Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori from Europe and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks by migration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck. Helicobacter pylori is a major human pathogen whose population structure is similar to that of its host. Here, the authors show that H. pylori has repeatedly spread out of Africa recently, replacing deleterious variants that accumulated during the original out of Africa migrations more than 50,000 years ago. H. pylori causes chronic, decades-long infections and is often acquired within the household, limiting the rate of its diffusion through human populations in comparison with more readily transmissible pathogens2. Genetic variation in H. pylori genome sequences shows a phylogeographic pattern similar to that of its host, consistent with an inference that human and bacterial genes are often spread by the same migrations6. However, the H. pylori population found in Europe and other parts of Eurasia is admixed, with many strains having more than half of their DNA attributable to populations closely related to those prevalent in Africa9. There is evidence for several recent human migrations out of Africa10, but together they have only contributed a small fraction of the ancestry of non-Africans. This discrepancy in ancestry proportions between the bacteria and their hosts implies that African H. pylori has been spread to Eurasia by movements of people that have left weaker signals in human DNA.HH. pylori have contributed extensive ancestry within parts of Eurasia, we assemble a collection of strains from Europe and the Middle East, from putative source populations in Africa, as well as from less-admixed strains in Asia. We infer a recent demographic history of the European and Middle Eastern strains that includes genetic drift, migration, and admixture from external sources. We show that there have been at least three admixture events from African source populations that have each contributed substantial ancestry. By examining the distribution of non-synonymous mutations in different populations, we conclude that there was a large and lasting increase in the frequency of segregating deleterious mutations during the out-of-Africa bottleneck associated with the initial spread of modern humans from Africa. When African H. pylori strains reached Eurasia due to later contact between humans, they, and the DNA they carried, had a fitness advantage and were able to spread. In the process, they reduced the mutational load in the newly admixed populations.To understand why African Based on fineSTRUCTURE clustering , implying that hpAsia2 is not a close surrogate for the pre-admixture population.The chromosome painting analysis supported previous findings that there is a North-South cline in the overall proportion of African ancestry and that hpAsia2 is a closer relative of the pre-admixture population than hspEAsia11, but are specific to individual ancestry components, rather than averaged across the entire genome.To investigate the demographic history of admixture further, we measured genetic drift profiles separately for each ancestry component Fig. . SpecifiGenetic drift profiles for the hpAsia2 and hspEAsia ancestry components showed a similar pattern across the four hpEurope subpopulations, as indicated by a near identical pattern of colors for these two ancestry components in Fig. 12 . However, although the out-of-Africa bottlenecks have had measurable impact on the pattern of segregating mutations within human populations18, there is less evidence that individuals from non-African populations have accumulated a larger burden of non-synonymous mutations when measured relative to an outgroup19. H. pylori shows much higher rates of genetic differentiation between geographical regions4 than its host13, which most likely reflects transmission bottlenecks during spread from person to person as well as the expansion of fit clones. Consequently, demographic bottlenecks that have had modest fitness consequences for humans can potentially have more substantial effects on the bacteria they carry.Population genetic theoryH. acinonychis outgroup  than would be predicted if they were random mixtures of African and Asian genomes with the proportions estimated by chromosome painting in Fig. We also observed a reduction of the mutational load by admixture and selection within hpEurope subpopulations. Bacteria from hpEurope subpopulations had dN/dS values with the H. acinonychis . We then investigated whether there was variation in overall Asian ancestry proportion (hpAsia2 + hspEAsia in the chromosome painting) associated with this score. Most sites were non-polymorphic and had a mutation score of 0.0, which was therefore used as a baseline and compared independently to positive and negative scoring sites. To allow for correlations between adjacent sites, statistical significance of the regression was assessed using a gene-by-gene jackknife (Methods).More specific evidence that admixture has reduced the burden of deleterious non-synonymous mutations was provided by tabulating the effect of mutations that accumulated in African and Asian populations on the ancestry of admixed bacteria in Europe and the Middle East Fig. . For eacH. pylori shows little evidence of codon usage bias21, so most synonymous mutations should be approximately neutral and can therefore be used as a control. A negative mutation score at non-synonymous sites was associated with a deficit in Asian ancestry, either in comparison with non-polymorphic sites or with synonymous sites with the same scores. The strongest regression slopes were estimated for the two Southern populations, and the weakest for hspNEurope , cell envelope biogenesis , and translation . However, the analysis did reveal overlap of low-admixture genes with genes that have highly differentiated SNPs within East Asia22, specifically HP0284 (mscS-1), encoding a mechanosensitive channel related to osmotolerance and HP0250 (oppD), encoding an oligopeptide permease. Since the same genes are often differentiated in different continents22, this overlap suggests that these genes may have been resistant to admixture due to local adaptation within Europe and the Middle East.Genes with extreme values of average ancestry proportion were involved in diverse, often central, cellular processes. For example, low African ancestry . Overall, this inspection suggested that the driving force for admixture within the core genome was increased fitness, provided by replacement of stochastic deleterious mutations genome-wide, rather than single pathogenesis-related genes allowing adaptation to new host environments.High-average African ancestry genes (>69%) likewise included those in central biological processes and stress tolerance, such as those of the Czc cation efflux system , purine salvage , and glycolysis . Most were not directly related to host interactions, although some with low average African ancestry might affect urease activity  left traces in the H. pylori of different origins can mix together and recombine within extended families31. Over time, extensive ongoing contacts between populations within Europe would be expected to homogenize H. pylori ancestry proportions both within and between locations. However, we lack quantitative information on transmission dynamics that might allow us to estimate mixture dates based on the properties of the ancestry clines we observe.H. pylori. For example, within East Asia, most strains appear to come from the hpEAsia population, with very little evidence of admixture. Lack of contact with Africans does not seem a sufficient explanation, given the large number of documented contacts between East Asians and other Eurasian populations within the last several thousand years. The high burden of H. pylori related gastric disease in the region is notorious and hpEAsia bacteria are known for distinctive variants at virulence associated loci including cagA and vacA32. It is possible that strains from this population have acquired a suite of adaptations that allows them to outcompete invading bacteria despite the large mutation load within their genomes. This raises the possibility that some of the non-synonymous mutations that rose to high frequency during the bottleneck may have allowed rapid adaptation, in other words a form of evolution by shifting balance33.An intriguing question is why some populations appear to have been resistant to invasion by DNA from African H. pylori seems to be an outlier amongst bacteria in many features of its biology30, including its slow rate of spread between human populations and its high mutation28 and recombination rates29. Our results suggest that recombination may save strains from rapid mutational meltdown but that deleterious mutations persist within populations, with the effects of bottlenecks enduring for millennia. The unusual properties of H. pylori make it a powerful model system for understanding how deleterious mutations interact with demographic processes and adaptive ones to mold diversity within natural populations.Helicobacter pylori whole-genome sequences was assembled, consisting of 213 newly sequenced isolates from Europe, Asia and Africa , Hannover Medical School, Hannover, Germany (MHH), Hellenic Pasteur Institute, Greece (HPI), Oita University, Japan (OiU), and University of Bath, UK (UBa) . The resulting core genome, which was collected with the same tool, contained 287,746 core SNPs from 979,771 variant sites.All isolates were mapped to the 26695 genome (NC000915.1) using the Snippy software v. 3.2-dev  chain, in order to conduct clustering of individuals based on the co-ancestry matrix.We inferred population structure among the strains based on the genome-wide haplotype data of the reference-based alignment to 26695 described above, using chromosome painting and fineSTRUCTUREhttps://github.com/pontussk/popstats) and specifying individual A as SouthAfrica7 (hpAfrica2), individual B as GAM260Bi (hspAfrica1WAfrica), individual Y as F227 (hspEAsia) and individual X. In this comparison, negative D-statistics imply more African ancestry in the strain designated as individual X than in F227. D-statistics values can be found in Supplementary Data D-statistics were calculated for strains assigned to each of the five ancestral populations hspEAsia, hpAsia2, hspCNEAfrica, hspENEAfrica and hspAfrica1WAfrica. D-statistics were calculated using popstats  function in R.We conducted chromosome painting of 646 recipient strains . For this purpose, we used ChromoPainterV2 softwareWe used the chromosome painting analysis to investigate shared genetic drift profiles. We calculated a separate drift profile for each pair of hpEurope subpopulations, including a within-population profile. For each profile, we calculated separate drift values for