaicSeeker 2.0 , a protein-coding gene modeled as an UV-induced DNA damage sensor in mice    . The ada pathway . MoreoveAF=0.22) . ZRANB3 d repair .RAD18, several examples of convergent evolution at the gene level were discovered. Notably, NRG1 involved in melanocyte development  we identified are involved in immune processes. It should be noted that the effects of material culture and traditions must also be considered, even if the strength of these influences cannot be easily quantified. Some TIA populations usually wear few clothes and their diets were rich in fish .We obtained the following data sets of present-day modern humans to contextualize the TIA samples : 1) the https://toppgene.cchmc.org/enrichment.jsp, last accessed December 29, 2021)  . Haploty9, 2021)   .We focused on the evolution patterns of some genes of particular interest, including genes involved in pigmentation-related pathways and ontologies defined by KEGG and Gene Ontology  were calculated between SNPs located within 500 kb from each other using PLINK v1.90  of an allele of interest, we identified the related haplotypes for this allele using the following procedures. First, we screened for the AF block involving this allele in each by 1) screening for the alleles at frequency f in the nonoverlapping 50-kb windows, 2) selecting out the windows with at least five alleles at frequency f, and 3) merging the adjacent windows and selecting the one with the target variant included. Second, we collected the union of the AF blocks identified in each population. Then, the haplotype networks were constructed and visualized at genomic regions of interest using NETWORK 10.1.0.0. To construct the haplotype networks, we selected 50% of the samples in populations with sample size >50, and make sure that the samples carrying the target alleles are included. We first calculated the median-joining network  following p is the empirical p value across the genome. When calculating the LSBL statistic, we chose Han Chinese (CHB) as an outgroup, and a neighbor population with the closest genetic relationship (measured by FST) as a reference for each representative TIA population: BEB for ADM, Burmese for MLS, and Dusun for PHI and PNG. An outlier SNP was defined as an SNP with an FCS among the 5% highest of the genome. A putatively selected genomic region was defined as a 100-kb window presenting a proportion of outlier SNPs among the 5% highest of all windows, in four bins of SNP numbers. Windows containing less than ten SNPs were discarded.We calculated iHS using n v1.2.0  for poly4 and 1.25 × 10−8, respectively, as recommended in the manual.Allele ages were estimated using the Genealogical Estimation of Variant Age (GEVA) (v1 beta) based on the mutation clock model . The inis) for variants in each category following s=−4.5 × 10−4 for GERP in 2–4, s=−4.5 × 10−3 for GERP in 4–6, and s=−1 × 10−2 for GERP >6. The total mutation load (L) is the sum of load for each locus , and the coding sequence region of MC1R in this study. To make it comparable across genes or gene sets, we averaged L by SNPs.Variants were grouped into three categories according to the genomic evolutionary rate profiling (GERP) score, corresponding to different deleterious levels . We assigned a selection coefficient ], the Department of Orang Asli Development  [JHEOA.PP.30.052.Jld 5(17)], as well as the district offices, village chief, and the chairperson of the Committee of Village Development and Security. Sample collection was also approved by the Biomedical Research Ethics Committee of Shanghai Institutes for Biological Sciences (ER-SIBS-261903). The family history, pedigree, and self-reported ethnicity were recorded via an interview using the local dialect. All procedures were in accordance with the ethical standards of the Responsible Committee on Human Experimentation  and the Helsinki Declaration of 1975, as revised in 2000.Not applicable.Molecular Biology and Evolution online.msab361_Supplementary_DataClick here for additional data file.


Population-specific random match probabilities ranged from a low of 10–115 to a maximum of 10–66. Mean informativeness (In) for ancestry inference was 0.355 (range 0.117–0.883). 65 novel SNPs were detected in 39 of the MHs using mMHseq. Of the 3018 different microhaplotype alleles identified, 1337 occurred at frequencies > 5% in at least one of the populations studied. The 90-plex MH panel enables effective differentiation of population groupings for major biogeographic regions as well as delineation of distinct subgroupings within regions. Open-source, web-based software is available to support validation of this technology for forensic case work analysis and to tailor MH analysis for specific geographical regions.Single-nucleotide polymorphisms (SNPs) and small genomic regions with multiple SNPs  are rapidly emerging as novel forensic investigative tools to assist in individual identification, kinship analyses, ancestry inference, and deconvolution of DNA mixtures. Here, we analyzed information for 90 microhaplotype loci in 4009 individuals from 79 world populations in 6 major biogeographic regions. The study included multiplex microhaplotype sequencing (mMHseq) data analyzed for 524 individuals from 16 populations and genotype data for 3485 individuals from 63 populations curated from public repositories. Analyses of the 79 populations revealed excellent characteristics for this 90-plex MH panel for various forensic applications achieving an overall average effective number of allele values (AThe online version contains supplementary material available at 10.1007/s00439-021-02382-0. For many years, the DNA markers for forensic practice have been short tandem repeat (STR) loci that are highly polymorphic with different numbers of repeat units at each locus  have been defined as small genomic regions of less