In 2013 both Saudi Arabia and Qatarlaunched genome projects with the aim of providing information for better diagnosis, treatment and prevention of diseases and, ultimately to realize personalized medicine by sequencing hundred thousands samples. These population based genome activities raise a series of relevant ethical, legal and social issues general, related to the specific population structure as well as to the Islamic perspective on genomic analysis and genetic testing. To contribute to the debate, the Authors after reviewing the existing literature and taking advantage of their professional experience in the field and in the geographic area, discuss and provide their opinions. In particular, the Authors focus on the impact of consanguinity on population structure and disease frequency in the Arab world, on genetic testing and genomic analysis  and on their regulations. A comparison between the Islamic perspective and the ethical, social and legal issues raised in other population contexts is also carried. In conclusion, this opinion article with an up-to-date contribution to the discussion on the relevance and impact of genomic analysis and genetic testing in the Arab world, might help in producing specific national guidelines on genetic testing and genomic analysis and help accelerate the implementation and roll out of genome projects in Muslim countries and more specifically in Qatar, and other countries of the Gulf. In 2004, the Arab population had an estimated size of more than 300 million people, contributing to 5 % of world populations, living in 22 countries across North Africa and West Asia, including the Middle East [The history of Arabs extends more than 5000 years. Around 3500 BC, Semitic-speaking people of Arabian origin migrated into the valley of the Tigris and Euphrates rivers in Mesopotamia, eventually becoming the Assyro-Babylonians. About 2500 BC, another group of Semites left the Arabian Peninsula and settled along the eastern shores of the Mediterranean; some of these migrants became the Amorites and Canaanites of later times. Starting 7th century, Arabs, proclaiming the new religion of Islam, ventured from the Arabian Peninsula and conquered the wide area from the Persian/Arabian Gulf to the Atlantic Ocean , 2. Todadle East . ResidenFigure The rates and trends of first cousin marriages and other consanguineous marriages can vary hugely within and between populations and communities. Many factors determine these variances and they include ethnicity, religion, culture and geography. Emigrant communities from highly consanguineous countries such as Turkey, Pakistan and the Maghreb that are now resident in Europe, North America and Australia continue with the tradition of consanguineous marriages .Consanguineous marriages are favored due to many sociocultural factors: the upholding of family groups and property, the better understanding of marital arrangements, the already established relationships with in-laws, and the monetary assets relating to dowry. Moreover, populations that favor consanguineous marriages believe that marrying within one’s family reduces health and financial risks . In the Consanguineous marriages are highly regarded and are used to preserve wealth. They also present the ideal chance to maintain cultural stability and values , 5. ConsConsanguineous marriage becomes possible when there is available close kin of comparable age with similar socioeconomic standing as well as a traditional system that advocates for or against distinct types of consanguineous marriage . In the In the Middle East, some regions have witnessed a decline in their consanguinity rates such as Jordan, Lebanon and Palestine. Several factors could have contributed to this decline: a more educated female population, smaller families offering a decreased number of marriageable close kin, growing urbanization and improved family finances. As the rates of infectious diseases decline, more attention is given to the health effects of consanguineous marriages. This increased public health awareness could have also contributed to the decline in consanguineous marriage rates –8.The decline in consanguineous marriage rates does not apply to all regions of the Middle East. Some Arab countries such as Qatar are seeing no significant reduction in their rates of consanguineous marriages. This could be attributed to the limited knowledge among the public regarding the nature of genetic risks that consanguineous marriages may present , 13, 14.In populations where consanguinity and endogamy are practiced, the probability that a husband and wife are carriers (heterozygotes) for the same gene mutation that has been passed down to them from a common ancestor increases. Furthermore, this husband and wife couple will probably have many children. Consequently the odds that one or more of their children will inherit two copies of the ancestral recessive mutation, one from each parent, is increased , 5. In pSome studies have shown a relationship between consanguinity and some genetic conditions and health problems such as phenylketonuria (PKU) , immunodA few studies have reported a lack of effects of consanguinity or even a mild protective effect on the risk of developing diseases like breast cancer. For example, Arab women whose parents are consanguineous displayed reduced breast cancer risk compared to those women whose parents are non-consanguineous , 25. ThiIt has been observed that no association exists between consanguineous marriage and Down’s syndrome. Nevertheless, it was noted that in some populations, the frequency of Down’s syndrome was elevated . ZlotogoThe connection between consanguinity and major congenital malformations remains controversial . It was Improving education about the health effects of consanguinity to help avoid feelings of guilt among the consanguineous populations.Educating the physicians on the importance of focusing on some specific diseases whose frequency might be increased due to consanguinity, for example: thalassemia, hearing loss, intellectual disability, etc.Offering premarital genetic screening for the most common population specific genetic diseases.The issue of consanguinity in Qatar and the Gulf region attracts much public attention. In many cases this issue is viewed negatively as a problem for both the family and the country. However, recent data clearly confirms that consanguinity has a minor impact on increasing disease frequency. Attention should be paid to:The term “genetic testing” refers to a series of techniques including molecular analysis of human nucleic acids (DNA and RNA). Genetic tests are used as a health care tool to detect gene variants associated with a specific disease or condition and for studying complex and quantitative traits such cardiovascular diseases, cancer, diabetes, etc. Other fields include (a) pharmacogenetics where genetic tests are used to predict the effectiveness of therapeutics and guide their administration, (b) nutrigenetics and lifestyle where tests are used to asses diet response, lifestyle habits, physical activity predisposition, etc. (c) and other non-clinical uses .Prenatal genetic tests are carried out on pregnant women to detect abnormalities/mutations in the fetus. Post-natal genetic tests are carried out after birth. Moreover, depending on their ultimate purpose, genetic tests can be divided into the following categories: (1) diagnostic, used to determine if the person is affected by a genetic disorder, (2) presymptomatic and predictive, when there is a family history of a genetic condition, undergoing genetic testing before symptoms actually appear may show if the person is at risk of developing that condition, (3) carrier testing, to detect the carrier status for recessive genetic diseases, such as thalassemia or cystic fibrosis, or if someone belongs to an ethnic group that has a high risk of a particular genetic disorder (e.g. Tay–Sachs disease in Jewish populations). The identification of a carrier status has relevant reproductive implications, (4) newborn screening, aimed at screening for certain genetic abnormalities that cause specific conditions such as PKU. In such cases, care and treatment can begin immediately following positive test results, (5) preimplantation testing, aimed, during an in vitro fertilization process, at diagnosing embryos for genetic defects in order to implant only those without them.Depending on the type of test to be carried out different sources of DNA can be used, such as blood samples, cheek and buccal swabs, saliva, amniotic fluid cells, chorionic villus sampling, fetal cord blood cells, etc.At the very early beginning, genetic tests were only available to detect chromosomal abnormalities . Then, with the development of the “Southern Protocol”, genetic testing also became available to investigate genes causing rare and inherited