This study was undertaken to gain insight in their perceptions, attitudes and knowledge. Two publically accessible databases were used to invite NGHPs providing cancer genetic services to complete a questionnaire. The survey assessed: sociodemographic attributes, experience in ordering hereditary cancer genetic testing, attitude, knowledge, perception of communication skills  and educational needs. Of all respondents , most have a positive view of their own information giving  and decision making skills (64–77 % depending on topic). NGHPs feel responsible for enabling disease and treatment related behavior (89–91 %). However, 20–30 % reported difficulties managing patients’ emotions and did not see management of long-term emotions as their responsibility. Correct answers on knowledge questions ranged between 41 and 96 %. Higher knowledge was associated with more confidence in NGHPs’ own communication skills . Although NGHPs have a positive view of their communication skills, they perceive more difficulties managing emotions. The association between less confidence in communication skills and lower knowledge level suggests awareness of knowledge gaps affects confidence. NGHPs might benefit from education about managing client emotions. Further research using observation of actual counselling consultations is needed to investigate the skills of this specific group of providers.Non-genetic health professionals (NGHPs) have insufficient knowledge of cancer genetics, express educational needs and are unprepared to counsel their patients regarding their genetic test results. So far, it is unclear how NGHPs perceive  Since the early 1990s the possibilities for DNA testing in cancer have rapidly expanded. Through the combination of pedigree risk assessment and genetic testing it can be determined whether a patient’s personal or family history of cancer has an underlying hereditary cause. Genetic information about cancer is complex and involves understanding risks and inheritance patterns. Many individuals find such complex information difficult to understand . ExplainOver the last decade, though, more genetic tests, especially in the United States, are being ordered by non-genetic health professionals, such as oncologists, gynecologists, and primary care providers , 5. The The six-function medical communication model of de Haes and Bensing  states tthemselves.Numerous studies have shown that non-genetic health professionals have insufficient knowledge of genetics, express educational needs and are generally unprepared to counsel their patients regarding genetic test results –14. AlsoThe current study was undertaken to gain insight into the attitude, knowledge and skills of NGHPs who provide genetic testing. Investigating the perceptions of non-genetic health professionals about the communication process will provide insight in their perceived barriers and challenges to genetic counseling and testing. If deemed necessary this could aid in the development of an intervention to support and enhance their communication skills.We also wondered if NGHPs are aware of their knowledge level. A study by Klitzman et al.  showed tHow do NGHPs providing genetic testing perceive their own communication behavior  in cancer gene testing?What education and/or training have NGHPs received with regard to communication about cancer gene testing and what are their needs in this regard?What is the level of knowledge NGHPs have about cancer gene testing and is it associated with self-perceived information giving skills?The primary research questions are:www.myriadtests.com/finddoc.php) and the National Cancer Institute (NCI) Cancer Genetics Services Directory (www.cancer/gov/cancertopics/genetics/directory) are publically accessible databases containing contact information for healthcare providers who have self-identified as cancer genetic service providers. At the time of this study, Myriad Genetic Laboratories was the sole provider of genetic testing for the BRCA1 and BRCA2 genes in the United States. The Find A Provider section on their website was a mechanism for patients to identify healthcare professionals in their communities who provided genetic testing. Listing on the website was completely voluntary and the list was managed by Myriad Genetic Laboratories. The database of the NCI lists professionals who provide services related to cancer genetics . Professionals must apply to be listed in this directory and must meet certain criteria. Inclusion in the directory does not imply an endorsement by the National Cancer Institute. Professionals listed are contacted yearly by the NCI through e-mail to verify and/or update their record information.The Myriad Genetics Find a Healthcare Provider website  healthcare professionals self-identified as providing cancer genetic services with contact information listed on the Myriad Genetics Find a Healthcare Provider or NCI Cancer Genetics Services Directory and (2) providers of care in one of four Midwest states . Individuals who met the eligibility criteria were sent a letter of invitation explaining the aim of the study along with a paper version of the survey and a return envelope. The letter of invitation also included a link to an online version of the survey. A reminder was sent after 2 weeks. Completion and return of the study questionnaire implied consent. Health professionals who completed the survey and reported they were trained as master-degree genetic counselors, advanced practice nurses in genetics, or medical geneticists were excluded from data analysis.Because of a low response rate we expanded the study to four more states . The procedure was the same. However, this time a prize draw for two Amazon gift cards of fifty dollars and the possibility to receive a report about the results of the study were added, trying to increase the response rate.The IRB of The Ohio State University approved the study.Questionnaire items assessed: 1) Sociodemographic characteristics, (2) Practice characteristics, (3) Information giving, (4) Decision-making, (5) Communication about disease and treatment related behaviors, (6) Managing emotions, (7) Education and (8) Knowledge. Table  SociodemDescriptive statistics were used to describe the study sample. Sum scores were calculated for Information giving Skills perception  was considered significant.We calculated reliability for the three subscales of the adapted Ideal Patient Autonomy Scale (IPAS) (see Table In the first mailing summer 2013) 151 invitations were sent of which 17 were not delivered due to incorrect address (returned to sender). In the second mailing (April 2014), we sent out 366 invitations of which 70 returned for incorrect address. In total, we received 49 questionnaires (11 % response rate). One filled-in questionnaire was removed because of many missing values, four others were removed because they were filled in by participants specialized in genetic counseling or medical genetics. Table 013 151 iNGHPs reported addressing most relevant topics when providing cancer genetic counseling. These topics included: benefits and limitations of close cancer surveillance , sharing test results with family members (91 %), benefits and limitations of prophylactic surgery (89 %) and confidentiality and privacy (89 %). The possibility of a negative psychological reaction to genetic testing was least often discussed (73 vs. 89–95 %).NGHPs positively perceived their own information giving skills with a mean score 53.91 (SD = 6.8) on a scale of 13–65. Individual items showed that NGHPs found it most difficult to inform a patient about a variant test result: 16 % (strongly) agreed, while 23 % neither agreed or disagreed. Also, informing minority patients  and lower educated patients  was perceived as difficult by some. In addition, 14 % found it difficult to ensure that patients understand the genetic test result (27 % neither agreed nor disagreed) and 14 % found it difficult to inform a patient about a positive test result (11 % neither agreed nor disagreed).On average, NHGPs’ opinion was that patients know best regarding decisions about genetic testing [mean score is 3.92 (SD = 0.50)] and patients should decide [mean score is 1.69 (SD = 0.59)]. Furthermore, opinions diverged as to patients’ right not to pursue genetic testing [mean score is 2.77 (SD = 0.80)]. Table Figure Ninety-eight percent (n = 43) of NHGPs agreed that it was their responsibility to discuss preventive behaviors with their patients, while only 2 % (n = 1) disagreed. Furthermore, 91 % (n = 40) agreed that it was their responsibility to remind patients about their screening, 2 % (n = 1) neither agreed or disagreed, while 7 % (n = 3) disagreed.Ninety-five percent (n = 42) of NHGPs were optimistic that genetic research will lead to significant improvements in the treatment of complex traits, while 5 % (n = 2) neither agreed or disagreed. Seventy-seven percent (n = 34) agreed that personalized risk information will motivate people to change their behavior, while 18 % (n = 8) neither agreed or disagreed and 5 % (n = 2) disagreed.Figure Seventy-one percent (n = 31) saw it as their responsibility to the manage emotions patients experience during genetic counseling, while 18 % (n = 8) neither agreed nor disagreed and 11 % (n = 5) disagreed with this. Thirty-nine percent (n = 17) saw it as their responsibility to manage the long-term emotions (>3 months) patients experience after a genetic test, while 30 % (n = 13) neither agreed or disagreed and 32 % (n = 14) disagreed.Fifty-five percent (n = 24) of respondents received specific training about how to communicate with patients about hereditary cancer. This training was received either through continuing medical education (CME) (36 %), a Myriad-sponsored course (29 %), fellowship training (9 %), medical school or residency (9 %) or others .Participants reported to educate themselves about the most recent advances in genetic testing with CME , journal articles , on genetic laboratory sponsored trainings , through colleagues  and on the job training .Ninety-one percent (n = 40) felt confident about taking a cancer genetic family history [(strongly) agree], while 9 % (n = 4) neither agreed or disagreed. Thirty-six percent (n = 16) used web-based tools for taking a family history. Main reasons for not using these tools are time (n = 6), unfamiliarity (n = 5), use a paper form (n = 5), prefer orally discussing family history (n = 3) and other (n = 8). Seventy-four percent (n = 31) used web-based risk assessment models. Of those not using web-based risk assessment tools 79 % (n = 11) would be interested to use them.Figure Sixty-eight percent (n = 30) agreed or strongly agreed that they were confident about their ability to interpret a variant cancer genetic test result, while 23 % (n = 10) neither agreed or disagreed and 9 % (n = 4) disagreed. Ninety-eight percent (n = 43) agreed or strongly agreed that they are confident about their ability to interpret a negative cancer genetic test result, while 2 % (n = 1) neither agreed or disagreed.The mean knowledge score was 6.5 (SD = 1.7). Table s = .33, p = 0.03, meaning that participants with more knowledge had a more positive view on their own information giving skills.There was a significant positive correlation between Objective knowledge and Information giving Skills perception, rIn contrast to known studies showing that providers are unprepared to counsel their patients –14, our To be able to interpret these findings we first need to address the low response rate (11 %) of our study. Our study may have suffered from response bias, as our respondents may be those who view this as an important process whereas those who do not may not have responded. Also, our data shows that respondents were individuals with a lot of experience in ordering cancer gene testing (47 % ordering more than 30 gene tests a year) and half of them received training regarding communication with patients about hereditary cancer. These findings suggest an overrepresentation of experienced and well-trained non-genetic health professionals. If the low response rate reflects a lack of interest, this is worrisome. Those who may not be as comfortable with communicating informed consent may not be answering and thus it is hard to address what they specifically need for resources. Furthermore, while all individuals in the databases identified themselves as ordering genetic testing we received many returned envelopes, suggesting that these databases were not as up to date or representative as may be suggested.So, how to interpret the positive view of NGHPs of discussing and ordering genetic tests for cancer? Especially, as the wealth of available research literature suggest that non-genetic health professionals experience difficulties in genetic counseling , 20–22. Our knowledge level seems to be higher than observed in most other studies where correct answers to knowledge questions regarding cancer gene testing in non-genetic health professionals ranged between 13 and 48 % –25 ). InAlthough most NGHPs in our study have an overall positive view, they report more difficulties managing emotions. In addition, they do not perceive management of long-term emotions as their responsibility. The majority of our respondents however are involved in long-term management of at-risk patients and should be prepared to address these emotions.genetic health professionals have moved, at least theoretically, from non-directive counseling to counseling based on principles of shared decision making [Slightly more than half of our respondents have received education regarding communication about genetic testing. The low percentage of NGHPs who received training is a reason for concern. In the last decennium n making –29. HoweOur study showed that the higher the knowledge level, the more confidence non-genetic health professionals reported in information giving skills. This association suggests that awareness of knowledge gaps affects health professionals’ confidence. Would identifying knowledge gaps in individual NGHPs be a helpful route towards enhancing quality and consistency of care among providers performing genetic risk assessment, counselling and testing? Interestingly, Prochniaks’ study suggests that confidence plays an important role in preference of physicians towards ordering genetic testing themselves or rather refer to a clinical genetic center . In theiOverall, NGHPs who participated in our study have a positive view on their attitude towards, knowledge of and skills in discussing and ordering genetic testing. However, our study raises several concerns about how well-informed patients and their families will be. Specific attention is needed for the consequences of genetic testing for family members and the interpretation of VUS. Continuing medical education should address these issues.


