Chlorops oryzae has become one of the major pests of rice in some regions, which has caused serious economic losses. To understand the genetic mechanisms of frequent local outbreaks and population expansion of C. oryzae, we analyzed the population genetic structure using two molecular markers, COI and ITS1 sequences. The results indicated that the C. oryzae populations experienced rapid expansion after a “Bottleneck effect” and the local outbreaks were probably caused by frequent gene flow among populations.Recently, Chlorops oryzae one of the major pests of rice in some regions. In order to understand the ecological adaptation of C. oryzae at the molecular level, and provide a scientific basis for formulating management strategies, we used two molecular markers, COI and ITS1 sequences, to systematically analyze the genetic structure of 31 populations. The higher haplotype diversity and lower nucleotide diversity indicated that the C. oryzae populations experienced rapid expansion after a “Bottleneck effect”. The results of the mismatch distribution, neutrality test , and haplotype network analysis suggested that the population has recently undergone an expansion. Although genetic differentiation among C. oryzae populations was found to have existed at low/medium levels , the frequent gene flow presented as well  was supposed to be responsible for frequent local outbreaks.Frequent outbreaks have made  Population genetic structure, the most basic genetic information of a species, represents the amount and distribution of genetic variation within and among populations. Moreover, it is the accumulation of the evolutionary history and the basis for the development of future evolutionary adaptations of a species ,2. PopulMeromyza saltatrix were studied in connection with the COI gene and morphological features . Ma. Ma38]. A total of 598 COI sequences  represeThe final aligned ITS1 sequence fragments were 607 bp. The average nucleotide composition was T = 31.2%, C = 14.8%, A = 36.5%, G = 17.5% and A + T = 67.7%. All sequences had a total of 20 polymorphic sites (3.29%), including 10 single variable sites and 10 parsimony informative sites. There were 8 transitions and 12 transversions, and the overall ts/tv bias was 0.639. The ts/tv rate ratio was observed to be higher with purines (2.402) than pyrimidines (0.116).C. oryzae populations had a total of 55 haplotypes, with an overall Hd of 0.346, k of 0.854, and Pi of 0.0012. The Hd, k, and Pi of each population ranged from 0.000–0.638, 0.000–3.985, and 0.0000–0.0052, respectively. LZ and NC populations had higher levels of genetic diversity   .C. oryzae populations had a total of 26 haplotypes, with an overall Hd of 0.750, k of 1.551, and Pi of 0.0026. The Hd, k, and Pi of each population ranged from 0.400–0.956, 0.400–2.911, and 0.0007–0.0048, respectively. Except for the YW and SZ populations, all populations had higher levels of Hd (Hd < 0.5) and lower levels of Pi (Pi < 0.005)  .C. oryzae population were performed based on COI and ITS1 sequences. The Tajima’s D and Fu’s Fs values of the total population were all negative, and all Fu’s Fs values were significant (p < 0.01)   . Besides 0.9050) .Genetic distances within and between populations were estimated based on COI and ITS1 sequences. For COI analysis, the inter- and intra-population genetic distance ranged from 0.0001–0.0060 and 0.0000–0.0056, respectively A, indicaC. oryzae populations was mainly from within populations, while less from among populations . Moreover, there was some degree of genetic variation within the overall populations  (The results of AMOVA suggested that the genetic variation in < 0.001) .p < 0.001).The Fst and Nm values between pairwise populations were calculated based on COI and ITS1 sequences . The FstThe Mantel tests based on both markers did not support a significant correlation between geographic distance and genetic distance, thus excluding the effect of distance segregation on genetic differentiation .To understand the relationships of identified haplotypes, the median-joining haplotype network was constructed . Among tGenetic diversity, which refers to the sum of genetic variation among populations within a species or individuals within a population , is a fuC. oryzae populations had higher haplotype diversity (Hd > 0.5) and lower nucleotide diversity (Pi < 0.005), suggesting that the population experienced a recent “Bottleneck effect” followed by a short period of rapid population expansion [Halyomorpha halys [Haplotype diversity and nucleotide diversity are the main indicators of genetic diversity. In this study, the ITS1 analysis presented that xpansion . Meanwhixpansion . This isha halys .38) implied that the population expansion occurred not so long ago as the Fu’s Fs values are more sensitive to recent population expansion [rg values [Historical population demographics is one of the core elements of molecular phylogeography. The analysis of historical population demographics is in favor of understanding the effects of external environmental factors on population development and distribution, and also provides a reference for developing pest management strategies . To thisxpansion . Likewisg values . Moreoveg values .Genetic distance, AMOVA , Fst valC. oryzae populations, which was in line with the previous study [It has been demonstrated that frequent gene flow can improve the population’s adaptability to the environment and cause outbreaks of pests . In thisus study . HoweverC. oryzae has been analyzed previously by Zhou et al. [C. oryzae as well, but using more geographic populations collected from a larger area, within which the ecological environment is more diverse and the annual occurrence generation of C. oryzae changes accordingly [C. oryzae populations have low or medium levels of genetic differentiation and experienced recent expansion events. However, disparities in the genetic diversity and experience of the “Bottleneck effect” were presented between two studies, probably due to the differences in sample size, molecular markers, and sampling locations.The genetic structure of u et al. . Likewisordingly . Both stC. oryzae has been subjected to highly toxic pesticides, such as furadan, oxamyl, and triazophos [C. oryzae might be caused by the abundant use of these pesticides. In addition, the recent expansion of C. oryzae may be related to changes in factors such as agroecological environments, tillage and cultivation systems, winter temperatures, and control agents [The “Bottleneck effect” refers to the dramatic variation of genetic structure owing to the sharp reduction in population size caused by deteriorated environmental conditions, such as farmland ecology and pesticide application levels . For theiazophos . We, thel agents .C. oryzae and farmland environments and farming practices may elucidate the causes of the “Bottleneck effect”, genetic differentiation, population expansion, and frequent outbreaks, thereby providing a theoretical basis for formulating management strategies.In the future, the investigations of the relationship between the population dynamics of C. oryzae populations suffered from a recent “Bottleneck effect”, followed by a rapid expansion. We also speculated that genetic differentiation and gene flow among populations are responsible for the geographical differences in the occurrence level.This study showed that


Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.The neglected tropical disease trichuriasis is caused by the whipworm  Trichuris trichiura is a soil-transmitted helminth that causes the neglected tropical disease trichuriasis in humans. Here, the authors produce whole genome sequences of modern and ancient samples from humans and non-human primates to characterise the genomic diversity and evolution of this pathogen.The whipworm  Trichuris trichiura, is a soil-transmitted helminth (STH) responsible for trichuriasis, a neglected tropical disease (NTD) estimated to affect as many as 500 million people worldwide1. As an intestinal parasite, an infection begins by the ingestion of embryonated eggs in contaminated food or soil; eggs migrate to the large intestine and hatch, after which emerging larvae burrow and establish an intracellular niche within intestinal epithelia2 where they develop into adult stages that can remain in situ for years3. Although low and moderate infection burdens typically remain asymptomatic, enabling efficient transmission in regions with poor sanitation such as endemic rural settings, chronic infections with high worm burdens cause a range of debilitating gastrointestinal symptoms and can lead to nutritional deficiencies and delays in physical and cognitive development, especially in children4. Infections with T. trichiura  are primarily treated with benzimidazole anthelmintics ; however, the efficacy of these drugs for the treatment of Trichuris is suboptimal when given as a single dose6 and few alternative drugs are available. In endemic regions, treatment is targeted towards pre-school and school-aged children, women of reproductive age, and high-risk working adults via annual or biannual mass drug administration (MDA) campaigns. In 2019, more than 613.1 million children, representing 60% of treatment coverage of children at-risk of STH infection, were treated with anthelmintics7. It is almost certain that this number will only increase in the near future, as STHs are targeted for elimination as a public health problem by 2030 in line with the WHO Sustainable Development Goals for NTDs8 and the WHO road map for NTDs 2021–2030.The human-infective whipworm, T. trichiura for millennia. Although now generally restricted to tropical and subtropical regions1, this parasite was once a globally distributed pathogen. Parasite eggs have been found in human coprolites (fossilised faeces) from archaeological sites dated back to 7,100 BC11, including locations in Europe and North America where autochthonous infections are now unusual16. However, whipworms are known to infect a broad range of mammals; over 70 species have been described within the genus Trichuris, and while generally host-specific, cross-host species transmission of individual parasite species has been reported, including between humans and pigs17, humans and dogs18, or humans and non-human primates23. Parasites that can interchangeably infect human and non-human hosts represent a credible challenge to control campaigns, as non-human hosts may act as a reservoir in which parasites can evade treatment and subsequently become a source of infections for treated populations. Understanding the historical and modern dispersal of T. trichiura in human and non-human hosts is likely required to achieve elimination. Further, the goal of eliminating STHs as a public health problem