each of the five ancestry components  compared with the reference genome (1.52 Mb).H. acinonychis as a reference, non-synonymous and synonymous mutations were called against representative strains from the African and Asian populations. For this analysis the ancestral populations were combined to reduce the ancestry components to either African (hpAfrica) or Asian (hpAsia). For each site, the frequencies of these mutations within each ancestral population were calculated to give a score between 0 and 1. These scores were then combined by subtracting the hpAsia scores from the hpAfrica scores to give a score between −1 and 1, where −1 = fixed in hpAsia and absent in hpAfrica, 0 = equal frequencies in hpAsia and hpAfrica, 1 = absent in hpAsia and fixed in hpAfrica. Thus, these scores represent the mutational load for each site in the ancestral populations. We then combined these site-by-site scores with the site-by-site chromosome painting data for the European populations so that each site had an estimate of ancestry component from both Asia and Africa, and a mutational score representing the mutational load in the ancestral populations.Using n − 1)*(j_slope-slope)). We then calculated the mean and confidence limits of these pseudovalues as mean ± quantile * se. All data manipulation and statistical analysis was performed in R 3.6.141. Packages from the tidyverse collection were used extensively, and ggplot2 was used for plotting.We then computed linear regressions of hpAsian ancestry against mutation score for each European population, and we did this for the positive (excess African) and negative (excess Asian) mutation scores separately. To confirm that the regression slopes were not driven by a small number of outlier genes we conducted a gene-by-gene jackknife by repeating the regressions but removing all the sites from a single gene each time. From this distribution of slope values we calculated the pseudovalues as pseudovalue = slope-, each accounting for 1/12 of the mutations. There was no back mutation and mutations had a multiplicative effect on fitness.We used SLiM v. 3.6At the beginning of the simulation, there was no variation in the population. These parameters resulted in an intermediate mutation load for each strain. Higher mutation rates would have led to excessive load in each bacterial generation, while smaller selection coefficients would have led to individual deleterious mutations behaving as if they were neutral.H. pylori has a high rate of import of tracts with mean around 400 bp46 but simulating larger tracts is more computationally efficient than simulating many tracts.In each generation, recombination happened between strains from the same population via the transfer of one segment from a donor strain into a recipient strain, with the length of the block taken from an exponential distribution. We used three different mean values, 0, 5000 and 50,000 bp. H. pylori6 but sizes substantially in excess of this are difficult to simulate due to memory and computation time issues. From generation 8000 onwards, migration from the non-bottleneck to the bottleneck populations happened at a rate of one strain every 500 generations. The simulations were completed after 15,000 total generations.A single population of size 10,000 evolved for 5000 generations, by which time the population was in approximate mutation-selection equilibrium. In total, 1000 strains were moved into a second population, which retained this size for 500 generations before expanding to size 10,000. This is substantially below estimated ancestral population sizes of 2,000,000 or more for Simulation output was analyzed using python and R. For each population, we calculated its average fitness, its fitness variance, its dN/dS to the ancestor and within population dN/dS. Subsequent to admixture, the ancestry proportion from each population was tabulated based on mutations that had arisen during the separate evolution of the populations prior to mixture. For sites without such mutations, their origin was assumed to be the same as the closest mutation of known ancestry, provided that the mutation was within 1 kb. Otherwise, its origin was recorded as unknown.https://github.com/EliseTourrette/Hpylori/tree/main/HpEurope.The SLiM, python and R scripts can be found at Further information on research design is available in the Supplementary informationDescription of Additional Supplementary FilesSupplementary Data 1–8Reporting Summary


As the most powerful tool for studying the origin and evolution of modern humans, mtDNA has great significance in the research of population genetics and evolutionary genetics. Here, we provide new insights into the maternal genetic history of the Daur ethnic group by generating complete mitochondrial genomes from a total of 146 Daur individuals in China. We also collected the published complete mitochondrial genome sequences of 5,094 individuals from 56 worldwide populations as reference data to further explore the matrilineal genetic landscape of the Daur ethnic group. First, the haplotype diversity was 0.9943 ± 0.0019 and nucleotide diversity was 0.0428 ± 0.0210. The neutrality tests of the Daur group showed significant negative values and the mismatch distribution curve was obviously distributed in a unimodal pattern. The results showed that the Daur ethnic group has high genetic diversity and may have experienced recent population expansion. In addition, the main haplogroups of the Daur population were haplogroup D (31.51%), M* (20.55%), C (10.28%), F (7.53%), and B (6.85%), all of which were prevalent in northern China. It probably implies the northern Chinese origin of the Daur population. The PCA, FST, and phylogenetic analysis results indicated that the Daur group formed a cluster with East Asian populations, and had few genetic differences with the populations in northern China. More importantly, we found that disease-related mutation sites of the mitochondrial genome may be related to ethnic groups, which may have important implications for the prevention and occurrence of specific diseases. Overall, this study revealed the complexity and diversity of the matrilineal genetic background of the Daur ethnic group. Meanwhile, it provided meaningful data for the research on the diversity of the human genome.Mitochondrial DNA (mtDNA) has the characteristics of maternal inheritance, high mutation rate, high copy number, and no recombination Mitochondrial DNA (mtDNA) is the only DNA that exists outside the nucleus of human cells. MtDNA has lower molecular weight and higher mutation rate than nuclear DNA . MitochoThe Daur ethnic group is one of the minority nationalities in northern China, mainly distributed in the Daur Autonomous Banner of Morin Dawa, Inner Mongolia Autonomous Region, and Qiqihar, Heilongjiang Province. The Daur language belongs to the Altaic language family. The Daur nationality is sparsely populated, and there are few studies on its mtDNA polymorphism. The few previous studies available have never performed complete mitochondrial genome sequencing of the Daur population. Therefore, the research on this subject is extremely necessary and significant. In recent years, there have been increasing studies on the genetic polymorphism of East Asian populations, especially Chinese ethnic groups . HoweverMany researchers pay more attention to the analysis of mitochondrial hypervariable region sequences. However, the mutations in the coding region also make an important contribution to the construction of maternal lineages. Therefore, sequencing of the whole mitochondrial genome will significantly improve the resolution for distinguishing differences between individuals or groups . On the A total of 146 samples were collected from unrelated healthy individuals of the Daur ethnic group in Qiqihar, Heilongjiang Province, including 71 males and 75 females. All of them have lived in Heilongjiang region for at least three generations according to the narrative. Written informed consent was obtained from all participants. This research was approved by the ethics committee of the Second Affiliated Hospital of Harbin Medical University . All methods were performed in a manner consistent with the approved protocols and in accordance with the relevant guidelines and regulations for human subjects research.DNA was extracted from sample blood using the QIAamp DNA Blood Mini Kit (QIAGEN) according to the manufacturer’s protocol.® DNA Library Prep Reagent Set for Illumina®. Library fragment selection and quality assessment were done using Agilent 2100 Bioanalyzer. Finally, the libraries were sequenced in a 2 bp × 150 bp paired-end mode on the Illumina Hiseq platform.At first, six pairs of primers were used for long-PCR amplification to enrich the mitochondrial genome. The primers are described in detail elsewhere . A totalhttp://bio-bwa.sourceforge.net/) (https://broadinstitute.github.io/picard/) was used to analyze the mapped information of each sample, including the ratio of duplicate reads resulting from PCR amplification and the average sequencing depth, etc. GATK (https://software.broadinstitute.org/gatk/best-practices/) (http://annovar.openbioinformatics.org/en/latest/)   to compactices/)  was usedlatest/) . Meanwhihttps://www.ncbi.nlm.nih.gov/genbank/). Ultimately, there are 5,240 mitochondrial genomes from 57 populations included in our research. Detailed information on the worldwide populations and cited references are listed in To further describe the complex matrilineal genetic landscape of the Daur ethnic group, this study sequenced and generated 146 mitochondrial sequences of the Daur group. In addition, we also searched a total of 5,094 complete mitochondrial sequences from 56 populations as reference data by two researchers. Among them, a total of 2,503 individuals from 26 populations were collected from the 1000 Genome Project. The whole mitochondrial sequences of the 30 populations were screened from published studies and then downloaded from GenBank (FST) were also generated by Arlequin ver 3.5.2.2. The haplogroups of the complete mitochondrial genome sequences for the Daur group were classified using HaploGrep2 based on PhyloTree build 17 (http://www.phylotree.org/index.htm). In addition, the haplogroups of 56 reference populations worldwide involved in this study have also been redefined. The frequencies of the mitochondrial haplogroups were calculated by direct counting. To reveal the relationship between mitochondrial polymorphism sites and disease of the Daur population, we annotated disease information based on MITOMAP (https://www.mitomap.org/) for variant sites. The chart of sequencing quality, pheatmap, and principal component analysis (PCA) were generated by R 4.0.3. The R packages used for PCA were “tidyr,” “dplyr,” and “ggplot2”. The phylogenetic tree was produced by MEGA. To