Thunnus thynnus; BFT) abundance was depleted in the late 20th and early 21st century due to overfishing. Historical catch records further indicate that the abundance of BFT in the Mediterranean has been fluctuating since at least the 16th century. Here we build upon previous work on ancient DNA of BFT in the Mediterranean by comparing contemporary (2009–2012) specimens with archival (1911–1926) and archaeological (2nd century BCE–15th century CE) specimens that represent population states prior to these two major periods of exploitation, respectively. We successfully genotyped and analysed 259 contemporary and 123 historical  specimens at 92 SNP loci that were selected for their ability to differentiate contemporary populations or their association with core biological functions. We found no evidence of genetic bottlenecks, inbreeding or population restructuring between temporal sample groups that might explain what has driven catch fluctuations since the 16th century. We also detected a putative adaptive response, involving the cytoskeletal protein synemin which may be related to muscle stress. However, these results require further investigation with more extensive genome-wide data to rule out demographic changes due to overfishing, and other natural and anthropogenic factors, in addition to elucidating the adaptive drivers related to these.Atlantic bluefin tuna ( This information is crucial to predict future demographic changes and thus promote sustainable fisheries management5. Studies of historical marine ecology offer an opportunity to learn and heed these past lessons7. In particular, genetic/genomic studies can infer past history from contemporary samples8, or directly test archaeological and archival samples9 for losses in genetic diversity, population restructuring, or adaptive responses to natural factors e.g., climate, or anthropogenic ones e.g., fisheries-induced evolution (FIE)10. A decade ago, Riccioni et al.11 were the first to investigate temporal demographic changes in the key species Atlantic bluefin tuna  using archival early-20th century samples and microsatellite markers. Here, we build on this work by genotyping archival and archaeological samples to extend investigations into the pre-industrial era, when fishing may have also had the potential to impact BFT.Overfishing has reduced numerous fish populations to remnants of their historical levels13, and inshore migration behaviour, that has made it vulnerable to overfishing. Recent genomic studies15 support the delineation of two BFT populations. These are a western Atlantic component that spawns predominantly in the Gulf of Mexico16, and an eastern Atlantic and Mediterranean component that spawns predominantly in the Mediterranean Sea17. Individuals of both populations migrate into the Atlantic Ocean to feed, including as juveniles18, and exhibit high-levels of mixing15. The role of additional contemporary and historical spawning areas i.e. the Slope Sea 16, the Bay of Biscay19, and the Black Sea21, are yet to be clearly defined, especially regarding the Slope Sea where connectivity between populations was observed15.BFT is a highly migratory pelagic top predator, characterized by its large size (up to 3.3 m in length and 725 kg in weight), slow maturation (between 4 and 8 years)25. However, reconstructions of 16th-20th century BFT trap catch records suggest abundance across the Mediterranean has been fluctuating for centuries27. Pelagic species are particularly susceptible to fluctuations in abundance since dynamic food and environmental conditions drive large variability in recruitment, but fishing magnifies poor recruitment and therefore population declines when large catches occur28. Multiple factors need to be taken into account to be able to interpret trap catch fluctuations as abundance29, though, it appears that catch numbers in the 16th and 18th century may be comparable to those during the industrial fishing of the last 50 years27. Hence, fishing appears to have been intense in this period.During the last few years, the eastern Atlantic and Mediterranean population of BFT has recovered to 1970’s levels following heavy overfishing that depleted spawning stock biomass, restructured the population toward younger individuals, and contracted the species range, in the late 20th and early 21st century3, Riccioni et al.11 were unable to detect losses in BFT genetic diversity when comparing contemporary and early-20th century samples. Likewise, no genetic erosion was observed following overfishing in the closely-related albacore 30, the Pacific herring 31, or the European anchovy (Engraulis encrasicolus)32. Even marine species  depleted to between 1 and 5% of their historical biomass appear to have retained genetic diversity33. However, several studies have noted genetic diversity declines or population losses following overfishing in Atlantic cod (Gadus morhua)36, Atlantic salmon 37, and Chinook salmon (Oncorhynchus tshawytscha)38. In addition, adaptive responses to size- 39, and sex- 40 selective harvesting, and environmental drivers (in Atlantic cod)41 have also been reported in studies using archival or archaeological fish samples. It remains unclear to what extent the inability of some studies to detect these differences results from the selection of genetic markers with low resolution, or the resilience offered by complex life history traits during times of population decline. A recent whole genome sequencing (WGS) study42, that did not detect genetic erosion or adaptive responses in two Atlantic cod populations following 20th century overfishing, may indicate, however, that the latter is the case for some populations.The current study investigates genetic variability in eastern Atlantic and Mediterranean BFT prior to both their 21st century population collapse, and record trap catches in the 16th and 18th century, using archived early-20th century specimens, and archaeological remains, respectively. Despite overfished species having an overall lower genetic diversity when contemporary data are comparedHere we test the hypotheses that the genetic diversity of BFT has declined and that their populations restructured following periods of intense fishing in the eastern Atlantic and Mediterranean. Further, we sought to identify adaptive responses that may be related to ecological or environmental conditions. To this end, our objectives were to genotype archaeological and archival specimens on a single nucleotide polymorphic (SNP) panel to (1) characterise their genetic diversity and population structure, (2) to compare those patterns to analogous ones from contemporary groups, and (3) to explore markers under putative selection and identify their associated function, if possible.11) were collected between 1911 and 1941 44, 2nd century BCE Tavira , and 4th–2nd century BCE Palacio de Justicia, 45. See Supplementary Materials We collected samples of contemporary, archival and archaeological BFT specimens for analysis as follows: Contemporary reference specimens , following the manufacturer's instructions. Quantification was performed using a NanoDrop 2000 . Negative controls indicated that no cross-contamination took place between samples.DNA was isolated from fin (adults) or muscle  of contemporary samples Table  as part 46. All bone specimens were sprayed with 1–2% sodium hypochlorite (bleach), left to soak for ten minutes, rinsed with distilled water and air-dried (as per47). Specimens were then mechanically cleaned using sandpaper, and the bleaching process was repeated. After, each specimen was exposed to UV light (254 nm) for 15 min before drilling to obtain ~ 200 mg bone powder. Bones that were too small for drilling were bisected, and their inner matrices were crushed.Archival and archaeological samples underwent ancient DNA (aDNA) extraction in sterile, PCR-free conditions at the Ancient DNA Laboratory of the Department of Cultural Heritage , as part of another study which investigated their species identification via barcoding49. Briefly, ~ 200 mg of bone powder from each sample was divided in two and placed into separate tubes. After an overnight incubation in EDTA  and proteinase K, lysates (1000 µl) of each sample were pooled and combined with 3000 µl binding buffer composed of guanidine thiocyanate (5 M), Tween 20 (0.05%), isopropyl alcohol (40% v/v), sodium acetate , and distilled water. This mixture was then centrifuged through a MinElute spin column , and washed twice with 720 µl PE buffer, before a final elution in 60 µl of distilled water.Isolation of aDNA was performed using a modified version of Dabney et al.® dsDNA HS (High Sensitivity) Assay Kit . Negative controls employed for each batch of samples extracted indicated an undetectable level of contamination (< 500 pg/ml).The total DNA obtained from each extraction was quantified using a Qubit30 that were polymorphic between contemporary sample groups (see14) or matched with gene functions. To identify protein association we blasted the flanking regions of these loci against sequences for Atlantic cod50, sea bass (Dicentrarchus labrax)51, BFT53 and an umbrella set of teleost sequences, on NCBI GenBank  samples contained sufficient quantities of DNA  for genotyping Table . SamplesSNP genotyping was conducted first using Fluidigm 96.96 Dynamic Array™ Integrated Fluidic Circuits  on the BioMarkHD034 platform . Historical samples were re-genotyped at a second facility using the Fluidigm EP1 platform  to assess genotype error rates. Genotyping employed two negative controls for each run, which confirmed no cross-contamination, and three positive controls , reporting identical genotypes. Similarly, 21 (7.5%) historical samples were extracted and genotyped twice and reported acceptable replicates at 97.8 ± 3.6% accuracy.54 as implemented in R v.4.0.355. A single clone was evident in the HIST archaeological sample group  between loci was tested using the R package Genepop57. Outlier loci were identified using Bayescan58 and OutFLANK v0.259 to obtain a neutral dataset and identify potential adaptive responses. Analysis was run excluding the western Atlantic sample group (GOM) between the following: all sample groups, pooled contemporary and historical sample groups, contemporary sample groups, and historical sample groups. Loci detected as outliers were removed from the dataset prior to demographic analyses and investigated as follows: gene association was inferred from the above blastn searches, and non-synonymous mutations were explored with the Expasy Translation tool as implemented online (https://web.expasy.org/translate). Default settings were used in the analyses. Significance was judged using the False Discovery Rate (FDR)60 approach at the 5% level, as calculated using 999 permutations.Deviation from Hardy–Weinberg equilibrium (HWE) was assessed at each locus using the R package Pegase, Ho), and the inbreeding coefficient FIS, were calculated for each sample group with the R package Hierfstat61. Significance of heterozygote excess was calculated with Genepop in R using the global excess method and default settings. Differences in aR, He, Ho and FIS between pooled contemporary and historical sample groups were assessed using unpaired t-tests in R. Significance was judged at the 5% level. Effective population size (Ne) estimates were calculated only for samples consistently scored across all 89 neutral loci, as summarised in Table 62 as implemented in NeEstimator v2.163 and an allele frequency