by MDA is threatened by the emergence of resistance to the benzimidazole compounds used to control them, as observed in other parasitic worms  frequently exposed to the drug24. Although evidence suggesting that resistance is emerging in T. trichiura is limited, understanding population connectivity within and between endemic regions will inform the likelihood and rate of resistance alleles spreading when they arise.Humans have been parasitised by T. trichiura. Using whole-genome sequencing data of modern whipworms collected from human and non-human primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits13, we describe broad-and fine-scale genetic structure, admixture, and population demographics of geographically and genetically distinct parasite populations. We also explore genome-wide evidence of local adaptation between different human worms and between human and animal worms. These genome-wide variant data will support future efforts toward implementing genomic epidemiology of this globally important STH.Here, we describe the first population genomic analysis of Papio hamadryas), two from colobus monkeys (Colobus guereza kikuyensis) and three from leaf monkeys (Trachypithecus francoisi). All modern samples were single worms, except for samples from Cameroon (n = 5) and Tanzania (n = 5) which were pools of eggs.We have generated whole-genome sequencing data from 44 modern and 17 ancient samples (Supplementary Data n = 17) derived from DNA extracted from pooled eggs were obtained from archaeological latrines and dig sites, primarily from Denmark (five locations) as well as from the Netherlands (two locations) and a single site in Lithuania, which individually have been dated to span the last thousand years , there was no significant impact of coverage variation on genetic diversity in any other population analysed  and KAM and ZWO in Holland (FST = 0.004), but more distantly located populations are more genetically distinct , suggesting little, if any, genetic connectivity to the populations in Denmark and Holland. However, this finding is consistent with the human genetic ancestry of Danish Vikings that predominantly travelled eastward, unlike Swedish Vikings that travelled more extensively into the Baltic region27. To understand the impact of time on these populations, we compared nucleotide diversity against the estimated age of the samples, revealing a small but significant loss of variation over time towards the present . The oldest and genetically distinct VIB samples were also among the most diverse; this relationship between diversity and time was robust to DNA damage artefacts , however, the trend was still negative but less pronounced and not significant when the VIB samples were removed . We note that this decline was well before modern interventions were used to control parasites such as T. trichiura and that poor sanitary conditions should have favoured transmission. However, the lower temperatures at northern latitudes would have significantly reduced eggs’ embryonation rate and viability in the environment, allowing for a maximum of one parasite generation per year. As a result, parasite population sizes may have undergone more significant fluctuations in such suboptimal conditions, including bottlenecks that would have reduced diversity relative to modern populations currently endemic.The generation of sequencing data from 17 ancient samples provided a unique opportunity to understand patterns of parasite diversity in Northern Europe over time. Measures of genetic differentiation between populations revealed some degree of isolation-by-distance, where geographically close populations were genetically indistinguishable, for example, COG and COA in Denmark  Fig. , broadlyT. suis and T. trichiura has been described previously in Ecuador28. We further quantified these admixture results using f-statistics on the nuclear variants  identified genetic similarities between parasites from China and Ecuador, which were distinctly different from those from Uganda17. Using baboon as an outgroup reduced our ability to measure more subtle differences between the closely related baboon, ancient, and Ugandan populations; to address this, we used Honduras as an outgroup, which provided greater resolution to show the closer relationship between the baboon and ancient samples, relative to comparison with the Ugandan samples.To quantify genetic admixture, we used Treemix to estimate migration between nodes under a maximum likelihood framework Fig. . Three mnts Fig. . Using bT. trichiura. Although we have made specific assumptions about important but unmeasurable parameters of the population demographic model — specifically, mutation rate and generation time — and therefore, the precision of these estimates is low, these data are consistent with bottlenecks in parasite populations that likely occurred as modern humans migrated out of Africa approximately 50–60 thousand years ago29. The intermediate f3 statistics between Uganda and China suggest a stepwise migration pattern, first from Africa to Asia and then from Asia to the Americas. This pattern would be consistent with human migration, the latter of which perhaps via a crossing of the Beringia land bridge that connected the two continents until approximately 11 thousand years before the present. This migration was distinct from that established in Europe, indicated by the lower degree of shared variation between ancient and Chinese samples but almost no sharing between the ancient and the Honduran samples. It has been argued that some intestinal parasites may not have survived the Beringia crossing due to unfavourable environmental conditions to sustain their lifecycle, and thus, the genetic connectivity between parasite populations may reflect additional migration to the Americas through trans-pacific or coastal routes9. To explore this here, we compared the proportion of variation shared between Ugandan, Chinese and Honduran samples. While a large proportion of variation was private  or shared by all three populations (26.8%), 3.2% of variants are shared between the Ugandan and Honduran populations that are absent from Chinese samples , which was consistent with the diversity expected (πX-linked/πautosome = 0.75) for an organism with a XXfemale/XYmale sex chromosome system. A genome-wide analysis of nucleotide divergence (Dxy) between populations revealed a strong correlation with nucleotide diversity within populations , we identified 373, 358, and 393 genes in region of high divergence (defined as the top 5%), respectively. Analysis of gene ontology (GO) terms (Supplementary Data p = 3.988e-2) and cation:chloride symporter activity . We extended this analysis to compare Ugandan and baboon samples, given their close genetic relationship despite being isolated from different host species. Genome-wide mean FST was intermediate (FST = 0.148) to that of the human-specific analyses, re-emphasising that despite divergence, baboon-infective whipworms are within the species-level of diversity expected of human-infective T. trichiura. Genome-wide, discrete regions of differentiation were observed, particularly on the sex-linked scaffolds , ISWI-type complex  and chromatin ; these terms are associated with chromatin remodelling and transcriptional regulation of developmental genes30 and may reflect selection on genes that require differential regulation during development between the different host species.Distinct geographic clustering of Ugandan, Chinese, and Honduran populations prompted us to examine evidence of diversifying selection that might indicate local adaptation within the human-infective parasites. Nucleotide diversity (π) varied between populations, with the highest diversity observed in Uganda (genome-wide median π = 0.0120), followed by China π = 0.0103), baboon (π = 0.0092), and then Honduras (π = 0.0079), consistent with a global dispersal of 3, baboonion Fig. . For eacTTRE_0000877201; Location: Trichuris_trichiura_1_001: 10684531-10686350) segregating within modern human populations; however, no variation within the codon positions P167, P198, or P200 that are typically associated with benzimidazole resistance was observed  and modern, non-European endemic regions16, and in Denmark, Trichuris infections were prevalent in children, particularly from rural areas up to at least the 1930s35. In contrast, the low genome-wide genetic diversity and declining nucleotide diversity over time of our ancient samples are consistent with a constrained and likely contracting population size. Whether this reflected actual population decline when the eggs were deposited, non-random sampling of genetically related parasite eggs within a defined archaeological site or non-random distribution of whipworm prevalence in the host population is unclear. However, the population demographics of these ancient northern European populations are likely different relative to populations found in more tropical conditions where it is currently endemic. The parasite development rate in the environment is temperature-dependent and decreases in cooler temperatures36, and below 15 °C, development arrests. This is particularly relevant for parasite populations such as those once endemic in northern Europe, as the proportion of the year during which favourable conditions for parasite development (and thereby transmission) decreases at increasing latitudes. Thus, suboptimal environmental conditions necessary for parasite development and infection may partly explain regional differences in parasite prevalence, including local regions of population decline and diversity. Genomic analysis of ancient parasites from southern Europe and Africa will likely shed light on the missing links connecting the northern European and African parasites analysed here and the relationship between genetic diversity and endemicity in response to a changing climate.Analyses of the genomic diversity of ancient parasites and their genetic connectivity to modern parasite populations unexpectedly revealed a close genetic relationship to Ugandan and Baboon samples. Unfortunately, the ancient samples consisted of pools of eggs rather than individual worms, limiting investigation of population demographics and estimated split times between ancient and these closely related modern populations. Epidemiological estimates of T. trichiura is specific to humans, baboons, and maybe some other non-sampled primates and emphasises that parasite demography will depend on its host. Population demographic analyses of human infective whipworms support the hypothesis that global parasite populations suffered severe population bottlenecks coinciding with human migration out of Africa and that differential proportions of genetic admixture between these