Myrmecophaga tridactyla, Pilosa, Linnaeus 1758)belongs to the mammalian order Pilosa and presents a large distribution along SouthAmerica, occupying a great variety of habitats. It is listed in the IUCN Red List ofthreatened species as Vulnerable. Despite threatened, there is a lack of studiesregarding its genetic variability. The aim of this study was to examine the geneticdiversity and patterns of genetic structure within remaining populations. We analyzed77 individuals from seven different populations distributed in four biomes acrossBrazil: Cerrado, Pantanal, Atlantic Forest and Amazon Forest. We sequenced twomitochondrial markers (control region and Cyt-b) and two nuclear markers (AMELY andRAG2). We found high genetic diversity within subpopulations fromNational Parks of Serra da Canastra and Emas, both within the Cerrado biome, withsigns of population expansion. Besides, we found a notable population structurebetween populations from the Cerrado/Pantanal and Amazon Forest biomes. This data isa major contribution to the knowledge of the evolutionary history of the species andto future management actions concerning its conservation.The giant anteater ( Myrmecophaga tridactyla Linnaeus 1758, is a mammalof the Myrmecophagidae family, order Pilosa   and appee.g. The main causes for the observed population decline are the deterioration and reductionof natural habitats e.g. , and extM.tridactyla populations progressively more isolated. It is known thatreductions in size and range of populations increase their vulnerability to stochasticextinction, leading in several instances to local extinction  or population structure driven by historical divergence.This information provides scientific resources for future management actions for theconservation of the species.In this study we present results on population genetic diversity and structure based onthe analysis of mtDNA and nDNA markers in different remaining populations across thedistribution range of M. tridactyla were collected between1994 and 2007 for nine Brazilian federal states , in 20 differentlocalities, except for seven samples that originated from a museum collection , which dated from 1957-1979. Sampled tissues variedfrom dry skin (museum), to hair, bone and soft tissues. The original samples werecollected from captive individuals of known origin, wild animals captured forecological studies, road-killed individuals, and museum collection vouchers. Alltissue samples were preserved in 95% ethanol, and stored in −20 °C. The samplescovered the populations of (i) Minas Gerais state , comprisingindividuals from the National Park of Serra da Canastra and other localities, (ii)Goiás state  comprising individuals from the National Park Emas, (iii)different localities in São Paulo state  and (iv) Mato Grosso state , all representative of the biome Cerrado (CE). Additionally, a population fromthe Atlantic Forest of Paraná state , from the Pantanal biome  ofMato Grosso and Mato Grosso do Sul states, and individuals from the Amazon Forest were also sampled. Sampling localities are displayed in TableS1 from Supplementary Material.Seventy-seven individuals of All biological material was collected with authorization for activity with scientificpurposes emitted by SISBIO/IBAMA under the accession number 15052-1.b gene (Cyt-b) using primersCytB-L and CytB-H (RAG2) was amplified with the set of primers RAG2-F220 andRAG2-R995 . Thermocycling conditions consisted of adenaturing step at 94 °C for 3 min, 35 cycles at 94 °C for 30 s, 50 °C for 40 s, 72°C for 30 s, and a final extension at 72 °C for 10 s. For nDNA markers, PCRamplification was done in 10 μL volumes containing 10X buffer, 1.5 μMMgCl2, 200 μM dNTPs, 0.5 μM of each primer and 0.5 unit ofPlatinum Taq® polymerase (Thermo Fisher Scientific). In bothcases, template DNA dilutions were used ranging between 20-100 ng/μL. Thermocyclingconsisted of a hotstart step at 95 °C for 9 min 45 s, 5-10 (touchdown) cycles of adenaturing step at 94 °C for 15 s, annealing at 49-54 °C for 30 s, extension at 72 °Cfor 80 s, and a final extension step at 72 °C for 3 min. All products were examinedon a 0.8% agarose gel stained with ethidium bromide, purified with a moidifiedpolyethyleneglycol (PEG) protocol , PolyPhred 5.04  algorithm inNETWORK v. 4.6 software ,Tajimas D, a test of selective neutrality  computed with pairwise differences, with 1000 permutations to testfor significance at the 0.05 level. Tajimas test of selective neutrality was used todistinguish between a random  and non-random evolving DNA sequencedataset, which may be caused by positive/balancing selection or by demographicfluctuations (expansions and contractions). When there is an excess of low frequencypolymorphisms in the dataset a negative D value results, which indicates populationsize expansions or positive selection  populations from all four biomes in distinct groups [CE][PT][AF][AM]; (ii)forested versus open vegetation biomes [AM+AF][CE+PT]; and finally (iii), using ageographic distance criterion: [CE+PT+AF] [AM].Software Arlequin v. 3.5  was usedutrality , populatelection . Since wRAG2) was used. Groupswere tested as follows: (i) HVI+Cyt-b [CEMG+CEGO][F]/ [CEMG+CEGO][M] and (ii) RAG2[CEMG+CEGO][F]/ [CEMG+CEGO][M].AMOVA was also used to test for sex biased dispersion, measured in the major sampledpopulations , grouped bypopulation and gender .For this purpose, mtDNA (HVI+Cyt-b) and nDNA  we conductedthe non-parametric Mantel's test, which correlates genetic and geographicaldistances. To access the correlation coefficient reliability, 10,000 replicates weredone. The test was performed in the software Alleles in Space, AIS vs. 1.0 . To explSequence data for mtDNA and nDNA markers are publicly available at GenBank, under theaccession numbers: KF543782-KF543820.Cyt-b. These fragments were analyzed jointly as one, totalizing1005 bp. After alignment, the sequences showed 29 haplotypes distributed along fourbiomes were shared among three or more populations  shared among three or more populations. Of these, all haplotypes except A1 wereshared with the PT population, and only one, R1, was shared with AF  shared among threeor more populations, and  with AF . Since oTajimas D for the entire sample set was −2.02 (p < 0.0001), indicating a likelydemographic expansion. Only populations CEMG and CEGO also showed significantnegative values of Tajimas D when analyzed separately .The mismatch distribution graphic presenteST values were low and non-significant between CEpopulations  and PT. Values were also low between populations AFand AM. Among CE populations, FST was significant only between CEMT andtwo other populations, CEMG and CESP. All CE populations except CEMT showedsignificant FST values against AM and AF . PT showed high FST valuesagainst AF and AM as well, but was significant only against AM . Considering mtDNA, only 2.99% of variation couldbe attributed to gender, and FST was not significant .In nDNA, similarly, 0.093% of the variation was attributed to gender groups, but wasnot significant FST = 0.0635, p- = 0.0861.The AMOVA test revealed the highest Fn groups . AMOVA ta between CEGO and CEMT, barrier bbetween CEMG and CEMT, barrier c between three CE regions and AF andbarrier d between PT and CEGO (e). CEMG, CEGO and CESPshowed no evidence of barriers to gene flow among them.The Mantel test of correlation between geographic and genetic distance was notsignificant  . Barrierand CEGO . When anMyrmecophaga tridactyla along Brazilian localities. We were able tosample populations from four biomes: Cerrado (CE), Pantanal (PT), Atlantic Forest (AF)and Amazon Forest (AM), constituting the largest description of giant anteater geneticdiversity available in literature to date, with mitochondrial and nuclear markers.Despite low amplification efficiency and limited sampling, the nDNA dataset corroboratedlargely the patterns revealed by mtDNA regarding CE, PT and AF populations.This work described the general patterns of genetic diversity variation in populationsof M. tridactylagenetic diversity. Despite the evidence of a barrier between CE and PT  was the genetically most diverse, followed by CEMG andCEGO, as shown by genetic parameters (haplotype and nucleotide diversity), despite thefew individuals sampled. The Pantanal biome is known to be composed by differentelements gathered from other biomes, such as shared fauna and flora . This miE and PT , estimatnificant , Table 5nificant . HoweverS compared to CEGO). Despite being represented by only fourindividuals, the region where it is located . This differentiation was also evidenced by the barrier analysis, where CEMTshowed to be separated from CE populations, but joined with AM when four barriers wereconsidered  suggests that IBD may benot the main factor generating genetic structuration in STvalues (e.g. At the same time, individuals from the AM population were most closely related toindividuals from AF, as demonstrated in the mtDNA haplotype network , and by Tvalues . Even thTvalues . FurtherM. tridactyla populations of the Serra da Canastraand Emas National Parks as strongholds of diversity, an important source for futuremanagement actions for the species. Besides, it showed a marked genetic structurebetween Cerrado and Amazon and Atlantic forests populations, representing a historicalbreak to gene flow, and high genetic similarity between Cerrado and Pantanalindividuals. We encourage further studies with widespread populations of this species,including specimens from other biomes outside Brazil, in order to better understand itsphylogeographic history and to be able to compare diversity indexes among suchpopulations, providing useful information for conservation actions towards the speciesat a continental level.The results presented in this study contributed to the understanding of the evolutionaryhistory and population dynamics of the threatened giant anteater. Our data pointed outthe importance of