disorders like thalassemia or sickle cell anemia. The availability of PCR protocols led to a spread of tests that are able to detect even single point mutations in a very large number of genes underlying many inherited diseases.Nevertheless, the positive detection rate of genetic testing varies largely from 10 to 80 % or more depending on several different limiting factors: clinical and genetic heterogeneity, accuracy and sensitivity of the technology used, etc. More recently, thanks to a series of technological improvements and new high-throughput protocols, the variety of genetic tests has greatly increased to also include multiple genes tests . As a maOnce a genetic test is carried out, the information should be accurately transmitted and explained to the patient and professional experts. Genetic test results may present various different situations, detailed below.The identification of a genetic cause, in other words, a positive diagnostic genetic test, will help physicians determine the right treatment (i.e. genotype–phenotype correlations) as well as develop a management plan which will allow relatives to receive accurate risk assessments from a genetic counselor. The genetic counsellor might help the couple in making more informed family planning decisions. In the case of predictive tests where a positive test does not necessarily mean that someone will get that disorder, it would be possible to make lifestyle changes that may decrease the risk of developing a disease or decrease its impact.A negative result means that a genetic alteration was not detected by the test. The absence of results can be explained by (1) the accuracy and sensitivity of the test which does not fully guarantee a positive outcome, and (2) the presence of large genetic heterogeneity hampering the mutation detection. Of course, if someone does not have the genetic alteration, this finding does not necessarily mean he or she will never get the disease. Moreover, in some cases, a genetic test may not be able to provide helpful information for the presence of DNA variants whose pathogenic role is doubtful. The use of TRS (and more broadly MPS) increases the possibility to detect such “doubtful” variants leading to increased uncertainty instead of clear-cut results. In these situations, follow-up testing may be necessary and such variants might be reinterpreted over time, as we learn more and potentially develop functional assays. This finding raises the question of whether there is a legal or moral responsibility to report such variants and eventually to recall, in the future, patients and their relatives and if so, with whom does the responsibility lie?In many societies subjects deal with the cultural fear of being associated with a genetic disease and the following possible stigmatization within the community. This finding might lead to social disadvantages such as (a) marriage refusal if subjects belong to families with a known genetic disease and (b) consideration of another marriage in presence of affected fetuses or children. All family members are affected by the negative connotations associated with the presence of a genetic disease especially female family members. In the absence of major and effective therapeutical protocols for inherited diseases, genetic testing can help in making earlier and presymptomatic diagnosis as well as preventing a genetic disease through a selective termination of an affected fetus, which, of course, raises continuous ethical debates in all societies including the Islamic ones. The Islamic schools of thought have agreed that abortion is possible and lawful if continuation of the pregnancy will endanger a mother’s life , 33. MorGenetic risks should be accurately explained during pre- and post-genetic test counseling, preventive options including PND should also be presented and discussed and a formal discussion about termination of pregnancy and the Islamic fatwa should take place.Improved educational plans should be developed to help improve the understanding of genetics and genetic-related diseases among the general population as well as the physicians.Where applicable networks of referral centers for genetic testing should be established or better improved to take care of the large number of genetic diseases present in the region.http://www.personalgenomes.org).Since the completion of the sequencing of the human genome, the demand for genetic analysis in the human health care system has increased, and molecular genetic diagnostics are urgently needed. However, many genetic diseases are molecularly and clinically heterogeneous, and until recently, the available techniques lacked the necessary ability to analyze several genes in parallel. The recently introduced MPS technology of next-generation sequencing now offers the unique opportunity to extend molecular genetic analysis by introducing this high-throughput technique, and by developing tailor-made medical re-sequencing approaches for gene identification and for the diagnosis of heterogeneous disorders. Whole genome sequencing (WGS) allows for the identification of all the possible variants at once, including chromosomal copy number variants. Protein-coding genes make up 1 % of the human genome but contain about 85 % of disease-causing mutations . Therefohttp://www.cincinnatichildrens.org/service/g/genetic-hearing-loss/default/).However, all these technological and diagnostic achievements have not yet led to updated and comprehensive guidelines and algorithms for the genetic diagnosis of many genetic diseases, which may be easily accessed by all the stakeholders involved: patients, clinicians, and geneticists. For example, since the publication of the “Genetic evaluation guidelines for the etiologic diagnosis of congenital hearing loss” by the American College of Medical Genetics , only spAs to Qatar, the recent launch of the Qatar Genome Project will “chart a road map for future treatment through personalized medicine” in the country. The ambitious goal of providing each citizen with his/her own genome, will lead to the discovery of almost all disease genes affecting the Qatari population, as well as a better understanding of the interaction between genes and the environment. These findings will translate into more accurate and personalized treatments as well as preventive plans and new lifestyle indications. Moreover, this project will pioneer the use of new genomic tools in consanguineous populations hopefully showing the great potential that these new tools have to reshape healthcare delivery in Qatar, and in a more broad sense, in other regional countries and inbred populations.Finally, beyond the rapidly evolving technical aspects, several key ethical issues arise in the clinical translation of MPS. In particular, the following issues should be taken into account:(a) Return of results, and in particular disclosure of ‘incidental findings’ (if such a term may still be used to refer to something we are searching for: (a) Green RC & Genomics ; (b) strThe use of MPS technologies  to answer specific clinical testing indications might lead, under some specific circumstances, to results beyond the scope of the test itself . Thus, stakeholders must agree and determine which findings should be disclosed, when and in what manner. A good example is the list of recommendations published in 2013 by the American College of Medical Genetics and Genomics. They include a series of conditions and genes that should be considered, on the basis of clinical outcome and therapeutical options, ‘mandatory’ to return.The presence of variants with uncertain functional role raises a series of open questions. Have the subjects/patients the ‘right’ to receive all of their genetic information or should they only be informed of data that are currently considered clinically relevant data? In any case, what about reinterpretation? Who is going to provide it and when, how to further report the results, etc.? Again stakeholders should agree in advance on what actions to take.So far, classical and standard models of informed consent for genetic testing have been widely used. They are mainly administered through a genetic counseling session carried out by clinical geneticists and/or genetic counselors. During the counseling, testing options, risks, benefits and limitations of genetic testing are discussed in detail. This approach is not feasible with MPS data because of the large amount of genomic information that is generated by MPS technologies. If we are to uphold the necessity to discuss thoroughly all the information obtained, then the process will be long and tiresome. One way of resolving this problem is to create very well trained and multidisciplinary teams which would include geneticists, physicians and bioinformaticians amongst others. In all cases, the consent form should be prepared accordingly and