Broad-scale population differentiation is primarily driven by an isolation by distance genetic structure model (49% of genetic variance), with clinal local adaptation corresponding to habitat bioregions. Signatures of selection were detected between bioregions, with no single region returning evidence of strong selection. The results of this study show that although the koala is widely considered to be a dietary-specialist species, this apparent specialisation has not limited the koala’s ability to maintain gene flow and adapt across divergent environments as long as the required food source is available.The Australian koala is an iconic marsupial with highly specific dietary requirements distributed across heterogeneous environments, over a large geographic range. The distribution and genetic structure of koala populations has been heavily influenced by human actions, specifically habitat modification, hunting and translocation of koalas. There is currently limited information on population diversity and gene flow at a species-wide scale, or with consideration to the potential impacts of local adaptation. Using species-wide sampling across heterogeneous environments, and high-density genome-wide markers (SNPs and PAVs), we show that most koala populations display levels of diversity comparable to other outbred species, except for those populations impacted by population reductions. Genetic clustering analysis and phylogenetic reconstruction reveals a lack of support for current taxonomic classification of three koala subspecies, with only a single evolutionary significant unit supported. Furthermore, ~70% of genetic variance is accounted for at the individual level. The Sydney Basin region is highlighted as a unique reservoir of genetic diversity, having higher diversity levels (i.e., Blue Mountains region; AvHe Specialist species evolve in stable environments to exploit available niches. However, specific adaptation to these niches, whether they be dietary or habitat specialisation, can make them more vulnerable to stochastic change than generalist species. Local persistence and dispersal rates of specialist species are strongly influenced by degree and type of ecological specialisation . This classification was first described in the early 20th century, based on skull morphology and skins alone  between its northernmost and southernmost populations, with intermediate phenotypes in the middle of its range  and Australian Capital Territory (ACT) but is not listed in VIC and SA. This dichotomy in conservation status is a reflection of the different overall population histories of koalas in these states and territories. Koala population declines have been observed across NSW and south east QLD, whereas some populations in VIC and SA are considered to be over-abundant. To prevent defoliation of preferred food trees, and subsequent starvation of koalas in these over-abundant populations, they are actively managed through translocation and fertility control  identified from restriction site-associated (RAD) fragments. SNPs were used for both population and phylogenetic analyses, whereas PAVs were only used in phylogenetic reconstructions. To ensure the highest quality loci were carried through to downstream analysis, the following sequence quality control and filtering measures were conducted. Raw sequence data in a fastq file format were obtained, and de-multiplexed according to individual barcodes. Each read was assessed for overall quality, and any reads containing base pair Q-scores <30 were removed. All reads were checked against existing sequences in the DArTdb database . KDCompute SNP calling was carried out by creating clusters of identical reads at a population (or dataset) level, with three nucleotide mismatches allowed, then similar clusters were matched together to identify polymorphisms within sequence reads as described by Wenzl et al. (2004) and Lind et al. (https://github.com/esteinig/dartQC). SNPs with an average read depth of <10  were identified across all populations using PLINK  in all populations, it was removed from the working dataset, SNPs, which deviated in only a single population, but were in HWE across the entire dataset were retained. Loci putatively identified as sex linked were removed from the final SNP dataset according to Kjeldsen et al. , using the fast read mapper function in Bowtie 2 , the average within- and between-bioregion FST values were calculated.In order to identify loci that are under selection, outlier analyses were conducted using a Bayesian approach, implemented within the program BayeScan 2.01 Foll . All Sou Jombart  using bo Jombart . Populatkerham’s  unbiasedkerham’s . To inveFinally, to assess if any putatively identified outlier loci were associated with genic regions, all identified outlier SNP sequence reads was mapped to the koala genome . Additionally, the region surrounding each marker (±2000 bp) was extracted and subsequently compared with publicly available genomic databases to identify any markers, which may be closely linked to functional regions (Supplementary Table corr), average observed heterozygosity (Ho), inbreeding coefficient (FIS), number of private alleles (Ap) and rare alleles  were calculated through the Genalex v6.502 analysis package  including average expected heterozygosity corrected for sample size (HeF-statistics (FST) , and Meirman’s approach in GenoDive (Meirmans and Van Tienderen 2004). However, as the Meirman’s values were similar, and the Meirman’s approach returned some negative, nonsignificant values, only Weir and Cockerham’s unbiased FST values are presented here  of the most informative individuals  to facilitate convergence of each run. Representative individuals were selected by retaining only a single individual from each minor cluster within the ML tree reconstruction. Bayesian analyses consisted of two runs of 100,000,000 generations each and eight independent chains. Heated chains were set to Temp = 0.10, with a 25% burn-in and a sampling frequency of 1000. Dirichlet prior states were set to 48:52, which were calculated based on observed frequencies of absence (“0”) and presence (“1”). Runs were completed if standard deviations between runs were below 0.05, and were independently assessed for convergence using Tracer v1.6  were removed from the dataset for violating filtering parameters, with low call rate being the primary factor. A total of 104 sex-linked markers  were identified and removed from the working dataset, and the remaining dataset of 5265 SNPs was then tested for conformity to HWE (three SNPs removed) and LD (659 SNPs removed). A final set of 4606 unique autosomal SNPs were retained for downstream analysis. A total of 22,022 PAV markers were initially identified across all individuals. Following filtering, a total of 6102 PAV markers were retained for use in phylogenetic reconstructions.FST across populations calculated using neutral loci was 0.18 (SD ± 0.06), with average FST for directional outliers being markedly higher . When NJ trees were constructed based on 1-proportion of shared alleles genetic distances for each locus type, branch lengths were slightly longer and more uniform among individuals using neutral SNPs when compared with directional outlier SNPs  and average between-bioregion FST was 0.20 (SD ± 0.11). Between-bioregion FST differences increased according to bioregion differences in a clinal pattern, whereby neighbouring bioregions displayed an intermediate average FST difference of 0.13 (SD ± 0.08), by comparison the greatest difference of 0.39 was observed between the most divergent bioregions (Sydney Basin (SYB) versus Brigalow Belt North (BBN); Table Weak to moderate signatures of selection were identified among populations investigated at both predefined FDR levels (FDR = 0.01 and 0.001). A total of 137 SNPs  were identified at FDR = 0.01, and 71 were identified at FDR = 0.001. Average NPs Fig. . HoweverNo putatively identified outlier loci were associated with annotated genic regions, and when flanking regions for each SNP were compared with existing genomic databases, only 20 markers fell within ±2000 bp of annotated gene region, with a matching identity and coverage of 100% (Supplementary Table corr) ranged from 0.10 to 0.20 (Table corr = 0.20), whereas the French Island population showed the lowest levels (Hecorr = 0.10). Percentage of polymorphic loci ranged from 37.7% (South Gippsland) to 82.5% (Campbelltown)  to 0.19 (Kangaroo Island). Average sMLH was highest in Port Macquarie (1.21), whereas Kangaroo Island displayed the lowest level (0.58). Frequencies of rare alleles within populations ranged from 0.04 to 0.18 across the species range, with Port Macquarie and Blue Mountains populations having the highest levels (Table Average observed heterozygosity (Ho) across populations ranged from 0.09 to 0.18, and average expected heterozygosity  were similar to those observed in Netview R plots at kNN 40–50  and phylogenetic trees (see below), admixture can be observed throughout NSW populations (with exception of the Lismore population), with higher levels of admixture observed within the Sydney region  returned the same results at each respective K value Figs. –f. Each mal Fig. , with ge= 4 Fig. . These cR2 = 0.49; Fig. The results of the IBD Mantel test revealed a moderate to strong correlation with genetic distance and geographical distance when evaluating all populations across the species range  compared with populations with more stable population histories . This indicated that these genetic bottlenecks may have skewed or inflated FST values , and overall average within the northern group (based on Netview R clustering) was lower than in the southern groups . Partitioning of genetic variance based on