Canis latrans) are based on one of two strategies: sampling many individuals using one or very few molecular markers, or sampling very few individuals using many genomic markers. Thus, a regional analysis of genetic diversity and population structure in eastern coyotes using many samples and several molecular markers is lacking. I evaluated genetic diversity and population structure in 385 northeastern coyotes using 16 common single nucleotide polymorphisms (SNPs). A region-wide analysis of population structure revealed three primary genetic populations, but these do not correspond to the same three subdivisions inferred in a previous analysis of mitochondrial DNA sequences. More focused geographic analyses of population structure indicated that ample genetic structure occurs in coyotes from an intermediate contact zone where two range expansion fronts meet. These results demonstrate that genotyping several highly heterozygous SNPs in a large, geographically dense sample is an effective way to detect cryptic population genetic structure. The importance of SNPs in studies of population and wildlife genomics is rapidly increasing; this study adds to the growing body of recent literature that demonstrates the utility of SNPs ascertained from a model organism for evolutionary inference in closely related species.Previous genetic studies of eastern coyotes ( Canis latrans) has colonized almost the whole continent in the last 100 years, with few exceptions (e.g. Long Island)2. The eastward range expansion was likely facilitated by widespread deforestation associated with agricultural development in the early 20th century and by the near extirpation of eastern wolves (Canis lupus lycaon orCanis lycaon) and red wolves (Canis rufus)5. The range expansion into northeastern North America advanced as two primary colonization fronts: the northern route through Ontario, where coyotes hybridized with resident eastern wolves, and the southern route through Ohio, where wolves were eradicated prior to coyote expansion6. Along with its geographic range expansion, the coyote also experienced a niche expansion by rapidly colonizing whole new biomes, including eastern temperate and boreal forests1. This complex scenario of colonization provides an interesting opportunity to explore the swift formation of population genetic structure following a rapid expansion in geographic distribution and ecological niche.Historically restricted to the open deserts and plains of central and western North America, the coyote  and eight microsatellite loci. They found no genetic structure in coyotes within Massachusetts or even within the broader region of northeastern North America. Instead, northeastern coyotes seemed to constitute one uniform population slightly differentiated from western coyotes. In another analysis of genetic variation in northeastern coyotes, Kayset al.5 identified three coarse phylogeographic areas: Ohio, the northeast zone, and a contact zone in western Pennsylvania and New York where the colonization front from Ohio has spread into the northeastern population. Although Kayset al. surveyed genetic variation in a dense geographic sample of 687 coyotes, they only used one genetic marker, the hypervariable mtDNA control region. vonHoldtet al.8 conducted a genome-wide analysis of North AmericanCanis species and detected population structure inC. latrans, but only at the broadest continental scale. Although vonHoldtet al. genotyped tens of thousands of loci, they only sampled 14 northeastern coyotes, making detection of finer levels of population structure in the region very improbable. Thus, all the previous studies of population structure in northeastern coyotes have adopted one of two strategies: sampling many individuals using one (mtDNA) or very few molecular markers, or sampling very few individuals using thousands of genomic markers. A regional analysis of genetic diversity and population structure in northeastern coyotes using many samples and many nuclear molecular markers is currently lacking.Recent analyses of population structure in northeastern coyotes have described a general lack of genetic differentiation among sampling localities, except at the coarsest geographic scales. Wayi.e., transformation rather than speciation)9. The detection of genetic structure largely depends on the type and number of molecular markers examined, their variability in the target population, the number of individuals sampled, and the spatial sampling scheme11. Single nucleotide polymorphisms (SNPs) have become a popular and inexpensive tool in the field of molecular population genetics. SNPs have properties that make them a superior alternative to other widely used genetic markers, such as microsatellites and mtDNA sequences, in evaluating genetic diversity and population structure14. Furthermore, the sampling scheme can greatly influence the location and composition of genetic clusters, especially in species that are continuously distributed across a landscape11, as are northeastern coyotes. Thus the conclusion of no population structure from previous analyses based on limited individual or genomic sampling may be imprecise.Genetic structure is a ubiquitous property of natural, domesticated, and human populations. Population genetic structure plays considerable roles in evolution, as both the basis and the consequence of local adaptation, the splitting of one species into two if the environments are markedly different, and the adaptability of a species as a whole across its range , Pennsylvania (N = 103), Vermont (N = 34), Ohio (N = 30), New Jersey (N = 14), New Hampshire (N = 11), Connecticut (N = 8), Massachusetts (N = 5), southern Quebec (N = 4), and Rhode Island (N = 2) . All sam16 . No Inst17. I used the programPLINK18 to compute observed and expected heterozygosity per locus in the subset of 17 northeastern and Ohio coyotes. I selected 16 unlinked SNPs, each on a different autosomal chromosome, with the highest observed heterozygosity, a measure of genetic variability in a population 20 using the University of California, Santa Cruz In-Silico PCR and BLAT web tools (http://genome.ucsc.edu).I selected molecular markers based on a genomics study that used the Affymetrix Canine Mapping Array to genotype 61,435 SNP loci in hundreds of wild and domestic canids, including 14 northeastern and 3 Ohio coyotesulation . Ascertaet al.5. I quantified DNA concentrations using a NanoDrop ND-1000 Spectrophotometer  and diluted the samples in water to attain concentrations of 5–30 ng/μl. I prepared four 96-well plates with template genomic DNA of 378 eastern coyotes, seven of which served as controls because they were already genotyped with the canine SNP microarray8. Two western coyotes from Washington and two western gray wolves (Canis lupus) from Yellowstone National Park served as additional controls; two wells containing only water served as negative controls.I extracted total genomic DNA from muscle or hide using the DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions, and also used DNA samples that were extracted in the New York State Museum as described in Kays2, and 1× Roche High Resolution Melting Master kit mix. The latter contains FastStart Taq DNA polymerase, dNTP mix, and ResoLight, a high resolution melting dye that fluoresces when DNA is double-stranded. During the melt curve analysis, the temperature increases very slowly to denature double-stranded DNA. Samples with variations in DNA sequence, even in one base pair, are distinguished by discrepancies in the shape of the melt curve, thus discriminating each of the two homozygous and the heterozygous genotypes. I processed the raw data using the Gene Scanning module of the Roche LightCycler software and followed Roche’s recommendations for evaluating data quality. The software automatically generates genotypes from the raw melt curve data. All SNPs were biallelic, so there are three possible genotypes per locus–AA, AB and BB–each forming three distinct clusters when signal intensity is plotted against temperature. I visually inspected all software-generated genotype calls and manually removed the ambiguous ones that did not conform to any of the three possible genotype clusters,SNP genotyping was performed at the GenoSeq Core laboratory in the University of California, Los Angeles, using a high resolution melting curve quantitative PCR assay on a LightCycler 480 thermal cycler . Each of the 16 SNPs was amplified and genotyped separately. DNA was amplified in a total volume of 10 μl, including 1 μl (5–30 ng) of genomic DNA, 0.2 µM of each primer, 4.2 mM MgClet al.8 making a total sample size of 385. I used PLINK to calculate average observed and expected heterozygosity, measures of genetic diversity, and deviations from Hardy-Weinberg equilibrium in the overall sample and in each of the three phylogeographic zones inferred by Kayset al.5 (Canis familiaris)17. I calculated average observed and expected heterozygosity using all 61,435 SNPs from the microarray because the vast majority of the loci in the microarray were ascertained by dog-dog comparisons17, and again using only the 16 high-heterozygosity SNPs genotyped in this study.To the 378 samples genotyped in this study, I added 7 more northeastern coyote samples genotyped by vonHoldtt al.5 . In orde21 to infer the most likely number of genetic populations. STRUCTURE implements a Bayesian algorithm to assign multilocus genotypes to genetic clusters by calculating the likelihood that a group of individuals constitutes a population. I tested whether finer population structure was detectable with the high-heterozygosity SNPs relative to the coarse structure detected with hypervariable mtDNA. I analyzed all 247 coyotes together to test whether more than three populations were detectable at the regional level, and then analyzed each zone separately to test whether more than one population was detectable within each zone. For all analyses, I used three replicate runs of 20,000 burn-in and 100,000 Markov chain Monte Carlo iterations, used the admixture ancestry model with correlated allele frequencies22, and set the number of populations from K = 1 to K = 8. I verified that alpha and likelihood statistics reached convergence during the burn-in period for each number of populations analyzed. I used STRUCTURE HARVESTER23 to evaluate the relative support for each value of K by plotting Ln P(D), the mean posterior probability of the data21, and ΔK, a quantity related to the second-order rate of change of the likelihood function with respect to K10. I used CLUMPP24 to align and average the three replicate cluster membership coefficient matrices, and ArcMap 10  to visualize the spatial distribution of genetic structure. I considered individuals with ancestry coefficients q > 0.8 as belonging to a specific cluster and individuals with all q < 0.8 as being admixed, consistent with recent genetic investigations ofCanis26. In order to corroborate inferences from the STRUCTURE analysis with a model-free approach, I conducted a principal components analysis (PCA) using the adegenet 1.3–4 package27 in R28. I also computed pairwise FST, the inbreeding coefficient within populations relative to the total, among the populations inferred by STRUCTURE and tested the significance of the differentiation by analysis of molecular variance (AMOVA) using 999 permutations in GenAlEx 6.530.To assess population genetic structure, I excluded all individuals with more than five missing genotypes, resulting in 247 coyotes . I used the program STRUCTURE 2.3U = 1771,P = 0.017). That is, the fecal samples had, on average, more missing or ambiguous genotypes than the tissue samples.I interrogated 16 SNP loci in 378 coyotes for a total of 6,048 expected genotypes . GenotypThe overall sample of 385 eastern coyotes had lower genetic diversity