threshold of 0.01. A random down-sampling to generate and analyse equal-size groups is summarised in Table e estimates are often unreliable at low sample sizes64, we calculated per locus round-robin estimates of minor allele frequencies in R (as per65) and plotted trajectories between temporal sample groups. We performed a hierarchical analysis of molecular variance (AMOVA) in Poppr, with 10,000 permutations to assess significance. AMOVAs were performed excluding the GOM sample group on the following levels: between periods; between sample groups; between samples ; and within samples.Allelic richness (aR), heterozygosity expected/observed  according to the function optim.a.score, based on an initial selection of all PC’s before refinement. Population structuring was also evaluated using STRUCTURE v.2.3.467, which implements a Bayesian clustering method to identify the most likely number of populations (K). We followed the Evano et al.68 method, and thus, we carried out 10 runs per each value of K ranging from 1 to 10. Runs used the locprior and admixture models and assumed correlated allele frequencies. Each run used 500,000 burn-in and Markov Chain Monte Carlo replicates. We estimated the ad hoc statistic ΔK in order to infer the most likely number of populations using STRUCTURE HARVESTER69. CLUMPAK70 was used to merge the 10 runs from the most probable K, and reported similarity scores > 95. We used a hierarchical approach to improve resolution due to the identification of 5 outliers  in two modern sample groups that constituted two distinct populations at K = 3 in the first run. Hence, these individuals were removed from the dataset and STRUCTURE was run a second time. Pairwise distances between sample groups were calculated with Nei's estimator of FST71 in the hierfstat R package, using 999 permutations to calculate the respective p-values, which were judged for significance under the FDR approach at the 5% level.A discriminant analysis of principal components (DAPC) was performed with the R package AdegenetOverall, 259 contemporary, and 123 historical  samples were analysed at 92 loci Table . No locip = 0.181, t(11) = 1.426), He  = 0.099) and Ho  = 0.578) between pooled contemporary and historical groups (Table IS) was rare within all sample groups  = 0.0791). The dataset lacked power to define reliable estimates of Ne using both methods for each sample group i.e., our CIs contained infinity until they were pooled  sample group was substantially separated Fig. . Considers Table . In addi25, and the 4th–15th century CE prior to a significant period of intense trap fishing marked by fluctuating catches27. If overfishing had resulted in a genetic bottleneck, we would expect to see significant decreases in minor allele frequencies, allelic richness, and observed heterozygosity72 for contemporary samples compared with historical samples. Therefore, we would also expect to observe an increase in inbreeding and a decrease in effective population size8, which we did not. The impact of overfishing on genetic diversity and allele frequencies has been observed in a variety of studies that directly test archaeological and archival samples38. At its most extreme, overfishing has been observed to restructure marine fish populations36, yet we found no evidence of genetic restructuring in BFT. Likewise, a recent study found that Atlantic cod had not been impacted by 20th century overfishing at the genomic level42. Our findings are similar to those of Riccioni et al.11 using microsatellite markers, though we did not observe significant sub-structuring within Mediterranean BFT as they did, and this is yet to be resolved to clarify alternative population structure hypotheses73. No recent genetic study, however, has detected population structure within the eastern Atlantic and Mediterranean BFT74. Perspectives from threatened populations of other taxa, inform us that a wide range of genomic responses are expected, along a continuous scale from resistance to collapse79, and recovery82. Despite differences between taxa, these data would suggest that there is likely no “one-size fits all” response to the depletion of marine fish populations, according to species life history traits and the extent and rate of overfishing.We found no evidence of genetic diversity loss or population restructuring in contemporary BFT sample groups of the eastern Atlantic and Mediterranean compared with those from the early 20th century CE prior to spawning biomass depletion and species range contraction72. This is plausible for BFT, though its western Atlantic population is smaller than the eastern Atlantic and Mediterranean population (ca. 1/10 the size) and was heavily fished itself since at least the early 19th century84. Connectivity with alternative spawning sites  remains poorly understood, and the unresolved frequency and duration of spawning at these locations means we cannot assess its effect on gene flow. Likewise, introgression occurs at a low rate between Thunnus species85 but could also act as a buffer. On the other hand, eastern BFT may be resilient towards genetic erosion due to their relatively large population size (enhanced by connectivity between spawning sites within the Mediterranean), and a long life cycle which promotes heavily overlapping generations17. In any case, our findings leave us with two possible explanations; either (1) overfishing was not severe enough to cause a genetic bottleneck in BFT, or (2) our observation of significant demographic changes was hindered by the methods we employed.The most common explanation for the maintenance of genetic diversity in threatened populations is that gene flow acts as a buffer25, and independent estimates e.g.,22 suggested that BFT abundance and range declined by 70% and 46–53%, respectively, between 1960 and 2010. However, there is debate on the extent of the population decline, where on one hand, impending population collapse was predicted in 200986, yet on the other, poorly understood population dynamics and incorrect assignments of catches has caused uncertainty in population estimates87. Hence, it is difficult to deduce whether we should expect to find evidence of a genetic bottleneck because the recent recovery of the population within just two generations from its lowest point in 200788 could suggest that either the population decline was not that severe, or that overfishing did trigger a severe population decline but BFT is remarkably resilient due to its complex life history traits.To address this first point, it is evident that BFT were overfished, at least in the 20th and early 21st century, if not as we suspect between the 16th–19th century. Studies by the management body ICCAT 29), which one might expect to be reflected in genetic diversity and structure. Climate is likely the largest regulator of recruitment and thus fish abundance90 and as a pelagic species, BFT are certainly no exception92. Therefore, one might expect to find evidence of fluctuating abundance—and potentially genetic diversity—that is merely exacerbated by fishing28. BFT’s Atlantic distribution varies with Atlantic multidecadal oscillation phases93, and thus gene flow and inbreeding is expected to vary accordingly because connectivity of populations is enhanced in warm years as ranges overlap, as attested by isotope data94. This is notwithstanding time-related effects driven by evolutionary processes i.e., mutation and genetic drift that we might expect to alter allele frequencies over time. Therefore, our observation of homogeneity between contemporary and historical BFT samples is somewhat striking. One might pose the question: at what rate should we expect to observe demographic changes at the genomic level? We analysed moderate sample sizes from 1911 to 1926 (~ 20 generations ago) and the 4th–15th century (~ 100 + generations ago), yet we did not detect time-related effects. Thus, to address this, even at conservative mutation rates lower than those shown for marine fish95, we would expect to observe changes in allele frequencies as a result of genetic drift alone.Nonetheless, fishing effort is not the only factor that influences catches and abundance e estimate CIs often contained infinity, suggesting that we have little power to make any inferences on Ne. In many cases Ne was strikingly lower in (supposedly unimpacted) historical samples than the empirical rule-of-thumb threshold of Ne (500) proposed to maintain long-term genetic diversity in marine populations72. In any case, Ne is often unreliable when using sample sizes such as ours64 and we caution the interpretation of our results for this reason. Additionally, our sampling strategy may have been limiting. For example, if genetic diversity had decreased following population declines  but was restored prior to our analogous archival samples of the early 20th century, or 2009–2012 contemporary samples, respectively. Species differ in their rates of genetic recovery according to their life history traits72, and as this rate is unknown in BFT, we cannot rule out this possibility.Moreover, our N90. However, genome-wide approaches are more likely to provide a better resolution to assess demographic impacts and adaptive responses. Assuming the availability of a reference genome, WGS approaches are increasingly cost-effective9, particularly for shallow sequencing102. This approach may also facilitate the recovery of data from arid Mediterranean specimens which were challenging to genotype herein due to their poor preservation46. Importantly, WGS would reduce ascertainment bias compared with SNP-genotyping. This is crucial where allele frequency distributions are used to infer demographic history, but also to scan for past targets of selection98. We were limited herein to detecting a single adaptive response in BFT: a potential change to the function of the protein synemin, which is a cytoskeletal protein that we speculate might be related to growth changes induced by size selective harvesting (FIE), although this remains to be tested. WGS studies able to detect additional loci under putative selection are ultimately required for the association of this response (and others) with natural or anthropogenic factors, in addition to discounting hitchhiking effects103.Clearly aDNA approaches offer utility to fisheries management because long-term trends are understudied and we lack fisheries-independent indices11. This may hint at BFT’s resilience which has been recently shown by rebounds in abundance25 and a return to previous habitats88. However, we acknowledge limitations in our dataset i.e., few markers and the potential for ascertainment bias, and suggest that future studies might benefit from obtaining WGS data to observe rare alleles and reduce bias. Genome-wide data will be especially necessary to investigate adaptive responses, such as the putative selection on the cytoskeletal protein synemin found herein, and associate these with natural or anthropogenic factors to elucidate the drivers of change.We identify that aDNA preserved within archival and archaeological fish remains has the potential to inform fisheries management by providing novel fisheries-independent baselines with which to observe unstudied long-term demographic and adaptive changes. We found no evidence that genetic diversity decreased or that populations restructured following several centuries of intense fishing, in line with a previous studySupplementary Information 1.Dataset S2.Dataset S3.


