Scutellaria floridana) is endemic to four counties in the Florida panhandle. Because development and habitat modification extirpated several historical occurrences, only 19 remain to date. To inform conservation management and delisting decisions, a comprehensive investigation of the genetic diversity and relatedness, population structure, and clonal diversity was conducted using SNP data generated by ddRAD. Compared with other Lamiaceae, we detected low genetic diversity (EH = 0.125–0.145), low to moderate evidence of inbreeding (FIS = −0.02–0.555), and moderate divergence (FST = 0.05–0.15). We identified eight populations with most of the genetic diversity, which should be protected in situ, and four populations with low genetic diversity and high clonality. Clonal reproduction in our circular plots and in 92% of the sites examined was substantial, with average clonal richness of 0.07 and 0.59, respectively. Scutellaria floridana appears to have experienced a continued decline in the number of extant populations since its listing under the Endangered Species Act; still, the combination of sexual and asexual reproduction may be advantageous for maintaining the viability of extant populations. However, the species will likely require ongoing monitoring, management, and increased public awareness to ensure its survival and effectively conserve its genetic diversity.The threatened mint Florida skullcap ( Genetic diversity is a fundamental component of biodiversity and has profound effects on ecological processes, including community structure and the ability of populations to recover from disturbance. Research in the field of conservation genetics has shown that loss of genetic diversity due to inbreeding reduces the ability of a population to persist in unstable environments . DespiteUnderstanding genetic factors such as diversity, reproductive strategies, and gene flow in rare plant species is especially crucial in an era of rapidly changing habitats. Due to their limited migration capabilities and often highly specialized adaptations to soil and moisture conditions, rare plants are especially vulnerable to the impacts of climate change and increasing land conversion due to human activities, and their survival will likely depend on their adaptive abilities . MaximizScutellaria floridana Chapman, Lamiaceae), a perennial wildflower endemic to four counties in the Florida panhandle  molecular markers to analyze genetic diversity among 197 samples collected from seven populations [S. floridana is vital to accurately determine their viability and inform the process of recovery under the Endangered Species Act (ESA).Only one study of genetic diversity in ulations . The autS. floridana represents a significant knowledge gap for the species. In addition, the type of clonal growth strategy, i.e., phalanx (closely spaced ramets with short internodes) or guerrilla (widely spaced ramets with long internodes) [Surveys have shown increases in the number of stems within several populations on managed lands over the past 10 years, and the majority of the remaining populations have good or excellent estimated viability . Howeverernodes) , is alsoWhile clonal propagation increases an individual’s chance of reproduction, increases population sizes, enhances longevity in disturbance-prone habitats, and allows reproduction when conditions are unfavorable for flowering or seed germination, it also poses issues for rare species and complicates conservation efforts ,17. WhenS. floridana. These are to (1) investigate changes in land use that resulted in population extirpation over the last 20 years, (2) assess the genetic diversity of several populations across the species’ range, and (3) determine the prevalence of clonality in the species. The conclusions drawn from this study will contribute to the recovery actions of S. floridana as specified in its recovery plan [S. floridana [This study has three objectives to help evaluate the current status of loridana . Our assS. floridana in 12 of the 17 accessible populations  and Sphagnum strictum , they all lacked an overstory of Pinus palustris (Longleaf Pine) and were located less than 100 m from major roadways. We were also unable to locate plants in one population that is located in a small area of St. Joseph Bay State Buffer Preserve  to the north of the main preserve. Several of the sites within the population were inaccessible due to fencing, but those that we could access were flooded by approximately 30–50 cm of water with an extremely thick understory of Cyrilla racemiflora (Titi) and little to no herb layer. Two of the populations that we attempted to visit were located in active rangeland or timber harvest operations in central Gulf County and were inaccessible. Of the 12 populations that contained actively growing individuals, Box-R Wildlife Management Area population 1  was the smallest, containing seven small patches (<10 stems/patch and <5 m2), while larger populations contained hundreds of stems. At the time of collection, we observed flowering individuals in several sites in the Apalachicola National Forest , one site in Tate’s Hell State Forest , one site in the BRWMA, and the only site in the Lathrop Bayou Habitat Management Area .We were able to locate individuals of ulations . Five po2 area that represents the entirety of S. floridana’s range experienced significant increases in medium- and high-intensity development . Deciduous and evergreen forests decreased by 33% and 14%, respectively, while mixed forest, shrubland, grassland, and herbaceous wetlands increased by 18%, 19%, 39%, and 22%, respectively. Approximately 22% of S. floridana’s range falls within the borders of public lands, including state forests, national forests, habitat management areas, and preserves. These areas experienced no significant change (>2%) in land cover during this time frame, suggesting that changes were restricted to private lands .The final dataset that we used for downstream analysis included 10,223 loci and 28,210 variable sites that were each present in at least 60% of individuals. The observed heterozygosity ranged from 0.04 to 0.14, with an average value of 0.11 . The obsFST = 0.05–0.15). Nine pairs of populations showed little to no differentiation from one another (FST ≤ 0.05), and six pairs of populations showed high divergence from one another (FST = 0.15–0.25). Overall, LB1, SJSBP1, SJSBP2, THSF1, and THSF2 all showed low divergence from one another as a group, with pairwise FST falling at or below 0.07 for all pairs. Five populations, ANF1, ANF2, ANF3, BRWMA2, and THSF2, also had high FST values relative to all other populations and particularly high divergence values (FST > 0.20) relative to ANF5 and BRWMA1 .Populations generally displayed moderate divergence from one another  as the most likely scenario. Lathrop Bayou, SJSBP2, BRWMA2, ANF1, ANF2, ANF3, ANF4, and THSF2 all contained individuals possessing unique ancestry proportions that were not present in any other population. The four remaining populations  were almost entirely composed of individuals with highly admixed ancestry estimates and did not display an identifiably unique pattern of ancestry .Cross-validation identified the optimal number of PCs to retain as 20. Of the eight possible discriminant axes, all were retained. The first two axes explained 33.7% and 26.1% of genetic variance, respectively. The DAPC results supported our findings of high admixture between populations and identified ANF2, ANF3, and BRWMA2 as genetically distinct with limited inter-population gene flow . PopulatWe identified clonal individuals in two 5 m diameter circular plots and identified a pair-wise genetic distance of 0.03 as the threshold below which individuals were considered to be clones . In the An analysis of the clonal diversity in the studied populations points out that 92% are multiclonal. There were one to 16 sampled clones in a single population , Table 2S. floridana using next-generation sequencing, ddRADseq. Our finding that about 62% of the sampled plants were unique genotypes suggests that the S. floridana populations consist of a mixture of both asexually and sexually reproducing individuals. This demonstrates that sexual reproduction is occurring in most populations, more than was previously thought based on field observations [We were able to describe genetic diversity and specifically assess the extent of clonal spread of rvations . Clonal Scutellaria floridana appears to possess moderately low genetic diversity and high levels of between-population differentiation in contrast to other members of the Lamiaceae of conservation concern [S. floridana study based on the AFLP marker [EH = 0.124–0.145 in this study versus EH = 0.145–0.184 in [S. floridana FST = 0.035 in [FST estimates in particular may be due to the different sampling scheme and greater number of samples and areas included in this study. For example, the two populations that we identified as the most differentiated and least genetically diverse, BRWMA1 and ANF5, were not included in the previous work. This likely led to an underestimation of population differentiation and an overestimation of genetic diversity in the previous study. concern ,22. CompP marker , we obseS. floridana populations , Florida’s Natural Heritage Program and state member of the NatureServe network, has previously documented currence . BecauseS. floridana that we located in the field are found on managed lands. The majority of these are located along the western edge of ANF in management areas designated as longleaf pine and slash pine adaptive management units that are maintained by prescribed fire [S. floridana are designated as wet prairie and are managed with prescribed fire at a 2–3 year frequency [Scutellaria floridana is found on the largest of these islands, Raffield Island. The primary management goal of this area has been to restore open-understory pine flatwood conditions using prescribed burns at a frequency of 1–2 years [Of the 19 populations previously identified as extant, we were unable to access two due to fencing and an active timber harvest operation. All of the extant populations of bed fire . Other pbed fire . Box-R Wbed fire . St. Josrequency . Lathrop–2 years .We conducted two trips in the spring of 2021 and collected a total of 294 plant tissue samples from 12 populations, representing 17 sites . We tookS. floridana was present, with a minimum distance of approximately 1 m between each sample . Samples that displayed adequate quality and reached a minimum DNA concentration of 20 ng/uL were then sent to Floragenex , where double-digest restriction site-associated DNA sequencing (ddRAD-Seq) was carried out. To summarize, DNA was first digested using the restriction endonucleases PstI and MseI. Samples were diluted for PCR amplification, and the product was used to construct a ddRAD-Seq library. The library was sequenced at the University of Oregon Genomics and Cell Characterization Core Facility (GC3F) on a NovaSeq 6000 with a SP100 chip, generating 118 bp single-end reads with a mean 27.5× effective coverage per sample. The sequence data were run through the pipeline STACKS (version 2.60) to assemble the short-read sequences from all the samples (via the process radtags program) and to align reads into loci that are genotyped (via the gstacks program) [We extracted total genomic DNA from our plant material samples using a protocol similar to that used by . Brieflyprogram) ,45. SingS. floridana’s range, on managed lands within its range, and within 500 m of each population.We analyzed changes in habitat over the past 20 years using 2001 and 2019 land cover rasters retrieved from the USGS National Land Cover Database. We used ArcGIS Pro (Esri ArcGIS Pro v. 2.9) to calculate the total area and change in area of each land cover category over the past 20 years across the entirety of FST for populations using the package StaMPP [FST and geographic distance between populations using the package ade4 [Floragenex filtered raw sequence data and identified and assembled loci using the Stacks pipeline . We usede StaMPP . To inveage ade4 . We estiage ade4  and perfFST and DAPC analyses), and signature of unique genetic ancestry  relative to other populations in this study. High to very high genetic value populations had high estimates of observed heterozygosity and low inbreeding coefficients, high or very high estimates of population divergence , and signatures of unique ancestry. Moderate genetic value populations have moderate estimates of observed heterozygosity and inbreeding coefficients, moderate estimates of population divergence, and exhibit signatures of unique ancestry. Finally, low genetic value populations have low estimates of observed heterozygosity, high inbreeding coefficients, moderate estimates of population divergence, and lack signatures of unique ancestry.The population genetic value was qualitatively assessed based on the level of a population’s observed heterozygosity, inbreeding coefficient, population divergence  that possess very low genetic diversity and evidence of inbreeding. If the high prevalence of clonality is driving the low genetic diversity in these populations, re-establishing disturbance regimes, especially prescribed fire, could stimulate flowering and potentially sexual reproduction and improve genetic diversity [S. floridana as a species and suggest that they be treated as the highest conservation priority, as their continued existence is vital to preserve the adaptive potential of the species.Overall, iversity . We idenScutellaria floridana will likely require regular monitoring and active conservation efforts, including public outreach, to avoid any further extirpation events. There is a possibility, however, that the S. floridana genotypes will persist in a population longer, since the guerrilla strategy tends to reduce the chance of extirpation, lessening the impact of stochastic events on the genetic structure of this species. The combination of sexual and asexual  reproduction may be advantageous for maintaining the viability of S. floridana populations.