reconstruct the demographic history for Daur samples, we performed a Bayesian skyline plot (BSP) using BEAST 1.8.4. The plot was visualized with Tracer v1.7.2.All of the complete mtDNA sequences in the FASTA format were aligned with rCRS using BioEdit software . The genIn the present study, 146 Daur individuals were sequenced successfully. To observe the sequencing quality clearly, we plotted a bar chart to show the depth of sequencing for all individuals. As shown in To research the genetic and variation characteristics of the Daur ethnic group, we calculated the related genetic diversity indexes . A totalp = 0.940) and HRI  of the Daur group showed the statistical test was not significant; it suggested that the hypothesis of group expansion cannot be rejected.The values of Tajima’s D (−2.503) and Fu’s Fs (−23.820) of the Daur group were calculated. Analysis of mismatch distribution was performed to reflect the historical dynamics of the Daur group, as shown in In order to further verify whether population expansion occurred in the Daur group, we performed the Bayesian skyline plot (BSP) to clarify the population expansion time. The distribution of each haplogroup reflects the basic composition of the genetic structure of a population. A total of 88 sub-haplogroups were classified among 146 complete mitochondrial genomes of the Daur group based on PhyloTree build 17. The detailed haplogroup classification results of each individual are shown in In order to research the distribution of known disease sites in the entire mitochondrial genome of the Daur population, and to grasp the specific maternal genetic markers of the Daur population more accurately, we screened out disease-related sites with a higher mutation frequency in the Daur population. According to disease information annotation based on MITOMAP, we found 71 reported mitochondrial genome loci related to disease in the Daur population, of which 17 loci with a minimum allele frequency higher than 0.05, accounting for 23.94% of the total detected known disease sites. The information of the 17 loci is listed in To determine the factors that may play a role in the mtDNA diversity, we performed an analysis of molecular variance (AMOVA) by grouping the 57 studied worldwide populations according to the different classifications  as shown in To investigate the genetic discrepancy between the Daur ethnic group and 56 other worldwide populations. Principal component analysis (PCA) based on haplogroup frequency was performed . Due to To observe the genetic relationship more clearly between the Daur group and other East Asian populations, we further performed PCA in the context of the entire Asian mtDNA . A totalFST values for the complete mitochondrial sequences between the Daur group and 56 reference populations were calculated as shown in FST values between the Daur group and the reference population ranged from 0.00402 to 0.46263. The lowest pairwise FST value was between the Daur group and TT (FST = 0.00402), followed by JPT (FST = 0.00464). The Daur group also showed lower FST values with SP (FST = 0.01728) and MON . In the comparison with the Daur group, the largest value was compared with IBS , followed by CEU . Meanwhile, all the pairwise FST values were visualized by a heatmap to show the genetic distance more clearly, as shown in To reveal the discrepancy in the matrilineal genetic landscape between the Daur ethnic group and other populations, the pairwise FST values (We conducted a phylogenetic analysis to further clarify the genetic relationship between the Daur ethnic group and other populations and generated a phylogenetic tree based on the T values . We stilMitochondria, as a useful genetic marker, reflect the characteristics of maternal inheritance and are suitable for population evolution analysis. The main purpose of the present study was to understand the maternal genetic diversity of the Daur ethnic group and to provide important genetic information for the study of human genome diversity.We analyzed the genetic variation in the complete mitochondrial genomes of the Daur ethnic group from 146 Daur individuals. The haplotype diversity  is a meaMitochondrial dysfunction and defects caused by mitochondrial DNA polymorphism are related to many diseases. It has been reported that the A10398G variant was probably related to metabolic syndrome , attentiThe results showed that the Daur ethnic group in this study produced 88 specific sub-haplogroups. The frequency of haplogroups varies with varying degrees among populations in different regions. Previous studies have shown that haplogroup D maintained a very high overall frequency among East Asian, North Asian, and Central Asian populations . HaplogrAMOVA was used to detect significant variation in the genetic structure of mtDNA among populations . We founAs for the PCA results, the close clustering between the Daur group and East Asian populations meant that there were almost no genetic differences between them. It confirmed the previous conclusion that the Daur ethnic group belongs to the East Asian branch. In the PCA for all Asian populations, we found the Chinese Heilongjiang Daur group had a close genetic relationship with JPT, TT, SP, DB, and LU2. The population genetic differences between East Asia and Southeast Asia seem to be less obvious to be detected. On the contrary, Daur and South Asian groups performed the farthest genetic relationship among all Asian populations. Meanwhile, the genetic structure of the Daur ethnic group was also well expressed by PCAs.FST provides important insights into the evolutionary processes that influence the structure of genetic variation within and among populations, and it is among the most widely used descriptive statistics in population and evolutionary genetics. The small FST value means that the allele frequencies in each population are similar. If the value is larger, that means the allele frequencies are different, indicating that the genetic distance is farther ( farther . AccordiFST values also revealed consistent results. The Daur group apparently congregated with East Asian populations and distributed in the nearest sub-branch with JPT. It revealed that there was little genetic difference between the Daur group and JPT; they had a close maternal genetic relationship. Our results may be due to the Daur sampling site in this study being located in northeast China, which is geographically close to Japan, and the recent introduction of the Daur genes into Japanese or their common maternal origin. In addition, the results revealed that the genetic distance between the Daur group and TT was relatively close. It may be due to the gene exchange and fusion between the Daur population and TT during historical development. The Daur ethnic group and MON also showed a close genetic relationship; they both belong to the Altaic language family. It is reported that the Daur ethnic group originated from the Mongolian ethnic group. The results of this study may further explain the view from the perspective of maternal genetics.At the same time, the phylogenetic tree generated based on the FST, and phylogenetic tree revealed that the Daur group was closely clustered with East Asian populations, especially in northern China. The Daur ethnic group showed a closer genetic relationship with TT, MON, JPT, and SP. We found that the specific disease-related mutation sites of the mitochondrial genome may be ethnic-related. Overall, the mitochondrial genome generated in this study would enrich the existing mtDNA database, actively promoting the research on the genetic diversity and population historical dynamics of the Daur ethnic group.Our research provided a complex and comprehensive maternal genetic landscape of the Daur ethnic group. First, we found that the Daur ethnic group has a high genetic diversity and may have experienced recent population expansion. According to the results, most of the haplogroups of Daur are prevalent in East Asia. It is confirmed that the Daur group belongs to the East Asian lineage and originated from north China. All results of PCA,


Clk is involved in the regulation of annual timing events and shows highly conserved sequence homology across vertebrates except for one variable region of poly-glutamine repeats. Clk genotype varies in some species with latitude, seasonal timing and migration. However, findings are inconsistent, difficult to disentangle from environmental responses, and biased towards high latitudes. Here we combine field data with a common-garden experiment to study associations of Clk polymorphism with latitude, migration and annual-cycle timing within the stonechat species complex across its trans-equatorial distribution range. Our dataset includes 950 records from 717 individuals from nine populations with diverse migratory strategies. Gene diversity was lowest in resident African and Canary Island populations and increased with latitude, independently of migration distance. Repeat length and annual-cycle timing was linked in a population-specific way. Specifically, equatorial African stonechats showed delayed timing with longer repeat length for all annual-cycle stages. Our data suggest that at low latitudes with nearly constant photoperiod, Clk genotype might orchestrate a range of consistent, individual chronotypes. In contrast, the influence of Clk on annual-cycle timing at higher latitudes might be mediated by its interactions with genes involved in (circadian) photoperiodic pathways.Timing is essential for survival and reproduction of organisms across the tree of life. The core circadian clock gene  In consequence, organisms have evolved internal  time-keeping mechanisms to anticipate changes in the environment that recur rhythmically with high precision. One such endogenous mechanism is the circadian clock that enables organisms to anticipate diel (i.e. within a 24 h period) changes5. Circadian clocks continue to run under experimental conditions of constant light or darkness, and in natural environments they are entrained by diel changes in light intensity and duration. The circadian core clock in many organisms is well characterised on the molecular level as a set of genes that regulate biochemical oscillations with a period of 24 h . For many species, for example fish, mammals and birds, we observe consistent variation in rhythmic behaviour on the level of the individual e.g., by being consistently early or late relative to the population8. Such so-called "chronotypes" have been linked to allelic variation in circadian genes, mostly, but not exclusively in humans9. The demonstrated links between behaviour and circadian gene regulators have made variation in rhythms particularly accessible to ecologists and evolutionary biologists10. Evolutionary interest so far has mostly focused on the gene Clk, a single copy gene that is