populations provide a plausible hypothesis by which parasites continued to infect modern humans as they distributed throughout the world. We note that one potential source of independent introduction of T. trichiura from Africa to the Americas resulting in shared parasite ancestry would likely be associated with the mass forced migration of Africans during the trans-Atlantic slave trade. We hypothesise that this genetic signature would be greater between West African (for which we have no samples) and American parasites than between Central or East African parasites as sampled here and that West African parasites would be more suitable to test the degree of genetic admixture and thus impact of different migration events on the genetic diversity of endemic American parasites present today. Collectively, our data reinforce the important role that human migration has played in the spread of modern helminths throughout the world39.Our genomic analysis of modern human and animal parasites further supports that T. trichiura will be influenced by the degree to which humans and animals occupy and directly share ecological niches, for example, the ground or water supply. Baboons are large ground-dwelling primates that can be found close to human activity and likely share a common source of infection of STHs such as T. trichiura. This is in contrast to smaller, tree-dwelling non-human primates, including leaf and colobus monkeys, that would have less interaction with humans. However, genome-wide analyses between human and baboon parasites reveal discrete regions of the genome that differ between hosts, potentially indicative of some degree of host adaptation. We note that whipworms were collected from only three species of captive non-human primates, and we lack sampling data from other apes that live in proximity to humans, particularly in natural settings. Further sampling of human- and a broader range of animal-infective parasites, particularly where cohabitation exists, combined with genome-wide analyses of selection, should provide further insight into the mechanisms by which host-specificity is maintained and cross-species transmission is tolerated.Comparative analysis of human and animal infective whipworms demonstrate that worms isolated from baboons likely represent a zoonotic reservoir of human-infective parasites in Africa. Unlike many zoonotic transmissions that involve a vector species, the likelihood of cross-species zoonotic transmission of a soil-transmitted pathogen like T. trichiura) is known to select genetic variation in β-tubulin conferring resistance to treatment. This is particularly evident in veterinary helminths40, where selection for benzimidazole resistance has rendered benzimidazole derivatives almost ineffective in some regions of the world, particularly against helminths of horses and sheep24. Emerging evidence suggests that MDA may be exerting similar selection pressure on STHs43. We found no evidence for selection within or surrounding the β-tubulin gene. However, we do not have any data on their phenotypic response or exposure history to anthelmintic treatment, and thus, we cannot determine whether the absence of known benzimidazole-resistance associated variants in our data indicates that: (i) all populations are phenotypically susceptible to anthelmintic treatment, or (ii) populations may or may not be phenotypically susceptible to benzimidazole treatment, but that selection on variation elsewhere in the genome, and not on variation in the β-tubulin gene, is responsible for drug response in T. trichiura. Future studies exploiting genome-wide analyses on parasite populations that are phenotypically well-defined for drug response44 are needed to define the genetic architecture of drug response in T. trichiura. It will be particularly important to disentangle known variation in the tolerance to benzimidazole drugs by T. trichiura46 from the emergence of loss of efficacy due to high drug exposure6, especially if the promise of using molecular diagnostics to monitor the emergence of anthelmintic resistance is to be realised50.Large-scale treatment of parasites using benzimidazole anthelmintics  after being informed about the study in English and/or the local language. Samples were collected in line with ethical considerations and clearances as follows:Trichuris sp. eggs were isolated from ancient latrine samples in which a metagenomic study has previously identified DNA from T. trichiura13. The eggs were isolated from ancient human latrines or deposit sites in Denmark (1000–1700 AD), the Netherlands (1350–1850 AD), and Lithuania (1550–1580 AD)  by mitochondrial genome and nuclear genetic markers53, were collected directly from the caecum of a human patient during surgery in Zhanjiang People’s Hospital in Zhanjiang, Guangdong Province, China (CHN_GUA). These worms were sent to the Department of Parasitology, Guangdong Medical University, for identification.Trichuris colobae n. Sp.54, were collected from the caecum during necropsy of Colobus guereza kikuyensis (Mantled Guereza or Eastern Black-and-White Colobus) (ESP_MAL [zoo]), which died of natural causes at the Fuengirola Zoo .Worms, previously defined as 23.Worms from baboons were recovered from the caecum during routine post mortem examination of animals culled for management reasons at Copenhagen Zoo, DenmarkSamples were collected as a joint effort by Ana Sanchez , Gustavo A. Fontecha and Gabriela Matamoros , Honduras) (HND_OLA and HND_SAL). Sample collection protocols were reviewed and approved by the Brock University Bioscience Research Ethics Board and Comité de Ética de Investigación, Maestría en Enfermedades Infecciosas y Zoonóticas, Facultad de Ciencias, UNAH.Trichuris spp. samples were collected during necropsy from the caecum of a captive Trachypithecus francoisi (François Leaf-Monkey), which was humanely euthanised due to acute gastric dilation (People’s Republic of China) (CHN_GUA [zoo])55.56. Permission was obtained from the Ministry of Health and the National Council of Science and Technology in Uganda, and the Danish Central Medical Ethics Committee recommended the study. A subset of worms was recovered after anthelmintic treatment from a Danish patient infected with T. trichiura from Uganda (UGA_DNK) as previously described3.Worms were recovered after anthelmintic treatment from the faeces of children in Uganda (UGA_KAB) as previously described6. Permission was obtained from the National Ethics Committee in Yaoundé, Cameroon (Sep 2011).Stool samples were collected with a focus on monitoring the efficacy of mebendazole for the treatment of soil-transmitted helminths6. Permission was obtained from the Ministry of Health, Zanzibar Revolutionary Government, Tanzania (July 2012).Stool samples were collected as previously describedIndividual worms were washed extensively in tap water or saline solution and preserved in 70% ethanol. When possible, the sex of individual worms was determined through microscopic examination before DNA extraction.13.Ancient samples were processed for egg extraction in a dedicated paleoparasitological laboratory at the Department of Plant and Environmental Sciences (PLEN), University of Copenhagen. Helminth eggs were concentrated by selecting particles based on flotation in high-density liquid sedimentation and size filtration. Briefly, 10–503 g from each sample was manually homogenised using a sterile scalpel blade before separating into 50 mL tubes, each containing ~10–15 g. TE buffer  was added to each tube to resuspend and then incubated for 3–6 days with daily mixing. Samples were pelleted (3000 RPM for 5 min), the supernatant was removed, and the pellet was resuspended in flotation buffer (FB) (glucose monohydrate 375 g/L+ sodium chloride 250 g/L). Samples were centrifuged (400–1000 RPM for 5 min), and the supernatant was sieved on stacked, disposable, autoclaved, 100 µm and 22.4 µm nylon filters. All tubes from a particular sample were processed on the same set of filters. Flotation, centrifugation and sieving were repeated for the pellet, and the filters were washed in demineralised water. The material was collected from the 22.4 µm, suspended again in FB buffer, centrifuged (400–1000 RPM for 5 min) and sieved on a new set of stacked 35.5 µm and 22.4 µm filters. Filters were washed in demineralised water, and sediments were collected from the 35.5 µm filter (most often termed ‘sample ID’ followed by ‘A’) and the 22.4 µm filter (most often termed ‘sample ID’ followed by ‘B’). Sample material was washed three times in molecular grade water; 10% was then used for morphological examination, and the remaining 90% for DNA extraction. Morphological examination of eggs was performed on microscope slides at 100× and 400× resolution, and quantification of eggs was performed either on fixed microscope slides or McMaster counting chambers. The egg samples were then processed for DNA extraction in dedicated aDNA laboratories at the Centre for Geogenetics (CGG), University of Copenhagen, following strict aDNA-specific requirements. DNA extraction was performed using the PowerLyzer PowerSoil DNA isolation kit  with minor modifications, including bead beating performed using Lysing Matrix I beads  for 90 s at 6.5× on the FastPrep-24  and DNA was eluted in 60 µl elution buffer. The complete protocol has been described previouslyTM DNA Purification kit  at the Department of Veterinary Disease Biology (VDB), UCPH following the manufacturer’s protocol with the following exception: worms were first homogenised in 300 µL lysis solution using a matching disposable plastic pestle and then incubated at 56 °C for three hours; (ii) for the colobus samples, DNA extraction from whole adult worms was performed at the Departamento de Microbiología y Parasitología, Universidad de Sevilla, Sevilla, Spain, using a DNeasy Blood & Tissue Kit  according to the manufacturer’s protocol; (iii) for the leaf monkey samples, DNA extraction was performed at the Department of Parasitology, Lanzhou Veterinary Research Institute, Lanzhou, China as previously described55. DNA (400 ng) was fragmented to approximately 300–700 bp using a Diagenode BioRuptor using 2 or 4 cycles of 15 s ON/90 s OFF at instrument setting HIGH. Fragmented DNA was concentrated using a MinElute Reaction Cleanup Kit , after which the DNA concentration  and library size  was determined.DNA from modern worm samples were prepared as follows: (i) for samples collected from stool following treatment with anthelmintics, directly from a human patient via colonoscopy, and from baboon, DNA was extracted from whole adult worms using the MasterPure13. DNA libraries were amplified in a single PCR reaction of 50 µL using: half of the prepared DNA library (12.5 µL), 5 U AmpliTaq Gold , 1× buffer Gold, 2 