Venturia inaequalis is a serious disease of cultivated apple worldwide. In this study, we collected 132 V. inaequalis isolates from Shaanxi, Gansu, Xinjiang, and the U.K. and analyzed their genetic diversity by using 13 microsatellite markers. Cluster analysis based on population structure and genetic distances suggested high similarity among the four regions. Population differentiation values ranged from 0.044 to 0.155, indicating there is a high level of kinship among the four regions. All isolates could be divided into 5 lineages with a 0.76 similarity coefficient. Among the four regions, Shaanxi had only one lineage, Group II; Gansu had four lineages, Group I, Group II, Group IV, and Group V; Xinjiang had all five lineages, Group I, Group II, Group III, Group IV, and Group V; and the U.K. had three lineages, Group I, Group II and Group IV. High molecular variance was detected for populations in the four regions, with 91% of the variance occurring within the populations and 9% among the populations. Structure analysis there are three common ancestors of these four regions. The results of the present study shed light on the genetic diversity of V. inaequalis in Shaanxi, Gansu and Xinjiang, which will lead to the development of more effective management strategies and new resistant apple cultivars through molecular marker-assisted selection.Apple scab caused by  Venturia inaequalis (Cooke) G. Winter is a serious disease of cultivated apple worldwide. Without appropriate control measures, crop losses in susceptible cultivars may reach up to 70% [V. inaequalis can also infect various rosaceous ornamentals, such as crabapples, loquat, and hawthorn [Apple scab caused by p to 70% . The patp to 70% . In addihawthorn . As mosthawthorn .Control strategies for apple scab has been aimed at primary ascosporic inoculum. In spring, protectant fungicides should be used to attenuate ascospore discharge and infection. In autumn, eradicant fungicides should be used to prevent pseudothecia development. A recent study revealed that ascospores contributed the most and overwintering conidia contributed approximately 20–50% as primary inoculum –9.V. inaequalis have been conducted in many regions around the world. and populations of V. inaequalis in Europe were more genetically diverse than those in other regions where apples were introduced [V. inaequalis populations provided evidence that South Africa most likely has V. inaequalis subpopulations linked to diverse climatic conditions in the coastal region compared with mountainous inland regions [V. inaequalis from coastal areas in Israel are genetically uniform and populations from the Golan Heights showed levels of genotypic diversity ten times as high than coastal areas. Since sexual reproduction in this pathogen has an obligate requirement for sustained low winter temperatures and in Israel only Golan Heights meets this condition. The result indicated that V. inaequalis does not reproduce sexually in regions characterized by the absence of low winter temperatures and is instead composed of clonal lineages [V. inaequalis isolates obtained from three different sites in Shaanxi Province of China by using simple sequence repeat (SSR) markers to analyze the structure of the disease-causing population. The result indicated that geographical factors have greater influence on genetic differences among populations than host cultivars [V. inaequalis isolates causing apple scab in 28 orchards and on five continents using 12 microsatellite markers showed that 88% of the genetic variation occurred within populations, which was consistent with extensive migration of the fungus among and within regions, while differentiation among regions was low  indicating that regions contribute a less diversity to populations [V. inaequalis isolates in Turkey employing random amplified polymorphic DNA (RAPD), no significant differences were observed between the isolates from two different geographic regions [V. inaequalis subpopulations linked to diverse climatic conditions of the Jammu region compared to the mountainous inland Kashmir region [Investigations of the genetic diversity of troduced –15. A mo regions . Howeverlineages . An studultivars . An exteulations . In an i regions . A recenr region . Based oIn China, the Loess Plateau, including Shaanxi and Gansu provinces, is the primary region of apple production in terms of acreage and yield, and crabapples in the Xinjiang Autonomous Region is considered to be the ancestor of modern apple cultivars. Since 1997, apple scab has been very common in the apple-growing regions of Shaanxi, Gansu and Xinjiang (part of Northwest China), where it causes serious yield losses . This diV. inaequalis populations in China was carried out in Shaanxi approximately ten years ago, and all isolates were collected only from this region [V. inaequalis in Northwest China remains unknown. Besides, the origin of the pathogen and the role of crabapple in the development of apple scab epidemics in Xinjiang is not fully understood. Therefore, this study aims to investigate the genetic diversity of V. inaequalis isolates collected from Shaanxi, Gansu, and Xinjiang by using microsatellite markers to determine the following: (a) whether there are genetic differences among the different regions in Northwest China; (b) whether apple scab isolates in Northwest China originate from the U.K.; and (c) the role that pathogens collected from crabapple forests in Xinjiang play in disease epidemics.The only research on the genetic variability of s region , 21. HowMalus sieversii (Ledeb.) Roem) forests. For each infected tree, 10 leaves with obvious sporulation spots were collected for fungal isolation and considered as one sample. A cork borer with a diameter of 0.5 cm was used to cut a disc with a lesion from each leaf. Each disc was transferred to a microcentrifuge tube, air-dried at room temperature, and stored at –20°C. Water agar  was used to obtain single-spore isolates. An infected leaf disc was added to 1.5–2 ml of sterilized distilled water and agitated thoroughly. Approximately 200 μl of suspension was collected from water agar and spread evenly, and the moisture on the surface of the water agar was air-dried in a sterile chamber. Then, plates were incubated at 18°C for 24 h. After approximately 24 h, the individual germinated conidia in the Petri dish were marked under the microscope. Marked areas were removed with a sterile needle and placed on potato dextrose agar (PDA). The plates were storaged at 20°C, and each colony was transferred to a fresh PDA plate after 2 to 3 weeks [S1 Table).Isolates for SSR analysis were collected from four counties  of Shaanxi Province in August 2016; five counties  of Gansu Province in September 2018; and three counties  of the Xinjiang Autonomous Region in September 2017 . The cou 3 weeks . All isoV. inaequalis isolates were inoculated on PDA medium and the medium was covered with Cellophane, stored at 20°C for 3 weeks. Approximately 30 mg of freeze-dried fungal mycelia was collected and ground in liquid nitrogen using a mortar and pestle. Then, 20 mg of the fungal powder was added to 2 mL centrifuge tubes. DNA was extracted with a Fungal DNA Extraction Kit . The quality of DNA was checked with a NanoDrop 2000C spectrophotometer. The DNA was diluted with distilled water and stored at −20°C.Mycelia of Isolates were genotyped by using 13 published SSR markers , 23. The2, 0.4 mM of each dNTPs, 0.05 U/μl Taq DNA polymerase), 1 μl of forward and reverse primers, respectively, 1 μl of DNA, adjusted to 25 μl with ddH2O. PCR reaction was performed under the following conditions: 30 cycles of 30 s at 94°C, 30 s at the specified annealing temperature (ranging from 50 to 55°C), and 20 s at 72°C. After 30 cycles, a final extension of 10 min at 72°C was performed. The PCR products were separated by Gel electrophoresis in Tris/acetic acid/EDTA (TAE) buffer solution with 3% agarose at 100 V for 1 h.For PCR amplification, a total of 25 μL of PCR mixture was prepared with 12.5 μl of 2X PCR Master Mix  [The PCR-generated bands were scored as ‘1’ (presence) or ‘0’ (absence) in a binary matrix. Matrix data were imported to NTSYSpc-Version 2.1. Unweighted pair group method with arithmetic mean (UPGMA) analysis was performed with genetic distances using a simple matching coefficient. All geographic populations were analyzed to determine which populations were genetically differentiated from each other . Nei’s garvest/) .V. inaequalis isolates were genotyped by 13 SSR markers used in the detection and all these markers were polymorphic and generated multiple alleles. The results obtained by POPGENE software for the genetic diversity parameters are presented in Vica10/154 was the most polymorphic, with 21 alleles. The effective number of alleles (Ne) ranged from 1.27 to 1.94, with an average of 1.62. Nei’s gene diversity (H) ranged from 0.21 to 0.48, with an average of 0.37. Shannon’s information index (I) ranged from 0.37 to 0.68, with an average of 0.55.Of the 457 scab-infected sample, 124 isolates was obtained from China. In total, 132 A total of 132 isolates from four regions  were screened with 13 polymorphic SSR markers. Two electrophoretograms are shown in With a similarity coefficient of 0.76, the 132 isolates were divided into five lineages. The results are presented in In terms of the number of lineages in each region , XinjianPT) values ranged from 0.044 to 0.155, indicating that between 4.4% and 15.5% of the variation occurred among the populations. Populations from the U.K. and Xinjiang were the least differentiated (ΦPT = 0.044), while populations from the U.K. and Shaanxi had the highest differentiation (ΦPT = 0.155) (The population differentiation (Φ= 0.155) .The lowest and highest genetic distance combination revealed population differentiation. High molecular variance was detected for populations in the four regions, where 91% of the variance occurred within populations and 9% among the populations .Structure analysis divided the isolates collected from the U.K. and the 3 regions in China into 3 populations (K = 3). Some isolates from Shaanxi and Xinjiang with