Coptodon zillii) in three different Egyptian aquatic environments: brackish (Lake Idku), marine (Al‐Max Bay), and freshwater (Lake Nasser). The habitat differences, environmental factors, and harvesting pressures are the main characteristics of the sampling sites. Three mitochondrial DNA markers  were used to assess population structure differences among the three populations. The population at Lake Nasser presented the highest genetic diversity , and the marine population of Al‐Max Bay the lowest  of the combined sequences. In addition, the phylogenetic haplotype network showed private haplotypes in each environmental habitat. Results presented here will be useful in aquaculture to introduce the appropriate broodstock for future aquaculture strategies of C. zillii. In addition, evidence of population structure may contribute to the management of tilapia fisheries in Egyptian waters.Recently, tilapia have become increasingly important in aquaculture and fisheries worldwide. They are one of the major protein sources in many African countries and are helping to combat malnutrition. Therefore, maintenance and conservation genetics of wild populations of tilapia are of great significance. In this study, we report the population genetic structure and genetic diversity of the redbelly tilapia ( The Nile tilapia, Oreochromis niloticus, together with O. aureus, Coptodon zillii, and Sarotherodon galilaeus are important aquaculture and/or fisheries species in Egypt  are widely distributed among tropical, subtropical, and temperate zones  has been reported in many studies  and sea levels affected the demographic population structure in Lake Idku and the north Delta lakes , and these data will be useful to assess population structure, which is needed for aquaculture and fisheries management.The goal of this study was to assess population structure in 22.1C. zillii  using a DNeasy Blood & Tissue extraction kit  following the manufacturer's protocol. PCRs were conducted using three primers for three regions of mtDNA: the D‐loop (noncoding) control region using universal primers CR‐A, 5′‐TTCCACCTCTAACTCCCAAAGCTAG ‐3′ and CR‐E, 5′‐CCTGAAGTAGGAACCAGATG ‐3′  for 45 s, and extension at 72°C for 1 min and a last extension step at 72°C for 10 min. The amplification products were examined by electrophoresis (1.5% agarose gel). They were purified with Exonuclease I and Alkaline Phosphatase Shrimp  and were incubated at 37°C for 20 min, followed by deactivation at 83°C for 30 min. Cleaned PCR products were sequenced using an ABI 3730 Genetic Analyzer at Fasmac Co., Kanagawa, Japan. Consensus sequences were edited and aligned using Geneious® 8.1.9  in deionized water. PCR cycling was performed in an Eppendorf thermocycler  using an initial denaturation step at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 45 s, optimum annealing (2.3H), haplotype diversity (Hd), the number of segregating sites (S), and nucleotide diversity (π). Neutrality tests based on Fu's FS were performed in the program Arlequin, version 3.5.2.2  was used to build the phylogenetic relationship among haplotypes of all markers  between geographic sampling sites using median‐joining (MJ) network algorithm  was estimated with 20,000 randomizations using IBDWS version 3.23 , 412 bp of D‐loop, and 439 bp of Cytochrome b (CYTB). In total, 8, 7, 3, and 17 mtDNA haplotypes were obtained from genes D‐loop, COI, CYTB, and combined sequences for the three populations of C. zillii, respectively  in each genetic marker are shown in Table Hd = 0.7717; π = 0.0046) and combined sequences . However, haplotype diversity and nucleotide diversity of the brackish water population in Lake Idku were higher than in the freshwater population based on COI  and lower using other markers  and Lake Idku . Overall, neutrality tests showed no signs of expansion for any marker in any site.The results of the neutrality test of Fu's Phylogenetic networks of the haplotypes were identified using the median‐joining algorithm for 8,7,3, and 17 haplotypes of D‐loop, COI, CYTB, and joined sequences, respectively Figures . The hapST values were estimated to assess the population genetic structure of C. zillii among the three aquatic environments (Table p < 0.05) ΦST values. Pairwise ΦST values ranged from 0.3796 to 0.8657 for the D‐loop and from 0.2943 to 0.5439 for COI (Table p < 0.05) ΦST values among all populations  between ΦST and geographic distance among all populations  revealed multimodal pattern among the observed distribution values Appendix . The isoAppendix .4Coptodon zilli, among three different types of Egyptian aquatic habitats  using three mtDNA markers . The genetic isolation of the freshwater population (Lake Nasser) from the brackish and marine populations might be due to anthropogenic factors such as the Aswan High Dam   based on both genetic markers (COI and D‐loop). Founder effects, predation, fishing pressure, and pollution  may all contribute to the observed low genetic diversity  indicating an excess number of alleles and contemporary population expansion. This pattern of population expansion may be due to demographic changes in Lake Idku (a semiclosed system). The demographics of Lake Idku are changing rapidly because the lake lost about 319.3 km2 of surface area between years 1800 and 2010  the Aswan High Dam was built in 1970, creating a barrier to exchange between populations on either side of the dam, and 2) the presence of various dendritic inlets (Khors) or side projections of the main lake  presented the lowest genetic diversity (0.1630C. zillii of Egyptian waters. Recently, in some countries, such as Ethiopia, construction of new dams on the Nile River has begun, and these are likely to further affect the diversity, distribution, and population structure of aquatic species in Lake Nasser and other Nile River populations. Therefore, the present study may serve as an important scientific resource for future studies to monitor the influence of existing and new dams. Such structures appear to have the ability to influence connectivity and subsequent genetic structure of the target populations. Our preliminary findings may support fisheries managers, for example, to understand the population structure and diversity of C. zillii in Nile waters. Loss of genetic diversity may lead to reduced resilience in the face of unforeseen potential ecological pressures, such as the emergence of diseases or susceptibility to pollutants. We hope that regional authorities will consider and/or monitor population diversity as part of adaptive management plans in this commercially important species.This study has clear implications for the fisheries management and aquaculture sectors for None declared.Taha Soliman designed and performed the experimental work such as sample collection, DNA extraction, and PCRs, performed data analysis, wrote, and reviewed the manuscript. Walid Aly and Reda M. Fahim collected the samples, designed experimental work, and reviewed the manuscript. Michael L. Berumen analyzed data and reviewed the manuscript. Holger Jenke‐Kodama provided the kits and chemicals and reviewed the manuscript. Giacomo Bernardi designed the experimental work, analyzed data, and reviewed the manuscript. Click here for additional data file. Click here for additional data file.


Non-CODIS STRs, with high polymorphism and allele frequency difference among ethnically and geographically different populations, play a crucial role in population genetics, molecular anthropology, and human forensics. In this work, 332 unrelated individuals from Sichuan Province  are firstly genotyped with 21 non-CODIS autosomal STRs, and phylogenetic relationships with 26 previously investigated populations  are subsequently explored. In the Sichuan Tibetan and Yi, the combined power of discrimination (CPD) values are 0.9999999999999999999 and 0.9999999999999999993, and the combined power of exclusion (CPE) values are 0. 999997 and 0.999999, respectively. Analysis of molecular variance (AMOVA), principal component analysis (PCA), multidimensional scaling plots (MDS) and phylogenetic analysis demonstrated that Sichuan Tibetan has a close genetic relationship with Tibet Tibetan, and Sichuan Yi has a genetic affinity with Yunnan Bai group. Furthermore, significant genetic differences have widely existed between Chinese minorities (most prominently for Tibetan and Kazakh) and Han groups, but no population stratifications rather a homogenous group among Han populations distributed in Northern and Southern China are observed. Aforementioned results suggested that these 21 STRs are highly polymorphic and informative in the Sichuan Tibetan and Yi, which are suitable for population genetics and forensic applications. In the past decades, comprehensive recognition of human STRs has provided insights into anthropology, archaeology, human forensics and population genetics5. Recently, non-Combined DNA Index System short tandem repeats (non-CODIS STRs) are attractive to genetic applications like population stratification analysis, regional population structure studies and forensic individual identification and paternity testing12. Non-CODIS STRs combined with previous commercial CODIS STRs amplification systems play an indispensable complementary role in the forensic applications: disentangling missing person cases, identifying victims, and solving the parentage testing cases with mutation. Genetic diversity of non-CODIS STRs in major ethnic groups in the East Asia, especially in China, has been explored17. However, the genetic architecture of geographically and linguistically distinct populations in Sichuan Province, the 5th largest and 3rd most populous province in China, remains uncharacterized.Short tandem repeats (STRs) are DNA sequences with a number of tandemly repeated short sequence motifs (2–6 bp), such as (ATCT)19. The population history of the Yi group remains controversial20. The main point is that the Yi migrated from southeastern Tibet through Sichuan and into the Yunnan Province and has the common ancestor with the Tibetan, Nakhi and Qiang peoples. Tibetan population is mainly resided throughout the Qinghai-Tibetan Plateau for hundreds of generations has genetic adaptations of distinct combinations of phenotype in high-altitude (>4000 m)23. Chengdu, the capital of Sichuan Province, is home to a large community of Tibetans, with 30,000 permanent Tibetan residents and up to 200,000 Tibetan floating population. In our previous study24, we have investigated the genetic polymorphism data of non-CODIS STRs in Sichuan Han population. However, until now no genetic diversity data about non-CODIS STRs was available for the Tibetan population and Yi population from Sichuan Province.Sichuan consists of two geographically distinct parts: the eastern part is mostly within the fertile Sichuan Basin and the western part consists of the numerous mountain range. Han Chinese (the majority of the province’s population) mainly reside in the eastern portion, while significant minorities of Yi, Tibetan and Qiang people reside in the western portion that is impacted by inclement weather and natural disasters. Yi population, also known as Lolo population, is the seventh largest of the Chinese officially recognized 55 ethnic minority groups and the Yi is the largest ethnic minority group in Sichuan24, the present study characterizes the genetic diversity of 21 non-CODIS STRs in Tibetan population  and Yi population  from Sichuan Province. Furthermore, other genetic data of 9,444 previously investigated individuals37 from 26 populations is used to investigate Sichuan and Chinese population genetic substructure using analysis of molecular variance (AMOVA), principal component analysis (PCA), multidimensional scaling plots (MDS) and phylogenetic analysis.In continuation to our previous studyPopulation genetic structures in East Asia are complex, especially in Chinese population stratification consisted of 56 Chinese officially recognized ethnic groups widely distributed in 34 administrative divisions. Different ethnic origin has been believed to have their special ethnic origin or common ancestors. Clearly understanding the genetic variation and forensic characteristics of different ethnic populations is indispensable in the forensic applications, especially in the paternity testing and individual identification. In the present study, a total of 332 unrelated individuals residing in Sichuan Province are genotyped using a multiplex assay amplifying 21 non-CODIS autosomal STR loci (AGCU 21 + 1 System). The detailed genotypes of 237 Tibetan individuals and 95 Yi individuals are presented respectively in Supplementary Tables In Sichuan Tibetan population, a total of 183 alleles are identified with corresponding frequencies vary from 0.0021 to 0.5401  and 26 previously studied populations, we first calculated the FPCA dissected the major factors accounting for the total variances of 28 Chinese populations using the Multivariate Statistical Package version 3.22 (MVSP) and SPSS software. Figure Subsequently, to evaluate the proportion of genetic heterogeneity and