Ursus arctos marsicanus (Uam), endemic to the Apennines of central Italy, and Ursus arctos arctos (Uaa), in the Italian Alps. Both populations are endangered and occasionally involved in human–wildlife conflict; thus, detailed management plans have been in place for several decades, including genetic monitoring. Here, we propose a simple cost-effective microsatellite-based protocol for the management of populations with low genetic variation. We sampled 22 Uam and 22 Uaa individuals and analyzed a total of 32 microsatellite loci in order to evaluate their applicability in individual identification. Based on genetic variability estimates, we compared data from four different STR marker sets, to evaluate the optimal settings in long-term monitoring projects. Allelic richness and gene diversity were the highest for the Uaa population, whereas depleted genetic variability was noted for the Uam population, which should be regarded as a conservation priority. Our results identified the most effective STR sets for the estimation of genetic diversity and individual discrimination in Uam , and Uaa  populations, which can easily be utilized by smaller laboratories to support local governments in regular population monitoring. The method we proposed to select the most variable markers could be adopted for the genetic characterization of other small and isolated populations.An assessment of the genetic diversity and structure of a population is essential for designing recovery plans for threatened species. Italy hosts two brown bear populations,  Conservation genetics has proved to be effective in evaluating species viability by providing estimates of heterozygosity and genetic diversity of populations ,2,3,4. IPopulation genetic characterization can be performed using different types of genetic markers, but microsatellite loci, or short tandem repeats (STRs), have been widely adopted in non-invasive genetic monitoring, as they are relatively cheap to characterize, and can be reliably amplified from eDNA. STRs have played an important role in ecological, evolutionary, and conservation research in recent decades, as they are particularly informative ,17. For Ursus arctos marsicanus  the loci commonly used by Italian laboratories ; (ii) a new set of 13 STRs, originally developed for high-throughput sequencing (HTS) for the large Pyrenean and Dinaric brown bear populations [Uam and Uaa populations with the highest resolution power for individual identification, i.e., the optimal set of STR markers to use in long-term monitoring projects (BEST).The present study is focused on using variability indices to develop an objective method for ranking polymorphic STR markers, choosing the panel of these markers with the highest statistical power in individual identification and genotype reconstruction, and testing the efficiency of these panels for small populations. The ultimate goal is to design efficient marker panels that can be used for inexpensive genotyping for the management of threatened species. Here, using traditional capillary electrophoresis and invasive and non-invasive samples of ulations  (NEW);  without the need to capture and handle individual animals. A minority of the bears were found dead, and their tissue samples were dissected from the carcasses, whereas the remaining bears were live-trapped for research purposes ,38 and r® Blood & Tissue Kit , using a robotic workstation for automated purification of DNA . Since the DNA obtained from non-invasive sampling is often diluted and deteriorated, a multiple-tubes approach, with positive and negative controls at each step, was used to prevent stochastic errors [® Multiplex PCR Kit  in Mastercycler® pro S  and Veriti™ 96-Well Thermal Cycler (Applied Biosystems™). PCR reactions were performed in a total volume of 8 µL, which comprised the following: 3.5 µL of 2× QIAGEN Multiplex PCR Master Mix , 0.7 µL of 5× Q-solution , a total volume equal to the sum of individual primer volumes of the fluorescently labelled primer mix (10 μM), 2.0 µL of 10–100 ng/µL DNA template, and adjusting volume to 8 μl with RNase-free water. We adopted the following thermal cycling conditions: initial denaturation for 15 min at 95 °C, for 45 cycles; 30 s at 95 °C, 90 s at the annealing temperature of the primer mix (Ta = 57 °C), 60 s at 72 °C and 10 min at 72 °C for final elongation. STR fragments were detected and sized using capillary electrophoresis on an ABI Prism 3130XL Genetic Analyzer DNA sequencer  at the ISPRA BIO-CGE lab. The electropherograms were collected by the Data Collection Software v.3.0. GeneScan-500 LIZ and GeneMapper® Software v. 4.1 (Applied Biosystems by Thermo Fisher Scientific) were used to calibrate and determine allelic size, respectively.All genetic analyses were carried out in the Unit for Conservation Genetics (BIO-CGE) at ISPRA, or the Animal, Environmental and Antique DNA Platform at the Fondazione E. Mach, using the same harmonized protocols . Total gc errors ,34,39. Mc errors . The micp-value, after Bonferroni correction, conforming to α = 0.05 [p-values (corresponding to 0.001) in multiple tests [MicroChecker 2.2.3  was usedα = 0.05 . False aα = 0.05 . The relα = 0.05 , with a α = 0.05 . GenAlExα = 0.05 ,45,46 wale tests . MM-Distle tests  was usedThe power of individual markers to discriminate individual multilocus genotypes ,49 was eUam and 15 in Uaa), NEW (13 loci from high-throughput sequencing for both populations), TOT , and BEST, including the probability of identity for the increasing number of loci ; the eqci ; the nuci ; and thci . UHe was 0.270  and 0.561  for Uam and Uaa, respectively. There was no evidence of the presence of null alleles or allelic dropout in either Uam or Uaa populations. Genotyping errors were found in six loci in Uam and in four loci in Uaa (Uam and 6 Uaa) produced particularly reliable genotypes with one ADO (rate: 0.24%) and two FA (rate: 0.49%) at one locus. In non-invasive samples (6 Uam and 17 Uaa), 28 ADO  and no FA were observed at 7 loci. The PID and PIDsib values for the NEW set of STRs showed that the most informative loci were UA25  in Uam, and UA16  in Uaa (Uam and at 11 loci (0.247) in Uaa, but even then, most of the genotypes differed by more than 3 loci. Hence, the MM distribution suggested that the Uaa population was more variable than the Uam population because most individuals within the populations differed by 11 and 6 loci, respectively  in Uaa . The misectively a,b.Uam) and [Uaa) (Polymorphism and genotyping errors of the CURRENT panel were extrapolated from  (Uam) anUam) and  (Uaa) (Tnd [Uaa) .Uam and Uaa, respectively (Uam and 20 out of 28 loci in Uaa were highly polymorphic (PIC ≥ 0.5).For the TOT set, the gene diversity (GD) was 0.40 and 0.61 for ectively . A totalUam and Uaa, respectively  and four (Uam) or seven (Uaa) tetra-nucleotide microsatellites. The mean PIC for selected loci was 0.45 and 0.64 for Uam and Uaa, respectively , the cumulative power of discrimination among siblings (PIDsib) was 5.3 × 10−03 and 5.4 × 10−05 for Uam and Uaa, respectively.Based on the comparative values of PIC, error rates, number of mismatches and PID across both populations, we selected a panel of 9 and 12 microsatellite markers for ly BEST, , which wectively . The cumUam, the BEST set of loci showed a weak signal of mismatches among genotypes , this set demonstrated higher MI values for both Uam and Uaa populations , the highest values of the genetic diversity indices  were reported for the BEST STR panel in both populations . The resulations .To reduce the dimensionality of genetic data and provide the smallest possible number of loci for optimal identification of individual multilocus genotypes, it is important to construct a set of appropriate markers. Marker sets are distinguished by the extent  of their informativeness, in turn depending on the degree of polymorphism. Many studies have used microsatellite panels for addressing issues related to endangered species ,62,63,64Uam, from 15 to 12 in Uaa), decreasing the risk of genotyping errors , and lowering the cost of genotyping.To select the most appropriate marker set, we used not only PID or heterozygosity values, but also other parameters, including allelic richness, gene diversity, distribution of allelic frequencies, presence of PCR errors , and indices such as PIC and MI. By applying this method to brown bear populations in Italy, we identified the BEST sets, reducing the number of markers  and the selection of a panel with compatible primer properties (Dye Set G5) also contributed to maximizing the amplification performance in multiplex PCRs by reducing bias between markers. In addition, the preferential use of tetranucleotide repeats allowed a reduction in the number of PCR-induced stutter sequences in the outputs of amplified microsatellites [We also show here that the traditional method currently used for STR genotyping, fragment analysis by capillary electrophoresis, was effective for microsatellite loci developed for high-throughput sequencing (NEW set), probably because the shorter markers (<210 bp) improved the amplification of degraded DNA. This is important, since HTS only becomes cost-effective when many samples are analyzed simultaneously. For smaller populations, this is not always possible, or desirable . To further reduce costs, the proposed BEST set of STRs could be optimized into multiplex PCRs. The use of markers with overlapping amplification products , and an ER of 1.78  in Uaa for 19 loci, corresponding to a mean ER of 