more long blue bars can be considered one population and to differ from others. Isolates from Gansu had the highest proportion of green bars. Isolates from the U.K. had the lowest proportion of blue bars .The results of principal coordinate analysis (PCoA) showed the division of lineages intuitively . The symV. inaequalis population structure is a prerequisite for developing management strategies. The population structure of V. inaequalis in Shaanxi, Gansu, and Xinjiang, especially the origin of the pathogen and the role of crabapple in Xinjiang in the development of apple scab epidemics, is virtually unknown. We first investigated the genetic variability of V. inaequalis isolates sampled from Shaanxi, Gansu and Xinjiang in Northwest China. These results are following those of the populations from Shaanxi, Gansu and Xinjiang were highly similar. These results are in accordance with former studies in China [V. inaequalis having a similar population structures. Our research results on genetic diversity were similar to those obtained in South Africa, indicating similar population structures of V. inaequalis under wet and cool climatic conditions [Apple scab causes serious losses in the yield and quality of apple. The study of in China , 21. Thein China . As a renditions .The result of AMOVA showed that 91% of the variance occurred within populations and 9% among the populations, indicating geographical location had a low contribution to population differentiation. and gene flow is more frequent in these regions. The result was corresponding to many previous works –16. ThisPopulation stratification inferred by using cluster analyses revealed that the 132 isolates clustered into five main lineages independent of isolate sampling location and host cultivar, and in STRUCTURE 2.3.4 analysis, four region share a common three ancestry, indicating a common genetic base among the isolates. These results are in accordance with previous reports of gene flow among apple-infecting scab isolates in Switzerland , PennsylV. inaequalis and thus contribute to its spread, given the occurrence of the disease in crabapple and commercial apple orchards. Under climatic conditions favorable for disease development, apple scab may sweep through apple-growing areas.In addition, we found that isolates from crabapple in Xinjiang included all the lineages determined by the UPGMA dendrogram at a similarity level of 0.76 . This reMalus floribunda 821 (a resistant breeding resource) without pathogen transport from Europe to North America was found [V. inaequalis isolates with multiple fungicide resistance were also found in America [Recently, new isolates that can overcome the apple scab resistance of as found . This isas found  that patas found , causing America . All of V. inaequalis populations causing apple and crabapple scab in Shaanxi, Gansu, and Xinjiang of Northwest China and the U.K. Our results showed that populations were highly similar among the four regions and indicated that 132 V. inaequalis isolates may share a common three ancestry. Our results revealed for the first time that crabapple forests could serve as inoculum reservoirs of the pathogen V. inaequalis, contributing to its spread to commercial apple orchards. Our study is a basic assay to improve the disease management strategies for the management of apple scab.This is the first report to use 13 microsatellite markers to measure genetic diversity in S1 Table(DOCX)Click here for additional data file.S1 Dataset(RAR)Click here for additional data file.


Globodera ellingtonae was originally described from populations collected in the United States. In the original description, ribosomal DNA loci from Globodera sp. collected in Chile and Argentina were similar to G. ellingtonae, suggesting this nematode originated in this region of South America. In an effort to find additional populations of G. elllingtonae, collection trips were conducted in 2017 and 2020 in the Antofagasta and Arica y Parinacota Regions in Northern Chile, respectively. Globodera sp. were more prevalent in Antofagasta  than in Arica y Parincota . The genomes of single cysts (N ≥ 3) from four fields were sequenced. Additionally, the genomes of the G. ellingtonae population from Oregon and a Globodera sp. population originally collected in Antofagasta Region but maintained in culture in France were also sequenced. Based upon a HSP90 sequenced data mined from WSG data, all of the populations from the Antofagasta Region were G. ellingtonae and grouped in a monophyletic clade. A population collected from the Arica y Parincota Region was identified as G. rostochiensis based upon HSP90 data. Genome-wide SNP patterns of the G. ellingtonae populations showed strong clustering based on geographic location indicating that G. ellingtonae has high genetic diversity within Chile. A phylogenetic tree derived from 168,354 binary SNPs in the nuclear genome showed separate but distinct clustering of the Oregon population and the population from Antofagasta maintained in France. The Oregon G. ellingtonae population subtended the Chilean clades and placed on a long branch representing approximately twice the genetic variation observed among all Chilean G. ellingtonae populations. The possibility remains that G. ellingtonae from Chile may be sufficiently diverged to constitute a new species from G. ellingtonae originally described from a population collected in Oregon. Globodera ellingtonae  sequencing on G. ellingtonae populations collected from Chile. Whole genome sequence data enables genome-wide comparisons of closely related populations and provides an increased ability to discern population structure beyond what is possible using marker loci (i.e. ITS and HSP90). Utilizing WGS data, we then explored population genetics among the G. ellingtonae populations and compared informative gene regions of these populations to available data.To date, there are sequences for four populations of rgentina . This is pallida . To expaIn April 2017, a collection trip was undertaken in the Antofagasta Region of Chile. Agricultural fields in Socaire, Camar, Talabre, Caspana, and Toconce were sampled. Another collection trip was conducted in March 2020 in the Arica y Parinacota Region. Agricultural fields in Azapa, Chapiquiña, Pachama, Belén, Putre, Visvíri, and Socoroma were sampled. The fields were either fallow or under production with a diversity of crops including potato, tomato, maize, chard, and quinoa. Seventeen soil samples were collected in 2017 and 16 in 2020.To collect samples, a trowel or probe (2.5 cm diameter) was used and soil was collected from multiple locations within a field to a depth of 10 to 15 cm and combined. The soil was placed in a paper bag. At the Servicio Agrícola y Ganadero Nematology Laboratory  soil samples were placed on trays and allowed to dry for at least a week. Cysts were then extracted from 250 g of soil using a modified Fenwick can . Cysts wGlobodera sp. populations were selected for sequencing. These were chosen because enough cysts were available, and cysts contained abundant eggs. These populations were from Putre, Talabre, and Socaire. The two populations from Socaire were collected from the same field but on different dates (B was collected on 23 October 2017 and H was collected on 25 April 2017). Cysts for sequencing were also obtained from a culture of Globodera sp. originally collected Calama, Chile in the Antofagasta Regoin and maintained on potato at Institut National de la Recherche Agronomique . The G. ellingtonae population from Powell Butte, Oregon, which was used for the original description of the species  following the manufacturer instructions for isolation from tissues. Additional cell wall disruption was done during the second step of the QIAamp protocol wherein sterile plastic pestles were used to crush cyst tissue and mechanically lyse eggs. Extracted DNA was sent to the Core Services Lab at the Center for Genome Research and Bioinformatics (CGRB) at Oregon State University  for Illumina library generation using the Nextera XT preparation kit . Each sample was uniquely barcoded and multiplexed in equimolar concentrations prior to sequencing on the Illumina HiSeq 3000 platform. Paired-end 150 bp reads were obtained from the CGRB along with associated sequencing quality information. Raw sequence data is available for download from the Sequence Read Archive (SRA) under BioProject PRJNA748409.https://sourceforge.net/projects/bbmap/).Visual inspection of sequencing quality reports resulted in the removal of one sample (Socaire B1) from additional analyses based on the overall poor sequencing quality metrics. The remaining samples were trimmed to remove bases with Phred quality scores < 20 and remove adapter sequences using bbduk (Globodera sequences available in Genbank. A single 1402 bp HSP90 sequence from G. ellingtonae (MK105568.1) was used as a reference to anchor the assembly of the locus from each sample using the software package SAUTE (Globodera and Cactodera (outgroup) from PopSet 1780190675  to facilitate comparison to other ge SAUTE . Sequenc80190675  were use v3.8.31 , trimmed v3.8.31 . The HSPG. ellingtonae genome assembly, GCA_001723225.1  measurements included (n = 32): body length, stylet length, tail length, and hyaline length all in µm. Cyst measurements included (n = 29): fenestra length (µm), distance from anus to nearest edge of fenestra (µm), number of cuticular ridges between vulva and anus, and Granek’s ratio . Additionally, individual G. ellingtonae J2 (n = 22) and cyst (n = 4) morphometric data collected in 2008 was obtained from Dr. Z. Handoo . Mean (minimum and maximum) G. ellingtonae morphological data from Argentina was also considered  had cysts in the Arica y Parinacota Region. Population densities of these samples were 10 and 60 cysts/250  g soil; both samples were collected from Putre. The populations collected from the Arica y Parinacota Region were morphologically identified as G. rostochiensis (data not shown). This identification was further confirmed in the HSP90 phylogeny which showed distinct grouping of the Putre population with identified G. rostochiensis