constrained from evolving fast11. The translated protein CLOCK is a key element in the positive arm of the circadian transcription-translation feedback loop12. Clk is highly conserved among vertebrates throughout most of its sequence, except for a C-terminal exonic region that contains an often-variable poly-glutamine  trio-repeat motif. Length variation in this region varies between species, but in many species also shows substantial variation between and also within populations13. This variable poly-Q region has been shown to influence the transcription activating potential of the CLOCK protein in Drosophila14 and mouse15, where truncated versions of the ClkTime is an essential dimension of the ecological niche of a species17, whose predictable change over the year provides an external calendar of increasing amplitude pole-wards, while being absent at equatorial regions. Annual changes in photoperiod can time key life cycle events, such as reproduction or migration, either directly or by entrainment of the circannual rhythm, for which photoperiod is the main synchronising cue18. Thus, molecular changes in clock genes may affect annual timing either directly via pleiotropic effects on circadian and circannual rhythms, or indirectly via influencing photoperiodic responses20. For example, in many organisms the circadian clock defines a photo-sensitive time window during which reproduction can be triggered or terminated17. Hence, individuals with genetic disposition for early versus late-entraining circadian clocks are expected to respond differently to a given daylength21. Recent evidence supports corresponding diel and annual chronotypes, e.g. for migration-linked behaviour in a songbird22. However, photoperiod-dependent links between diel and annual time-keeping can be complex, since daylength increases in spring, but decreases in autumn.Circadian clocks, and the molecular mechanisms that control them, are also involved in annual time-keeping, in conjunction with circannual clocks. Their main role is measurement of photoperiod (daylength)Clk genotype and annual-cycle traits. For example, poly-Q repeat length correlated with timing of migration in fish24 and birds26, timing of moult27, as well as latitudinal clines and breeding phenology in some species30. However, these results varied between study systems and a consistent pattern is lacking, e.g. no such correlation was detected between poly-Q repeat length and latitudinal clines in other bird and fish species33. Furthermore, no correlation was found for timing of breeding32 and migration34 in other bird species, and some species were altogether lacking detectable genetic variation at the poly-Q locus35.So far, some studies have indeed demonstrated a correlation between 36, especially when evaluating genotype–phenotype association of complex behavioural traits that are generally assumed to be polygenically controlled. Furthermore, circadian genes have broad, highly pleiotropic effects, similar to “house-keeping” genes10, and there is scepticism whether selection for a particular trait  would modify genes with such broad functions, rather than acting on more specific physiological pathways38. In addition, caveats have been raised against investigating single candidate genes that are part of wider pathways, like the Clk gene39, and genotype–phenotype associations based on polymorphism of these genes. The reasons for the observed inconsistency between studies are so far not clear.This lack of consistency has raised critical responses towards candidate gene approachesClk gene in different species emphasize that results from these candidate gene studies cannot easily be generalized across taxa25. For different organisms, the role of Clk likely differs, for example due to differences in circadian organization, in genetic architecture, in photoperiodic environments, or in demands on time-keeping41. Thus, in some organisms diel and annual timing might correlate, whereas in others separate and independent regulation may be beneficial42. Furthermore, most studies are correlational, so that genetic contributions to timing are entangled with a host of responses to environmental conditions43.Inconsistent findings on the role of the Clk genotype–phenotype associations using a widely discussed subject, avian annual-cycle timing, by a combination of experimental approaches and comparison of closely related taxa. In birds, variation in allelic diversity and repeat length has been focally studied in the context of environmental and behavioural seasonality, specifically breeding latitude, annual-cycle timing and migratory distance. With increasing latitude and thus intra-annual amplitude in day length, reproductive seasons are delayed and shortened, moult timing is shifted, and migratory distance generally increases44. Convincing support for a relationship between Clk genotype and annual-cycle timing, however is scarce46.Here, we address uncertainty over 38. We compared closely related taxa within a species complex across wide latitudinal breeding ranges and used a common-garden experiment to disentangle inherited timing from environmental responses43.Aiming to shed light on so far inconsistent results, we developed an experimental design that paired biogeographic associations with controlled experiments under common-garden conditions in captivityClk poly-Q polymorphism and breeding latitude, migration and annual-cycle timing in a well-studied songbird complex with a trans-equatorial distribution range, the stonechat , Saxicola torquatus , Saxicola dacotiae (Canary Islands), Saxicola stejnegeri (Japan), and Saxicola maurus (Kazakhstan). Henceforth, we here use the term “population” to refer to any group of individuals belonging to these taxa, except where otherwise stated. We compared Clk genotypes of nine populations with breeding ranges spanning 55° of latitude, including resident, short- and long-distance migratory populations from Europe, Asia and equatorial Africa (Table 48). In addition to their latitudinal variation these populations differ in their annual timing of breeding, moult and migration. Furthermore, stonechats breeding at the equator experience a constant 12:12 h light/dark cycle throughout the year , through autumn migration to spring migration. We investigated Clk-related differences in timing with the following key objectives:Clk genotype affects annual-cycle timing, we expect allelic diversity to vary with latitude and migration behaviour (here quantified as mean migration distance). We tested this hypothesis using all nine populations. If individuals within the population differ in timing or in their photoperiodic exposure, we would expect to find high allelic diversity in Clk. Evolutionarily, such differences could result from fluctuating selection on timing. For increasing breeding latitude and migration distance, we might expect increased allelic diversity due to fluctuating mortality linked to arrival timing and wintering latitude52. In equatorial populations, constant photoperiodic conditions, but inter-annually variable breeding opportunities could either lead to canalisation and favour fixation of an optimised circadian Clk genotype, or, conversely, diversified time-keeping and hence, a broad range of genetically determined chronotypes.Investigating geographic patterns of allelic diversity. Assuming that Clk allele lengths affect annual timing, we expect to detect differences in allele lengths with breeding latitude and migration distance in the nine populations of wild birds. Equatorial populations may again deviate from patterns at higher latitudes because of the absence of photoperiodic change.Investigating associations between repeat length and population-level timing. If Clk genotype and individual-level timing of annual cycle traits. We test directly for genotype–phenotype associations using the four captive populations kept under identical photoperiodic conditions. Because Clk allele lengths might affect annual timing via photoperiodic time measurement, we examine whether relationships between repeat length and timing reverses between autumn (decreasing daylength), and spring (increasing daylength). Finally, because the role of Clk in annual timing might differ between populations, e.g., due to changes in photoperiodism, we test for population-specific relationships.Investigating associations between A subset of four of these populations was studied under a common garden setting; specifically: residents (Kenya), partial migrants (Ireland), short-distance (Austria) and long-distance migrants (Kazakhstan). This approach required to keep all main known modifiers of timing consistent between populations. Thus, photoperiod, the main synchroniser of annual cycles, was set to simulate European daylength, under which all populations perform appropriate, population-specific annual activitiesClk poly-Q allelic lengths variation in nine breeding populations and identified eight length variants ranging from 8 to 15 poly-Q repeats  found at medium to high frequencies  as well as within population gene diversity (defined as unbiased gene diversity per sample and locus by Goudet53). We analysed Clk gene diversity across the geographical range of stonechat populations in the context of breeding latitude and migration distance, two factors that were correlated, albeit not significantly . Equatorial populations from Africa and the Canary Islands showed lower levels of diversity for both measures compared to all other populations; highest diversity levels were found in the Irish population . When we excluded the Canary Island population and restricted our analysis to continental populations, lowest levels of gene diversity were found in the African populations breeding close to the equator . This analysis tentatively evaluated if the pattern we observed in Clk gene diversity is due to selection in that particular region of the genome or the result of whole genome elevation of nucleotide diversity. When adding autosomal pi as a covariate to our models for this subset of populations, we neither found a correlation of Clk gene diversity with breeding latitude or migration distance, nor with genome-wide nucleotide diversity .We thus additionally analysed the five populations for which genome-wide nucleotide diversity  was available ; see Supplementary Material, Fig. We detected no significant relationships of breeding latitude or migration distance of our nine stonechat populations with Clk genotype, characterised as the mean number of poly-Q repeats at the variable locus, and timing of different focal traits we used the four captive populations and included all available data from a common-garden experiment47  and frequency of eight different allelic variants is high compared to other passerines. For example, a couple of earlier studies on a variety of study species