0.094 per locus (TOT set). Increasing the number of loci may decrease ER, generating false unique genotypes, and leading to an overestimated population size [Previous studies have shown that on the one hand, estimates of heterozygosity based on a few loci provide poor estimates of genome-wide genetic variation, and may not allow the differentiation of individuals, potentially leading to the underestimation of population size . On the ion size .U. arctos) is currently listed at a global level as ‘Least Concern’, but there are many small, isolated populations that are categorized as ‘Critically Endangered’; for example, both Italian populations have been placed on the Italian vulnerable species list (Criterion D). In fact, we estimated relatively low genetic diversity in the studied Italian bear populations, especially with regard to Uam, with three loci being monomorphic. The Uam population was characterized by an even lower genetic diversity compared to Uaa , confirming the results reported in [This study successfully identified optimal STR panels that perform reliably using invasive and non-invasive samples, which are useful for monitoring and protecting threatened brown bear populations in the future. The adopted workflow could be replicated in any small population affected by low genetic variability for which reliable and effective long-term genetic monitoring could be helpful to detect changes in patterns of variability, or to confirm the effectiveness of management practices. On the IUCN Red List of threatened species, the brown bear (orted in ,34. Sinc


The Middle East plays a central role in human history harbouring a vast diversity of ethnic, cultural and religious groups. However, much remains to be understood about past and present genomic diversity in this region. Here we present a multidisciplinary bioarchaeological analysis of two individuals dated to the late 7th and early 8th centuries, the Umayyad Era, from Tell Qarassa, an open-air site in modern-day Syria. Radiocarbon dates and burial type are consistent with one of the earliest Islamic Arab burials in the Levant. Interestingly, we found genomic similarity to a genotyped group of modern-day Bedouins and Saudi rather than to most neighbouring Levantine groups. This study represents the genomic analysis of a secondary use site with characteristics consistent with an early Islamic burial in the Levant. We discuss our findings and possible historic scenarios in the light of forces such as genetic drift and their possible interaction with religious and cultural processes (including diet and subsistence practices). Ancient genomic and archaeological data combine to identify a surprisingly early Islamic burial in modern day Syria. In the later Chalcolithic period, evidence of distinctive cultural practices and associated population movements highlight the dynamic history of the region, especially in the Southern Levant8. Further, genomic studies from the Bronze to Iron Ages in the Levant also report admixture and population movements, suggesting some degree of continuity with modern populations11. On a more recent timescale, studies from the medieval period12 and modern populations14 describe genetic structure and the role played by culture and religion in the formation of these structures. Notably, a study on medieval individuals from Lebanon previously identified as Crusaders12 demonstrated their ancestry to be either European or local as well as an admixture between Europeans and Near-Easterners. These signals cannot be detected in modern Lebanese groups, suggesting that they were only transient. This provided evidence of genetic signatures of historical religious events such as The Crusades which saw considerable movement from Europe to the Middle East, admixture with local populations and eventually, their dilution over time. Genetic analyses of modern Lebanese populations13 suggest that population movements linked to the spread of religions like Islam in the past millennium led to stratification in the Levant. Further south, data from modern Yemenis combined with other Middle Eastern groups found little correlation between genetic structure and geography14. It is thus clear that the present distribution of genetic diversity in the Middle East is the result of convoluted processes with culture as an additional level of complexity.The Middle East has an unparalleled place in human history. From the out-of-Africa movements of modern humans and admixture with Neandertals, to the spread of agriculture and emergence of civilisations, it has been at the crossroads of genetic as well as cultural history for millennia. While archaeological and historical sources provide instrumental insights into socio-cultural aspects, ancient DNA (aDNA) enables the recovery of past genetic information, filling crucial gaps in our understanding of population history. A number of aDNA studies from different time periods in the Middle East have provided a general overview of the genetic history in this region. These include descriptions of the earliest local farming groups from the Neolithic15. However, the Arabization and Islamisation of the area did not fully take place until the last decade of the seventh century led by ‘Abd al-Malik16. As such, the Aramaean, Byzantine and Christian legacy interacted with the new Arab Islamic rule and cultural values for decades. The collapse of the caliphate (750) and transfer of the political centre to Iraq caused political marginalisation and economic decline in Syria-Palestine17 . This area became the political centre of the empire with the founding of the Umayyad caliphate in Damascus in 661Ancient DNA analysis is a powerful tool to provide a genomic snapshot of this dynamic period, giving insight into past demographic processes of a currently conflicted and inaccessible territory of the Levant. However, while much of the focus of Near-Eastern Archaeology has been on funerary remains, few Islamic burials have been investigated as such studies might be considered as harming or disturbing the dead. We present an archaeogenomic analysis of two Umayyad Era individuals found at a prehistoric site in modern-day Syria, with no connection to an Islamic cemetery, but buried with indications of Islamic funerary rituals  at a depth coverage of 0.16× and 6.15×, respectively , projecting the two newly sequenced Umayyad Era individuals along with 262 published ancient individuals from the Near East, Western Europe, North and sub-Saharan Africa on a broad set of modern Middle Eastern, Arabian Peninsula, European and North African groups. The two UEB individuals fell between modern genetic variation in the Middle East and Arabian Peninsula and are shifted towards the latter Fig. . FurtherADMIXTURE34 analysis  but only in low amounts in the UEB individuals and was absent in any modern population from the Middle East/Arabian Peninsula which showed high values of the second component maximised in Bedouin B. At K = 6, another component emerged in Bedouin B and was present in high to moderate proportions in groups like Saudi/Bedouin A/Yemenite Jews and Middle Eastern populations, respectively. The UEB individuals also harbour high proportions of this component along with small amounts of the Neolithic Levant component seen at K = 5.To gain insight into the genetic composition of ancient and modern populations, we ran an unsupervised sis Fig. . Therefof3 statistics35 to increase resolution on population affinities between the two UEB samples and modern Bedouins, Saudi and Yemenite Jews, as indicated by PCA and ADMIXTURE analysis. We observed high shared genetic drift with Bedouin B and Saudi , Table snpAD36). Compared to the pseudohaploid data available for the other ancient individuals, these diploid genotypes allowed for a more fine scale analysis of the relationship between syr013 and modern populations. We used Beagle 4.137 to analyze sharing of tracts of identity by descent (IBD) between syr013 and present-day populations. Consistent with other results, the highest number and total length of IBD tracts was shared with multiple Bedouin B individuals and a single Saudi individual in the Human Origins 2.0 dataset  testing whether ‘X’ is an outgroup with respect to the group constituted by UEB individuals and ‘A’ or if there is an excess of allele sharing between ‘A’ and ‘X’ or the UEB individuals and ‘X’. When we tested Bedouin B and Saudi as candidates for groups ‘A’ and ‘X’, we found that no population configuration using this topology was consistent with the data (Tables Z = 6.4) while Saudi were rejected as a sister group due to an excess of allele sharing between the UEB and Bedouin B (Z = 13.8). Testing the UEB individuals as an outgroup to Saudi and Bedouin B revealed this to be a scenario consistent with the data (Z = 0.19) confirming that the two UEB individuals form their own group and are not directly matched by any of the modern populations in our reference data.We then tested different scenarios to investigate the most likely population affinities between the sequenced UEB samples and modern regional populations, performing D statistics of the form a Tables –S8. Bedo38. Within the Levant itself, groups such as Syrians, Palestinians and Jordanians have also been suggested to have higher African ancestry relative to nearby populations14  as potential sources of ancestry. We found that most ancient groups do not require all four sources, but that few of them can always be modelled as a mixture of Levant_N, Iran_N and either