must reflect this broad and complex situation. Alternatively, an individual undergoing MPS may be requested to complete a short online course explaining the advantages and disadvantages of this technology.Reiterating earlier discussion, genomic analysis may generate a wealth of new genetic information with clinical relevance. Some of this genetic data may also impact the patient’s relatives. For example, MPS data may identify a predictive risk factor for cardiovascular disease without any relevance to the patient: the patient’s biochemical and clinical parameters are normal, the patient leads a healthy lifestyle, etc. However, this data may be of possible value to other relatives who might have completely different lifestyle habits and additional genetic and or environmental risk factors. This finding is even more relevant in inbred populations such as the Qatari population where kinship is very high and genetic results from one individual can be used, at least in part, to draw conclusions related to his/her relatives.Huge efforts should be made related to informing and educating the public as to the great potential benefits arising from data, but also on the possible negative repercussions for the individual and his/her family should the data be casually and widely shared.Most likely, in the near future, genomic analysis will oblige the society to reevaluate the current standards of medical confidentiality and privacy, a situation similar to the extensive use of internet and how that has changed our perceptions of personal space and privacy. Henceforth, substantial resources should be allocated to rapidly develop appropriate policies, guidelines and rules.In the USA, three different federal agencies play a role in the regulation of genetic tests: Centers for Medicare and Medicaid Services (CMS), Food and Drug Administration (FDA), and the Federal Trade Commission (FTC). The CMS is responsible for regulating all clinical laboratories performing genetic testing, ensuring their compliance with the Clinical Laboratory Improvement Amendments of 1988. The FDA has the largest authority in terms of regulating the safety and effectiveness of genetic tests. Finally, the FTC aims at regulating genetic test advertising to ensure that it is not false or misleading. Regulations on genetic testing were and are still provided by a series of reports and acts such as: (1) NIH-Department of energy task force on genetic testing recommendation in 1997; (2) secretary’s advisory committee on genetic testing report in 2000; (3) secretary’s advisory committee on genetics, health and society report in 2008; (4) the laboratory test improvement act in 2006; (5) genomics and personalized medicine act in 2007; (6) genetic information nondiscrimination act in 2008; (7) framework for regulatory oversight of laboratory developed test (LDTs) in 2014; (8) FDA notification and medical device reporting for LDTs always in 2014.In the EU there is a lack of specific genetic legislation. Nevertheless, some general principles are clearly defined: genetic data pertaining to health is ‘sensitive data’ under the EU data protection directive, and is thus to be treated confidentially. Likewise, discrimination based on genetic features is prohibited in the EU member states. Despite this legislative deficiency at EU level, several national legislations have been approved so far. The first explicit law including genetic testing was enacted in France in 1994 . Soon after, specific legislations have been approved in Norway, Spain, Italy, Germany, Austria, Portugal, Sweden and Switzerland. In any case, most countries have applicable provisions for genetic testing in the general legal framework governing other medical, laboratory and professional activities in biomedicine, and other legislation, such as in vitro fertilization and PGD, data protection, patient rights, and discrimination.In Australia the provision of pathology services is regulated by the department of health and aging administration of the funding schemes for public health is by Medicare. Under the provisions of the health insurance act 1973 (Cth) (HI Act), only accredited laboratories are eligible for Medicare benefits for only those tests or services listed on the Medicare benefits schedule. Accreditation of testing services is administered by the national association of testing authorities (NATA), the only national accreditation body endorsed by the Commonwealth government to assess laboratory competency. The accreditation scheme is based on guidelines issued by the national pathology accreditation advisory council (NPAAC). Before providing a genetic test through the Medicare benefits it should be assessed in compliance with the NPAAC guidelines. Genetic testing services generally comply with, but are not legally bound to the ethical aspects of human genetic testing information paper and guidelines on genetic research released by the national health and medication research council (NHMRC).China’s regulatory position on genetic tests was quite unclear until March 2014 when the China food and drug administration (CFDA) acted to impose regulatory constraints on medical institutions (and other providers) related to the provision of clinical genetic tests. As a first measure CFDA issued a ban on all medical applications of ‘gene sequencing technology products’ such as disease prevention  and diagnosis, treatment and monitoring, health status assessment and genetic risk factor prediction. It applies to manufacture, import, sale or use (including software) of MPS technology and genetic testing.According to a quite recent survey from UNESCO , only Lebanon has written a law specifically formulated to regulate genetic testing (No. 625 of 2004). For all the remaining Arab countries, the issue of the regulation of genetic testing has been only partially addressed. In particular, many Arab countries, Bahrain, Tunisia, UAE, Saudi Arabia, Palestine, Oman, Kuwait, Algeria, Egypt and Jordan, have referred to genetic testing in relation to other procedures such as premarital screening, reproductive medicine, forensic medicine, biobanking, etc. or directly to worldwide accepted international guidelines and regulations. In some other cases, such as Syria and Morocco several laboratories perform genetic testing despite the absence of specific legislation. As regards to Qatar, quite recently the Shafallah Genetics Medical Centre has produced a formal operation manual that includes guidelines on genetic testing and release of information, stating that genetic data should not be given to insurance companies, employers, schools, or governments, except after obtaining the full informed consent of the person tested. Prenatal diagnosis should be offered to those who need it, but without applying any pressure on parents to agree to such testing. Strong attention should be paid in providing protection to minors and disabled persons. Genetic counselling should be given in a compassionate and professional manner, offering guidance and allowing individuals and families to make an informed choice.Till present, the Department of Biomedical Research of the Supreme Council of Health in Qatar has prepared several policies and guidelines related to genetic testing, however, these guidelines are only present in a specific policy on diabetes mellitus.A summary of the web links for some of the above mentioned agencies is given in Table The absence of clearly defined policies and regulations related to “sensitive data”, discrimination, data protection, and privacy in Qatar means that each genetic test provider center must develop its own policies based on international regulations governing this type of testing. In that effect, this current draft may be used as a helpful review.Qatari academic and research centers together with the other major players in Qatar, should collaborate to work on producing specific national guidelines on genetic testing and genomic analysis to be approved and released by the department of biomedical research in the supreme council of health. This legal framework will help accelerate the implementation and roll out of the Qatar genome project.This opinion article provides an up-to-date contribution to the discussion on the relevance and impact of increasing knowledge on genomic analysis and genetic testing in the Arab world. This new data can potentially form a new resource from which scientists and physicians can bring fresh insights to the world genomics community with a strong impact on understanding the genetic bases of mendelian as well as complex diseases . In this light, this paper might help in producing specific national guidelines on genetic testing and genomic analysis and further help accelerating the implementation and roll out of genome projects in Muslim countries and more specifically in Qatar, and other Arab countries of the Gulf.