populations . Limited morphological data was available for some populations thereby restricting the number characters available to use in a character phylogeny. Of the characters available, all displayed overlapping ranges with no discriminatory power among populations. A summary of morphological measurements of G. ellingtonae populations reported in the literature  or presence on the highly abundant mitochondrial genome (COI and cytB). In contrast, these attributes are detrimental to de novo assembly as within-genome copy variation often confounds assembly methods  using microsatellite data, the whole-genome SNP data revealed that the genetic diversity in the eviously . In facteviously , likely eviously . The INRG. ellingtonae from other Globodera species utilizing a conserved 10 bp deletion in the chorismate mutase (CM) gene. Initial assessment of the INRAE Antofagasta population by G. ellingtonae. However, based on our sequencing data, nucleotide alignment of the probe site for the cultured population showed the conserved deletion is present as are the forward and reverse priming sites suggesting the TaqMan assays should classify the population as G. ellingtonae; this is further supported by the HSP90 phylogeny. The Oregon population also possess the discriminatory deletion however the sequence similarity with the other populations across the entire 2kb CM alignment was only 98.5%. The possibility remains that the G. ellingtonae population from Chile may be sufficiently diverged from G. ellingtonae populations from Oregon to constitute a new species. Further sampling of Globodera in southern South America is warranted to understand the genetic diversity of G. ellingtonae and the relationship to the Oregon population. Additionally, the use of WGS to explore genomic difference among Globodera populations will become more valuable as more plant-parasitic nematode genomes become available.


Human Y-chromosomal haplogroups are an important tool used in population genetics and forensic genetics. A conventional method used for Y haplogroup assignment is based on a set of Y-single nucleotide polymorphism (SNP) markers deployed, which exploits the low mutation rate nature of these markers. Y chromosome haplogroups can be successfully predicted from Y-short tandem repeat (STR) markers using different software packages, and this method gained much attention recently due to its labor-, time-, and cost-effectiveness. The present study was based on the analysis of a total of 480 adult male buccal swab samples collected from different regions of Bosnia and Herzegovina. Y haplogroup prediction was performed using Whit Athey’s Haplogroup Predictor, based on haplotype data on 23 Y-STR markers contained within the PowerPlex® Y23 kit. The results revealed the existence of 14 different haplogroups, with I2a, R1a, and E1b1b being the most prevalent with frequencies of 43.13, 14.79, and 14.58%, respectively. Compared to the previously published studies on Bosnian-Herzegovinian population based on Y-SNP and Y-STR data, this study represents an upgrade of molecular genetic data with a significantly larger number of samples, thus offering more accurate results and higher probability of detecting rare haplogroups. The paternally inherited non-recombining portion of the Y chromosome (NRY) is the material of choice when it comes to tracing the paternal lineage of populations . The potA single Y haplogroup represents a group or a family of Y chromosomes related by descent or ancestry, and each such haplogroup is determined by a specific set of Y-SNPs, which makes them extremely important when it comes to better understanding of past migrations and demographic processes that shaped modern populations . Since YPopulation genetic studies are of high importance in Bosnia and Herzegovina B&H;  as they A previously published study observing SNPs was published using 256 samples . A laterThe Ancestors in us – Genetic Heritage of Bosnia and Herzegovina (Bosnian: Preci u nama – Genetičko blago Bosne i Hercegovine), designed and implemented by Mladinska knjiga  according to manufacturer’s recommendations, while DNA quantification was assessed on Qubit® 2.0 fluorometer . PCR amplification of target 23 Y-STR loci was performed using PowerPlex® Y23 System  following manufacturer’s recommendations. Amplicons were detected on an ABI PRISM® 310 Genetic Analyzer . Raw data analysis was performed using ABI PRISM® Data Collection software, while the final Y-STR DNA profiles were generated using GeneMapper® ID v3.2 software. Allele frequencies and haplotype data obtained in this way are currently submitted for publication elsewhere .Y-chromosomal haplogroup prediction using allele frequencies on 23 Y-STR loci was performed using Whit Athey’s Haplogroup Predictor v5, an algorithm based on the Bayesian-allele-frequency approach , 2013.in silico Y haplogroup assignment from Y-STR data even in relatively small and heterogeneous populations such as the B&H population. Therefore, we have decided to use this tool in this study. Additionally, for the samples where prediction accuracy was lower than 90%, we have used the NevGen algorithm (The results of Y haplogroup prediction using Whit Athey’s Haplogroup Predictor program are summarized in lgorithm  as a verPrediction accuracy was estimated to be 100% in 456 cases. For the remaining 24 samples (5%), prediction accuracy varied between 52.2 and 99.9% . Out of High prevalence of haplogroup I with its sublineage I2a (43.13%) was expected, when considering previously published literature . In threHaplogroup R, with its major sublineage R1a, was the second most abundant in the study population of B&H with frequency of 14.79%. In the previous Y-SNP-based study, the haplogroup R accounted for around 14% of all Y chromosomes with an even distribution among three ethnic groups, namely 15% in Bosniaks, 14% in Serbs, and 12% in Croats . In the Haplogroup E was detected in the present study through its sublineage E1b1b (14.58%), which is characteristic for European male individuals . The preRare haplogroups N, L, and T , as well as Hg Q (with frequency of 0.21%) were detected in this study, and are mainly uncharacteristic for the area of the Balkans as they were not detected previously. In fact, haplogroups L, T, and Q were detected for the first time in the B&H population. Haplogroup N has most probably expanded from Northern Eurasia to Eastern Europe and is present in low frequencies in the Balkans and Central Asia . PreviouRst values and calculated based on the results of the same set of markers from other populations from the YHRD database, indicates that the Bosnian-Herzegovinian population does not deviate significantly from the neighboring populations of Serbia, Croatia, and Slovenia, while it stands out drastically from the populations of Belgium, Italy, Germany, Austria, and the Czech Republic .Additionally, analysis of 23 Y-STR loci on the currently studied population of B&H (unpublished data) quantified through The current study performed on a significantly increased sample size generally confirmed the previous main haplogroup balance. However, it offered a more detailed understanding into the genetic diversity of the population of B&H and provided insight into rare and unusual haplogroups. This includes L, T, and Q, which have been detected as part of the region’s population genetic diversity for the first time. Therefore, it can be concluded that this study improves the current findings regarding the population and presents this information in a more reliable way.https://yhrd.org/, YA003787.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.NJ and TK participated in laboratory work, data analysis, and manuscript drafting. SD conducted data analysis and visual representation of the data. LB, LS, RK, VŠ, and PP performed laboratory work and data analysis. VH participated in study design. AA participated in laboratory work, data analysis, and manuscript preparation. DM conceived the research idea, designed the work, and participated in data analysis and manuscript preparation. All authors contributed to the article and approved the submitted version.LS and RK were employed by the company ALEA Genetic Center (AGC) in Sarajevo, Bosnia and Herzegovina. VŠ and PP were employed by the company Genos Ltd. in Zagreb, Croatia.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pachyphytum cesium (Crassulaceae), an endemic species from central Mexico threatened by human activities and climate change. To increase our knowledge of the species, we analyze the diversity and genetic structure of all known populations of P. caesium to change their current genetic status and propose conservation strategies. The results indicate that this species presents low to moderate levels of genetic diversity and incipiently structured populations due to low genetic flow. We found that genetic parameters are essential to the conservation strategies and status vulnerability reclassification. Based on our results, we propose reclassifying the conservation status of the species in danger of extinction; hence a series of conservation strategies are provided to prevent its disappearance.Species decline has accelerated in recent decades, with rare species often being the first to go extinct, mainly due to low population sizes. This study worked with Pachyphytum caesium is an endemic species with extremely small populations in central Mexico. This work analyzes the diversity and genetic structure of Pachyphytum cesium (Crassulaceae) to change the conservation status and propose conservation strategies. Twelve dominant ISSR markers were used to describe the genetic diversity of all known populations. Additionally, we carried out two simulations to explore how the loss of individuals or the local populations extinction affect the genetics parameters of the species. The average results indicated moderate levels of genetic diversity , parameters that decreased significantly when simulations were performed in P. caesium. Additionally, a genetic structure of the populations was found with low gene flow (Nm = 0.92). Genetic parameters are negatively affected by the loss of individuals and the local extinction of populations. Based on our results, we propose to reclassify the conservation status of the species in danger of extinction, hence a series of conservation strategies are provided to prevent its disappearance.Genetic studies in rare species are important to determine their patterns of genetic diversity among populations and implement conservation plans aimed to reduce extinction risks.  