Indian cultural influence is remarkable in present-day Mainland Southeast Asia (MSEA), and it may have stimulated early state formation in the region. Various present-day populations in MSEA harbor a low level of South Asian ancestry, but previous studies failed to detect such ancestry in any ancient individual from MSEA. In this study, we discovered a substantial level of South Asian admixture (ca. 40–50%) in a Protohistoric individual from the Vat Komnou cemetery at the Angkor Borei site in Cambodia. The location and direct radiocarbon dating result on the human bone  indicate that this individual lived during the early period of Funan, one of the earliest states in MSEA, which shows that the South Asian gene flow to Cambodia started about a millennium earlier than indicated by previous published results of genetic dating relying on present-day populations. Plausible proxies for the South Asian ancestry source in this individual are present-day populations in Southern India, and the individual shares more genetic drift with present-day Cambodians than with most present-day East and Southeast Asian populations. Anatomically modern humans arrived in MSEA approximately 50,000 years ago2. The early to mid-Holocene hunter-gatherers associated with the Hoabinhian archaeological tradition  were modelled as a deeply diverged East Eurasian lineage, related to present-day Andamanese and to MSEA foragers such as Jehai3. During the Neolithic period , ancient MSEA populations exhibited an admixed genetic profile between the deeply diverged East Eurasian lineage and East Asians4. The same genetic mixture is typical for some present-day groups speaking Austroasiatic languages, such as the Mlabri4. The genetic structure of Bronze Age individuals from Northern Vietnam (dated to ~ 2000 BP) suggests an additional wave of migration from Southern China4.The high ethnolinguistic diversity of Mainland Southeast Asia (MSEA) reflects the complex population history of this region5). Archaeological evidence revealed that the cultural interaction between Indian and local MSEA people began by the fourth century BCE, creating synergies that influenced the formation of early states in MSEA7. Various present-day MSEA populations, especially those highly influenced by Indian culture, harbor a low level of South Asian admixture: for instance, Bamar, Cham, Khmer, Malay, Mon, and Thai10. Using methods based on autosomal haplotypes, this genetic admixture was dated to around the 14th c. CE (1194 CE–1502 CE) in a Khmer group from Cambodia11). In a later study, the admixture date for the Khmer group from Cambodia was confirmed (12th–13th cc. CE), and admixture date estimates for a wider range of present-day SEA groups were obtained: they lie between the 16th c. CE for Bamar and the 4th cc. CE for Giarai (Jarai) from Vietnam10. Previous ancient DNA studies, although of very limited extent due to poor ancient DNA preservation in tropical climate, did not detect South Asian ancestry in any ancient MSEA individuals4.Debates over the origins of early states in MSEA have hinged on the nature of contact with South Asia for more than 50 years . A direct radiocarbon date on the human bone  was also obtained for the individual AB M-40 by Lipson et al.4.Genetic data for the male child from the Vat Komnou cemetery  were first reported by Lipson et al.3, Lipson et al.4, and this study) onto these PCs. The PC1 vs. PC2 plot  support the existence of the West Eurasian genetic component in the individual AB M-40/I1680 as all f4-statistic values are positive, with |Z| > 2 in most cases  show results   is illustrated in Fig. lot Fig.  reveals f3-statistics f3 . Values of the statistics f4 are all negative, with most of absolute Z-scores higher than 2  groups and present-day Cambodians share additional genetic drift as compared to the Protohistoric individual AB M-40/I1680. On the other hand, statistics f4 are all positive, with most absolute Z-scores higher than 2  and the Protohistoric individual AB M-40/I1680. However, when we replaced Kinh from Mallick et al.24 with a much larger Kinh group from the 1000 Genomes Project25, the f4-statistics showed a mix of positive and negative values  suggest that substantial South Asian admixture is present in the individual AB M-40/I1680, which sets him apart from 25 other Southeast Asians for whom genetic data of acceptable quality was reported in the literature4.To sum up, these PCA and f3-statistics, f3 and f3, where X are ancient individuals from Southeast Asia, present-day ESEA populations, Tibeto-Burman-speaking groups from India and Nepal, and Kusunda from Nepal  and Vellalar26 (South Indians) as alternative SAS surrogates, and Atayal and Dai27 as alternative ESEA surrogates. Most present-day and ancient groups from ESEA demonstrate a linear relationship of genetic drift sharing with the South Asian and East Asian surrogates. f3-statistics for various groups, namely the ancient individual AB M-40/I1680 from Cambodia, Tibeto-Burman-speaking populations (Tibetan and Sherpa) and Kusunda from Nepal, Austroasiatic-speaking (Birhor and Kharia) and Tibeto-Burman-speaking (Riang) groups from India, do not conform to this trend line  with a single reference population  for the Protohistoric Cambodian individual AB M-40/I1680, Ami is the only East Asian surrogate, and Irula, Mala, and Vellalar are the only non-East Asian surrogates that result in plausible models  using a els Fig. . Remarkaels Fig. . The facqpAdm models as a two-way admixture of East Asians and a deeply diverged East Eurasian lineage (represented by Onge and Papuans in the set of reference populations) , Malaysia , Thailand (Th531), and Vietnam  can be interpreted in the light of our 10. However, the qpAdm reference populations used here and in the previous study10 are different. To avoid an unforeseen bias which may cause the excess of South Asian ancestry in the Protohistoric Cambodian individual AB M-40/I1680, we estimated the proportion of South Asian admixture with the same set of surrogates and qpAdm “right” populations for the ancient individual and present-day Cambodians. Since no plausible model was found for present-day Cambodians when we used the reference population set from the analysis above, we replaced Ami with Dusun and performed qpAdm analysis with the new set of reference populations , as estimated in a previous studyons see “”. The reons see “.4, but we generated additional data to increase the sensitivity of the analyses in the current study. South Asians were not considered as a potential ancestry source for MSEA groups in the original study, which explains the discrepancy between our results and those by Lipson et al.4. For instance, PCA in Fig. 4 lacks any West Eurasian groups, and another PCA in Fig. 4 lacks any South Asian groups except for the Andamanese. Nevertheless, according to the latter PCA4 the individual AB M-40/I1680 is shifted towards Europeans and Andamanese as compared to all present-day and most ancient MSEA individuals.We report new archaeogenomic data from a radiocarbon-dated individual AB M-40/I1680 from Cambodia  that serves as a key data point for dating the beginning of the South Asian gene flow into Southeast Asia. This individual was previously analyzed by Lipson et al.32. Strontium isotopic analysis of this individual suggests limited evidence of human mobility33. Since the individual AB M-40/I1680 was a young child the strontium isotope data do not rule out the possibility of his parents’ mobility. Nevertheless, outgroup f3-statistics  were generated for just 21 individuals from Cambodia, Laos, Malaysia, Thailand, and Vietnam dated to the 5th c. BCE and later4. As suggested by archaeological evidence7, we expect to find traces of interactions with South Asia starting in the 4th c. BCE.South Asian admixture is present in various present-day MSEA populations, especially those heavily influenced by Indian culture10. Notably, the proportion of South Asian ancestry in the ancient individual from Cambodia  is considerably higher than in the present-day majority population of the country and in any present-day MSEA population investigated in the literature . In this study, we also demonstrate that the ancient individual from Cambodia harbors an approximately three times higher proportion of South Asian ancestry as compared to the present-day Cambodians, as