than expected , even thDogs appeared to be the most genetically diverse when the diversity of the five different canid groups was estimated using all 61,435 SNPs from the canine microarray. The genome-wide ascertainment bias was towards dogs: the expected heterozygosity of dogs was almost twice that of western coyotes. But the ascertainment bias reversed when heterozygosity was measured using only the 16 selected SNPs: coyotes appeared to be the most genetically diverse, with northeastern coyotes having a very high expected heterozygosity, whereas dogs appeared the least genetically diverse .ST among the three genetic clusters varied from 0.08 to 0.10 and all were significant in the AMOVA framework (P = 0.001). This indicates that there are three primary genetic subdivisions in the broad sampling area. The three groups did not correspond to the Ohio, contact, and northeast zones previously inferred by mtDNA .”First, thank you for the opportunity to review Javier Monzón’s article, “et al. (2011)vonHoldt, while also asserting the need for addressing ascertainment bias prior to the use of SNPs in non-model organisms.I found this to be a well-written, engaging, and thoughtful manuscript with important contributions to the use of SNPs in studies of population structure and in elucidating the genetic structure of the northeastern coyote. The author adeptly targets a need for using multiple markers across multiple individuals for proper inference. The author furthermore supports the use of the canine SNP array in studies of coyotes, following the recommendation ofI have no major reservations, rather a few minor comments regarding clarification of the methods along with some questions and considerations for future discussion and research.Methods:With such a large sample of high quality tissue available for the analysis, why were scats (n=6) included in the analysis? Were the scats important for adequate sampling across the study area?et al’s. (2011) coyote specimens were included in the initial selection of the 16 SNP panel used in subsequent analyses. Why were only 7 of vonHoldtet al.’s (2011) samples included in the analysis of diversity and structure?Fourteen of vonHoldtResults:Kays et al. . It would be interesting to see the Ho and He for the population clusters identify by the current STRUCTURE analysis .Using the SNP panel, the author reports the observed and expected heterozygosity for the three putative population zones identified byWhat are the pairwise Fst values for two genetic clusters also supported by the STRUCTURE analysis?Discussion:some geographic structuring,” at least based on Figure 2c. The author does a thorough job of listing the factors and processes that may be involved in explaining a lack of geographic structure, as well as the discrepancy between the current nuclear analysis of population structure and the previous mtDNA analysis .There is ample evidence of genetic structure; however, I’m not sure that I would agree with the author’s suggestion that there is “et al. 2004Morin). This might allow one to detect patterns of genetic structure without any obvious geographic patterns.A possible further explanation for the lack of geographic structuring could be that a SNP panel - with such high heterozygosity - may actually reflect older mutations that have had a longer time to move around the landscape via dispersal . Presumably the same clustering patterns would remain if the samples were analyzed at thousands of genome-wide SNPs that have not been selected for their polymorphism - i.e. presumably more, less polymorphic loci should show the same substructure.This is a very well-written, interesting article on utilizing highly-polymorphic SNPs to identify cryptic substructure in a population of eastern coyotes. The analyses are comprehensive  and the data do seem to support fine-scale population substructure. If it weren't for the extensive analyses I would find it very difficult to believe that 16 SNPs (even highly polymorphic ones) could find such fine-scale structure because it has been estimated that it take at least 15x more SNPs than microsatellites to identify structure in populations , but I wonder if social structure (i.e. family/pack groups) may contribute to the genetic patterns found.  The biological/ecological reason for the clustering remains unclear . The author provides plausible explanations for the structure , coyote and gray wolf from Kayset al. 2010  correspond with genetic structure based on SNPs.This would provide important information to help understand how similarities/discrepancies in genetic structure are distributed across space. The author describes the SNP results relative to the areas of Ohio, the contact zone and the northeast zone from           et al. 2011vonHoldt suggest a bias against coyotes, although coyotes may at least in some regions, have higher genetic diversity than wolves considering their range expansion and increase in population size. It might be worth noting that sequencing coyotes directly could provide a more in-depth view of genomic regions under selection. Although I agree with the author’s conclusion about the utility of using a closely related and well-studied species, alternate methods such as genotyping-by-sequencing could be recommended as possibilities for future research.  The relatively low genetic diversity for coyotes in Table 3 and the results fromst sentence “Historically restricted to the open deserts and plains...”. Though the coyote is widely regarded as a species of western and central North America, it is difficult to exclude the possibility that it could also have been present farther east prior to European colonization and landscape modifications.  Introduction, 1rd paragraph:Introduction, 3Population genetic structure plays considerable roles in evolution... the splitting of one species into two if the environments are markedly different”.  In addition to adaptation, it may be important also to mention genetic drift, and subsequent development of incompatibilities between isolated populations.- The sentence “The detection of genetic structure largely depends on the type and number of molecular markers examined”: Here you may want to mention explicitly the temporal resolution of markers with different mutation rates (as you have done in the Discussion).- The sentence starting with “SNPs have become a popular and inexpensive tool”: perhaps “increasingly affordable” or similar would be better here?    - “rd paragraph, sentence:“...cryptic genetic structure of Northeastern coyotes, especially as they have only inhabited the region for 30-80 years”. The author notes earlier in the text that coyotes in the northeast zone may descend from a few founders, which likely arrived via a northern route through Ontario, whereas a second colonization front occurred farther south through Ohio.et al. 2011Bierne proposed that intrinsic (environment-independent) genetic incompatibilities may coincide with ecological boundaries, as these environmental transition zones often “trap” the tension zones resulting from e.g. assortative mating. Such processes might be influencing the contact zone between the southern and northern colonization fronts, and interact with the selective pressures associated with forested, agricultural and urban habitat, and deer density, noted inMonzón 2012 (PhD Dissertation).    Discussion, 3The article is well written, and the author has done a good job explaining the main limitations of the work and outlining why findings should be interpreted with caution. I have one suggestion on supplementary data that could be included for an additional perspective on genetic structure, and some minor comments on the manuscript text:   I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Remote sensing (RS) of plant canopies permits non-intrusive, high-throughput monitoring of plant physiological characteristics. This study compared three RS approaches using a low flying UAV , with that of proximal sensing, and satellite-based imagery. Two physiological traits were considered, canopy temperature (CT) and a vegetation index (NDVI), to determine the most viable approaches for large scale crop genetic improvement. The UAV-based platform achieves plot-level resolution while measuring several hundred plots in one mission via high-resolution thermal and multispectral imagery measured at altitudes of 30–100 m. The satellite measures multispectral imagery from an altitude of 770 km. Information was compared with proximal measurements using IR thermometers and an NDVI sensor at a distance of 0.5–1 m above plots. For robust comparisons, CT and NDVI were assessed on panels of elite cultivars under irrigated and drought conditions, in different thermal regimes, and on un-adapted genetic resources under water deficit. Correlations between airborne data and yield/biomass at maturity were generally higher than equivalent proximal correlations. NDVI was derived from high-resolution satellite imagery for only larger sized plots (8.5 × 2.4 m) due to restricted pixel density. Results support use of UAV-based RS techniques for high-throughput phenotyping for both precision and efficiency. High-throughput phenotyping, particularly through the application of remote sensing tools, offers a rapid and non-destructive approach to plant screening . Satellite imagery has the advantage of covering large areas instantaneously, but generally does not offer the spatial (sub-meter) and temporal (weekly/daily) resolution required for breeding experiments. Low level, airborne remote sensing measurements have the advantage in that resolution is at plot level while at the same time providing the possibility of instantaneously capturing multiple plots at a practical breeding scale at a high temporal resolution  in the Sonoran desert, close to Ciudad Obregon, NW Mexico . Environmental and management details of this area are given in  and dry biomass weight (gm−2) were estimated at maturity for each plot following the methods described in Pask et al.  was recorded at ground level using the Sixth Sense LT300 handheld infrared thermometer. Measurements were made along each of the plots from a distance of ~0.5 m above canopy, angled to avoid bare soil (about 60° to nadir) and directed specifically at the part of the plot most exposed to the sun , when cloud cover was minimal and at times of low wind speed . Table where Aerial imagery was collected via the AscTec Falcon 8 Unmanned Aerial Vehicle (UAV) Figure . The 8-r2, including the whole of the CIMMYT research station.Satellite imagery was obtained from the commercial Digital Globe WorldView-2 (WV-2) satellite, taken on 6th April 2013. The imagery includes an 8-band multispectral image (bands between 396 and 1043 nm) and a panchromatic image (447–808 nm), with a spatial resolution of 0.46 and 1.85 m respectively. The georeferenced, unprocessed images cover ~25 kmTable Processing was carried out using ENVI version 5.0 . Radiometric distortions, e.g., lens vignetting, were solved by applying a cross track illumination correction, removing any broadband variation without affecting narrowband features. Geometric distortions are corrected using a “warping” procedure, by which an image is resampled to match the geometry of a “base” image or a vector map via the selection of Ground Control Points (GCPs). Images are subsequently mosaicked together by identifying overlapping regions within images.TM4 (≈ 760–900 nm) and TM3 (≈ 630–690 nm) denote the Landsat bands.A camera specific mask is applied to the image/mosaic of each trial, via pixel band ratio thresholds, to differentiate between vegetation and non-vegetation pixels, aiming to remove any non-vegetation pixels, such as soil, as different materials can be distinguished by their pixel signal. An algorithm is then applied for the automatic detection of plots using