AMOVA tests revealed that among individuals and within individuals accounted for most of the genetic variation  independent of the groupings applied to populations. Among-population variance ranged from 14.3% to 20.4% between analyses, reflecting moderate population differences corresponding to other analyses. Among groups as the source accounted for the least amount of genetic variance, with the largest variance of 13.6% recorded when populations were grouped together based on genetic similarities and optimum number of clusters . Although a historical north/south separation is observed within the Sydney Basin   (Brakefield and Reitsma Sciurus vulgaris)   . This pattern was also observed in a recent study using microsatellite markers, where the South Gippsland region was observed to be distinct from other Southern populations . Animals that persisted in this region may have carried remnant diversity, and genotypes, which were lost elsewhere in southern regions as a result of widespread hunting, may have been retained in these populations.Ecological history appears to have a direct effect on contemporary genetic diversity levels, with all southern populations (populations sampled from VIC and SA) displaying clear reductions of diversity Table . HistoriFST values in this region are likely inflated due to repeated genetic bottlenecks and random genetic drift effects . Genetic relationship patterns clearly show that most mainland Victorian koalas are very similar to those from French island Figs.  and 3, bOf the regions sampled in this study, groups of populations that were sampled from areas surrounding protected habitats  appeared to have higher levels of admixture, and generally higher diversity values than those surrounding suburban areas Fig. ; Table 1FIS values close to zero, which is consistent with outbred populations . Despite the small number of animals that colonised these populations, . The French Island population has been used repeatedly to supplement mainland Victorian and South Australian populations. These translocations are concerning as French Island displays one of the lowest heterozygosity values across Australia (Ho = 0.11), and the high FIS values observed here should also be noted, as any further translocations from this island may impact diversity levels in the target population, particularly if the target population was originally sourced from French Island. Similarly, the Kangaroo Island population is of particular concern, as in addition to it returning the highest inbreeding value, it also has the lowest observed heterozygosity value (Ho = 0.09), lowest average standardised individual multilocus heterozygosity (sMLH = 0.59) and the highest IR value (IR = 0.83). These populations have been highlighted as having reduced genetic diversity in the past , and the Blue Mountains (FIS = 0.1) that returned positive FIS values. However, Netview R clustering indicates that population substructuring is present in these populations , which in extreme scenarios can lead to outbreeding depression Frankham . From a Currently koala populations are managed based on arbitrary geographic distances, with translocations and movement of animals often restricted to local government boundaries, or prohibited completely . These populations could be considered to be reservoirs holding substantial species diversity. However, the effects of local adaptation between bioregions should not be ignored, because movement of animals into unsuitable habitats may result in overall reduced fitness Frankham . Despitehttps://datadryad.org//resource/doi:10.5061/dryad.1683r1sData available from the Dryad Digital Repository: Supplemental material - informationSupplementary Figure 1Supplementary Figure 2. a)Supplementary Figure 2. b)Supplementary Figure 3Supplementary Figure 4Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


Portunus pelagicus Linnaeus, 1758) is one of the commercially exploited crab fishery resources in Vietnam. This is the first study to provide a broad survey of genetic diversity, population structure and migration patterns of P. pelagicus along the Vietnamese coastline. The crab samples were collected from northern, central and southern Vietnam. Here, we used a panel of single nucleotide polymorphisms (SNPs) generated from restriction site-associated DNA sequencing (RADseq). After removing 32 outlier loci, 306 putatively neutral SNPs from 96 individuals were used to assess fine-scale population structure of blue swimming crab. The mean observed heterozygosity (Ho) and expected heterozygosity (He) per locus was 0.196 and 0.223, respectively. Pairwise Fst and hierarchical AMOVA supported significant differentiation of central and northern from southern populations (P<0.01). Population structure analyses revealed that P. pelagicus in the south is a separate fisheries unit from the north and center. Contemporary migration patterns supported high migration between northern and central populations and restricted genetic exchange within the southern population. In contrast, historic gene flow provides strong evidence for single panmictic population. The results are useful for understanding current status of P. pelagicus in the wild under an environment changing due to natural and anthropogenic stresses, with implications for fisheries management.The blue swimming crab ( In winter, currents flow in a North East-South West direction while in summer, ocean currents flow from the South West-North East [The tropical to subtropical Vietnamese coastal zone is divided into the Gulf of Tonkin in the North, the central coast, the southeast coast and the Gulf of Thailand in the South ,2. The erth East  with therth East ,5–7.Portunus pelagicus) is a scavenging tropical marine species, widely distributed in the Indian and Pacific oceans, the East coast of Africa, the Mediterranean Sea and southern Japan [The blue swimming crab  [nd Japan ,19,20. And Japan , global nd Japan . Gillnetctively) .A crab management plan for Vietnam is in place. However, due to unsystematic application of management measures (the minimum landing size and the closed season), and lack of demographic information, management is considered ineffective ,23. ReceP. pelagicus throughout its distribution range. In the early 2000s, Yap et al. (2002) [P. pelagicus have been reported as previously recorded by Lai et al  [P. pelagicus in Vietnam, although limited published studies examining genetic structure of marine organisms have indicated high connectivity in the dynamic and complex Vietnam East Sea waters [Several studies have revealed different population structuring of . (2002)  and Sezm. (2002)  detected. (2002) ,47 using. (2002) ,49. In bal (2010  identifi. (2016)  found di. (2016)  applied a waters ,53.P. pelagicus in Vietnam, to better understand fine-scale population structuring and gene flow along the Vietnamese coastline and to provide data on resilience and sustainability for fisheries management.The goal of this study was to develop SNPs using RADSeq, previously not accomplished for Table 1).Blue swimming crabs were collected along the north-south geographical temperature gradient: Cat Ba Island—Hai Phong City; Ha Long Bay—Quang Ninh Province (northern population), Nha Trang Bay and Van Phong Bay—Khanh Hoa province; Song Cau and Tuy Hoa—Phu Yen province , and Phu Quoc Island, Rach Gia City—Kien Giang Province (southern population)  were presented in ® 2.0 Fluorometer (Invitrogen). Selected DNA templates  were then purified using AMPureXP (Agencourt) beads using a 2:1 template to bead volume ratio with the beads left in.Genomic DNA was extracted from preserved tissue samples using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions, and treated with RNase (100 mg/mL) to remove residual RNA. Extracted DNA was eluted three times (100 μl elution/time) to get better DNA quality. All elutions were assessed using gel electrophoresis (1% agarose gel). The best elution  was selected to determine the concentration by QubitPurified DNA from each crab individual was simultaneously digested with two restriction enzymes: MboI and Sau3AI (NEB). Each digestion was performed in 25 μl reactions: 2.5 μl SmartCut Buffer (10X), 0.5 μl MboI and 0.5 μl Sau3AI (5 unit/μl), and 21.5 μl of DNA template (eluate from the beads). Digestions were incubated at 37°C for 3 h to overnight, and then 65°C for 20 min, cleaned with PEG solution , and eluted with 20.1 μl Illumina Resuspension Buffer.2O and 5.625 μl DNA libraries. Biorad thermocyclers (Icycler) were used under the following temperature program: initial denaturation at 95°C for 3 min, followed by 8 cycles of 98°C for 20s, 60°C for 15s and 72°C for 30s. Final extension was done at 72°C for 5 min and the soaking temperature was set to 4°C.Cleaned digestions were inserted directly into the Illumina TruSeq nano DNA library Prep kit following the Sample Preparation v2 Guide starting with the “Perform End Repair” step for one-third volume reactions (Supplement S1 ). DigestPCR products (The 400–500 bp fragments of which 120 bp are the ligated adapters) were inspected using a 1.5% agarose gel with ethidium bromide and bands were visualized under UV transilluminator. PCR products were purified using SP Beads (1:1), and quantified using qPCR. DNA libraries were sequenced as paired-end 100 bp runs on HiSeq 2500/4000 system (Illumina) in Texas A&M University Corpus Christi Genomics Core Laboratory, USA.de novo reference assembly in Rainbow v2.0.2 [SNP detection was implemented by dDocent v2.0 pipeline . At firsw v2.0.2  and CD-Hw v2.0.2 ,58 basedw v2.0.2 ,60 with w v2.0.2 . SAM filw v2.0.2  and outp≤ 10), INDEL loci (this step decomposed insertion and deletion genotypes), Hardy-Weinberg Equilibrium (HWE with p < 0.001), mean quality score (Q > 