homogeneity attributable to Chinese population stratification, we calculated the Nei’s genetic distances for total 378 pairwise groups  and paternal genetic signatures (Y-Chromosome)48. Despite these large-scale efforts in investigating patterns of natural selection, estimating individual ancestry and predicting the evolutionary history in Chinese populations based on SNPs, InDels, and CODIS STR loci50. The human genetic variation of non-CODIS STRs in the Sichuan Yi and Tibetan populations remains unexplored. In this study, a total of 237 Chinese Tibetan individuals and 95 Yi individuals from Southwest China are genotyped using AGCU 21 + 1 PCR amplification kit. In addition, the population differentiation analyses also included 9,444 individuals in 26 groups from 23 distinct administrative divisions or 14 ethnic groups that are genotyped using this same kit in the previous studies. The final data set is made up of 21 non-CODIS STR loci genotypes in 9,776 individuals from 28 Chinese populations. We have analyzed the genetic variation and population structure of the aforementioned populations via analysis of molecular variance, PCA, MDS and phylogenetic analyses.China is currently populated by over 1.3 billion people who belong to at least 56 Chinese officially recognized linguistically and ethnically different groups. Genetic studies of Chinese populations from different minority ethnic groups are of great interest due to China’s complex demographics, large population size and complex geographical characteristics. Additionally, clearly identifying and detecting the modern human evolution, origin and demographic history have been a resurgence of interest in population geneticists and medical geneticists due to successfully analyze the ancient nuclear and mitochondrial DNA sequences of Neanderthal and DenisovanTM STR kit51 , HuaxiaTM Platinum PCR Amplification kit52 , PowerPlex® Fusion kit53  and so on, are widely used in the forensic human identification, paternity testing, and DNA database construction in criminal investigations or missing persons cases. CODIS STRs amplification systems with the limitation of improving the forensic efficiency when used them as a complementary to each other in the complex forensic cases. However, simultaneously testing the 21 non-CODIS STRs included in the AGCU 21 + 1 kit can minimize adventitious matches, increase discrimination power and facilitate data sharing in the cases with mutation, degraded sample cases and deficiency cases of paternity testing. Several measures of genetic diversity  and forensic statistical indexes of 21 non-CODIS STRs  are relatively high in Sichuan Yi and Sichuan Tibetan populations in the present study. Most previous genetic studies based on a set of SNPs or STRs located on sex Chromosome show similar results55. But some researchers illustrated the lack of enough combined power of discrimination (0.99999999995713) and power of exclusion (0.97746) in Yi ethnicity56. However, the CPEs in the new studied populations and previous studied Han group24 are over 0.99999 and the CPDs are larger than 0.9999999999999999999. Our findings in these two investigated populations in combination with the previous studied Sichuan Han group24 demonstrated that twenty-one non-CODIS STRs included in AGCU 21 + 1 PCR amplification kit are highly discriminative and informative in diverse ethnic populations residing in Sichuan Province, West China, and should be used as a complementary tool in complicated paternity cases . Besides, it can also be integrated into the new panel examined using the massively parallel sequencing platform, such as Ion S5 XL and Illumina-Miseq sequencer. This study also provides the first batch of genetic diversity information of 21 non-CODIS STRs in two ethnic groups and enriches the Chinese non-CODIS STRs reference databases.Recently, large number of commercial kits included the overlapped 13 CODIS STRs, such as GlobalFiler57. Among Han Chinese populations, no significant differences are observed in different populations defined by geographic boundary (Yangzi River), which is identified by the Y-STRs and high density SNPs panel57. However, a slight North-South gradient difference can be vaguely identified and not a significant North-South genetic distinction. The identified genetic similarities and differences among Chinese populations are a valuable technique for identifying accurate disease risk gene in genome-wide association study, avoiding a spurious association, and detecting more ethnicity-special ancestry informative markers in forensic ancestry inference.The results observed in this comprehensive population comparison reveal that significant genetic differences are identified between Han Chinese populations and some minority ethnic groups, especially predominantly in Tibetan and She populations. Besides, our analyses of phylogenetic relationship reconstruction and MDS indicated that Han Chinese population is homogenous based on autosomal genetic makers compared with sex-inherited genetic markers58. Yi population, as we expected, keeps a relatively distant genetic relationship with the Tibetan population residing in Sichuan. Besides, these two Tibeto-Burman-speaking populations keep a relatively genetically distinct relationship with our investigated Sichuan Han population24 although all three groups have a close geographical position, which is accordance with different ethnicity origin, cultural background. It is strange to find that Sichuan Yi and Yunnan Yi keep a distant genetic relationship which may be influenced by the culturally different of three subgroups of Yi . In the future, large-scale population genetic history studies from different administrative divisions based on different high-density genetic marker sets (even whole genome sequence of archaic or present-day human DNA) will be needed to investigate and elucidate the origin, migration of the Sino-Tibetan-speaking ethnicity groups.Tibetan and Yi populations belong to the Tibeto-Burman-speaking subfamily in the Sino-Tibetan languages and the previously investigated Southwest Chinese Han population belongs to another subfamily (Chinese). Tibetan, as a most representative group, is genetically adapted to extreme hypoxia, and has been the genetic subject for multidisciplinary Studies. Our results reveal that two Tibetan populations distributed in different geographic positions  have a strong genetic affinity, however, keep a far genetic relationship with other populations. These features are consistent with previous findings revealed by genetic studies based on high-throughput genotyping data and genome sequence dataIn summary, we sampled 332 individuals from two minority ethnic groups to assess the genetic variations of 21 non-CODIS STR loci and combined these samples with 9,444 individuals previously investigated from 26 Chinese populations to explore Chinese population structures. Our results demonstrated that this panel of STRs is highly informative and polymorphic in the Sichuan Tibetan and Yi population, and can be widely used as a tool for personal identification and parentage testing in forensics. Additionally, the estimate of genetic differentiation  suggested that the Sichuan Tibetan population and Sichuan Yi keep the close relationship with Lhasa Tibetan and Mongolian population, respectively, but being relatively isolated from other ethnic groups, especially within Han Chinese populations. The results obtained from PCA, MDS and phylogenetic analyses also demonstrated that genetic differences among Han Chinese and minorities widely exist and Han Chinese populations are homogeneous in different geographical divisions.This study was approved by the institutional review boards of Sichuan University. All participants signed informed consent statements prior to participation. Human blood samples were collected upon approval of the Ethics Committee at the Institute of Forensic Medicine, Sichuan University. All the experimental procedures and the methods for each procedure were carried out in accordance with the approved guidelines of the Institute of Forensic Medicine, Sichuan University.Unrelated blood samples were collected from 237 unrelated Tibetan individuals  recruited from Chengdu and 95 Yi individuals  recruited from Liangshan Yi Autonomous Prefecture, Sichuan Province. All individuals had been required to be the indigenous inhabitants or with a recent ancestor residing in the corresponding sample collection region at least three generations.Human genomic DNA was extracted using PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The quantity of the DNA template was determined using Quantifiler Human DNA Quantification Kit on a 7500 Real-time PCR System (Thermo Fisher Scientific). DNA samples were normalized to 1.0 ng/μL and stored at −20 °C until amplification.PCR amplification was performed following the manufacturer’s protocol on a ProFlex 96-well PCR System (Thermo Fisher Scientific). The PCR system was a 25 μL reaction volume containing 10 μL of Reaction Mix, 5 μL of Primers 21 + 1, 0.75 μL C-Taq and 1.0 ng of template DNA. The thermal cycling conditions consisted of an initial step at 95 °C for 2 min; followed by 30 cycles of 94 °C for 30 s, 60 °C for 60 s, and 65 °C for 90 s; and a final extension at 60 °C for 60 min.Amplification products were separated and detected on the Applied Biosystems 3130 Genetic Analyzers following the manufacturer’s recommendations. One microliter of PCR products or allelic ladder was added to a mixture containing 9.5 μL of deionized Hi-Di formamide and 0.5 of μL AGCU Marker Size-500 (AGCU ScienTech Incorporation). The mixture was injected at 1.2 kV for 16 s and electrophoresed at 13 kV for 1550 s with a run temperature at 60 °C. Allele allocation was carried out with GeneMapper ID 3.20 analysis software using the allelic ladder and the set of bins and panels provided by the kit.59. Allelic frequencies and forensic parameters, including the polymorphism information content (PIC), power of discrimination (PD), power of exclusion (PE) were calculated using the modified PowerStat V12 spreadsheet (Promega)60.In order to evaluate the forensic efficiency of this non-CODIS STR panel for application in the Sichuan Tibetan and Yi, genotype data of 332 unrelated individuals were analyzed. The observed heterozygosity (Ho), expected heterozygosity (He), the exact test of Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were estimated and performed using Arlequin 3.5.2.237 comprising of 9444 individuals were retrieved to investigate the Sichuan and Chinese population genetic substructure. These reference populations include Salar (n = 120) residing in Qinghai Province, Tibetan (n = 104) from Lhasa in Tibetan autonomous region of Tibet, Tujia (n = 107) from Hubei Province, Bai (n = 106) from Yunnan Province, Han (n = 202) from Ningxia Hui Autonomous Region, Uyghur (n = 502) from Aksu region in Xinjiang Uyghur Autonomous Region, Kazakh (n = 114) from Ili region in Xinjiang Uyghur Autonomous Region, She (n = 154) from Fujian Province, Mongolian (n = 523) from Inner Mongolia Autonomous Region, Han (n = 220) from North China, Han (n = 275) from Guanzhong region in Shanxi Province, Yi (n = 110) from Yunnan Province, Li (n = 504) from Hainan Province, Han (n = 506) from Guangdong Province, Han (n = 501) from Hunan Province, Han (n = 225) from Huadong region of China, Han (n = 459) from Beijing Municipality, Han (n = 481) from Zhejiang Province, Russian (n = 114) from Inner Mongolia Autonomous Region, Han (n = 1030) from Shandong Province, Han (n = 207) from Liaoning Province, Han1 (n = 1136) from Henan Province, Yugu (n = 180) from Gansu Province, Xibe (n = 226) from Xinjiang Uyghur Autonomous Region, Han2 (n = 970) from Henan Province and Han (n = 368) from Sichuan Province. Population comparison was conducted using the principal components analysis (PCA), multidimensional scaling plots (MDS) and phylogenetic analysis. PCA was conducted using the Multivariate Statistical Package version 3.2261 and the SPSS software62  based on the allele frequency distribution of 21 non-CODIS STRs in 28 Chinese populations. Genetic distance (Nei’s) was calculated using Phylip-3.695. The MDS was performed in SPSS and a Neighbor-Joining tree was constructed in Mega 7.063 based on Nei’s genetic distance matrix64.Additionally, 26 previously investigated populations65. Control DNA 9947A (AGCU ScienTech Incorporation) and ddH2O were used as positive and negative controls respectively for each batch of genotyping.All experiments were conducted at the Forensic Genetics Laboratory of the Institute of Forensic Medicine, Sichuan University, which is an accredited laboratory (ISO 17025), and has been accredited by the China National Accreditation Service for Conformity Assessment (CNAS). We strictly followed the recommendations of Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM)Supplementary Figures S1-S4 and Supplementary Tables S1-S9