Argiope bruennichi, deposited in two large German natural history collections. We tested the influence of different factors on the utility of specimens for genotyping. Our results show that not the specimen’s age, but the museum collection is a major predictor of genotyping success. These results indicate that long term storage conditions should be optimized in natural history museums to assure the utility of collections for DNA work. Using historical material, we also traced historical genetic and morphological variation in the course of a poleward range expansion of A. bruennichi by comparing contemporary and historical specimens from a native and an invasive population in Germany. We show that the invasion of A. bruennichi is tightly correlated with an historical increase of genetic and phenotypic variation in the invasive population.Natural history collections house an enormous amount of plant and animal specimens, which constitute a promising source for molecular analyses. Storage conditions differ among taxa and can have a dramatic effect on the success of DNA work. Here, we analyze the feasibility of DNA extraction from ethanol preserved spiders (Araneae). We tested genotyping success using several hundred specimens of the wasp spider,  Natural history museums provide a rich source for historical DNA studies. DNA from museum specimens can be used for taxonomic assignments –2, phyloSuch range expansions and biological invasions have been frequently observed in recent decades and often attributed to global climate change or increasing global trade . PopulatArgiope bruennichi  has rapidly expanded its distributional range since about 1930, from warm Oceanic and Mediterranean regions into increasingly Continental climate zones , marker type , marker size (long/short), sex/stage , distance of the museum collection to the sampling site [km], collection site, museum collection (Naturkundemuseum Berlin or Senckenberg Museum Frankfurt), and country of origin. As the design of the study was not orthogonal, i.e. some of these variables were strongly correlated, we excluded sex/stage, collection site, and country of origin, from the analysis. As several measurements (markers) were made on the same individual, we used Generalised Estimating Equations (GEE) with exchangeable correlation structure and binomial error structure. GEE estimates a population average model and accounts for repeated measures by specifying a working correlation structure . The linhttp://dx.doi.org/10.5061/dryad.r8n7c “Files: Sampling localities, Prosoma width, Alignment of complete historical COI sequences, Contemporary COI alignment”). While the latter study analysed the species’ range expansion on a wide geographic scale (across Europe), here we focused on two geographic regions, which are particularly well represented in our museum specimens: north-eastern Germany (primarily the states of Berlin and Brandenburg) and south-western Germany . The latter correspond to the historical native range edge of the spider, while the former to the first records of invasion. Contemporary material from both regions is easily accessible, making these native and invasive populations particularly interesting to study temporal genetic changes.We analysed a subset of published datasets of the mitochondrial COI gene, ~1200 bp) and morphological measurements (prosoma width) from an invasive and a native population (from [00 bp andWe distinguished specimens collected into (1) before the range expansion (i.e. before 1930), (2) during the early phase of the expansion (> 1930 ≤ 1960), and (3) after the species had started to massively expand (> 1960). Due to little genotyping success in very old native populations, we analysed combined mitochondrial data for all specimens from before 1960 for South-western German populations. DNA sequences were aligned using MEGA 6  under deWe tested for the influence of six factors on genotyping success in 397 specimens. All study variables in interaction with one another had significant effect on the genotyping success . The intThe main effect of the museum collection was highly significant. Genotyping success, disregarding the marker type or size, was 4.5 times higher from material deposited in the Naturkundemuseum in Berlin than from material deposited in the Senckenberg Museum Frankfurt . The intWe analyzed contemporary and historical COI sequences of 291 specimens from the first invasive population of the species in north-eastern Germany and the historical native range edge in south-western Germany. Our analysis shows a clear association of increasing genetic variation and the ongoing invasion . Before p < 0.001, p < 0.05). We did not find any significant change of body size variance over the studied time period in the native population . Although we found differences in morphological variation, we did not find significant changes of the mean body size in any of the studied populations . However, invasive spiders are generally smaller than native ones .In parallel to the increase of genetic variation after 1930, the body size variance of invasive populations increases markedly between 1930 and 1960  can be amplified in nearly 100% of samples. Molecular historical studies on spiders should thus rely on short PCR fragments. Indeed, such fragments can be sufficient for taxonomic or phylogeographic studies [Due to higher copy number, mitochondrial fragments outperformed nuclear markers in our analysis. As expected, a specimen’s utility for molecular analysis does also depend on its age. However, the age was among less important predictors of genotyping success. We also did not observe a clear threshold after which a specimen is no longer useful for DNA analysis as has been found in insects . Even anConsidering the strong effect of museum collection on genotyping success, it is highly advisable to perform initial test extractions on samples, to estimate the utility of a whole collection for historical DNA analysis, before embarking on large scale genotyping projects.A. bruennichi is a highly dispersive species, we did not find a comparable admixture and phenotypic change in native populations. This is surprising, as the analyzed south-western and north-eastern German populations are located next to each other. A selection against introgressing variation might be causative for the observed difference. While novel variation might be advantageous in continental Northern European climate, it could swamp adaptations in native populations [The success of range expansions and biological invasions has often been associated with genetic admixture of formerly isolated lineages . Such adulations . Howeverulations .S1 TableGenotyping success is indicated by 1 for success and 0 for failure.(XLSX)Click here for additional data file.


Genetic heterogeneity was evaluated with observed heterozygosity (HO). Best-fitting spatial autoregressive models were identified, using socio-demographic variables as covariates. Spatial analysis included surface interpolation and geostatistics of observed and predicted patterns. Prediction accuracy was quantified. Spatial autocorrelation was detected for both socio-demographic and genetic variables. Augsburg City and eastern suburban areas showed higher HO values. The selected model gave best predictions in suburban areas. Fine-scale patterns of genetic heterogeneity were observed. In accordance to literature, more urbanized areas showed higher levels of admixture. This approach showed efficacy for detecting and analyzing subtle patterns of genetic heterogeneity within small areas. It is scalable in number of loci, even up to whole-genome analysis. It may be suggested that this approach may be applicable to investigate the underlying genetic history that is, at least partially, embedded in geographic data.Aiming to investigate fine-scale patterns of genetic heterogeneity in modern humans from a geographic perspective, a genetic geostatistical approach framed within a geographic information system is presented. A sample collected for prospective studies in a small area of southern Germany was analyzed. None indication of genetic heterogeneity was detected in previous analysis. Socio-demographic and genotypic data of German citizens were analyzed (212 SNPs;  Accurate assessment of genetic heterogeneity is relevant to manifold fields, ranging from clinical research, pharmacogenetics, and statistical genetics, over forensic sciences up to evolution . In plaUncovering the genetic basis of complex traits remains an immense and urgent challenge in genetic epidemiological research. Great efforts are set to establish well-designed cohorts and large control samples, intended to serve as basis for genetic epidemiological studies. Besides restricting recruitment to individuals of uniform ancestry, a common strategy applied to efficiently gain a representative sample of the inspected population and to control for potential unknown population substructure is to collect samples in smaller geographical areas, usually in medium to large urban centers e.g., –9)..9]).Even well-characterized or supposedly homogeneous regions may still account for subtle genetic structure with potential geographical components , 10. SloMost available well-standardized methods in geographical genetics  were devWith the aim of unveiling modest amounts of population substructure in a small, admixed area we (a) searched for subtle patters of genetic heterogeneity and (b) explored potential predictors of the observed patterns. To this end, we combined statistical genetics with spatial statistics (geostatistics) within the framework of a Geographic Information System (GIS). A GIS provides a computational environment designed for spatial analysis of geographic data, therefore the most suitable framework to detect, to model, and to analyze the geographic variation of genetic diversity.E − 5% heterozygotes deficit) measured in terms of FIS values , HO ∈ .In order to run HO values were created for the following data sets: HO(ALL), and HO(GER). Interestingly, since the KORA S4 genotypes conform a control population pool for genetic studies ).ion (cf. ).Taking these findings of the KORA S4 sample altogether we can state that fine-scale spatial genetic variation may be assumed in the study area. Our results indicate that patterns of genetic heterogeneity can be present in small regions within Germany. In conclusion, it may be stated that the presented genetic geostatistical approach has the potential of being a powerful tool for detecting, modeling, and analyzing spatial patterns of genetic heterogeneity even within populations inhabiting small regions.Supplement 1 describes the KORA S4 sample.Supplement 2 presents additional analysis of spatial genetic differentiation based on conventional measures.Supplement 3 presents an analysis of genetic differentiation between urban and peri-urban areas.Supplement 4 provides a sensitive analysis of the effect on genetic variation of immigrants in the KORAS4 Survey.Supplement 5 summarizes results of additional multivariate analyses of spatial genetic differentiation.Supplement 6 describes the KORA S4 marker set.Supplement 7 provides an analysis of genetic relatedness of individuals included in the KORAS4 sample.