than ~ 300 bp with two or more polymorphisms, usually SNPs, resulting in at least three common haplotypes in the population  in the 30 populations studied (average Ae > 5.08). Analyses of frequency variation among populations showed that some of the loci had significant variation among populations. To be of maximal value in forensics as well as in other areas of research, a panel of loci needs a broad set of reference population frequencies. To that end, we have now assembled and analyzed sequence-based data on 4009 individuals in 79 populations for these 90 microhaps. These results also demonstrate the value of microhaplotypes for biomedical and anthropologic studies of human populations.We previously presented a panel of 90 microhaplotypes evaluated using data for 26 populations extracted from the 1000 Genomes (1 KG) project data  is a measure that standardizes the information among diverse populations for their different frequencies among the multiple alleles . As such, it is the number of equally frequent alleles that would yield the same heterozygosity as the observed set of alleles with diverse frequencies. This measure is good for evaluating multiallelic loci (such as microhaplotypes) for individualization and mixture analysis. Informativeness (In) for measuring allele frequency differences among populations was calculated according to Rosenberg et al.  iterations. The result with the highest likelihood of the 20 runs was selected to illustrate the results for a given K value.To help visualize clustering of individuals in populations, we used version 2.3.4 of the STRUCTURE software  on the matrix of haplotype allele frequencies for all 90 microhaplotype loci in the populations relevant to each analysis.To help visualize clustering of populations, we used Principal Component Analyses (PCA). We used the XLSTAT 2019 software , and sequence bases . Any sample that failed this QC was removed from further analysis. The first QC metric (sample coverage), defined as the number of reads per sample, was used for detecting samples that failed in the multiplex PCR. An average read depth across 384 samples was 705,536 reads per sample. Eight out of 384 samples had lower read depth coverage of less than 150,000 reads and were flagged for further analysis of amplicon and base coverage . The second QC metric (amplicon coverage) was used to identify samples with partially failed amplification, such as individual amplicons that may have been insufficiently covered despite an overall normal read count for that sample. For each sample, we obtained the number of amplicons that had > 0.2-fold the mean amplicon coverage and used a threshold of 2 standard deviations below the mean to flag samples for review. This metric identified 4 samples with poor amplicon uniformity . The third QC metric (base pairs) assessed base coverage for each sample, reasoning that if base coverage was sufficiently high, even samples with lower amplicon uniformity could be analyzed further. Five samples had a lower base coverage (< 75% of bases with 100 × reads per nucleotide per amplicon). Three samples failed QC at all three levels and were removed from the analysis, while the other samples flagged in one of the three QC steps yielded interpretable results in sequence analysis. Thus, final analyses are based on data for 381 individuals  in the last five sequencing runs, each of which involved sequencing of 48 individuals. Thus, sequencing of a total of 240 individuals contributed to this number. These are the right-most 62% of the reads in supplemental Fig. S1. Some variation in read numbers occurred among the five runs considered, but the variation in reads per locus was consistent; the distribution of the number of loci by the number of reads is given in Fig. S2. We note that except for 13 loci, there were more than 500 reads per allele per locus per individual. Only one locus, mh01KK-001, averaged fewer than 100 reads per allele with 75.3 reads per allele. In general, coverage per locus exceeds the clinical exome sequencing standard of 80×. It is unclear whether the differences in reads per locus per individual are inherent to the locus or are inherent just to the sequence or concentration of the specific primer pair used for the sequencing. A future effort will be made to better balance across loci to assure a higher minimum number of reads for all loci.In summary, the mMHseq 90-plex data for the sequenced individuals from 16 populations are available at the Scharfe lab mMHseq website and have also been deposited in the Zenodo archive (see Data Availability). Our previous study  and variation among populations (In). Figure n by average Ae for the total of 79 populations. Some of the markers rank very high by both criteria. The six MHs that are highest for Ae are shaded and included in Table e across loci for populations that are farther from Africa is evident in Fig. e ranging from 3.0 to more than 6.0 , at a single locus is the sum over all the possible genotypes in the population of the squares of the genotype frequencies. In other words, it is the expected frequency (probability) for the population of, having randomly selected one individual, another unrelated individual will have that same specific genotype. For multi-locus genotypes, RMP becomes the product of the individual locus probabilities. It is often used in criminal cases to note how unlikely it is that someone else has the same genotype as a defendant. The RMP values are quite small for these 90 MH loci. However, RMP is population-specific and has a dramatic difference of 50 orders of magnitude depending on the population  across the 79 population samples likewise shows considerable variation by locus . K = 7 shows that these loci can begin to distinguish among the sub-Saharan Africans. Yet, when all 79 populations were analyzed up to K = 