pre-defined parameters by the user, for example the plot size in pixels, and distance between plots in pixels. An average across all bands for each pixel is calculated and pixels within each plot that exhibit high variance are removed, to eliminate any non-vegetation pixels that the mask may have missed, as well as pixel mixing effects. The average of each plot at each band is then taken to derive the target indices at plot level Figure . For theTI was calculated using the sum of the green and blue bands of the plot averaged values of the processed images acquired from the recorded analog video:The processed images collected from the thermal camera onboard the UAV were used to derive a temperature index, which relates to the CT of each plot. The temperature index TG and TB are the averaged “plot” values at the green and blue bands respectively.where The Fast Line-of-sight Atmospheric Analysis of Spectral Hypercubes (FLAASH), an ENVI atmospheric correction tool, was applied to the satellite imagery used here. FLAASH incorporates the Moderate Resolution Atmospheric Transmission V4 (MODTRAN4) radiative transfer model to simulate the spectral radiance at pixel sensor level using user defined input variables. For a detailed description on the methods used by FLAASH see Adler-Golden et al. .The panchromatic  and the multispectral imagery  were fused together to create a single high resolution multispectral image, via the ESRI “pan-sharpening” algorithm, using ESRI ArcMap 10.1  for SAS  compared to those of the hot irrigated trials, also made up of elite lines . When separated into groups, there was significant difference between the UAV and proximal derived correlations with yield and biomass for the following groups CT/thermal index , yield , biomass , and NDVI .When considered together, correlations between the UAV derived indices and yield/biomass were significantly different to the equivalent proximal sensed correlations (P = 0.06) was observed between the proximal and UAV phenotypic correlations between yield and biomass, probably associated with the greater biomass correlations, particularly those of the UAV (see Table P = 0.08) between the proximal and UAV phenotypic correlations between yield and biomass for both NDVI and the thermal index/CT. This can be attributed to the higher correlations for the UAV observations. No interaction was observed for the DRT environment, this can be partly explained by un-adapted material in the genetic resource trial, as explained above.The phenotypic correlations were separated into the three environments  and a multi-factor ANOVA was performed. For the OPT environment, a significant interaction , water vapor in the atmosphere, background noise and measurement geometry , the approach lends itself well to routine measurements including for growth analysis, to measure the evolution of stress, and the application of regression e.g., (Lopes and Reynolds, Despite the promising results presented here for NDVI derived from the satellite measurements, it is probably not the most effective tool for this application. While satellite imagery has the advantage of covering vast areas, resolution restricts its application to measurements in which target objects are of a larger scale than the small plots typical of genotypic screening. Furthermore, it is difficult to obtain satellite imagery at frequent time intervals and the option to adjust timing of measurement to avoid cloud cover or other inclement weather conditions is absent.The fact that the estimates of CT and NDVI were generally better associated with performance traits when measured by UAVs compared to proximal data, under both heat and drought stressed conditions, and in advanced lines as well as unimproved genetic backgrounds, confirms the value of the UAV approach in breeding for climate change, where a new generation of breeding lines must be developed based on extensive screening of plant genetic resources.MT collected data, carried out the data analysis, drafted the article and carried out revisions. MR created the experimental design, made suggestions for the data analysis, helped with the draft and carried out critical revisions. SC made suggestions for the data analysis, helped with the draft and carried out critical revisions. All authors gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diaperis boleti(L.) (Coleoptera: Tenebrionidae), a fungivorous saproxylic beetle that inhabits sporocarps ofLaetiporus sulphureus(Bulliard) Murrill  on trees growing in highly-fragmented agricultural landscapes. Isolation by distance was tested with spatial autocorrelation analysis of kinship  and correlating matrices of genetic and geographic distances with the Mantel test (population-based approach). These results were compared with the results obtained for saproxylic beetles living in the same landscape but differing in ecological preferences. It was shown that the species dependent on sporocarps of wood-decomposing fungi had higher variability, lower individual inbreeding, and less intensive isolation by distance pattern than saproxylic beetles living in tree hollows. It was also demonstrated that spatial autocorrelation analysis of kinship is a more sensitive approach for detecting fine-scale spatial genetic structure than the Mantel test.Living in unstable habitats is expected to decrease the intensity of isolation by distance in populations through the need for frequent movements of individuals. Insects associated with fruiting bodies of fungi therefore are supposed to have weak spatial genetic structure of populations compared with those living in more stable habitats. With the use of an amplified fragment length polymorphism technique, this study investigated the isolation by distance, inbreeding, and genetic diversity in The abilities of organisms to disperse among subpopulations and to colonize distant habitat patches are important for metapopulation survival, especially in landscapes subjected to a strong fragmentation . TherefUnfortunately, empirical results showing a link between ecological specialization and dispersal abilities of insects are scarce  (Coleoptera: Tenebrionidae), a beetle that inhabits fruiting bodies of wood-decomposing fungi. IBD is a phenomenon characterized by increasing genetic divergence due to decreasing gene flow with increasing geographic distance between individuals or populations  genetic markers were used to examine isolation by distance (IBD) oflations . In a pD. boletiis a widespread species, occurring in almost all of Europe (except extreme northern areas); it is reported also from Northern Africa and the Middle East  Murrill (Fomitopsidaceae),Piptoporus betulinus(Bulliard) P. Karsten (Fomitopsidaceae),Fomitopsis pini-cola(Swartz) P. Karsten (Fomitopsidaceae),Fomes fomentarius(L.) Fries (Polyporaceae) (cf.http://www.mycobank.org). The complete life cycle takes one year; the beetles emerge in the summer and survive the winter as adults (L. sulphureus) is also one year, beetles must colonize a new habitat patch every year.le East . The sp bodies . As the bodies :Laetip adults . BecausD. boletiwere collected from fruiting bodies ofL. sulphureusgrowing on roadside trees in rural avenues , followed byQuercus robur,Fraxinus excelsior, andAcer platanoides, andBetula pendula, Carpinus betulusandSalix alba. Other trees  have a share of <1%  to >200 km. Samples were collected to provide a large number of pairwise comparisons between genotypes for different geographical distances. Although the goal was to collect at least 10 individuals from each sporocarp, in most cases a smaller number of individuals were encountered and so as many as possible were collected . To avoEcoRI  and 5 UTruI (MseI isoschizomer) (Fermentas), 1.5 U T4 DNA Ligase (Fermentas), 1× T4 DNA Ligase Buffer (Fermentas), 0.05% BSA, 50 mM NaCl, 0.5 pmol/mL E-Adaptor, and 5 pmol/mL M-Adaptor. Reactions were carried out at room temperature overnight and then diluted five times with H2O to obtain PCR matrices (pre-matrix DNA) for pre-selective amplification.Genomic DNA was extracted from insect tho-races using the Insect Easy DNA Kit (EZNA)  following the manufacturer’s protocol. The AFLP analysis followed the protocol developed byPre-selective amplifications were carried out in 10 mL total volume. A pre-selective PCR mixture contained 2 mL pre-matrix DNA, 1× Qiagen Master Mix , 0.5 mM E-primer (E+A), and 0.5 mM M-primer (M+C). Amplification was carried out using the following program: 72°C for 2 min, 20 cycles of 94°C for 20 sec, 56°C for 30 sec, and 72°C for 2 min, and finally 60°C for 30 min. A product of pre-selective PCR was diluted 20 times to obtain a PCR matrix for selective amplification (sel-matrix DNA).—1°C per cycle) for 30 sec, and 72°C for 2 min, 20 cycles of 94°C for 30 sec, 56°C for 30 sec, and 60°C for 30 min. Both pre-selective and selective amplifications were carried out using PTC200 thermocycler .Selective amplifications were carried out in 10 mL total volumes, consisting of 3 mL sel-matrix DNA, 1× Qiagen Master Mix, 0.5 mM FAM-labelled E-primer  and 0.5 mM M-primer  . PCR reThe products of selective amplifications were sized using automated capillary sequencer ABI3130XL  and the manufacturer’s Genescan 3.7 software. AFLP profiles were then subjected to visual assessment to eliminate outlier samples in Genotyper 3.7 software (Applied Biosystems). Then, all peaks in a profile within the range 60-400 bp were automatically labelled, and bins were created based on all labelled peaks. Automatically created bins were visually checked to ensure that the bin was centered on the distribution of peaks within the bin. Bins with low polymorphism (3% < frequency of dominant haplotype <97%) were omitted from the further study. To reduce the occurrence of homoplasy , bins w(Hj),andWright’sFor the purposes of analysis, it was assumed that the beetles collected from one sporocarp formed a distinct group of individuals (“population”). For each population, after estimating allelic frequencies with a Bayesian method, assuming a non-uniform prior distribution of allele frequencies , 10 km, 20 km, 40 km, 80 km, 160 km, and >160 km. Within these distance classes, the numbers of pairwise comparisons among individuals were as follows: 624, 480, 861, 1161, 2759, 3079, and 216, respectively.To test for the relationship between genetic and geographic pairwise distances, Mantel’s test was used to calculate the correlation between matrices of genetic distances [linearized and log-transformed geographic distances . This pConfidence intervals around the average estimates of kinship coefficients for a given distance class were obtained from standard errors calculated by jack-knifing data over loci.Sp = - b1 /(1 -f(1))index was estimated (Vekemans and Hardy 2004), whereb1is a slope of a log-linear regression between observed kinship and a distance between individuals, andfis the average kinship estimated for the first distance class.Under IBD, given drift-dispersal equilibrium, kinship is a linear function of logarithm of distance between individuals . TherefSpatial genetic structure (SGS) may arise because of IBD or spatial variation of selective forces affecting distribution of alleles of specific loci ; therefAFLP markers were analysed under the assumption of complete dominance (binary data). Hence, inference about inbreeding needs simultaneous estimation of allele frequencies and the inbreeding coefficient, based on phenotypes. The estimation was carried out using a Bayesian method introduced by(F).Each sampled individual is characterized by an individual inbreeding coefficient(Fi)that by assumption follows a beta distribution. Generally, a probability of observing a multilocus phenotype is a function of (unknown) allele frequencies and (unknown) individual inbreeding coefficient . Using a Markov Chain Monte Carlo approach , I4A estimates simultaneously marginal posterior distributions of the average inbreeding coefficient and allele frequencies given provided phenotypic data. Our estimates were obtained after 100,000 steps, after 10,000 burn-in steps. Because the method requires initial guesses on the priors, which are shape parameters of beta distribution(aandb)such, thatF = a / (a + b),to avoid a dependence of final results on these guesses, analyses were conducted starting from three initial sets of parameters. Each set determines the same mean(F= 0.5) but implies very different shape (and variance) of the prior distribution.The method assumes that a sample of individuals, genotyped at AFLP loci, is randomly taken from a population characterized by an unknown average inbreeding coefficientSTF–outlier method, only one marker was recognized as being under positive selection and one under stabilizing selection. These two loci were removed from further analyses, leaving 81 putatively neutral loci. On average, 86.2% loci were polymorphic within populations. The unbiased expected heterozygosity for all populations ranged from 0.302 ± 0.015 to 0.386 ± 0.023, with an average value of 0.347 ± 0.019 . The Mantel test did not detect any significant correlation between matrices of genetic and geographic distances of populations. Nonetheless, more sensitive individual-oriented spatial autocorrelation analysis revealed that there was a relationship between genetic similarity of individuals and geographical distance. As expected based on the theory of isolation by distance, beetles that lived in close spatial proximity were more genetically similar than expected under a random distribution of genotypes. This pattern suggests a fine-scale genetic structure withinD. boletiwhere proximate sites are genetically more alike than more distant ones. Average pairwise kinship coefficient was estimated to be 0.0386 (SD 0.005) for individuals collected from a single fruiting body ofL. sulphureus. Kinship coefficient significantly exceeded zero for the two first distance classes .classes . The poclasses , i.e., All three analyses executed with different prior distributions (see Methods) resulted in consistent estimates, which were slightly but significantly greater than zero . Because intensive gene flow may be responsible for homogenizing spatial genetic structure, the presence of IBD could be surprising in a species with expected high mobility. It should be noted however that spatial clustering of related genotypes is also found in other mobile insects depending on ephemeral resources. A population survey of the forensically important black blow fly,Phormia regina(Meigen) , found that adults arriving together at a carrion bait shared a much higher proportion of alleles than would be expected from a random sample (Ochlerotatus notoscriptusSkuse (Diptera: Culicidae), indicating restricted gene flow and IBD effect  andProtaetia marmorata(F.). Because the study was conducted within the same region as the present study, such comparisons are justified. BothO. barnabitaandP. marmorataare exclusively associated with wood mould in tree hollows. Because large hollows develop in tree trunks during several decades, and some hollow trees could last for hundreds of years, durability of such a habitat is estimated for tens to hundreds of years (O. barnabita (Sp= 0.016 ± 0.002), while IBD in less specialised and more commonP. marmoratawas intermediate(Sp= 0.006 ± 0.003) betweenO. barnabita and D. boleti (Sp =0.0016 ± 0.0007). In the case ofO. barnabitaandP. marmorata,observed differences in SGS were influenced not only by habitat stability but also species population characteristics, among which the most important seems to be niche breadth . It could be hypothesized that the weakening of gene flow may enhance the role of genetic drift at a local scale, therefore decreasing local effective population sizes and increasing the loss of genetic variation.Nonetheless, IBD inf years , which breadth . SimilaBolitophagus reticulatus(L.) (Coleoptera: Tenebrionidae) in the fragmented forest was three times greater than in the continuous one, strongly indicating a genetic isolation effect of habitat fragmentation. However, it was unresolved whether landscape fragmentation increased genetic differentiation, because agricultural fields presented barriers to movement or more indirectly through gradually decreasing gene flow with increasing distance . Methods applied to infer IBD inB. reticulatus did not find any statistically significant trend in genetic differentiation with distance and, hence, there was no clear evidence for IBD either in the fragmented or in the continuous area (However, direct comparisons between species may be hindered by differences in responses to the habitat fragmentation, which is often regarded as the strongest factor affecting SGS. The spatial scale at which species perceive fragmentation varies with intrinsic species properties, like trophic level, dispersal ability, body size, and rarity . For a us area . One maus area . TherefD. boletiusing molecular markers. Compared with beetles living in more stable tree habitats, higher genetic diversity and weaker IBD pattern in a fungivorous species was demonstrated, in concordance with theoretical predictions. It was also demonstrated that the spatial autocorrelation analysis of kinship might be more sensitive in detecting fine-scale spatial genetic structure than the correlating matrices of genetic and geographic distances based on the Mantel test.This is the first study of the spatial genetic structure of


Nyctereutes procyonoides) in north-eastern and central Europe. This invasive species is of management concern because it is highly susceptible to fox rabies and an important secondary host of the virus. We hypothesized that the large number of introduced animals and the species’ dispersal capabilities led to high population connectivity and maintenance of genetic diversity throughout the invaded range. We genotyped 332 tissue samples from seven European countries using 16 microsatellite loci. Different algorithms identified three genetic clusters corresponding to Finland, Denmark and a large ‘central’ population that reached from introduction areas in western Russia to northern Germany. Cluster assignments provided evidence of long-distance dispersal. The results of an Approximate Bayesian Computation analysis supported a scenario of equal effective population sizes among different pre-defined populations in the large central cluster. Our results are in line with strong gene flow and secondary admixture between neighbouring demes leading to reduced genetic structuring, probably a result of its fairly rapid population expansion after introduction. The results presented here are remarkable in the sense that we identified a homogenous genetic cluster inhabiting an area stretching over more than 1500km. They are also relevant for disease management, as in the event of a significant rabies outbreak, there is a great risk of a rapid virus spread among raccoon dog populations.The extent of gene flow during the range expansion of non-native species influences the amount of genetic diversity retained in expanding populations. Here, we analyse the population genetic structure of the raccoon dog ( Non-native species pose a great threat to the integrity of natural systems and are of evolutionary interest because genetic processes may play a role in their establishment and spread. Because genetic founder effects can be overcome by high propagule pressure, a positive association between introduction effort and invasion success has been reported –3. The eNyctereutes procyonoides) is a canid with a high reproductive rate, short generation times, high population turnover and a generalised diet )The results presented here have great relevance for disease management. First, the extent of its genetic homogeneity suggests the lack of any substantial landscape barrier to dispersal. Secondly, the absence of a (strong) IBD pattern, as well as some direct evidence from our assignment results, suggests frequent long-distance dispersal. This is in line with the speed of the historic spread of the species, as well as with several field studies reporting raccoon dog dispersal over large distances in relatively short period of time e.g. ,75,76), ,76, 75,7At present, it is impossible to know whether a pattern of high population connectivity and frequent long-distance dispersal, together with the resulting homogenous genetic structure, will be maintained after the end of the species spatial expansion. While perhaps species-specific, there are examples in the literature of reduced dispersal in high-density populations . BecauseS1 FigThe red star indicates the geographic coordinate for each pre-defined population  and the black lines the least-cost distance separating the populations .(TIF)Click here for additional data file.S2 FigPop 1 = Source population , Pop 2 = Eastern Poland; Pop 3 = Brandenburg/Saxony; Pop 4 = Schleswig-Holstein; Pop 5 = Denmark.(TIF)Click here for additional data file.S3 FigPlot of the number of genetic clusters tested against their estimated log-likelihood. STRUCTURE was run using the admixture and correlated allele frequencies models.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


With the expansion of novel chromosome testing, a career as a certified genetic counselor has been gathering a lot of attention. However, few people certified as a genetic counselor after completing postgraduate courses are able to find employment as a genetic counselor, and their salaries are quite low. It is also questionable whether or not such newly graduated genetic counselors, who have limited life experience and knowledge, can fully understand family issues and properly perform counseling sessions. To address these issues, a wide range of education and training may be necessary. In this study, we examined current problems in genetic counseling education in Japan, and proposed effective measures to address these problems. Toward creating a new society, we are currently establishing a national qualification system and cultivating qualified professionals capable of providing patients with accurate information on chromosome and genetic testing. In addition, these professionals could encourage younger generations to have an interest in genetic counseling. I also hope that these professionals will work not only in Japan but all over the world. Early on, in Europe and the United States, genetics education was conducted in terms of human genetic diseases. According to WHO guidelines, genetics education from elementary school is recommended. In Japan, students have mainly studied Mendel’s law –3.Many years have passed since the completion of the Human Genome Project. Based on the information obtained from the project, personalized medical practice, specific gene tests, genetic tests, and pharmacogenomics have developed in Japan –11. A nuIn Japan, promotion of genetic counseling  as defined by the American Society of Human Genetics (ASHG) began in the 1970s. At that time, genetic counselors were mostly basic researchers, medical researchers, and physicians  . HoweverIn response to these developments, the Japanese Society of Human Genetics (JSHG)  and the In Japan, various practical guidelines have been established –11. Of pIn order to become a genetic counselor in Japan, it is necessary to graduate from a 4-year university course, have operational experience as a professional of medical welfare of 5 years or longer, and take courses on human genetics, counseling, psychology, bioethics, statistics, physical education, role playing, and other subjects in a graduate school in principle. After completing a thesis and graduating from the school, it is possible to take a certification examination to become a genetic