Crotalus oreganus) occurs in British Columbia, Canada, where it is federally classified as threatened and restricted to five geographic regions. In these areas, Western Rattlesnakes hibernate (den) communally, raising questions about connectivity within and between den complexes. At present, Western Rattlesnake conservation efforts are hindered by a complete lack of information on genetic structure and degree of isolation at multiple scales, from the den to the regional level. To fill this knowledge gap, we used Genotyping‐in‐Thousands by sequencing (GT‐seq) to genotype an optimized panel of 362 single nucleotide polymorphisms (SNPs) from individual samples (n = 461) collected across the snake's distribution in western Canada and neighboring Washington (USA). Hierarchical STRUCTURE analyses found evidence for population structure within and among the five geographic regions in BC, as well as in Washington. Within these regions, 11 genetically distinct complexes of dens were identified, with some regions having multiple complexes. No significant pattern of isolation‐by‐distance and generally low levels of migration were detected among den complexes across regions. Additionally, snakes within dens generally were more related than those among den complexes within a region, indicating limited movement. Overall, our results suggest that the single, recognized designatable unit for Western Rattlesnakes in Canada should be re‐assessed to proactively focus conservation efforts on preserving total genetic variation detected range‐wide. More broadly, our study demonstrates a novel application of GT‐seq for investigating patterns of diversity in wild populations at multiple scales to better inform conservation management.Delineation of units below the species level is critical for prioritizing conservation actions for species at‐risk. Genetic studies play an important role in characterizing patterns of population connectivity and diversity to inform the designation of conservation units, especially for populations that are geographically isolated. The northernmost range margin of Western Rattlesnakes  to reveal significant population structure and limited connectivity among populations of the at‐risk Western Rattlesnake at their northern range periphery in Canada. Our results demonstrate a novel application of GT‐seq and suggest that the current conservation unit status for this species in Canada warrants re‐examination to better inform proactive management. Although broadly distributed throughout the western half of the United States, this snake is classified as “threatened” by the Canadian Species At Risk Act and protected under the British Columbia (BC) Wildlife Act  to characterize the distribution of genetic variation within and among Western Rattlesnake populations in Canada and neighboring Washington (USA). Given the protected status and venomous nature of the animal, we largely employed minimally invasive sampling (MIS) using cloacal swabs, as it reduced handling time and limited disturbance to individual snakes. Although MIS is often suboptimal for use with many GBS approaches , cloacal swabs (n = 552), buccal swabs (n = 5), and skin sheds (n = 4), from 36 dens across all five of the informally recognized regions of the Western Rattlesnake range in Canada, namely the Thompson‐Nicola (TNI), Vernon (VER), Okanagan‐Similkameen (OKS), Midway (MW), and Grand Forks (GF), as well as from neighboring Washington (WA), USA , sex, weight (g), and age class for individual snakes when possible. For the present study, we defined adults as those snakes with a SVL measurement of >540 mm either at the time of sampling or postsampling, based upon recent estimates for sexually mature Western Rattlesnakes  following the manufacturer's protocols (including RNAse A treatment). Only slight variations were applied for the shed, buccal, and cloacal swab samples. For the shed skins, ~160–180 mg of each shed underwent a surface clean with a 3% bleach solution. After bleach treatment, we then washed the sheds with ethanol and left them to air‐dry completely. Once dry, we manually pulverized each sample and placed them into separate 2‐ml tubes. Proteinase K and lysis buffer were added to each tube, and samples were incubated for 48 hr at 56°C. The rest of the extraction procedure followed the manufacturer's protocol (including RNAse A treatment).K for 1 hr at 56°C. The rest of the extractions then followed the manufacturer's protocol (including RNAse A treatment). Once eluted, we quantified all DNA extracts using a Qubit™ 3.0 Fluorometer and Qubit™ dsDNA High Sensitivity Assay kit (Invitrogen).For the cloacal and buccal swab samples, we extracted DNA from the lysis buffer in which the swabs were stored. 180 μl of buffer from each sample was incubated with 20 μl of Proteinase 2.3https://github.com/GTseq/GTseq‐Pipeline). The GTseq_Genotyper_v3.pl script was used to call genotypes for each individual sample, and then, GTseq_GenoCompile_v3.pl was used to compile all the individual genotypes. This resulting output file was then converted into a PED file format  markers previously developed and optimized in Schmidt et al. . A totaln = 17) as the proportion of discordant genotypes across all loci successfully genotyped for both replicates. To reduce bias due to sampling potentially highly related individuals at dens, we used ML‐RELATE . Those individuals and SNP loci with >30% missing data were removed from the dataset. Loci with a minor allele frequency <0.05 and those that deviated from Hardy–Weinberg Equilibrium (HWE) in >60% of sampling units were also removed. We calculated genotyping error for repeated samples that passed data quality filters (2.4K = 1–8 (the number of a priori geographic regions + 2), each with a burn‐in of 500,000 and a run length of 1,000,000 MCMC steps. The resulting output was then summarized using STRUCTURE HARVESTER  among sampling regions and den complexes as implemented in GENETIX 4.05  and correlated with estimates of pairwise θ. We then repeated this test using den‐level pairwise geographic and genetic distances within each geographic region containing >2 dens to evaluate more fine‐scale patterns of connectivity.To further examine connectivity across the range, we tested for patterns of isolation‐by‐distance Wright,  among alWe also assessed the amount and direction of contemporary migration among den complexes with BAYESASS 3.04 , expected heterozygosity (HE), nucleotide diversity (π), and the proportion of variable sites (P) as implemented in ARLEQUIN  for each den complex with GENETIX  in a den complex were conducted in R  was 0.36%. After repeated samples and those individuals with an inferred parent in the dataset (n = 27) were removed, a total of 461 individuals were used for subsequent population genetic analysis with an average depth of 332x (range: 23x‐1113x) and 3.82% missing data (range: 0.00%–29.87%).A total of 608 samples  were genotyped, with an average of 341,838 raw sequencing reads per sample. After quality filtering for 30% missing data, minor allele frequency > 0.05, and HWE, 505 samples and 308 loci remained in our dataset. Mean genotyping error across all repeated samples  were detected in the TNI region   were found within each of the VER , OKS , and WA  regions, respectively, for a total of 11 genetically unique or geographically isolated den complexes. Among sampling regions, all values of pairwise genetic differentiation (θ) were significant (p < .001) and ranged from 0.059 to 0.321 (Table θ ranged from 0.0459 to 0.526 (p < .001)  Figure , while tr = −.038, p = .50)  or GF  regions; however, a significant pattern of isolation‐by‐distance was detected among dens in VER , OKS , and WA . Estimates of recent migration found significant gene flow from the OS den complex to WA‐S and from OS to WL. All other estimates of gene flow were low (m < 0.1) and not significant  and the lowest in the TNI‐C den complex (Ho = 0.290) (Table FIS = 0.017) (Table p = .994), while the least polymorphism was found in the TNI‐E den complex (p = .302) ; however, nucleotide diversity was significantly and negatively correlated with latitude   Table . A signi7) Table , while aAt the individual den level, estimates of heterozygosity tended to be higher than those calculated at the den complex level Table . Values rQG = .781), while MW was the least related (rQG = −.047) . At the broad scale, the five main geographic regions  in Canada represented three unique genetic clusters Figure . In partθ Table . Overallθ Table . This isθ Table  that corθ Table . This seθ Table , after wθ Table .At a finer scale, we found evidence for low and generally nonsignificant estimates of genetic migration, with no significant pattern of isolation‐by‐distance among all 11 den complexes that we defined across the distribution Table . While mCrotalus horridus; Clark, Estimates of relatedness within and among den complexes also indicated that the behavior of these snakes may influence levels of migration and population connectivity. Snakes sampled within a given den complex were found to be more related than snakes among complexes within a geographic region. At a finer level, snakes sampled at the same den were found, in some instances, to be more related to one another than snakes across dens within a given den complex. This pattern of relatedness suggests that snakes are denning near related individuals and may be mating locally with snakes from nearby dens, while exhibiting some form of kin recognition to avoid inbreeding. Kin discrimination behavior has been previously demonstrated in captively raised Timber Rattlesnakes (Sistrurus catenatus) in Ontario, Canada  as defined by Lowe and Allendorf , other factors may be contributing to the genetic differentiation detected between these den complexes. In addition to geographic distance between these sites, there are several high‐elevation mountain peaks within the Pasayten Wilderness in Washington (USA) that are situated between OS and WA‐N, which may be inhibiting snake movement. In order for gene flow to occur between these groups, snakes would have to successfully traverse over these mountains at elevations >2,000 m, well above the elevation at which this species has been shown to occur in studies across its range in North America  between the TNI‐W01 and TNI‐W02 dens, suggesting that underlying substructure may be responsible for the reduced heterozygosity of the den complex as a whole. As for the WA‐S den complex, the only significant (p < .05) pairwise differentiation detected was between the WA‐S02 and WA‐S03 dens (θ = 0.0813). Rather than genetic substructure influencing heterozygosity at the den complex level in WA‐S, relatedness among sampled individuals may be leading to a “Family Wahlund Effect”  and regional scales. The identification and subsequent preservation/restoration of key movement corridors and critical habitats can help to encourage connectivity, whether it facilitates individual movements or the gradual expansion of existing populations. However, this approach would be challenging in landscapes where land ownership is primarily private and where strong legislation does not regulate land use to favor species at risk conservation and recovery. Furthermore, increased public outreach and education will be necessary to minimize human–snake conflicts if these animals are to re‐establish in areas from which they have been extirpated due to development.Alsophis antiguae; Daltry et al., Vipera berus; Madsen, Ujvari, & Olsson, An alternative approach for managing populations separated by unsuitable habitat or more distinct barriers could be to artificially increase gene flow through translocation. For example, in the north and central OKS region near the City of Kelowna Figure , we wereOverall, the continued genetic monitoring of Western Rattlesnakes in this system can allow conservation managers to track changes in genetic diversity and population connectivity over time, as well as evaluate the outcomes of management efforts. The genotyping method employed in this study ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology ; Writing‐original draft (lead); Writing‐review & editing . Purnima Govindarajulu: Conceptualization (supporting); Funding acquisition ; Resources (supporting); Writing‐review & editing (supporting). Karl W. Larsen: Conceptualization (supporting); Data curation (supporting); Funding acquisition ; Resources (supporting); Supervision (supporting); Writing‐review & editing (supporting). Michael A. Russello: Conceptualization (lead); Formal analysis (supporting); Funding acquisition ; Investigation (supporting); Methodology (supporting); Project administration (lead); Resources ; Supervision (lead); Writing‐original draft (supporting); Writing‐review & editing .