16, the African clustering looks identical to the K = 7 pattern . In contrast, the East Asia pattern became much more complex at K = 16. This panel of 90 loci is capable of more refined STRUCTURE clustering when subsets are analyzed separately. When the 21 African and Southwest Asia populations were analyzed as a group, K = 6 showed five clusters within sub-Saharan Africa . All other populations are primarily distributed according to PC2. To separate those non-African populations better, a separate analysis was done omitting all of the sub-Saharan populations Fig. . This anThe tree analysis of Tau genetic distances on all 79 populations involved evaluations of a total of 294 different additive tree structures of which 31 had no internal negative segments. The best of these 31 is shown in Suppl. Fig. S8. There are two small negative segments connecting the two mostly West African populations (ACB and ASW) to the African branch of the tree. This is an indication that these do not conform to the underlying assumption of an additive tree for which only random genetic drift has caused divergence of populations. Indeed, these two populations are admixed and do not meet the assumptions, but were included as part of the 1 KG set of populations.In general, many of the clusters of populations are similar to those seen in the STRUCTURE and PCA analyses. The South Asians are divided into four different clusters in the tree. One is close to the European and SW Asia cluster; the others are more differentiated.The utility/value of a locus in forensics can relate to at least four different questions: individualization, ancestry inference, kinship analysis, and mixture resolution. Individualization is often noted as the random match probability (RMP) reflecting the low likelihood that a match between evidence and an accused individual would have occurred by chance alone. Ancestry inference can be pursued as the identification of the population for which the probability of the observed genotype is highest  are a consequence of the high Ae values (3.00–6.25) across all 79 populations among the 90 microhaplotypes , but K = 6 provides a convenient basis for summarizing aspects of the data such as the MHs with the highest regional Ae values. Table S3 summarizes the top 20 MHs ranked by Ae value for each of the six biogeographic regions defined in Fig. e values for the 20 highest loci are lower for the non-African regions with the smallest for the Pacific populations, but the decrease is not great compared to the overall decrease seen in Fig. e in more than one broad region of the world. Only 6 of these 38 loci occur in all six biogeographic regions  in 79 populations and the highest average Ae (9.708 and 9.750) in the 79 populations.The large number of MH alleles varying in the six biogeographic regions are illustrated in Fig. 212 Fig.  and mh05212 Fig. , with thn markers require a reference database to determine allele frequencies for calculating RMP values and for use in forensic attempts to identify the population ancestry of the donor of a DNA profile. This study provides reference data on 79 population samples. Several of those populations are smaller than ideal for forensic reference, but as seen in Fig. K = 6 and K = 7 define Mendelian populations of considerable size in some cases. It is clear that an amalgam of European population samples in one STRUCTURE cluster is as valid a reference population as a forensic reference population such as “U.S. White”.High IThe PCA and STRUCTURE results presented show that the extensive genetic variation in the 79 populations analyzed with the 90 MH panel can both differentiate clear population groupings for major geographical areas of the world as well as delineate distinct subgroupings of populations, especially when analyses are restricted to particular biogeographic regions.K values > 6. The new marker data do provide new information on some of the populations as discussed and also presented separately for African and East Asian populations below.There were no real surprises in the population relationships seen in STRUCTURE analyses and PCA. Indeed, as noted earlier, several other sets of markers on similar collections of populations have shown similar relationships  which is a higher K value than the likelihood increases warrant, but illustrates the general pattern for subdivisions of the six major regions. For Africa, the change from K = 7  but with the Ethiopian Jews showing differing patterns at higher K levels. The North African and Southwest Asian populations became a separate cluster from the Europeans at K = 9 and the cluster persisted through higher K values. The South-Central Asia cluster separates off the Pakistani populations with a distinct admixture component at K = 13 and that distinction remains through K = 16. Three of the South-Central Asia populations show inconsistent patterns of clustering after K = 13. In contrast to the small refinements of the African and European patterns, the East Asian patterns became more subdivided with increasing K value, as discussed below. The Oceania populations show several different patterns at the different K values.The six main clusters of populations seen in Fig. K = 5 and K = 6  cluster and the Southeast Asia  cluster. That pattern persisted through K = 16. If it has any meaning, it is probably that the Han Chinese are intermediate in a North-to-South cline in far East Asia and not that they are individually admixed of those flanking populations. At K = 9, the Atayal became distinct. At K = 10–16, the Khanty became distinct and usually (for K = 10 to 14) group with the Buryat and Yakut; in both cases, they remained distinct through K = 16 . Oceania showed inconsistent clustering among the populations except for the consistent clustering of the two Melanesian populations together.The most