estimated by qpAdm with the same set of reference populations . Collectively, these data suggest some level of Indian cultural influence in the Mekong Delta in the 1st–3rd c. CE. The only plausible South Asian genetic sources for the individual from Funan, inferred by the qpAdm method, are populations from Southern India. Even though the results suggest that South Indian populations are by far the most plausible surrogates for this individual, we caution that the actual ancient sources possibly had a genetic profile different to the present-day South Asian populations we used for the qpAdm analysis.The location and date (1st–3rd c. CE) of the individual AB M-40/I1680 fit the early period of Funan, an early state in the territories of Cambodia and Vietnam. Chinese written sources documented that the Funan dynasty was established by an Indian Brahmin named Kaundinya and a local princess named Soma38) remains a mystery. The term “Funan” is based on the modern Chinese pronunciation. Whether the local pronunciation was “biunâm”, resembling the old Khmer word “bnam”, meaning “mountain,” remains an unresolved question5. Continuities in architectural style, imagery, and settlement forms from the Protohistoric through later Angkorian period suggest that some residents of Funan spoke old Khmer. Outgroup f3-statistics  share more genetic drift with the ancient individual AB M-40/I1680 than present-day Cambodians , respectively10, but the date of the ancient individual AB M-40/I1680 is much older . Indeed, true dates of admixture events, especially its starting date, do not necessarily fall into the range of admixture dates estimated from similar present-day populations from the same or proximate locations. Our results suggest that some South Asians migrated to MSEA and intermarried with local people before or at the early stage of state formation. These South Asians may have influenced the expansion of Indian culture and the establishment of Indian-style states . aDNA data from MSEA are scarce, so further data collection is necessary to trace the incorporation of South Asian ancestry into the populations of this region.Changmai et al.14. Despite site disturbance, the presence of 33 primary burials, most interred in the same orientation (with head pointing southwest), suggests the excavated portion (approximately 0.03 percent of the mound) was part of a designated communal burial area.Archaeological field investigations at the Vat Komnou cemetery  were undertaken by the Lower Mekong Archaeological Project (LOMAP) in 1999 and 2000 as a collaboration between the University of Hawai ‘i-Mānoa (USA), the Ministry of Culture and Fine Arts (Kingdom of Cambodia), and the Royal University of Fine Arts (Kingdom of Cambodia). A total of 111 individuals were sorted and analyzed from 57 burial features excavated from excavation unit AB7, a 5 m by 2 m unit near the edge of the cemetery mound. Many of the burials were commingled  and lack clear grave cuts. The extensive commingling of the burials is due, in part, to past disturbances of the stratified nature of this densely packed cemetery, often resulting in subsequent interments in the same location4) from the Vat Komnou cemetery, is the same individual as AB40 in Lipson et al.4. The individual was sorted from commingled remains of at least five different individuals. The skeletal remains for AB M-40 included cranial and postcranial elements of a male child whose age was estimated to be 5–7 years at the time of death based on clavicular diaphyseal length, the degree of epiphyseal fusion, and dental calcification and eruption. The cranial elements include the right and left parietals, occipital, frontal, right zygomatic, inferior maxillary bones, left temporal, and the petrous portion of the right temporal. The mandible was missing the right ramus. A left clavicular shaft and a thoracic vertebra represent the postcranial remains for this individual. DNA was obtained from the petrous portion of the right temporal bone .AB M-40/I1680, a Protohistoric Period male individual  in addition to 7 analyzed by Lipson et al.4, which increased the coverage for autosomal targets on the 1240K SNP panel16 from 0.047 × to 0.061 × and increased the number of usable SNPs from 54,221 to 64,103 1240K sites . We kept only autosomal SNPs from the 1240K panel16 and filtered out ancient individuals who have data for fewer than 50,000 autosomal SNPs. Details of the dataset composition are available in Suppl. Table In the present study, we generated two single-stranded sequencing libraries for this individual . We computed principal components for present-day populations from Central, East, Southeast, and South Asia, Andamanese Islands, Siberia, and Europe. We then projected the ancient Southeast Asian individuals onto the principal components using options "lsqproject: YES" and "shrinkmode: YES".We performed PCA using PLINK v.1.90b6.10 with options "-indep-pairwise 50 5 0.5" . The LD-pruned dataset included 523,650 SNPs. We performed ADMIXTURE analysis22 using ADMIXTURE v.1.3 . We first tested 4–12 hypothetical ancestral populations (K from 4 to 12) with tenfold cross-validation and five independent algorithm iterations for each K value. The CV errors are almost indistinguishable for K from 6 to 10 and grow for K values above 10 .We performed LD-pruning using qp3pop" from the R package "ADMIXTOOLS2"45 with default settings (https://uqrmaie1.github.io/admixtools/index.html). For outgroup f3-statistics in Fig. f3, where X are present-day populations from East and Southeast Asia.We used the function "f3-biplots in Fig. f3  and f3 , where X are ancient individuals from Southeast Asi or, present-day populations from East, Southeast, and South Asia; SAS are South Asian surrogates ; and ESEA are East and Southeast Asian surrogates .For the f4-statistics using the function "qpdstat" from the R package "ADMIXTOOLS2"45 with default settings (https://uqrmaie1.github.io/admixtools/index.html). For the plot in Suppl. Figs. f4, f4, and f4, respectively.We computed qpWave and qpAdm analyses we fixed Mbuti as R1 in a matrix of statistics f4.We used a set of 13 populations as “outgroup” (“right”) populations: 12 diverse worldwide populations  + one of the following South Asian populations . All the South Asian surrogates were composed of at least three individuals. In total, we had 14 alternative sets of reference populations. For consistency, in all qpWave modeling using the "qpwave" function from the R package "ADMIXTOOLS2"45. We considered a pair composed of the target individual  and one of the 12 reference populations  as "left populations". We assigned the remaining 12 reference populations as "right populations". We used a cut-off p-value of 0.05 and tested both "allsnps = TRUE"  and "allsnps = FALSE" settings . Overall, we tested 168 group pairs with qpWave per one target group per one "allsnps" setting.We first performed pairwise qpWave models for all the ancient MSEA targets was fitting the data, we further tested 2-way admixture models using qpAdm with a “rotating” strategy31. All possible combinations of two reference populations (except for Mbuti) acted as surrogates for the target. The remaining 11 reference populations were assigned as "right populations". We defined a model that meets two following criteria as a "plausible model": (1) p-value is higher than 0.05; (2) inferred admixture proportions ± 2 standard errors lie between 0 and 1 for all ancestry components. We tested qpAdm models using the "qpadm" function from the R package "ADMIXTOOLS2". We tested both "allsnps = TRUE" and "allsnps = FALSE" settings. In total, 924 models per target per one "allsnps" setting were tested.As none of the qpAdm analysis). The set of right populations consists of Mbuti, Japanese, Yi, Onge, Papuan, Pima, Karitiana, Druze, Adygei, Sardinian, and Orcadian.For comparing South Asian ancestry proportions in the Protohistoric Cambodian individual AB M-40/I1680 and in present-day Cambodians, we replaced Ami with Dusun in the set of reference populations. We modeled the ancient individual and present-day Cambodians as a 2-way admixture of Dusun and one of the following South Asian populations: Irula, Mala, and Vellalar (plausible South Asian sources for the Protohistoric Cambodian individual in the previous Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.