PD-L1. In this review, we focus on the oncogenic role of STAT3 in human lymphoma and highlight potential therapeutic intervention by targeting JAK/STAT3 signaling.Signal transducer and activator of transcription 3 (STAT3) is an important and the most studied transcription factor in the Janus kinase (JAK)/STAT signaling pathway. STAT3 mediates the expression of various genes that play a critical role in many cellular and biological processes, such as cell proliferation, survival, differentiation, migration, angiogenesis, and inflammation. STAT3 and associated JAKs are activated and tightly regulated by a variety of cytokines and growth factors and their receptors in normal immune responses. However, abnormal expression of STAT3 leads to its constitutive activation, which promotes malignant transformation and tumor progression through oncogenic gene expression in numerous human cancers. Human lymphoma is a heterogeneous malignancy of T and B lymphocytes. Constitutive signaling by STAT3 is an oncogenic driver in several types of B-cell lymphoma and most of T-cell lymphomas. Aberrant STAT3 activation can also induce inappropriate expression of genes involved in tumor immune evasion such as  Signal transducer and activator of transcription 3 (STAT3) was discovered in 1993 by a biochemical study, in which STAT3 functions as an acute phase response factor and binds to its consensus DNA motif in nuclear extracts from the liver cell line HepG2 upon stimulation with the cytokine IL-6 . Since tSTAT3, a key transcription factor in the JAK/STAT signaling pathway, regulates expression of genes that control various cellular and biological processes, including cell proliferation, survival, differentiation, migration, angiogenesis, inflammation, and immune responses ,8. STAT3STAT3 and other STAT proteins contain six domains: a conserved amino-terminus, a coiled-coil domain, a DNA-binding domain (DBD), a linker domain, the SH2 domain, and a carboxy-terminal TAD  12]. Th. Th12]. STAT3 gene has two splice sites close to its 3′ end, which create four STAT3 splice variants, termed α, β, ΔS-α, and ΔS-β  DLBCL cells . ConstitSomatic STAT3 mutations are largely restricted to hematological disorders . STAT3 mDLBCL is a type of aggressive non-Hodgkin lymphoma, accounting for 30–40% of newly diagnosed cases . Gene exNFKB2, NFKBIA, NFKBIZ, CXCL13, and TRAF1), JAK/STAT pathway genes , antiapoptotic pathway genes , and PI3K/AKT/mTORC1 pathway genes    35,77].,77.NFKB2nd STAT2 ,79,80. Tnd STAT2 .EBI3), and genes involved in regulating cellular metabolism  [We also performed genome-wide analysis in both ABC DLBCL and normal activated B-cells and identified 1014 STAT3 targets that are specific for ABC DLBCL, including the immune checkpoint molecule PD-L1, the transcriptional activator MEF2B, the hematopoietic cell kinase (HCK), the Epstein–Barr virus-induced genes 3 ( SCARB1) . PD-L1 i SCARB1) . In DLBC SCARB1) . High le SCARB1) ,84. Seve SCARB1)  and BCR- SCARB1) .In conclusion, the JAK1/STAT3 signaling pathway plays an important role in the pathogenesis of DLBCL. Constitutively active STAT3 regulates expression of genes that are involved in multiple oncogenic processes in ABC DLBCL, including promoting cell survival, proliferation, and migration, reprogramming metabolism and facilitating tumor immune evasion. Targeting JAK1/STAT3 signaling represents a promising therapeutic strategy in ABC DLBCL.STAT3 is phosphorylated in 20–40% of mantle cell lymphoma (MCL), an aggressive, rare form of B-cell non-Hodgkin lymphoma ,88. ConsJAK2 in primary mediastinal B-cell lymphoma and HL [Classical Hodgkin lymphoma (HL), a common cancer in young adults, is characterized by the presence of Reed-Sternberg cells, which harbor various recurrent genetic lesions, including chromosome 9p24 amplification  and mutations in genes in the JAK–STAT and NF-κB signaling pathways  ,95,96,97a and HL . Targetia and HL .MCL-1, BCL2, and BCL-XL due to STAT3 activation promotes MM cell survival [Multiple myeloma (MM) is a cancer of plasma cells  in the bone marrow, accounting for 10% of all hematological malignances . Hallmarsurvival ,111. STAsurvival ,113,114.survival . Severalsurvival ,118,119.The JAK/STAT signaling pathway mediated by the common γc receptor-dependent cytokines IL-2, -4, -7, -9, -15, and -21 plays critical roles in T-cell immune responses . AbnormaAberrant expression and constitutive activation in several types of B-cell lymphoma and in almost all T-cell lymphomas render STAT3 as a promising potential therapeutic target. STAT3 inhibitors have been developed to interact with the STAT3 domains, with a major focus on the SH2 domain and the DBD . The STATo date, no STAT3 inhibitors have yet been approved to treat cancer by the Food and Drug Administration (FDA), but many early-phase clinical trials are ongoing . AZD9150As STAT3 is downstream to several cytokine and growth factor receptors and their associated JAKs, blocking these receptors by monoclonal antibodies or inhibiting JAKs by small molecular inhibitors represents a promising therapeutic option in both B- and T-cell malignancies. Ruxolitinib has been approved for the treatment of myelofibrosis  and polyBecause of the crosstalk between the JAK/STAT3 signaling pathway and many other oncogenic pathways  in B- and T-cell lymphoma ,121, comSGK1, a target gene of STAT3, by its inhibitor synergizes with AKT inhibitors in the killing of ABC DLBCL cells [Our recent studies suggest several potential combination therapeutic strategies in DLBCL ,70,77. IAlthough STAT3 expression is properly controlled in normal immune responses, constitutive activation of STAT3 is common in human lymphoma. Aberrant activation of STAT3 promotes tumor cell survival, proliferation, and migration through regulating gene expression in multiple oncogenic pathways. In addition, constitutive signaling by STAT3 plays an important role in evasion of immune surveillance. Targeting JAK/STAT3 signaling inhibits tumor growth and enhances antitumor immune responses. Therefore, STAT3 is a promising therapeutic target in lymphoma. Despite significant efforts, no STAT3 inhibitors have yet been approved in the clinic setting. Future perspectives include developing more potent and selective STAT3 inhibitors for cancer therapy.