A Troublesome Inheritance. A letter http://cehg.stanford.edu/letter-from-population-geneticists/signed by 143 scientists, including 16 members of the PLOS Genetics Editorial Board, and PLOS founder Mike Eisen, criticized the book in the New York Times Book Review on August 8, 2014. As there is widespread interest in this issue from our Editorial Board, we have invited Marcus Feldman to publish his book review in PLOS Genetics.There has been considerable debate about the representation of human population genetics in a recent book by journalist Nicholas Wade: In 1969, Arthur Jensen ignited a decades-long debate when he wrote that it is a “not unreasonable hypothesis that genetic factors are strongly implicated in the average Negro–white intelligence difference” The Bell CurveThe Bell Curve elicited a flood of strong criticisms of the data used, the statistical analyses, and the policy inferences The errors in Jensen's choice of data A Troublesome Inheritance, we are now in a genomic age, where individual differences at the level of DNA can be detected. The early chapters present a hodgepodge of historical ideas about race, aggression, and genetics. On page 57, Wade gives an inkling of what will come in the last half of the book: “important aspects of human social behavior are shaped by the genes” and “these behavior traits are likely to vary from one race to another, sometimes significantly so” As pointed out by Nicholas Wade in the first half of A Troubled Inheritance from results on worldwide variation in DNA polymorphisms, namely microsatellite polymorphisms (The Rosenberg-Feldman studies) Whereas inferences on the causes of human behavioral variation referred to above were based on correlations between relatives, on pages 97–99 Wade develops his arguments for the genetic basis of social behaviors in the second half of Using data from 15 protein genes, R. C. Lewontin in 1972 From the data and analyses of worldwide molecular genomic variation, Richard Lewontin and I amplified the conclusions of Lewontin and Barbujani et al. as follows: “The repeated and consistent results on the apportionment of genetic diversity…show that the genes underlying the phenotypic differences used to assign race categories are atypical of the genome in general and are not a reliable index to the amount of genetic differentiation between groups. Thus, racial assignment loses any general biological interest. For the human species, race assignment of individuals does not carry with it any general implication about genetic differentiation” A Troublesome Inheritance, Wade's erroneous interpretation of its significance for racial differences becomes the basis for his entry into the “speculative arena at the interface of history, economics, and human evolution” (page 15). In the second half of the book, he claims that differences among continents in economic development, social institutions, and social behaviors are based in genetics. This classic correlation–causation error cannot be excused on the grounds that Wade is just speculating; continents can be distinguished genetically; they also have different economic and social histories. One cannot conclude, as Wade does, that the former causes the latter.Even though the between-continent fraction of genetic variation is small, as the reader discovers on leaving the first half of The first paragraph of chapter seven summarizes Wade's process of inference: “Each of the major civilizations has developed institutions appropriate for its circumstances and survival. But these institutions, though heavily imbued with cultural traditions, rest on a bedrock of genetically shaped human behavior; and when a civilization produces a distinctive set of institutions that endures for many generations, that is the sign of a supporting suite of variations in the genes that influence human social behavior.” I will focus on two of the studies invoked by Wade to justify his totally unfounded claims that differences in the societies of different continents  are due to their genetic differences.The first is by Gregory Clark, an economic historian who studies changes in interpersonal violence, literacy, the propensity to save, and the propensity to work in the English population from 1200 CE to 1800 CE Against the argument that changing culture may have been involved in the 600-year process, Wade states (page 160) that these “behaviors emerged gradually over several centuries, a time course more typical of an evolutionary change than a cultural change.” To justify his claim that 600 years is enough time to have produced “significant changes in social behavior” (page 161) of the English, Wade leans on experiments by Belyaev, who artificially selected silver foxes for tameness. The strength of this selection was extreme: “typically not more than 4 or 5 percent of male offspring and about 20 percent of female offspring have been allowed to breed” IQ and Global InequalityWade devotes almost four pages of chapter seven, the longest chapter in the book, to IQ. After claiming (page 190) that “intelligence is almost certainly under genetic influence,” he goes on to discuss the relationship between wealth and IQ and invokes the work of Richard Lynn and Tatu Vanhanen, in particular their book The Mankind Quarterly, described by the famous psychologist Leon Kamin as a “vulgarly racist” Lynn is known for his work as an associate editor of Wade gives the appearance (page 192) of care in interpreting Lynn and Vanhanen: “It is hard to know which way the arrow of causality may be pointing, whether higher IQ makes a nation wealthier or whether a wealthy nation enables its citizens to do better on IQ tests.” However, from his statement (page 203) about “the strong heritability of intelligence” and his belief  that in England “the children of the rich carried with them inheritance for the same behaviors that made their parents rich,” we can only assume that Wade believes there is a genetic basis for both IQ and wealth. His “arrow of causality” has two points, with genetics responsible for both IQ and wealth.This section of the book is redolent of the claims of Jensen, as well as Herrnstein and Murray, mentioned at the beginning of this review. It also harks back to claims by Taubman Why Nations Fail by Acemoglu and Robinson Wade goes even further than proposing a genetic basis for continental variation in wealth; he would have us believe that differences in economic and political institutions among populations have a genetic basis. On page 196, he criticizes the book Wade is using “evolution” here to mean the production and maintenance of genetic differences, and “variations in human behavior” is his euphemism for racial, and hence (in his understanding) genetic differences. He appears to backtrack slightly in the final chapter (pages 240–241), where he poses the paradox “that people as individuals are so similar yet human societies differ so copiously.” His resolution of the paradox is that these societal differences “stem from the quite minor variations in human social behavior…that have evolved within each race during its geographical and historical experience.” Again, “evolved” must be understood in genetic terms: it is “because of their institutions—which are largely cultural edifices resting on a base of genetically shaped social behaviors—that the societies of the West and East Asia are so different.”A Troublesome Inheritance are advised to heed the admonition by Guedes et al. concerning Ashraf and Galor: “bold claims on the basis of weak data and methods can have profoundly detrimental social and political effects” We can juxtapose Wade's conclusions on the genetic basis of racial differences in wealth, economies, and institutions with those of Ashraf and Galor on a similar topic. Their claim was that the high and low molecular genetic diversity characteristic of African and Native American populations, respectively, “have been detrimental for the development of these regions,” while “the intermediate levels of diversity associated with European and Asian populations have been conducive for development” A Troublesome Inheritance. I have no trouble with the existence of human genetic variation. It is Wade's dangerous interpretation, however speculative, of the meaning of human genetic variation that is indeed troublesome.Wade's premise is that molecular population genetics has shown sufficient variation between continents to define races. He argues (page 126) that these genetic differences are responsible for differences in individual social behaviors that “undergird” societal institutions, which themselves differ among races. Ironically, the molecular genomic data that have become available in the last fifteen years—the very data that Wade argues we need to bravely acknowledge—explain in terms of admixture, gene flow, demographic change, etc., why human genetic variation is arrayed the way it is. Echoes of the hereditarian arguments about racial difference in IQ and the reductionist arguments of sociobiology and evolutionary psychology resound in