30), max-missing , and number of variants . Secondary filtering steps included keeping loci based on allelic balance (AB > 0.3), mean mapping quality (0.9 < MQM/MQMR < 1.05), and proportion of alternate alleles (0.05 < PAIRED/PAIREDR < 1.75). Putative SNPs were submitted to rad_haplotyper (https://github.com/chollenbeck/rad_haplotyper) to remove possible paralogs, and one SNP filtering to get the validated SNP panel.SNP calling was performed using Freebayes v0.9.21  with defOur final filtered panel of SNPs was run in BayeScan v2.1  under de2) calculated using the ‘LD’ function in the R package ‘genetics’[LD was measured as the squared pairwise correlation coefficient between loci , effective numbers of alleles (Ne), expected (He) and observed (Ho) heterozygosity, and inbreeding coefficients (GIS) were calculated for each sampled population and over all populations across the Vietnam coastline using GenAlexv6.5 [Numbers of alleles  steps, using the correlated allele frequencies admixture model. The optimal value of K was evaluated using the Evanno method [We tested for population connectivity and structure in the program Structure v2.3.4 ,78 usingo method  by Struco method . A Discro method . This anNe generations in the past [6. After optimization, the maximum mutation-scaled effective population size (θ) prior was set at 0.1 while the maximum mutation-scaled migration (M) prior was set at 20,000. Five hypotheses of migration among populations were tested: (1) symmetric migration rates between all sites (Panmixia Model), (2) non-symmetric migration rates between all sites (Full Model) (3) migration between all sites only from the north to the south (North-South Model), (4) migration between all sites only from the south to the north (South-North Model), (5) migration occurring only between neighboring, north-center sites but no migration between south population (South Separate Model). The most likely model was chosen using the Bezier ln produced by Migrate-N according to Beerli et al. (2009) [Historic gene flow between populations was estimated using the Bayesian inference implemented in MIGRATE-n v3.6.11 . MIGRATEthe past ,83. Samp. (2009) . To eluc. (2009)  of R pac. (2009) . Gene fl. (2009) .Ethics Statement: All crab were collected from fish markets or through normal fishing activities and therefore within the guidelines of approved IACUC procedures, and did not need sampling permission in Vietnam. This study did not involve protected or endangered specieshttp://www.geome-db.org/).Data Archiving: Upon acceptance, the unmodified sequence data in FASTQ format used in this research along with corresponding metadata will be uploaded and archived in the publicly accessible Genomic Observatories Metadatabase . LD network presented one selected outlier cluster (SOC) including 32 loci  . The outlier loci detected by BayeScan were included in the SOC of LD network. In total, 32 loci were removed from the SNP panel and the 306 remaining loci were assumed to be neutral.BayeScan identified thirteen SNPs as outliers  from the panel of 338 putative SNPs used to detect selection footprints  and effective number of alleles (Ne) of the populations were 1.930 and 1.354 respectively. Average observed (Ho) and expected heterozygosity (He) were shown across all populations, ranging from 0.166–0.216 (mean 0.196) to 0.211–0.246 (mean 0.23), respectively. Inbreeding coefficients ranged from 0.154 (Center) to 0.265 (South), with an overall GIS for all individuals at 0.168. % Polymorphic sites were highest in the central (98.22%), and lowest in the southern population (88.46%).Genetic diversity of Table 2) following removal of 16 individuals (S2 Table). Both full and half siblings (22 pairs) occurred abundantly within the southern population, while the other sibling pairs occurred in remaining sampling sites (Table 2).Analyses of genetic relationships between individuals revealed 23 pairs of putative half siblings and 5 pairs of putative full sibling  and with two data set  showed the majority of the variation (80.91–87.5%) in P. pelagicus was found within individuals, and highly significant in all cases . The proportion of variance explained by differences among populations (FST) were larger in the two-populations  than in the three-pops arrangements . It is clear that related individuals contributed to the percentage of variation according to different clustering of populations, however, in all cases the difference were highly significant (P<0.001). With all arrangements and two datasets, among individuals within populations (FIS) differentiation were not significant.AMOVA results  in all arrangements, and data sets (Table 4). In three-population clustering, the southern population showed more differentiation with the northern  than the central . However, connectivity was observed between northern and central populations in all cases .Pairwise K of 2 as chosen by the Evanno method, also showed a clear distinction between the south and the remaining two populations. The similar patterns were observed either with related pairs or related individuals removed from SNPs panels. The southern population was assigned to a first lineage with high certainty  of “green” lineage, and central exhibiting a mixing of “green/red” with percentages of 82.5/17.5 and 80/20 .The STRUCTURE analysis, plotted with a Fig 3B). In the dataset with removed related individuals, the northern and central populations were somewhat separated .The Discriminant analysis of principal component (DAPC) showed a clear distinction between the southern population from northern and central populations in both neutral SNPs data sets (Table 5). The analyses disclosed that there was no mating restriction between crab individuals in the history supposed to be over 1000s of years [P. pelagicus populations range from 0.1 to 1 . Among these, asymmetric directional migration seems to have occurred from southern to northern and central populations, however, bootstrap analysis (nbs<0) showed that directional migration was not significant. Migration from northern to central, however, involved significant asymmetric migration (nbs>0) .Historic migration results strongly supported the Panmixia model based on the highest Bezier approximation score (ln = -115475.9) in which migration was maintained among all sites with random mating between crab individuals . Structure and DAPC analyses clearly divided the populations into two subdivisions . Outlier SNPs, which represented higher genetic differentiation, and respected providing better resolution to detect fine-scale population structure, identified the same patterns as neutral loci, suggested neutral loci themselves may reflected geographical adaptation  along the coast from the Gulf of Tonkin up to the Gulf of Thailand and vice versa. However, all analyses revealed the consistent patterns of non-connectivity from the south to the remaining P. pelagicus populations.The fine scale population structure of swimming crab, applicable in fisheries management was investigated in both putative neutral and outlier loci. Overall, the current analysis based on SNP panels  all showed similar results. The genetic patterns appear to indicate that ptation  [P. pelagicus from the south, while the extensive migration occurring along the northern and central coasts. The migration relative rate (Nm) indicated 10 fold greater migration between northern and central populations than from these sites to the south. What makes this more interesting is that significant asymmetric migration from northern to central populations . Monsoon-induced currents and eddies reported in the central coast [The Vietnamese coastline in the East Sea is influenced by seasonally complex water circulations, which result in upwelling and anticyclonic/cyclonic eddies along the south and central coasts ,4,89. In3/s) ,94 were al coast . These hal coast  and obstal coast ,95, whicTable 2). This heterozygosity deficiency is also recorded in P. pelagicus populations in Malaysia [P. pelagicus in Vietnam, high level of inbreeding and relatedness, and significant genetic differentiation may indicate that local recruitment originates from a limited pool of successful reproductive adults, and reflect somewhat the pressure of overexploitation on crab populations.In term of genetic diversity, the lowest value was detected in the southern population, in concordance with high inbreeding coefficient (0.265) as well as related pairs , or creating an artificial population structure (when included related pairs). However, the two data sets analyses give the same structure, so we can also confirm an accurate reflection of results for a true phenomenon in this species. P. pelagicus can therefore be considered two fisheries and conservation management units. The factors driving current connectivity patterns of P. pelagicus are complex, and cannot accurately be identified. P. pelagicus is likely at risk from inbreeding and subpopulation isolation, and subsequently poor adaptive potential. The management for this species should be careful to ensure that overfishing and habitat degradation do not further affect the vitality of existing populations. Immediate actions such as a seasonal ban on catching crabs in the autumn and late summer to increase successful spawning [This was the first study to apply the powerful technique of over a hundred SNP markers to infer the natural and/or manmade barriers to gene flow in  lobster  and gian lobster , shown u lobster , RADseq-ntiation ,45,47 asntiation ,51. Highntiation , while cntiation , and a lntiation . In casespawning , establispawning , and coaspawning ,33. Morespawning  should bP. pelagicus highlighted the important of conservation genetic studies using advanced genomics for information-lacking geographic zones such as Vietnam East Sea. These results also provide important baseline measures of diversity that can be used for future genetic surveys as well as for monitoring responses of P. pelagicus for environmental changes and temperature rises due to climate change.This study of S1 FigNumber of bootstraps (nbs) were presented along the links.