Although the clinical benefits of medical genetic testing have been proven, there has been limited evidence on its economic impact in Thai setting. Thus, this study aimed to evaluate the economic impact of genetic testing services provided by the Center for Medical Genomics (CMG) in Thailand.Cost-benefit analysis was conducted from provider and societal perspectives. Cost and output data of genetic testing services provided by the CMG during 2014 to 2018 and published literature reviews were applied to estimate the costs and benefits. Monetary benefits related to genetic testing services were derived through human capital approach.The total operation cost was 126 million baht over five years with an average annual cost of 21 million baht per year. The net benefit, benefit-to-cost ratio, and return on investment were 5,477 million baht, 43 times, and 42 times, respectively. Productivity gain was the highest proportion (50.57%) of the total benefit.The provision of genetic testing services at the CMG gained much more benefits than the cost. This study highlighted a good value for money in the establishment of medical genomics settings in Thailand and other developing countries. Medical genetic testing is usually used to examine any changes of the chromosomes and genes, which could provide important data for doctors to determine a patient’s chance of developing severe adverse drug reactions (ADRs), the failure of treatment, or a genetic disorder . Such daIn 2005, the Thai Pharmacogenomics Project was initiated by the Faculty of Medicine, Ramathibodi Hospital with partial support from the Thailand Center of Excellence for Life Science (TCELS) to identify the genetics that could affect drug response, metabolism, ADR, and toxicity on certain key diseases , 8. LateBased on the viewpoint of the CMG as a provider, providing medical genetic testing services not only generated their income, but was also cost-saving in the country by avoiding sending examinations abroad which charged a higher fee. Moreover, according to the perspective of people who received genetic testing with positive results, the services can help prevent morbidities and mortalities due to early detection and effective intervention. This leads to increased economic benefits, in terms of cost avoidance from healthcare utilization in the future and income earnings or productive gain owing to longer survival. In line with the WHO’s recommendation, there is a significant need to generate economic evaluation evidence on medical genetic testing services to support investments in establishing medical genomics settings towards promoting a more cost-effective health care system , since sCost-benefit analysis (CBA) was used to estimate the costs and benefits after the provision of medical genetic testing services by the CMG in Thailand based on a societal perspective, which considers all costs and benefits incurred by everyone in the society according to the Thai Health Technology Assessment (HTA) guidelines . MoreoveDirect benefits included: (1) revenue generated from genetic testing services calculated by the total number of each service multiplied by its unit price obtained from the CMG; and, (2) cost-saving from genetic testing services at the CMG calculated by the difference between the costs of genetic testing provided by genetic testing agencies overseas, such as the United States, United Kingdom or Canada, and those provided by the CMG in Thailand. The prices of genetic testing services performed by agencies overseas were obtained through literature reviews and available price list from their agencies’ websites. These were adjusted to Thai baht using an exchange rate referred from the Bank of Thailand  . The S1 t is the expected income to be received in year t, r is the discount rate, and t is the number of years expected to live without death:Indirect benefits included cost avoidance and productivity gain due to genetic testing. Cost avoidance, which is the expected cost of treatment for diseases or conditions that could be avoided by receiving genetic testing services, was calculated by the number of people receiving genetic testing with positive results multiplied by the cost of treatment for diseases, which might occur in the future per case. We assumed that they received medical care and treatment throughout their lifetime period. Therefore, future costs incurred more than one year were adjusted using a discount rate of 3% based on the Thai HTA guidelines . MoreoveSince genetic testing can help prevent different types of diseases or conditions that are predicted to be avoided in the future, cost avoidance and productivity gain for each genetic testing were calculated except for whole exome and genome sequencing as they are still being researched and have not been disclosed to the public. When people living with HIV have poor response to antiretroviral (ARV) drugs or do not respond to them, doctors have two options: (1) use second-line ARV drugs; or, (2) test for HIV drug resistance to help select appropriate drugs. If HIV-resistant genes or positive test results were found, the doctor would have to change to second-line ARV drugs to prevent treatment failure and the spread of drug resistance in the community and country levels. If negative test results were found, the doctor could still prescribe first-line ARV drugs, which are cheaper as compared with the second-line drugs. Since the first-line ARV therapy is cheaper than the second-line ARV therapy for patients who do not have HIV-resistant genes, this can reduce the cost of treatment. Therefore, cost avoidance can be calculated by the difference in treatment costs for all patients changing to the second-line ARV drugs and those for patients with negative test results receiving the first-line drugs. To calculate the treatment costs, we assumed that the first-line ARV therapy was the combination of tenofovir (TDF) 300 mg, emtricitabine (FTC) 200 mg, and efavirenz (EFV) 600 mg every 24 hours. Meanwhile, the second-line ARV therapy consisted of TDF 300 mg every 24 hours, zidovudine (AZT) 300 mg every 12 hours, and lopinavir/ritonavir (LPV/r) 400/100 mg every 12 hours based on the guidelines for the diagnosis and treatment of people with HIV and AIDS  and expeScreening the embryos for chromosomal abnormalities or genetic diseases will increase the chances of successful pregnancies. The genetic diseases that can be avoided include beta-thalassemia, Gaucher's syndrome, DiGeorge-Velocardiofacial syndrome, polycystic kidney disease, neurofibromatosis type 1, hemophilia B, spinal muscular atrophy, Duchenne muscular dystrophy, Marfan syndrome, glutaric acidemia IIB, osteogenesis imperfecta types I–IV, Hallervorden-Spatz syndrome, Ellis-van Creveld syndrome, and multiple congenital anomalies-hypotonia-seizures syndrome 2, etc. Screening chromosomal abnormalities or genetic diseases before implantation in the uterus can prevent the recurrence of genetic diseases in a family whose first child has a genetic disease. It can also reduce the burden of medical expenses on the care for patients with genetic diseases and the psychological impact on mothers when deciding to terminate pregnancies, as well as increase the chances of having children in families who are ready to have children. We assumed that the treatment for genetic diseases would be provided to patients for the rest of their lives. Cost avoidance was then calculated by annual treatment cost multiplied by a patient’s survival for each genetic disease. However, there was no productivity gain from preimplantation genetic testing.Screening for BRCA1/2 genes to detect mutations in breast cancer and ovarian cancer can help inform the risk of family members who are considered to be at high risk for developing breast and ovarian cancer. Therefore, those at high risk would have the opportunity to enter the disease surveillance program, such as mammogram for genetic breast cancer, resulting in reducing the risk of death from the early detection of the disease and avoid the cost of treatment for breast and ovarian cancer at an advanced stage. Cost avoidance was calculated by the treatment cost of breast and ovarian cancer at an advanced stage which could be avoided in the future. We assumed that the treatment for patients with breast and ovarian cancer at an advanced stage would be given throughout their lifetime period. Productivity gain was calculated by the annual income of patients multiplied by the number of life months gained for those with BRCA1/2 testing compared to those without.Noninvasive Prenatal Testing (NIPT) is genetic testing of fetal abnormalities, such as Patau's syndrome, Edward's syndrome, and Down syndrome caused by more than one trisomy pair of 13, 18 and 21 chromosomes, respectively. NIPT screening has a direct benefit in preventing the occurrence of recurrent genetic diseases in families whose first child has a genetic disease. Besides, it can reduce the risk of miscarriage and amniotic fluid drilling, which is the traditional standard method, and reduce medical expenses on the care of patients with genetic diseases. Cost avoidance was the expected cost form the treatment of these genetic diseases, which might occur in the future. However, there was no productivity gain from NIPT.Hereditary cardiomyopathy panel testing using NGS technology will help provide the information on the family specific mutations and the risk of family members who are considered at high risk for developing heart disease due to genes associated with myocardial infarction (MI) and major arteries (cardiomyopathy). The most common type is dilated cardiomyopathy , which cThe expected cost of unnecessary second-line drugs that could be avoided in the future due to genetic testing for HIV drug resistance was approximately 621 million baht. Moreover, the detection of HIV drug resistance will help select appropriate ARV drugs for HIV patients who do not respond to the first-line regimen. This could prevent death or extend the expected life expectancy resulting in a productivity gain of 2,964 million baht. Furthermore, preimplantation genetic testing for aneuploidies resulted in the cost that can be avoided in the future from the treatment for genetic diseases caused by chromosomal abnormalities of 960 million baht. The top five diseases with cost avoidance from the treatment of genetic diseases were Gaucher's syndrome (500 million baht), DiGeorge-Velocardiofacial syndrome (186 million baht), hemophilia B (82 million baht), polycystic kidney disease (63 million baht), and osteogenesis imperfecta types I-IV (58 million baht).Screening for BRCA1/2 gene mutations in breast and ovarian cancer using NGS technology could avoid a treatment cost of 42 million baht for patients with advanced breast cancer (37 million baht) or advanced ovarian cancer (5 million baht). Moreover, the detection of BRCA1/2 gene mutations in breast and ovarian cancer with NGS technology could reduce the risk of death due to early detection of the diseases and extend the expected life expectancy leading to a total productivity gain (51 million baht) for breast (44 million baht) or ovarian (7 million baht) cancer patients. Providing Thai NIPT yielded a total cost avoidance of 77 million baht from the treatment of genetic diseases or chromosomal abnormalities, e.g., Down syndrome (48 million baht), and Patau’s syndrome and Edward’s syndrome (29 million baht). Hereditary cardiomyopathy panel testing by NGS could help reduce the risk of death due to cardiomyopathy or extend the expected life expectancy which could generate an additional income of 130,360 baht.Of all genetic testing services at the CMG, the total cost avoidance of genetic testing for preimplantation genetic testing for aneuploidies was the highest (960 million baht), followed by HIV drug resistance (621 million baht), and Thai NIPT (76 million baht). The total productivity gain of genetic testing for HIV drug resistance was the highest , followed by BRCA1/2 gene mutation screening for breast and ovarian cancers (51 million baht).The results of cost-benefit analysis of genetic testing services provided by the CMG during 2014 to 2018 showed that the total cost of CMG establishment was 103 million baht; whereas the total benefit was 5,994 million baht . TherefoOver the past decades, significant advances in the field of medical genetic testing have been observed in Thailand . HoweverThe