only showed four different poly-Q variants including barn swallows  and pied flycatcher 26 while five different poly-Q variants were reported in other species including tree pipit  and nightingales 26, blue tits 28 and great tits 35. One possible explanation for the high variation observed in the stonechats could be that our study comprised populations that varied not only in breeding latitude, but also in their migratory behaviour.Clk gene diversity, whereby genetic variation was reduced in populations breeding at the equator. This is particularly interesting in a chronobiological context as the scope for light entrainment at the equator dramatically differs from seasonally varying light-dark cycles at higher latitudes. The two equatorial breeding populations from Kenya and Tanzania, which experience constant 12-h days throughout the year, differed from all other populations included here by significant reduction in Clk gene diversity. Previous research on African stonechats has revealed that life-cycle timing shows robust annual cycles, both in free-living birds and under restrictive experimental conditions. Captive African stonechats held under constant 12-h light–dark cycles retained clear circannual rhythms of breeding condition and moult for several years59. Thus, although African stonechats have retained the ability to respond to changing photoperiod49, they appear to rely on an endogenous timing mechanism. Hence, the lower variability in Clk repeat length could be signature of selection facilitating adaptation to a constant photoperiod in the birds’ environment resulting in a putatively optimised and less variable genotype59.Across stonechats, we found a latitudinal pattern in 60 are less rigid, and the substantial changes in photoperiod they experience may play a dominant role for their annual time-keeping61. Hence, greater variability in Clk repeat length might have resulted from fluctuating inter-annual selection for photoperiodic timing, for example, due to rough winters or sudden weather changes52. Thus, we speculate that breeding populations in Germany, Austria, Spain and Ireland require integration of photoperiodism20, and thereby a higher degree of genetic diversity in Clk genotype.Circannual rhythms of European stonechat populations that live permanently in the northern hemisphereClk locus are instead caused by random drift. While similar Clk gene diversities in Austrian, German and Irish stonechat populations could be due to ongoing gene flow between these populations , phylogeny cannot explain the similarly high Clk gene diversity we find in Kazakh stonechats, which diverged over 2.5 mya55. Furthermore, the pattern in genetic diversity we observed for the focal Clk locus differs from a genome-wide characterisation across populations, which showed similar levels across all populations except for Kazakhstan, where levels were elevated . However, when we included genome-wide diversity of five of the nine populations as a covariate, we no longer observed a significant relationship between Clk gene diversity and latitude. We thus cannot exclude that the higher Clk gene diversity at higher latitudes could also be a result of higher genome-wide diversity. However, power for these results from only five populations was low, and results might be driven by the high genome-wide diversity in Kazakhstan54. The other populations showed similarly low diversity values, although the Austrian and Irish populations breed at similar latitude as the Kazakhstan population. The Kazakhstan stonechat has a vast breeding range and high effective population size63, which may have caused its outlying, elevated genome-wide diversity54. Conversely, despite its low genome diversity, the Irish population shows high Clk gene diversity. It is tempting to speculate that the particular diversity in the Clk gene is associated with the partial migratory nature of the Irish population62. Among partial migrants, the resident fraction of a population experiences different photoperiods, and typically breeds earlier in the season, compared to the migrant fraction47.We consider it unlikely that the observed genotype–phenotype patterns for the focal Clk gene diversity, independently of breeding latitude. The only indicative effects of migratory phenotype are the differences in level of heterozygosity between migrants and residents , the consistent results suggest that 65. More broadly, genetic contributions to annual cycle timing have been shown in several common garden studies, in quantitative genetic analyses and in breeding experiments61. Heritability (h2) estimates from captive migratory songbirds were medium to high for onset of migratory restlessness in blackcaps Sylvia atricapilla (0.34–0.45) and garden warblers Sylvia borin (0.67 onset for spring and autumn migration); heritability estimates for termination of migratory activity however were lower . Heritability estimates of migration timing traits from the wild are scarce, and estimates of repeatability and heritability are generally moderate, but sometimes lower, for example in Collared flycatchers 65. It is thus clear that to a varying degree, flexibility and non-genetic factors, such as learning, state and ontogenetic factors, also contribute to individual variation69. Inherited timing programs need to integrate information from multiple physiological pathways, such as metabolism and photic input70. Hence, evolutionary change in annual cycle timing can involve several pathways including, but not limited to, genes with known circadian roles such as Clk71.Overall, our results fit well with evidence that circadian clock gene variants can also be relevant for coordination of annual timing, but their roles and effects depend on species, populations and time of yearSaxicola complex, which also differed between populations72. Our new data suggest the possibility that the underlying genetic basis for individual timing might differ between populations from different latitudes. In equatorial populations, variation in annual chronotype might be partly regulated through a limited range of variants in the gene Clk that exert major effects, whereas at higher latitudes, Clk variants may mainly act in conjunction with photoperiodic pathways, and hence show weaker genotype–phenotype associations. Despite the lack of a mechanistic framework and reservations regarding the candidate genes approach, our results provide important and novel insight into understanding the possible genetic basis of annual-cycle timing.Previous findings from the captive stonechat populations used in this study indicate high individual consistency and high heritability estimates of annual timing traits in the Clk gene diversity in a large dataset of 950 records from 717 individuals distributed over a wide geographical range, confirming findings in other species. Making use of a common garden setting our study highlights that the relationship between Clk polymorphism and annual-cycle timing in captive stonechats depended on population and on time of year. Our findings also allow us to speculate that in populations that live under unchanging photoperiods of the equator, Clk genotype may be less variable, but exert strong association with annual chronotype. Conversely, at higher latitude, other evolutionary forces may favour Clk gene diversity to be higher, but its association with phenotype may be obscured by additional molecular inputs into annual timing that furthermore depend on time of year.In conclusion, we found a latitudinal cline in 50. In captivity, differences in timing between free-living populations largely persist when the birds are kept under common-garden captive conditions50, and even resident populations from equatorial Kenya display migratory restlessness, albeit at a low level50. Combined, this makes stonechats an ideal system to study associations of Clk gene polymorphisms with different processes. Here, we capitalise on the following phenotypically and geographically distinct populations to study Clk gene polymorphism within one species complex . Different populations exhibit a variety of migratory behaviours  and 52 individuals originating from Tanzania ], which are year-round residents. We further included a resident island population (n = 61) endemic on Fuerteventura, Canary Islands, Spain .We studied two African mainland populations [Saxicola rubicola ssp: (1) one population (n = 217) sampled in Austria , (2) one population (n = 47) from north-west Germany , (3) one population (n = 184) from Ireland , and (4) one population (n = 93) from mainland Spain . Birds from Austria and Germany are obligatory medium-distance migrants that spend the winter in Mediterranean regions including North Africa. Irish stonechats are partial migrants that either stay on the breeding grounds year round or migrate over short distances75. Spanish birds originate from a population of non-migratory stonechats .We further included four populations of European stonechat Saxicola maurus ssp: one population (n = 150) from Kazakhstan  and a second population (n = 11) from Japan . Kazakh birds migrate to India, southern continental China and North East Africa76, while Japanese birds migrate from Hokkaido over a continental path to winter in mainland southeast Asia77.Lastly, we included two populations of long-distance migrants, Eurasian stonechat Clk poly-Q repeat length. Most samples (n = 504), including birds from populations in Austria (n = 130), Ireland (n = 112), Kazakhstan (n = 90) and East Africa , originated from a common-garden experiment that Eberhard Gwinner initiated in 1981 at the Max Planck Institute for Ornithology in Andechs, Germany, and continued until 200578. These samples were thus collected from birds born over a long time span, including some individuals that were born in captivity, and others were brought in from the wild. African and European Stonechats hatched between 1982 and 2004, with similar spread over time, whereas Irish and Siberian stonechats hatched between 1997 and 2005. Additional genotype samples (n = 213) were collected from birds sampled in the wild at their breeding grounds. Specifically, these included populations from the Canary Islands56, Spain , Germany79 and Japan77. These samples were collected between 1998 and 2017.We successfully genotyped 717 individuals from the nine study populations for variable https://arriveguidelines.org).All methods were carried out in accordance with relevant guidelines and regulations, all experimental procedures were approved and conformed to the relevant regulatory standards under permit (Number: 55.2-1-54-2531-119-05) by the Ethics committee of the state of Upper Bavaria. The study was carried out in compliance with the ARRIVE guidelines (Clk gene using a polymerase chain reaction (PCR) amplification protocol and lengths characterisation of the