mM MgCl2, 0.25 mM dNTPs, and 0.2 mM of primers , for 8–16 cycles depending on library strength. Libraries were sequenced using 100 bp single-end  or 100 bp paired-end  chemistry on an Illumina HiSeq 2000/2500 platform at The Danish National High-Throughput DNA Sequencing Centre (now Centre for GeoGenetics Sequencing Core).DNA sequencing libraries were prepared for all sample types, modern and ancient DNA extracts, using the NEBNext DNA Sample Prep Master Mix Set for 454  kit with a modified protocol previously described for ancient samples57 (version 2.3.0); for both SE and PE reads, adapters were removed and N-bases trimmed, and for PE reads, read 1 (R1) and R2 reads were collapsed where possible. Where multiple sequencing lanes of data were generated, trimmed read sets were merged before mapping.Raw reads were first processed using AdapterRemoval258 (version 0.7.17-r1188) to an unpublished but significantly improved reference genome of T. trichiura (available here: https://github.com/stephenrdoyle/ancient_trichuris/tree/master/02_data). Originally described by Foth et al.59, the new assembly used here is larger (80.57 Mb vs 75.49 Mb), more contiguous (N50 = 11.3 Mb [vs 0.07 Mb] & N50n = 2 [vs 265]), and in fewer scaffolds overall (n = 113 vs 4156) relative to the published version. Three linkage groups are defined via synteny with the Trichuris muris genome60 and are designated by the following prefixes in the reference assembly: Trichuris_trichiura_1 , Trichuris_trichiura_2 , and Trichuris_trichiura_3 . Scaffolds unassigned to linkage groups have the prefix Trichuris_trichiura_00. For the ancient samples, trimmed SE reads were mapped, whereas for the modern samples, trimmed PE and SE reads (merged PE and trimmed SE) were mapped independently and subsequently merged. For all samples, duplicate reads were identified using Picard MarkDuplicates .Mapping was performed using BWA-MEMhttps://github.com/pontussk/PMDtools)61. This analysis revealed bias in the terminal 2 bp, particularly in the ancient samples; to account for this, the mapped reads were trimmed by 2 bp for all samples (ancient and modern) using bamUtils trimBam62 (version 1.0.14). Genome coverage was determined using samtools bedcov (version 1.9) in 100 kb windows per scaffold; sex was determined using the coverage ratio between the sex-linked and autosomal scaffolds. To explore putative causes of mapping variation, we ran Kraken263 (version 2.1.2) to estimate the degree of contamination in the raw sequencing reads. This analysis showed that while each sample contained a small degree of contamination evidenced by hits to the Kraken database (minikraken2_v1_8GB), it did not explain overall variation in mapping. Quality control and quantitative comparison between samples were undertaken at each stage of the pipeline and visualised using MultiQC64 (version 1.8).Putative deamination damage  was assessed using PMDtools . To improve efficiency, each task was split by sample and by scaffold and run in parallel before merging to produce the final call sets.Variant calling was performed with GATK Haplotype Caller (version 4.1.4.1), first by generating per sample GVCF files , followed by joint genotyping of the sample cohort. Heterozygosity was adjusted from default settings based on estimates from raw reads using GenomeScopehttp://lh3lh3.users.sourceforge.net/snpable.shtml); regions of the genome where overlapping 35-mers mapped uniquely and without 1-mismatch were retained (75.02% of the genome was retained).The raw output was split into mitochondrial and nuclear variants and then split again into single nucleotide variants (SNV) and indels and filtered independently. A hard filtering approach was employed based on removing relevant tails of variant distributions  on the following quality metrics: QUAL, DP, MQ, SOR, FS, QD, MQRankSum, and ReadPosRankSum. Variants were further filtered to ensure they met the following criteria: minimum and maximum alleles = 2; minor allele > 0.02; Hardy Weinberg Equilibrium = >1e-6 (nuclear variants); per sample missingness > 0.8; per genotype depth > 3. Finally, variants were removed if they were found in regions of the genome where the reference genome-guided mapping of kmers (k = 35) was poor or not unique, determined using SNPable  to identify variable sites.The ancient samples underwent an additional round of variant calling using a pool-seq framework, using samtools mpileup and popoolation2 mpileup2sync 67 (version 3.15). These analyses were performed on both mitochondrial and autosomal nuclear variants separately and with and without the colobus and leaf monkey samples, which were outliers in all analyses.Broad-scale genetic relatedness between samples and populations was explored by principal component analysis using the R package SNPrelate68 , from which pairwise identify-by-state (IBS) matrices were analyses using multidimensional scaling. This approach also used genotype likelihoods per variant site , which are more sensitive to poorer quality and low coverage data.To explore the impact of sample heterogeneity on broad-scale relatedness and population structure, we subsampled the BAM files selecting a single allele at each variant site using ANGSDGenetic differentiation between ancient samples was performed using popoolation2 fst-sliding.pl (–pool-size 1000 --window-size 15000 --step-size 15000 --min-count 1 --min-coverage 2 --max-coverage 2%).T. trichiura , T. suis , T. discolor (“JQ996231 [https://www.ebi.ac.uk/ena/browser/view/JQ996231]”), T. ovis (“JQ996232 [https://www.ebi.ac.uk/ena/browser/view/JQ996232]”) and that of the Leaf Monkey, Trichuris sp. (“KC461179 [https://www.ebi.ac.uk/ena/browser/view/KC461179]”) reporting only the best hit. The published genome with the most hits was then used as a reference in guided assemblies using the mitochondrial baiting and iterative mapping approach, MITObim (version 1.8)69. Subsequent manual curation of the assemblies was performed to remove redundant, overlapping sequences at the ends of the mitochondrial contigs, and the starting position was adjusted to the first codon of the COXI gene. For the ancient samples, consensus mitochondrial genome sequences were previously called for ten samples  that provided 100% bases with coverage to the “KT449826 [https://www.ebi.ac.uk/ena/browser/view/KT449826]” T. trichiura reference sequence13. Consensus sequences were annotated using the MITOS Webserver70. Assembled mitochondrial genomes were aligned together with all Trichuris spp. reference genomes available at NCBI using mafft (version 7)71, with the global-pair setting at 1,000 iterations. A neighbour-joining tree was built using the Jukes-Cantor nucleotide distance model and 1,000 bootstraps in CLC Sequence Viewer 7 (Qiagen). The mitochondrial genome of Trichinella spiralis (“NC_002681 [https://www.ncbi.nlm.nih.gov/nuccore/NC_002681]”) was used as an outgroup.Preprocessed sequence data from modern samples were mapped to published mitochondrial genomes of 72. Briefly, genotype likelihoods were extracted using vcftools , after which NGSadmix (-P 4 -minMaf 0.05 -misTol 0.9) was run over a range of K values from 2 to 10. The optimal value of K was determined by iteratively running NGSadmix as above five times, changing the seed value on each run. The log-likelihoods of each run  were used to determine the optimal value of K using Evanno’s method73 on the Clumpak web server74.Admixture was determined using NGSadmix from the ANGSD package75 (version 1.12) was performed on the nuclear dataset, which included leaf and colobus monkey samples as outliers. Variants were first filtered using vcftools (--max-missing 1), followed by further pruning to minimise variants in linkage disequilibrium. Customs scripts (ldPruning.sh & vcf2treemix.sh) were modified from https://github.com/speciationgenomics/scripts. The optimal number of migration edges was estimated using the R package OptM (https://cran.r-project.org/web/packages/OptM/).Treemixf-statistics were calculated using ADMIXTOOLS76qp3Pop (version 435) to calculate outgroup f3 data. All combinations of source 1 and source 2 populations were determined, using either baboon or Honduras samples as the outgroup. Customs scripts (convertVCFtoEigenstrat.sh) were modified from https://github.com/joanam/scripts. Standard error was calculated using a weighted block jackknife, using three blocks. For each comparison, a Z score was determined to test the deviation from 0 ; a Z score of three or greater was deemed significant.77 (version 1.15.2). For each population, variant sites present in all individuals were extracted using vcftools (--max-missing 1), after which smc++ vcf2smc was run per scaffold. Estimated population sizes were fit to the data for all scaffolds using smc++ estimate, which used the nematode C. elegans mutation rate of 2.9e-9 mutations per site per generation78 as a proxy for T. trichiura mutation rate, which is currently unknown. Finally, the effective population size per generation was scaled based on an estimated generation time; while unknown precisely, the prepatent period has been estimated to range between 13 to 16 weeks3 and, therefore, we chose a period of three months or four generations per year. As the ancient samples were a pooled population of eggs rather than individual parasites, they violated the assumptions of the model and were, therefore, removed from the analysis.Population demographics were determined using SMC++79.The kinship between samples within populations was assessed using vcftools (--relatedness2) and NGSRelateST and Dxy was determined using pixy80 (version 1.2.6.beta1) in 20 kb non-overlapping sliding windows. The input to pixy was an all-sites VCF containing both variant and non-variant sites, which was derived from the per sample gVCF files generated by GATK (described above). To improve the visualisation of the genome-wide comparisons, analyses were restricted to scaffolds in chromosomal linkage groups, representing 89.83% of the whole genome assembly. Mitochondrial nucleotide diversity of the ancient samples was assessed by generating individual pileup files from the multisample mpileup, followed by analysis using npstat81 .Genome-wide nucleotide diversity per population and pairwise FT. trichiura assembly available in WormBase ParaSite82  using liftoff83 (version 1.5.1). By retaining gene identifiers from the original assembly, cross-validation of gene hits within WormBase ParaSite could be performed. In total, 8,451 of 9,650 gene features (~88%) were transferred; of the genes that did not transfer, ~80% were classified as contamination based on hits to the uniprot_reference_proteome database using DIAMOND84 (version 0.9.14).The annotation of the unpublished genome assembly used in this analysis was not available at the time of analysis. To identify genes within outlier regions of the genome-wide analyses, we performed a liftover of existing gene models and gene identifiers from the published version of the ST outliers using gProfiler85, applying a g:SCS multiple testing correction method and a significance threshold of 0.05, restricting the complete geneset to only genes that had successfully been transferred from the original annotation. GOterms for T. trichiura genes can be obtained from WormBase ParaSite using BioMart or can be used directly within gProfiler by selecting the T. trichiura genome version and uploading the list of gene identifiers.Gene ontology term (GOterms) analysis was performed on the top 5% of FAnalysis of variation in β-tubulin was performed using vcftools (--site-pi --maf 0.01), using a bed file of exon coordinates derived from manual curation of the gene.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