Astrocaryum aculeatum, a palm tree incipiently domesticated from upland ecosystems in the Brazilian Amazon, is especially adapted to anthropized areas. The pulp of the fruit, obtained by extractivism, is consumed fresh by the Amazonian population. The objective of the study is to evaluate the diversity and genetic structure of the natural populations of A. aculeatum, exploited by extractive farmers in Amazonas, Brazil, seeking to suggest conservation and management strategies for this species. A total of 218 plants were sampled in 15 populations in 14 municipalities in the state of Amazonas, evaluated by 12 microsatellite loci. A total of 101 alleles were observed. The means of the observed heterozygosities (OH = 0.6390) were higher than expected (EH = 0.557), with high levels of heterozygotes in the populations. The fixation index in the loci and populations was negative. The STF (0.07) and AMOVA showed moderate population structure. Bayesian analysis indicated the grouping k = 4 as the most adequate. There is a high genetic diversity in populations, with a moderate genetic structure due to possible historical events, which could be related to the process of subpopulation formation, possibly presenting three historical moments: before and after the beginning of deforestation and today. The conservation and management policies of this species must be carried out at a watershed level. Astrocaryum aculeatum Meyer), a palm tree of the Arecaceae family, exists in the western and central areas of the Brazilian Amazon. It is distributed in the states of Acre, Mato Grosso, Rondônia, Roraima, part of Pará, and Amazonas [Dasyprocta azarae and Myoprocta sp., which end up behaving as secondary dispersers (zoochoric) of the species itself [The tucumã-do-Amazonas , is the main available means of production. It is irregularly offered, in both quantity and quality, to the market of Amazonas, the largest state in Brazil. However, if we analyze the extractive exploitation based on the protection of the Amazon forest ecosystem [A. aculeatum [Extractive exploitation of cosystem , the concosystem . The ecocosystem . This isculeatum .A. aculeatum are paramount for the establishment of adequate strategies in the use of this genetic resource. This may be the starting point to obtain a response from current natural populations and it may suggest how to increase the efficiency of domestication and selection at multiple stages of the genetic improvement program for this species. Natural populations of A. aculeatum have a great genetic variability of traits, such as plant height, fruit yield and quality, size, pulp yield, flavor, fiber, and oil content [A. aculeatum by Ramos et al. [Astrocaryum, including A. aculeatum, originating in the state of Pará [The knowledge of the parameters of interpopulational and intrapopulational diversity and genetic structure of natural populations of  content ,15. Howe content . SSR mars et al. . They has et al.  and to a of Pará .A. aculeatum Meyer exploited by extractive farmers in the state of Amazonas, Brazil, using microsatellite markers. The following questions were posed: (a) what is the level of genetic variability among the sampled genotypes in the evaluated natural populations of tucumã-do-Amazonas? (b) How is this diversity structured? (c) What is the role of hydrographic basins in structuring populations? (d) Is there isolation by distance between populations? The answer to these questions will enable the improvement and efficiency in the management, use, and conservation of this natural resource.Considering that this species has not yet been domesticated and aiming to contribute to the management, use, and conservation in situ and ex situ of the genetic resources of this species, this research aims to evaluate the diversity and genetic structure of natural populations of EHW) (p > 0.005) (EH) varied between 0.096 for locus Aac02 and 0.87 for locus Aac04. In most loci, the observed heterozygosities (OH) showed higher values than those of EH, with the exception of the loci Aac06 (0.63), Aac07 (0.65), Aac09 (0.49), and Aac14 (0.61). OH ranged from 0.10 for locus Aac02 to 0.97 for locus Aac12. The mean of all loci for OH was 0.64, higher than the mean for HE (0.59).Of the 12 microsatellite loci used, the primers Aac01 and Aac13 were excluded because they presented monomorphism in the plants sampled. The ten analyzed loci were in Hardy–Weinberg equilibrium (> 0.005) , and theOH showing higher values than the EH in 14 populations. There was a single exception for the Itacoatiara population, which had a similar value (0.55) to OH and EH. The OH ranged from 0.55 for the populations of PF– Rumo Certo and Itacoatiara to 0.74 for Presidente Figueiredo–Estrada Balbina km 42 . The EH ranged between 0.42 for the PF–Rumo Certo and 0.59 for the Iranduba population. The mean of OH of all populations was 0.64, thus, superior to EH, with 0.54. The fixation index (f) presented values below zero, which shows an excess of heterozygotes  to 5.1 for the Humaitá population . The valozygotes .EHW evaluated from the ten loci in the 15 populations showed that 37 interactions between locus and population do not adhere to EHW and that the loci Aac03 and Aac10 are those with the greatest lack of adherence to EHW in populations. However, the loci Aac02, Aac11, and Aac14 showed EHW for all populations. Most loci segregate independently because in the linkage equilibrium analysis, they presented percentages of linkage disequilibrium (LD) that varied between 2.22% for the populations of S.S. Uatumã and Iranduba and 24.44% for the population of Borba.The analyses also showed a total of 17 private alleles observed in 13 populations. These were obtained for the loci Aac02, Aac04, Aac06, Aac07, Aac09, Aac10, and Aac14 and distributed in different populations . The anaITF = –0.0714) and the estimate of inbreeding due to the reproductive or intrapopulation system (ISF = –0.1521) were lower in relation to inbreeding due to subdivision (STF = 0.0700). The fixation indices STF were outside the upper and lower limits of bootstrapping, which indicates that the estimates are significantly different from zero  to the probabilities of each pairwise comparison .When populations were grouped according to their proximity to a watershed  and compch other . The paie values . Howeverp = 0.001). The remainder of the genetic variation was observed between populations , which means that there is a moderate percentage of differentiation between populations  through the Bayesian analysis performed by the software structure, two possible forms of grouping were observed: K = 3 and K = 4 , group II , group III (PF–Rumo Certo), and group IV   .The cluster analysis classified the populations into three groups, which somewhat corroborate the Bayesian analysis . The firIn the second group of the dendrogram, the populations of Novo Aripuanã, Borba, Humaitá, and Manicoré belong to the Madeira River watershed, showing that there is a flow of genetic material between the populations of this watershed. The population of Humaitá presents a greater genetic similarity with the population of Manicoré. The population of Borba grouped with the populations of group II in the Bayesian analysis , and notThe third group classified the population of the Rumo Certo community in the municipality of Presidente Figueiredo, which has a different genetic constitution from the others. This event can be explained by the fact that this population was isolated by the Balbina Dam and because this population is in an area that is a small island within this dam.A. aculeatum in the state of Amazonas. The numbers are comparatively higher than those found in other studies, such as Astrocaryum murumuru and A. paramaca [Euterpe edulis [A. aculeatum), for populations in the state of Pará [f), which shows negative values in populations and an excess of heterozygosity, with higher values of OH, compared to EH [A. aculeatum presents reproduction by allogamy [Astrocaryum mexicanum [Geonoma schottiana [Phoenix dactylifera [Oenocarpus bataua [OH over EH of A. aculeatum shows a high diversity compared to Euterpe oleracea [A. aculeatum and A. murumuru, only for A. paramaca [This study showed a high mean number of alleles per locus and high levels of genetic diversity for natural populations of paramaca , Euterpee edulis , includi of Pará . This hied to HE . This inallogamy  and doesallogamy . We ask exicanum  or even hottiana , Phoenixtylifera , and Oens bataua , showed oleracea . Howeverparamaca .A. aculeatum evaluated are geographically distributed in two types of climate, Af and Am, according to the Köppen–Geiger climate classification [The presence of private alleles in the populations sampled suggests the importance of genetic conservation for this species. A special management in populations with this allelic difference is suggested . An examfication .EHW model. However, the EHW algorithm does not consider the performance of evolutionary forces, as it is a referential model, except for those imposed by the reproductive process itself [The behavior of plants in a given habitat always aims at local adaptation, which would be caused by the contribution of conditional neutrality at many independent loci interacting to influence fitness, with alleles from different constraining environments being favored at different loci . This wos itself .A. aculeatum within the domestication process is important information, although anthropic extraction may affect the genetic diversity of the species in the future in the process of recruiting new plants in the seedling bank [The high genetic diversity observed for ing bank . This ising bank .STF), Wright’s F statistics indicated the existence of a moderate genetic structure [A. aculeatum sampled. Most of the genetic variability sampled is found within populations by the inbreeding information observed within subpopulations due to the reproductive system (ISF). This information is corroborated by AMOVA results, which show that the greatest concentration of diversity occurs within populations. This result may be strongly influenced by the characteristics of the species and by the ability to disperse its genetic material [Due to genetic differentiation because of subdivision , which deposit seeds in the vicinity of plants [Before deforestation, or in climax-type succession forests,  species . In addi species . Thus, b species . This do species . However species ,2, and sf plants , and alsf plants . This sef plants .A. aculeatum, specifically in areas where the species was present in the climax forest before being cleared. This formation and expansion in deforested, anthropized areas could be mainly related to the zoochoric dispersion of pyrenes , allowing them to be present in the seed bank of climax forests before being disturbed, since the soils of tropical forests are often considered as the final place where plant diaspores are deposited [A. aculeatum trees, as is the case of several species of palm trees that are called secondary and invasive species in the Amazon, such as Astrocaryum acaule, Attalea humilis, Bactris maraja, and Lepidocarium tenue [A. aculeatum is a species capable of withstanding high soil temperatures, resulting from these fires [The second moment could be related to era after the beginning of deforestation in the Amazon in 1960, marked by the process of formation and expansion of large or small natural populations of eposited , startineposited . This prum tenue . Anotherum tenue , reducinum tenue . Temperaum tenue . HoweverA. aculeatum, as they are possibly new populations, are still in the process of adaptation and differentiation from each other, since even within continuous populations, environmental heterogeneity may provide an excellent dimension of the genetic structure with the evolution of local adaptation [The third moment is the current