results suggested that the provision of genetic testing services by the CMG would gain more benefit than cost in a Thai society. Furthermore, of all types of benefit from genetic testing services, productivity gain was the highest proportion, followed by cost avoidance, cost-saving, and revenues. In addition to generating revenues for the CMG, genetic testing services can help reduce costs and budgets for the Thai government and society since there is no need to use overseas services, which are more expensive. Moreover, genetic testing could result in treatment cost avoidance in the future and productivity gain in society due to its life prolongation for high-risk people. When the total benefit was classified by the type of genetic testing, genetic testing for HIV drug resistance yielded the highest benefit. It can be explained by the fact that the CMG provided the highest number of genetic testing services for HIV drug resistance since the cost of service is covered by all health insurance schemes in the country. This implies that all HIV patients can get access to the service . NeverthOn the other hand, based on the CMG’s perspective, which considered only the revenue as the benefit, it is noted that genetic testing services would gain little benefit over cost as they are under a governmental, university-based hospital where high mark-up prices are usually not observed. Our study suggested that the establishment of CMG as a medical genomics setting would be a good value for money and worth for investment in Thailand. Furthermore, these findings could be used to support policy decision-making on the establishment of a medical genomics setting to provide genetic testing services in other developing countries, where there is tendency to implement under the UCS but may have resource constraints on health care services.Nevertheless, the implementation of a medical genomics setting in the national health policy depends on many factors, such as socio-economic status, medical genetic infrastructure, international collaboration, law and regulation, cultural perspective and medical care ecology of the country , 6, 18. Interestingly, among the medical genomic applications for personalized medicine, genetic screening and testing for genetic diseases and prenatal screening were widely applied, as they were necessary to focus not only on survival, but also on health, growth and development of newborns . TherefoNotably, the limitations of this study were needed to be addressed. First, due to the lack of data available in Thailand, data on some parameters were obtained from published literatures in international countries. Specifically, to calculate cost-saving from the tests being sent abroad, their prices were obtained from literature reviews or price information available on the websites of genetic testing agencies in the United States, United Kingdom, or Canada as we could not find the price information disclosed from the agencies that provide genetic testing services in Asian countries. It should be highlighted that this could lead to an overestimated cost-saving, since most people usually did not pay the advertised price which could be negotiated and discounted. In addition, a price reduction under a service provider agreement could result in a decrease in benefit to the government. Therefore, future studies using the actual costs found from Asian agencies should be further investigated. Second, medical expenses and productivity gain were calculated throughout the patient's lifespan as we assumed that they would receive treatment and could earn income for the rest of their lives. Consequently, this might lead to an overestimation of the benefit. Moreover, providing NIPT to pregnant women could facilitate a productivity benefit by not having to spend time for caring a child with genetic disorders. Nevertheless, productivity gain was not accounted for, since it was beyond the scope of this study. Third, we did not take into account the costs resulting from other diseases, such as chronic non-communicable diseases, which might occur as patients would have longer life expectancy due to receiving genetic testing services. Therefore, this may lead to an overestimation of the benefit. Fourth, we included the total cost of the operation at the CMG as the cost for managing genetic testing services. However, this cost may be overestimated since the CMG also provides other services, such as training, arranging academic conferences, and networking with other national and international organizations. Fifth, we estimated the economic impact of the provision of genetic testing services at the CMG only, which may not be a representative of all genetic testing centers in Thailand. However, we expect that the benefits would be higher than the costs if we estimate for all governmental genetic testing settings. Lastly, apart from the economic impact of medical genetic services, other impacts based on the perspectives of patients, healthcare personnel and policy makers were not considered considering that the CMG has performed research and provided genetic knowledge and techniques through training and academic conferences for medical personnel, scholars, researchers in both domestic and international organizations. This would result in academic advancement, which could affect patient care, realize the importance of genetic research, and drive health policy. Thus, further research is warranted to explore these impacts as these can demonstrate the usefulness of the services, in terms of clinical outcomes and mental health of service recipients, healthcare decision-making, research, and knowledge dissemination.Our study suggested that the provision of genetic testing services at the CMG would gain more benefit than cost in Thai society. It highlighted that the establishment of the CMG would provide good value for money and would be worth for the investment in Thailand. These findings could be used to support policy decision-making on the establishment of medical genomics settings to provide medical genetic testing services not only in Thailand but also in other developing countries.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Teratoscincus keyserlingii, is one of nine members of the genus. This species is distributed in Iran, Afghanistan, and Pakistan, with a small isolated population in the United Arab Emirates (UAE), where it is classified nationally as Critically Endangered. Within its Arabian range, anthropogenic activity is directly linked to the species’ decline, with highly localised and severely fragmented populations. Here we describe the evolutionary history of Teratoscincus, by reconstructing its phylogenetic relationships and estimating its divergence times and ancestral biogeography. For conservation implications of T. keyserlingii we evaluate the genetic structure of the Arabian population using genomic data. This study supports the monophyly of most species and reveals considerable intraspecific variability in T. microlepis and T. keyserlingii, which necessitate broad systematic revisions. The UAE population of T. keyserlingii likely arrived from southern Iran during the Pleistocene and no internal structure was recovered within, implying a single population status. Regional conservation of T. keyserlingii requires improved land management and natural habitat restoration in the species’ present distribution, and expansion of current protected areas, or establishment of new areas with suitable habitat for the species, mostly in northern Abu Dhabi Emirate.Effective biodiversity conservation planning starts with genetic characterization within and among focal populations, in order to understand the likely impact of threats for ensuring the long-term viability of a species. The Wonder Gecko,  Arid and semi-arid regions cover almost 20% of the world’s land surface and are important for understanding global biodiversity patterns –4. BiodiThe United Arab Emirates (UAE) in the Arabian Peninsula, similar to other oil producing countries of the Middle East, is developing rapidly. As a result of this extensive development, human populations are increasing, threatening wildlife populations and natural habitats ,10. RapiOne of the most widely used indicators for assessing the health of ecosystems and their biodiversity is the conservation status of flora and fauna, which is also an important component of priority-setting exercises for species conservation. Reptiles  comprise a major element of the global biodiversity  and consTeratoscincus keyserlingii Strauch, 1863, in the UAE provides particular evidence for strong anthropogenic pressures : 13.9–25.1 Mya), most likely in South-West Asia (63% probability) with subsequent splits to north and north-west . A sister-taxa relationship between T. bedriagai and T. microlepis is recovered in the species tree and biogeographic analysis, but not supported in the concatenated mtDNA-nucDNA tree or at all recovered in the COI tree. Teratoscincus microlepis shows high intraspecific diversity and is divided into two distinct lineages, which split around 5.6 Mya (95% HPD: 2.6–9.6 Mya) and have a genetic distance of 8.3% in COI and 8.5% in ND2 . The remaining species comprise a well-supported clade diverging approximately 4 Mya (95% HPD: 2.4–6.2 Mya), yet relationships among them are not supported in all trees.The ML and BI phylogenetic trees of datasets , the timdatasets , and thedatasets  show a s% in ND2 . A sisteTeratoscincus keyserlingii is paraphyletic with respect to T. mesriensis, and shows great intraspecific diversity, with four (I–IV) and five (A–E) distinct lineages in the concatenated mtDNA-nucDNA and COI datasets, respectively. These lineages comprise specimens from Pakistan (lineage I/A), Iran , and Iran and the UAE (lineage IV/E). Genetic distance among these lineages of T. keyserlingii ranged between 3.4–9.3% in COI and 5.1–9.4% in ND2. Lineage IV/E of T. keyserlingii comprised all the specimens sampled from across the UAE. These specimens clustered together with one specimen from southern Iran collected near the city of Bandar Abbas on the shores of the Strait of Hormuz . The total number of pre-filtered loci was 34,487, which dropped to 33,123 after applying all quality control filters, and to 3,912 in the final dataset, after applying a conservative filter of “all loci present in all samples”. Most loci were found to be monomorphic , while 282 included one SNP, 56 had two SNPs, 28 had three SNPs and only four loci were found to include the maximum observed number of four polymorphisms. Consequently, the final concatenated ddRAD dataset was 136,177 bp long and included a total of 494 SNPs and 370 unlinked SNPs (uSNPs).For the 26 specimens of -4. Tajima’s D test of neutrality resulted in a value of -2.233. Basic F-statistics estimated from the uSNPs dataset showed a significant departure from the HW equilibrium in less than 5% of the loci (p<0.05), but an excess of homozygosity in most of them. Average FST and FIS values over all loci were 0.19 and -0.18, respectively. The individual inbreeding coefficient (FIC) ranged between 0.44–0.52.Percentages of polymorphic loci and polymorphic sites in our concatenated ddRAD dataset were approximately 0.095 and 0.004, respectively, and the estimated π value was 1.28x10ST values were similarly high (0.11–0.53).The optimal number of clusters obtained from DAPC was K = 1 . STRUCTUTeratoscincus accomplishes the task of combining broad taxonomic and geographic sampling for time-calibrated species tree inference to investigate the origin and evolution of the genus, together with including valuable information gained from genomic data for the population structure of T. keyserlingii in the UAE.The coupled phylogenetic-genomic approach carried out in this study for Teratoscincus species and identify natural groupings within taxa. Congruent phylogenetic structure and monophyletic groups of species were recovered in previous studies of Teratoscincus [T. microlepis and our non-monophyletic focal species, T. keyserlingii. Sister-species topology is mainly supported in the species tree and the biogeographic reconstruction, though with only weak or no support in the concatenated mtDNA-nucDNA and COI trees. This is especially evident in geographically proximate species, and at least in one sympatric location, of T. microlepis and T. bedriagai. Divergence time estimations using the