Monopterus albus) is one of the most widely distributed freshwater fish in China. In this study, we identified the single nucleotide polymorphisms (SNPs) of M. albus from 19 wild populations in China using restriction-site associated DNA sequencing (RAD-seq), and used SNP markers to investigate the swamp eel the genetic diversity and population genetic structure. A total of 8941794 SNPs were identified. Phylogenetic and principal component analysis suggested that the 19 populations were clustered into four groups: The Jiaoling County (JL) and Poyang Lake (PYH)populations in Group Ⅰ; the Chengdu City (CD), Dali City (YN), Eli Village (EL), Dongting Lake (DTH), Huoqiu County (HQ), and Chaohu Lake (CH) populations in Group Ⅱ; the Puyang City (PY), Chongming Island (CM), Tai Lake (TH), Gaoyou Lake (GYH), Weishan Lake (WSH), Haimen City (HM), Hongze Lake (HZH), Baiyangdian Lake (BYD), Dagushan (DGS), and Pinghu City (PH) populations in group Ⅲ; and the Lingshui County (LS) populations in Group Ⅳ. All 19 populations may have evolved from four ancestors. The genetic diversity was relatively high in CM, GYH, and HM; and low in LS, EL, and JL. The LS, and CM populations had the highest and lowest differentiation from the other populations, respectively. These findings provide new insights for germplasm resources protection and artificial breeding of M. albus.The Asian swamp eel ( Monopterus albus), which is commonly found in rice fields, muddy ponds, and swamp areas  are a highly popular type of marker . Then, we analyzed their genetic differentiation, population genetic structure, and genetic diversity based on the SNP data, with the aim of accelerating the process of molecular breeding in swamp eels.In this study, we identified SNP markers from 19 A total of 137 wild eels were collected from 19 sampling points in China . One GraUsing the method detailed by via their unique nucleotide barcodes. The read quality was examined using Fastp software -realignment and mark-duplication, and identifies variants across all samples simultaneously using the HaplotypeCaller program in GATK 4.1 . The ML bootstrap (MLBS) analyses were conducted using FastTree with 1,000 replicates under the GTR + CAT model. We also performed principal component analysis (PCA) based on SNP data from 137 individuals. This analysis was carried out in the GCTA software  algorithm in the software .To cluster each ancient genome in the study populations, we used the unsupervised maximum-likelihood clustering algorithm in the ADMIXTURE package . For theSTACKS pipeline, with default parameters (π).The POPULATIONS program in the rameters ; 2013, wA total of 8941794 SNPs were detected in the 19 eel populations, and the number of SNPs in the four detected genotypes in each population are shown in http://dalexander.github.io/admixture). When the K value was 4, the population genetic structure was closest to the real situation. According to The genetic diversity indices of the 19 eel populations were shown in M. albus lacks typical morphological characteristics, e.g., pectoral, pelvic, and caudal fins; therefore, molecular markers are effective to study its phylogeny and taxonomy  evolved from one single ancestor. However, in another study, the JX population was shown to be a common descendant of both ancestors and to share the genetic information of clade A with other populations . In fact, during our survey in Gushan county, we found that the local population had little enthusiasm for eels as food. Therefore, it can be deduced that the genetic diversity of M. albus was almost unaffected by artificial introduction.Previous study indicated that the lineages of coastal ulations . In thislineages . This reM. albus collected from the Yangtze River Delta, including Tai Lake, Hongze Lake, Gaoyou Lake, Haimen City, and Chongming Island, had the highest genetic diversity, with a Pi value ranging from 0.15 to 0.19. Moreover, these populations shared the genetic characteristics of both inland ancestor Ⅱ and coastal ancestor Ⅲ. This feature was similar to the results of other studies  and islands (LS). There was a close relationship between southwest populations  and the Anhui population. The coastal M. albus were distinctly differentiated from the inland populations. Therefore, it can be estimated that the genetic diversity of wild population was high and the germplasm resources were excellent. Further measures should be taken to protect these good wild resources and to convert them into artificial breeding parents, to make sustainable use of the wild germplasm resources of eel.In conclusion, there was significant genetic differentiation of


Ramonda includes three Paleoendemic and Tertiary relict species that survived in refugial habitats of the Balkan Peninsula  and the Iberian Peninsula (R. myconi). They are all “resurrection plants,” a rare phenomenon among flowering plants in Europe. Ramonda myconi and R. nathaliae are diploids (2n = 2x = 48), while R. serbica is a hexaploid (2n = 6x = 144). The two Balkan species occur in sympatry in only two localities in eastern Serbia, where tetraploid potential hybrids (2n = 4x = 96) were found. This observation raised questions about the existence of gene flow between the two species and, more generally, about the evolutionary processes shaping their genetic diversity. To address this question, genetic markers (AFLP) and an estimate of genome size variation were used in a much larger sample and at a larger geographic scale than previously. The combination of AFLP markers and genome size results suggested ongoing processes of interspecific and interploidy hybridization in the two sites of sympatry. The data also showed that interspecific gene flow was strictly confined to sympatry. Elsewhere, both Ramonda species were characterized by low genetic diversity within populations and high population differentiation. This is consistent with the fact that the two species are highly fragmented into small and isolated populations, likely a consequence of their postglacial history. Within sympatry, enormous variability in cytotypes was observed, exceeding most reported cases of mixed ploidy in complex plant species (from 2x to >8x). The AFLP profiles of non-canonical ploidy levels indicated a diversity of origin pathways and that backcrosses probably occur between tetraploid interspecific hybrids and parental species. The question arises whether this diversity of cytotypes corresponds to a transient situation. If not, the question arises as to the genetic and ecological mechanisms that allow this diversity to be maintained over time.The genus  Alyssum  includes three species—R. myconi (L.) Rchb., R. nathaliae Pančić & Petrović and R. serbica Pančić. All three species are considered relicts of the Tertiary  indicates that the three species may be paleopolyploids  with small enclaves in E Serbia (the Black Sea watershed). In contrast, R. serbica is mainly present in the areas of the Adriatic watershed  and to a smaller extent in the Black Sea  and the Aegean watersheds  and genome size (2C = 5.14 pg) were found at both locations  to generate genetic profiles (using AFLP markers) of individuals within these two zones to test whether there is gene flow between the two species and to gain further insight into the genetic mechanisms that might explain the observed cytotype diversity; (3) to assess the genetic diversity and population structure of both species and their genetic differentiation using AFLP markers on a sample of monospecific populations covering most of their range in the Balkans. Knowledge of these genetic patterns is essential to provide a reference situation for the analysis of gene flow between the two species and especially in the two local areas of sympatry.The aims of the present study were: (1) to investigate the variation in genome size within the two locations of sympatry between Ramonda nathaliae and R. serbica from monospecific populations, and a significant sample of individuals from the only two sites of sympatry known to date, both located in E Serbia  as an addition to previously published chromosome numbers. This was done using pretreatment with 0.002 M 8-hydroxyquinoline for 4.5 h at 16°C, followed by cold fixation in 3/1 (v/v) ethanol/acetic acid for 24–48 h, hydrolysis for 14 min at 60°C, staining with Schiff reagent  as an addition to previously published results, as well as in two localities where these species grow in sympatry. Total nuclear DNA amount was determined by flow cytometry according to Pisum sativum cv. Long Express (2C = 8.37 pg) and Lycopersicon esculentum cv. Roma (2C = 1.99 pg) were used as internal standards. Nuclei were stained using propidium iodide, and measurements were done by Elite ESP flow cytometer  equipped with argon laser. Both methods are described in detail by Genome size was estimated for the first time in ten monospecific populations . The material collected from individuals in sympatry consisted of leaves from 26 individuals potentially corresponding to R. serbica, 29 to R. nathaliae, and 40 individuals with ambiguous phenotypes at Oblik, and 11 individuals potentially corresponding to R. serbica, nine to R. nathaliae, and 35 individuals with ambiguous phenotypes at Radovanski Kamen. At each location of sympatry, sampling was conducted along a transect across the entire population. We systematically avoided sampling adjacent plants to eliminate the possibility of sampling clonal individuals since vegetative reproduction also occurs  according to the manufacturer’s instructions. AFLP analyses were performed according to the method described by Fragments of leaves dried in silica gel weighting 21–26 mg were used for DNA extraction, which was performed using the NucleoSpinThree primer combinations were selected for the full analysis (fluorescent dyes in parentheses): in both