striking result for the 79 population analysis is that at K = 7  and restricted (21) sets of populations.Similar population groupings are seen in the PCA results . The Khanty from northwest Siberia is a clearly distinct population in this analysis. Note that in the full global context, it was intermediate between the Europeans and East Asians. We chose 21 population samples from Western Siberia to the Pacific omitting the South Central Asian samples that were a clearly distinct cluster in Fig. = 7 Fig. . The KorK = 5 that is not seen in any of the results for all 79 populations. In contrast, the East Asians by themselves cluster in ways that are similar  to the clustering of all populations at K levels up to K = 16. We do not fully understand the cause in this case of the different patterns. We know that different markers are most relevant to different regions; the magnitude of the allele frequency differences is undoubtedly relevant. How well this regional inconsistency in finer clustering generalizes to other datasets is unknown at present.Overall, these 90 microhaplotype markers are a powerful set for population relationships, but it was impossible from these analyses to determine when a subset of populations would provide an answer not inferable from the full set of populations. The Africans, in the separate 21 population analysis, clearly show clustering at e values of this set of MHs should be especially informative, because the probability of allele sharing identical by state can be much less than sharing identical by descent for close relatives. However, no direct test has been done. Recent papers by Puente et al.  as seen at locus mh05KK-170 in Bennett et al. . Howevere and high In. This has resulted in loci with, on average, greater extent to encompass more SNPs. Eliminating the loci with the lowest Ae and/or In values globally should improve the efficiency of the panel. However, a careful analysis should be undertaken to assure that the lowest In marker for all populations is not providing significant differentiation of some population(s). We generated exploratory STRUCTURE runs from K = 2 to K = 8 for 79 populations after excluding 19 MH with In ≤ 0.25. The cluster patterns of the highest likelihood runs for the 71 MHs were all very similar to those obtained with all 90 MH. The most noticeable difference occurred at K = 7 where the Biaka from central Africa clustered with the West African groups instead of the East African cluster. Some of the excluded MH markers undoubtedly have value in differentiating among the sub-Saharan groups. Given the high level of informativeness of the panel for obtaining results at 90 loci, efficiency is not an issue. Rather, any pruning would allow space for adding additional marker loci with higher values, including some of the best of the loci identified by others, e.g.,  and the expanded set of populations studied from around the world revealed a highly informative set of markers that has characteristics that can serve a range of forensic, medical, and anthropological applications. Additional useful microhaplotypes will likely emerge from other and future studies Below is the link to the electronic supplementary material.


Eriosyce (Cactaceae) might present a greater specific diversity since much of its species richness might be hidden in morphological species complexes. The aim of this study was to investigate species delimitation using the molecular data of the globose cacti “E. curvispina”, which harbor several populations of unclear evolutionary relationships. We ran phylogenetic inferences on 87 taxa of Eriosyce, including nine E. curvispina populations, and by analyzing three plastid noncoding introns, one plastid and one nuclear gene. Additionally, we developed 12 new pairs of nuclear microsatellites to evaluate the population-level genetic structure. We identified four groups that originated in independent cladogenetic events occurring at different temporal depths; these groups presented high genetic diversity, and their populations were genetically structured. These results suggest a complex evolutionary history in the origin of globular cacti, with independent speciation events occurring at different time spans. This cryptic richness is underestimated in the Mediterranean flora of central Chile, and thus unique evolutionary diversity could be overlooked in conservation and management actions.Unraveling the processes involved in the origin of a substantial fraction of biodiversity can be a particularly difficult task in groups of similar, and often convergent, morphologies. The genus  The planet’s biodiversity is declining at unprecedented rates . MediterThe underestimation of diversity may be due, in part, to the occurrence of species that are sufficiently distinct based on molecular characterizations, but which have been classified as a single nominal species because they are at least superficially morphologically indistinguishable . EstimatEriosyce sensu lato has a complex taxonomic history with a high level of uncertainty, evidenced by the long history of taxonomic changes since its description more than 100 years ago. Species complexes, which include several populations, were historically assigned species ranks, but are currently considered single species. Eriosyce curvispina, with dozens of published names, is one of the most taxonomically complex species within the family )s nested , and devE. curvispina complex has a globular stem form, with slightly curved prickles; the forms of the flowers are also very similar, but the colors differ from place to place, from lemon yellow to reddish, as shown in The E. curvispina complex, we added 33 samples to a backbone phylogenetic data matrix of the genus published elsewhere [Horridocactus and 15 new individuals of the sister clade Neoporteria, all from field collected