(TIF)Click here for additional data file.S1 TableCarapace width (CW) and weight (W). Abbreviation for sampling locations as shown in (DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Musk deer (Moschidae), whose secretion is an expensive and irreplaceable component of traditional medicine, have become endangered in the wild due to habitat fragmentation and over-exploitation. In recent years, China has had success in the artificial breeding of forest musk deer, thus relieving the pressure on wild populations. However, many farmed populations are experiencing degradation, and little genetic information is available for conservation management. In this study, we selected 274 individuals from three typical captive populations (originated from the Ta-pa Mountains (Tp), the midrange of the Qinling Mountains (Ql) and the Western Sichuan Plateau (WS), respectively) to evaluate the genetic variations. A total of more than 3.15 billion high-quality clean reads and 4.37 million high-quality SNPs were generated by RAD sequencing. Based on the analysis, we found that captive forest musk deer populations exhibit a relatively low level of genetic diversity. Ql displayed a higher level of genetic diversity than the Tp and WS populations. Tp and WS had experienced population bottlenecks in the past as inferred from the values of Tajima’s D. There were high levels of heterozygote deficiency caused by inbreeding within the three populations. Population structure analysis suggested that the three populations have evolved independently, and a moderate amount of genetic differentiation has developed, although there was a low level of gene flow between the Ql and Tp populations. Furthermore, the average quantities of musk secreted by musk deer in the Tp and WS populations were significantly higher than that in the Ql population. The present genetic information should be considered in management plans for the conservation and utilization of musk deer from captive breeding. Moschus berezovskii) (FM Deer), alpine musk deer (Moschus chrysogaster) (AM Deer) and Siberian musk deer (Moschus moschiferus) (SM Deer). Among these, the FM Deer is the main species used in breeding programs. Whereas, the production from captive populations remained constant over the subsequent four decades until the beginning of the present century (There are six musk deer (Moschidae) species in the world . However century . Since tMusk deer breeders have accumulated a rich body of experience in all aspects of breeding management  . SNPs haIn our study, a total of 274 FM Deer samples collected from three farms and used to estimate the level of genetic diversity and population structure using RAD sequencing. The three farmed populations originated from different regions that together nearly cover the areas of origin of the captive FM Deer. In addition, we evaluated whether there were differences in musk secretion among the three populations. The results of this study provide informative data for the conservation and utilization of musk deer, and the results should be helpful for breeders attempting to develop a healthy genetic breeding strategy.Ear tissue samples of 274 FM Deer were obtained from three captive farms from one company, with 59 individuals from the Tp farm located in the southernmost area of Shaanxi, 192 individuals from the Ql farm located in the southwest of Shaanxi and north of the Qinling Mountains, and 23 individuals from the WS farm located in the north of Sichuan province ; Table 1The three farms are independent and do not exchange musk deer with each other. The 274 individuals were all adult males aged 2.5-6.5 years and were selected randomly from productive groups in each farm. FM Deer from the same farm had similar feeding conditions. Samples collection was under the permit from the Forestry Department and conformed to the National Wildlife Conservation Law in China.Meanwhile, 28 AM Deer for which there were records of musk secretion were selected randomly from the above farms, with 6, 6 and 16 individuals from the Tp, Ql and 16 WS farms, respectively . All of DNA was isolated from ear tissue samples using the traditional method of SDS and proteinase K referred to in EcoR I and then heat inactivated at 65°. Various P1 adapters, each with a unique 4-8 bp molecular identifying sequence (MID), were then ligated to designated individuals, which were then pooled in groups and randomly sheared to DNA fragments. Sheared DNA was purified, eluted, and separated using gel electrophoresis, and a DNA fraction corresponding to 300-700 bp was excised and purified. After end repair, purification, and elution, dATP overhangs were added to the DNA fraction. A paired-end P2 adapter containing T overhangs was ligated to the sheared, size-selected, P1-ligated, and pooled DNA template with a specific adapter. The ligated material was then purified, eluted, and subjected to PCR enrichment. RAD products were sequenced using 150 bp pair ends and the data for each individual were extracted according to the specific MID.RAD-seq libraries were constructed using a protocol adapted from All libraries underwent high-throughput sequencing using an Illumina HiSeq 2500 platform at the Gene-denove Bioinformatics Institute .Quality trimming is an essential step to generate high confidence of variant calling. Raw reads would be processed to get high quality clean reads according to three stringent filtering standards:Removing reads with ≥ 10% unidentified nucleotides (N);Removing reads with > 50% bases having phred quality scores of ≤ 20;Removing reads aligned to the barcode adapter.http://catchenlab.life.illinois.edu/stacks/) on the basis of sequence similarity of the first read  , the Burrows-Wheeler Aligner (BWA) was used to align the clean reads from each sample against the RAD tags with the settings ‘mem 4-k 32 -M’, -k is the minimum seed length, and -M is an option used to mark shorter split alignment hits as secondary alignments . Variantπ (the nucleotide diversity), θω (Watterson’s estimator) and FST (population differentiation) using an approach described by IS  and FIT  according to Nei (1983). Deviation from Hardy-Weinberg equilibrium (HWE) was assessed using the PLINK (version 1.9) software  was recorded as “0 g.” The individual’s annual average quantity of musk was used for the comparative analysis of musk secretion among Tp, Ql and WS farms. The comparison didn’t include individuals with an average musk yield of 0 g. Kolmogorov-Smirnov tests were performed to verify the distribution of musk secretion quantity data for each population. When the data showed a normal distribution, one-way ANOVA was used to test for differences in the quantity of musk secretion among the three farms, and the Kruskal-Wallis H test was used when the data deviated from a normal distribution. All of the data were presented as mean ± SD. All of the statistical tests were carried out using SPSS 20.0 software, and the significance level was set to 0.05.https://figshare.com/s/5a9129a307d00c94f61d). Figure S1 and Table S1 contain detailed SNP and filtered SNP statistics. Figure S2 and Table S2 contain detailed InDel statistics. Table S3 contains statistics information of re-divided groups. Supplemental material available at Figshare: https://figshare.com/s/5a9129a307d00c94f61d.All raw sequences of the 274 FM Deer have been deposited in the NCBI Short Read Archive with project accession: SRP161974. Supplementary information have been upload to the figshare  high quality clean reads were generated by quality trim. A total of 188,761,630 stacks were yielded by cluster of clean reads basis of sample-specific MID, in which the average number of stacks per individual was 688,911, with amounts ranging from 148,853 to 1,369,592. The average depth achieved was 5.32 × per individual, varying from 4.196 × to 7.362 ×. In addition, FM Deer’s SNP calling was performed from 274 samples, and 4,371,985 high-quality SNPs were identified, with nearly twice the number of transitions  than transversions  . Insertions and deletions ranged from 1-44 bp and 1-69 bp in length, respectively, of which a substantial proportion  of the InDels were relatively small (1-6 bp), with only 2.14%  greater than 20 bp in length .