studies, EcoRI-AAC/MseI-CTC (FAM) and EcoRI-CAG /MseI-CTC (VIC) were used, whereas the third primer pair was different: EcoRI-ACA/MseI-CTC (NED) was used in the first AFLP experiment (monospecific populations only), and EcoRI-CAG/MseI-CTT (VIC) in the second experiment (hybridization study).Amplified fragments were detected using the ABI Prism 3,100 sequencer on the Gentyane platform  and analyzed by GENESCAN 1.1 software (Applied Biosystems). DNA fragment detection results were indicated as present (1) or absent (0) by GeneMapper software version 4.0. Only unambiguous peaks in the size range of 50 to 500 bp were scored, with the minimum peak height set at 30. Each peak from each profile was checked by eye and its status changed accordingly. Finally, singletons  were removed to increase the reliability of the AFLP data.R. nathaliae and two from R. serbica, were used for the preliminary repeatability test. They were duplicated from the same amplification and then, genotyped with the same mixture of formamide and molecular weight marker. In this way, the reproducibility of the detection of the AFLP profiles was estimated (97.15%). In the second test, twenty individuals were amplified twice with two independent mixtures and the percentage of amplification reproducibility was 99.07%.To ensure the best possible reliability of AFLP markers, all samples from each AFLP experiment were managed in a single manipulation . Two types of repeatability tests were performed. Four individuals, two from Because the samples from the two AFLP experiments were managed separately, the two datasets were not combined for the population genetic analyses to ensure better reliability of the analyses.Ramonda species. For small sample sizes, nonparametric tests were used . For pairwise comparisons, p values were corrected according to the Holm correction for multiple testing. Student t-test and Welch test (when homoscedasticity was rejected) were used for mean comparisons for larger sample sizes (n > 15).Statistical analyses of genome size were performed in R4.1.2. For monospecific populations, each species was analyzed separately because genome size values differ greatly between the two AFLP-SURV 1.0  was usedGenetic dissimilarity between individuals and populations was estimated using Shared Allele Distance  with POPhttp://cmpg.unibe.ch/software/arlequin35/Arlequin35.html). To test for correlations between geographic distance and genetic variance, a Mantel test was performed using the same software. The significance of Φ-statistics was tested based on the distributions of Φ-statistics calculated under the null hypothesis from 1,000 virtual samples obtained by random permutations of the original data set.The distribution of genetic variation  was estimated by analysis of molecular variance (AMOVA) performed in Arlequin 3.5.1.2 (K), where K varied from 1 to 12, with a burn-in period of 50,000 iterations followed by a run length of 120,000 iterations of the Markov chain. The most probable number of groups was determined using the method of The genetic structure of the monospecific and the two populations of sympatry was studied using genetic model-based and non-genetic model-based methods. For the first type of analysis, Bayesian inference of genetic clusters and assignment of individuals was performed using STRUCTURE v.2.3.4 . STRUCTUIn addition, and to evaluate the robustness of the classification used, a Discriminant Analysis of Principal Components DAPC,  implemenQ-values) estimated by the Bayesian clustering analysis in sympatry and monospecific populations, according to ploidy levels inferred from genome size.Finally, in order to integrate cytogenetic data and genetic inferences, we provided distributions of ancestry coefficients values .As expected, chromosome number showed that all individuals sampled in monospecific populations of R. nathaliae and 7.83 pg in R. serbica , pairwise Nemenyi comparison tests yielded only three significant comparisons, all involving the population from Mt. Kozjak (N8) with slightly larger GS compared to other populations. There were also significant differences in genome size values between populations of R. serbica . However, this was the result of differences between populations with the smallest (Mt. Timfi and Lazareva Reka) and populations with the largest (Nisava and Crna Reka) genome size values.The average 2C DNA value was 2.33 pg in R. nathaliae, and 235 fragments were private to R. serbica. However, none of these fragments was fixed for alternative alleles in the two species . Low frequency fragments dominated in both species. The number of fragments detected was moderately higher in R. serbica than in R. nathaliae (938 in the former and 866 in the latter), a trend expected due to the larger genome size of R. serbica.In the first molecular study, a total of 1,077 AFLP fragments were obtained with the three primer pairs. Once singletons excluded 163 fragments were present only in R. serbica (mean Hj = 0.105) and R. nathaliae , and the highest values were found in R. nathaliae from Matka in North Macedonia (B) and R. serbica from Mt.Timfi in Greece (L).Within-population genetic diversity was moderately similar in Φct = 0.718, see Φsc = 0.318). However, AMOVA analyses for individual species showed that population genetic differentiation was much higher in R. nathaliae (Φst = 0.432) than in R. serbica (Φst = 0.244).AMOVA and PCoA analyses showed that most of the genetic diversity within the entire sample are explained by genetic differentiation between the two species, as would be expected in the case of truly independent evolutionary lineages  and Demir Kapija (D) and the highest between Mt. Suva Planina (A) and Matka (B) , at the southern edge of the species’ range, and Godulja (P) in SW Serbia, were genetically the most distant from the others  on one side and southwestern populations from Greece, North Macedonia, and Montenegro  on the other , but was significant in R. serbica . However, this relationship is mainly due to the differentiation between northern and southern populations of this species. Overall, geographic distance between populations explained their genetic distances poorly, as can be clearly seen for northern populations G and F on the one hand, and M, N, O, and P on the other.Total genetic diversity (Ht) was equally low in both species with values of Ht = 0.116. Among the populations of atka (B) . In R. se others . PCoA anhe other , 2B. TheR. serbica between two populations in E Serbia (Radovanski Kamen (F) and Mt. Rtanj (G)) that were only 50 km apart, and also between two geographically very close populations (I and J), both from Mt. Rumija in Montenegro.DAPC analysis also showed that the vast majority of individuals of both species could be unambiguously assigned to their population of origin . There wR. serbica individuals from several populations  showed a slightly intermediate genetic profile between the two species . The GS of this group was also not significantly different from the mean genome size of R. nathaliae estimated from monospecific populations . Thus, the plants in this group certainly corresponded to diploid individuals.Individuals from both locations of sympatry showed tremendous but discontinuous variability in genome size . A firstR. serbica , it remained within the range of variation of R. serbica. Pairwise comparisons showed that only the comparison with the Lazareva Reka population was at the threshold of significance (data not shown). Therefore, this group of individuals was likely hexaploid at both sites of sympatry.A second group of individuals had a mean genome size of 7.98 pg (range between 7.48 and 8.45 pg). This group showed no significant difference between Oblik and Radovanski Kamen . Although the mean GS of this group at the two sites was significantly higher than the mean of the monospecific populations of cf. Class I in Ramonda species. This supported that these individuals are interspecific tetraploid hybrids. It is noticeable that a few individuals exhibited a combination of morphological characteristics of the two species   had a higher genome size than the hexaploid R. nathaliae and tetraploid interspecific hybrids  that were used as references in addition to the individuals sampled at the two sites of sympatry . After rR. serbica and R. nathaliae would have been expected because of the genome size and ploidy level of R. serbica. In fact, the distribution of the difference in fragment frequencies between the two species was clearly centered at zero  (A strong contrast in AFLP fragment frequencies between = 0.049) . Thus, tΦct = 0.548; Φct = −0.009) was observed between diploid and hexaploid individuals from the two sites in sympatry, as also evidenced by the strong mixing of individuals from both ploidy levels in the dendrogram (Φst = 0.22 for diploids and Φst = 0.10 for hexaploids). Consistent with this result, clusters of individuals from each location of sympatry, regardless of their ploidy level, are clearly visible in The strong genetic differentiation between the two species, already reported in the large-scale geographic study, was confirmed when only monospecific populations were considered (ndrogram . The tetK = 2 (data not shown). This result was to be expected, since species differentiation is the factor that contributes most to the genetic structure of the population in the entire data set. Indeed, from the data of monospecific populations, it appears that the two genetic clusters correspond to one of each species , had an intermediate ancestry coefficient . All but one of them were sampled in the Oblik site of sympatry. The remaining 18 diploid individuals had very high ancestry coefficients to R. nathaliae, as expected based on their assumed ploidy level . In contrast, among hexaploid individuals, five of the total 30 individuals, all from Oblik, showed strong ancestry to R. nathaliae, contrary to what was expected based on their genome size.The two sites of sympatry showed a very different and more complex genetic pattern, similar to what was observed for genome size. The main difference with the monospecific populations was that there was much less agreement between the genome size values (and thus the inferred ploidy level) and the AFLP profiles. In fact, when 2ytotypes . SurprisR. serbica populations. This also testifies to the high genetic heterogeneity of tetraploid plants. The mean ancestry value of the 40 individuals belonging to the right side of the distribution was 0.80, very close to the value of 0.75 expected in the case of a first-generation hybrid resulting from a cross between hexaploid R. serbica and diploid R. nathaliae. However, ten individuals, all from Oblik . Their mean ancestry value to “serbica” genetic cluster was 0.05 (range: 0.01–0.2), very