Ecological and genetic studies are essential to implement conservation plans, especially in endangered, rare and endemic plant species ,2,3, in Currently, there are various methods of DNA-based markers for the genetic analysis of plant populations, the most common being amplified-fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), microsatellites (SSR), and single nucleotide polymorphisms (SNPs); each varies in polymorphism quantification, repeatability, resolution power and analysis cost ,10,11. AAlthough genetic studies are important toward the species conservation, these results are rarely included in the Red List of threatened species of the International Union for Conservation of Nature’s (IUCN) methodology ,15,16,17Nowadays, habitat loss and climate change are two of the major causes of the current biodiversity crisis, as they cause not only cascading extinctions ,2,3, butPachyphytum, an endemic genus of the Crassulaceae [Pachyphytum caesium Kimnach & Moran is a cliff-dwelling species endemic to the dry tropical forest of Aguascalientes in central Mexico, and has been categorized as PSESP due to its highly specialized habitat, small and fragmented populations, and a high vulnerability to environmental changes [Habitat loss has been particularly worrying in central Mexico, due to land-use change, as more than 80% of its original vegetation has been transformed into agriculture and cattle raising ,24. Thissulaceae . Knowledsulaceae ,27,28,29 changes . Based o changes  classifiThe MER is a tool that is applied exclusively in Mexico, as part of the Mexican Official Standard (NOM-059-SEMARNAT-2010) . This toPachyphytum caesium reported by Clark-Tapia et al. [Because an appropriate estimation of extinction risk is one of the key components of any conservation plan, in this work we evaluated the diversity and genetic structure of the currently know populations of a et al. . In addiP. caesium are located in this region [2, representing 3.18% of the state’s total surface [The study area is located in central Mexico, between 22°40′00″ N and 102°30′00″ W in the southwestern portion of the state of Aguascalientes . The onls region , whose n surface .Pachyphytum caesium (Crassulaceae) is a succulent perennial plant growing in the north-central region of Mexico [f Mexico ,28. A gef Mexico ,28,29 wif Mexico . Populatf Mexico . The areP. caesium can be propagated through asexual reproduction, from the leaves or stems that are detached (Pers. Observations).This species presents two flowering periods per year (from February to April and from September to November). The reproduction is sexual, and the observed pollinators are bees and hummingbirds. The fruit produces numerous small (<1 mm) brown seeds. However, their reproductive biology is unknown, and more studies are needed . AdditioPachyphytum caesium was carried out between 2009 and 2013 as new populations were found. We collected young leaves of 20 individuals per population, for a total of 120 individuals. Distance separation between collected individuals was 20 m to avoid kinship. To access each site and collect the tissue, we used a rock-climbing technique with a Petzl stop descender. Subsequently, we stored the collected biological material in a plastic bag. Once in the laboratory, samples were stored at −70 °C to preserve it until used.The tissue collection of DNA was extracted following the Qiagen DNeasy Plant Kit protocol . For the evaluation of the quality and quantity of genetic material obtained from the 120 individuals, electrophoresis was performed in 1% agarose  gels in TBE buffer 0.5X, and stained with Midori Green Direct  for an average of 40 min at 90 V. Subsequently, these gels were visualized with UV light in a transilluminator equipment Gel Doc XR + System .P. caesium, we opted for ISSR, a dominant marker with reasonable reproducibility and polymorphism than other markers, as well as a robust pattern in the analysis of genetic diversity [P. caesium individuals; these were selected for their high-polymorphic states and repeatability , effective number of alleles per population (Eff_num), and private alleles per population (Ie) were determined as the total number of observed bands (It) using GenoDive v.3.0 . In addiMoreover, we calculated the following genetic diversity parameters: (1) percentage of polymorphic loci with 95% criterion (%P) and (2) expected heterozygosity (He)  using thWe calculated the population structure in six ways: (1) the level of genetic differentiation among populations was obtained with the estimator Gst , using tAdditionally, we used (4) the principal component analysis (PCA) to analyze genetic variation and possible associations in the allele frequencies of the sampled populations using a covariance matrix . (5) We P. caesium. It was performed using the software STRUCTURE v.2.3.4 [Finally, (6) we used a Bayesian model-based clustering approach to identify the genetic structure in populations of  v.2.3.4 . We used v.2.3.4 , which i v.2.3.4  to deterP. caesium shows a pattern of isolation by distance, a Mantel test with 10,000 permutations was performed with the APE package [To evaluate whether  package , using t package . Subsequ package .In 2018, we returned to the previously collected populations and found a drastic decrease in abundance (≥50% in two sites), in addition to an extinct local population reported by Clark-Tapia et al. . Given tPachyphytum caesium. This tool used four normalized criteria to assess risk categories of species: (A) breadth distribution; (B) habitat status; (C) intrinsic biological vulnerability ; and (D) human impact. Numerical values of each criterion are assigned in ascending order of risk, which are subsequently added up. Each criterion is evaluated qualitatively, and the total score is calculated by adding the normalized results from the four. The higher the total score value, the higher the risk of extinction of the assessed species. According to the MER [Based on the method for evaluation of the risk of extinction of species in the Mexican legislation  , we esti the MER , species50) and local extinction (2020eex). In addition, we integrated ecological and genetic data, considering average values (2020e + g) and two simulations, one with loss of individuals (2020e + g50) and the other with extinction (2020e + gex). For comparison, we also applied parts of the IUCN Red List system [We analyzed the MER for two years (2005 and 2020e) based on the ecological and genetic knowledge of the species. We used 2005 because we only had basic knowledge (breadth distribution and habitat status). In 2020e, we analyzed the MER criteria using only ecological knowledge  and ecolt system . This met system .Pachyphytum caesium that presented the highest number of alleles was Presa Cebolletas (96), and the lowest was Presa Malpaso and Mesa Montoro . The total number of alleles did not change when performing the simulation with random extraction of 50% of the individuals or the local extinction of two populations (Presa Cebolletas and Mesa Montoro). However, the number of private alleles decreased significantly   .On the other hand, the highest levels of genetic diversity were found in Presa Cebolletas , and the lowest in Mesa Montoro , with a low population average . The expected heterozygosity was significantly more affected by the simulated extraction of individuals than by the local extinction of two populations . Furtherp < 0.001) partitioning among the populations, of which 43% was attributed among populations and the rest 57% within populations. The coefficients of genetic differentiation among populations Gst and Fst were 0.471 and 0.443, indicating that more than 44% variation was presented among the populations, which is indicative of population structuring. Identical values of variation between populations  were obtained after the random extraction of individuals or the local extinction of populations.The analysis of molecular variance (AMOVA) showed a significant  explained 23.98% of the accumulated variance, where the first component explained 13.56% of the total variation, while the second explained 10.42%. The analysis generated four well-defined population groups, including the association between Puente Cuates, Presa Malpaso, and Río Gil, which is closely associated with Mesa Montoro a.In the PCAs obtained with the simulations of the extraction of individuals b and theFinally, discriminant analysis of principal components (DAPC) detected that the optimal number of principal components to generate the model was two. This result generated a model of two discriminant functions  where a grouping pattern between populations like the PCA was obtained and there was a strong structuring between populations a.With the simulations of individual extraction b, the grP. caesium populations , and mean (similarity score) were detected at K = 2 by Evanno’s method using the total individuals and simulated 50% of populations, indicating that two groups could be distributed across all the ulations a,b. A deulations . Under tulations c.Gene flow between the populations was Nm = 0.92 migrants per generation for all individuals or considering 50% of the individuals per population. However, Nm decreased dramatically to 0.633 when we simulated a local extinction of two of the six populations.p = 0.29; 999 permutations), or 50% of the individuals , and the local extinction of two populations .Mantel’s correlation analysis found no correlation between genetic and geographic distances considering the six populations  (The MER showed four results according to the criteria and knowledge used in the method: (1) with basic knowledge of two criteria (A: breadth distribution and B: habitat status), the