situation for populations. It would be marked by the process of secondary dispersion, carried out by humans, leading to the establishment of new populations. In the collections of tucumã-do-Amazonas in different watersheds, the production of agricultural products already domesticated, incipiently domesticated, or collected in an extractive way in the Amazon forests by Amazonian farmers is commercialized among these populations. They use river transport mainly for this purpose, transporting genetic material to other areas of the Amazon. This indicates that this external connectivity variable is a vector of dispersion that affects the movement of seeds, which makes the plant persist, expand, and colonize new habitats . Thus, taptation .A. aculeatum are relatively new and that they have a vicariant structure.The possible historical moments and the beginning of the evolutionary process of adaptation could be confirmed by the clusters obtained in the Bayesian analysis. It happened mainly in the only grouping of the population of PF–Rumo Certo. This event could be related to the isolation by vicariance of this population because it is located on an island within the Balbina Dam. The beginning of the construction of this infrastructure was in 1981. The dam was closed on October 1, 1987, leading to the formation of a reservoir that presents a reticulated interconnection arrangement between backwaters, that is, a labyrinth of channels between approximately 1500 islands and 60 tributaries . It alloRegarding the management of the species, this first study on the diversity and genetic structure of tucumã-do-Amazonas indicates that the conservation of the species should be carried out mainly at the level of watersheds, as the results obtained in the different analyses indicated. Studies should also be carried out at the level of conservation in situ/on farm and at the ex situ level in order to start the process of domestication and improvement of plants through different accessions of genotypes in these watersheds. It allows improvements to knowledge and genetic materials for the benefit of traditional farmers in the Amazon and future ventures.A. aculeatum collected by extractive farmers from the Amazon rainforest to supply their products, especially to the market of Manaus, the capital of the state of Amazonas. Fourteen municipalities were selected seeking to fill most of the main hydrographic basins within the state of Amazonas to carry out the respective samplings. The municipalities selected for this study were: Nova Olinda do Norte, Borba, Novo Aripuanã, Manicoré, and Humaitá on the Madeira River; Presidente Figueiredo, S.S. Uatumã and Urucará on the Uatumã River; Iranduba and Manaquiri on the Solimões River; Maués and Silves on the Maués-Açu and Urubu Rivers, respectively; Itacoatiara on the Amazon River; and Manaus on the Negro River , under the project titled “Assessment and selection of superior genotypes of peach palm for palm heart (Bactris gasipaes Kunth. var. gasipaes Henderson) and generation of technologies for cultivation of tucumã (A. aculeatum G. Mey) in the State of Amazonas”.The plant material collected was a leaflet, which was stored in a previously identified zip lock plastic bag containing silica gel until it could be transported and stored at –20 °C. These collections were carried out following the rules of SisGen , record n°. A00C6EC. Samples of progenies of the evaluated plants were accessed and currently constitute the ex situ germplasm bank of this species, located at the experimental field of Embrapa Amazonia Ocidental, in Iranduba, Amazonas, Brazil (coordinates: 3°14′56′′ S and 60°13′20′′ W). The access to the genetic heritage of –1 to be used for amplification. The 218 samples were amplified by polymerase chain reaction (PCR) using 12 microsatellite loci  developed for A. aculeatum [2, 0.16 µM of primer forward and M13 spray (FAM or NED dyes) [DNA was extracted according to the CTAB 2× (Cationic Hexadecyl Trimethyl Ammonium Bromide) cationic detergent protocol described by Doyle and Doyle  and quanculeatum . PCR reaED dyes) , 0.32 µMSample genotyping was performed by capillary electrophoresis on the ABI 3130XL Genetic Analyzer Automatic DNA Analyzer . The ET-550 ROX size standard  was used to determine the size of alleles. Amplified fragments were observed and analyzed using the GENEMAPPER v. 4.0 software .TA), average number of alleles/locus (A), observed heterozygosity (OH), expected heterozygosity (EH), fixation index (f), and Hardy–Weinberg equilibrium (EHW). These parameters were calculated using the function divBasic of the package diveRsity [LD), number of private alleles (Ap), and the number of null alleles were calculated using the functions LD, Nm_private, and the null of the package genepop [EHW and LD were performed by Fisher’s exact test with 100,000 permutations. The significance level (p < 0.05) of EHW and LD was adjusted with Bonferroni correction [The genetic diversity of each sampled population was obtained using the total number of alleles , inbreeding index in subpopulations due to the reproductive system (ISF), and genetic differentiation due to subdivision (STF) using the algorithms of Weir and Cockerham [STF pair by pair. A matrix at the population level and another matrix between the hydrographic basins formed by the respective populations located on the banks of each hydrographic basin or located close to it were built  with 20,000 bootstrappings, using the function diffCalc of the package diveRsity [To verify the existence of genetic structure in Wright’s  F statisockerham . Lookingre built . The caliveRsity .STF, in terms of populations and geographic distance, were used to perform the Mantel test [mantel.rtest of the package ade4 [The pairwise matrices tel test ,61, seekage ade4 ,63,64,65age ade4 .The degree of genetic variation according to hierarchical levels between and within populations was analyzed by analysis of molecular variance (AMOVA) , as implK) was set from 1 to 20, and for each K, twenty iterations were performed, with a Burn-in of 100,000 followed by 500,000 iterations Markov Chain Monte Carlo (MCMC). The number of clusters was estimated using the data probability Ln (ln Pr(X|K)) for the different values of K [K selected, a consensus was reached of iterations performed in this cluster through the CLUMPP v. 1.1.2—Cluster Matching and Permutation Program [For the genetic structure analysis, a Bayesian analysis was performed to determine the number of clusters within the set of samples evaluated using the software structure  configurues of K . With th Program . With th Program , the graA dendrogram was also constructed with the average linkage method between groups (UPGMA = unweighted pair group method using arithmetic averages) at the population level using the Nei’s genetic distances . The conA. aculeatum populations used by extractive farmers shows that there is high genetic diversity within populations. However, the genetic structure of this species is moderate and occurs, in part, as a function of watersheds. The groupings obtained in the analysis of genetic structure are important for the conservation and management of the species, allowing directing management policies to the watersheds of the Amazon.The evaluation of


Ormosia hosiei is a tree species native to China that has been extensively used for ornamental and furniture purposes due to its valued timber. The mating system has substantial impact on genetic diversity and structure of plant natural population. Such information should be considered when planning tree planting for forest restoration. Here we used 12 microsatellite markers and described the mating system of single families and the population genetic diversity of O. hosiei. A high level of genetic diversity was observed in both adults and progenies, although slight differences existed among populations and their progenies, with the expected heterozygosity ranging from 0.763 to 0.794. Overall, O. hosiei displayed a predominantly outcrossed mating. The estimate of multi-locus outcrossing rate (tm) was high with low variations among families, ranged from 0.997 to 1.000. The value of tm-ts, ranged from 0.000 to 0.139, indicated that biparental inbreeding occurred in progenies. Therefore, to obtain a reasonable genetic representation of native tree species and prevent problems associated with inbreeding depression, we suggested effective in situ conservation by replanting seedlings, but seedling production for restoration purposes may require a much larger sampling effort than is currently used. Moreover, it is necessary to conduct further multiple population and multi-year experiments to verify our conclusions. Genetic diversity and its genetic structure of a tree population are closely related to life history traits , in whicRobinia pseudoacacia  were calculated using the mating system program (MLTR), described by Ritland [Acacia visco. The 95% confidence interval (CI) of each parameter was obtained from 1000 bootstrap replicates. The bootstrap analysis at the population level was carried out using adult trees as the units of resampling and at the family level using individuals with families as the resampling units.The mating system of  Ritland , based p Ritland . Biparen Ritland , as tm-t Ritland  in the am, ts, rp) were used to estimate other demographic and genetic parameters (Nep). The effective number of pollen donors over maternal trees was calculated from the paternity correlation by the equation Nep = 1/rp (m) [In the above analyses, mating system parameters (t1/rp (m) .All 12 microsatellite loci analyzed were polymorphic, with a total of 117 alleles observed in total sample (246). The number of alleles, heterozygosity and fixation index exhibited a rather high variation among locus . In the R) in progeny-JXMTS was vaguely lower, whereas in terms of ZJBD population and adult-JXMTS, it was similar. The average fixation index (F) was positive and significantly different from zero in all populations, ranging from 0.217 (p <0.05) in adults to 0.484 (p <0.05) in progeny from JXMTS, showing inbreeding.Comparing adults and progenies from both populations, the average number of alleles was lower in adults than observed in progenies for both populations and a slightly higher in JXMTS than that in ZJBD population . The obsst = 0.030 at locus SSR2 to 0.162 at SSR1. Genetic differentiation was also significant between populations and progenies except between adults-JXMTS and progenies-JXMTS (p = 0.051), and adults-JXMTS and progenies-ZJBD, (p = 0.059), the genetic differentiation between adults-JXMTS and adults-ZJBD was highest (Fst = 0.115), with the lowest (Fst = 0.041) between adults-JXMTS and progenies-ZJBD.Analysis of population genetic differentiation with Equations (1) and (2) was summarized in m) and single-locus outcrossing rate (ts) was high in both populations (O. hosiei are mostly outcrossers. The mean multi-locus rate (tm) was higher than the single-locus outcrossing rate (ts). The estimated biparental inbreeding rate (tm-ts) was low in two populations, and the value in the ZJBD population was greater than in the JXMTS population and significantly different from zero, suggesting some tendency to mating between relatives in two populations and a higher inbreeding rate existing in the ZJBD population than that in the JXMTS population.According to the instruction provided in MLTR, null alleles at relevant loci in both populations and each family were counted in estimating the outcrossing rate. Results are shown in ulations . These rt) differed between the two populations. For ZJBD population, the rt estimate was high (rt = 0.910). However, for JXMTS population, the rt estimate was low (rt = 0.108), it is compatible with the limited variations estimated tm among progenies (p(m) or rp(s)) was significant in JXMTS (rp(m) = 0.142, Se = 