Metallura tyrianthina.The ridges and valleys of the Andes create physical barriers that limit animal dispersal and cause deterministic local variation in rainfall. This has resulted in physical isolation of animal populations and variation in habitats, each of which has likely contributed to the evolution of high species diversity in the region. However, the relative influences of geographic isolation, ecoclimatic conditions, and their potential interactions remain poorly understood. To address this, we compared patterns of genetic and morphological diversity in Peruvian populations of the hummingbird m) between persistently humid and semi-humid environments was nearly 20 times higher when the habitats were contiguous (m = 39.9) than when separated by a barrier, the Cordillera de Vilcanota (m = 2.1). Moreover, the population experiencing more gene flow exhibited a lesser degree of bill length divergence despite similar differences in climate.Phylogenetic and variation partitioning analyses showed that geographic isolation rather than climatic dissimilarity explained the greatest proportion of genetic variance. In contrast, bill length variation was explained by climatic seasonality, but not by genetic divergence. We found that mutation-scaled migration rate  contains supplementary material, which is available to authorized users. In sum,Ecological speciation occurs when ecological factors, regardless of gene flow, catalyze reproductive isolation by natural selection . In the Given the lack of clear support for an exclusive role of geographic isolation or ecological speciation in driving Andean diversification, a better understanding of the speciation process in the Andes will likely come from addressing how geographic and ecological isolation operate in concert , 15. TheMetallura tyrianthina to understand the drivers of spatial diversity in functional morphology and genetics. The geological history of the Peruvian Andes generated a topographically complex landscape, with glaciated cordilleras >5000 m in elevation interspersed with deep river valleys  for evidence of long term trends. Runs ran for 105 steps of burnin and at least 107 steps post-burnin, sampling every 100 steps to generate 105 genealogies. For all runs, we employed a geometric heating scheme with 50 heated chains.To assess patterns of genetic divergence among sampling localities we calculated all pairwise Fst-values across the four sampled populations for each of the four loci in Arlequin v.3.5 . Secondlingbirds . These mm et al.  hypothesion size . Based os et al.  by settiND2 sequences were 1041 bp in length and included 76 informative sites (7.3 %). A 90 bp indel in the AK1 intron and a 15 bp indel found in the Bfib7 intron were removed before any analyses were conducted, leaving: 337 bp of AK1 (four informative sites–1.2 %), 629 bp of Bfib7 (five informative sites–0.8 %), and 594 bp of MUSK (four informative sites–0.7 %). There were no internal stop codons, indels, or anomalous substitution patterns in the ND2 sequences that might indicate amplification of pseudogenes. However, we excluded one sample (FMNH 433155) because it did not align with other sequences and BLAST returned ambiguous matches with a variety of hummingbird species. Mitochondrial and nuclear intron sequence data have been deposited on GenBank, accession numbers: KU527140-KU527416.M. t. septentrionalis, on the west side of the central Andes (clade B), which is 0.24 % divergent from a northern clade of M. t. smaragdinicollis on the east side of the central Andes (clade C); clade D of southeastern Peru, which is isolated from clade E (0.91 % divergent), of the upper Apurímac and Urubamba valleys, by the Cordillera de Vilcanota; and finally, an Ayacucho population (clade F) that is bounded by the Mantaro River to the north (0.67 % divergent) and the Aprurímac River to the south (0.79-1.04 % divergent). Some structure was also detected within clade C potentially due to the influence of the Huallaga Valley or isolation by distance. Fst values largely corresponded to corrected pairwise differences among clades  were recovered in the Bayesian phylogeny Fig. . This stred Fig.  and onlyMetallura tyrianthina. When DA was used as a metric for genetic distance, 33.9–85.3 % of the variance was explained by geographic isolation alone compared to the 0.9–40.3 % attributable to certain climatic dissimilarities among localities. In no instance did climatic variation explain patterns of genetic variation better than geographic distance  than climate (0.78–12.87 %). However, less of the overall variance was explained by geographic isolation and, using the same permutation test, none of the variance explained by geographic distance was found to be significant when climate was accounted for (and vice versa).Results of variation partitioning analyses corroborated a strong role for isolation by geographic distance, but not ecological isolation, in shaping patterns of genetic variation in Peruvian ce Table . Moreovep = 0.015; R2 = 0.024); however, body mass was also found to contribute very little to bill length variation in M. tyrianthina relative to climatic factors in an AICc analysis .The model including an interaction between PC1 of seasonality and PC1 of precipitation, plus their main effects, was the single best model explaining bill length variation, containing 77.8 % of the Akaike weight. The second best model consisted solely of the PC1 of seasonality . Although a number of models were competitively ranked with PC1 of seasonality  all of these models include PC1 of seasonality as a nested component and were thus considered uninformative  than across the Cordillera de Vilcanota (0.336). Significant genetic structure was also found between the two humid sites  across all loci and for ND2 and AK1 between the two drier sites (Urubamba and Paucartambo).nt Table . Signifim) based on all four loci across both humid to semi-humid comparisons were found to be greater than zero .IMa2 analyses of populations separated by the Cordillera de Vilcanota and between the two semi-humid sampling localities showed good evidence of convergence with ESS values >150 and all three runs exhibiting similar values for all parameters. By contrast, analyses of the populations along the Manu Road and comparisons between the two humid-site populations did not exhibit evidence for convergence  after sampling 250,000 genealogies despite similar results being obtained across each of the three independent runs. Migration rates . Although bill length in Paucartambo was slightly longer (average 0.47 mm) than birds from the adjacent humid slopes at Pillahuata, this result was not significant when corrected for multiple comparisons (p = 0.216). There were no differences