WHG or Mota, . The UEB individuals can be modelled either as a two-way mixture of Levant_N (61 ± 6%) and Iran_N (39 ± 6%)  Table , or also%) Table . Interes%) Table . In addiLevant_N 1 ± 6% an40. Consistent with this explanation, when we also tried to model the UEB individuals as a two-way mixture of already published Levantine individuals more proximate in time and Mota, we obtained high standard errors for Mota-related ancestry in all models and found only a few successful single-source models from published Levantine individuals  and could be explained by different scenarios. For instance, the Neolithic Levantine ancestral background could have been diluted by the introduction of African-related ancestry later, i.e. after the UEB individuals. This might also account for the observation that none of the other historical Levantine individuals from earlier time periods seem to produce a fitting model with Mota as an ancestral source. In fact, the only ancient individuals that could be successfully modelled with African-related ancestry are Neolithic Moroccansls Table . Furtherls Table , we foun41. However, population-level genetic and archaeological data are required to make conclusive inferences about large-scale migrations and religious/cultural stratification especially since it is possible that the Arabian peninsula-related ancestry was present in the Levant long before the UEB individuals lived. It is worth noting that the historical UEB individuals are the population with the lowest amount of genomic data, restricting a precise estimation or even a full rejection of the presence of European or African ancestry in these individuals, thus affecting ancestry estimates. However, even if we cannot fully reject the presence of any African ancestry, all analyses consistently suggest a lower level of African ancestry in the UEB individuals. While the reduced resolution for the historical UEB individuals prevents us from making more reliable inferences at this stage, the genetic differences observed here could potentially suggest a slightly different trajectory and contacts with other groups in the long term compared to Saudi and Bedouins as well as the effect of recent history on population structure. Similar results were also obtained from models repeated with data from UEB individuals restricted to damaged sites  and results showed a lower value of CND than syr005 and syr013  as expected for an unrelated pair of individuals. As low genetic diversity could be the result of small population size and/or inbreeding between individuals, we analysed runs of homozygosity (ROH) to determine the extent of inbreeding. In the syr013 individual, we observe both high numbers and cumulative lengths of ROH segments in syr013 compared to other Middle Eastern populations 013 Fig. . Howeverons Fig. . Consistern Fig. . These fLCT (encoding lactase) in introns of the gene MCM6, which serves as an enhancer for LCT transcription:49 −13910C/T, −13915T/G, −14010G/C, −13907C/G, and the rare variant −14107G/A. Lactase persistence is known to be an autosomal dominant trait, thus the presence of a single derived allele is sufficient for milk digestion. Interestingly, nine reads in sample syr013 were found mapping to the SNP −13915T/G, out of which five were the derived allele and four were ancestral which indicates that syr013 was heterozygous and lactose tolerant. Despite the long history of camel herding in this region beginning with its domestication more than 6000 years ago50, syr013 being heterozygous marks the earliest observation of this variant in genomic data. Lactase persistence could not be tested for sample syr005 as no reads covered the sites of interest. Nevertheless, this finding again draws a connection to the Arabian Peninsula as this variant is common in modern Arabian (for e.g. has a frequency of 72–88% in Saudi50) populations as well as in pastoralists that have traditionally relied on the Arabian camel (Dromedary camelous) for milk consumption and show high levels of lactase persistence phenotypes 50. We also tested other autosomal dominant or recessive conditions reported to occur frequently in Arabs47  but no pathogenic alleles were found in syr013  with high animal protein intake. Consumption of food from an arid location or where manuring was practiced are also possible explanations for the high nitrogen values. Without comparative faunal or human data from the period and location, it is difficult to ascertain the extent of these effects. We contextualised our data with human stable isotope regional datasets from historical/proto-historical periods ), with the exception of a Medieval individual with European ancestry (δ15N = 11.3‰) excavated from Lebanon12. It is possible that this individual hailed from Europe and had a different diet from those in the Middle East (before being buried there). The monastic community of St Stephens tends to have higher nitrogen values than the other datasets δ15N . The high nitrogen values for this site have been attributed to animal protein consumption51. The Bedouin dataset has comparable (and higher) nitrogen isotope ratios to our two individuals (δ15N 10–17.3‰), however, less negative δ13C values. The high nitrogen values of the Bedouin are attributed to 'considerable amounts of animal protein' including blood, milk (products) and meat, and the high δ13C ratios are attributed to the habitation of and resource exploitation of predominantly C4 or mixed C4-C3 ecosystems56. Compound specific amino acid carbon isotope values were obtained58 to investigate whether the high nitrogen values were influenced by the intake of aquatic resources. The δ13CGLY-δ13CPHE values for the individuals are between 10 and 13‰59, δ13CVAL-δ13CPHE values around 0 to 1‰60 and the δ13CLEU-δ13CPHE values are negative , but obtained from an almost exclusively C3 ecosystem .We attempted to infer dietary consumption patterns in these individuals using stable isotope analysis, using collagen samples extracted from the petrous bone Table . The bulve Table ; these v53 and the data from refs. 52, who, like us, used petrous bone. Jørkov et al.62 have argued that the isotope signal from petrous bone reflects some childhood diet, reporting slightly lower δ13C values (<0.3‰) and slightly higher δ15N values (<0.8‰) in petrous bone compared to those of rib and femur samples (at a population level). Even if this offset applies to our dataset, these differences would not affect the dietary interpretation of the two individuals from Tell Qarassa.The majority of stable isotope data discussed herein were measured from collagen samples extracted from cortical long bone and ribs, with the exception of four samples taken from molars63 conclude that there is no strong evidence that Islamic diet has a distinct isotopic signal that distinguishes it from contemporary Christian diets (e.g. ref. 64), despite some well-known cultural differences . They highlight that further research is required to investigate such distinctions63 and certainly further work is required in the Middle East to expand our knowledge of dietary stable isotopes in the Umayyad Era.This dietary interpretation is intriguingly consistent with the Bedouin ancestry components of the individuals and the presence of the lactase persistence variant in syr013. Dietary stable isotope studies of ancient Islamic individuals have been widely studied in Iberia , but less extensively elsewhere. Lopez-Costas and Alexander65. We screened our sequences for DNA from different known pathogens and find potential traces of different bacterial species that can cause infections in syr005  for DNA preservation. The Middle East and the Arabian Peninsula are pivotal regions in the timeline of human history and an increasing number of aDNA studies have attempted to understand their genetic history. Although there have been successful studies14, given the poor conditions for DNA preservation, this process is proving to be slower than in more environmentally favourable regions of the world. Nonetheless, given its historical importance, each newly recovered DNA sequence adds an important piece to the genomic and cultural puzzle of a war-stricken region currently under difficult access.The continuous development and improvement in ancient DNA methodologies and molecular techniques is constantly pushing the temporal and geographic limits of aDNA recovery, exploring older time periods70. However, the absence of contemporary genetic data from this period limits resolution on finer substructure among such groups. Additionally, while genomic methods are a powerful tool to analyse an individual’s ancestry and infer past demography and population dynamics, genetic datasets often use regional, cultural and/or archaeological affiliations for genotyped individuals. Thus, while it seems that the UEB individuals are genetically similar to some nomadic groups, determining their exact cultural affiliation is a question that cannot be answered through genomic analysis.We have been able to infer that the two Umayyad Era individuals buried on top of a prehistoric site represent individuals that were genetically close  to a subgroup of modern-day Bedouins ('Bedouin B') from the Negev desert in Israel as well as Saudi from the Arabian peninsula in the Human Origins 2.0 dataset. Several sources document the existence of historical nomadic groups that either occupied the Tell Qarassa region and/or migrated from the Arabian Peninsula to Syria during the Umayyad Era71. The Tell Qarassa graves do not