0.057) and in ZJBD (rp(m) = 0.213, Se = 0.067; rp(s) = 0.276, Se = 0.087), suggesting a high probability that a randomly chosen pair of progenies from the different arrays was closely related. The obvious difference between rp(m) and rp(s) (rp(s)-rp(m) = 0.063, Se = 0.026) in the ZJBD population indicated the existence of substructure. The effective number of pollen donors was 7.042 in the JXMTS population and 4.695 in the ZJBD population. Thus, the result may be interpreted, assuming that all plants blooming in the year of seed collection would act as pollen donors. In all cases, fixation index in progenies (Fo) was positive, ranging from 0.072 to 0.094, indicating a certain extent of inbreeding in two populations. Fixation in adult trees (Fm) was similar with that estimated for their progenies, but was significant from zero in two populations, suggesting Hardy–Weinberg deviations in the populations.The correlation of tm within progeny arrays  ranged from 0.000 (JXMTS1) to 0.139 (ZJBD2), with the mean of 0.047 over the seven families, suggesting some tendency to mating among families. The positive correlation of paternity at multiple loci was significant in ZJBD1, ZJBD2, and ZJBD3, indicating that the same fathers in pollination were partially shared in these families but not in JXMTS and ZJBD4. The correlation of outcrossed paternity (rp(m)) exhibited a quite high level of variation among families; family ZJBD2 had the highest value (0.352) and family JXMTS2 had the lowest value (0.097). The effective number of pollen donors (Nep) was greater than that estimated in populations, but had the same ranking patterns as Nep in populations.Generally, all the families showed high tm values in both populations with low variations among the families, ranging from 0.997(ZJBD3) to 1.000(JXMTS) . The estO. hosiei, a species endemic to southern China, by using nuclear microsatellite markers. Mountains, rivers and even forest fragments are often rich in endemism, but these habitats are commonly at risk of degradation, causing a reduction of genetic diversity of its populations [O. hosiei is endemic to the southern region of China, and most, if not all, populations of species were distributed along the river, they are in a highly particular environment with precipitation and anthropogenic influence, but we did not find low genetic diversity in the populations sampled adults and progeny cohorts (Embothrium coccineum (He = 0.21), a very common tree in relatively recently fragmented landscapes of southern Chile and a self-incompatible species, and equivalent to that of Acacia pennata (He = 0.57), Copaifera langsdorffii (He = 0.85), and Dalbergia nigra (He = 0.74), which belong to Leguminosae [O. hosiei can also be considered high, which is consistent with the studies that were based on ISSR [O. hosiei populations, even though small with only a few adult plants, has not yet been influenced by habitat fragmentation and the level of genetic diversity is not necessarily synonymous with the rarity or endemicity of a species. Similarly, genetic diversity of O. hosiei did not vary with current population size, which may be related to longevity and recent fragmentation. Moreover, although the species’ clonality has not yet been formally studied, we observed evident clonal reproduction on our field investigation.Genetic diversity is the sum of genetic variations among all individuals in a species, which is critical to their long-term survival and reflects the adaptability of a species to the environment and its potential for modification . In thisulations ,43. Here cohorts . We obseuminosae ,44,45. A on ISSR ,24. ThesBerchemiella wilsonii and Tsoongiodendron odorum [R), a measure of genetic variation somehow different from gene diversity, is more sensitive to bottlenecks, demographic changes, reflects better part fluctuations in population size and is highly dependent on effective population size [R were all relatively high, although, according to AR, the genetic diversity of the adults was slightly lower than that of the progenies. Our results showed an excess of homozygotes in both parent and progeny cohorts (F = 0.217~0.484), and the proportion of homozygotes among progeny cohorts was higher than that among parents. We observed a certain proportion of albino seedlings in progeny cohorts, which strongly suggests the existence of inbreeding depression. It should be noted that the progeny populations analyzed in this study were not grown in completely natural conditions, and the seeds were mechanically processed before germination and germinating in appropriate environmental conditions, which excluded the inbreeding depression during seed set and germination. Therefore, we underestimated the extent of inbreeding depression, which resulted in a higher proportion of homozygotes.The genetic diversity of progenies  was higher than that of adults, but with no significant difference, which caused by the number or density of plants flowering in bearing year. Variation in density of plants flowering within populations may cause fluctuation of outcrossing rate and gene flow. The progeny cohorts produced by populations with clustered plants flowering may be lower outcrossing rate than cohorts produced by populations with high incidence of flowering. This is due to the expression of lethal recessive alleles when homozygosity increases in low density flowering plants or in small populations . Many ren odorum ,47. In cion size ,49,50. IO. hosiei was moderate, with Fst = 0.073 among watersheds with SSR and 0.116 among nine populations [st = 0.095~0.156 [st = 0.115), adults and progenies (Fst = 0.041~0.094), and progenies (Fst = 0.060) suggests that there is little restriction of gene flow (Nm > 1) among populations. Nevertheless, within-population inbreeding seems to follow distinct patterns in adults and progenies. The adult JXMTS population exhibited a higher fixation index (0.118) than progenies (0.072), while there was an opposite phenomenon in the ZJBD population, perhaps reflecting pre-fragmentation Wahlund effects and thus the existence of a former population structure. Reduced fixation index of progeny in the JXMTS population corelates with the observed high outcrossing rates.Previous studies demonstrated that population differentiation of ulations , and wit95~0.156 ,24. The O. hosiei. The multi-locus outcrossing rate obtained in the present paper are comparable to those reported for other leguminous species with different molecular markers as well as for different levels  and average single-locus outcrossing rate (ts) can be used to infer inbreeding other than self-fertilization. Single-locus outcrossing rates are expected to be biased by any inbreeding in addition to self-fertilization. Therefore, the mean of such single-locus outcrossing rate is estimated to be lower than the multi-locus rate when mating among relatives occurs [O. hosiei indicated that this expectation was generally positive with the value of 0.000–0.139. The positive difference between tm and ts may be explained by mating among relatives or any other kind of genetic structure, which may be caused by the seed dispersal system. On the one hand, studies have shown that the seed of O. hosiei are spread by rats, water and gravity. Water flow and Edward’s long-tailed rats (Leopoldamys edwardsi) make a positive effect on seed dispersal, due to their accumulation and distribution of large quantities of seeds, while gravity and Chinese white-bellied rats (N. confucianus), hoard even more seeds in their nests, leads to a high relatedness of neighbor trees that are the most probably involved in reciprocal mating [The comparison between multi-locus (ts occurs . Comparil mating .m) and single-locus outcrossing rate (ts) of families in the JXMTS population was higher than that of in ZJBD population, which may be related to a few factors: different environmental factors or different pollinators; genetic relationship and geographic distance between mother plants and pollen donors [O. hosiei, but this is not always the case in other species. For example, species such as P. elegans mainly blossom annually, but some plants do not blossom year after year; thus, the proportion of flowering plants to adult plants ranged from 6% to 80%, and it was 33% and 80%, respectively, for two consecutive years in the same population, but the outcrossing rates were almost unchanged [O. hosiei were similar to studies on Magnolia stellata and T. odorum [O. hosiei seeds is only 3.4–5.1%, but after mechanical breaking, it can be as high as 94.7%. We also observed that the proportion of albino seedlings differed in different families, ranging from 0% to 13.2%, which indicated that an inbreeding decline occurred at different development stages. In Pinus pinaster, the size and crown of the seed trees were the main factors that affected the outcrossing rate among individuals. In this study, the breast diameter ranged from 37.3 cm to 125.5 cm and the crowns varied greatly, which could have resulted in the differences in the outcrossing rates among individuals. Considerable differences in outcrossing rates were detected among the investigated families. The multi-locus outcrossing rate  few plants flowering in the year of seed collection and low population density of the species, at three individuals per hectare (Investigated by author); few plants flowering and low population density is assumed to be related to reducing pollen diversification [The effective number of pollen donors (Nep) per family suggests that few paternal parents contributed pollen to each progeny in the analyzed generation, indicating inbreeding or biparental inbreeding. The correlated mating found in the fication ,54, leadO. hosiei in populations, both adults and progenies of O. hosiei had high genetic diversity, indicating that habitat fragmentation and anthropogenic disturbance could not have a significantly negative effect on these populations. Results from the multi-locus outcrossing rate of O. hosiei generally exhibited a predominantly outcrossing system. However, inbreeding may occur, as indicated by the comparison between tm and ts. The outcrossing rates varied among populations and individuals, which may be a consequence of different pollinator behavior caused by differences in environmental conditions and differences in pollen sources within the populations (density of adult trees). Limited seed dispersal and close genetic relationships of individuals in a smaller range lead to the occurrence of inbreeding, therefore, we suggested effective in situ conservation by artificial plantation establishment. For rehabilitation of disturbed populations, such as ZJBD and JXMTS, enrichment planting should be undertaken with materials collected from as many maternal trees as possible to prevent inbreeding depression due to possibility of mating among close relatives. The suggestion will not completely avoid inbreeding in the samples because inbreeding is determined by the natural mating system, which is out of our control. However, part of the inbreeding may be controlled at the stage of collecting seeds and seed nursery, by increasing the distance of seed collection and evicting albinism, low vitality, or other abnormal seedlings. Our results also imply that it is necessary to prevent human destruction of marginal or isolated individuals that will result in gene flow obstruction. Since we only analyzed seven families in two natural populations in a single year, it is necessary to conduct further multiple and multi-year experiments to verify our conclusions.Despite fragmentation of Therefore, for typical fragmented populations of other species, such as species with high outcrossing rate and genetic diversity, carrying out effective in situ protection by artificial plantation to promote natural regeneration and expansion of the existing population is proposed.