in bill length between the two humid sites (p > 0.4).Cusco populations on the drier, west-facing slopes had longer bills than those on the humid, east-facing slopes Fig. . Bill leMetallura tyrianthina, a result consistent with studies of the Genus Metallura as a whole [M. tyrianthina as in many other taxa [Adelomyia melanogenys [. This suggests that hummingbird bill morphology does not evolve steadily over time, but diverges only in response to differential ecological pressures.Phylogenetic, network, and AMOVA analyses all point to an important role for topographic barriers in structuring genetic diversity in  a whole , 94 and  a whole , 95–97).her taxa , 97, 98.anogenys . Topograanogenys ). This sM. tyrianthina, patterns of plumage variation in the species appear to vary in association with topographic barriers and the degree of genetic divergence. A high Andean ridge separates the similar looking subspecies M. t. smaragdinicollis and M. t. septentrionalis, and the Marañón valley corresponds to turnover between the red-tailed subspecies, M. t. tyrianthina, and the two purple-tailed subspecies occupying the rest of Peru [ND2 locus; whereas divergence between red and purple-tailed subspecies distributed in similar environments across the Marañón valley was ~2.2 %. Concordance between tail color and genetic divergence in M. tyrianthina is consistent with the positive correlation between the extent of plumage and mtDNA divergence across the Marañón valley, based on multiple passerine genera [Although we found discordant patterns of genetic and morphological divergence in  of Peru . Plumagee genera . Plumagee genera ).Metallura tyrianthina occupying habitats characterized by semi-humid montane scrub were found to have significantly longer bills than birds in humid forest habitats  is likely conservative considering that the climate data is coarsely interpolated from unevenly distributed weather stations in the Peruvian Andes. In the Andes, consistent patterns of morphological divergence occurring in association with ecological dissimilarities have been found in Adelomyia hummingbirds [Glyphorhynchus woodcreepers [Adelomyia melanogenys, where 57 % of the variance in bill length was explained by precipitation seasonality, minimum temperature of the coldest month, and mean elevation [M. tyrianthina contribute to mounting evidence that the ecological differences generated by the topographically complex Andean landscape play an important role in promoting local adaptation and functional diversity in Andean taxa [Populations of ats Fig. . This maats Fig.  and seasats Fig. . Our estingbirds , 65 and creepers , 64. A slevation  especiallevation –102. Patean taxa , 33, 65.Anolis species [Glyphorynchus spirurus in Ecuador found that the Andean ridge separating eastern and western populations played the largest role in structuring genetic divergence. Whereas genetic variance within eastern or western populations was explained largely by ecological variation [M. tyrianthina, negative results for this test can be difficult to interpret due to factors such as insufficient time for divergence to occur at neutral loci or inappropriate ecological variables selected [The variation partitioning approach we used to test for IBE revealed that geographic distance, not climatic variation, explained the greatest proportion of genetic variance. These results provide additional evidence for an exclusive role of geographic isolation in promoting genetic divergence at the neutral loci studied. Our results resemble a similar study in Trinidadian guppies where no influence of ecological dissimilarity on genetic variation was detected . However species  and a me species  found coariation . Althougselected . Other dselected , consistselected . Thus, oIn a wide array of taxa, mismatches between morphology and neutral genetic diversity have been interpreted as evidence for phenotypic divergence with gene flow , 105–108We found an inverse relationship between bill length divergence and gene flow, with greater divergence in bill length found across the Cordillera de Vilcanota than the Manu Road Fig. . A similM. tyrianthina colonized the central Andes from the north [An alternative explanation for differences in bill length between Urubamba and Paucartambo is that they reflect differences in the amount of time spent in semi-humid environments. In support of this possibility, migration-rate estimates from IMa2 can be spuriously high, according to two recent simulation studies , 112. Bohe north , 51, 113Glyphorynchus spirurus [Although subtle morphological divergence in the face of gene flow has been demonstrated in other Andean birds (e.g. spirurus ), phylogspirurus , 43, 51.M. tyrianthina and other Metallura species [Metallura tyrianthina. First, longer bills in drier, less diverse environments could be caused by competitive release, analogous to bird populations on depauperate islands which tend to exhibit divergent bill morphologies as part of a more generalist phenotype [M. tyrianthina has a shorter bill than most other hummingbird species in the humid forests of east-facing Andean slopes. Longer bills might allow M. tyrianthina to exploit a greater variety of flowers on rain-shadowed slopes that harbor fewer competing nectarivore species. Second, diversifying selection on bill length could be exerted by flower species that have different corolla shapes, a co-evolutionary process that has been well-documented in other hummingbird species [M. tyrianthina because striking floral species turnover coincides with the climatic gradients along which it occurs.Repeated evolution of longer bills in drier, more seasonal habitats suggests adaptation by natural selection, although the mechanism by which increased bill length may increase fitness in these environments has yet to be tested. Given that climatic variation likely covaries with a number of other potential ecological pressures, pinpointing the mechanistic basis of inter-population variation in bill length would require rigorous field observations and experimentation. Climate could play a direct role in driving bill length differences; however, bill length variation in  species  directly species . Additio species , 116 hum species . Insteadhenotype –120. M.  species –57, 121;Metallura tyrianthina. Across the Peruvian distribution of this species, we found that topographic barriers play the dominant role in structuring effectively neutral genetic diversity, whereas climatic variation shapes patterns of bill morphology. Moreover, analyses across four sites in the department of Cusco indicated that topography promotes genetic structure even across small spatial scales (<20 km) and bills are longer on rain-shadowed, west-facing slopes. This fine-scale analysis also revealed how gene flow constrains functional divergence, suggesting that topography and climatic variation interact to promote Andean diversification. If functional divergence driven by climatic variation permits coexistence upon secondary contact [Metallura, the east-to-west precipitation gradient may have played an important role in the diversification of several avian genera that contain narrowly distributed and geographically variable taxa in the central Andes . Indeed, analyses comparing speciation rates among Andean and adjacent lowland lineages have found overall higher speciation rates in the Andes, a pattern driven in part by genera such as Cranioleuca [Spinus [Andean ridges and valleys hinder connectivity among populations and also create local climatic variation that imposes differential selection pressures , 46. We  contact , it coul contact . These p contact , 123. Banioleuca , 125 and [Spinus . TopograThe bill length dataset supporting the results of this article are included within the article ). Mitochondrial and nuclear intron sequence data have been deposited on GenBank, accession numbers: KU527140-KU527416.