π and θω, and the values were lower compared with that of the Ql population . Positive values of FIS were also found in the three populations, with values ranging from 0.34 (WS) to 0.56 (Ql) (Table S2).A total of 256,953 SNPs were generated according to the missing rate < 0.2 and MAF ≥ 0.05 (Table S2). The SNPs were then pooled into three populations, with 241,411 from Tp, 255,774 from Ql and 245,889 from WS. Of these SNPs, a high proportion deviated from HWE in each population  than the others (Tp vs. Ql: 0.081 and Tp vs. WS: 0.088), corresponding to the relationship shown above. The PCA revealed a similar result, dividing the individuals into three groups. The WS population was clearly separated from the other populations in the first two PCs, while the Ql population was sandwiched between the other populations and overlapped with the Tp population, suggesting a higher similarity of genetic background between Ql and the others  and WS (8.7%) populations had a higher proportion of individuals that secreted 0 g or unqualified musk compared with the Ql population (5.2%).vs. Ql, P = 0.013; WS vs. Ql, P = 0.047; Tp vs. WS, P = 0.806) (P > 0.05) among these populations (The average quantities of musk for the FM Deer populations were as follows: Tp-F (12.82 ± 4.76 g) > WS-F (12.51 ± 4.79 g) > Ql-F (10.99 ± 4.79 g), and the amount in both Tp and WS populations was significantly higher than in the Ql population (Tp = 0.806) . To furtulations .The musk deer captive breeding industry is thriving in China, and many private farms have been built during the past decade. Nationally, western China has long been the main area for the musk deer breeding industry, especially the Shaanxi and Sichuan Provinces. In our study, three typical captive populations from the two above provinces were selected to evaluate their genetic structure. The present research could provide effective guidance for FM Deer population management.The level of genetic diversity is closely related to a population’s viability, meaning that a population with a higher level of genetic diversity has greater adaptability, conducive to its long-term survival . The popAccording to the filter criteria of P < 0.01). Admittedly, this may have been affected by the genotyping accuracy, but in our research the large sample size could effectively remedy the genotype error caused by the relatively low sequencing depth. Many other factors could have caused the deviation from HWE, including inbreeding, genetic drift, Wahlund effects (presence of population substructure) and natural selection . Additionally, the higher levels of deviation from HWE in the Ql and Tp populations may be explained by Wahlund effects   . In our ST values ranging from 0.05 to 0.15 indicates a medium level of differentiation between populations (ST varied from 0.065 (between WS and Ql populations) to 0.088 (between WS and Tp populations). This suggests that a medium level of differentiation between these populations, and that this was likely to be partly shaped by historical factors and not entirely caused by genetic drift with captive breeding. Geographic isolation was one possible reason for the genetic differentiation among the three populations . The Tp and WS farms were established 30 years earlier than the Ql farm, and the longer period of directional selection may have generated more homozygous genes contributed to musk secretion, resulting in an increase of musk production. Furthermore, differences in nutrition levels, especially in crude protein, also affect the quantity of musk secretion . We found that the P group  had the lowest level of genetic diversity. Furthermore, the values of Tajima’s D in the L group  and the P group were significantly higher than those in other groups, which may be associated with the accumulation of low-frequency detrimental mutations in these groups. A population with lower genetic diversity and a higher detrimental mutation tends to be more susceptible to pathogens, and the resulting disorders may be the best explanation for the phenomenon  Inbreeding should be avoided to some extent, and individuals displaying traits of inbreeding depression should be weeded out immediately. The kinship of all individuals (data unreleased) identified in our studies could be useful for avoiding inbreeding. (2) The three populations should be treated as distinct units. Genetic evaluation is necessary before introduction, and we advise against optional introductions between these regional populations. (3) In practice, nutrition levels, especially for crude protein, should be supplemented in the farms. Managers should balance the genetic diversity and musk production, and establishing a preserved population and a breeding population may be a feasible way to relieve the conflict.In summary, this research has provided the first description of genetic variability and differentiation in three forest musk deer populations that originated from different geographic areas. Our results reflect the genetic status of the musk deer industry in China. There was a relatively low level of diversity in captive forest musk deer and a medium level of genetic differentiation had developed between captive populations. Populations in the Qinling Mountains could be used as a valuable gene pool in genetic improvement. These results provide insight into genetic improvement and conservation for the musk deer.


LCT and the HLA, two well-known targets of selection, at which 40‒50% of the Canarian genome is of recent African descent according to our estimates. Putative selective signals were also identified in these regions near the SLC6A11-SLC6A1, KCNMB2, and PCDH20-PCDH9 genes. Taken together, our findings provide solid evidence of a significant recent African admixture, population isolation, and adaptation in this part of Europe, with the favoring of African alleles in some chromosome regions. These findings may have medical implications for populations of recent African ancestry.Despite the genetic resemblance of Canary Islanders to other southern European populations, their geographical isolation and the historical admixture of aborigines (from North Africa) with sub-Saharan Africans and Europeans have shaped a distinctive genetic makeup that likely affects disease susceptibility and health disparities. Based on single nucleotide polymorphism array data and whole genome sequencing (30×), we inferred that the last African admixture took place ∼14 generations ago and estimated that up to 34% of the Canary Islander genome is of recent African descent. The length of regions in homozygosis and the ancestry-related mosaic organization of the Canary Islander genome support the view that isolation has been strongest on the two smallest islands. Furthermore, several genomic regions showed significant and large deviations in African or European ancestry and were significantly enriched in genes involved in prevalent diseases in this community, such as diabetes, asthma, and allergy. The most prominent of these regions were located near  The Canarian Archipelago consists of seven main islands located in the Atlantic Ocean ∼100 km from the northwest African coast. By no later than 2,500 years B.P.  and untiAlu insertions have shown that this three-way admixture model encompasses a prominent EUR ancestry (62–78%) and as much as 20–38% NAF and 3–10% SSA ancestries  of samples from all seven islands. We also assessed these data to evidence loci showing large deviations in ancestry with respect to the average of the genome, putative targets of selection and disease associations. In addition, because the population offers a uniquely challenging admixture scenario , this study secondarily evaluated the performance of two of the fastest and most accurate methods of local ancestry estimation for multiway admixtures .r2 threshold of 0.5) from 416 individuals  were used for the analyses. The leading principal components (PCs)  from the principal component analysis (PCA) including reference populations evidenced the intermediate position of Canary Islanders between the NAF and EUR populations (separated by PC2) and their more distant relationships with SSA, separated by PC1 , a total of 100,175 SNPs (d by PC1 . In addiP < 3.1 ×10−69). Using ALDER, we also estimated the number of generations since the last admixture event. The results were consistent across comparisons and indicated that the admixture event took place ∼13.6 ± 0.7 generations ago (429–495 years BP), which is within the timescale of the historical conquest of the archipelago in the XVth century.We next examined whether Canary Islanders as a whole can be considered an admixed population based on formal tests. To do so, for each pair of reference populations considered as surrogates for the true ancestral populations, we used ALDER to calculate a weighted two-locus admixture LD statistic based on the decay  online aK varying from 2 to 7 . This NAF-related ancestry was also evident in Canary Islanders. Other ancestry clusters emerged for K > 4, which generally reflected both further ancestry sharing or unresolved clustering in NAF and Canary Islanders and additional ancestry subdivisions among NAF populations compatible with the mixed pattern of ancestry components evidenced elsewhere    online. K = 4, the overall ancestry proportions among Canary Islanders were, on an average, 22% NAF and 3% SSA (r2 threshold set at 0.5). However, in this study, which comprised a larger and more diverse sample of Canary Islanders, we found a wider NAF-related ancestry interval of 14.9–29.9%, and a slightly higher SSA-related ancestry of as much as 9.2% . Similarly, balancing the number of individuals from the reference populations (n = 75 for each) resulted in similar ancestry estimates (averages of 26% NAF and 1% SSA). Overall, these results demonstrate that the Canary Islanders are closely related to EUR but show substantial influences from NAF and distant relatedness with SSA.Based on this evidence, we interpreted the EUR-, NAF-, and SSA-related fractions indicated by the ADMIXTURE analysis as the admixture proportions in Canary Islanders. For d 3% SSA . These ed 3% SSA  despite  as 9.2% . The useP = 0.160), although the SSA-related ancestry differed significantly between the eastern and western islands . However, the largest differences were observed between the islands that were conquered first and more peacefully by the Spanish  and those in which the conquest took longer  . This observation agrees with findings based on mitochondrial DNA (mtDNA) lineages that suggest increased African affinities on the former islands  and western islands   are common to all world populations, are longer than expected, and have profiles that can indicate distinctive demographic histories of the population and of inbreeding . Here, wP < 1.0×10−3 for all pairwise comparisons) except La Palma. Interestingly, La Gomera and El Hierro showed average total ROH lengths of 114 and 134 Mb. The