species was classified in special protection category (2005). (2) Adding the ecological knowledge about intrinsic biological vulnerability and human impact , the status of the species was elevated to threatened (2020e). (3) After incorporing the genetic knowledge into intrinsic biological vulnerability , the status of the species was further elevated to in danger of extinction level (2020e + g). Although the inclusion of genetic factors in both simulations also generated a status of danger of extinction, the total score in vulnerability was higher (2020e + ge + gex) .50 and 2020e + g50), and (3) the critically endangered status achieved with simulations of local extinction (2020eex and 2020e + gex).On the contrary, the IUCN system showed three conservation statuses: (1) the status of vulnerable for years with basic knowledge (2005), ecological (2020e), and ecological-genetic (2020e + g). (2) The endangered status obtained in the simulations with a decrease in population size ; however, it shows an absence of sexual recruitment despite producing seeds. For this reason, we suggest that the small populations’ size of ulations . The hisulations  and is rulations . Its higulations . Furtherulations ,64. ThisDraba aizoides, a cliff-dwelling species of the Northern Franconian Jura and Swabian Alps in Germany due to the decrease in the population number of adults and seedlings. Our results suggest a similar scenario of affectation at the genetic level, considering the simulation results with loss of individuals and local extinction. It is known that the populations of P. caesium have decreased due to changes in land use and sport climbing [Land-use change or anthropogenic activities have been documented to affect the distribution and size of a population and species’ genetic diversity ,23,54,65climbing ; howeverPachyphytum caesium, like other endangered or endemic species, such as Megacodon stylophorus (Gentianaceae) [Rhodiola alsia (Crassulaceae) [P. caesium populations have a fragmented distribution due to land-use changes, with a discontinuous distribution associated with cliffs in the remnants of the tropical dry forest in the state of Aguascalientes [Pachyphytum makes its comparison difficult. However, it is possible with other endemic plants or PSESP that inhabit cliffs, including members of the Crassulaceae family. In these species, it is common to find a high genetic differentiation between populations, e.g., [An incipient level of among-population genetic differentiation was revealed in anaceae)  and Rhodulaceae) . The genulaceae) ,54. P. calientes . The lacs, e.g., ,54,61,66Pachyphytum caesium, the absence, or low gene flow, as a factor that could homogeneize allelic frequencies [P. caesium populations, added to the allelic loss due to the decline in their populations and local extinctions, increases the genetic drift effect and allows the action of other evolutionary forces as inbreeding. If this is so, new molecular studies (SNPs) are also required to know aspects of inbreeding, genetic landscape, and locate local and functional genotypes to provide greater certainty in the conservation of the species.In quencies ,67, togequencies ,69. GeneEven though there is a discrepancy in the conformation of groups between the Minimum spanning network, PCA and DAPC analyses showed between 3 and 4 groups, concerning STRUCTURE that obtained two groups, or three in the simulation without two populations. At landscape level, the cliffs from Río Gil, Presa Malpaso, and Puente Cuates have greater connectivity between them as they are within the same micro-basin and are more likely to exchange migrants. Therefore, they have a greater association and overlap extensively in the analyses. In contrast, the three remaining populations have been isolated in recent decades with less probability of genetic exchange.P. caesium populations were fragmented, leading to isolation and genetic drift phenomena as reported in other endemic species [Cluster analysis and STRUCTURE clustering showed a clear geographic pattern suggesting that, in the past,  species . A possi species , furtherP. caesium, a result found in other studies, which also emphasizes the need to consider genetic studies.This study highlights the importance of using genetic data in the risk analysis of a species, as suggested by Garner et al. , VictoriLike the MER, the risk is increased in the IUCN by modifying the population size and distribution area; two key criteria used by these methods assign threat status ,30. AlthP. caesium, precautionary conservation measures should be appropriate because the risk of extinction is higher for small populations [In PSESP, as in ulations ,23, whiculations  and the ulations . That isP. caesium is not subject to federal protection in the short term, its vulnerability to adapt to future environmental changes increases, mainly due to a decrease in private alleles, e.g., [The immediate conservation strategy is to insert the species on the IUCN red list and the list of species at national risk, in addition to the studies of the species’ vegetative propagation and reproductive biology which have already begun. The establishment of rappelling routes and the prohibition of this activity in some areas where the species is found is a pending activity. Given the possibility that s, e.g., , and reds, e.g., , which wPachyphytum caesium exhibit moderate to low levels of genetic diversity, a strong genetic structure of their populations, and low gene flow. Genetic drift is an evolutionary force that negatively affects the genetic population structure and depends on the demographic stability of the species. The simulations of extraction of individuals and the local extinction of populations negatively affect genetic diversity, which should be considered for population conservation. According to ecological and genetic data, populations of P. caesium are at risk of extinction following the MER. We highlight the importance of using genetic data in MER, which is why we suggest its use in the IUCN Red List methodology. Finally, genetic data can support future endangered species conservation planning.Our study found that


Etheostoma caeruleum) is a member of this ichthyofauna and has a disjunct distribution in glaciated and unglaciated environments west of the Mississippi River. Based on glacial history of the region, there are different expectations on the observed spatial genetic structure of populations in these environments. The aim of this study was to utilize genome‐wide SNP data to compare the population genomic structure of the rainbow darter in river networks with disparate glacial histories; the Volga River in the glaciated upper Mississippi River basin and the Meramec River in the unglaciated Ozark Plateau. Individuals were sampled from localities within each river system at distances dictated by the organismal life history and habitat preferences. Riverscape analyses were performed on three datasets: total combined localities of both rivers and one for each river independently. The results revealed a lasting influence of historic glaciation on the population genomic structure of rainbow darter populations. There was evidence of population expansion into the glaciated northern region following glacial retreat. The population genetic signature within the Volga River did not fit expectations of the stream hierarchy model, but revealed a pattern of repeated colonization and extirpation due to cyclic glaciation. The population within the unglaciated Meramec River adhered to the stream hierarchy model, with a directional order of genetic diversity based on the life history and habitat preferences of the species. These results demonstrate the importance of considering the geologic and climatic history of a region as well as the life history of an organism when interpreting spatial genetic patterns.Periodic glaciation during the Quaternary period shaped the contemporary riverscape and distribution of freshwater fishes in the Mississippi River drainage of central North America. The rainbow darter ( The aim of this study was to compare the population genomic structure of the rainbow darter in river networks with disparate glacial histories. The results support the hypothesis that historical glacial cycles during the Quaternary period influenced the observed genetic signature of rainbow darter populations in drainages of central North America. They support the prediction that the populations in the glaciated environment in the north does not fit expectations under the stream hierarchy model. Understanding the influences on the evolution of spatial genetic structure of populations in riverine environments is a unique problem that involves a specific set of questions, which are described as riverscape genetics  and Meramec  rivers were selected as systems for comparison. Both waterways are historically known to contain abundant resident populations of E. caeruleum individuals . A two‐adapter process was utilized to create DNA libraries from the digested samples, with 10–15 individually barcoded samples allocated to each Illumina library pool. P1 adapters with unique barcodes were ligated to the specific SbfI overhang sequence exposed during the digestion process. Samples were then pooled, sheared using a sequence of cyclic sonication (Diagenode Bioruptor), and size selected into approximately 300–400 base pair (bp) lengths using magnetic beads in PEG/NaCl buffer  was run to create a consensus sequence catalog, generate SNP loci, and formulate candidate alleles for individual fish  as an optimization target and filtering step to ensure only SNP loci shared by a minimum of 80% of all individuals in a population are retained . The Bayesian assignment test was performed using the software package Jombart, .fastStructure makes use of efficient algorithms to employ a Bayesian framework model for the inference of the total genetic clusters (K) within the data, and assignments of fish to a cluster based on individual genotypes without a priori definitions  following Rousset  than the Volga River localities  among all 10 localities in the combined river dataset showed a substantial amount of genetic distance between the Meramec and Volga River localities with values ranging between 0.3453 and 0.4345 and an average of 0.4083 ; Data curation ; Formal analysis (lead); Writing – original draft (lead). Tony Gamble: Data curation (supporting); Formal analysis (supporting); Writing – original draft (supporting). Peter B. Berendzen: Conceptualization (lead); Data curation ; Formal analysis (lead); Funding acquisition (lead); Writing – original draft .Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