counselor, and those who pass become qualified.Genetic counselors work in genetic service settings, such as universities, pediatric clinics, and general hospitals –11. TheyAging of the Japanese population is rapidly progressing; conditions such as dementia and other medical concerns of elderly people are increasing demands for nursing care. When such people become patients, it is extremely difficult to explain chromosome and gene analysis, diagnosis, and treatment in ways they can comprehend. Moreover, patients of any age may become anxious, thus further inhibiting their ability to comprehend complex genetic information. Therefore, it is imperative for paramedics  that play roles in team medicine to be knowledgeable about genetic testing, genetic conditions, and genetics, and also have the skills for explaining this information to patients. In Japan, it is not sufficient just for physicians to provide accurate and straightforward information to patients in order for them to make a decision. In addition to physicians, support by paramedics may be important considering the length of time they spend with patients. The medical fee structure was recently revised in Japan, and genetic counseling and chromosome and gene testing became widely covered by health care insurance. For example, the fee for genetic counseling is 5,000 yen for use of services once per month (about 60 U.S. dollars). Thus, these services are likely affordable for many patients.As described above, Japanese were previously ashamed of having a handicapped child and they tended to be pessimistic about the child’s future. Moreover, many people who have no one with a congenital abnormality in their lives regard congenital abnormalities as unusual and not of concern to them. Most Japanese, even paramedics, may not know that congenital abnormalities are the cause of 1/3 of neonatal deaths . In poinWhen chromosomal aberration additionally occurs in translocation carriers, it is difficult to determine the theoretical probability. Some patients seek genetic counseling about chromosomal aberration due to influences of drugs and radiation. However, it is impossible to identify what chromosomal aberration is induced by which environmental factor, excluding specific chromosomal aberrations, because fertilized ova with chromosomal aberrations are frequently produced in any individual , 19.When an abnormality is detected by genetic testing, pregnancy termination is considered; however, no legal provision currently exists concerning fetuses before 22 weeks of pregnancy in Japan, and therapeutic abortion is performed based on lawful reasoning in accordance with the Maternal Protection Act. The decision is between doctors and their patients . In geneHereditary diseases that the general public has never heard of now create anxiety, and concerned individuals visit many hospitals. Hospitals in Japan have increasingly opened departments specializing in hereditary diseases, such as gene therapy and hereditary disease treatment. Several departments specializing in hereditary diseases have developed websites offering information about facilities providing genetic counseling and containing the latest genetic medical information. For example, “Iden Net”  is mainlRecently, the position of genetic nurse has been established in the UK, and training for this position has begun in Japan. According to WHO guidelines, not only physicians but also all health care providers, such as nurses and public health nurses, are advised to have the ability to provide genetic counseling irrespective of their professions. According to WHO, the profession and population ratio of medical geneticists in the advanced countries is about 1:220,000, and about 1:3,700,000 in developing countries and Japan. Therefore, Japan needs to accelerate further training .Genetics is not taught in Japanese high schools , 29. In Owing to the advice of the ASHG  and the Chromosome and genes analysis technologies have been advancing rapidly, such as the PCR and genome-wide association study (GWAS) . RecentlGiven such advancement, human genetics must be studied by healthcare providers engaged in chromosome and genes analysis as well as the general public of not only East Asian countries but also countries throughout the world.I have provided some proposals about the improvement of the quality of genetic counseling education Table . Since mIn Japan, employment of genetic counselors is a critical problem. It has been difficult for genetic counselors to find desired employment. Because they are not required to obtain a national qualification, their position is not well accepted in the medical field. In addition, salaries are quite low for genetic counselors. Therefore, it is necessary to improve their work situation and modifications are required in order to meet the current needs of Japanese society.In order to address the problems described in Table The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhipidura cyaniceps), White-browed Shama (Copsychus luzoniensis), and Lemon-throated Leaf-Warbler (Phylloscopus cebuensis). Each species has two described subspecies within Greater Luzon, and a single described subspecies on Greater Negros/Panay. Each of the three focal species showed a common geographic pattern of two monophyletic groups in Greater Luzon sister to a third monophyletic group found in Greater Negros-Panay, suggesting that common or similar biogeographic processes may have produced similar distributions. However, studied species displayed variable levels of mitochondrial DNA differentiation between clades, and genetic differentiation within Luzon was not necessarily concordant with described subspecies boundaries. Population genetic parameters for the three species suggested both rapid population growth from small numbers and geographic expansion across Luzon Island. Estimates of the timing of population expansion further supported that these events occurred asynchronously throughout the Pleistocene in the focal species, demanding particular explanations for differentiation, and support that co-distribution may be secondarily congruent.Phylogeographic studies of Philippine birds support that deep genetic structure occurs across continuous lowland forests within islands, despite the lack of obvious contemporary isolation mechanisms. To examine the pattern and tempo of diversification within Philippine island forests, and test if common mechanisms are responsible for observed differentiation, we focused on three co-distributed lowland bird taxa endemic to Greater Luzon and Greater Negros-Panay: Blue-headed Fantail ( Widespread lowland rainforest bird species are of great interest for studying phylogeographic structure, which links genetics and geography , 2, 3, 42, e.g. ),2, e.g. , 34, 44,2, e.g. ). Howeve2, e.g. ).The Philippine archipelago provides a natural model to test for genetic differentiation in continuous habitats within islands. With the exception of Palawan and some of its offshore islands which were apparently united to the Sunda Shelf (reviewed in ), the PhRhipidura cyaniceps, Rhipiduridae), White-browed Shama  and Lemon-throated Leaf-Warbler . These taxa are co-distributed within the Greater Luzon PAIC, which includes the present-day islands of Luzon, Polillo, Catanduanes, and Marinduque; and in the Greater Negros-Panay PAIC, which includes Negros, Panay, Cebu, Ticao, and Masbate islands. Each species is common in forested habitats on these islands, and they are regularly found together in mixed species flocks. Both inter- and intra-island plumage differences have been documented in these species, as suggested by recognized subspecies throughout their range , and single subspecies are endemic to Greater Negros-Panay.The dominant paradigm for explaining the high diversity levels in the Philippines has been based on the arrangement of exposed land among aggregated islands during Pleistocene sea-level changes (reviewed in ), predic0) corresponding to the classical PAIC paradigm, in which genetic differentiation is expected to be partitioned between clades restricted to PAICs; alternative hypotheses involving genetic differentiation as a result of different colonization events, resulting in unrelated clades in a single PAIC (H1), and within-island genetic differentiation, in which sister clades are distributed in a single PAIC (H2).We used tools from phylogenetics and phylogeography to estimate historic demographical patterns in these co-distributed Philippine passerines. Given the extensive effects of Pleistocene climatic changes in Southeast Asia , 52, 53,R. cyaniceps is also found on Tablas Island (Romblon Island Group). Neither R. cyaniceps nor C. luzoniensis are present on Cebu  are a monophyletic group, thus we included samples of the other two endemic Philippine species. Finally, because the monophyly of P. cebuensis has been questioned, with some authors lumping this species and P. olivaceus into a single taxon [P. cebuensis [P. ijimae and P. coronatus as additional outgroups [Because species-level relationships of ern Asia , 20. Out studies ,56, showle taxon , 58, we ebuensis . We alsoutgroups .http://www.qiagen.com/). Markers selected for this work have been used widely in bird systematics and biogeography, and include the entire second subunit  and third subunit  of nicotinamide adenine dinucleotide dehydrogenase. These markers were amplified with the external primers L5215 and H6313 for ND2 [DNA was extracted from frozen tissue using Proteinase K digestion procedures following the manufacturer’s protocols , glyceraldehyde-3-phosphodehydrogenase intron 2 , and beta-Fibrinogen intron 5 FIB5 565 bp). The and FIB6  as well  bp. The and FIB6  and Myo2and FIB6 , 68, as Genomic DNA was amplified using 5-primeTaq DNA polymerase under standard PCR thermocycling protocols and visualized in agarose gels stained with ethidium bromide. Resulting products were cleaned with ExoSAPIT  and the purified products were cycle-sequenced with ABI Prism BigDye v3.1 terminator chemistry. Cycle-sequenced products were purified with ethanol precipitation, and sequenced on an ABI 3730 automated sequencer. Sequences were aligned using MAFFT , as impl7 generations and sampled every 500 generations. Stationarity of the MCMC chains was assessed in Tracer v1.5.0 [Phylogenetic relationships were reconstructed from the concatenated dataset for each taxon through Maximum Likelihood analysis (ML) as implemented RAxML 7.0.3 , which ar v1.5.0 , after wr v1.5.0  by usingAll population parameters were estimated from the mitochondrial dataset (ND3 and ND2) for each species. Genetic diversity was assessed using indices of haplotype diversity (Hd) and nucelotide diversity (π), with samples grouped by present-day island boundaries .R. cyaniceps and P. cebuensis, but with the Bicol Peninsula clade in C. luzoniensis. These arrangements suggest particular dynamic biotic processes in the Zambales region, for which we conducted separated analyses for the populations of the three species. As a relative measure of divergence between populations, we estimated Nei’s genetic distance values (Dxy), using DNAsp v5 [Following the phylogenetic results, Luzon samples were further divided according to the obtained clades, corresponding to northern Luzon and the Bicol Peninsula. However, the geographic structure in these clades showed the Zambales region (in western Luzon) samples grouped with the northern Luzon clade in both DNAsp v5  with a JDNAsp v5 , betweenFst. For the AMOVA analysis, samples were arranged in groups corresponding to the clades found in the phylogenetic analyses. The significance of the AMOVA results was assessed through 10,000 non-parametric permutations. The parameter Fst was calculated through pairwise differences between haplotypes; significance of the Fst parameter was also assessed with 10,000 