specimens. We mainly collected the material from roots or flowers and kept it in CTAB-NaCl (hexadecyltrimethylammonioum bromide-sodium chloride) buffer (2%:22%) to transport to the laboratory and store at 4 °C until the extraction.To analyze the evolutionary relationships of the lsewhere . SpecifiEriosyce curvispina from nine populations: Putaendo, El Escorial, Tilama, Choapa Valley, Limahuida and Laguna Verde , Farellones , Ocoa  and Los Molles-Pichidangui , all collected as described in For the microsatellite analysis, we sampled 150 individuals of the complex rpl32-trnL, trnH-psbA and trnL-trnF), one plastid gene (ycf1) and one nuclear gene (PHYC) following the protocol described by Guerrero et al. [For DNA extraction, we used 40–50 mg of root or flower tissue that first was pulverized into a fine power using an automatic homogenizer, and then total DNA was extracted using a DNeasy Plant Kit . For the phylogenetic analysis, we amplified three noncoding chloroplast markers . Sequences for each marker were automatically aligned using Muscle and then checked manually. The outgroup consisted of 19 species, mostly from the core Notocacteae. Each marker was aligned separately and then concatenated. A microsatellite region in the ycf1 dataset was excluded (450 bp) due to ambiguous alignment in this region. The best partitions and molecular models were evaluated using PartitionFinder v.2.1.1, as described by Guerrero et al. [We utilized 117 sequences , assembrpl32-trnL and trnH-psbA and GTRINVGAMMA for the rest of the markers. Convergence was monitored using the standard deviation of split frequencies, and when this value stabilized below 0.01, it was considered a strong indication of convergence. The associated likelihood values, effective sample size (ESS) values, and burn-in values of the different runs were verified with the program Tracer v1.7.1 [Bayesian inference of the concatenated matrix was performed using Mr.Bayes v3.2.7 , and unlr v1.7.1 . Trees wr v1.7.1  . These samples were collected between Los Molles and Pichidangui localities (Latitude −32.2 and Longitude −71.47). Sequencing libraries were prepared using a Nextera XT DNA kit, and then sequenced using an Illumina® MiSeq Next Generation Sequencer with an output of 15 million fragment reads at AutralOmics facilities . After ary 2022  and Prinary 2022  softwareary 2022  was usedary 2022  generateary 2022 ) search Eriosyce DNA samples to find the most polymorphic ones. Finally, we choose 12 loci: PS5, PS9, PC2, PC11, PM10, PA12, PM8, PC10, PM6, PC7, PM7 and PA6  and 0.5 uL of each primer at 5uM. The reverse primers were 5′fluorescently labeled with either 6-Carboxyfluorescein(6-FAM), 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein (VIC), 2′-chloro-5′-fluoro-7′,8′-benzo-1,4-dichloro-6-carboxyfluorescein (NED) or PET .From the information described above, we choose dinucleotide and trinucleotide sequences common in the four pools. Afterwards, a set of 52 primer pairs were tested with different  and PA6  to perfo®  and co-running with a 100 bp DNA ladder . The amplified PCR products were sent to the Unidad de Secuenciación y Tecnologías Ómicas at the Pontificia Universidad Católica de Chile  for genotyping. They were analyzed on ABI PRISM 3500 XL (Applied Biosystems) using the size standard Genescan LIZ 500 . Then the loci from dinucleotides repeats, were scored manually and analyzed using Geneious Prime®  with Microsatellite plugin taking only the fragments over 100 pb.The PCR’s were performed in a thermo-cycler  programmed as: 1 min at 94 °C for initial denaturation, followed by 35 cycles of 98 °C for 00.05 s, primer specific annealing temperature (58 or 62 °C) for 00.05 s min, 72 °C for 00:40 s, and final extension at 72 °C for 1:45 min. The PCR products were checked on 1% agarose gels in 1X Tris-boric acid-EDTA buffer running a mixture of 5 ul of PCR product with 2 ul of 6X loading buffer  with GelRedFis) and linkage disequilibrium (LD) with Genetix 4.05; Fst with FreeNA [We estimated allelic frequencies, polymorphic loci (%P), expected heterozygosity (He) and observed heterozygosity (Ho) with GenAlEx 6.51b2 ; the fixh FreeNA ; and theh FreeNA  Table 2Fis and ls (referred as DK) against a range of K values and selecting the true K based on the maximal value using the Evanno method [The population’s genetic structure was evaluated using Structure v2.3.4 ; this apo method  , trnL-trnF contributed 1084 bp (15%) and trnH-psbA contributed 439 bp (4%), while the plastid gene ycf1 contributed 930 bp (20%) and the nuclear gene PHYC contributed 1034 bp (locus with 31% of variable sites).The concatenated matrix with the five markers included 4841 bp of aligned sequences for 117 individuals, of which 2440 were variable for the complete matrix and 1958 for the ingroup . Of the E. curvispina complex; we found that its members clustered in four pairs of taxa distributed across three different clades within Horridocactus in four groups  from Andean populations Putaendo and Escorial. Within Groups I and II, divergences were dated 3.3 ± 2.0–4.9 Ma and dated 2.7 + 1,6–3.8 Ma, respectively. Non-putative members of the E. curvispina complex were retrieved in Clade B, which is composed of species with the geophyte growth form (northernmost distribution within Horridocactus). Within Clade C, Group III of E. curvispina harbors populations from Choapa Valley, Tilama and Limahuida, which were placed together in a branch sister to E. limariensis; this divergence occurred at dated 1.2 + 0.5–1.9 Ma. Two more members of the E. curvispina complex (Ocoa and Farellones) originated at 1.4 ± 0.4–2.1 Ma (Group IV), and were placed sister to E. aspillagae, which was the species with southernmost distribution.Sequence data yielded a well-supported phylogenetic hypothesis of the Horridocactus clade . Bayesiaps I-IV, . These gps I-IV, . The