variable region. PCR amplification was carried out in a 10 µl total volume using a previously published primer set Johnsen et al.28 . PCR conditions were optimised for stonechats following conditions published in Liedvogel et al.29: Amplification reaction conditions for Taq DNA polymerase catalysed PCR for the stonechats: Mg2 +  = 2 mM; 95 °C/2 min; 95 °C/30 s, 56.8 °C/30 s, 72 °C/30 s, 40 cycles; 72 °C/5 min, 4 °C hold. PCR products were prepared for capillary electrophoresis by adding HIDI-formadide-LIZ 500 mixture. Length polymorphism of amplified products were analysed using geneious version 10.1.3. Repeat counts of lengths characterised samples was confirmed by Sanger sequencing of 50 samples across populations using an adapted version of a previous published protocol29 with following primers: forward: 5′-TTTTCTCAAGGTCAGCAGCTTGT-3′; reverse: 5′-CTGTAGGAACTGTTGCGGGTGCTG-3′). Amplification conditions using Taq DNA polymerase for Sanger sequencing were optimised for the stonechat material: Mg2 +  = 2 mM; 95 °C/2 min; 95 °C/30 s, 61.1 °C/30 s, 74 °C/30 s, 40 cycles; 74 °C/5 min, 4 °C hold. Nucleotide sequences of with Exo/SAP purified PCR fragment were determined with BigDye Terminator ready reaction mix, version 3.1 (Applied Biosystems) under standard sequencing conditions according to the manufacturer’s protocol29.Genomic DNA was isolated from blood (n = 706) and feathers (n = 11) using a salt extraction protocol and diluted to a working concentration of 25 ng/µl. Genomic DNA samples were genotyped for length polymorphism in the variable poly-Q repeat region of the Clk genotype as mean allele length  and did additional sensitivity tests using minimum and maximum allele length in our models. Results from these sensitivity tests were qualitatively the same, therefore we only present results from models with mean allele length in the main text. Genotype frequency data for all populations were tested statistically for deviation from Hardy–Weinberg equilibrium using GENEPOP 4.2 . We adapted to use the same Markov chain parameters that were used in previous studies29: dememorization , batches  and iterations per batch (10 000). For each population we calculated observed heterozygosity as a measure of Clk allelic diversity as the proportion of observed number of heterozygotes of the total number of individuals. To account for the differences in sample size we used the program FSTAT version 2.9.3.253 to calculate Fsdiv, an index of unbiased gene diversity. Results of gene diversity remained similar when we excluded captive-bred individuals from our dataset  from publicly available whole genome re-sequencing data for five of the included stonechat populations . To identify if differences in gene diversity were due to differences in breeding latitude or migratory phenotype, we ran linear models predicting gene diversity by covariables latitude, migratory distance and pi.To examine whether the Clk gene length by running linear mixed effects models with Clk mean repeat length as response variable, and either latitude or migratory distance as predictor variable as well as origin as a random effect.Within individuals, we tested for an effect of latitude or migratory distance on 47. Briefly, stonechats were hand-raised in the Max Planck Institute for Ornithology, as offspring of captive or wild stonechats belonging to the Kenyan, Austria, Irish or Kazakh populations (for detail see47). The birds were kept indoors under simulated natural daylength changes in south Germany . Here we include all birds with available phenotype and genotype data .Data on migratory phenotypes were previously presented for our four captive stonechat populations80. A precise description of how moult was recorded was published previously72. In brief, immature stonechats were regularly checked for body moult in 19 plumage areas. The number of plumage areas moulting provided a moult score for each date to determine the timing of onset and completion of moult72.Postjuvenile moult in stonechats is the first moult after hatching and equates to the first prebasic moult81, although in stonechats and other songbird species, resident populations also show some migratory restlessness47. A detailed description of how migratory restlessness of the stonechats was recorded and characterised is published elsewhere50.We used nocturnal migratory restlessness behaviour as proxy for migratory timing. Migratory restlessness, the nocturnal activity of captive birds during migration periods, generally mirrors the migratory timing of free-living conspecificsClk genotype and annual-cycle phenotype we ran linear mixed effects models including mean Clk repeat length, origin, sex and a two-way interaction of Clk repeat length with origin as fix effects and year and individual as random effects in our comparisons with migratory timing. For our analysis of postjuvenile moult we added hatch date and a two-way interaction of origin and hatch date as we expected a potential effect of hatch date on moult timing as it had been demonstrated in a previous study72. Random effects were not used in this analysis, since we only had values from 1 year.To investigate correlation between 83 using packages ‘lme4′84 and ‘sjPlot’85.We tested the genotype effect on timing of postjuvenile moult, spring and autumn migration, as response variables, using three different time-points, i.e. onset, peak and end (given as Julian date). Analysis was performed in RStudio interface of R version 3.3.38 was initially excluded. However, Q8 genotype for this individual (as classified by size fragment determination) was confirmed by Sanger sequencing, giving us no reason to exclude it from the analysis.All trends remained unchanged in alternative models where one individual with a rare short poly-Q repeat length of QSupplementary Information.


Procapra gutturosa) population has shown a considerable range of contractions and local extinctions over the last century, owing to habitat fragmentation and poaching. A thorough understanding of the genetic diversity and structure of Mongolian gazelle populations in fragmented habitats is critical for planning effective conservation strategies.The Mongolian gazelle (Cytb) to compare the levels of genetic diversity and genetic structure of Mongolian gazelle populations in the Hulun Lake National Nature Reserve (HLH) with those in the China-Mongolia border area (BJ). The results showed that the nucleotide diversity and observed heterozygosity of the HLH population were lower than those of the BJ population. Moreover, the HLH and BJ populations showed genetic differentiation. We concluded that the HLH population had lower genetic diversity and a distinct genetic structure compared with the BJ population.In this study, we used eight microsatellite loci and mitochondrial cytochrome b (The genetic diversity of fragmented Mongolian gazelle populations, can be improved by protecting these populations while reinforcing their gene exchange with other populations. For example, attempts can be made to introduce new individuals with higher genetic diversity from other populations to reduce inbreeding.The online version contains supplementary material available at 10.1186/s12864-023-09574-0. Globally, severe human disturbances can easily lead to fragmentation of natural landscapes, resulting in fragmentation of the distribution of many animals . Small p2, owing to habitat destruction caused by human disturbance and other factors ) and observed heterozygosity (HoBJ = 0.593 [SD = 0.224]) in the samples from BJ than in those from HLH . The highest number of effective alleles (Ne = 7.010) and expected heterozygosity (He = 0.857) were found at the OArFCB304 locus in the BJ population  revealed 28 haplotypes  network based on pes Fig. .In the STRUCTURE analysis, the delta K values were highest when K = 2, indicating two genetically heterogeneous clusters Fig. . We furtConsistent with the results of the STRUCTURE analysis, PCoA clustering demonstrated a clear separation between the two populations Fig. . The AMOCytb.Genetic diversity affects the adaptation of populations to environmental change . Small pCytb analyses revealed low levels of genetic diversity in the HLH population, whereas the BJ population exhibited higher levels of genetic diversity. The nucleotide diversity in the HLH population was 0.00401, which was lower than that of the Mongolian gazelle population in a previous study (0.05000) [Procapra species , it was found to be lower than that of P. przewalskii around Qinghai Lake in China (0.01500) and P. picticaudata in Tibet (0.08100) [Moschus moschiferus  and Cervus nippon , the HLH population showed a low level of genetic diversity  [Antilope cervicapra, π = 0.00704; Cervus elaphus hanglu, π = 0.008); however, the BJ population exhibited higher nucleotide diversity  than other ungulate species [Axis porcinus population may have been due to historical gene flow, which guarantees a high level of genetic diversity [Microsatellite and 0.05000) . When th0.08100) –35. In 20.08100) . However0.00401) , 38. The species , 40. As iversity . The lowiversity . The abiiversity . Populativersity , 45. TheIn addition to the reduced genetic diversity, our findings showed evidence of genetic differentiation between the HLH and BJ populations. Both PCoA and STRUCTURE analyses demonstrated a clear delineation between the two Mongolian gazelle populations on the basis of allelic composition, implying genetic isolation. Previous studies have shown that natural landscapes, such as mountains, rivers and deserts can act as ecological barriers to gene flow between populations –48. In aCytb. In comparison with the BJ population, the HLH population showed lower genetic diversity and a distinct genetic structure. Low genetic diversity and small population sizes increase the risk of extinction of Mongolian gazelle populations in fragmented habitats. Therefore, increasing the population size and preventing inbreeding are crucial for protection of Mongolian gazelles. Our findings indicate the need for long-term program to monitor the dynamics of the Mongolian gazelle. In addition, for long-term survival of the HLH population, a certain number of Mongolian gazelles should be introduced from other populations.We estimated the genetic diversity and structure of Mongolian gazelle populations in fragmented habitats by using microsatellites and Below is the link to the electronic supplementary material.Supplementary Material 1