close to the value obtained for R. nathaliae individuals from monospecific populations.The bimodal character of the distribution of the ancestry coefficient distribution was also observed for tetraploid individuals , althougom Oblik , formed R. serbica but higher (between 5.61 and 6.55 pg) than potential 4x individuals  were unambiguously assigned to R. nathaliae.Apart from these three groups of individuals, eight individuals from Radovanski Kamen had rather mixed genetic profiles with a GS lower than It is noticeable that individual assignment posterior probabilities inferred by the DAPC analysis and ancestry coefficients inferred through the Bayesian approach were highly correlated .Both GS and AFLP patterns showed that more complex genomic structures than first-generation interspecific tetraploid hybrids are present at both sites of sympatry.Ramonda species. Although n = 36 to be the most frequent haploid chromosome number in R. nathaliae, later both R. nathaliae is a diploid with 2n = 2x = 48 chromosomes, and this is confirmed by broad and detailed cytogenetic studies by R. serbica based on the following reports: 2n = 72 in R. serbica individuals collected in Sićevo Gorge in E Serbia . It is therefore likely that the very low genetic diversity of this population is the result of a very small effective size, due to the combined effects of small size and potential selection.This pattern of variability is expected in long-living plants with geographically isolated populations and probably with limited seed dispersal, as is the case in  species . The ovePyrenees  and H. rd Greece , 2015. IR. nathaliae and R. serbica may have occurred in the middle or last Pleistocene. This divergence is one of the youngest divergence times among Gesneriaceae species estimated in this study. This could also mean that the genus Ramonda is a relict of the Tertiary, but that the individual species are actually younger.The current ranges of these two species do not overlap, and only two narrow areas of sympatry have been observed so far . This obAster amellus L., for example, diploid and hexaploid cytotypes are usually found in separate locations, while a few parapatric and one mixed-ploidy population have been observed. Based on fine-scale cytotype screening, the authors concluded that there is no evidence for the existence of tetraploid plants  probably as a consequence of the exclusion of minority cytotypes (Ramonda is the case of Senecio carniolicus Willd. (syn. Jacobaea carniolica (Willd.) Schkrank), an autopolyploid complex with diploid and hexaploid plants as the most widespread ploidy levels. In contrast to Ramonda species, a high number of mixed ploidy populations were found in this species  are also observed, but at very low frequency. The authors noted that there should be barriers limiting the production and/or maintenance of interploid hybrids, to which the distinction between 2x and 6x habitats contributes. Lower fitness of progeny from crosses between diploids and polyploids also clearly contributes to limited gene flow between cytotypes  belonging not to one but to two different species. This makes hybridization more difficult and the genome constitution of the hybrids extremely diverse and sometimes difficult to explain. The case of Ramonda documented in this article therefore stands out from most studies of mixed-ploidy populations. It is interesting because, first, it shows enormous variability in genome size at micro-level in the only sites of sympatry between two species. This variation in GS allows us to hypothesize that the observed ploidy levels are also diverse, ranging from at least 2x to 8x (or possibly up to 10x), including odd ploidy levels such as at least 3x and 5x. For example, the class III of genome size  of tetraploid individuals, resulting in potential stabilization of the hybrid genome, a phenomenon already suggested by several authors  appears to be very high in these two sites of sympatry compared with most previously reported studies  the presence of individuals with an intermediate genome size compatible with the expected value for F1 tetraploid hybrids given the genome sizes of the two parental species; (ii) the presence of tetraploid individuals combining morphological features of the two species; and (iii) the observation that diploid and hexaploid individuals at the two sites of sympatry differed genetically much less (based on AFLP markers) than in monospecific populations. Several factors make this hybridization between the two species in sympatry possible. Both species flower at the same time. Although the flowers are slightly different, they are most likely pollinated by the same pollinators . This latter possibility would imply that synapsis occurs between the chromosomes of the two species, which remains to be proven. However, in that case, one would expect to observe a continuous distribution of ancestry coefficients within the tetraploid group, from “nathaliae” to “serbica” and all possibilities in between. Data reported here showed a different pattern with a clear bimodality in this distribution. The coexistence of divergent genomes in interspecific hybrids is considered as a major mechanism of molecular and phenotypic novelty. This is explained by the huge structural and functional variation caused both by the change in ploidy level, if any, and by novel genetic interaction effects generated between the divergent genomes that merge  gametes of R. nathaliae may also be involved in the production of tetraploid individuals that possess only chromosomes of R. nathaliae by autopolyploidy. The existence of such tetraploid individuals could also open the possibility of obtaining 6x progeny with only R. nathaliae genomes, the presence of which is suggested by the AFLP data. This hypothesis deserves attention because such tetraploid “nathaliae” would have inherited the ability to produce unreduced gametes  from their R. nathaliae diploid parent.The high number of tetraploid plants and their high AFLP diversity, similar to that of diploid and hexaploid plants in both sites of sympatry, suggests that hybridization between the two species is not an uncommon event in these sites. Alternatively, it is possible that only a few founders of tetraploid hybrids are at the origin of all currently observed tetraploid plants and that preferential breeding among plants of this cytotype has provided the opportunity for genetic recombination between the genomes of erge see , limitinerge see . It is tnditions . In addinditions  and therdization . SecondlTherefore, the AFLP profiles of the tetraploid individuals may indicate the existence of an autopolyploid-allopolyploid mixture at both sites of sympatry. They also suggest that there could be some barriers to reproduction between the two tetraploid hypothetical types, since otherwise tetraploid individuals with a much higher genome admixture would arise in the progeny from such crosses, which was not observed. However, the few individuals in our sample that exhibit such an intermediate AFLP profile do not allow us to rule out this last possibility. If this is indeed the case, the tetraploid level could also be a bridge between the two species, reinforcing interploidy gene flow in sympatry.x gamete from R. nathaliae and a 3x gamete from a hexaploid. These pentaploid individuals could also play a role as mediators of gene flow, but without forming an independent evolutionary unit  and a hexaploid, or even from the union of an unreduced 2ary unit .x plants could produce haploid gametes with one R. serbica complement, a pathway for the production of 2x progenies with the genetic feature of R. serbica, an intriguing pattern we observed in our data.Obviously, other pathways could be invoked to explain all patterns of GS and AFLP profiles observed in this study. For example, it has been observed  that hybR. nathaliae, but also in triploids and tetraploids. In particular, a more detailed genetic study of the genetic profiles of the plants found at the two sites of sympatry, based on large-scale sequence data, should be useful to assess the extent of genome mosaicism with respect to each cytotype. This would help to assess the extent of genetic introgression between the two species and the potential for recombination between their genomes. However, it is already clear that hybridization between the two species is occurring and that, at the very least, backcrossing (in which tetraploid hybrids give rise to pentaploids) is possible. Unfortunately, backcrosses with one or both parental species often result in the appearance of individuals whose morphology is difficult to distinguish from the parental species, and identification of hybrids based on morphology becomes very difficult or impossible , unless there were counteracting factors . Vegetative reproduction may also contribute to their maintenance (Our data (both cytotypes and AFLP markers) therefore suggest that the first-generation interspecific hybrids are viable and possibly fertile. It has already been shown that pollen grains from tetraploid individuals are viable, although somewhat less so than pollen from parental cytotypes in sympatry . On the ulations , which culations . The larntenance . It is antenance .Ramonda in the Balkan Peninsula. The turbulent geological past of the Balkan Peninsula, together with climatic fluctuations throughout the Mediterranean, has influenced the distribution, ecology, speciation and genetic diversity of Ramonda species, leading to the formation of isolated populations in refugial habitats. During migrations favored by interglacial periods, a secondary contact zone between R. nathaliae and R. serbica formed in a narrow area in E Serbia. In this newly formed sympatry, an intense hybridization process takes place between the two species, leading to the occurrence of different ploidy levels (from 2x to > 8x) and individuals with mixed morphological characteristics, suggesting that not only crosses between the parental species occur, but also backcrosses and possibly duplication of the whole genome. These two sites of sympatry are places for a huge genetic mixing. Moderate pollen fertility, but low seed germination and low dispersal potential suggest that the effects of hybridization are limited to the sites of sympatry. However, the rich mixture of ploidy levels shows that these relict species, which have preserved a specific survival strategy, are not evolutionary “dead ends” and raises the question of possible ecological and evolutionary consequences of this phenomenon. These two locations constitute therefore an open-air laboratory for evolutionary and interspecific recombination studies.The present study shows the complex influence of various factors on the evolution of two representatives of the genus The original contributions presented in the study are included in the article/SS-Y, BS, VS, and TR designed the study. ML, SS-Y, BS, VS, TR, AS, MN, and GT collected plant material. ML and SS-Y performed cytometric measurements and chromosome counts. ML, AS, and FL performed molecular laboratory work and provided data for statistical analyses done by TR and DH. ML, TR, and SS-Y drafted the manuscript. All authors read, revised, and approved the final version of the manuscript.This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (No. 451–03–68/2022–14/200178), the International Franco-Serbian “Pavle Savić” project, and the Federative Institute of Research (IFR 87 La Plante et son environnement) at Gif-Orsay. The l’Institut Français de Serbie provided grants to ML.