concatenated mtDNA-nucDNA dataset were generally similar though older, as expected, from the species tree.Our emphasis on geographic range-wide diversification patterns enabled us to estimate relationships among almost all described oscincus ,45,66, soscincus –35,46,67Teratoscincus correspond to mountain ranges uplifting in Asia caused by the Indo-Eurasian and Arabian-Eurasian tectonic collisions  in the middle Pliocene [T. roborowskii to the Turpan Basin in China. The western population diverged into northern T. scincus and southern T. keyserlingii, likely as a result of the Kopet-Dagh mountain uplifting along the Iran-Turkmenistan border in the early Pliocene [Geographic limits of the major phylogenetic groupings of llisions . These cs  restricted to the Hamun-e Jaz Murian Basin in southeast Iran, where it is sympatric with T. keyserlingii, spanning across the southern provinces of Kerman and Sistan and Baluchistan. A second lineage (lineage I/A) is widespread in eastern and southern Iran, southern Afghanistan and western Pakistan. Mitochondrial genetic distance values between these lineages are greater than between the two recognised species T. roborowskii and T. przewalskii  [T. microlepis from our study exhibits characters belonging to the two species . Since T. sistanensis was described based on morphological data alone and the lack of additional distinguishing characters known, further studies are needed to investigate these taxa, considering the type localities of the two species.Taxonomic concerns are raised regarding ewalskii . These lcrolepis . These tanensis) . Each liT. keyserlingii, with respect to T. mesriensis, and the presence of five distinct lineages within it. The lineages recovered within T. keyserlingii, including that of T. mesriensis, diverged in the Pliocene and Pleistocene, and are geographically structured within Iran and neighbouring countries. For both T. microlepis and T. keyserlingii we hypothesise that the tectonic consequences of the northward motion of the Arabian plate and collision with the Eurasian landmass, resulted in intense geological instability and uplift of mountain ranges, as well as associated climatic changes prevailing in the Miocene to Pleistocene, most likely prompted these diversifications [A second case is the non-monophyly of ications . A positications . We therT. keyserlingii cluster together in lineage IV/E with one specimen from southern Iran, near the coastal city of Bandar Abbas. Low levels of genetic divergence between the coastal southern Iran sample and the Arabian population leads us to hypothesise that Arabia was colonised from southern Iran relatively recently, during the Pleistocene. Connections between Arabia and Iran have been recorded for several reptile taxa, including Acanthodactylus [Mesalina [Hemidactylus [Pristurus [Pseudocerastes persicus [T. keyserlingii. Several UAE specimens differentiate by a single mutation in the mitochondrial data indicating rapid recent expansion across the UAE.The UAE specimens of dactylus , MesalinMesalina ,84, Hemidactylus ,86, and risturus . As has persicus , colonizpersicus ; anotherT. keyserlingii population lacking any internal geographic structure, hence representing a single population. In fact, this pattern is in agreement with the genetic variation measures (percentage of polymorphic loci and polymorphic sites) that highlight the extremely low genetic diversity within the studied system. Recently, a study that also used a ddRAD approach estimated within-population π values for more than 80 lizard populations ranging from 2.14x10-4 to 3.99x10-3 [T. keyserlingii in the UAE is much lower than the lowest estimated value in the aforementioned study. Accordingly, Tajima D test of neutrality returned a negative value of -2.233. Negative values are indicative of recent selective sweeps or, more possibly in our case, population expansions after a recent bottleneck or a founder event, which is congruent with our divergence time estimation of Arabian colonization during the late Pleistocene.Genetic structure analysis with DAPC shows the UAE .99x10-3 . Our estT. keyserlingii in the UAE, while the latter detected three sub-populations. The DAPC analysis does not require a model of admixture and describes the relationship between the population clusters, optimizing the variance between groups while minimizing variance within groups. In this sense, it is expected to perform better when organisms exhibit continuous spatial population structure [Results from DAPC and STRUCTURE seem contradictory at first glance: the former identified no genetic structure within tructure . On the tructure .T. keyserlingii in the UAE. Firstly, where several values of K give similar estimates of LnP(X/K), as here for K = 1 and K = 3, the smallest value is often “correct” [IC) per individual in our sample showed high values of FIC>0.45 , but, butT. ks . This genetic inference therefore has significant implications for conservation management.Biodiversity conservation starts with knowledge of genetic diversity within and among populations. A high level of genetic diversity in a population is thought to increase the probability of surviving a disastrous event or extinction . Thus, iT. keyserlingii distribution included within these protected areas, showing that it did not reach the strict Aichi biodiversity conservation target of 17% and only slightly surpassed the less restrictive 12% target. Their study indicated an immediate problem regarding habitat loss of T. keyserlingii and its low presence in protected areas. Currently, three protected areas  incorporate areas of T. keyserlingii distribution in the UAE, three include past localities of the species , and the rest are either too small and scattered, or located beyond the natural habitat of the species  [T. keyserlingii may be extended to southern Dubai and northern Abu Dhabi Emirates. As protection of the environment is being recognised as an essential component of the natural development policies in the UAE, one achievable proposition is expansion of current protected areas , or establishment of new areas with suitable habitats for T. keyserlingii . These spatial zones are of critical importance to the natural preservation of the species in the UAE. In this regard, improved land management and natural habitat restoration in disturbed areas are also plausible efforts to improve the quality, connectivity, and permeability for future dispersal of different species, as well as preserving the biological diversity and stability of the ecosystem [The UAE currently includes 42 protected areas, covering 16.6% of the terrestrial area of the country , albeit  species . Unfortu. (2019)  indicatecosystem .T. keyserlingii–in Abu Dhabi and Sharjah Emirates. The goal of many ex-situ conservation agendas, such as captive breeding or re-introduction programs, is retention of a high percentage of genetic diversity from wild populations, until its potential re-introduction to natural habitat to ensure the long-term viability of the founder populations [x-situ conservation programs have to be carefully integrated within in-situ programs, so that captive populations can constitute an insurance against the loss of natural populations and a source for re-introduction projects [T. keyserlingii, captive breeding and other ex-situ conservation programs should not be considered alone as efficient conservation measures without additional in-situ measures [In the UAE there are currently two breeding centres for endangered Arabian wildlife, including ulations –103 Click here for additional data file.S2 FigT. keyserlingii from the UAE used for the ddRAD analyses. The Bayesian values do not improve as the clusters increase from 1 to 10 implying K = 1.Analysis based on 26 individuals of (DOCX)Click here for additional data file.S3 Fig(A) The optimal clustering was identified using Evanno’s method (ΔK plotted against K) at K = 3. (B) The division or runs is also given .(DOCX)Click here for additional data file.S1 TableTeratoscincus keyserlingii specimens used for the ddRADseq analyses (n = 26).Localities of specimens from the UAE are indicated next to the country’s name and in (DOCX)Click here for additional data file.S2 TableInformation including the primers used, with their orientation, sequences, references, and PCR conditions.(DOCX)Click here for additional data file.S3 TableCOI  and ND2 , and within each taxon or lineage .Values derived from the mitochondrial gene fragments of (DOCX)Click here for additional data file.S1 Appendix(ZIP)Click here for additional data file.S2 Appendix(RAR)Click here for additional data file.


Capra ibex) population of the Bargy massif, reservoir of a virulent outbreak of brucellosis. We analysed the polymorphism and associations with disease resistance of the MHC Class II Drb gene and several non-MHC genes  involved in the innate immune response to Brucella in domestic ungulates. We found a very low neutral genetic diversity and a unique MHC Drb haplotype in this population founded few decades ago from a small number of individuals. By contrast, other immunity-related genes have maintained polymorphism and some showed significant associations with the brucellosis infection status hence suggesting a predominant role of pathogen-mediated selection in their recent evolutionary trajectory. Our results highlight the need to monitor immunogenetic variation in wildlife epidemiological studies and to look beyond the MHC.While it is now broadly accepted that inter-individual variation in the outcomes of host–pathogen interactions is at least partially genetically controlled, host immunogenetic characteristics are rarely investigated in wildlife epidemiological studies. Furthermore, most immunogenetic studies in the wild focused solely on the major histocompatibility complex (MHC) diversity despite it accounts for only a fraction of the genetic variation in pathogen resistance. Here, we investigated immunogenetic diversity of the Alpine ibex ( Often neglected, inter-individual variation in host susceptibility plays a key role in this dynamics since, depending on their biology , their spatial or social behaviours and/or their genetic characteristics, some individuals contribute far more than others to disease spread2. Therefore, identifying individuals and their characteristics that are most responsible for disease transmission is an important step for improving epidemiological surveillance and management of wildlife diseases3.The epidemiological dynamics of infectious diseases results from a complex interplay between the pathogen, the host and the environment4. Pathogen-mediated selective pressures shape the genetic components of host immunity and give rise to inter-individual variation in resistance to infectious diseases5. This immunogenetic variation is particularly well documented in humans and domestic animals but much less in wild animals. Furthermore, most studies focused solely on the adaptive branch of the immune system, particularly on the major histocompatibility complex (MHC)7. Although there is no doubt that MHC plays a key role in individual susceptibility to many diseases8, it accounts for only a fraction of the genetic variation in pathogen resistance9. Before the adaptive immunity intervenes in the host response, other genes involved in the innate immunity such as the Toll-like receptors (TLRs) genes are major players in the host frontline defense against a wide range of microparasites including vectors of zoonotic diseases such as Coxiella brunetii (Q fever) or Borrelia sp. (Lyme disease)12.Host genetic and immunogenetic characteristics are rarely investigated in wildlife epidemiological studies despite evidence that inter-individual variation in the outcomes of host–pathogen interactions is at least partially genetically controlled13. Populations that went through severe bottlenecks may show low levels of genetic diversity at the MHC  or TLR loci . However pathogen-mediated balancing selection may in some cases buffer the effect of drift to maintain polymorphism17. Several not-mutually exclusive mechanisms may act in synergy to maintain immunity-related diversity, the three more widespread being: the “negative frequency-dependence”, the “heterozygous advantage” and the “fluctuating selection”. The “negative frequency-dependent selection”  hypothesis suggests that the fitness-advantage of alleles is inversely