represent a traditional Muslim context, i.e. a traditional Muslim cemetery and – from the available evidence – do not seem to have been located near a permanent settlement of the period. This suggests that it is possible that these two individuals were transient Muslims in the region. The absence of trauma to the bones and the young age of the deceased suggests that they could have died from disease, possibly the Justinian plague which ravaged the Middle East from 541 AD to 749 AD recurring in cycles72. Specifically, the dates of the burials may be linked to the outbreak of 79 AH (698 AD) which was reported in Syria by as-Suyuṭī73. However, we did not find conclusive evidence of pathogens and the exact cause of their death remains difficult to pinpoint.The archaeological context proves to be slightly more informative regarding their burial characteristics, i.e. orientation towards Mecca, separate graves and wrapping practice which appears to be indicative of these individuals possibly being early adherents of Islam in a Christian-majority region74. Our results suggest the early presence of Muslim Arabs in the Syrian countryside. Extensive additional sampling from different groups in this region is crucial to understand the extent of their genetic structure today and to potentially identify relatively genetically isolated populations, which could have implications for population genetic and clinical studies. The Middle East is a region with a complex history and a diverse ethnic and genetic composition, yet our current understanding of the genetic structure in the past and present appears to have only scratched the surface.The genomic ancestry of the two individuals buried at Tell Qarassa in the late seventh or early eighth century offers a glimpse of early Islamic society in Syria. This study provides further insight into the possible re-use of prehistoric burial sites by Muslim groups. On a general level, this burial provides additional indications for the early adoption of specific Islamic burial rites which were followed even in remote locations. At present, there are no examples of genetic studies from the region which relate to this period, the only genetic data related to early Islamic burials is the study of two individuals from the south of France77. Located on the shores of an ancient dried-up lake, it is described to consist of multiple sites or so-called ‘Tells’, one of which (Northern Tell) contains remains from Pre-Pottery Neolithic B to Late Chalcolithic settlements, while the other (Southern Tell) holds evidence of Early Bronze to Iron Age remains. Evidence of a Natufian settlement has also been found in close proximity78. The village of Qarassa is a Druze community today. Pre-Pottery Neolithic mortuary practices have been described from this site, shedding light on such practices in this period19. The individual syr005 was found laid on his back in a decubitus supine position, although the lower limbs were slightly flexed and placed on their right side. The burial was oriented east-west, with the head at the west, facing south. Further, individual syr013 was placed on her right side in a lateral decubitus position oriented east-west, with the head at the west, facing south. The distribution of the skeletal elements suggests that both bodies were wrapped before burial20. When soft tissues of the body decay faster than the wrapping, it can create either temporary or semi-permanent spaces around the putrefied body, yielding some skeletal movements at the disarticulated joints before wrapping decay . These individuals were located very close to each other. However, intensive archaeological fieldwork has not yielded evidence of further burials from this period at the site. Therefore, the archaeological record does not support that these burials belong to a cemetery for a specific community. Permits for archaeological samples processed in this study were obtained from the General Directorate of Antiquities and Museums (DGAM), the relevant authority of the Arab Republic of Syria. The remains are curated by DGAM and have been deposited at the Archaeological Museum of As-Suwayda (Syria).The archaeological site of Tell Qarassa, located in modern-day Syria Fig. , is prom80 at the Molecular Archaeology Laboratory at La Trobe University using cold 0.6 M HCl and yields are recorded in Table 81, and the IntCal13 calibration curve82. Radiocarbon results for individuals syr005 and syr013 were 666–768 Cal AD 2σ , and 665–766 Cal AD 2σ , respectively. The collagen stable isotope values and quality parameters were also measured at Waikato and are presented in Table 57 and ref. 58. In brief, 1 mg of collagen was hydrolysed under vacuum for 24 h at 110 °C and the hydrolysate was dried in a rotary vacuum concentrator and frozen until required for analysis. The sample was resolved in Milli-Q water with an internal standard (2-amino isobutyric acid). Instrumental analysis was carried out using a three-phase method similar to that described in ref. 58 with 10–15 µg of hydrolysate delivered to the column. Isotope values were calculated relative to standard CO2 gas peaks delivered throughout the analytical run, with the CO2 calibrated against USGS-40.Collagen was extracted from two petrous bones following a modified Longin method83, with an incubation of 24–48 h, using the MinElute column Zymo extender assembly replaced by the High Pure Extender Assembly  and performed twice for each sample. Further, blunt-end Illumina multiplex sequencing libraries were prepared84 resulting in two double-stranded libraries per sample. Library amplifications were performed as in85 using indexed primers84 and 4−11 cycles . All extraction and library preparation steps were conducted at the dedicated ancient DNA facility at Stockholm University. A total of four DNA libraries were shotgun sequenced on a HiSeq X10 sequencing platform (150 bp paired-end reads) at the NGI Stockholm.Prior to DNA extraction, the petrous bones were UV irradiated (6 J/cm2 at 254 nm) and the first millimetre of bone surface abraded using a Dremel™ tool. DNA was extracted from a 100–200 mg piece of the bone using a silica binding methodBWA aln86 with non-default parameters −l 16500 −n 0.01 −o 2. Data were merged at the library level using samtools v1.587. PCR duplicates were collapsed using a modified version of FilterUniqSAMCons_cc.py88. Different libraries for one individual were then merged into one bam file using samtoolsv1.587. Reads shorter than 35 bp, showing more than 10% mismatch with the reference and/or a mapping quality score below 30 were discarded. Biological sex was inferred using two different approaches30.Sequenced reads were mapped to the human reference genome build 37 (hs37d5) using 26. X-chromosomal contamination was estimated for the male individual syr005 using the approach implemented in ANGSD28. VerifyBAMID27 was used to estimate autosomal contamination in both samples. It uses a hypothetical ‘true genotype’ model and checks whether reads in a bam file are more likely to match a single individual or result from a mixture of other samples, such as a closely related/a different individual.Contamination for the two individuals  was assessed on three different levels: mitochondria, X-chromosome and autosomes. Two methods were used to estimate mitochondrial contamination89 and HaploGrep90, two web-based applications that use the Human Phylogenetic Tree91 to identify the Y-haplotype of sample syr005, reads mapping to the Y chromosome with minimum base and mapping quality of 30 were compared to biallelic substitution SNPs from the International Society of Genetic Genealogy . We excluded A/T and G/C SNPs to avoid strand misidentification and C/T and A/G SNPs to avoid post-mortem damages.Mitochondrial haplogroups were identified using Haplofind1 as well as 272 published ancient individuals from the Levant, Europe, Caucasus, North and sub-Saharan Africa98, resulting in a total of 2854 individuals and 541300 SNPs that passed quality control filters from the Human Origins dataset. We generated pseudohaploid representations of the ancient individuals by randomly drawing one allele from the samtools mpileup87 output at each SNP site, restricting the analysis to minimum mapping and read qualities of 30 and coded transition sites as missing to avoid post-mortem damage.For population genetic analyses, we merged the ancient samples with modern genotype data of the Human Origins 2.0 datasetsmartpca99 using the options lsqproj and shrinkmode. The first PCA included 73 modern populations from North, West, South, Central and East Europe, Caucasus, Turkey and the Middle East, North and North-east Africa, the Arabian Peninsula, along with 262 published ancient samples from the Levant, North and sub-Saharan Africa and the two ancient UEB individuals (N = 1321) from this study. In order to identify the closest modern populations to the UEB individuals, we generated a second PCA with an increased resolution by reducing the original set of modern populations. For this second PCA we excluded divergent groups such as Europeans, Somalis and was limited to 37 modern groups in the Middle East, Turkey and Caucasus henceforth the Middle East, along with ancient individuals . PCA plots were generated using GNU R.A principal component analysis was conducted using ADMIXTURE34 for model-based clustering analysis. All data were haploidized by randomly picking one allele per individual at each SNP. Next, the dataset was thinned by pruning out SNPs in linkage disequilibrium