Molecular genetics has been extremely useful in determining the relation between animal populations and documenting the degrees of genetic variation found within them. The present study was undertaken to evaluate genetic diversity and the relationships between the three camel populations reared in Egypt: Maghrabi, Sudani, and Baladi using mitochondrial 16S sequences and other breeds of camels in the world.Blood samples were collected from camels belonging to these three populations. Genomic DNA was extracted from the collected blood samples and subjected to PCR using specific primers for mitochondrial 16S region. The amplified products were purified using DNA purification kit to remove residual primers and dNTPs. Sequencing was performed in the Macrogen Incorporation. The amplified products were submitted to GenBank/NCBI under accession numbers OM 278349 and OM 278350Camelus dromedarius populations revealed a strong relationship between them.Sequencing was done on the partial mitochondrial 16S amplified fragments at 530 bp. This amplified area had two haplotypes. There was one substitution (G/A) at nucleotide 309 of the amplified segment. The nucleotide (π) and Hd stand for haplotype diversity, respectively, at 0.00008 and 0.042, and the average number of pairwise nucleotide differences, k, is 0.042, according to Fu’s Fs statistic and Tajima’s D, which is −1.10686. Genetic distance percentages between the three populations under study range from 0.000 to 0.0312. A phylogenetic analysis of Egyptian camel populations and other This study suggests that the 16S rRNA sequencing in mitochondria plays a critical role in genetic variation studies and analysis of phylogeny between camel populations and breeds. Camelus dromedarius, Bactrianus, Lama glama, Vicugna pacos) and wild . Around 37 million camelids are kept globally [lama, alpaca, guanaco, and vicuna) are found mostly in Peru, Chile, and Argentina and have their natural home in the Andean highlands.Camel is a valuable livestock animal particularly in dry and semidry regions which extend in many African countries . The proglobally , with thIn Egypt, the number of camels in 2019 was about 119,885 belonging to five populations: Maghrebi, Somali, Sudani, Baladi , and Mowaled. At the molecular level, three populations  were found to have a high degree of genetic purity with a lower degree of mixing, so they are considered pure populations . These pThe mitochondria are essential organelles that perform oxidative phosphorylation to produce adenosine triphosphate (ATP), as well as other functions in metabolism, cell signalling, cell cycle regulation, cell differentiation, proliferation, and apoptosis . BecauseCamel ecotype is not well defined in Egypt, and very little information is available about its genetic background. Hence, this study was conducted to find out phylogenetic connection and genetic variation among three camel populations reared in Egypt  using partial mitochondrial 16S region.16 s gene and phylogenic relationships between our tested camels and other breeds of camels in the world to be available for selection of superior animals for breeding.The goal of this study is to identify the genetic conservation between three camel’s breeds reared in Egypt using mtDNA 16S F: 5′-GCTATAGAGAAAGTACCGTAAG-3′16S R: 5′-TCATATTAACATTATTGCTTC-3′Blood samples of Maghrabi camel were kindly supplied by King Mariout Research Station. While blood samples of Sudani and Falahi  were collected from camel market in Berkash, Giza, Egypt. Blood samples were collected from healthy males and females of the three camel breeds. These samples were drawn from the jugular vein of camels into sterile interior plain glass tubes after the addition of EDTA disodium salt (EDTA-Na2) as anticoagulant in each case. Blood samples were used to isolate genomic DNA using phenol/chloroform procedure previously described by Wajid et al. . The extThe amplification was carried out with initial denaturation at 95 °C for 5 min was followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 45 s, and final extension at 72 °C for 7 min. To eliminate residual primers and dNTPs, the amplified products were purified using a DNA purification kit  according to the manufacturer’s instructions. The Macrogen Incorporation did the sequencing .Individual haplotype mutations were identified by aligning mitochondrial 16S sequences from tested camels using the BioEdit software . DnaSP 5For more illustrative phylogenetic relations, three phylogenetic methods were used in this study: neighbor joining, maximum likelihood, and minimum evolution.PCR amplified 530-bp fragments from mtDNA 16S mtDNA region of tested camel populations. These amplified fragments were electrophoresed on 1.5% agarose gel  with single-nucleotide polymorphism (SNP) (G/A) at nucleotide no. 309 of 16S amplified region in tested camel populations  and haplotype diversities (Hd) in the studied samples were 0.00008 and 0.042, respectively, while the average number of pairwise nucleotide differences (k) was 0.042, Fu’s Fs statistic: −1.602, and Tajima’s D: −1.10686. There were no insertions or deletions observed in any of the 530-bp sequences. The average percentages of the four nitrogen bases were 39.8% (39.7 to 39.9%) for adenine (A), 26.8% (26.6%) for thymine (T), 16.2% (16.4%) for cytosine (C), and 17.2% (17.4 to 17.0%) for guanine (G) (G). In the three camel populations, the mtDNA 16S region displayed a substantial A+T bias (66.6%).The two detected haplotypes were submitted to GenBank/NCBI under accession numbers OM 278349 and OM 278350. Alignment of several sequences yielded a consensus length of 531 sites , KU605079 (Sudan), KU605077 (Dubai), KU605078 (Kenya), KU605076 (Austria), N632608 (Morocco), KU605072 , KU605075 (Arabian Peninsula), MH109985 (Kazakhstan), MH109974 (Russia), and MH110002 (Iran). On the other hand, the tested sequences revealed a homology with MH109929 (Mongolia), MH109942 (China), and KU666457 (Vienna) at 97.34%, 97.16%, and 97.15%, respectively.C. ferus (Vienna), and hap 2 of the examined species had the greatest genetic distances (0.0312) in Egyptian camels. There were no genetic distances (0.0000) detected between hap1 (Egypt) and each of camel populations in Iran, Pakistan, Sudan, Austria, Morocco, Dubai, Saudi Arabia, and Kenya and Bactrian camels in Kazakhstan and Russia.Table The phylogeny methods used in the present study showed the same relations among camel populations with some different in support values. All camel haplotypes were separated into two distinct clusters; according to the results, the populations from different geographic regions  belonged to one cluster while the second cluster divided into two lineages, the first lineage included ferus populations from Mongolia and Vienna, while the second lineage included Bactrian camel population from China Figs. , and 6.FMitochondrial DNA (mtDNA) reflects maternal inheritance, and it is valuable for determining genetic variety between animals. It is used to examine genetic variation in dromedaries and to confirm their breeding discrimination and strategies. Between Mongolian domestic and wild Bactrian camels, there was a 1.9% nucleotide variation in the mitochondrial regulatory regions (CR) , 30. AlsC. dromedarius compared with other camelid species showed that the modest differences in length were primarily due to differences in the control region. This discovery explains why the regulatory region was first employed to explore genetic variation in mammals due to its quick evolutionary pace compared to protein-coding genes and rRNA [The present genetic data of and rRNA .Camelus dromedarius (African breed) than the Maghrebi breed, which separated with Camelus ferus in one clade and indicating that they share a common ancestor. Furthermore, the present results agree with finding of El-Seoudy et al. [The results revealed the presence of two haplotypes with low genetic differences, and proven Baladi population is more closely related to Sudani population than the Maghrebi population. This finding agrees with the result of Dowidar et al.  who repoy et al.  who repoy et al.  indicateOur results revealed that the partial sequences of mtDNA 16S in the three tested populations are rich in AT content with an average of 66.6%. These findings support earlier studies that discovered an imbalance in the A+T base composition of mtDNA , 33 and The results revealed only one nucleotide substitution was identified in the 16S regions of sixty sequences of tested camels, and no deletion or insertion of mutations was found. To determine the degree of genetic difference across and within camel populations, the phylogenetic tree and haplotype network were constructed. The two haplotypes, Hap 1 and Hap 2, were shared with fourteen reference populations with a consensus length of 531 sites.D-tests, which differ slightly in their approach. The D-test of Tajima [In this study, the average number of pairwise nucleotide differences, k: 0.042 and Fu’s Fs statistic: −1.602 with D: −1.10686 of Tajima, was discovered. The population expansion was determined using Fu’s Fs and Tajima’s f Tajima  is calcuf Tajima  is basedOur results showed that camel populations are slightly different in terms of haplotype and nucleotide diversity, with differences of 0.042 and 0.00008, respectively. Ming et al. , who stuPresent result has found little differences in dromedary camel populations, as evidenced by very low nucleotide diversity 0.00026) and moderate to high haplotype diversity 0.725). When compared to other animals that are more carefully bred for a specific function, these diversity indices may reflect a significantly lower selection pressure on camels 6 and mod. Moreove25. When In the present study, an evolutionary tree based on the mtDNA 16S was constructed for better understanding of genetic variation between and within Egyptian camel populations. The phylogenetic tree explained the closest pairwise genetic distance between the two detected haplotypes and camel populations from other fourteen countries as reference populations. The results documented that there is no pairwise genetic distance between Sudani and Baladi populations where they have the same haplotype and the most genetic distance was found between the Baladi, Sudani, and Maghrebi breeds; it suggests that the Baladi and Sudani breeds are more closely linked and share common origins. This result is supported by the bootstrap value of 1000% which is a benchmark to determine the level of accuracy of the phylogeny tree . These fMitochondrial DNA has evolved into a highly effective tool for species identification and forensic science owing to the large number of copies in each cell and the lack of recombination with paternal mtDNA. Current study’s findings validated the 16S sequence’s relevance and effectiveness in measuring genetic variety among camel populations. This data could be utilized as a starting point for developing additional proposals for genetic enhancement and conservation strategies for Egyptian camel genetic resources.