Paramuricea clavata. Using eight populations separated from tens of meters to hundreds of kilometers, which were differentially impacted by recent mortality events, we identify 25 °C as a critical thermal threshold. After one week of exposure at this temperature, seven of the eight populations were affected by tissue necrosis and after 30 days of exposure at this temperature, the mean % of affected colonies increased gradually from 3 to 97%. We then demonstrate the weak relation between the observed differential phenotypic responses and the local temperature regimes experienced by each population. A significant correlation was observed between these responses and the extent of genetic drift impacting each population. Local adaptation may thus be hindered by genetic drift, which seems to be the main driver of the differential response. Accordingly, conservation measures should promote connectivity and control density erosion in order to limit the impact of genetic drift on marine populations facing climate change.The differential response of marine populations to climate change remains poorly understood. Here, we combine common garden thermotolerance experiments in aquaria and population genetics to disentangle the factors driving the population response to thermal stress in a temperate habitat-forming species: the octocoral  Among these impacts, the increase in mass mortality events and disease outbreaks in marine species is particularly worrying because it severely threatens the structure and functioning of ecosystems and may significantly alter the provision of ecosystem services4. Generally, positive anomalies in seawater temperature have been concomitant with mass mortalities in affected regions8. These mortalities differentially affected species, populations and individuals at multiple spatial scales12. For instance, field surveys conducted during mass mortality events in 1999 and 2003 in the Northwestern Mediterranean (NWM) revealed that, within the impacted species (mainly anthozoans and sponges), individuals separated by less than one meter showed varying responses ranging from severe to a complete absence of injuries while populations separated by a few to hundreds of kilometers displayed either similar or highly contrasting degrees of impact7. These differential responses might be explained by the complex interactions between populations and their local environments15. In particular, environmental conditions, such as past and present thermal contexts, and biological processes, such as acclimatization or genetic adaptation, may be important drivers of the response of organisms to thermal stress17. Accordingly, improving our understanding of the influences of environmental and biological factors on the response of populations to thermal stress is necessary to anticipate the consequences of climate change.The current global warming trend is affecting marine ecosystems in several ways, such as by decreasing ocean productivity, altering food web dynamics, shifting species distributions and reducing abundance21. Acclimatization implies physiological changes that allow an organism to maintain a functional phenotype when facing an environmental stressor21, while local adaptation entails genetic changes driven by divergent selection22, which corresponds to spatial variations in natural selection resulting from local environmental conditions. Consequently, divergent selection may drive local populations to evolve traits that are advantageous in their local environments20. Nevertheless, neutral evolutionary forces such as migration and genetic drift 23 can also affect phenotypic divergence and thus influence differential responses. Migration can counteract divergent selection and limit local adaptation, while genetic drift may hinder local adaptation18.Acclimatization and local adaptation are two of the main biological processes modulating population responses to environmental variation, and they may drive the divergence in the phenotypic responses to thermal stress observed among populations26 . For instance, differential acclimatization to thermal stress was demonstrated in crabs27, whereas local adaptation to thermal conditions has been suggested in corals15 and seagrasses28. Focusing on temperate octocorals, studies conducted on the Mediterranean red coral, Corallium rubrum, demonstrated the existence of differential adaptive abilities of populations depending on the depth of origin of the individuals and of their thermal history29. Local adaptation or acclimatization were considered to explain these results  . P. clavata is a key species of Mediterranean coralligenous assemblages and is one of the species most affected by recent mass mortality events35. We carried out common garden experiments in aquaria using eight populations separated by tens of meters to hundreds of kilometers within the NWM  in common garden experiments performed in aquaria; and 2) to evaluate the role of environmental conditions  and biological processes  in the differential responses of populations to thermal stress by combining colonies tissue necrosis data and population genetic analyses.In this study, we characterized the response to thermal stress of a temperate coral, the red gorgonian a Risso, 826  Average annual temperature cycle (mean ± SD of daily T) at 20 and 40 m depths for Medes, Calanques and Scandola are shown in Fig. First, it is worth noting the differences inside localities between the 20 and 40 m depth (warm period median temperature range 18.8–21.2 °C vs. 15.5–17.1 °C for 20 and 40 m depth respectively) Fig. . The mosst quartile value of 16 °C)  and the maximum temperature (26.9 °C) Fig. . This lo°C) Fig.  which ar°C) Fig.  where me°C) Fig. .Even though at 40 m depth differences in mean temperature are attenuated, there are still clear patterns in thermal regimes. Medes is the warmest locality in depth, taking into account the median (17.1 °C) and maximum temperature values (24.9 °C) as well Fig. . Medes ath percentile, considering the warm period of all available years). This representation of extreme temperatures reinforces the patterns observed on thermal regimes at seasonal scale in Fig. Finally, extreme hot conditions of the warm period were characterized. Figure From a heat stress perspective, information analyzed here account for important differences in the thermal regimes and extreme hot temperatures distribution both among depths  and between localities (same depths).Indeed, global Kruskal-Wallis statistical test evidenced significant differences  between temperature regimes . Pair-wise comparisons (using Dunn-Sidak’s approach) accounted for significant differences among all pairs of localities and depths .P. clavata as 25 °C because after one week of exposure, 20 colonies from seven of the eight populations (8%) were affected by necrosis. After 30 days of exposure at 25 °C, the mean % of affected colonies increased gradually from 3% to 97%  was observed after the 8P. clavata populations dwelling in contrasting thermal regimes, and these results do not fully support our hypotheses. Considering the response to temperature of populations from different localities, the results were opposite to those expected. Scandola which was clearly the warmest locality at 20 m depth was the most affected while Calanques, which was the coldest locality both at 20 and 40 m depth , was the least affected  for Calanques 2 shallow to 0.090 (95% CI: 0.058–0.127) for Calanques 1 shallow  was 0.25 (95% CI: 0.007–0.436), and the pairwise PST values ranged from 0 for Calanques 3 shallow vs. Calanques 1 shallow, Calanques 3 shallow vs. Calanques 1 deep and Calanques 1shallow vs. Calanques 1 deep  to 0.235 (95% CI: 0–0.521) for Calanques 2 shallow vs. Scandola . Accordingly, genetically isolated populations seemed to be more impacted by thermal stress , colonies were affected by necrosis from the first day, and most of them suffered extensive damage in less than 5 days. Other experimental studies also indicated that similar short-term exposure to 25 °C was critical for triggering necrosis in P. clavata39. At lower temperatures (23–24 °C), 49 days of exposure were required for the first signs of necrosis to be observed35. Overall, these results are consistent with in-situ temperature surveys conducted during the mortalities events in 1999 and 2003 in different areas of the NWM37. They are also supported by previous experimental studies focused on other anthozoans affected during these events. Indeed, in experimental conditions, 25 °C was identified as critical temperature for aposymbiotic species such as the red coral Corallium rubrum and the yellow gorgonian Eunicella cavolini32.Our study indicated that 25 °C is a critical temperature for 37, we suggest that the shallow  populations of P. clavata are living near their upper thermal limit. This hypothesis is strengthened by the fact that Mediterranean gorgonians, in general, and P. clavata, in particular, shift their distribution to greater depths in warmer areas41. Specifically, Linares et al.42 noted this upper depth limit distribution on the latitudinal scale with populations inhabiting from 10–15 m at 42° 22′N and from 30–35 m at 37° 38′N associated to the changes in the temperature conditions. Coupling this information on geographic and bathymetric species distributions to projected ocean warming and to population response variability would enable the identification of areas that are particularly vulnerable to climate change45 and the design of efficient specific conservation measures45.Since temperatures higher than 25 °C are rarely observed in the studied localitiesP. clavata dwelling in warm conditions did not exhibit a higher resistance to thermal stress than those found in colder conditions. This result contrasts with previous studies conducted in various marine species suggesting that populations from warm habitats were more tolerant to thermal stress than populations from cold habitats (e.g. refs PST) and the neutral genetic divergence among populations (FST) may be due alternatively to selective or