using PLINK v1.9100 with a window size of 200 kb, a step-size of 25 and a squared correlation (r2) threshold of 0.4. Following this step, 59509 of 541300 variants were removed. ADMIXTURE was run for ten iterations with different random starting seeds, five-fold cross validation and the number of ancestral populations (K) was varied from 2 to 10. Results of admixture were visualised using Pong101. We ran unsupervised analyses to study the assignment of genetic clusters in the larger set of 73 modern populations from Europe, Middle East, Arabian Peninsula, North Africa and Caucasus and the ancient individuals grouped into 28 population labels determined from their respective studies (N = 1321).We used qpAdm102 from the ADMIXTOOLS35 package with the options allsnps: YES, details: YES and summary: YES to run various modelling scenarios for ancient and modern Levantine groups. First, we modelled the UEB individuals, other ancient and modern Levantine groups as a mixture of four basal ancient populations, namely Neolithic Levant, Neolithic Iran, Western European Hunter-Gatherers and Mota  and their subsets. Secondly, we modelled the UEB individuals as a mixture from ancient Levantines more proximate in time (i.e. Iron/Bronze Age and later periods) along with Mota from Africa39. Thirdly, we modelled modern Middle Eastern groups as a mixture of the UEB individuals and Mota. Finally, all models that involve UEB individuals were also run by restricting the models to damaged reads. For this, a likelihood-based approach implemented in PMDtools103 was used, restricting to sequences with a PMD score of at least 3. For all models, we chose 6 outgroups ('right' populations): Mbuti, Sweden Hunter-Gatherers92 (SHG), Kostenki14104, Ust-Ishim105, CHG95 and Neolithic Anatolia92. We attempted to implement suggested recommendations for qpAdm106 and tried to minimise combinations of ancient and modern groups as references, as well as to keep the same set of sources and references for all targets.We used f3 and D statistics107. Population genetic summary statistics were calculated using POPSTATS108 with sub-Saharan Mbuti rainforest hunter-gatherers as an outgroup. Standard errors were estimated using a weighted block jackknife procedure.To detect shared genetic history between the sequenced samples and various Middle East populations, we used 42. CND is a measure of genetic diversity within a population based on a comparison of nucleotide differences between two individuals. We used transversion sites that are polymorphic in the Human Origins 2.0 SNP array. Standard errors were estimated using a block jackknife procedure.To estimate genetic diversity in our data, we used conditional nucleotide diversity (CND)109. Neither of these were an option for this study as we are faced with two individuals from a unique population. Instead, we sampled two distinct reads per individual at each SNP site with at least two reads resulting in two pseudohaploid representations for syr005 and syr013. These representations should behave as identical twins ('pseudo-twins') in a pairwise comparison, meaning that they should show half the genetic distance of an unrelated pair from the same population. Hence, we can compare individual distances to distances between syr005 and syr013 to assess their genetic relationship.Commonly used approaches for estimating a degree of genetic relationship requires some type of reference data to set a baseline for relatedness. This is usually obtained either by using allele frequencies from modern populations or pairs of unrelated individuals from the same population50. We also looked for common recessive disorders described in Arab populations from the OMIM (Online Mendelian Inheritance in Man) catalogue110. Information about SNP positions and the phenotype was taken from dbSNP111 and SNPedia112. A pileup file was created in samtools from .bam files with a high base quality (q > 30) as well as mapping quality (Q > 30) and the occurrence of variants known to be involved in LP and other disorders was checked.Given the observed Arabian ancestry in the sequenced samples, we looked at phenotypic variants that might be expected in this region. For instance, lactase persistence (LP) has been described in the Arabian PeninsulasnpAD version 0.3.436, a diploid genotype caller dedicated to work with ancient DNA to call diploid genotypes for syr013. Reads were first realigned around indels using GATK113. The genotype calls were restricted to reads with mapping quality of at least 30 and base quality of at least 30, calculating an error profile for the first and last 5 bp of each read. Downstream analyses were then restricted to positions with a genotype quality of at least 30 and between 5 and 22 reads sequencing depth at each position.We used PLINK100 with parameters -homozyg-density 50, -homozyg-gap 100, -homozyg-kb 500, -homozyg-snp 100, -homozyg-window-het 1, -homozyg-window-snp 100, -homozyg-window-threshold 0.05 and -homozyg-window-missing 25 for the Human Origins dataset. For the SGDP dataset, the analysis was restricted to 6,383,589 sites with a minor allele frequency of at least 0.01 and no missing data after adding syr013. Therefore, -homozyg-window-missing 25 was set to 1 for the SGDP dataset.Runs of homozygosity was estimated for modern populations and the diploid calls of syr013 using Beagle 4.137. We used Human Origins 2.0 as well as the Simons Genome Diversity Project (SGDP46) to compare results between the HO panel and the SGDP data, which was filtered to remove missing sites from syr013. Beagle’s IBD was run using VCF files with the following parameters: ibdtrim = 30 and 150 for the Human Origins and SGDP datasets respectively; ibd = true; and impute = FALSE, for all chromosomes. We selected segments with a LOD score >3 and length >1 cM for both panels.Segments showing identity-by-descent between the UEB individual syr013 and modern human populations were determined using Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Urocyon littoralis catalinae) sampled in 2007–2008 have a high prevalence of ceruminous gland tumors, which was not detected in the population prior to a recent bottleneck caused by a canine distemper epidemic. The disease appears to be associated with inflammation from chronic ear mite (Otodectes) infections and secondary elevated levels of Staphyloccus pseudointermedius bacterial infections. However, no other environmental factors to date have been found to be associated with elevated cancer risk in this population. Here, we used whole genome sequencing of the case and control individuals from two islands to identify candidate loci associated with cancer based on genetic divergence, nucleotide diversity, allele frequency spectrum, and runs of homozygosity. We identified several candidate loci based on genomic signatures and putative gene functions, suggesting that cancer susceptibility in this population may be polygenic. Due to the efforts of a recovery program and weak fitness effects of late-onset disease, the population size has increased, which may allow selection to be more effective in removing these presumably slightly deleterious alleles. Long-term monitoring of the disease alleles, as well as overall genetic diversity, will provide crucial information for the long-term persistence of this threatened population.Small effective population sizes raise the probability of extinction by increasing the frequency of potentially deleterious alleles and reducing fitness. However, the extent to which cancers play a role in the fitness reduction of genetically depauperate wildlife populations is unknown. Santa Catalina island foxes ( As threats to wild populations increase due to habitat destruction and climate factors, understanding the genetic consequences affecting small, isolated populations can inform conservation practices to mitigate the continued loss of genetic diversity. When the effective population size is small, both inbreeding and stochastic processes such as genetic drift can increase the frequency of deleterious alleles. Increased genetic load is particularly difficult to eliminate from small populations because drift hinders the power of selection to remove weakly and moderately deleterious alleles, as has been observed in island foxes ,2. TheseStudies of cancer in wildlife species present unique challenges, but research is beginning to uncover the impact of cancer on wildlife populations ,12,13. PHPSE2 [RAS11A activation in Tasmanian devils [Few genetic studies of wild populations have attempted to identify polymorphisms putatively associated with cancer susceptibility ,17,18,19HPSE2 , and facn devils ,21,22,23Urocyon littoralis) (Urocyon cinereoargenteus) that was thought to have colonized the three northern Channel Islands near the coast of southern California about 10,000–16,000 years bp, with the first evidence of colonization of the southern three islands by mainland or island populations 2000–4000 years bp [Urocyon littoralis dickey) was subsequently discovered for the major histocompatibility complex (MHC) and was hypothesized to be maintained by balancing selection [The island fox  A is a spyears bp ,25. Moreyears bp . The numyears bp ,27,28. Fyears bp ,30. Subsyears bp . High geelection . Howeverelection . As wellelection . Lastly,election .Staphylococcus pseudintermedius, an opportunistic pathogen, has been shown to influence the sustained inflammation associated with tumor development [SCA foxes have a high prevalence  of ceruminous gland carcinoma