Phelotrupes auratus, which inhabits montane forests and exhibits three color forms , diverged into five local populations  in the Kinki District of Honshu, Japan, based on the combined interpretation of genetic cluster and color‐form data. Here, we estimated the demographic histories of these local populations using the newly assembled draft genome sequence of P. auratus and whole‐genome resequencing data obtained from each local population. Using coalescent simulation analysis, we estimated P. auratus population divergences at ca. 3800, 2100, 600, and 200 years ago, with no substantial gene flow between diverged populations, implying the existence of persistent barriers to gene flow. Notably, the last two divergence events led to three local populations with different color forms. The initial divergence may have been affected by climatic cooling around that time, and the last three divergence events may have been associated with the increasing impact of human activities. Both climatic cooling and increasing human activity may have caused habitat fragmentation and a reduction in the numbers of large mammals supplying food (dung) for P. auratus, thereby promoting the decline, segregation, and divergence of local populations. Our research demonstrates that geographic population divergence in an insect with conspicuous differences in traits such as body color may have occurred rapidly under the influence of human activity.Knowledge of population divergence history is key to understanding organism diversification mechanisms. The geotrupid dung beetle  Phelotrupes auratus , the geotrupid dung beetle with three color forms diverged into five geographic population groups based on the combined interpretation of genetic cluster and color‐form data. We estimated the demographic histories of these population groups using the newly assembled draft genome sequence of P. auratus and whole‐genome resequencing data obtained from the groups. The evolutionary histories of wild organisms have been shaped largely by past climatic and geographic events. For example, environmental changes that occurred during the last glacial period strongly influenced species' distribution patterns. The combined interpretation of past climatic and geographic events and molecular population genetic data has provided a powerful means to elucidate the evolutionary history of organisms over relatively long time scales such as the Quaternary Hewitt, , 2004. IGenetic variation between local populations is determined by the balance of the homogenizing effect of gene flow and the diversifying effect of local adaptation Endler, . GeograpPhelotrupes auratus (Motschulsky). It exhibits notable variation in its structural metallic body coloration, which is provided by a simple multilayer reflector structure , east/red (ER), south/indigo (SI), south/green (SG), and south/red (SR)—and that geographic color variation was maintained by hybrid transition zones with steep genetic clines  or transition zones without genetic differentiation. In the latter zone type, we inferred the action of strong selection on different body colors in different regions. However, as the function of body color in P. auratus is unclear, the inference that color differentiation is maintained solely by selection despite frequent gene flow was questionable. Obscure genetic differences between sister populations may have been caused by very recent population divergence, even if they are separated by barriers to gene flow. Therefore, the lack of detection of genetic differentiation in our previous study may be attributable to the low resolution of the RAD‐seq data and/or the methodology of our population clustering analysis. Analysis using a larger genomic dataset, such as that provided by whole‐genome resequencing, may provide new insights into geographic color diversification in P. auratus.Here, we focused on the geotrupid dung beetle P. auratus and analyzed genome‐wide single‐nucleotide polymorphism (SNP) data obtained by whole‐genome resequencing of individuals sampled from five local populations. We found that the five local populations diverged recently—within several thousand years, under the influence of human activity—and that no substantial gene flow had occurred among the diverged populations. Our findings reveal an unexpected influence of human activity on population differentiation through the divergence of a conspicuous trait in this insect species.In this study, to construct robust demographic history models and estimate the dynamics of effective population sizes, gene flow, and divergence times among populations, we constructed a de novo draft genome of 22.1P. auratus adults were collected using traps baited with horse dung at six locations around Lake Biwa in the Kinki District, Honshu, Japan  for ONT PromethION long reads and fastp , following the manufacturer's protocol. The quality of DNA samples was assessed with Qubit (Thermo Fischer Scientific), NanoPhotometer (Implen), and 2200 TapeStation (Agilent) instruments. For genome assembly, the genomic DNA of an SI individual (SI03) was sent to GeneBay, Inc. (Yokohama) for library construction and long‐read sequencing using the ONT PromethION system (Oxford Nanopore Technologies). Short‐read sequencing was performed with 150‐bp paired‐end reads in three runs on an Illumina HiSeq X Ten sequencer (Illumina) at Macrogen Japan. For genome assembly, a single sample (SI03) was sequenced using one lane of the HiSeq X Ten sequencer. For resequencing, 10 samples from the SI and ER groups and 15 samples from the WR, SG, and SR groups were separately sequenced using two lanes of the HiSeq X Ten sequencer. Library construction for SI and ER samples was performed at Macrogen Japan and that for WR, SG, and SR samples was performed at Kyoto University using the NEB Next Ultra II FS DNA Library Prep Kit for Illumina . Adapters on raw reads were trimmed using Porechop  based on the result of the 31‐mer count obtained with Jellyfish v2.3.0  at the scaffold level using Illumina HiSeq X short reads and ONT PromethION long reads by a hybrid assembly procedure using MaSuRCA 3.4.2 . The average read depth was 16.02, excluding SI03 . SNP calling was conducted for each sample using bcftools 1.12  populations in the current geographic distribution. To simplify the simulations, we assumed that each population had a constant size with no growth after population divergence; to disregard recombination rates by minimizing linkage disequilibrium among the SNPs used in the analysis  and was thus longer than the estimated size, possibly due to insufficient removal of haplotigs. Gene prediction resulted in the detection of 30,643 transcripts in 28,887 protein‐coding regions from the draft genome; 30,252 (98.72%) transcripts were annotated in the BLAST search. BUSCO score quality assessment revealed 1810 (85.2%) complete single‐copy, 226 (10.6%) duplicated, 30 (1.4%) fragmented, and 58 (2.8%) missing BUSCOs. Variant calling against the draft genome for all resequencing data (n = 27) resulted in the assembly of a dataset with 9,184,739 SNPs in 8838 scaffolds . We used this dataset in the subsequent analyses with and without SNP pruning for LD. After LD pruning, 247,662 SNPs remained in the dataset .The genome size of 3.2K) was 2 based on cross‐validation error values ; individuals were divided into one group including the WR and ER populations and another including the SI, SG, and SR populations. However, individuals from WR and ER were clearly discriminated at K = 3  formed different clades. Within the southern populations, one SG individual (SG05) was included in the clade of SI individuals, and one SG individual (SG06) was included in the clade of SR individuals. The population structural analysis Figure  showed t3 Figure .3.3We first estimated the demographic history of each population and the divergence times between pairs of populations based on the SNP data using SMC++. The temporal changes in the effective population size were consistently similar among all populations until approximately 50,000 years ago Figure . The estNem) was much lower than unity (10−10 to 10−7) for all cases  set to 30,000 generations  ago, based on the results of the SMC++ analysis. Parameter estimation using the demographic model revealed the following demographic histories. WR and A1  diverged 3830  years ago, ER and A2  diverged 2086  years ago, SR and A3 (MRCA of SI and SG) diverged 607  years ago, and SI and SG diverged 195  years ago Figure . Althougs Figure , suggests Figure .44.1P. auratus. The SMC++ results revealed long‐term historical changes in the effective population sizes. The similarity in the patterns of demographic change among populations may reflect the low degree of genetic differentiation among populations until recently  and the presence of hybrid zones between WR and SG and between ER and SR  coincides roughly with the beginning of the rapid increase in the human population in Japan due to the introduction of rice farming, which provided a stable food resource  and documents mentioning local products from the mid‐Edo era (1730 s) have shown that sika deer and other large mammals such as wild boars had reduced their distribution areas by the 18th century . However, carcasses of the these beetles are often found in the feces of mammals such as raccoon dogs ; data curation (lead); formal analysis (lead); investigation (lead); writing – original draft (lead). Teiji Sota: Conceptualization ; funding acquisition (lead); project administration (lead); supervision (lead); writing – review and editing (lead).The authors declare that there is no conflict of interest.Table S1:Table S2:Table S3:Click here for additional data file.