proportional to their frequency in the population as a result of the host-pathogen18 or prey-predator co-evolutionary dynamics19. The “heterozygote advantage” hypothesis predicts a correlation between individual heterozygosity and fitness or disease susceptibility21. The “fluctuating selection” hypothesis23 suggest that population genetic diversity is maintained when spatially and/or temporally varying directional selection occurs24.Genetic variation at the MHC and other immunity-related genes encoding pathogen recognition receptors is shaped by both neutral  and selective processes. The relative importance of these evolutionary forces depends on both the population demographic history that influences the strength of the genetic drift, and the (multi)pathogen challenge that determines the type and intensity of selection processesCapra ibex) population from the Bargy area in France. This ibex population from the northern French Alps has undergone a major outbreak of brucellosis (Brucella melitensis). Until very recently, European wild ungulates were seen as dead-end hosts for this pathogen25 and all previous outbreaks of brucellosis in wild ruminants spontaneously faded out28. However, the situation in Bargy is alarming since the disease has affected an unprecedented high proportion of the population . Brucellosis persisted at high levels in the following years despite intense culling of animals which led to the removal of 40% of the population . Beyond the conservation and ethical issues, this outbreak has raised public health and economic concerns since, for the first time, cattle and humans were infected by the wildlife reservoir30. In this context, it was critical to identify the drivers of pathogen persistence in this population and individual characteristics favoring its spread. Previous works showed that host-infectiousness vary significantly across age and sex and among socio-spatial units within the Bargy massif29. However, genetic factors of susceptibility to brucellosis in Alpine ibex have never been investigated while several associations with innate gene polymorphism have been discovered in domestic ungulates32.Here, we investigated the neutral and immunity-related genetic diversity of the Alpine ibex  involved in the innate immune response to Brucella sp. in domestic ungulates36. The second objective was to investigate associations between individual brucellosis infection status and genetic/immunogenetic profile. We first assessed the role of multi-locus heterozygosity at neutral microsatellites as a proxy of inbreeding (heterozygosity-fitness correlation). We then looked at potential associations with the heterozygosity status (heterozygous advantage hypothesis) and/or specific haplotype of immunity-related genes. In both domestic and wild ungulates, brucellosis is well-known to cause abortion and reproductive failures38 with known impacts on individual fitness. Therefore, these associations may reflect pathogen-mediated balancing or directional selective pressure on immunity-related gene polymorphism.The first aim of this study was to assess the neutral and immunity-related genetic variation of the Bargy population. We screened the neutral microsatellite diversity to test the hypothesis that the intrinsic susceptibility to brucellosis of ibex at Bargy results from a particular low genetic diversity in comparison with other reintroduced ibex populations. We also evaluated the polymorphism of the commonly used E was 0.43 ± 0.17 (mean ± standard error) and mean NA was 3.12 over all microsatellite loci. Neutral variation did not vary significantly before  and after  the culling operations that occurred in 2013–2015 .The tests for Hardy-Weinberg equilibrium, after correction for multiple testing, revealed that SR-CRSP07 STR locus showed a significant excess of homozygotes. This marker was thus removed from further analysis. HMhc-Drb gene on 144 individuals revealed a unique haplotype in Bargy corresponding to CaIb-DRB*1 haplotype, previously isolated by Schalschl et al.39 and Grossen et al.40. The Sanger sequencing of the three Tlr genes generated an average of 2,169 bp of sequence covering almost the entire coding region of the genes (92% on average). Tlr genes exhibited seven SNPS among which five were non-synonymous. We isolated 1 SNPs for Tlr1 and revealed two functional haplotypes (i.e. encoding different amino acid sequences) including a rare haplotype (5%) observed in only 14 individuals. The Tlr2 gene exhibited three SNPs (two non-synonymous) and showed three functional haplotypes with a common haplotype (Tlr2a = 72%) and two haplotypes with low frequencies (Tlr2b = 15% and Tlr2c = 13%). Lastly, we found three SNPs (two non-synonymous) in Tlr4 gene and identified four functional haplotypes among which three haplotypes showed similar moderate frequencies ranging between 25% and 35% while the fourth one was rare (9%). Expected heterozygosity (HE) (given differences in haplotype frequencies) varied greatly among Tlrs ranging from 0.09 for Tlr1 to 0.71 for Tlr4  with frequencies of 75% and 25% respectively (HE = 0.37).The Illumina sequencing of the exon 2 of the SLC11A1 genotype and brucellosis status  had a lower probability to be seropositive than heterozygous individuals . Tlr1 heterozygous status was also associated with ibex brucellosis status  or of the heterozygous/homozygous status (heterozygote advantage) . The probability to be seropositive was lower in males than in females and reached a maximum in the core area of the massif   Table .Figure 140. By contrast, other immunity-related genes involved in the innate immunity such as Tlr genes  and Slc11A1 have maintained polymorphism despite serial founder events occurring throughout the Alpine ibex reintroduction history. We revealed significant associations between the polymorphism of some of these genes (Tlr1 and Slc11A1) and Brucella infection status, which may suggest a role of pathogen-mediated selection in their recent evolution.The aim of this study was to assess the neutral and immunity-related genetic diversity of the Alpine ibex population of the Bargy massif, wildlife reservoir of a persistent and virulent outbreak of brucellosis. As expected, this population established a few decades ago from a very limited number of founders (<15 ibex) shows very low genetic variation at neutral microsatellite loci and exhibits a single MHC Class II haplotype similarly to most ibex populations of the Western AlpsE) of ~40% and an average number of alleles (MNA) per microsatellite loci <4, the ibex population of Bargy shows particularly low levels of microsatellite genetic variation, compared to values reported in goat breeds  and in other wild European ungulate populations . However, these differences may arise because the microsatellite loci used in the different species underwent different ascertainment bias. Our results are consistent with the levels previously observed in other Alpine ibex populations reintroduced in Switzerland during the XXth century42 and in particular, for the Mont Pleureur population (HE = 0.42) from which the Bargy founders came from. Low genetic diversity might reduce individual fitness and population demographic performance due to reduced evolutionary potential44. For example, in the Gran Paradiso ibex population, there is a direct relationship between multi-locus microsatellite heterozygosity, male body mass and gastrointestinal nematode infestation  (i.e. Heterozygosity-fitness correlation)35. In our case, we did not observe any relationship between the genome-wide heterozygosity (MLH) of ibex and their Brucella infection status. This is in agreement with the recent study of Brambilla et al.45 that did not find any effect of neutral heterozygosity on infectious keratoconjunctivitis symptoms, another bacterial disease recurrently affecting Alpine ibex populations. Similar low levels of neutral genetic diversity were observed in other brucellosis-infected ibex populations (Grand Paradiso) in which the prevalence level has remained low and infection vanished spontaneously26. Therefore, contrary to our hypothesis, the low level of neutral diversity at Bargy is in itself not sufficient to explain its potential higher sensitivity to brucellosis compared to other ibex populations. However, a growing number of studies illustrated the poor predictive power of microsatellite loci to evaluate functional diversity and showed the importance of accounting for immunogenetic variation to get a better understanding of the population’s adaptive response potential to pathogen-mediated selection47.With an expected heterozygosity  play a fundamental role in vertebrate innate immune defense against intracellular and extracellular micropathogens including viruses, bacteria, protozoa or fungi50. In particular, these genes are involved in the recognition and immune response to pathogenic bacteria that recurrently cause outbreaks such as Mycoplasma sp. (causing pneumonia or infectious kerotoconjunctivitis infections)52 or Brucella abortus54. Contrary to our expectation and despite serial founder events throughout the species reintroduction history, the three studied Tlr genes and the SlC11A1 have all maintained several haplotypes while the MHC Class II DRB (second exon) is monomorphic in Bargy. There are very few studies that investigate Tlr polymorphism in ungulates populations: levels of Tlr haplotype diversity in Bargy ibex  appears similar to those reported for the same genes in roe deer populations (NH = 3–5)17, domestic goat (NH = 1–4)55 or cattle (NH = 2–5)56. Tlr4 showed four functional haplotypes (i.e. encoding different amino-acid sequences) including three alleles with moderate to high frequencies (>25%). This balanced polymorphism which contrasts with the very low neutral and MHC diversity in Bargy, suggests that balancing pathogen-mediated selection may have favoured the maintenance of genetic variation as showed in other free-ranging animal species58. Furthermore, it is worth noting that the presence of balanced polymorphism does not mean that the gene is involved in brucellosis resistance, which can be demonstrated only by studying associations between immunogenetic patterns and pathogen prevalence59.Our study is the first to our knowledge to reveal immunity-related polymorphism in non-MHC genes in Alpine ibex. Previous work on ibex focused exclusively on MHC Class II geneset al.35, we did not find evidence of correlation between multi-locus heterozygosity at neutral microsatellites and brucellosis infection status . This means that a higher individual multi-locus heterozygosity (a proxy of the level of inbreeding) does not confer a resistance advantage regarding Brucella infection. In contrast, we found associations between single immunity-related genes  and ibex infection status hence supporting the “local effect” hypothesis in agreement with Brambilla et al.45. These authors observed a lower susceptibility of MHC Drb heterozygous to infectious keratoconjunctivitis (heterozygote advantage hypothesis) while we revealed here an effect of specific alleles. Ibex carrying two copies of the less frequent allele of the Slc11A1 gene were less likely to develop the Brucella infection, which suggests that this “resistance allele” is recessive. Associations between a specific Slc11A1 allele and Brucella infection has been already reported in domestic goat36 and water buffalo31. Moreover, in an in-vitro experiment on water buffalo’s monocytes infected by several Brucella species including B. melitensis, Borriello et al.60 demonstrated that some Slc11A1 genetic variants confer a higher Slc11A1 mRNA expression and a higher ability in controlling the intracellular replication of the Brucella. The “resistant allele” (A330) showed similar frequency in the Bargy and Aravis populations (25% and 21% respectively) but is much more frequent in other Alpine populations (e.g. 54% in Belledonne massif (northern French Alps), 40% in Grand Paradiso National Park – results not shown). Further comparison with other recently reintroduced ibex populations should be performed to investigate whether the particularly low frequency of this resistant variant in Bargy may have favored the emergence of this brucellosis outbreak together with other, non-genetic factors such as spatial ibex distribution and abundance.In contrast to Brambilla Tlr1 (96%) conferred a resistance advantage against Brucella