and adenoma  that appear to be associated with inflammation induced by chronic ear mite (Otodectes) infections . Ceruminelopment . Ear mitelopment . Howeverelopment , though elopment . AlthougHere, we test the hypothesis that the remarkably high incidence of ceruminous gland tumors in SCA island foxes is the result of genetic variants for cancer susceptibility that differ in frequency between cohorts using whole-genome sequencing of affected individuals (cases) and long-lived unaffected individuals (controls) from SCA, and control individuals from SCLE. We predict that the alleles that increase cancer susceptibility are deleterious and, therefore, relatively young and that they rose in frequency in the SCA population as they rebounded from the recent population bottleneck. Within the genomic regions of implicated loci, we expect reduced nucleotide diversity in cases, increased differentiation in allele frequencies between cases and controls, and reduced heterozygosity in cases. Along with these population genomic signatures, we use functional gene annotation from the domestic dog to identify candidate genes that contribute to cancer susceptibility in the SCA island fox.n = 12), and control individuals from SCA that did not develop tumors (n = 11), see [n = 9). Two of the SCLE individuals were previously published [We sequenced the genomes of 32 island foxes from Santa Catalina Island and San Clemente Island. The dataset included cases as individuals with carcinoma 11), see  for agin1, see  fWe obtained existing tissue and blood samples collected for a previous study . DNA wasReads were trimmed using trimmomatic  and aligUrocyon and Canis genomes. SNV with a depth of coverage less than 60% or greater than 250% of the average for the individual were excluded since they may represent interspecies paralogy or copy number variation between domestic dog and island fox. SNV with minimum genotype quality of less than 20 and sites called in fewer than 95% of individuals or a minor allele frequency less than 0.01 were removed from further analysis. The number of segregating sites and mean coverage per individual were calculated using VCFtools [Variant filtration was necessary to ensure sequencing data with high accuracy were used. Qualimap2  was usedVCFtools . We usedVCFtools  and PRIMVCFtools ,45. IndiVCFtools  based on2 > 0.90 with all other SNV in a 50-SNV window) using PLINK, which reduced false autozygosity calls by removing redundant markers in SNV-dense regions and making SNV coverage more uniform. Coordinates of putatively neutral regions in the canine genome were obtained from Freedman et al. [To assess genetic clustering, we applied two methods to the dataset of putatively neutral loci pruned for linkage disequilibrium (LD). Datasets were LD-pruned using PLINK v2.0 (-indep-pairwise 50 5 0.2) . The datn et al. . First, n et al. .−9/site/generation [−8/site/generation  and 2.0neration . TherefoD in cases would indicate an excess of rare variants compared to the controls, as has been performed in multiple heritable disease studies [ST for each comparison pair . All were calculated using a sliding window approach in VCFtools. Z-score transformations were performed using custom R scripts using the SciPy library . We calculated each statistic with a window size of 100 kb, step size of 10 kb, and at a finer scale assessment using 50 kb windows and 1 kb steps.We calculated several population genetic statistics to test for deviation from neutral expectation. Using Tajima’s D, we measured the departure from neutral evolution for each cohort with the hypothesis that a smaller Tajima’s  studies ,53,54. WThe cross-population composite likelihood ratio test XP-CLR was employed to compare allele frequency differentiation between comparison pairs to identify regions of elevated differentiation . XP-CLR ST and differences in allele frequency. The top 1% outlier windows for the difference in nucleotide diversity between cases and controls (∆π) were examined for potential candidate genes. We expect that cases would have lower π values than the SCA controls in genomic regions associated with cancer susceptibility. Therefore, we only examined windows in which this was the case. SNVs within these candidate windows were annotated using SnpEff, and resulting gene annotations were used for gene ontology (GO) enrichment analysis using g:Profiler  with theST and ∆Tajima’s D if they were in the top 1% of normalized values. The intersection of the outlier peaks for FST, ∆Tajima’s D, and ∆π were chosen as possible candidate regions. We also examined comparisons between cases and both control populations (SCA and SCLE). We selected the top 1% outlier loci (50 kb windows) for ∆π between cases and both control populations (SCA and SCLE), as well as the intersection of the top 1% for case/SCA control and case/both controls (resulting in 8 outlier loci). We also selected outlier peaks for each comparison for FST and difference in allele frequency at individual SNV within each region. SNVs within these regions with an allele frequency difference greater than 0.45 (n = 38) were selected. The closest gene to each SNV was used as input for gene ontology (GO) enrichment analysis using g:Profiler [The resulting candidate regions were examined further for implicated SNV by assessing allelic FProfiler . AdditioProfiler . https://rdrr.io/cran/detectRUNS/, accessed on 1 November 2021). Using a sliding window-based method in “detectRUNS”, ROHs were detected using a 50-SNV window to scan the genome of each individual using the LD pruned dataset. The proportion of overlapping homozygous windows to call an ROH was 0.05, with a maximum of 2 missing and one heterozygous SNV allowed within each ROH. To minimize the number of false positive ROH, the minimum number of SNVs to call an ROH was set to 2 and the minimum length of an ROH to 100 kb. A minimum density of one SNV per 50 kb and a maximum of 100 kb gap between consecutive SNV was imposed. The inbreeding coefficient, F, was estimated for each individual using FROH in the R library “detectRUNS”. The R programming environment  was usedAfter we detected ROH using a sliding-window method using a 50-SNV window (see above), we used “detectRuns” to calculate the proportion of times  each SNV falls within an ROH and plotted each proportion against the SNV position along the genome. We defined candidate loci as SNVs that were within ROHs in more than 75% of cases and less than 75% in the two control groups. HLA-DRB1 (ENSCAFG00000000806), DLA-DQA1 (ENSCAFG00000000812), DLA_DRA (ENSCAFG00000000803), and DLA-DQB1 (ENSCAFG00000000814). The resulting candidate SNVs were examined further for differentiation by assessing allelic FST and ∆ allele frequencies between cases and controls. Additionally, we assessed if these mutations were within TFBSs.Several genes belonging to the major histocompatibility complex (MHC) were analyzed. Filtered SNV were selected if they were annotated with the following genes: We obtained high-quality sequence reads with a per-individual average unfiltered yield of 46,553 ± 11,194 Mb . After r2) across all autosomal SNV decays below a threshold of 0.2. We found that populations of SCA (n = 23) and SCLE (n = 9) had moderate levels of LD  ~80 kb [To assess LD patterns, we estimated the physical distance at which the pair-wise genotypic association (r) ~80 kb . LD esti) ~80 kb  and may To assess the genetic partitions based on our neutral SNV dataset, we used complementary analyses of genetic clustering patterns. The first two component axes from PCA show two distinct clusters that correspond with the sampling location B. On PC1We used SMC++ to estimate historical population sizes and the divergence time of these two populations D. The SCST, ∆π, and Tajima’s D identified multiple regions across the genome with differences between groups  for selection in cases and controls, respectively. The highest scores for selection in cases on chromosomes 13 and 37 and the highest scores for selection in SCA control on chromosomes 5 and 27  after removing loci where controls had lower π than cases, 17,667 SNV remained  and “moderate” . After removing SCLE individuals, the top 1% outlier loci for ∆π between cases and SCA controls were all categorized as “moderate” impact  were annotated, resulting in proximity to 17 genes (n = 2) of SLAMF8 (SLAM family member 8) were highly differentiated between case and controls. This gene is responsible for lymphocyte activation. Three SNVs (2 intronic and 1 upstream) were found in FCRL6 (fc receptor such as 6), a receptor expressed by cytotoxic T and NK lymphocytes that have been implicated in immune-stimulating cancers as well as those involving PD1 (programmed cell death protein 1) [To identify implicated SNV, we examined allelic F regions . No fixe17 genes . Of thesotein 1) . This geotein 1) . Three Sotein 1) , had a dThese seventeen genes were used for gene ontology (GO) enrichment analysis. We found two significantly enriched GO terms, both of which were related to the regulation of superoxide anion generation (GO:0032928 and GO:0032930). The two genes that define the significant GO terms are Decapping MRNA 1A (DCP1A) and C-Reactive Protein (CRP). We found two intronic variants with allele frequency <0.45 and 2 upstream variants within the TFBS of DCP1A, which is a biomarker used for gastric , colorecp = 0.3182; A total of 55,317 ROH were identified with an average of 1728.7 (±787.3) ROH per individual with a minimum of 90 and a maximum of 3283 ROH per