permutations. Finally, as an additional measure of gene flow, we calculated the number of migrants per generation (Nm). Because some studies have shown that populations of R. cyaniceps and C. luzoniensis in the Greater Negros-Panay PAIC and the Romblon Island Group are evolutionarily distinct from those in Greater Luzon [Fst and Nm values followed the guidelines in Hartl and Clark [Genetic structure in the three species was explored using three-way AMOVA and er Luzon , 55, 81,er Luzon . Interprnd Clark .Fs statistic [s was determined using a p-value of 0.02 as suggested by Fu [D statistic [s and Tajima’s D were calculated by constructing 1000 coalescent simulations in DnaSP v5 [We tested whether populations of the three species had experienced demographic changes by calculating Fu’s tatistic , which ied by Fu . We alsotatistic , anotherDnaSP v5 .2, which is better suited for small sample sizes [2 significance was estimated through 1000 coalescent simulations for the different clades  of each species in DnaSP v5 [t of potential step-wise expansion from a relatively small, but constant population to a large population of size Ɵ1 over t generations in the past, with t = τ/2u, where τ = age of expansion  and u = 2μk, where μ = mutation rate and k = the length of the sequence [−9 substitutions/site/year (s/s/y), which is supported by a large dataset of studies on passerine birds [−9 s/s/y, which is a ND2 specific mutation rate calculated from mockingbirds in the Galapagos Islands [-9 s/s/y, which accounts for uncertainties about mitochondrial substitution rates [Population history was further inferred by plotting mismatch distributions , 87 and le sizes . R2 signDnaSP v5 . For clane birds , 2.7 x 1 Islands , and a fon rates , 92. Somon rates  often leon rates . Thus, won rates . Confideon rates , as implon rates .http://www.fluxus-engineering.com), assigning equal weights to all variable sites and with default values for the epsilon parameter (Ɛ = 0).Finally, because it has been suggested that bifurcating trees may not always fully represent intraspecific phylogenies due to the coexistence of ancestral and derived haplotypes in a given sample , we alsoR. cyaniceps, 2198 characters for C. luzoniensis, and 2429 characters for P. cebuensis. Sequences for R. cyaniceps, C. luzoniensis, and P. cebuensis are deposited in GenBank  dataset were identical to those obtained from the complete dataset in oniensis  and P. cebuensis . NuclearR. cyaniceps and P. cebuensis, but with the Bicol Peninsula clade (including Catanduanes Island) in C. luzoniensis. One additional monophyletic grouping included the Tablas island samples of R. cyaniceps, as previously reported [ML and BI results were largely congruent in all three cases Figs , and 5. reported .R. cyaniceps and C. luzoniensis, consistent with recent systematic treatments in which these populations have been recognized as full species ctively, . High lectively, .2 values  showed a bimodal pattern, suggesting either that for both species, there has been a relatively constant effective population size in the past [R. cyaniceps and C. luzoniensis showed a multimodal pattern, suggesting either population bottleneck or persistence of small and isolated populations, which seems highly probable as this PAIC is presently partitioned in smaller islands fragments, in comparison to Greater Luzon PAIC. However, these results are only preliminary and should be taken with caution, given the sample size and geographic coverage, which prevented conclusive demographic inferences, especially for P. cebuensis.Mismatch distributions  and Ramo2 values  showed sttleneck . The Zamthe past , or probFst values [Fst values (>0.72) were also obtained for within-PAIC comparisons, particularly involving Northern Luzon and the Bicol Peninsula populations in the three species. Values of Fst from populations in Zambales and Northern Luzon showed no significant genetic structure in R. cyaniceps and P. cebuensis, but were moderate (0.21) for C. luzoniensis populations from the Zambales and Bicol Peninsula. Gene flow was low [R. cyaniceps in northern Luzon and the Zambales (4.11 migrants per generation), and between populations of C. luzoniensis from Zambales and Bicol (1 migrant per generation). Population expansion in Northern Luzon was supported by significantly negative values obtained for Fu’s Fs [R. cyaniceps and P. cebuensis  and early to middle Pleistocene for P. cebuensis (0.8 mya). In the Bicol Peninsula, estimates of population expansion strongly suggest two different events. For C. luzoniensis, estimates suggest mid to early Pleistocene events, whereas populations of R. cyaniceps may have expanded in the late Pleistocene.Although estimated population expansion dates are heavily dependent on the mutation rates used, all of them and their respective confidence intervals fall within the Pleistocene . Median R. cyaniceps and C. luzoniensis, 40 and 41 mutational steps respectively) to low (3 in P. cebuensis) numbers of mutational steps separated Greater Negros-Panay populations from Luzon populations. Luzon birds were grouped in two different haplotype clusters in R. cyaniceps and C. luzoniensis, in which 19 and 7 mutational steps, respectively, separated the Bicol Peninsula clades from Northern Luzon clades. Haplotype networks in the three species showed a star-like pattern, suggesting historical demographic expansion after isolation [C. luzoniensis were observed in the Mingan Mountains in eastern Luzon, where analyses showed representatives of north and south haplotype groups. One of the haplotypes nested with the Bicol peninsula samples, the other nested with Northern Luzon samples . Althou0), phylogenetic trees of the three species showed monophyletic sister groups in Greater Luzon and Greater Negros-Panay PAIC. The pattern departs from strict PAIC expectation within Luzon, with substantial structure discovered in this clade in all three species (H2). Populations in each of the species showed levels of genetic divergence higher than those proposed for the recognition of full species [P. cebuensis. Genetic differentiation within Luzon is particularly evident in R. cyaniceps and C. luzoniensis, where high Fst values are coupled with a low estimated number of migrants, suggesting the existence of a past barrier to gene flow within the island. Additionally, the association of samples from the Zambales region either with northern Luzon (in R. cyaniceps and P. cebuensis), or the Bicol Peninsula, as in C. luzoniensis suggests that this region in western Luzon has had a dynamic biotic history, in which lowlands may have connected at different times with Northern Luzon or the Bicol Peninsula lowland rainforests.Genetic differentiation in the three lowland passerine species in this study also corresponds to a mixed model. Consistent with the PAIC paradigm  gene flow in populations within the same PAIC , 54. ResOur study adds to the growing body of evidence showing that speciation and genetic differentiation may occur within a single island, probably as a consequence of the Pleistocene climatic changes. Recent studies for birds (reviewed in ) and mamResults in this and other studies have repeatedly suggested that the current taxonomy for the Philippine biota does not accurately reflect diversity in the archipelago, which means that current species diversity estimates are better interpreted as conservative , 115. BiP. cebuensis (1.3% average). Populations of the other two taxa in Greater Negros-Panay both show deep genetic differentiation from Luzon populations and diagnostic characters that allow them to be recognized as full species [Phylogenetic patterns showed two main clades: one grouped all of the Greater Luzon samples; the other grouped all of the Greater Negros-Panay samples. High genetic distances and Fst values indicate substantial differentiation between these populations in all focal species, with the exception of  species , 55.R. cyaniceps, the two recognized subspecies cyaniceps and pinicola were included in the same clade, suggesting that morphological variation may be clinal [pinicola is mainly restricted to pine forests [C. luzoniensis. Samples from Luzon Island were included in two sister clades, with populations from northern Luzon sister to those from western, central, and southern Luzon (Bicol Peninsula), and Catanduanes Island. Deep genetic divergence  and high Fst values  also suggest long isolation and consequent differentiation. Finally, the only case where taxonomy apparently agrees with our work is in P. cebuensis. The two recognized subspecies luzoniensis and sorsogonensis seem to match the monophyletic groups obtained; this subespecific arrangement is supported by a low genetic differentiation  but high Fst values .Additional genetic variation was also discovered within the bounds of a single PAIC. In Greater Luzon PAIC, phylogenetic analyses showed two well-supported monophyletic sister groups. This arrangement apparently agrees with current taxonomy, as there are two recognized subspecies for each taxon , 57; howe clinal  and even forests , 117. ThR. cyaniceps and C. luzoniensis found in Luzon Island. It has been suggested that evidence from a single dataset may not be enough, underscoring the need for additional evidence such as morphological and song characters [C. luzoniensis revealed that birds from Bicol and the Polillo islands differ from the northern Luzon birds in the size of the white spots on the undertail , suggesting that diagnostic characters are present. In R. cyaniceps, birds from the Bicol Peninsula apparently have darker plumage [P. cebuensis, which has the lowest genetic divergence but clear diagnosable characters; subspecies in Luzon differ in the amount and brightness of yellow in the throat and undertail coverts, being brighter in the Bicol Peninsula sorsogonensis [Throughout the northern Philippines, molecular studies in birds have found values of genetic divergence ranging from 2.7% to 13.8% between populations of the same taxon, suggesting that species boundaries in a number of avian species should be revised , 119. Tharacters , 121. In plumage . This sigonensis .Species delimitation may be controversial due to different philosophies and species concept applicability . This maS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The first documents mentioning Jewish people in Iberia are from the Visigothic period. It was also in this period that the first documented anti-Judaic persecution took place. Other episodes of persecution would happen again and again during the long troubled history of the Jewish people in Iberia and culminated with the Decrees of Expulsion and the establishment of the Inquisition: some Jews converted to Catholicism while others resisted and were forcedly baptized, becoming the first Iberian Crypto-Jews. In the 18th century the official discrimination and persecution carried out by the Inquisition ended and several Jewish communities emerged in Portugal. From a populational genetics point of view, the worldwide Diaspora of contemporary Jewish communities has been intensely studied. Nevertheless, very little information is available concerning Sephardic and Iberian Crypto-Jewish descendants. Data from the Iberian Peninsula, the original geographic source of Sephardic Jews, is limited to two populations in Portugal, Belmonte, and Bragança district, and the Chueta community from Mallorca. Belmonte was the first Jewish community studied for uniparental markers. The construction of a reference model for the history of the Portuguese Jewish communities, in which the genetic and classical historical data interplay dynamically, is still ongoing. Recently an enlarged sample covering a wide region in the Northeast Portugal was undertaken, allowing the genetic profiling of male and female lineages. A Jewish specific shared female