oldFst = Wright´s F-statistics) were significantly different from zero. The population in Ocoa has little genetic variation, shown by the lowest fixation index, observed and expected heterozygosity, and levels of polymorphism of 67%, also present a fixation index, Fis = 0.0990 (see The 12 nuclear simple sequence repeats (SSRs) markes revealed that the groups previously identified in the phylogenetic inferences differ in their genetic diversity and structure . We dete0990 see .E. curvispina Putaendo and E. curvispina Escorial) and Group III . Group I presented substantial genetic differentiation compared to its closely related Groups II and III. Group IV (E. curvispina Ocoa and Farellones) showed a higher genetic divergence, as the population of E. curvispina at Ocoa is the most differentiated genetic group . These results are consistent with the DAPC analysis, where the cluster formed by E. curvispina from Putaendo, El Escorial, Farellones, Tilama and Valle del Choapa was observed, and with the result of Structure, K = 6 ; two of them harbor E. curvispina populations, which in turn were distributed into four distinct groups (I–IV). This revealed a more complicated evolutionary scenario that is not consistent with the existence of a taxonomic complex, because it presents independent cladogenetic events. Additionally, our study strongly suggests that the members of Groups I to IV originated at different time spans, supporting that divergence mechanisms produced deep and shallow speciation events across the evolutionary time.Phylogenetic inferences retrieved a well-supported phylogenetic tree revealing evolutionary lineages within the section E. curvispina at Los Molles and Laguna Verde presented an estimated divergence of 3.3 Ma; additionally, DAPC of SSRs data support the existence of the population genetic structure, suggesting that there is a hard barrier between these two coastal populations that constrain gene flow, reinforcing differences. In contrast, the Andean populations  of Clade A showed high levels of divergence in the phylogenetic tree  in two independent cladogenetic events. Group III occupies valleys of north central Chile, while species members of Group IV occupy areas further south within the transversal mountain ranges and the Andes. Both habitat types have experienced major vegetation changes over the last million years following glacial cycles ,56,57,58Neoporteria, another group of globose cacti, diverged along the coast but did not show levels of deep divergence [Members of section vergence ; this divergence , most ofvergence ,67. The E. curvispina” has experienced a habitat reduction, increasing the chances for inbreeding, plant extirpation and genetic diversity reduction.The population at Ocoa showed the lowest fixation index, and the lowest genetic diversity among populations, congruent with the fact that it is an isolated population, and thus gene flow with other populations is less likely. This isolation may be caused by the fact that the native xerophitic vegetation remains in island-like hills, and in the last two centuries, the surrounded matrix has been transformed into an anthropized landscape due to different activities such as cereal crops, fruits, livestock, and housing ,69. In cThese results support inferences about the historical and modern eco-evolutionary processes that have contributed to the observed diversity patterns of this group, as the combination of geographic factors (topography and climatic variations) added to pollination guild variation, and in more recent times agricultural activities, may be major factors molding the genetic diversity of these globular cacti. Despite the wide distribution and importance of the arid and semi-arid zones, phylogenetic and population genetics studies are still scarce ,71,72, eEriosyce, and that the Eriosyce curvispina complex is a polyphyletic group lumping at least four evolutionary lineages. We identified four groups that originated in independent cladogenetic events occurring at different temporal depths; these groups presented high genetic diversity, and their populations were genetically structured. These results improve our understanding on the origin of an endemic rich group of cacti, allow us to more clearly determine its biodiversity, and suggest that a fraction of the flora of the Mediterranean Central Chile biodiversity hotspot may be cryptic. This information is mandatory for accurate extinction risk assessments and the efficient design of conservation actions, avoiding overlooking highly threatened species in an increasingly anthropized landscape.The phylogenetic reconstruction with chloroplast DNA (cpDNA) and nuclear DNA markers showed strong relationships of the taxa within


Protobothrops mucrosquamatus. In total, 16 polymorphic microsatellite loci were screened in 150 snakes  covering most of their distribution. Microsatellite analysis revealed high genetic diversity in P. mucrosquamatus. Bayesian clustering of population assignment identified two major clusters for all populations, somewhat inconsistent with the mitochondrial DNA phylogeny of P. mucrosquamatus reported in previous research. Analyses based on 92 sex‐determined and 37 samples of P. mucrosquamatus from three small sites in Sichuan, China  consistently suggested female‐biased dispersal in P. mucrosquamatus, which is the first example of this pattern in snakes. The female‐biased dispersal patterns in P. mucrosquamatus may be explained by local resource competition.Dispersal plays a vital role in the geographical distribution, population genetic structure, quantity dynamics, and evolution of a species. Sex‐biased dispersal is common among vertebrates and many studies have documented a tendency toward male‐biased dispersal in mammals and female‐biased dispersal in birds. However, dispersal patterns in reptiles remain