infection. However, this resistance advantage is relative: although 92% of ibex in Bargy are homozygous for this allele, 37% are still seropositive. Therefore, individuals that carry out this allele have a lower probability to get infected after a contact with the bacteria, but do not show a complete resistance. Such relationship between Tlr1 genotype and Brucella infection has been already evidence in Cattle by Prakash et al.32. Tlr1 interact with Brucella sp. by recognizing specific pathogen-associated molecular patterns. Individuals carrying the rare allele (Tlr1b) may have a decreased ability to recognize Brucella and thus to trigger an adequate immune response. Further work is needed to clarify the functional role of these immunity-related genes against brucellosis in Alpine ibex and in particular how these polymorphisms affects the detection and the replication of the bacteria . Particularly, in vitro studies of ibex white blood cells and their response to Brucella36 would help to confirm a variation linked to immune genes and to identity cellular pathways to resistance.Our results also suggest that the most frequent haplotype of Slc11A1 and Tlr1) and brucellosis infection status in Alpine ibex. However, this does not in itself provide evidence for directional pathogen-mediated selection because we did not actually demonstrate that these alleles confer a selective advantage. Seropositive ibex were euthanized, so there is no data available on the impact of B. melitensis infection on survival and reproduction rates in this population. However, brucellosis is well-known to cause abortion and reproductive failures in other domestic and wild ungulates38. So we can reasonably hypothesize that B. melitensis infection actually affects the fitness of ibex in Bargy and may ultimately lead to selection on these genes.In agreement with our hypothesis and the literature on domestic ungulates, we showed significant associations between immunity-related genes  involve micropathogens  and MHC Class II often plays a minor role in the immune response against these pathogens49. Our study highlights the need to look beyond MHC class II and to explore the diversity of other candidate immunity-related genes5, in particular MHC class I, cytokine and Toll-like receptors genes that are centrally involved in the innate immune response against micro pathogens in humans and domestic species.Genetic monitoring is widely recognized as a valuable tool for the management and conservation of populations65 is recommended to prevent inbreeding depression and increase both genome-wide45 and immunity-related diversity47. Further, these results raise the question of the interplay between sanitary management and population genetic structure. Here, the test-and-cull management procedure was expected to affect the genetic structure of the population, by selectively eliminating infected individuals carrying specific alleles. In the same line, some authors suggested that artificial selection of specific Slc11A1 or Tlr genotypes in breeding programs may help to increase population natural resistance and limit spread of brucellosis in domestic ungulates32. However, it is important to keep in mind that natural populations are exposed to multi-pathogen pressures (e.g. brucellosis and Q fever in the Bargy population) and that most immunity-related genes are involved in the immune response against several pathogens. Therefore, artificially selecting a specific genotype to increase population resistance to a pathogen may lead to the loss of key haplotypes in the host-control of other diseases, particularly in species with very low genetic diversity. Incorporating data on immunogenetic variation in epidemiological models should help to predict how variation in allele frequency may affect the spread dynamics of the disease, and how disease management may affect genetic structure of the population.In a conservation framework, our results call for immediate management actions to increase the neutral and immunity-related diversity of the Bargy ibex population, whose census size had been halved  over the monitoring period. Translocations of unrelated individuals from the Gran Paradiso source population or from other genetic units of Alpine ibex66. Since the discovery of the outbreak in 2012, the population has been continuously monitored by Capture-Mark-Recapture (CMR)29. Using GPS data on marked individuals, Marchand et al.29 demonstrated that females are structured in five distinct socio-spatial units . Animals were captured by dart-gun xylazine-ketamine anesthesia  following protocols described in Lambert et al.67. During captures, test-and-cull was implemented on the basis of serological tests  as part of management measures decided by the French Authorities . Seronegative individuals were marked and released while seropositive ones were euthanized. All captured animals were handled by professionals from the French Game and Wildlife management agency  according to the ethical conditions detailed in the specific accreditations delivered by the Préfecture de Paris , by the French Minister of Ecology, Sustainable Development and Energy  and by the Préfecture de la Haute-Savoie  in accordance with the French environmental code (Art. R421-15 to 421-31 and R422-92 to 422-94-1). Euthanasia was performed by veterinarians in accordance with the requirements of these accrediting authorities, and slaughtering operations were performed in accordance with accreditations delivered by the Préfecture de la Haute-Savoie . We hence confirm that we treated animals in accordance with relevant guidelines and regulations and that our protocols were approved by the National Committee for Nature Protection (CNPN in French), a group of independent experts elicited by the ministry of the environment and in charge of evaluating capture/destruction of the protected species.Alpine ibex were reintroduced in the Bargy Massif (France) during three release events in 1974 and 1976 comprising a total of 14 released individuals  translocated from the Mont Pleureur population in Switzerland29.In addition to CMR monitoring, French Authorities implemented selective culling of animals with observed clinical signs and mass indiscriminate slaughter of individuals of five years and more: culling operations occurred between autumn 2013 and spring 2014 (N = 256 killed) and then in autumn 2015 (N = 70). The population size dropped from 567 individuals (95% CI: [487–660]) in 2013 to 310 [275–352] in 2014 and 277 [220–351] in 201568. In particular, since 2014, the rapid Laminar Flow Immune-chromatographic Assay (LFIA)  was performed in the field. This test had been validated on ibex samples by the EU/OIE/FAO and National reference laboratory . Further details on the validation method and the congruence of the FLIA with reference tests  were provided in Anses (2014). Seropositive ibex were considered as infected by B. melitensis, but not necessarily infectious at the time of capture67.Brucellosis serologic status of individuals was evaluated using a range of serological tests42 . The same individuals were also typed at a microsatellite located at the 3′UTR of the Solute Carrier Family 11 member A1 (SLC11A1) gene using the following primers: Fw- TCTGGACCTGTCTCATCACC and Rv- ACTCCCTCTCCATCTTGCTG70. The SLC11A1 gene, formerly known as natural resistance-associated macrophage protein 1 (NRAMP1) gene, is involved in the innate resistance to intra-cellular pathogens71. Polymorphism in microsatellites of the 3′ UTR of the gene were associated with resistance to Brucella infection in water buffalo Bubalus bubalis36 and in the domestic goat31, a species closely related to the Alpine ibex. In particular, some SLC11A1 genotypes have a higher ability of controlling the intracellular replication of Brucella in vitro60.DNA was extracted from blood using the DNeasy Blood and Tissue kit (QIAGEN). All individuals were genotyped at 25 polymorphic putatively neutral microsatellitesMhc-Drb and Tlr genes for a subset of the individuals (N = 146) sampled between 2012 and 2014. The second exon of the Mhc-Drb class II gene encoding the ligand-binding domain of the protein was amplified and sequenced using Illumina MiSeq system following the procedure detailed in Quéméré et al.17. Haplotypes and individual genotypes were identified using the SESAME barcode software72. Tlr genes  were genotyped using the two-step procedure described in Quéméré et al.17. A pilot study on 30 individuals was first performed to identify polymorphic sites (SNPs). We screened almost the entire coding region of the three Tlr genes (92% in average) including the leucine-rich extracellular region of receptors involved in antigen-binding. Details on primer sequences and accession numbers are provided in Table 73.We also genotyped E) and the number of alleles/haplotypes (NA) as implemented in GENETIX software v4.05.274. For each locus, a test for Hardy–Weinberg proportions was performed based on 1000 permutations. To allow comparison with other ibex populations42 and between the two monitoring periods (before and after culling operations), we used FSTAT version 2.9.375 to calculate the allelic richness (sNA), a standardized measure of number of alleles corrected for differences in samples size. Lastly, standardized multi-locus heterozygosity at neutral microsatellites (MLH) was calculated for each individual as the ratio of its heterozygosity to the mean heterozygosity of the population76. Our set of microsatellite loci showed significant identity disequilibrium , (e.g. a measure of covariance in heterozygosity), a condition for detecting heterozygosity-fitness correlations. Both MLH and g2 were calculated using the R package inbreedR77. To assess whether management operations  had led to a decrease of the genetic diversity, we performed a comparison (using Student’s t-tests) of the allelic richness (sNA) and expected heterozygosity (HE) of ibex sampled before (between autumn 2012 and autumn 2013) and after (years 2016–2017) the slaughtering operations that occurred between autumn 2013 and autumn 2015.The genetic diversity of both microsatellites and immunity-related genes was evaluated by calculating the expected heterozygosity  of the binomial family (using a logit link function). The serological status of each individual was coded as 1  or 0 .vs homozygote) and the “haplotype advantage” hypothesis by considering associations between the infection status and the number of copies  of Tlr or Slc11A1 haplotypes with frequency >10%. Concerning non-genetic factors, following Marchand et al.29, we included the effects of ibex sex as a categorical variable and age as a continuous variable in years (linear and quadratic terms). We also fitted a “year” effect as a categorical variable to test for varying epidemiological situations across years and a “location” effect because seroprevalence has been shown to vary among socio-spatial units29.We fitted models testing for the effect of genetic effects, while taking into account other biological and environmental factors that possibly affect pathogen exposure. We tested the “heterozygosity fitness-correlation” hypothesis  by fitting individual standardized multi-locus microsatellite heterozygosity (MLH) as a fixed effect. For each immunity-related gene, we tested the “heterozygote advantage hypothesis” by fitting a binary fixed effect . We conducted an initial exploration of our data to ascertain their distribution and spread, to identify outliers and examine relationships between variables81. We used variance inflation factors (VIF) to assess which explanatory variables were collinear, and should be retained in the analyses 81. To minimize multicollinearity, the effects of the most frequent haplotype of a gene and its heterozygosity status were evaluated in separate models.We used a multimodel inference approaches to establish which explanatory variables had an effect, averaged over plausible modelset al.79 and using the “zero method” (i.e. a zero is substituted into models where the parameter is absent). Analyses were performed using the package lme482, MUMIN v1.7.783 and AICcmodavg v1.2584 in R version 3.3.3 .Model averaged parameter estimates are provided with their unconditional standard errors (SE) and 95% confidence intervals, after averaging model with ΔAIC < 7 (relative to the best model) as recommended by Burnham Table S1— S5Table S6.Table S7.