conclusions remained unchanged when considering only ROHs >1.6 Mb for the average total ROHs estimates , whereas the differences disappeared when using the average total of ROHs ≤1.6 Mb (lowest P = 4.5×10−3). Given that variant ascertainment of the array is less of a problem for ROHs >1Mb (P = 6.7×10−3).An exploration of average total ROHs by island again indicated El Hierro and La Gomera as population outliers. The genomes from these two islands showed the largest number of fragments in ROHs and the longest average total ROH length , the latOHs >1Mb , we inteP < 5.0×10−10 in all cases) . Many local ancestry methods perform well in two-way admixture scenarios with large genetic differences between the ancestral populations . However, the difficulty increases with the number of ancestral populations , particularly if a small to moderate degree of differentiation exists between some of the ancestral populations . Given that this study constitutes the first time that the patterns of ancestry blocks in Canary Islander genomes have been assessed, we first evaluated two of the fastest and most accurate methods of local ancestry estimation for multiway admixtures, ELAI and LAMP-LD, both of which have been fruitfully utilized with Hispanic populations . We compl cases) . Howeverl cases) . A leastl cases) , ELAI prP < 3.0×10−8). Based on these block length estimates and the ADMIXTURE proportions calculated for all Canary Islanders, we would expect averages of ∼181 EUR-, 82 NAF-, and 13 SSA-related blocks per haploid genome , which are lower than but within the range of the estimates previously suggested for African–Americans  and chr6 . The two peaks of NAF-related ancestry enrichment were located on chr2  and chr6 . Peaks associated with SSA-related ancestry were situated on chr3 , chr6 , and chr13 . These regions span several Mbs (range of 1.2–12.3 Mb), meaning that, while the observed ancestry deviations may indicate adaptive processes, any adaptation may be unrelated to the gene harboring the lead SNP. However, it is likely that the LCT and HLA loci caused the peaks on chr2 and chr6. One regulatory variant of LCT is the major determinant for lactase persistence in EUR populations and underlies the strongest signal of selection in the human genome identified to date  in a subset of individuals for which WGS data was available. Deviations from neutrality at three locations were revealed by at least one test for the three loci, with frequent support for more than one hit obtained for some regions after accounting for the number of comparisons . These three locations were as follows: in the intergenic region between the SLC6A11 and SLC6A1 genes on chr3 , near KCNMB2 on the same chromosome , and in the intergenic region between PCDH20 and PCDH9 on chr13   . Interesarisons) , bone trarisons) , and astarisons)  reside iarisons) . This obarisons) .Table 3LCT variants have been linked with lactase persistence in other populations, we then explored whether any of those variants existed in this population and could offer a potential explanation for this peak. By accessing the WGS data available for a subset of 14 subjects, we found that the only detected variant position associated with lactase persistence was rs4988235 , which is the major determinant for lactase persistence in Europe. The frequency of this lactase persistence allele in Canary Islanders  is intermediate between the frequency reported in other central and northern EUR populations (60–80%) and that in NAF populations (24%) (http://www.ucl.ac.uk/mace-lab/resources/glad). Therefore, we suggest that the prevalence of lactase nonpersistence alleles in Canary Islanders and NAF populations likely explains the chr2 peak in Canary Islanders. In fact, a recent ancient DNA study of pre-Hispanic human teeth from a small sample of five Guanche people from Tenerife and Gran Canaria also suggested that the dominant phenotype was lactose intolerance   . Regulation of inflammatory response, the complement pathway, telomere maintenance, and antigen processing and presentation were among the pathways significantly enriched  but ranked lower in the results , the largest so far identified in southwestern EUR populations . FurtherDespite the profound impact of the European immigration starting in the XVth century, our results indicate that significant genetic footprints of African ancestry persist in the current inhabitants of the Canary Islands. Our data also imply that gene flow between the island populations must have been high to maintain the relative homogeneity observed for the African ancestries, despite the significant discontinuity favoring African affinities in the populations of El Hierro, La Gomera, Fuerteventura, and Lanzarote. Strikingly, such a pattern was also revealed in mtDNA studies of independent samples , suggestLCT, paralleling this scenario, where genetic affinities with African populations remain regionally preserved today. Independent studies and our own data support that putative selective processes are the cause of such autosomal affinities. However, given that the bulk of human adaptation occurs via selection on standing variation  (HLA-C)   and HIV- (HLA-C) , and the (HLA-C) , asthma  (HLA-C) .To the best of our knowledge, this is the first study to evaluate the proportion of the genome in ROHs in this population. Our results support distinct degrees of isolation and consanguinity between the seven islands, which are most extreme in the two smallest islands, La Gomera and El Hierro. Because of isolation, the inhabitants of these islands would be enriched in low-frequency functional variants  that canLorenzo et al. 2006It is important to declare some limitations of our study. First, scarce genomic data are available for NAF populations in public databases . As a coIn conclusion, here we have provided a genetic dissection of population ancestries and isolation in Canary Islanders at an unprecedented level. We estimate that up to 34% of Canarian genomes are of recent African descent and that the geographical distribution of ancestries still reflects historical events. Local ancestry estimates enabled the identification of Mb-size chromosome regions with higher-than-expected African affinities, most likely involving putative adaptive signals. Our results suggest that these observations may have implications for the major health disparities affecting the population. In addition, genetic testing and genetic mapping studies of diseases in Canary Islanders should take local ancestry into account. Finally, because the adaptive signals were previously described in populations from Africa and America, our conclusions could also have repercussions for the identification of disease loci in other recently admixed populations.P < 1.0×10−6) were excluded, leaving a total of 516,348 SNPs. To maximize SNP density in the downstream analyses, we relied on the 1KGP Phase 3 data to extract the data sets serving as EUR and SSA sources (n = 289) as well as Yoruba Nigerian (YRI) people (n = 108) as the representatives for EUR and SSA sources, respectively. To perform sensitivity analyses of the results, random subsamples of 75 individuals from each set of EUR, NAF, and SSA samples were alternatively used as well as other data sets from 1KGP, namely the Gambian Mandenka  and Sierra Leone Mende  data sets. The NAF representative grouping (n = 125) was gathered from samples with origins in North and South Morocco, Occidental Sahara, Algeria, Tunisia, Egypt, and Libya that had previously been genotyped with the Genome-Wide Human SNP Array 6.0 (Affymetrix) (P < 1×10−6 in at least one population. The intersection of data sets and postfiltering (of SNPs located in mtDNA or the sex chromosomes) left a total of 114,567 SNPs for downstream analyses .The sample of Canary Islanders consisted of 429 unrelated subjects who self-declared as having two generations of ancestors born on the same island of the Archipelago. Samples were selected from a large cohort study entitled “CDC of the Canary Islands” , which i sources . Accordi sources , the pre sources . This inymetrix) . Using PPrincipal Component Analysis (PCA) was performed using PLINK v1.9 . ALDER vK) and used a 10-times cross-validation with random seeds to estimate the best predictive K. A subsample of 100,175 SNPs from EUR, NAF, SSA, and Canary Islanders was used for this assessment. This subset resulted from excluding with PLINK those SNPs in LD  or located in regions of long-range LD according to hg19 positions . The ADMIXTURE results were also assessed for the effects of downsampling the number of samples from the reference populations and the use of alternative SSA surrogates other than YRI (GWD and MSL). To provide further support to the ADMIXTURE results, we assessed the goodness of fit of the ADMIXTURE model to the underlying genomic data based on the patterns of haplotype sharing between individuals using badMIXTURE v0.0.0.9000 (https://github.com/danjlawson/badMIXTURE), whose residuals provide information on the ancestral relationships between the population groups. Statistical differences in ADMIXTURE ancestry estimates between islands and regions were assessed by Wilcoxon test.We used ADMIXTURE v1.22 , which uP < 2.4×10−3 considered significant).To further explore the population history and isolation of Canary Islanders, ROHs were calculated with PLINK based on a previously described sliding window approach  considerLocal ancestry blocks across autosomes were inferred by using LAMP-LD v1.0  and ELAIZ-score statistic at each SNP position as a measure of the deviation in the local ancestries with respect to the average ancestry in the genome. This scan was conducted for the three ancestries separately, and an excess of locus-specific ancestry in a