Moschus berezovskii) is a species that is currently classified as endangered and found in China and Vietnam. In order to prevent their extinction, China initiated captive breeding programs in the 1950s. Maintaining the high genetic diversity of the population is a crucial factor for ensuring the sustainable and rapid growth of these captive populations. Mitochondrial DNA (mtDNA) possesses a unique lineage of matrilineal evolution, and the control region (CR) is considered to be one of the most popular molecular markers for conducting a genetic diversity analysis and determining maternal lines. In this study, we assessed the current genetic diversity status of 338 individuals from seven captive forest musk deer populations located in the Shaanxi province using mtDNA CR, and the results showed that the genetic diversity was high. We also made full use of previous mtDNA CR data clearly defining 65 haplotypes with their frequency and concluded with about 90 maternal lines. The analysis revealed no significant genetic differentiation among the populations, and the populations might not have experienced rapid population expansion. The current study represents the most comprehensive research of genetic diversity of captive forest musk deer to date, and will be helpful to preserve and enhance the genetic diversity of the captive forest musk deer populations.Forest musk deer (Moschus berezovskii) are endangered ruminants whose adult males secrete musk. China has been breeding forest musk deer artificially since the 1950s in an effort to restore wild populations, with Shaanxi and Sichuan provinces as the two main sites for captive breeding. Genetic diversity is a significant indicator that determines the long-term viability and status of a population, particularly for species at risk of extinction. In this study, we analyzed the current genetic makeup of seven captive forest musk deer populations in the Shaanxi province, using the mitochondrial DNA (mtDNA) control region (CR) as the molecular marker. We sequenced 604 bp of mtDNA CR, with an average content of A+T higher than G+C. We observed 111 variable sites and 39 different haplotypes from 338 sequences. The nucleotide diversity (Pi) and haplotype diversity (Hd) were 0.02887 and 0.908, respectively. Genetic differentiation between these populations was not significant, and the populations might not have experienced rapid growth. By combining our sequences with previous ones, we identified 65 unique haplotypes with 26 rare haplotypes and estimated a total of 90 haplotypes in Shaanxi province captive populations. The Shaanxi province and Sichuan province obtained 88 haplotypes, the haplotypes from the two populations were mixed together, and the two populations showed moderate genetic differentiation. Our findings suggested that captive forest musk deer populations in the Shaanxi province had high genetic diversity, with a rich founder population of about 90 maternal lines. Additionally, managers could develop genetic management plans for forest musk deer based on the haplotype database. Overall, our study will provide insights and guidelines for the conservation of genetic diversity in captive forest musk deer populations in the Shaanxi province.Forest musk deer ( The process of genetic diversity ensues through the mutation and aggregation of genetic material during the evolution of a species . As the Moschus spp.), indigenous to East Asia with solitary nature and high stress, are renowned for the secretion of musk by adult males. At one point, China housed the largest population of musk deer, accounting for 70% of the total population with an estimated 2.5 million wild musk deer in the 1950s [Musk deer (he 1950s . MiserabMoschus berezovskii), one of seven musk deer species and the most widespread in China, continues to face a perilous situation, with its population listed as Endangered on the IUCN Red List [The forest musk deer , Fumin , Pianzaihuang , Haixing , Liangdang , Hongda , and Guanling . Except for LD, which is situated in the Gansu province but where the forest musk deer population has been introduced from the Shaanxi province, all other populations are located in the Shaanxi province. In the process of sampling, we adhered to the principle of random sampling and did not know the genetic relationship of each population. We stored the samples in a laboratory refrigerator at a temperature of −20 °C until DNA extraction.We obtained feces, tissue, and blood samples from a total of 338 forest musk deer in seven captive populations: randomly sampled from Feng County  using the primers F: 5′-CAACTAACCTCCCTAAGACTTCAAG-3′ and R: 5′-CCAAATGTATGACAGCACAGTTATG-3′. The PCR amplification volume was 25 μL, including 12.5 μL 2 × Taq PCR Master Mix (Dye), 30–50 ng DNA template, 1 μL forward primer, and 1 μL reverse primer. The conditions were performed as follows: initial denaturation at 94 °C for 5 min, followed by 34 cycles of PCR , and a final extension of 72 °C for 8 min. PCR products were examined by electrophoresis on a 1.0% agarose gel. All reagents and labware used are listed in Fst), analysis of molecular variance (AMOVA), Tajima’s D (reflects long-term population events), and Fu’s Fs (relatively sensitive to recent population events) statistics were carried out with Arlequin 3.5  [p < 0.05; the extremely significant level was set at p < 0.01; p > 0.05 indicates no significant difference.Individual mtDNA CR sequences were edited by the SeqMan Pro program in DNASTAR Lasergene version 7.1   and thenzerland) . The levIn order to build the existing haplotype frequency of captive forest musk deer in the Shaanxi province and infer all haplotypes, mtDNA CR sequences from captive forest musk deer in the Shaanxi province were downloaded from NCBI, accession: JX499255-69 , MH04734Upon editing and multiple sequence alignment, the effective sequence length of mtDNA CR for 338 forest musk deer was 604 bp. The nucleotide composition was typically A+T-biased, with an average content of A+T (63.3%) higher than that of G+C (36.7%). The total number of sites, excluding those with gaps or missing data, was 597, with 7 sites containing alignment gaps or missing data. Invariable sites were 486, variable sites were 111, and the total number of mutations was 114. Singleton variable sites with two variants were 5, while parsimony informative sites with two variants were 103. There were no singleton variable sites with three variants, but there were 3 parsimony informative sites with three variants. Variable sites with four variants were absent. The Pi was 0.02887, and the K was 17.236. In total, 39 haplotypes were defined for the 338 individuals, and the Hd was 0.908. The HD population had the highest Pi, while the LD population had the lowest Pi. PZH had the highest Hd, whereas HX had the lowest Hd. There was no significant difference between Pi and HD in various populations. The information regarding haplotype variation sites is illustrated in We obtained a total of 65 known haplotypes, and the total haplotype richness at the sampling site, estimated using the sample-based Chao-1 estimator, is 90 (95% CI: 75–130). The range of genetic distance, according to the Kimura 2-parameter model, between each haplotype was 0.002 to 0.131, and the average genetic distance was 0.041. A total of 88 haplotypes of forest musk deer were obtained by combining the existing haplotypes from Shaanxi and Sichuan provinces, and 7 haplotypes were shared between the two populations. Based on genetic distance, we constructed an NJ tree with 1000 bootstrap replicates . All hapData from this study and the Sichuan population were used in the genetic structure study, and Fst between each of the populations and p-values. The analysis results display that HX and FM, as well as HX and LD, were highly differentiated, with the Fst value of more than 0.15. Most of the populations showed moderate differentiation from each other, while a few populations exhibited low differentiation. Specifically, the Shaanxi and Sichuan populations demonstrated moderate differentiation from each other.The AMOVA involved the separation of variation between populations (Va) and within populations (Vb), followed by a significance test . The resD and Fu’s Fs did not achieve statistical significance  [Dama mesopotamica) [Elaphurus davidianus) [Captive breeding followed by reintroduction is an indispensable technique to safeguard endangered species ,34. Thiss ferus) , Persianotamica) , and Peridianus) . Its puridianus) ,39.This was the largest sampling range research on the genetic diversity of captive forest musk deer populations in the Shaanxi province, aiming to objectively evaluate genetic diversity and the total number of haplotypes. Pi is defined as the average number of nucleotide differences at each site between randomly selected DNA sequences in a given population. Hd refers to the frequency at which two different haplotypes are randomly selected from a sample. Pi and Hd are commonly used to measure genetic diversity, with higher values indicating a greater polymorphism in the population and a more abundant genetic diversity . The Pi We conducted the analysis of the frequency of known haplotypes; of the 65 haplotypes, 26 haplotypes were rare haplotypes. In populations with high genetic diversity, rare haplotypes are at risk of being lost when the carrier dies. Therefore, increasing the frequency of rare haplotypes in the population is crucial. One noteworthy issue is that multiple individuals share the same haplotype, except for R and PZH populations, on account of an unbalanced representation of the haplotypes affecting the genetic diversity . The forThe genetic structure analysis indicated that high genetic variation exists within populations, but low genetic differentiation exists between populations . With liD and Fu’s Fs values are negative and the difference is statistically significant (p < 0.05), the sequence contains more change in nucleotide sites than the neutral evolutionary model, which might indicate that the population has experienced a history of expansion. With a nucleotide mismatch analysis, the mismatch distribution curve had a single peak, which accords with the theoretical distribution expected by the population expansion model [D and Fu’s Fs did not reach statistical significance, and the mismatch distribution curve presented multiple peaks; this meant that there was no significant population expansion of forest musk deer in the Shaanxi province. This may have something to do with the habits and reproduction of forest musk deer. Forest musk deer have strong territorial behavior and are highly stressed [Tajima  believedon model . In thisstressed . Even afTo sum up, our research aimed to evaluate the genetic diversity of seven captive forest musk deer populations in the Shaanxi province and trace maternal lines. Our findings showed that the genetic diversity was abundant and the differentiation among each population was not very high. Furthermore, the populations did not undergo a fast population expansion. We also identified about 90 maternal lines and created a haplotype database. We firmly believe that our study will aid the breeding and conservation of captive forest musk deer in the Shaanxi province.