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Felis silvestris subspecies) and the peoples of developing agrarian societies in the Fertile Crescent. As humans transitioned from hunter-gatherers to farmers ~12,000 years ago, bold wildcats likely capitalized on increased prey density . Humans benefited from the cats’ predation on these vermin. To refine the site(s) of cat domestication, over 1000 random-bred cats of primarily Eurasian descent were genotyped for single-nucleotide variants and short tandem repeats. The overall cat population structure suggested a single worldwide population with significant isolation by the distance of peripheral subpopulations. The cat population heterozygosity decreased as genetic distance from the proposed cat progenitor’s (F.s. lybica) natural habitat increased. Domestic cat origins are focused in the eastern Mediterranean Basin, spreading to nearby islands, and southernly via the Levantine coast into the Nile Valley. Cat population diversity supports the migration patterns of humans and other symbiotic species.Cat domestication likely initiated as a symbiotic relationship between wildcats ( Felis silvestris catus; Felis catus  genotypes  sequences of 2604 base pairs from F.s. bieti) suggest the noted introgression of this wildcat with random-bred cats in China does not explain the distinctive genetics of Far Eastern and Western European random-bred cats; further, the agricultural center near the middle Yangtze and Yellow Rivers is likely not a second domestication site for cats  swabs, Flinders Technology Associates (FTA) Cards , gonads from neuter clinics, and donated EDTA whole blood samples. DNA isolations were conducted following the manufacturer’s protocol using the method appropriate for the sample, including QIAamp DNA blood mini kits, Qiagen DNA Easy kits , organic extractions or methods for FTA card blood spots  from a previous study included random-bred cats from 17 locations (Supplementary Tables F.s. lybica) were collected as part of other studies from Western Sahara, Morocco, Tunisia, and Mauritania, and provided as extracted and whole genome amplified DNA  has been previously published  were selected to represent all autosomes from the 1.9× coverage cat genomic sequence, which were defined by one Abyssinian cat  was performed for the SNP data with the smartpca program from the EIGENSOFT package . Removal of admixed locations was done to strengthen the relationship between modern samples and ancient processes by removing more recent admixture events. For the SNP data, 24 sample location populations were analyzed, not including the F.s. lybica and the wildcat hybrid populations. For the STR data, the same individuals from the SNP dataset were used resulting in 22 populations. The two populations lost due to no STR genotypes were from Spain and Portugal. Isolation by distance was tested with a Mantel test between calculated matrices of geographical distances (geodesic in meters from latitude and longitude coordinates) and Cavalli-Sforza and Edwards chord genetic distances with the adegenet and geodist R packages  , four African wildcats (F.s. lybica), and 10 putative hybrids of domestic and European wildcats (F.s. silvestris)  genotyped for both SNPs and STRs, 1018 cats were genotyped for STRs only and 130 cats were genotyped for SNPs only. The four African wildcats were genotyped for both SNPs and STRs, while the 10 putative hybrids of domestic and European wildcats were only genotyped for the SNPs. The random-bred cats represent over 40 countries including over 85 sampling sites (Supplementary Tables The genotyped cat samples consisted of 1987 random-bred cats (F.s. lybica), which are considered the progenitor subspecies for the domestic cat, are positioned mainly with the felines from the Near East, near the center of the PCA space. However, the putative wildcat hybrids in the SNP dataset cluster peripherally from the random-bred felines, and appear more closely related to the felines from Western Europe, including cats from the Americas, which could be due to introgression among the populations.The PCA of the SNP and STR data sets have similar patterns Fig. . PrincipST = 0.078; SNP FST = 0.088). On average, the local populations had a deficit of heterozygotes of 6–9%  whereas the total worldwide random-bred population had a deficit of heterozygotes of 15–16% .The San Marcos Island  population in the STR dataset diverges from the European and American felines, reflective of a small, isolated island population. Both PCA reflect genetic divergence due to geographic separation, but the populations form a cline rather than clear geographical clusters. Southeastern and East Asian cats are located at one periphery of the distribution, as are the Near Eastern and Mediterranean cats in another, with the Western European cats in the third. Only 8–9% of the total genetic variability could be attributed to differences among the cat populations , supporting admixture within the 22 target populations. The sample population from Lahore, Pakistan has the lowest z-score of −4.9 and had 56 significant z-scores with other populations that are highly indicative of admixture. Populations most frequently contributing to significant admixture as parent  populations include Thailand, Vietnam, the wildcat hybrids, and Asyut, Egypt.The allele frequencies of the cat populations were analyzed to calculate f3 statistics  for random-bred cats in the contiguous United States of America, suggesting multiple importations of felines into the USA and little geographical structure in the genomic data.Since population structure analyses suggest a single population with possible differentiation due to geographic separation, isolation by distance was formally tested among the sample location populations in Europe, Africa, Near East, Middle East, and Asia for which evidence of admixture was not observed from F.s. lybica, a negative relationship is identified; as the genetic distance from F.s. lybica increases the observed heterozygosity decreases  remain a behaviorally semi-domesticated species that can quickly revert to a wild state. While they have a low survival rate in the wild, their high reproductive capacity increases population size  Near East, (2) China, and (3) Southeast Asia against the null hypothesis of a single domestication center. This focused sampling could also identify distinct populations indicative of admixture with wild relatives. However, despite this intensive sampling, only the Near East is suggested as a site of cat domestication indicating a pattern of dispersal outwards from regions like the Levant and the Nile Valley, while elsewhere in the world lacks this pattern  have many studies focused on the concern of introgression with free-roaming or partially-free-roaming random-bred cats , suggested some geneflow between this wild species and domestic cats, but not sufficient to explain the genetic difference between Far Eastern and Western domestic cats. Although a few Asian wildcats (F.s. ornata) were included in the Chinese study, the four cats were sampled from one site and specimens from wildcats from the Near East and the Indus Valley were not available (Yu et al. European wildcats (Although cats and agricultural species serve very different purposes to humans, the geographic patterns of admixture in cats are a near-perfect reflection of admixture and migration in cattle populations, such as along the Silk Road and in the Americas (Decker et al. F.s. lybica. The origin of domestication for F.s. catus is suggested as the coastal regions of the Mediterranean Basin of the Fertile Crescent where cats have high observed heterozygosity and a short genetic distance to the progenitor subspecies. As highly agrarian societies developed, domesticated cats then spread down into the Nile Valley where cultural integration of felines into society slightly decreased heterozygosity and increased the genetic distance from the initial founders. The slightly lower diversity could also be an influence of ancient cultural selections. Mummified Egyptian cats have control region mtDNA mitotypes specific to the mitotype G of contemporary Egyptian cats and a mitotype D highly common in Near and Middle Eastern populations but one mummified cat also had a common mitotype C that has a worldwide distribution (Kurushima et al. Overall, worldwide random-bred feline populations exhibit low levels of genetic differentiation even when geographically separated; however, populations on the peripheries of migration can be genetically differentiated. Populations were significantly isolated by distance; among populations, the genetic distance increased as the geographic distance increased. Observed heterozygosity was higher in populations located near the Mediterranean Basin of the Fertile Crescent where archeological evidence points towards the first human-cat interactions. In addition, these populations have a shorter genetic distance to the progenitor species F.s. ornata from Iraq, Iran, the Indus Valley region, and Northwestern India could further explain the genetic variation seen in cat populations. Genetic and archeological studies from pre-farming cats would be an important addition in further clarifying the cat domestication process. The patterns of genetic diversity and differentiation observed in worldwide random-bred cats parallel those of other species, especially humans once they became farmers, suggesting human history is written in the DNA of domesticated species.This study infers the relationships, dispersal, admixture, and genetic distances among worldwide random-bred cats from patterns of genetic polymorphisms, which were unlinked, randomly identified, and assumed to be neutral. Population bottlenecks and effective population sizes cannot be evaluated in the current study. Additional studies including data from various wildcat species/subspecies, particularly Supplementary TablesSupplementary FiguresSupplementary File 1Supplementary File 2