Genetic diversity defines the evolutionary potential of a species, yet mounting evidence suggests that epigenetic diversity could also contribute to adaptation. Elucidating the complex interplay between genetic and epigenetic variation in wild populations remains a challenge for evolutionary biologists, and the intriguing possibility that epigenetic diversity could compensate for the loss of genetic diversity is one aspect that remains basically unexplored in wild plants. This hypothesis is addressed in this paper by comparing the extent and patterns of genetic and epigenetic diversity of phylogenetically closely related but ecologically disparate species. Seven pairs of congeneric species from Cazorla mountains in south-eastern Spain were studied, each pair consisting of one endemic, restricted-range species associated to stressful environments, and one widespread species occupying more favourable habitats. The prediction was tested that endemic species should have lower genetic diversity due to population fragmentation, and higher epigenetic diversity induced by environmental stress, than their widespread congeners. Genetic (DNA sequence variants) and epigenetic (DNA cytosine methylation variants) diversities and their possible co-variation were assessed in three populations of each focal species using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MSAP). All species and populations exhibited moderate to high levels of genetic polymorphism irrespective of their ecological characteristics. Epigenetic diversity was greater than genetic diversity in all cases. Only in endemic species were the two variables positively related, but the difference between epigenetic and genetic diversity was greater at populations with low genetic polymorphism. Results revealed that the relationship between genetic and epigenetic diversity can be more complex than envisaged by the simple hypothesis addressed in this study, and highlight the need of additional research on the actual role of epigenetic variation as a source of phenotypic diversity before a realistic understanding of the evolutionary relevance of epigenetic phenomena in plant adaptation can be achieved. Genetic diversity is generally considered the chief determinant of evolutionary change, but epigenetic diversity is now recognized as another layer of heritable variation with potential adaptive consequences. Epigenetic diversity could sometimes  alleviate the loss of genetic diversity and provide an ‘evolutionary backup’ mechanism for wild plants. This study compares genetic and epigenetic diversity in seven congeneric species pairs with restricted and widespread distributions in south-eastern Spain. Results suggest that higher epigenetic diversity could alleviate the loss of genetic diversity in some populations of endemic plants, but also other plant features can be essential to understand the relationship between genetic and epigenetic diversities. The genetic diversity of species and populations has multiple implications for their ecology, evolution and survival. For example, reduced genetic diversity arising from inbreeding, fragmentation, bottlenecks or founder effects have been long known to pose threats on long-term survival of species and populations . An incrArabidopsis thaliana it has been recently shown that epigenetic variation is under selection and contributes to rapid phenotypic adaptive responses in absence of consistent genetic changes , one of several mountain units comprising the Baetic Ranges complex in south-eastern Iberian Peninsula . This NaAnthyllis ramburii and Anthyllis vulneraria  and the manufacturer’s protocol. Genetic and epigenetic analyses were conducted on the same DNA extracts.MseI + 3/PstI + 2 primer pairs that resolved more reproducible and easier to score bands were selected on each genus and AFLP analyses were conducted using them (seeGenetic fingerprints were obtained for each plant using amplified fragment length polymorphism (AFLP) markers . The AFLHpaII and MspI in parallel runs in combination with another restriction enzyme (here the frequent cutter MseI is used). HpaII and MspI are two isoschizomers that recognize and cleave the same tetranucleotide sequence 5′-CCGG, but differ in their sensitivity to the methylation state of cytosine. HpaII can cut at sites that are either non-methylated or contain one methylated external cytosine, whereas MspI cuts non-methylated sites and those with one or two methylated internal cytosine  technique .Amplified products from both AFLP and MSAP protocols were analysed on an ABI PRISM 3130xl DNA sequencer, and fingerprint profiles were scored manually by visualizing electrophoregrams with GeneMapper 5.0 software. All primer combinations used with each species were merged in a single binary data table to generate finally one genetic (AFLP) and two epigenetic (MSAP) raw data matrices (see below). We assessed the repeatability of banding patterns for each species by repeating the entire AFLP/MSAP protocol in a number of randomly selected samples. For each species an average of 11.2 % (AFLP) and 25.8 % (MSAP) of the samples were used as replicates  fragments present in both profiles (1/1), indicating an unmethylated state; (2) fragments present only in MseI/HpaII profiles (1/0), indicating hemi-methylated CHG sites; (3) fragments present only for MseI/MspI (0/1), indicating hemi- or fully methylated CG sites; and (4) absence of fragments in both profiles (0/0), representing an uninformative state caused either by different types of methylation, or due to restriction site polymorphism. Under the ‘Mix1’ scoring scheme the MSAP profiles are transformed into two binary data sets: one data set of methylated epiloci (hereafter M-MSAP) which scores conditions (2) and (3) as 1 and all other conditions as 0; and a data set of unmethylated epiloci (U-MSAP) which scores condition (1) as 1 and conditions (2) and (3) as 0. All plants sampled were characterized epigenetically by presence–absence scores for U- and M-type MSAP markers. In total 1450 U-MSAP and 2197 M-MSAP markers were scored in the whole group of 1088 plants sampled, with a mean per individual plant of 103.6 (range = 54–181) and 156.9 (range = 95–213), respectively, for U-MSAP and M-MSAP markers.The MSAP profiles were analysed with the R script ‘MSAP_calc’  was calculated according to where ual loci . The rarn is the number of markers, six is the binary state of the ith marker in individual x (either 1 or 0) and k is the total number of individuals in the population data set (i.e. within the population sample). In the denominator the number of occurrences of the ith marker in the total population data set is calculated. Population rarity index (RI) was estimated as the average of individual values. Calculations were carried out using ‘AFLPdat’  were modelled as binomial processes. Residuals were graphically inspected for linearity and homoscedasticity, and normal distribution of residuals was not rejected (P > 0.05) in any of the fitted models presented. Statistical significance of the two fixed effects and their interaction on response variables was determined in all cases with ordinary likelihood ratio tests using the ‘anova’ function from the R ‘stats’ library  and epigenetic (U-MSAP and M-MSAP) diversity indices obtained in each population for the group of restricted and widespread species included in our study are depicted in Z = 12.09, P < 0.0001; and Z = 8.060, P < 0.0001) and widespread species . However, opposing trends were found between restricted and widespread species when U-MSAP and AFLP markers were compared. Specifically, U-MSAP markers had higher values of polymorphic fragments than AFLP markers  in restricted species, but the opposite pattern occurred in widespread species  and widespread species . However, U-MSAPs had more private markers than AFLPs only in widespread species  but not in restricted species  and M-MSAP markers  or in M-MSAPs , and significantly lower in U-MSAPs than in M-MSAPs . In summary, we were able to detect significant differences between restricted and widespread only in one group of epigenetic markers (U-MSAP), particularly for PPOL and PPRIV, but not in genetic markers or in epigenetic methylated markers (M-MSAP).Results of the linear and generalized mixed models  indicate species . No signr = 0.374, 0.653 and 0.555; and P = 0.095, 0.0013 and 0.0089, for PPOL, SI and RI, respectively), or for the group of unmethylated (U-MSAP) markers ; or U-MSAP type of markers , concurring with the pattern found in restricted species  correlation between most of the diversity indices measured for AFLP markers, on one side, and on the other for the group of methylated (M-MSAP) markers  and 75.7 % . All species studied here thus exceeded the population average value (41.6 % ± 21.5) reported by In this study, the average polymorphism in AFLP markers per population ranged between 52.9 % , and was higher than polymorphism in those cytosines that are most commonly unmethylated (U-MSAP), which varied between 52.0 and 76.5 % . Epigenetic diversity was thus lower for U-MSAP than M-MSAP markers, and populations of endemic plants were more variable as regards the two epigenetic markers. Contrary to our expectations, however, populations of endemic and widespread species did not differ in their average epigenetic diversity. Although our sampling design simultaneously accounted for species relatedness and geographic location we cannot discard however that other factors important for plant population evolutionary dynamics such as population size or isolation level, which were not taken into account in our study, must also be operating and could have influenced our epigenetic (as well as genetic) diversity estimates. Recent models suggest, for instance, that greater rates of spontaneous epimutations can increase adaptation to local conditions and can help maintain polymorphisms in small and peripheral populations , although the sign of divergence was not consistent. Such intriguing results suggest that a deeper analysis with more species within genera but differing in other traits such as habitat requirements, lifeform, population size or isolation could be helpful to better understand the effect of species-specific plant traits in epigenetic diversity as previously suggested for genetic diversity  than genetic (AFLP) polymorphism in the whole sample, which was consistent with the idea that substantial epigenetic variation at shorter spatial distances might contribute significantly to population epigenetic diversity see also . In partariation .We found that all populations studied exhibited moderate to high levels of genetic polymorphism. However, contrary to our expectations, populations of restricted endemic and widespread species did not differ in average genetic or epigenetic diversity in our study region. The concurrent estimation of genetic and epigenetic diversity in replicated population samples of each species confirmed that levels of polymorphism at frequently methylated cytosines (M-MSAP markers) were always higher than polymorphism at genetic (AFLP) markers in both restricted endemic and widespread species. Importantly, correlation analyses showed that only in the group of species with restricted distribution did populations with higher epigenetic diversity tend to have also higher levels of genetic variation, although the difference between epigenetic and genetic diversity was greater at populations with low genetic polymorphism. This result is partly consistent with our expectation that a higher epigenetic diversity could possibly alleviate the lower genetic diversity expected in populations of endemic plants. Further work is needed to understand the effects that other intrinsic plant traits (e.g. lifeform) and population features  may have in comparative multispecies patterns of genetic and epigenetic co-variation in this Mediterranean hotspot region or elsewhere.The following additional information is available in the online version of this article—Table S1. Primer combinations used for amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) protocols.Table S2. Replicated samples and scoring error rates in the amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) protocols.Table S3. Genetic and epigenetic diversity estimates obtained in each population for the 14 study species.Table S4. Summary of the genetic and epigenetic diversity estimates for the group of restricted endemic and widespread species.plaa013_suppl_Supplementary_FilesClick here for additional data file.Financial support for this study was provided by grants CGL2013-43352-P and CGL2016-76605-P from the Spanish Ministry of Economy and Competitiveness.C.A., C.M.H. and M.M. conceived the idea and designed the study; C.A. and M.M. gathered plant material from natural populations; E.L. and P.B. isolated all DNAs and performed all AFLP and MSAP analyses; M.M. performed data analysis and prepared all figures and tables. C.A., C.M.H. and M.M. wrote the manuscript. All the authors read and approved the manuscript.None declared.