neutral processes48, the low and mostly similar pairwise PSTs and FSTs values combined to the positive correlation between the population-specific FST and the level of necrosis at the end of the experiment support the influence of genetic drift. Accordingly, we hypothesize that the differential responses to thermal stress in P. clavata is mainly driven by genetic drift, a stochastic and neutral evolutionary processes.The populations of 29 while, a genetic survey of P. clavata populations recently impacted by a mortality event demonstrated a positive link between the extent of genetic drift and the level of tissue necrosis49. Moreover, this hypothesis is consistent with model predictions51 and the results of a meta-analysis52. Indeed, the influence of genetic drift on local adaptation is expected to be stronger in species with restricted connectivity and low effective population size52 as reported in P. clavata54. Considering these life history traits are shared by other Mediterranean anthozoans56, the lack of local adaptation to thermal conditions may be widespread in these species questioning their abilities to deal with on-going climate change.In line with this hypothesis, recent studies suggest that genetic drift may have a dominant impact on phenotypic differentiation in Mediterranean octocorals. The level of genetic drift was suggested as a limiting factor in the adaptation of red coral populations to their local environmentsPST computations59. Genomic studies60 are thus needed to further decipher the respective roles of selective and neutral processes on the evolution of P. clavata and similar anthozoans in the environmental shift.One should highlight the limitations linked to the 35, thermodependent pathogens61, sex53). Taking this information into consideration should improve our capacity to more accurately characterize the vulnerability of marine coastal ecosystems. In this sense, we addressed the importance of the assessment of genetic drift on populations of P. clavata at different spatial scales, which is especially urgent in areas where warming is expected to reach critical temperatures, such as the North Western Mediterranean62.Understanding the response of populations to climate change is a central topic in conservation biology as a way to better assess species viability in the currently changing ocean. Our study highlights the potential impact of genetic drift in the response to thermal stress, which complements the picture of previously identified modulating factors   was the model species chosen for this study. This species is considered a key species of Mediterranean coralligenous assemblages and is also one of the species that has been most affected by recent mass mortality events34.The red gorgonian 37), 2) their differential degrees of impact during the recent regional mass mortality events37 and 3) their association with distinct genetic clusters54.We considered three localities separated by hundreds of kilometers within the NWM region: the Medes Islands , Calanques  and Scandola  Fig. . These tAt each locality, two populations dwelling at shallow (20 m) and deep (40 m) depths, and thus separated by tens of meters, were sampled Fig. . In Cala37. Here we provide a description of the main attributes of thermal regimes of the different localities and depths where the populations were sampled through the analysis of hourly temperature time series provided by TMEDNet (http://www.t-mednet.org). The longest possible period for which data was available at the 3 sites for a given depth were considered, i.e. 11 years (2004–2014) for 20 m depth and 8 years (2004–2011) for 40 m depth. From these long term series, average annual cycle and statistics on the distribution of hourly records were calculated. Annual cycle is based on daily average temperatures. For each julian day, mean ± SD were calculated. Boxplots were used to summarize information on central tendency, dispersion and extreme values of hourly temperature records: first, to characterize conditions during warm months, from July to end of October (hereafter warm period); then, to characterize distribution of extreme hot temperatures during the warm period (from 90th percentile).Temperature regimes of the studied localities were characterized in previous studies18) in aquaria with the selected Paramuricea clavata populations. We performed four independent common garden thermotolerance experiments in aquaria, considering for each, four different temperatures respectively: 25 °C, 26 °C, 27 °C and 28 °C . All the experiments were conducted between July and November of 2009 with apical tips (between 5 and 7 cm in length) of healthy, mature red gorgonian colonies (20–40 cm in height63). Apical tips (hereafter colonies) were randomly sampled in the study localities by SCUBA diving after 2009 spawning occurred. All the colonies were sampled in June 2009 except for those of the 28 °C experiment which were sampled in November 2009  to be transported to experimental aquarium facilities at the Institute of Marine Sciences-CSIC . Experimental plates were built up by three PVC rectangular pieces (5 × 30 cm in size) and two rubber layers. Each PVC piece had 10 holes (1 cm in diameter) to place the colonies. The colonies were fixed in the experimental plates without using any putty, instead the colonies were hold mechanically thanks to the perpendicular cuts previously done in the rubber layers (one cut in each layer) see Fig. .The maximum transportation time was of 36 h for the colonies collected in Scandola  while the minimum time was of several hours for the colonies collected in Medes . The temperature in the coolers was monitored regularly and seawater ice packs were used when necessary to maintain the water temperature between 15–18 °C. Once in the aquarium, colonies were maintained at 16–17 °C until the experiments started  in the buffer tank with submersible resistance heaters and was regulated by temperature controllers (Aqua Medic T controller). All the tanks were equipped with submersible pumps to facilitate water circulation within the tanks. Temperature was registered in the Control and Treatment aquariums with Tidbit Stowaway autonomous temperature sensors every half an hour.In the Control, seawater was maintained at 16 to 17 °C. We decided to set this temperature as control temperature because it corresponds to low temperatures during the summer period in all localities and it was also the Control temperature used in previous experimental studies with corals of the same study regionDepending on the experiment, 15 to 30 colonies were used per population and per Control/Treatment set Table . The exp2/branch linear cm35) was used. Overall the results showed that colonies placed in the Control followed similar variations in surface area than those in the field  and the other with phytoplankton diatom Tetraselmis chuii (chl-a = 3.3 ± 0.4 ug/l) . After the addition of food, the tanks were maintained in closed system mode for 5 h. During this period, water circulation within tanks was supplied by submersible pumps.In the Control and Treatment sets, colonies were fed two days per week, one day with nauplii of in situ studies of food abundance and prey capture at short time scales in benthic suspension feeders65. The results of sampling food abundance near the organisms and their gut contents demonstrated that organisms commonly acquire available resources in short time pulses. There was estimated that these pulses of food abundance occurred between 2 and 3 times per week on average65, so that was simulated in the performed experiments. The period of 5 h was chosen on the basis of the duration of the pulses of food abundance in the field65 and because previous experimental studies with P. clavata indicated that 5 h is an appropriate period, after which food abundance reaches similar levels prior to starting the feeding66. Furthermore, we measured food concentrations before, during and after the feeding events, and we arrived to the same results (results not shown). To obtain an estimate of food concentration during the experiments, we measured the particulate organic carbon (POC) concentration which is the most integrative descriptor of food conditions66. The POC concentration throughout the experimental treatment was 323 ± 50 ug C/l. POC values that characterize coastal waters in the NW Mediterranean are on the order 300 ug C/l68, hence, provided food concentrations were similar to those observed in the summer period in the field.The strategy of supplying pulses of food instead of a constant supply was based on the results of 66.Overall, the frequency and amount of food together with the maintenance of the system in a close mode after feeding, guaranteed that colonies were supplied with a surplus of food as observed in previous experimental studiesFor all of the experiments, the response variable was the level of tissue necrosis, which was visually monitored daily and assessed as the percentage of the colony exhibiting dead tissue (from clear grey to black) or denuded axis see Fig. . From thParamuricea clavata and to evaluate the importance of environmental and neutral genetic factors in the response of P. clavata populations to thermal stress. Four experiments considering the following temperatures were carried out: 25, 26, 27 and 28 °C (see next subsection).The common garden experiments were designed to assess the thermotolerance thresholds of P. clavata. The first experiment consisted of a stepwise temperature augmentation from 18 °C to 22 °C by 2 °C per week and then by 1 °C per week until 25 °C, which was identified as the critical temperature for P. clavata (see Results). Once this temperature was reached, the conditions were kept constant for 30 days (until at least 95% of the colonies were affected by tissue necrosis).We carried out four experiments to evaluate the thermotolerance thresholds of To explore the response to thermal stress above this temperature, we performed three additional experiments with constant temperature of 26, 27 and 28 °C. These temperatures were attained after one day of gradual augmentation from 18 °C to the treatment temperature, and the experiments were conducted until at least 95% of the colonies were affected by necrosis. The 26, 27 and 28 °C experiments had a total duration of 13, 6 and 4 days respectively Table . The 25,P. clavata, for each experiment, we calculated the mean (±SE) daily percentage of affected colonies across populations.For each population and experiment, we reported the daily percentage of affected colonies. Then, to characterize the thermotolerance features of P. clavata populations, we considered the common garden experiment at 25 °C because this temperature was identified as critical for P. clavata (see Results).To explore the influence of environmental and genetic factors on the response of 37 (see Results section), we expected shallow populations to be less affected by thermal stress than those in deeper water. Regarding the localities, the Scandola and Calanques populations should be the least and most affected, respectively, while the Medes population should present an intermediate response. To analyze these hypotheses, we focused on the response to temperature of six populations coming from three localities and two depths with contrasting thermal regimes: Medes shallow and deep, Calanques 1 shallow and deep and Scandola shallow and deep37. The two populations from Calanques 2 and Calanques 3 shallow were not included to maintain a balanced