poorly understood. In this study, we explored the genetic diversity and dispersal patterns of the widely distributed Asian pitviper  Protobothrops mucrosquamatus. Microsatellite analysis revealed high genetic diversity in P. mucrosquamatus. Analyses based on sex‐determined samples suggested female‐biased dispersal in P. mucrosquamatus, which is the first example of this pattern in snakes. The female‐biased dispersal patterns in P. mucrosquamatus may be explained by local resource competition.In this study, we explored the genetic diversity and dispersal patterns of the widely distributed Asian pitviper  The generated library was sequenced on the Illumina HiSeq 2000 platform to produce paired‐end reads. The quality of the raw reads was assessed using FastQC v.0.11.9 , 12.5 µl of 2 × T5 Super PCR Mix (PAGE) , and 10 µl of nuclease‐free water. The cycling conditions included a hot start pre‐denaturation of 95°C for 4 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 61–63°C (according to each primer pair) for 30 s, extension at 72°C for 30 s, post‐extension at 72°C for 10 min, and heat preservation at 10°C.The successfully optimized microsatellites were used to evaluate the genetic diversity of The PCR product size was measured on an ABI 3730xl DNA Analyzer (Applied Biosystems) according to each forward primer labeled with fluorescent dyes  and data were obtained with GeneMapper v.4.0 (Applied Biosystems). All samples were read at least three times to reduce artificial error.P. mucrosquamatus , expected heterozygosity (He), observed heterozygosity (Ho), and polymorphic information content (PIC) of each microsatellite marker  was analyzed for various genetic diversity indices. Based on the mitochondrial DNA phylogeny of 2.4K) varied from K = 1 to K = 10, with a burn‐in of 100,000 and MCMC repeats of 1,000,000 with 10 iterations. Results were collated using Structure Harvester v.0.6.94  were performed using GenAlEx v.6.5  , relatedness (r), mean assignment index (mAIc)  from the SCV and SWC populations were used to evaluate sex‐biased dispersal. We assessed sex‐biased dispersal from three small sites in Sichuan  in China using a two‐sided test. With reference to Goudet , Goudet To further verify the results of sex‐biased dispersal, we analyze data from the 92 sex‐determined individuals and three small sites separately, we calculated and compared relatedness values between the sexes using COANCESTRY v.1.0 with five moment and two likelihood estimators Wang, .33.1P. mucrosquamatus individuals, 16 microsatellites were successfully optimized with polymorphic and call rates above 90% across all samples. Statistics calculated for the 16 polymorphic microsatellite loci across the sampling localities are listed in Table Based on genotyping of 25 microsatellites in 150 The overall level of polymorphism detected in the 16 loci was high, with total alleles of 364 and average number of alleles (Na) of 22.75 (ranging from 13 to 37). Ho varied from 0.480 (YM‐3) to 0.899 (YM‐20), with an average of 0.764. The highest He value was 0.951 (YM‐11) (average 0.891). The highest PIC value was 0.945 (YM‐11) (average 0.879). Statistics for the 16 polymorphic microsatellite loci for total dataset are listed in Table 3.2P. mucrosquamatus populations, the coancestry relations of the populations were analyzed based on a Bayesian clustering model. The independent clustering of all samples recorded the highest ΔK value at K = 2  in southern China and Myanmar Vietnam came from the same ancestral population (blue), while 1 population in southwestern China (SWC), the genetic information is mainly from another ancestral group (red). The two clusters displayed different population membership to that reported previously based on mtDNA , the majority of individuals revealed low probabilities of being assigned to any particular clusters  and Linear Discriminant 2 (LD 2) separated among P. mucrosquamatus cluster . SWC population were roughly at the same level with respect to LD 2, and HN, VM and SCV, TW populations were above and below them, respectively. AMOVA of the five populations showed that 82% of the variation was found among individuals, with only 4% found among populations (see Appendix Fst) was high in HN, VM, SCV, and SWC populations compared to the TW population. Fst values between VM and SCV, SWC populations, and SCW with SWC population were low, suggesting low genetic differentiation among them , Hs , and vAIc values  compared to males, but lower Fst, mAIc, and r values (Table Fis (0.1113 vs. 0.0347), Hs (0.8174 vs. 0.7907), and vAIc values (14.6314 vs. 12.5667) compared to males, but lower Fst, mAIc, and r values (Table p < .05)  , high Ho (0.480–0.899), and high He (0.713–0.951) (Table Coronella austriaca)       Table , but areP. mucrosquamatus, different from previous mtDNA‐based findings  when the temperature rose  ; Data curation (lead); Formal analysis (supporting); Methodology ; Validation ; Visualization (supporting); Writing – original draft ; Writing – review & editing . Qin Liu: Conceptualization (supporting); Data curation (supporting); Formal analysis (lead); Investigation ; Methodology ; Software (lead); Visualization ; Writing – review & editing . Ya‐yong Wu: Conceptualization (supporting); Data curation (supporting); Formal analysis (lead); Investigation . Peng Guo: Conceptualization (lead); Data curation (lead); Formal analysis (supporting); Funding acquisition (supporting); Investigation (supporting); Methodology (supporting); Project administration (lead); Visualization (supporting); Writing – original draft ; Writing – review & editing (supporting). Kong Yang: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing – original draft ; Writing – review & editing (supporting).Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.Appendix S6Click here for additional data file.