lineage (HV0b) was detected between the community of Belmonte and Bragança. In contrast to what was previously described as a hallmark of the Portuguese Jews, an unexpectedly high polymorphism of lineages was found in Bragança, showing a surprising resistance to the erosion of genetic diversity typical of small-sized isolate populations, as well as signs of admixture with the Portuguese host population. Chuetas, an isolated Mallorcan community from the Moslem period, 10–13th centuries .The settlement of Jewish groups in Iberia undoubtedly occurred long time ago, although the exact date of their appearance is still today uncertain. The oldest archeological evidence of the Jewish presence in Iberia known so far, was recently found in the south of Portugal (Silves) with a chronology of 390 CE . Jews were at that time a demographically non-negligible minority with a very heterogeneous social status . Over thFrom the 12th to the end of the 13th century, the geography of Judaism changed significantly with the Christian settlement policies, to which the first Jewish medieval colonies owe their existence . From thnew-Christians or Conversos  and mitochondrial DNA (mtDNA), has played a central role in disclosing the demographic events that have shaped modern human population structure . Both geThe search for a common Middle Eastern origin of contemporary Jewish male lineages started with Hammer and Skorecki’s landmark genetic study of the Cohanim , a priesSubsequent studies  have sugA high-resolution Y chromosome haplotype analysis on unrelated Israeli and Palestinian Moslem Arabs showed a common pool for the male lineages. However, some significant differences were also detected between Jews and Arabs, suggesting a recent divergence of the Arab clade from the common ancestral population .The research on the Jewish Priestly lineages, Levites and Cohanim was again addressed by A very recent study  of the hFigure 1.Sephardic Jews were also included in studies of the Y chromosome phylogeography , in the Several studies refer to the putative contribution of Sephardic Jews to the Y chromosome genetic pool of particular geographic regions, like the Azores and Madeira islands  and alsoThe profile of male lineages in Portugal was drafted in a study comprising 663 male samples from the 18 administrative districts of Portugal and a typical western European composition was demonstrated by the high frequencies of haplogroups R1b1a-M269 (57.7%), I-M170 (6.1%), G-M201 (5.5%), and E1b1b-M81 (5.6%), as well as a Middle Eastern influence, denoted by the presence of J-12f2.1 lineage 10.4%; . PossiblThese samples were included in a larger group of self-defined Sephardic Jewish males not only from the Iberia Peninsula but also from other countries that received Jewish exiles after the decrees of expulsion in the 15th century. This group of self-defined Sephardic Jews was treated as a single group, therefore the inference of a genetic profile for the Portuguese Jews was not possible from the published data. In the following, we will focus on the results obtained exclusively for the 16 Jewish samples of Belmonte, obtained upon request to the authors .Figure 2. The Y chromosome SNPs analyzed allowed the definition of just three different lineages in Belmonte Jews: eleven individuals were classified as J-12f2.1, four as R1b1a-M269 and one as G-M201, with a frequency of 68.8, 25 and 6.2% respectively. The analyses of the STRs DYS19, DYS388, DYS389I-II, DYS390, DYS391, DYS392, DYS393, DYS438, DYS439, DYS385a, and DYS385b revealed a total of only four distinct haplotypes. In the R1b1a-M269 haplogroup two different haplotypes were detected, diverging one from the other by one mutation step (DYS389II), inside the J-12f2.1 haplogroup all the eleven individuals presented exactly the same haplotype, reflecting very low levels of genetic diversity among this Jewish community.The genetic profile of the Portuguese Jewish and non-Jewish male lineages can be seen in A completely different picture of the Portuguese male Jewish lineages was, however, brought to light when the descendants of the crypto-Jews from Bragança district were analyzed . In thisFigure 2).The SNPs typed allowed the discrimination of 10 different haplogroups and the analysis of the Y-STR loci revealed 41 different haplotypes. The most frequent haplogroups found were R1b1a-M269, J-12f2.1, and T-M70, adding up to 80.7% of the total sample  and T-M70 (15.8%). However, the high haplogroup diversity combined with the high (intra-haplogroup) haplotypic diversity are extremely surprising, as they show exactly the opposite of what is expected, namely a deep genetic diversity loss. Although inbreeding practices were sustained by the Portuguese crypto-Jewish communities, in the light of the obtained results it seems that its effects were less pronounced in the Bragança district compared to Belmonte, due probably to complex mating strategies and/or a very heterogeneous genetic pool in their origin.Haplogroup R1b1a-M269, representative of Western Europe, is the most common lineage found in the Portuguese general population 57.7%;  while inHaplogroup R1b1a-M269, emerges as the most frequent lineage in European individuals. Its distribution displays an increasing gradient moving from east to west , being tRST genetic distances between R1b1a-M269 Jewish haplotypes from Bragança district and those from Portugal  from the one found in Bragança.Haplogroup J-12f2.1 has a Middle Eastern origin and includes two groups, the J1-M267 and the J2-M172. Lineage J2-M172 is more common and is widely spread over Europe, particularly in the Mediterranean basin . HaplogrThis haplogroup is referred to as being predominant in diverse referenced Jewish populations , reachinThe high frequencies of J-12f2.1 haplogroup found in both groups of Portuguese Jews, compared to the non-Jewish Portuguese host population, could therefore represent part of the genetic pool of the ancestral Sephardic population that established the first Jewish settlements in Portugal. For this lineage, again, no exact matches were found between the haplotypes of Belmonte and Bragança district. Three mutational steps apart, one haplotype of Belmonte matches five individuals from Bragança, Carção, Argozelo, and Vilarinho dos Galegos.The presence of the mutation M70 defines haplogroup T. Its origin is attributed to the Middle East  and fromIn Portugal it accounts for just 1.6%  but reac10831.2 with 1.8%, and G-M201 with 3.5%.Several other haplogroups were detected in the NE Portuguese Jews with residual frequencies, namely E1b1b-M78 with 3.5%, E1b1b-M81 with 5.2%, I-M170with 3.5% R1b1-P25 with 1.8%, R1a-SRYG-M201 was also detected in Belmonte (6.2%) at about the same frequency as in the non-Jewish Portuguese population. The first work dealing with the maternal (mtDNA) lineages in Sephardic populations dates from 1986 . The autLater , HVRI seFigure 3.Similar results were found in later works , includiChuetas , with a presumable Sephardic Jewish ancestry were analyzed by Conversos), and samples from Portugal and Brazil, also consistent with a speculated Sephardic ancestry.Complementary strategies have also been used to study this possible Sephardic contribution in a more recent work . The anaFurther insights regarding the Sephardic signature inside T2e haplogroup and the genetic affinities of the T2e Northern Mexican samples were presented later . The mitIn parallel with the analysis of the male counterpart (Y chromosome), the mtDNA variation in Portugal was used to investigate the maternal heritage in the current Portuguese genetic landscape. The first detailed report on the Portuguese mtDNA, was done by Figure 4.The possible contribution of Sephardic lineages to the female Portuguese genetic pool was investigated as previously stated , 2010 neThe Belmonte community presented a very low diversity, with only two lineages detected, and all samples inside each haplogroup presented the same haplotype. The distribution of the haplogroups is uneven, since one of them, HV0b haplogroup, stands out with a frequency of 93.3%. On the other hand, the Bragança Jewish sample presented a much higher haplogroup diversity (32 haplogroups) as well as intra-haplogroup diversity (45 haplotypes). The haplogroups that stand out in the Bragança Jewish sample, when compared with the Portuguese host population, were: HV0b, N1, T2 and U2, adding up to 38.6%.The single mtDNA lineage shared between the two analyzed Portuguese Jewish series, Belmonte and Bragança, is HV0b. The expansion of the haplogroup HV0, including the haplogroup V, had been proposed to have an Iberian origin, after the last glacial maximum, and nowadays can be also found in North African populations. However, little is known about the distribution of the HV0b clade. According to the CR mutations, the specific defining combination variants  can be found at EMPOP forensic database in nine individuals, including one from Northern Africa; five from Central and North America; two from Central Europe; and one from Southern Portugal. Moreover, a recent paper focused on mtDNA variation in Andalusia (Southern Spain),  reportedLooking deeper into the variability of the HV0b clade through the comparison of complete mtDNA sequences from Bragança Jews (seven), Belmonte Jews (one) and GeneBank (two), reveals a shared common private variant in the coding region (8520G) of the Portuguese Jewish samples. Moreover, the Bragança samples cluster together, sharing a variant (10644A), which seems to have arisen locally. Although new complete mtDNA sequences are needed for a better understanding of this particular clade, the results available so far support the hypothesis that at least the HV0b-8520G haplotype is a Sephardic Jewish founding lineage.Another interesting clade that deserves further attention is T2e1. This clade was described as one of the founder lineages of the Bulgarian Sephardic community , and as Similar results regarding shared haplotypes between samples from Sephardic and Ashkenazi origin were found inside U2e1a1 sub-clade. All four U2e1a1 samples from Bragança Jews share the same haplotype with a Jewish Ashkenazi sample from Moldova , presentThis pattern of shared haplotypes between Sephardim and Ashkenazim samples, firstly described by In conclusion, the demographic processes underlying the genetic pool of the Portuguese Crypto-Jews descendants studied so far, are much more complex than would be expected under the classical model of extreme inbreeding and drift, with consequent loss of genetic diversity. The contrasting patterns observed in Bragança community and Belmonte are enough to sustain that whatever the results of future studies, no simple and uniform evolutionary model will accommodate the sharp heterogeneity already observed.Notwithstanding this difference, both groups display a genetic pool clearly showing contributions of European and Near Eastern lineages, in accordance with a significant persistence of a Jewish heritage, translated in a conscience of belonging to a distinctive community. This ancestry was detected within both male and female lineages, indicating that introgression from and admixture with the host population does seem to have been significantly gender biased.Moreover, the high genetic diversity found in Bragança demonstrates that there was neither a low number of founder lineages, nor a significant reduction of effective population size as indeed occurred in Belmonte. It remains to be explained how this resistance to genetic erosion, as expected in endogamous, small sized populations, was achieved, that is to say, what mating strategies were undertaken by these communities which ensured a steady gene flow between them, counteracting the expected inbreeding.New data from recombining genetic markers in the line of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.