design in the analysis (see below).Environmental factors. We postulated that populations from depths/localities with colder thermal regimes will be more affected by thermal stress than populations subject warmer thermal regimes. In particular, and bearing in mind the temperature regimes of the selected localities and depths70 with Locality (three levels) and Depth (two levels) as fixed factors. A multivariate approach, taking into account tissue necrosis from three days covering the whole experiment  as the multivariate dependent variable was considered69. Post-hoc pair-wise PERMANOVA tests were performed to identify significant differences between pairs of localities. Complementarily, we ran a second PERMANOVA taking into account the six populations but accounting for only one factor (Population) that was considered fixed. The aim of this one-way analysis was to obtain pair-wise (through post-hoc pair-wise tests) results for all of the populations without restricting comparisons within depths or localities. The same multivariate approach of the previous experiment was considered. The colonies from the Control sets were not considered in these analyses because they remained healthy (no tissue necrosis) until the end of the experiment. For all of the tests, 9999 permutations were achieved. PERMANOVA analyses were performed in PRIMER v671 in conjunction with the Windows PERMANOVA + module72. For a more detailed description of the statistical analyses, we refer to Supplementary Methods To assess statistical differences in tissue necrosis in the Treatment between localities and depths, we performed a two-way multivariate PERMANOVA74, and one way to characterize this interaction is to estimate and compare the neutral genetic divergence to the phenotypic divergence for the considered trait58. Through this characterization, we aimed to obtain the first insight into the role of population genetic features on the observed differential responses to thermal stress.Biological processes. Phenotypic divergence between populations, such as the differential response to thermal stress, results from the interaction between neutral and adaptive processes54 DNA extraction and genotyping protocols are detailed in Supplementary Methods Neutral genetic divergence: Before initiating the experiments, a small tissue sample was taken from each apical tip and stored in 96% ethanol and at −20 °C for subsequent genetic analysis. Two hundred and forty colonies belonging to the eight populations were genotyped using six microsatellite loci  following75 using the default parameters. These analyses showed that the two populations from Medes  and the two populations from Scandola  were not significantly genetically different . Accordingly, we grouped Medes shallow and Medes deep in the Medes genetic pool and Scandola shallow and Scandola deep in the Scandola genetic pool, so the following analyses were conducted considering six different genetic pools .As a preliminary step, we identified the different genetic pools  within our dataset by conducting exact tests of the overall genotypic differentiation among populations and between all pairs of populations in GENEPOPθ, the Weir & Cockerham estimator of FST76 in FREENA77. We also calculated the population-specific FST as implemented in GESTE78 for the six genetic pools. This method measures how genetically distinct a population is compared to all of the populations combined and provides insight into the relative impact of genetic drift and gene flow on the differentiation of each population78.We estimated the neutral genetic divergence and respective 95% confidence intervals (95% CIs) by computing the global and pairwise differentiation between the six genetic pools using PST80, which is defined as PST = cσ2GB/(cσ2GB + 2h2σ2GW), where σ2GB and σ2GW represent the among-sample (in this context one sample refers to one genetic pool) and the within-sample variance components for the considered phenotypic trait, respectively; h2 represents the assumed additive genetic proportion of the differences between individuals within samples (“narrow-sense heritability”); and c represents the proportion of the total variance presumed to occur because of additive genetic effects across samples81. We considered the null assumption that the genetic architecture of the trait  remained equal across samples with c/h2 = 181. Among- and within-sample variances were computed based on PERMANOVAs, which were conducted considering “the level of tissue necrosis of the 10th, 20th and 30th experiment day” of the common garden experiment as a multivariate dependent variable and “genetic pool” as the only random factor .Phenotypic divergence: The global and pairwise phenotypic divergences were estimated between the six genetic pools using MSB and MSW, respectively) from the PERMANOVA output. The within-sample variance (σ2GW) was directly estimated based on the within-sample MSw. The between-sample variance (σ2GB) was estimated as σ2GB = (MSB − MSW)/n0 with n0 being a weighted average of the sample size for each comparison:a is the number of samples to be compared, and ni is the number of individuals in the ith sample83. Negative variance values were set to zero84.Variances were estimated by extracting the within-sample and between-sample mean squares  and phenotypic (PST) estimates of divergence. A significant relationship between the two estimates of divergence suggests that the pattern of phenotypic divergence is mainly explained by genetic drift85 or, alternatively, that isolation by adaptation occurs with divergent selection driving the evolution of the phenotypic trait and the differentiation at neutral loci87. To disentangle the two hypotheses, we then compared the PST (under c/h2 = 1) and FST estimates for each population pair. If divergent selection is involved in the observed patterns, we expect the PST estimates to be significantly higher than FST58. The comparison of the estimators was based on their 95% CIs.Comparison between genetic and phenotypic divergences: We conducted a Mantel test (n = 1000) to analyze the relationship between pairwise genetic .The PERMANOVA (adonis function), Supplementary information


Gambusia holbrooki and G. affinis) around Mobile Bay, AL. The region has been studied episodically, over decades, making it perfect for addressing temporal dynamics and for providing a deeper understanding of the genetics of these periodically reclassified fishes (as species or subspecies). We used six microsatellite markers to assess the current population structure and gene flow patterns across 19 populations of mosquito fish and then compared our results with historical data. Genetic evidence demonstrates that the current hybrid zone is located in a similar geographic region as the historical one, even after three decades. Hybrid fish, however, demonstrate relatively low heterozygosity and are genetically distinct from western and eastern mosquito fish populations. Fin ray counts, sometimes used to distinguish the two species from one another, demonstrate more eastern (G. holbrooki) phenotype fish within the molecular genetic hybrid zone today. Mosquito fish are globally invasive, often found on the leading edge of flooded waters that they colonize, so the impact of hurricanes in the wake of climate change was also evaluated. An increase in the frequency and intensity of hurricanes in the hybrid region has occurred, and this point warrants further attention since hurricanes are known to move these aggressive, invasive species into novel territory. This work contributes to our classical understanding of hybrid zone temporal dynamics, refines our understanding of mosquito fish genetics in their native range, evaluates important genotype–phenotype relationships, and identifies a potential new impact of climate change.Hybrid zones allow for the investigation of incipient speciation and related evolutionary processes of selection, gene flow, and migration. Interspecific dynamics, like competition, can impact the size, shape, and directional movement of species in hybrid zones. Hybrid zones contribute to a paradox for the biological species concept because interbreeding between species occurs while parental forms remain distinct. A long‐standing zone of intergradation or introgression exists for eastern and western mosquito fish ( A mating barrier breakdown then occurred between the sympatric benthic and lentic three‐spined sticklebacks (Gasterosteus aculeatus). A substantial uptick in hybrid frequency raised concern about native species loss and the formation of a new hybrid swarm  for North America. The basin contains 40 endemic species  in the Mobile Bay, AL region. Both species are highly aggressive, globally invasive freshwater fishes, native to the southeastern United States. G. affinis has a native range west of the hybrid zone into parts of TX and NM, and north through several mid‐western states. G. holbrooki has been considered native eastward of Mobile Bay, south throughout peninsular FL, and north up the eastern seaboard to NJ. These fishes have been introduced to every continent except Antarctica, and to over 50 countries  . In 1939, G. holbrooki and G. affinis were reclassified as a single species  hybrids. All seven locations were southern creeks, meaning fish were migrating northward from the region closer to the Gulf of Mexico  hybridized with G. affinis males (ZZ), the F1 generation produced was 100% male (XZ), but when G. affinis females (WZ) hybridized with G. holbrooki males (XY), the F1 generation was, on average, 50–75% male  and 25–50% female   structure were evaluated and compared to historical data  of 22.1About 30 individual fish were collected (from 2011 to 2014) for molecular analysis from each of 19 populations in the native range Figure  so in thTotal genomic DNA was extracted from tail  muscle tissue using the Qiagen DNeasy Blood and Tissue Kit . Samples were assessed for quality and quantity using a Thermo Scientific NanoDrop 1000 spectrophotometer . Polymerase chain reaction (PCR) was run for six microsatellite loci using fluorescently labeled primers for Gafμ 1, Gafμ 2, Gafμ 3, Gafμ 4, Gafμ 6, and Gafμ 7  genetic analyzer. Forward primers were fluorescently labeled with FAM or HEX dye. Peak calls were made manually using GeneMapper v. 3.7 and Peak Scanner 1.0 (Applied Biosystems) and rounded to the nearest whole number. Bins were created and subsequently compared to previously reported microsatellite reads for MICRO‐CHECKER v. 2.2.3 was used to assess the accuracy of allele scoring by checking for stutters, large allele dropouts, and null alleles   values were produced to determine the correct number of clusters (K) for use with STRUCTURE. All runs were conducted with a burn‐in period of 100,000  iterations. Individuals adhering to their respective assigned STRUCTURE cluster by >80% agreement of associated genotype were considered to be in their assigned cluster ) and change in FST values. Mantel tests were run in R using ade4 and vegan packages to detect spatial autocorrelation between matrices . A partial Mantel test was run with FST values, STRUCTURE 2.3.4 clusters, and GPS coordinates to test whether isolation by distance results could have been falsely positive due to clustering  molecular analyses included all markers (discussed elsewhere).To compare genotype and phenotype, each individual was assigned to a category (species or hybrid) based upon fin ray count and then compared to the genetic cluster assignment , considering