segment was considered significant if a large deviation was detected . In those regions showing large ancestry deviations, genetic diversity was evaluated on the basis of the mean SNP heterozygosity estimates and the mean number of subjects with SNPs from those regions that were contained in ROH stretches. The statistical significance of the differences in these regions compared with data from the whole genome was assessed by Wilcoxon rank sum tests. Enrichment analyses were conducted for all regions with ancestry deviations together based on the peak regions defined by a Z score > 3 on hg19 using the Genomic Regions Enrichment of Annotations Tool (GREAT) .We used the method proposed by Zhu  to estim (GREAT) . A hyperLCT on chr2 and the HLA genes on chr6), we accessed data from deep WGS from a subset of 14 individuals (two per island) to further explore if the other regions showing deviations also harbored putative signals of selection. Briefly, DNA samples were processed with a Nextera DNA Prep kit with dual indexes following the manufacturer’s recommendations . Library sizes were checked on a TapeStation 4200 . The concentration of each library was determined by a Qubit dsDNA HS Assay . As a control, a PhiX DNA sample (1%) was also sequenced with the samples. Samples were sequenced to an average depth of 36.5× (range 24–45×) with paired-end 150-base reads on a HiSeq 4000 instrument (Illumina). Reads were preprocessed with bcl2fastq v2.18 and aligned to hg19 with BWA-MEM 0.7.15-r1140 (http://broadinstitute.github.io/picard). Variant calls were obtained with HaplotypeCaller in GATK (v3.7) (http://teidehpc.iter.es/en). Sequencing data are available in http://www.iter.es/wp-content/uploads/2018/09/chr3_data_from_Canary_Islanders_MBE-Guillen-Guio-et-al.2018.vcf_.tar.gz for chromosome 3 and in http://www.iter.es/wp-content/uploads/2018/09/chr13_data_from_Canary_Islanders_MBE-Guillen-Guio-et-al.2018.vcf_.tar.gz for chromosome 13.Given that deviations of local ancestry often contain well-known targets of selection   followinLCT that are involved in lactase persistence as reported previously in the literature: rs4988235, rs41525747, rs41380347, and rs145946881 . Statistical significance was calculated using Hudson’s “sample_stats” software (http://home.uchicago.edu/rhudson1/source/mksamples.html).WGS data from Canary Islanders were used to extract the genotypes for the four described SNPs located upstream of 45946881 . Further and PBS  in 10-kbFor the simulations, we used msHOT software . As para./msHOT 28 5000 -t tbs -r tbs 10000 -I 4 0 0 0 28 -n 1 1.68202 -n 2 3.73683 -n 3 7.29205 -n 4 3.73683 -g 2 116.010723 -g 3 160.246047 -ma x 0.881098 0.561966 0 0.881098 x 2.79746 10 0.561966 2.79746 x 0 0 1 0 x -es 0.0005 4 0.1 -ej 0.0005 5 2 -en 0.0005 4 0.068 -ej 0.0135 4 1 -ej 0.028985 3 2 -en 0.028985 2 0.287184 -eM 0.028985 28 -ej 0.197963 2 1 -en 0.303501 1 1 < random_thetas-rhos.txt > output_file.mshttps://genome-euro.ucsc.edu). Uncertainty in the estimates of theta and rho was considered by generating a random and normally distributed set of values for these parameters, with mean equal to the estimated value and variance equal to the mean, from which we sampled a pair of values in each simulation.The initial values of theta were inferred from the comparison of human and chimp orthologous sequences for each 10-kb region using a mutation rate based on the average number of substitutions per site (with the Jukes and Cantor correction) as assessed by DnaSP v5.1 . The rho−8, and a range of background false negative rates (FNRs) of 0.0–0.3 for variant discovery. Considering the broad range of FNRs assumed, this analysis yielded a uniform Ne in the range of 470–560 for the Guanche population.To estimate the Ne of Canary Island aborigines in pre-European times, we accessed the publicly available Guanche genome  with the largest depth-of-coverage (3.9×) from Molecular Biology and Evolution online.Supplementary DataClick here for additional data file.


Rattus tanezumi is a common commensal rat and an important host animal of bubonic plague in South China and Southeast Asia. The northward dispersal of this species in mainland China has been reported in recent decades, along with more recent intercontinental expansion. Population genetics of R. tanezumi in mainland China were studied to explain the relationship between dispersal history and the ancient and modern transportation networks of China.R. tanezumi populations from Yunnan have highest genetic diversity and populations from Tibet with lowest genetic diversity. 18 populations can be divided into four clusters, the first cluster including populations from southwest Yunnan, the second including two populations of Tibet, the third for populations from middle and east of mainland China, and the forth for two populations from north Yunnan. Both microsatellite and mtDNA data reveal that the populations from coastal areas are closely related to populations from Yunnan, whereas populations from Tibet are closely related with populations from Sichuan.In total, 502 individuals belonging to 18 populations were collected from 13 provincial areas. Nine microsatellite loci and two mtDNA sequences were analyzed. The results indicate that R. tanezumi in mainland China depended on shipping transportation, with subsequent expansion from coastal areas into Central China occurring along the Yangzi River. Further, the linkages between populations in Tibet and Sichuan point to a modern era introduction via the Chuan-Zang highway, rather than along the Tea Horse Ancient Road.The results suggest that early dispersal of The online version of this article (10.1186/s12863-019-0714-3) contains supplementary material, which is available to authorized users. Rattus tanezumi  (Rodentia: Muridae), is a common commensal rat in East and Southeast Asia. From South China to Southeast Asia, R. tanezumi can be found in indoor and outdoor habitats. In outdoor habitats, it is the dominant pest rodent in rice fields, causing crop damage before harvest [R. tanezumi will migrate to residential areas and even indoor areas for food [R. tanezumi can carry a number of different pathogens including zoonotic agents of public health concern. Thus, R. tanezumi is regarded not only as a pest related to agriculture but also as a host of several zoonoses, including plague (Yersinia pestis), haemorrhagic fever with renal syndrome (Hantaan virus), and leptospirosis (Leptospira) [The Asian house rat,  harvest . After tfor food , causingtospira) –7.R. tanezumi was not noted until recently after Huang et al. reported the northern border of the distribution area of R. tanezumi in China as the Yellow River [R. tanezumi was found in provinces north of the Yellow River, including Hebei [R. tanezumi northeast of Qinghai, a northwestern province of China [R. tanezumi in north China was easily recognized because this species can be distinguished from another commensal rat, R. norvegicus, with morphological characters. However, the expansion of R. tanezumi out of Asia was not recognized until genetic methods were used in congeneric species identification [R. rattus species group. With molecular genetic methods, Bastos et al reported the presence of R. tanezumi in 13 locations in South Africa [R. tanezumi were also found in California in the United States and in Madagascar [R. tanezumi [The range expansion of ow River . From thng Hebei  and Shanng Hebei , and in of China . The colfication  because dagascar . Based otanezumi .R. rattus in the western Indian Ocean is congruent with human colonization events in this area [R. exulans is also a good example and has been used to imply the history of human colonization on Pacific islands [Rattus norvegicus was not reported at Xinjiang before the 1970s because of the Gobi Desert between Xinjiang and Central China. In the 1970s, colonies of R. norvegicus were first found in railway stations of Xinjiang, after the railway was built [R. norvegicus spread to other cities of Xinjiang with railway and highway traffic. Because the dispersion of R. tanezumi was reported only in recent decades, the dispersal routes of this species and how these relate to human activity is not known.The extension of the geographic range of commensal rats is considered a result of human activity. The phylogeography of his area . The dis islands , 18. RatR. tanezumi on mainland China, especially from Yunnan to Tibet, could be explained by population genetics analysis of rats.In ancient China, because of the complex landscape of Southwest China, commercial transportation in this area  usually depended on caravans. Starting approximately 1200 years ago (Tang Dynasty), a famous caravan road system for tea, salt and horse trading developed in Southwest China, which is now called the Tea Horse Ancient Road. This road system linked Sichuan, Yunnan and Tibet  in 13 provinces covering almost the entire range of R. tanezumi in China .Samples belonging to 18 populations were collected at 22 locations (2+ Plus) , 2.5 mM of each dNTP , 10 pmol of each primer, and 0.75 U of LA Taq polymerase . The 5′ end of the forward primer was labelled with a fluorescent dye . Cycling conditions were as follows: 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 30 s at the annealing temperature (TA), and 30 s at 72 °C, and then 5 min at 72 °C. The length of the DNA fragments was analyzed manually in GeneMapper v. 4.0 (Applied Biosystems).All samples were genotyped for 9 microsatellite loci developed for this research , number of effective alleles (Ne), Shannon’s information index (I), number of private and locally common alleles, allele richness (Rs), observed heterozygosity (Ho), unbiased expected heterozygosity (He) and positive inbreeding coefficient (FIS) using Fstat