Camellia tunghinensis, which is one of the species with the lowest natural distribution area in the sect. Chrysantha (golden camellia) of the family Theaceae. We investigated the genetic diversity and population structure of 229 samples from wild and ex situ populations using genotyping by sequencing (GBS). Core germplasm was constructed from these samples. The results showed that wild C. tunghinensis exhibited high genetic diversity, with observed heterozygosity of 0.257–0.293 and expected heterozygosity of 0.247–0.262. Compared with wild populations, the genetic diversity of ex situ populations established by transplanting wild seedlings was close to or even higher. However, the genetic diversity of those established by seed or cuttings of a few superior trees was lower. The Admixture analysis revealed that the structure of the ex situ populations derived from seeds and cuttings was relatively simple compared with the ex situ populations derived from transplanted wild seedlings and wild populations. These results suggested that direct transplanting of wild seedlings was more conducive to preserving the genetic diversity of endangered plants in the wild. In addition, wild populations demonstrated a small differentiation (mean FST = 0.044) among themselves, possibly due to long‐term and frequent gene flow between the wild populations. In contrast, moderate differentiation (mean FST > 0.05) was detected among ex situ populations and between ex situ and wild populations. This may be the combined result of the absence of gene flow pathways and strong selection pressure in various ex situ environments. Finally, 77 core germplasms were extracted from 229, likely representing the genetic diversity of C. tunghinensis. This study provides future strategies for the ex situ conservation and management of the golden camellia species and other rare and endangered plants.Whether ex situ populations constructed in the limited nursery resources of botanical gardens can preserve enough genetic diversity of endangered plants in the wild remains uncertain. Here, a case study was conducted with  It refers to the off‐site management of species whose survival and reproduction in their native habitat are seriously threatened  revealed the effect of selection on genetic diversity, and they found slower rates of linkage disequilibrium (LD) decay and lower levels of nucleotide diversity in domesticated cultivars and landraces compared with wild walnuts  is a section of evergreen shrubs or small trees and is the only section with yellow flowers in the family Theaceae, also known as the “queen of camellias” and “giant panda of the plant world”  markers  according to the manufacturer's instructions. The DNA quality was assessed using 1% agarose gel electrophoresis and a Nanodrop2000  spectrophotometer. The DNA was quantified using Qubit3.0 (ThermoFisher), ensuring that each sample met the following criteria: total mass >3 μg, concentration > 30 ng/μl, and OD260/OD280 = 1.80–2.00. The quality‐checked DNA was double‐digested using an enzyme combination of g et al. . Next, t2.3Camellia sinensis reference genome  using BWA v0.7.12 , expected heterozygosity (HE), nucleotide diversity (π), and inbred lineage coefficient (FIS) were calculated by Stacks v2.59  set from 1 to 20. The optimal K value was determined based on the minimum cross‐validation (CV) error  between the nine populations were calculated using VCFtools. As the ex situ populations were artificially constructed and it was known that there were gene flow barriers between them, three ex situ populations were excluded from the subsequent gene flow analysis. Treemix v1.12  were generated from 229 le Table . The clele Table .3.2C. tunghinensis was calculated using 134,613 SNPs. Among the nine populations, GXG2 displayed the highest HO (0.297) while the lowest was observed in GXG3 , followed by GXG1 (0.275), and the lowest was in GXG3 (0.211). The size ranking of the π in each population was consistent with the HE  and two ex situ populations (GXG1 and GXF) exhibited FIS coefficients greater than 0, suggesting the possibility of inbreeding. In addition, all populations had positive Tajima'D values, suggesting that C. tunghinensis may have experienced population shrinkage or equilibrium selection. The LD analysis showed that GXF, GXG1, and the four wild populations had comparable LD decay  and offspring populations  in wild C. tunghinensis were calculated based on SSR markers . Previous investigations have shown that SSR markers estimate heterozygosity two to three times higher than SNP markers  and wild type (HO = 0.229) C. sinensis  were higher than those in the wild populations Table . In term4.3C. tunghinensis, the structure of the ex situ populations derived from seeds and cuttings was relatively simple compared with the ex situ populations derived from transplanted wild seedlings and wild populations  and marginally moderate differentiation between the ex situ populations (mean FST = 0.051). Wild C. tunghinensis is found naturally in four populations within a distribution area of less than 100 hm2. These trees show limited dispersal distances with seedlings primarily growing around the mother tree, mostly due to the large fruit size. However, the geographical distance between the wild populations was relatively close and the pollinators  of golden camellia species could easily accomplish cross‐population pollination, which is also consistent with the Admixture and Treemix inferences. Therefore, the low degree of differentiation seen between the wild populations was not unexpected. In contrast, there were no pathways for gene flow between the ex situ populations, and at the same time, they needed to adapt to the local ex situ environment. Differentiation between the ex situ populations and between ex situ and wild populations would be expected to increase with time (Wei & Jiang, C. tunghinensis populations, there are artificial barriers between the ex situ populations and it is thus necessary to strengthen cooperative breeding programs between different public organizations. However, it is still unclear whether blind crosses between plants from different populations will result in inbreeding or outbreeding depression (Lin et al., C. tunghinensis plants and provide useful support for subsequent inbreeding and outbreeding trials.An understanding of population structure is vital for identifying the correct conservation and management strategies (Pan et al., s Figure . We furt Wright, . Using t4.4The construction of core germplasm can significantly improve the efficiency of the management and utilization of germplasm, thus reducing costs. This concept is heavily applied in plant resource collection and utilization (Miao et al., C. tunghinensis germplasms. Based on experience in the agricultural field (Li et al., In this study, 77 core germplasms were extracted from 229 1. In the absence of genetic diversity surveys, priority should be given to the direct transplantation of wild seedlings for introduction and the collection of material from different maternal lines whenever possible. It is important to back up these transplanted wild seedlings so that they can survive well in the ex situ environment Hoban, , for exa2. Based on an in‐depth understanding of the genetic diversity of the populations, both populations and individuals within the populations that need to be prioritized for conservation should be identified, and we strongly recommend the selection of individuals that require ex situ conservation by constructing core germplasms to improve the efficiency of conservation.3. Genetic diversity should be used as an important indicator for assessing ex situ conservation and continuous attention should be paid to it. For example, after an ex situ population has been successfully established, the dynamics of genetic diversity, especially between generations, should be recorded through genetic monitoring (Diaz‐Martin et al., The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Figures S1 and S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.