also private and semiprivate alleles identified for each species. Due to the presence of potential null alleles in some of these populations for two loci (Gafμ 4 and Gafμ 6), every individual's genotype was assessed for four loci , allowing for designation as pure 2.3G. affinis alleles. The Scribner and Avise  was plotted for the Western Atlantic Ocean and Gulf of Mexico NOAA  and eval was plot33.1K = 19) showed that four populations were out of HWE for Gafμ 6, and two populations were out for Gafμ 4. Due to potential stuttering and disequilibrium for four populations for Gafμ 6, all further genetic tests were conducted twice: (1) all populations and all loci, and (2) all populations and all loci, excluding Gafμ 6. Given the similarity of results, we report results for all populations and all loci since statistical significance did not vary between (1) and (2).Power analyses of sample size prior to this assessment indicated that 30 mosquito fish per population were sufficient to identify the important genetic relationships within and among populations  in STRUCTURE demonstrated three genetic clusters Figure a. The wee Figure c.r = .6677, p = .001); those from the regular Mantel test (two matrices) were not .For the western cluster, no individuals deviated genetically from the cluster. For the hybrid cluster, 41 individuals (23%) deviated genetically, with the vast majority (90%) of these (37 of 41) individuals being from the two most peripheral populations of the hybrid cluster (4‐MA and 9‐AC). For the eastern cluster, about a quarter (24%) of the individuals (67 of 280) deviated genetically, with most mismatches actually corresponding to the western cluster. Results from the partial Mantel test (three matrices) were highly significant (H0) differed significantly among the three genetic clusters identified , although other population genetic variables did not, including allelic richness (AR) , average inbreeding , and average subpopulation structure . Comparison of populations within and among clusters demonstrated that the highest allelic diversity was present in eastern mosquito fish.Observed heterozygosity  was a clear mix of ‐MA was as Figure  and was s Figure  and a clThe Garza–Williamson (G‐W) index represents the ratio between total number of alleles and allelic range. Particularly low G‐W values may reflect reduced effective population sizes (Ne), or bottlenecks. The G‐W index was lowest for the hybrid cluster, intermediate for the western cluster, and highest for the eastern cluster populations Table .n = 2) private alleles, the hybrid cluster an intermediate number (n = 11), and the eastern cluster the most (n = 21). This same rank held true for the average number of private alleles per population  in the hybrid zone, and each of the other western cluster populations  that were not different from one another (Table Applying the same pairwise comparisons to the eastern edge of the hybrid cluster produces a similar result: Comparing the second most easterly population in the hybrid cluster (8‐AB) to the three nearby eastern cluster populations  produces greater differentiation (3.2n = 4 of 6 hybrid populations), fish with G. holbrooki fin ray counts vastly outnumbered fish with western or hybrid fin ray counts  had G. holbrooki fin ray counts (Table n = 10/108) had hybrid fin ray counts , most females (78%) had western fin counts, fewer females had hybrid counts (17%), and very few (4%) had eastern counts. Genetically, however, nearly half of these fish (48%) contained a mix of western and hybrid cluster alleles (~50% each), about a third (30%) actually had only hybrid cluster alleles, and about a fifth (22%) had western cluster alleles. Male fin counts were also largely western (86%), less eastern (14%). Genetically, these males were also mixed hybrid and western cluster alleles (50% of each). So in this population, western fin counts were dominant, but hybrid alleles abounded.However, in the middle of the hybrid cluster , the dominant fin count for females was eastern (88%); the remaining fish had an equal frequency of western and hybrid (6%) fin counts. Yet genetically, all of these females had hybrid genotypes. The majority of males in this region (87%) also had eastern fin counts, although a few were western (13%). Yet all of them had hybrid genotypes. Thus, eastern fin counts dominated most of the hybrid zone, despite hybrid genotypes.In the population on the eastern edge of the hybrid cluster (9‐AC in AL), most females (84%) had western fin counts, with 11% hybrid and 5% eastern fin counts. Yet, (61%) of these fish had hybrid cluster genotypes. Just over one‐third of these fish (39%) had mixed hybrid  alleles. The males in this population all had western count fins, but were of mixed genetic origin . Here, the western fin count was most abundant, again despite the high frequency of hybrid cluster genetics.In the eastern cluster populations, nearly all (91%) females had eastern fin counts, but a few (9%) had hybrid counts. Most females (83%) were comprised of eastern alleles, but some were mixed hybrids (17%). Males here all had eastern fin phenotypes, except for one fish in AL; these males had mostly (67%) eastern cluster alleles, but some (33%) were mixed hybrids.3.3G. holbrooki alleles a bit further into the hybrid zone on the eastern edge, than in the past  patterns do not match genetic ones closely for hybrids. This point is important relative to historical data for comparative purposes, but also because these “species” are frequently introduced for mosquito abatement and species identification has occurred by fin count and geographic origin.G. holbrooki counts, and less hybrids, appear now  counts present in populations dominated by G. affinis , with ~67% of the variance in storm frequency explained by time. The frequency of intense hurricanes (categories 3–5) has doubled, up from 17 events in the 1980s, to 35 in the 2000s. The decade from 2000 to 2010 had the highest record of intense hurricanes since the 1900s. The vast majority of storms hit the Gulf of Mexico from the southeast, with wind gusts and flooding moving northwesterly. Since 1985, seven major storms, including Hurricanes Ivan, Andrew, Katrina, Dennis, Opal, Elena, and Jeanne, have substantially impacted the hybrid region. Empirical data demonstrate the movement of G. holbrooki during hurricanes  persist in LA, hybrid fish are found in MS and much of southern AL, and eastern mosquito fish (G. holbrooki) are found in southern‐central AL, and throughout FL. The region where hybrid cluster fish are found is largely similar geographically to that which has been identified previously a number of times as a region of intergradation. The hybrid cluster is currently substantially different from the two parental populations, and distinct differences exist for the most easterly and westerly populations of the hybrid cluster, relative to the other hybrid populations, with a large proportion of eastern and western alleles present in the peripheral populations of the hybrid cluster.Three decades have elapsed since comprehensive allozyme work drew Wooten et al.  to conclOur results also support a hierarchical island model over an isolation by distance model, suggesting that demes within neighborhoods exchange more migrants than they do with other neighborhoods Wright,  which coG. affinis territory, we may have found more private alleles in this species, too.Recurrent changes in sea level have clearly impacted freshwater fish populations. When sea level rose historically, the only fresh water habitat was a few points of higher elevation, meaning some reproductive isolation could have occurred for extant subpopulations, producing semiprivate alleles. Once seas receded, if neighborhood‐level gene flow was the model of movement, subsets private alleles may have migrated to novel local habitat. Since FL was almost twice the size it is today, the relatively greater landmass and expansive habitat near historic refuge may have contributed to the high number of private alleles arising. If we had sampled additional Gambusia holbrooki populations were shown to have higher heterozygosity than G. affinis previously which remains true today, and can contribute to fitness advantages  between MS (5MB) and populations of western fish in LA, combined with the fact that this MS population has the lowest heterozygosity of all populations studied, indicates reduced gene flow between these regions relative to others, which is important with respect to understanding hybrid zone boundaries.ges Orr, . The relges Orr, . The relFST = 0.135 and 0.178), which is less than this current hybrid region, but also consistent with our overall results  between G. affinis and G. holbrooki, so understanding more about this region is timely.Genetic structure within species across mosquito fish populations has previously been reported to be fairly low ; however, counts were poorly correlated with genetic data in the hybrid region. Individuals with alleles from both species had fin ray phenotypes that could not be predicted based upon semiprivate alleles, or genetic match to cluster. Males, even those that had hybrid alleles, tended to have eastern mosquito fish fin counts, but not always. Previous work indicated that when G. holbrooki and G. affinis were crossed, F1 fish only had G. holbrooki fin ray counts, leading Hubbs, G. holbrooki fin ray counts did not dominate in hybrid populations. They also found evidence of nonrandom segregation, also supported by Scribner and Avise . However, in one population genetically comprised largely of western and hybrid alleles, the western fin phenotype did dominate. This could suggest that fin count is polygenic and/or controlled by a form of incomplete dominance, where dominant alleles are found more often in the “true” species, with the G. holbrooki phenotype dominant over G. affinis. Future phenotypic research on fins should include comprehensive assessment of ventral and dorsal counts , a male bias also results, but not all (but 50–75%) progeny are male. These males may survive in the hybrid region but might not successfully colonize further east because they are outcompeted by pure G. holbrooki  genetic variation is explained within populations, with a much smaller amount (6.9% and 9.5%) explained at the population or group level (respectively). Semiprivate alleles from both species are found in the hybrid region and there are fewer alleles overall in the hybrid region. However, identifying whether reproductive isolation exists between any clusters remains warranted since even single genes, adaptive in two parental species, can drive hybrid inviability , populations to move inland, or upstream, while potentially invasive warm water fish, like topminnows and sunfishes (Fundulus sp. and Lepomis sp.), have expanded their ranges  fish species, Gambusia being the most abundant by far (76% of the total number of fishes in the pools, Caillouët et al., G. holbrooki exhibited a twofold increase in the St. Sebastian River, FL (Paperno et al., Regarding future directions, climate change has impacted coastal freshwater systems in the past 30 years through sea level rise and increasing temperatures, which have forced cool water species, like catfish (Mandrak, . Recent Mandrak,  and NOAALastly, it is worth mentioning that humans contribute to mosquito fish movement. Frequent introductions for biological control of mosquitoes has occurred Rupp,  and ≥65 None declared. Click here for additional data file.