V2.9.3.3 (https://www2.unil.ch/popgen/softwares/fstat.htm) and GenAlEx 6.41 [t-Test was used to determine whether two sets of data were significantly different from each other in SPSS 16.0 [The genetic diversity of every locus was characterized by estimating number of different alleles (lEx 6.41 . StudentPSS 16.0 . Fstat wPSS 16.0  was usedFST/(1-FST) on the natural logarithm (Ln) of straight-line geographic distance. Two approaches were used for testing genetic bottlenecks at the microsatellite loci for each population, we calculated M, a ratio based on the number of alleles to the allelic size range [Isolation by distance (IBD) was estimated with Mantel’s test in Web Service of IBD , using tze range  in Arleqze range , and hetze range  to assesze range , Stepwisze range , and Twoze range , which wze range .R. tanezumi, two Bayesian model-based cluster analyses were contrasted: Structure [Κ) in the dataset without prior information of the sampling locations. A model in which allele frequencies correlated within populations was assumed . The software was run with the option of admixture, allowing for some mixed ancestry within individuals, and α was allowed to vary. We used 20 independent runs for each value of Κ (K = 1 to 10), with a burn-in period of 50,000 iterations and 250,000 replications. The method of Evanno et al. [K, based on the second-order rate of change of the likelihood function between successive values of K. The results of 20 replicate runs for each value of K were combined using the Greedy algorithm of Clumpp 1.1.1 [K were displayed graphically using Distruct v1.1 [F-statistics [FST for statistical significance was also tested in Fstat to evaluate genetic differentiation between populations. The number of putative migrants (Nm) per generation between populations was estimated from pairwise FST, Nm ≈ (1-FST)/4FST [To identify the population genetic structure of tructure  and Genetructure . Structutructure  was firso et al.  was usedpp 1.1.1 , and sumuct v1.1 . Differeatistics  computedatistics . PairwisST)/4FST . CriticaST)/4FST .The second method relied on the spatial cluster model implemented in the Geneland package , 41. FolThe Neighbour-Joining (NJ) tree was computed with Powermarker 3.25 , based oIn total 502 individuals were used to explore sequence polymorphism in the mitochondrial COI gene and D-loop region. The mitochondrial COI gene was used not only in population genetics analysis bust also as DNA barcoding at first in specimens identification. DNA samples were used as templates to amplify a 702-bp fragment of COI and a 546-bp fragment of D-loop according to Robins et al. . PCR proS), the number of haplotypes per sample (Hp), haplotype diversity (HpD), the average number of nucleotide differences (K) and nucleotide diversity (π) were computed with DnaSP v5 [Sequences were aligned using Muscle  within MDnaSP v5 . NeutralR. tanezumi in Genbank [Rattus rattus , R. pyctoris(JF499340), R. argentiventer(HM217484), R. exulans (KC617851), R. andamanensis (KY605368), R. norvegicus , R. nitidus  and Mus musculus  were added into dataset, and Cricetulus migratorius  was set as outgroup in the construction of ML and BI trees.The phylogenetic relationships of mitochondrial sequences and haplotypes were performed using Bayesian inference (BI), maximum likelihood (ML) and median-joining network (MJ) methods as implemented in Mrbayes 3.2.5 , Raxmlgu Genbank –55 (AddiNa) as 21 (Rt1), 26 (Rt2), 27 (Rt3), 33 (Rt4), 16 (Rt5), 19 (Rt6), 26 (Rt7), 30 (Rt8) and 24 (Rt9)  was almost twice the minimum Rs in the LHS population from Tibet (6.14). The observed heterozygosity (Ho) for each locus ranged from 0.28 (Rt4) to 1.00 (Rt9); the minimum Ho was in LHS (0.58) and the maximum Ho in CHQ (0.82) (Table He) for each locus ranged from 0.37 (Rt1) to 0.94 (Rt4); the minimum He was in LHS (0.67) and the maximum He in RUL (0.91) . In general, populations in Yunnan showed a higher genetic diversity than other regions, while the genetic variability of populations in the Tibetan areas and SJZ was low.A total of 502 individual rats collected in this study from 18 populations were successfully genotyped at all 9 microsatellite loci. All loci were polymorphic, showing the number of alleles (2), 27 Rt, 33 , reflecting heterozygosity deficits. The populations in Yunnan (Southwest China) had the highest number of loci in departure from HWE (21 out of 28), LOY sample had 3 loci in departure from HWE, WZS sample had 2 loci in departure from HWE, SHH and LEZ populations had 1 locus in departure from HWE respectively  comparisons did not conform to Hardy-Weinberg expectations after sequential Bonferroni correction, and the deviations were associated with a positive inbreeding coefficient  were at linkage disequilibrium after sequential Bonferroni correction, and all three such pairs were found in SHH and LOY populations . No pair of loci appeared in LD in more than one population, suggesting genetic independence between loci.M ratio was low and indicated historical bottleneck in all populations. Significant deviations from mutation-drift equilibrium conditions under the IAM model were found in all populations except the SJZ, CHS, and LHS populations. In contrast, the test for each population gave non-significant results under the SMM model. According to the TPM model, the RUL population showed heterozygote excess in both percentages of stepwise mutations, and the results for the LOY and QUZ populations suggested a heterozygosity excess only in 80% of stepwise mutations, which indicated that bottlenecks would have occurred recently under pressure of rodent control in these populations.Tests for genetic bottlenecks yielded mixed results Table . The M rK = 3). Most individuals of LIH, XGL, QNJ, BAS and RUL populations belonged to Cluster I; almost all individuals of WZS, QUZ, SHH, CHQ, XIS, CHS, SJZ, XIY, and SIC populations were of Cluster II; and individuals of NYC and LHS were members of Cluster III. While populations of LOY, LEZ were composed with individuals of clusters I and II (Table The significant deviations from HWE with heterozygote deficiency and the presence of linkage disequilibrium suggest the presence of population subdivision within the samples (the Wahlund effect). Bayesian analysis divided all individuals into three clusters (posterior probability of Bayesian clustering Ln (D) likelihood score optical for II Table a, which FST across all loci was 0.064 and was highly significant (P < 0.001). We then tested the genetic heterogeneity among populations within and between the clusters. We chose populations in which more than 70% of individuals were assigned to cluster I or II for analysis. Cluster I included 5 populations , while cluster II included 9 populations . Within each cluster, pairwise FST was moderate: 0.020 (CHQ-CHS) to 0.077 SHH-SIC) in cluster I and 0.022 (LIH-RUL) to 0.048 (LIH-XGL) in cluster III. Among the clusters, a higher level of differentiation was demonstrated, with FST from 0.051 (QUZ-QNJ) to 0.168 (LIH-LHS). Nm estimates among populations were higher (2.980 to 12.005) within clusters and lower (1.24 to 4.49) among clusters . The run with the highest average posterior density was selected . Cluster 1 included three populations from Southwest of Yunnan , and cluster 4 included two populations from North of Yunnan (XGL and QNL). Cluster 2 was constituted by populations from Tibet (LHS and NYC). And cluster 3 was composed of other populations from south, east and middle of China , while for cluster I and II, the correlations were stronger . The correlations for cluster III were not significant . These results suggest that geographic distance weakly contributes to the genetic differentiation in R. tanezumi populations.Tests of isolation by distance were performed for each population cluster and for all the populations together. For all populations, weak correlations were detected between genetic and geographic distances , while the lowest indices occurred in the WZS population from Hainan . The SHH and LHS populations from Shanghai and Tibet also showed lower genetic diversity. The most frequent haplotype, H1 (48.1%), was detected in all geographic populations except LIH and RUL. For the D-loop region, the polymorphism was much higher than in COI. There were 60 segregating sites defining 70 haplotypes. The highest nucleotide diversity indices were similarly among the QNJ population in Yunnan , while the lowest indices occurred in the XIS population from Hubei   but not statistically significant, while Fu’s Fs statistic was significantly negative .Nucleotide sequences of mtDNA COI (702 bp) and D-loop 546 bp) were obtained for 502 6 bp wereThe Median-Joining Networks show a significant geographical component to variation. For COI, the network shows three distinct haplogroups. Haplotypes from non-Yunnan regions form a star-like pattern centred at H1, and haplotypes from southwest and northeast regions of Yunnan form two star-like patterns centred around H10 and H17, respectively. Furthermore, H4 (only found in LEZ) and H24 (found in both QNJ and LOY) are most closely related to centre H17  compared with other populations, with the two populations from Tibet (NYC and LHS) having the lowest values  have a low number of alleles and effective alleles Additional files 2:Table S2. List of haplotypes used in the phylogram of Fig. Additional files 3:Table S3. Genetic variability at nine microsatellite loci in R. tanezumi populations from China. (XLSX 17 kb)Additional files 4:Table S4. Pairwise genetic distance (FST) and gene flow (Nm) for 15 populations based on microsatellite data. (XLSX 11 kb)Additional files 5:Table S5. The haplotype networks of COI and Dloop sequences. (XLSX 64 kb)Additional files 6:Figure S1. STR data. (TIF 6056 kb)