individual . The aven = 18, SCA: n = 151, and SCLE: n = 99). Of the 18 ROHs in cases, 13 of these were not found in the control populations and were considered candidate ROHs. Two of these regions, on chromosome 4 . “moderate” impact variants included 39 missense mutations. “low” impact variants consisted of seven synonymous variants and one splice site variant. MHC regions have notably high levels of diversity that can lead to difficulties with alignment, particularly between species . The aveST was 0.31, with an average value of 0.004234. The highest value of the difference in allele frequency was 0.48 (position 2208261), with an average value of 0.08027. Position 2208261, which had the highest FST and delta allele frequency, is an intergenic variant between DLA-DQB1 and DLA-DQA1. Of the 675 SNVs, three SNVs were located within TFBSs .We examined the difference in allele frequency between cases and SCA controls at SNV within the MHC region . No fixen = 12) when compared to individuals who are unaffected on the same island (n = 11), as well as a closely related population with mite infections that do not result in tumorigenesis (n = 9). The sample size does not lend itself well to traditional genome-wide association studies, and therefore an outlier approach was undertaken in the context of selection against deleterious variation as the populations rebound from severe population bottlenecks. Multiple genes implicated in cancers have been shown to be under selection in mammals [Urocyon grey fox genus.This work uses genome scans based on allele frequency to identify patterns of variation enriched in foxes with ceruminous gland epithelial tumors  between island foxes . HabitatSubstantial morbidity and mortality in many wildlife species may be due to cancer ,13. In dAlthough it is well supported in applied conservation that there are often positive effects of population augmentation with divergent immigrants , it is oEvolutionary rescue (adaptation resulting from existing genetic variation) may be effective in reducing cancer susceptibility, especially since the population size has recovered from the recent bottleneck. We found no evidence of candidate loci fixed for alternative alleles between groups, suggesting that the population maintains allelic variation that could allow for an adaptive response to the disease, even if the selection is relatively weak on each locus. This implies that attempts to increase adaptive potential by translocating individuals from other islands are unlikely to have a large benefit. We suggest further field and genetic monitoring illuminate trends in cancer prevalence as well as simulations of purifying selection and drift to predict the future course of population-level susceptibility under different scenarios. Urocyon.An increasing body of knowledge is emerging with regard to conservation and management of adaptive potential ; yet, th


Coryphaena hippurus, is one of the most important species caught in the Tropical Eastern Pacific (TEP). However, the lack of consensus about the existence of genetically differentiated populations in the area has hindered the adoption of management strategies to ensure its viability.Marine species constitute commercially important resources, and knowledge about mechanisms that shape phylogeographic patterns and genetic structure provides valuable information for conservation. The dolphinfish, C. hippurus in the TEP, evaluate the extent of connectivity between dolphinfish populations, infer potential barriers to gene flow, and test for signals of contemporary and historical demographic expansions.We assessed genetic variation and phylogeographic structure using two mitochondrial genes and 14 nuclear DNA microsatellite loci. Population genetic tools were used to characterize the spatial distribution of genetic variation of Hs = 0.66–0.81). Notwithstanding, positive FIS and low effective population size (Ne = 77.9–496.4) were also recorded.Mitochondrial DNA sequences showed genetic homogeneity across locations in the TEP, as well as a strong signal of population expansion dated to the late Pleistocene. In contrast, nuclear microsatellite markers resolved four genetically distinct groups with a remarked genetic differentiation between the most distant locations, at the northern and southern boundaries of the species’ range. High mean genetic diversity was found at all localities (Ne values are of conservation concern and must be considered in fishery management plans.The distribution of genetic variation could be related to expansion-contraction cycles following seasonal temperature changes at transitional areas, promoting population subdivisions. However, we cannot rule out the effect of oceanographic dynamics to the observed patterns. Although this marine species remains highly abundant despite commercial exploitation, the low  HoweverC. hippurus in the TEP, (2) evaluate the connectivity of the dolphinfish locations, (3) infer barriers to gene flow, and (4) estimate past and contemporary changes in effective population sizes to provide insight on possible associations with climate variability. The motivation of this study was to provide genetic information that is necessary to define the dolphinfish population connectivity with ample spatial sampling and more informative markers to gain knowledge on this highly exploited species.In this study, we combined the analysis of two mitochondrial segments and 14 nuclear microsatellite loci to (1) characterize the spatial distribution of genetic variation of Muscle tissue samples from dolphinfish individuals were opportunistically collected from 2003 to 2006, from artisanal fishing boats operating in the TEP, including the Gulf of California ; Fig. 1.AY135025–AY135028, and AY189832), 13 described by 10.5281/zenodo.6787993).Nineteen microsatellites were genotyped using primers for five loci  designed by Robert Chapmann et al. 2005 , and a third M13 primer labeled with a fluorophore  was included in the reaction to bind to the forward primers. Forward, reverse, and florescent-M13 primers were used in 1:10:10 proportions, respectively, and were added to the reaction mix according to the protocol described by PCR amplification reactions were performed in 10 μl containing 10–100 ng of DNA, 1X PCR buffer , 0.2 mM of dNTPs, 0.2 μM of each primer, 2.5 mM of MgCl4 randomizations and a confidence interval (CI) of 95%. Departures from Hardy-Weinberg equilibrium (HWE) for each locus were examined by the exact test method. We also tested for linkage disequilibrium (LD) between pairs of loci. For both tests we used GENEPOP 4.7.5 software . PCR amplifications for ND1 consisted of 95 °C for 5 min followed by 35 cycles of 1 min at 95 °C for denaturation, 1 min at 58 °C for annealing, and a final extension at 65 °C for 3 min, with a final extension at 72 °C for 10 min. Amplicons were purified and sequenced on an ABI 3730xl® automated sequencer by Macrogen Inc. . The thermal cycling profile for CYTB consisted of an initial denaturation step at 95 °C for 2 min, followed by 35 cycles at 95 °C for 1 min, 52 °C for 1 min, and 65 °C for 1 min, with a final extension of 3 min at 65 °C. Mitochondrial sequences were edited and aligned using ClustalX 1.8 (MZ725384–MZ725448). The two loci were analyzed independently because of inconsistency in the amplification of many individuals. Because of this, we could not concatenate the sequences from the two loci.We amplified fragments of mitochondrial gene ND1 and cytochrome B (CYTB) using the primers reported by talX 1.8 . The comHo), mean gene diversity (Hs), and allelic richness (Ar). Gene diversity and inbreeding coefficient (FIS) were estimated using the R 3.5.3  using the function allelic.richness using the same R package.For nuclear microsatellites, genetic diversity was assessed in terms of total number of alleles (Na), private alleles (Pa), observed heterozygosity , number of polymorphic sites (p), and haplotype (h) and nucleotide (π) diversity were estimated using ARLEQUIN v. 3.5.2.2  and their respective P-values were calculated based on Tamura and Nei distance corrected by the gamma shape parameter; we used 1 × 104 permutations and fixed a significant level of 0.05 in ARLEQUIN.An isolation by distance (IBD) model was evaluated from the microsatellite data using a Mantel test to estimate the correlation between genetic and geographic distances using the istances  and comp package . The allckage  was obtained using the function find.clusters and selecting the best-supported number from the lowest Bayesian Information Criteria (BIC) value. After cross-validation, 49 PCA eigenvalues were retained, containing 78% of the variance. In addition, we evaluated genetic structure using the Bayesian framework from STRUCTURE 2.3.4  were obtained from STRUCTURE HARVESTER . The biogeographic provinces were the: Californian (BM), Cortez , Panamic (CH and EC), Peruvian (PE) and the ocean islands (OC). The oceanographic regions were: the upwelling from the west coast of Baja California (BM), the Cabo San Lucas front (PL and CSL), upwelling of the continental margin of the Gulf of California (GY and MAZ), the Tehuantepec upwelling (CH), Peruvian upwelling (PE); considering Ecuador (EC), and the Oceanic sample (OC) as two distinct groups. AMOVA analyses were implemented in ARLEQUIN using the number of different alleles; significance of F-statistics was determined by 1 × 104 permutations.To explore different hypotheses of population structure within the TEP based on microsatellites, we performed