Amanita exitialis is a poisonous mushroom and has caused many deaths in southern China. In this study, we collected 118 fruiting bodies of A. exitialis from seven different sites in Guangdong Province in southern China and investigated their genetic relationships using 14 polymorphic molecular markers. These 14 markers grouped the 118 fruiting bodies into 20 multilocus genotypes. Among these 20 genotypes, eight were each found only once while the remaining 12 were each represented by two to 54 fruiting bodies. Interestingly, among the 12 shared genotypes, four were shared between/among local populations that were separated by as far as over 80 km, a result consistent with secondary homothallic reproduction and long-distance spore dispersal. Despite the observed gene flow, significant genetic differentiations were found among the local populations, primarily due to the over-representation of certain genotypes within individual local populations. STRUCTURE analyses revealed that the 118 fruiting bodies belonged to three genetic clusters, consistent with divergence within this species in this geographic region. Interestingly, we found an excess of heterozygous individuals at both the local and the total sample level, suggesting potential inbreeding depression and heterozygous advantage in these populations of A. exitialis. We discuss the implications of our results for understanding the life cycle, dispersal, and evolution of this poisonous mushroom. Amanita /4, stF values of 0.05–0.15, 0.15 < stF < 0.25, and >0.25 are indicative of moderate, high, and very high genetic differentiation among populations, respectively. If neutral genetic markers are used and assuming constant population size, the degree of genetic differentiation among geographic populations would be closely related to gene flow and to genetic drift within and between populations. In general, mN values > 1.0 indicate that the populations are unlikely to be completely differentiated. When mN < l.0, increasing genetic differentiation among populations over time is expected due to genetic drift [A. exitialis, the observed stF value was 0.19, corresponding to a high level of genetic differentiation among the local populations. On the other hand, shared genotypes were common among the sites. However, the shared genotypes were not uniformly distributed among different local populations. Instead, differential over-representation of certain genotypes was common in each of the seven local populations, causing significantly biased allele frequencies for one or the other allele at each of the 14 marker loci and contributing to the high-level differentiation among the local populations. Indeed, if clone-corrected data were used, the overall stF value would decrease drastically to <0.05 . At present, the reasons why certain multilocus genotypes are more broadly distributed than other genotypes are not known. However, both the age of the founding population and the location-specific selection forces could have contributed. For example, an older founding population and a more favorable mushroom growth and fruiting environment may lead to the production of more fruiting bodies from a bigger genetic individual with the same genotype. The fixation index ic drift ,39, and Agaricus bisporus var. bisporus [A. exitialis in nature (Based on the observed basidia structure where most basidia had only two basidiospores each, the most likely mode of reproduction is secondary homothallism, similar to that of the commercial button mushroom bisporus . In secobisporus . These bn nature .Candida tropicalis [A. bisporus [A. exitialis that upon germination, can mate with homokaryons with compatible mating types to form fertile heterokaryons and increase heterozygosity. All three mechanisms could contribute to the heterogeneous distribution of heterozygosity in the natural population of A. exitialis, with excess heterozygosity in certain regions of the genome while lower than expected heterozygosity in other parts of the genome.However, we should note that if secondary homothallism was the only mode of reproduction, heterozygosity would likely decrease over time due to meiotic crossing over and segregation during basidiospore formation, causing lower observed heterozygosity than expected heterozygosity over time. However, as was shown in opicalis . In the bisporus ,44. In tThe geographical distribution of genotypes provides important information for understanding the biogeography and evolution of populations and species, including for tracking the origin and dispersal routes of specific genotypes and lineages. Between Guangzhou city and Shenzhen city, there are several mountains, rivers, densely populated cities, and other geographic barriers. However, in our study, one genotype “d” was broadly shared between these two cities separated by over 80 kilometers. Both wind-aided dispersal and anthropogenic activities could have facilitated the dispersal of self-fertile basidiospores between the two cities. In this case, the TreeMix analysis revealed that the dispersal was most likely from site D in Guangzhou to site G in Shenzhen . HoweverA. exitialis is commonly associated with Castanopsis fissa, an evergreen tree in Fagaceae that is broadly distributed in southern China and Southeast Asia. This evergreen tree species has been considered the host species of A. exitialis. However, in most of the seven sites, fruiting bodies of A. exitialis were found growing under a mixture of trees, including a different evergreen species, Lithocarpus glaber. L. glaber is distributed in Japan and China. At present, the importance of other trees, such as L. glaber in the growth and reproduction and in ectomycorrhizal associations with A. exitialis awaits further investigation.In Guangdong Province, A. exitialis genotypes. Temporally, both identical and different genotypes of A. exitialis were found within the same site between years during our sampling period from 2007 to 2012. This is not surprising given the secondary homothallic reproduction of this species and its ectomycorrhizal association with trees that may last many years. Our results are different from those in Amanita manginiana where no identical genotype was found between two years of sampling at a site in Sichuan Province, southwestern China [A. manginiana is a heterothallic species with four spores per basidia, as well as has small and short-lived genetic individuals in nature. In contrast, as shown here, A. exitialis has a secondary homothallic life cycle and likely has large long-lived genetic individuals. The spatial and population genetic features of A. exitialis identified in our study could help prevent future mushroom poisoning. For example, warning signs containing pictures of A. exitialis fruiting bodies should be installed at specific locations within each site to explicitly warn people to not pick this poisonous mushroom.In this study, our focus was on spatial distributions of rn China . The rea