Languages and genes arguably follow parallel evolutionary trajectories, descending from a common source and subsequently differentiating. However, although common ancestry is established within language families, it remains controversial whether language preserves a deep historical signal. To address this question, we evaluate the association between linguistic and geographic distances across 265 language families, as well as between linguistic, geographic, and cranial distances among eleven populations from Africa, Asia, and Australia. We take advantage of differential population history signals reflected by human cranial anatomy, where temporal bone shape reliably tracks deep population history and neutral genetic changes, while facial shape is more strongly associated with recent environmental effects. We show that linguistic distances are strongly geographically patterned, even within widely dispersed groups. However, they are correlated predominantly with facial, rather than temporal bone, morphology, suggesting that variation in vocabulary likely tracks relatively recent events and possibly population contact. Early explorations on the association between languages and genes indicated that patterns of linguistic diversity paralleled those of genetic diversity. Most studies used pairwise distance measures of genetic and linguistic dissimilarity between populations in order to statistically compare the significance of their association12345791112351315For much of recent human evolution, genetic drift—changes that are due to stochastic rather than directed processes—is considered to be the primary mode by which hominin populations became differentiated182122232428Indeed, while genetic drift may be one of the primary evolutionary modes of differentiation in modern human populations, gene flow is an important factor that can act to reduce diversity among populations. Gene flow between populations increases their similarity via the exchange and mating of individuals. Similarly, active communication between speakers of different languages can lead to borrowing, ultimately making these languages appear more similar to each other. As such, it is not only the diversity within populations that must be examined, but also the diversity between populations. In addition to the negative correlation between intra-population diversity and increasing distances from Africa, a serial founder model also predicts a positive association between inter-population differences  and geographical separation 2032Over the last decade, important progress has independently been made in both skeletal shape analysis and historical linguistics. In the former case, advances have allowed for better quantification of variation between populations and species, proving useful in the assessment of phylogenetic affinities of previously contentious taxonomic categories  with vocabulary distances. We defined a purely data-based vocabulary distance measure (L) between languages. From this we derived an aggregate linguistic distance measure between language families (F), defined as the arithmetic mean of the pairwise distances between their component languages. The aggregate geographic distance G between two families was likewise calculated as the arithmetic mean of the corresponding distance between the component populations. This allowed us to compare the variation across N = 265 language families and isolates. Since genetic and skeletal phenotypic distances between populations are significantly correlated with land-based G, a significant association between F and G would imply a linguistic spatial patterning consistent with a serial founder model.To test the hypothesis that vocabulary information can be used for reconstructing population history at a deep temporal scale, we first quantified the association of geographical distances (L) and, in turn, evaluate the association of L with G and with biological distances. For L, we used the weighted alignment method using vocabulary dataPST) using cranial shape data. The PST measure is analogous to the genetic distance measure FST, with an underlying assumption that the phenotype reflects the net effect of polygenic inheritance5152PST values derived from it should correlate with L to a greater degree than with other cranial regions if vocabulary distances indeed parallel neutral genetic distances through a deep temporal scale. We statistically assessed the associations between PST, L, and G using Mantel and Dow-Cheverud correlation tests.Secondly, we sampled eleven populations from Africa, Asia, and Australia  in orderG and F was significant ; thus, geography explains ~8% of variance between language families. In the association between L and PST, the whole cranium had the highest correlation and, independently, all of its regions showed statistical significance  both lexical and grammatical variables, (ii) distinct skeletal phenotypic variables, and (iii) various genomic polymorphisms for the same populations would be ideal. Likewise, further empirical work and simulation approaches that vary the rate of anatomical and lexical characters under diverse evolutionary models will serve to validate or falsify this hypothesis.The rate of change of vocabularies is estimated to be between 3–4 times faster in comparison to changes in their grammar34L and face PST might be broadly attributed to epigenetic effects on phenotype. For example, by extension of the known association between environmental variables and facial phenotype3637While not mutually exclusive, other interpretations can be offered for our results. First, the association we found between PST values41We caution that interpretation of our results is bound to the limitations of our dataset and study design. In particular, landmark configurations in our study capture diverse anatomical characters to varying degrees. For example, while the landmark configurations for the face and temporal bone both comprise thirteen landmarks each, the neurocranium only comprises eight. More generally, the face and temporal bone configurations capture more anatomical details than the neurocranial configuration. Thus, the best comparison is between the face and the temporal bone, which indeed results in a statistically significantly different association with language. An additional limitation to our study is that we did not consider differences in population size or divergence time, which can be informative in understanding the effect of drift experienced by populations and which can serve to contextualize pairwise Having already made substantial progress since the modern evolutionary synthesisN = 265  and 1 indicates complete genetic differentiation—PST values contrast in that they can exceed the 1 threshold when using heritability and population size estimates. In spite of this, FST and PST are highly correlated under neutrality and in the absence of selection5152PST values for each cranial region are reported in Finally, we used the selected PCs to calculate L) were computed from the Automated Similarity Judgment Program (ASJP) database49Linguistic distances  in hant-mano do not. This asymmetry is captured by the sum of the weights of the aligned sounds corresponding to the two alignments. For hand-hEnd, this sum is 4.80 while for hand-mano it is −11.85, so the former pair is a much better candidate for reflecting common descent than the latter. In the second step, the similarity between two doculects is quantified as the degree to which the distribution of string similarities between synonymous word pairs exceeds the distribution of string similarities between non-synonymous pairs. The distance between two doculects is defined as a linear function of the similarity with negative coefficient that has 0 and 1 as theoretical minimum and maximum, respectively.Distances between word lists were computed following the two-step procedure detailed in ref. L) between population pairs was calculated as the arithmetic mean of all linguistic distances between candidate doculects assigned to each population (results reported in F) were computed as the arithmetic mean of all linguistic distances between sampled doculets. We followed the classification of languages into families according to the World Atlas of Language Structure (WALS)Some ethnographic records were available for the cranial collections, but in most cases the languages spoken by the individuals could not be uniquely identified. ASJP contains meta-data for each doculect, such as geographic location and expert classifications. We chose a group of candidate doculects from the ASJP database for each population, using a combination of three heuristics. First, subpopulation information consisting of ethnic affiliation was used to narrow down the space of candidate languages. For instance, the South India population is specified as ‘Tamil’; hence Tamil is the only candidate doculect for it. Second, if the population was from islands , only doculects from these islands were considered. Third, whenever no more specific information was available, the candidate doculects were those ASJP doculects whose geographic coordinates (according to the ASJP meta-data) are situated within a distance of at most 500 km from the population in question. The lists of candidate doculects for each population are given in the G) between populations following the method from refs We computed land-based geodesic distances (L or F) and cranial phenotype (PST) distances, as well as between these and geographical distances (G). We used partial Mantel tests to evaluate the association of L and PST when controlling for G. Correlations between distance matrices were computed as Spearman’s rank correlation coefficients, as the dependencies between linguistic distances and both cranial phenotype and geographical distances are non-linear. In order to assess whether the PST values of a given cranial segment were statistically correlated more with L than the PST values of another cranial segment, we applied the Dow-Cheverud testG. In all cases, two tailed p-values were determined with 10,000 permutations. Likewise, Bonferroni corrections were applied to p-value results of the subset landmark configurations, including a correction for multiple model comparisons and a sequentially rejective correction78p-value value, then at α = 0.025 for the next lowest p-value value, and finally at α = 0.05. We applied these corrections to the Dow-Cheverud results but note that p-value significance thresholds in such a test have conventionally been set at α = 0.05 .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.