The genetic diversity in three populations of creole sheep managed by indigenous communities of the central region of Veracruz, Mexico, is reported from blood samples of 90 sheep, taken from the herds of indigenous families in the municipalities of Tehuipango, Astacinga, and Tlaquilpa, Veracruz, Mexico. The genomic DNA of the sheep was evaluated using four microsatellites amplified by PCR and visualized on polyacrylamide gels. The four microsatellites were polymorphic, observed heterozygosity was lower than the expected level, and the indices of endogamy indicated a slight diminution of homozygotes and the variation was hosted at the individual level. The evaluated sheep present a genetic diversity that is conserved across endogamic crosses, for which reason the design of a plan of protection and use of these sheep populations would permit their sustainable management.p < 0.05), presumably this means that they do not deviate significantly from H–W predictions and there was slight genetic differentiation (STF = 0.025), along with a slight decrease in homozygotes (ISF = −0.021). According to the analysis of variance, 99% of the total variation was hosted at the individual level. It is concluded that the three creole sheep populations still present genetic diversity at the four loci and non-random pairings have occurred.In the indigenous communities of central Veracruz, herds of creole sheep have been established and managed through traditional practices of crossing, but their genetic characteristics have never been examined in order to evaluate their state of endogamy, and to help the management programs to protect this genetic resource. The objective of the present study was to characterize the genetic diversity of three populations of creole sheep managed by indigenous communities in the central region of Veracruz, Mexico. Indigenous family producers of creole sheep were located and blood samples taken from 90 individual sheep from the municipalities of Tehuipango, Astacinga and Tlaquilpa, Veracruz. In the laboratory, the genomic DNA was extracted and genetic diversity characterized using four microsatellites  amplified by PCR and visualized on polyacrylamide gels. The four microsatellites were highly informative (PIC = 85%) and presented values of 0.6 to 0.81 of heterozygosity, with an average number of 16 alleles. According to the Hardy–Weinberg equilibrium model, three of the loci were not significant ( Livestock production was introduced to Mexico during the colonial period and has become a socioeconomically productive activity . Ovine lThe creole sheep breeds are those populations that have adapted to the rural and cultural environments in which the herds are managed but still present certain traits of the original breeds introduced to Mexico during the colonial period . Among tStudy and diagnosis of the genetic diversity of creole sheep are necessary in order to determine the status of the genetic variability of the populations , take men = 90 sheep) under study were found in the municipalities of Tehuipango , Astacinga  and Tlaquilpa , located in the central region of Veracruz, Mexico . Genotyping of each individual was conducted with the following four microsatellite type molecular markers: OarFCB128, SRCRSP9, ILSTS5, and ILSTS11 , with the following parameters: migration at 250 V for 1.5 h, running buffer TBE 1X , and 25 ng of 20 bp molecular weight marker (Sigma-Aldrich) placed every 10 samples. Each fragment was detected using silver staining [®, Jerusalem, Israel). The weight of each band in bp was determined with the software GelAnalyzer version 19.1 in order to construct the matrix of molecular data.To determine the size in bp of the product of each amplification, electrophoresis was conducted in non-denaturing polyacrylamide gels and using 3 µl of each reaction per well in gels prepared with 19:1 acrylamide:bisacrylamide  in the Mstaining . Each geISF, ITF and STF) as well as analysis of molecular variance (AMOVA), which were processed with the software packages GenAlEx V.6.503 [The Hardy–Weinberg Equilibrium (HWE) of each marker was evaluated using a Chi-squared test, and the markers were also examined in order to qualify their polymorphism and obtain the parameters of population diversity . These p V.6.503  and Powe V.6.503 . The sof V.6.503  was usedThis study found significant deviations from the HWE in some of the loci studied, and suggested non-random pairings in the populations of creole sheep. ILSTS11 locus was the only one in the HWE loci/marker or similar between only and in the three populations, while the rest of the microsatellite loci were found to be in HWE in only one of the three populations: locus ILSTS5 was found in equilibrium for population 1 (Astacinga) and the loci OarFCB128 and SRCRSP9 were found in equilibrium in population 2 (Tehuipango) . The fouThe allelic frequencies per microsatellite  as the lSTF) was 0.025 (ILSTS11), 0.011(ILSTS5), 0.029 (SRCRSP9), and 0.034 (OarFCB128) revealing that the sheep of the high mountains of central Veracruz present what is considered low genetic differentiation, suggesting minimal changes in the genetic structure of the population (ISF) for each locus was negative: −0.234 (ILSTS11), −0.150 (ILSTS5), −0.248 (SRCRSP9), and −0.221 (OarFCB128), all one can conclude is there is an excess of heterozygotes, indicating a greater incidence of non-random pairings among related individuals. The degree of genetic differentiation  were identified. The first of these were grouped as population 1 (Astacinga) and population 2 (Tehuipango), indicating that these populations presented a high degree of genetic similarity, while the second group was represented by population 3 (Tlaquilpa), which was genetically differentiated from populations 1 and 2 .Finally, the AMOVA showed that the greatest genetic variation was provided by the individuals of the population (99%), and that there was almost null variation (1%) among individuals of the different sheep populations evaluated .The HWE showed that the frequencies of the microsatellites evaluated in the three populations of sheep have not remained stable among generations, since these sheep originate from a population with a low number of founding individuals, or that experienced little random breeding. The lack of equilibrium detected in the loci could have been motivated, in part, by the need of indigenous producers for crossing with rams that present phenotypic characteristics of commercial value  or by thThe four microsatellite loci evaluated in the creole sheep presented more than one allele . The micStudies of genetic diversity have reported values of allelic diversity and allele numbers similar to those found in this study. In a population of Nigerian sheep, alleles of 96 to 130 bp were recorded at the loci OarFCB128 . In a stThe numbers of alleles observed per locus in this study exceeded those reported in a study of characterization of Colombian creole sheep, reported 11 different alleles for ILSTS11, 12 alleles for ILSTS5, and 12 alleles for OarFCB128 , who ideWithin genetic improvement programs, knowledge of the genetic diversity of creole sheep is the basis for reproductive and productive success in populations for sustainable management . In the STF index revealed that the creole sheep present low genetic differentiation, suggesting a genetic structure that is becoming unstable at population level [ISF) for the four microsatellites evaluated, indicating a decrease in homozygous individuals. In a study of genetic characterization of sheep in Colombia, reported ISF values higher than 0, which was attributed to the fact that the breeds could be inbred or subjected to human selection through management in order to acquire certain phenotypic characteristics [In this study, the on level . This meeristics . The sheeristics .The results also identified two groups: the first of which included population 1 (Astacinga) and population 2 (Tehuipango), while the second consisted only of population 3 (Tlaquilpa). The group that included populations 1 and 2 showed that these populations have a high degree of genetic similarity, which can be attributed to the fact that the physical proximity of their regions has facilitated the occurrence of crossing between these populations. Finally, it is possible that the allelic variability of the creole sheep evaluated has been selected as a function of the indigenous group or family that manages the herd, which has produced the small genetic differentiation. The creole sheep populations should still maintain sections of genome that comprise a large part of their identity; however, given the limited number of loci examined, this situation remains to be demonstrated.Genetic diversity, based on the genotyping of four microsatellite markers in three populations of creole sheep managed by indigenous communities in the central region of Veracruz, showed a large catalog of alleles. The results contribute to the genetic knowledge of these sheep required in order to control situations through reproductive management. For the purposes of survival, it is essential to protect and maintain these populations. Furthermore, for the purposes of use it is required to fix phenotypic characteristics such as wool production in the populations of Astacinga and Tehuipango; rams and ewes of these populations that express these desired phenotypic characteristics should be crossed. Moreover, if it is desired to increase the height of the cross, rams from Tlaquilpa can transfer this characteristic over a few generations, given the degree of endogamy presented by the populations.