hierarchical analyses of molecular variance (AMOVA) grouping locations according to (a) the biogeographic provinces proposed for resident fishes , and (b)4 iterations. Then, we implemented BAPS 5.4 (K (from 1 to 5). The admixture analysis  is a critical parameter when evaluating population conservation status for management planning. We estimated contemporary Ne for each population group obtained by TESS3 from microsatellite data (single population sample approach) using the bias-corrected version of the LD method  based on microsatellite data, we implemented BOTTLENECK 1.2.02  under three mutation models: the infinite allele model (IAM), two-phase mutation (TPM) with a variance of the geometric distribution = 0.36 as suggested for microsatellites by the authors, and the stepwise mutation model (SMM).To detect recent reductions in effective population size over a relatively short period  and the Sum of Squared deviations (SSD) were used to determine the fit to the expected curve under sudden population expansion. Because the CYTB fragment failed to reach convergence in ARLEQUIN, we obtained mismatch distributions in DnaSP 6.0.7  exceeded 200 for each parameter. We plotted the BSP with their associated CIs (upper and lower highest posterior density at 95%) using TRACER.The historical demography of dolphinfish was inferred from several approaches, considering the whole sample of mtDNA sequences as a single population. First, mismatch distribution analysis  was condSP 6.0.7 . We expe6.0.7  were missing in all individuals from one or three sampled localities . Similarquencies . As we fHo), which over all localities ranged from 0.64 to 0.80 (Hs) across localities ranged from 0.66 to 0.81. Mean allelic richness (Ar) ranged from 5.2 to 10.3, averaging 6.4. Although Chiapas (CH) had the highest Ho and Ar values, Ho, Hs, and Ar exhibited wide variability among sampling locations  exhibited a range of plausible values that suggested some degree of heterozygote deficiency, especially for Guaymas (GY)  ranged from 75 to 150 , and all to 0.80 . The meaocations ; therefomas (GY) .h) diversity was higher than nucleotide diversity (π) for both fragments; h ranged from 0.81 (PL) to 0.96 (CH) for ND1 and from 0.85 to 0.94 for CYTB; π ranged from 0.003 (PL) to 0.005 (CH) for ND1 and from 0.004 to 0.005 for CYTB. Bahía Magdalena (BM) exhibited the lowest number of haplotypes for both fragments (nh = 12 and 8), whereas Guaymas (nh = 33) and the Oceanic sample (nh = 21) had the highest estimations for ND1 and CYTB, respectively  mtDNA-ND1 sequences from 364 individuals from the ectively . The 65 r = −0.16, P = 0.66). The mean Jost’s D between localities  suggested low allelic differentiation, whereas values of GST  indicated very low and significant differences in allele frequencies across sample sites. Nonetheless, comparisons involving Bahía Magdalena (BM), Peru (PE), and Ecuador (EC)   exhibiteK value of 6–7. However, as the variance notably increased for K > 4  was an isolated population, with a second well-defined cluster consisting of Punta Lobos (PL), Cabo San Lucas (CSL), Guaymas (GY), Mazatlán (MAZ), and the Oceanic samples (OC)  . Furtherles (OC) . These sles (OC)  and 1D. les (OC)  and 1D.K from K = 1 to K = 2, and reached a plateau with K = 3  values from the STRUCTURE analysis increased when increasing th K = 3 . The ΔK  samples . Becausey values . Individy values .FCT = 0.03; P = 0.004). This contrasts with the lack of significant differences of groupings based on dominant oceanographic processes and coastal regions (see Methods) . It is noteworthy that the clustering results from TESS3 coincided fairly well with the biogeographic provinces proposed for the TEP, suggesting the influence of differences in environmental conditions among provinces in shaping the genetic structure.The hierarchical AMOVA grouping by biogeographic provinces using the microsatellite results revealed that ~93% of the genetic variation was explained by differences within populations . HoweverK) given the data was K = 2. However, no geographic structuring was detected for either of the mitochondrial fragments .Using microsatellite data, BARRIER analysis identified genetic boundaries with good statistical support >65%; , which cNe suggested, in general, low values of Ne for C. hippurus within the TEP. Estimates ranged from 77.9 to 496.4 , supporting the occurrence of a sudden demographic expansion and past fluctuations in population size. Tajima’s D and Fu’s Fs both suggested departures from neutrality for ND1  and CYTB . Demographic Bayesian reconstructions from the mtDNA-ND1 gene suggested a growth of the dolphinfish population that started approximately 126,000 years before present  or biparentally inherited  molecular markers has been used separately, but had never been combined. Likewise, the sampling at different geographical scales has provided inconsistent patterns of genetic differences. This is the first study to apply mitochondrial DNA and nuclear microsatellite markers and a sampling of almost the entire latitudinal range of C. hippurus in the Tropical Eastern Pacific to infer historical and contemporary factors determining the genetic variation of dolphinfish populations. Based on nuclear microsatellite markers, it was possible to identify four significantly divergent genetic groups within the TEP. Although genetic population subdivisions were low, different lines of evidence confirm the observed differentiation pattern. In addition, the analysis of the mtDNA data strongly suggests that the population has undergone expansion.Knowledge of stock composition and population dynamics of exploited species is highly relevant for sustainable fisheries management . In the Without apparent barriers to dispersal for marine species having a pelagic larval stage, we would expect genetically homogeneous populations. However, the low differentiation that has been reported does not necessarily imply high levels of gene flow among populations , even foe.g., temperature and salinity) and chemical properties   and Peru (PE), trients) . Considetrients) , it is ptrients) , boostintrients) .e.g., driftwood, detritus, and other flotsam), with which dolphinfish are strongly associated, showing both homing and high levels of site fidelity , which could be facilitated by possible annual migration route during winter  reflects that the deep-water zone does not constitute insurmountable barriers separating populations . This pattern is unlikely to be attributed to a high frequency of null alleles because loci were extensively tested for genotyping artifacts that could produce null alleles. Loci Chi389, Chi853, and Chi967 showed null allele frequencies of about 25% in only one population (one of each); however, loci had a minor effect on GST and Jost’s D estimates. Also, no evidence for significant allele drop-out was found using MICROCHECKER. Likewise, a Wahlund effect also seems unlikely because no cryptic genetic structure was detected using clustering analysis. Therefore, we suggest that the positive values of FIS and the low effective population size constitute a warning that the dolphinfish populations are vulnerable to fishing pressure.Our estimates of contemporary ong term , raisingNe estimations have been documented for other similar species such as X. gladius in the TEP. Estimates of Ne for X. gladius ranged from 100 (or lower) to 1,000 individuals and are comparable to those reported for C. hippurus in this study. The notably decreased Ne for C. hippurus parallels the remarkable decline in X. gladius fishery  and nucleotide (very low) diversities estimated for the mtDNA and the star-like phylogeny. Previous studies suggested that habitat reductions or bottlenecks are associated with low nucleotide diversity resulting from a limited number of shared sequences and numerous low-frequency haplotypes separated by few mutations . This ar Pacific .C. hippurus populations could be due to extensive region-wide gene flow  proportional to the length of each color. Population names are in i.e., the lowest BIC). (D) The uppermost hierarchical level of the genetic partition by K [black line in (D)].(A) Cross-validation score from TESS 3 in which smaller values mean better runs. (B) The proportion of estimated ancestry with Click here for additional data file.10.7717/peerj.14389/supp-2Supplemental Information 2K = 2 of Coryphaena hippurus based on a fragment of the mitochondrial gene NADH subunit 1  and Cytochrome B  in the Tropical Eastern Pacific. Population names are in Plots from Bayesian assignment analysis for Click here for additional data file.10.7717/peerj.14389/supp-3Supplemental Information 3Yellow lines represent the three main geographic barriers indicated with letters a, b, and c. Voronoi tessellation is shown in green, and in color blue the corresponding Delaunay triangulation of samples (dots and numbers in red). Black numbers indicate bootstrap support. Populations label code: 1 = Bahía Magdalena (BM); 2 = Punta Lobos (PL); 3 = Cabo San Lucas (CSL); 4= Guaymas (GY); 5 = Mazatlán (MAZ); 6 = Chiapas (CH); 7 = Oceanic sample (OC); 8 = Ecuador (EC); 9 = Peru (PE).Click here for additional data file.10.7717/peerj.14389/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14389/supp-5Supplemental Information 5First probability test and then, when H1 = heterozygote deficitClick here for additional data file.10.7717/peerj.14389/supp-6Supplemental Information 6Linkage disequilibrium between pairs of loci.Click here for additional data file.