Eriocheir sinensis is an economically important decapod crustacean in China. Despite a widespread distribution and production in China, the resources of E. sinensis have experienced a dramatic decline in the past decades. Here we describe a new set of novel polymorphic microsatellite loci to facilitate the investigation of genetic structure and artificial breeding.The Chinese mitten crab E. sinensis was developed from EST databases. The number of alleles per locus ranged from three to twenty. The observed and expected heterozygosities ranged from 0.047 to 0.932 and from 0.047 to 0.935, respectively.In this study, a set of 30 novel polymorphic microsatellite markers for E. sinensis.These informative microsatellite markers will be useful in studies of genetics, genomics and marker-assisted selection breeding in  Eriocheir sinensis is one of the most economically important aquaculture species in China [E. sinensis have experienced a dramatic decline in the past decades due to overfishing and water pollution [E. sinensis is almost completely from artificial breeding. Unfortunately, like many other cultured species, the aquaculture performance of E. sinensis has declined significantly. In order to protect genetic diversity and prevent population degradation, understanding population genetic structure and genetic connectivity among populations and making a genetic linkage map are necessary.The Chinese mitten crab in China  due to iollution . In ChinE. sinensis [E. sinensis (2n = 146) [E. sinensis.Microsatellite markers provide a powerful tool in genome researches due to their wide distribution, codominant inheritance and high polymorphism. To date, approximately 83 microsatellite markers have been developed and applied for n = 146) . In thisE. sinensis ESTs obtained from the GenBank database (2013) were screened using SSRIT program [A total of 17067  program  that wasE. sinensis individuals were randomly captured from Xieyuan Fishing Company in Qilihai region in Tianjin City, China. Genomic DNA was extracted from the leg muscles using a modified phenol-chloroform protocol [Taq DNA polymerase (Takara), 1× PCR buffer, 0.2 mM dNTP mix, 1 μM of each primer set, 1.5 mM MgCl2 and about 100 ng template DNA. The PCR profiles for all loci were an initial denaturing at 94 °C for 3 min, followed by 35 cycles of 1 min at 94 °C, 1 min at the annealing temperatures listed in Table Sixty cultured protocol . PolymerE. sinensis. The number of alleles at each locus ranged from three to twenty with an average of 8.767 alleles per locus ; GenBank accession numbers see Table The microsatellite sequences are available through the National Centre for Biotechnology Information (


We applied a quantitative genetic method to derive the first heritability estimate of survival under elevated CO2 conditions in a metazoan. Specifically, we reared offspring, selected from a wild coastal fish population , at high CO2 conditions (∼2300 μatm) from fertilization to 15 days posthatch, which significantly reduced survival compared to controls. Perished and surviving offspring were quantitatively sampled and genotyped along with their parents, using eight polymorphic microsatellite loci, to reconstruct a parent–offspring pedigree and estimate variance components. Genetically related individuals were phenotypically more similar  than unrelated individuals, which translated into a significantly nonzero heritability (0.20 ± 0.07). The contribution of maternal effects was surprisingly small (0.05 ± 0.04) and nonsignificant. Survival among replicates was positively correlated with genetic diversity, particularly with observed heterozygosity. We conclude that early life survival of M. menidia under high CO2 levels has a significant additive genetic component that could elicit an evolutionary response to OA, depending on the strength and direction of future selection.Assessing the potential of marine organisms to adapt genetically to increasing oceanic CO Unfortunately, such in vitro approaches are largely unfeasible in metazoans with substantially longer generation times. In vitro selection for OA-relevant genes was demonstrated via analysis of single nucleotide polymorphisms (SNPs) in purple sea urchin Strongylocentrotus purpuratus larvae cultured under different CO2 levels , the rate of evolutionary change (ΔZ) can be estimated with the breeders equation  could be measured following selection over multiple generations, but as with experimental evolution, this is practical mostly for organisms with short generation times  to be distinguished  crosses that are separately reared under standardized laboratory conditions. Unfortunately, this can necessitate an impractically large number of experimental vessels , because (i) it is an ecologically important, annual forage fish along most of the western Atlantic coast, (ii) elevated CO2 levels have previously been shown to reduce early life survival in this and related species  and the genotype of each larva, which were used to construct and analyze a parent–offspring pedigree. We then tested the null hypothesis that genetically related offspring were no more phenotypically similar  than unrelated offspring. Other genetic parameters such as observed heterozygosity or allelic richness were quantified for each replicate to examine the relationship between genetic diversity and offspring survival in high CO2 environments.To explore the approach, we reared newly fertilized offspring from wild-caught parents at COM. menidia at the beginning of the spawning season (25 April 2013) from a tidal salt marsh on the North shore of Long Island . Males and females were caught with a 30 × 2 m beach seine, then transported to our laboratory facility (Flax Pond Marine Laboratory) and held overnight in separate temperature-controlled baths . The next morning, 29 females were strip-spawned by gently squeezing their hydrated eggs into a large shallow container containing seawater-activated sperm of 42 males and a large sheet of window screen (1 mm mesh). Within 15 min, fertilized eggs attached to the screen via uncoiled chorionic filaments, in contrast to unfertilized eggs that could later be gently rinsed off. The window screen was randomly cut into many small pieces, and within 2 h after fertilization, 3–6 randomly selected pieces totaling 100 embryos were suspended into each of 10 replicate rearing containers (20 L) preset for the high CO2 treatment  until the end of the experiment.We collected ripe, adult 2 and air (air only for controls) directly to the bottom of each rearing container via air stones. Target pH for high CO2  and control levels  were monitored daily with an Orion ROSS Ultra pH/ATC Triode and Orion Star A121 pH portable meter, which were calibrated regularly using three-point National Institute of Standards and Technology (NIST) traceable pH references. In addition, discrete water samples were taken once from each replicate/treatment  and analyzed for dissolved inorganic carbon  with an EGM-4 Environmental Gas Analyzer , temperature, salinity, and first and second dissociation constants of carbonic acid in seawater  for subsequent genomic DNA extraction. Frequent siphoning was critical, because fish larvae decompose beyond recognition within hours after death (at 24°C). The pattern of daily posthatch mortality was typical for early larval fish, with mortality peaking soon after hatch (1–4 dph), but then declining exponentially over the remaining days of the experiment  were individually transferred to tissue lysis solution for DNA extraction. To specifically evaluate the resistance of the population to elevated CO2, only hatched larvae of the 10 high CO2 replicates were sampled for genetic analyses. Unhatched embryos were recorded but not genotyped. To validate the assumption that high CO2 was a significant source of mortality in the experimental replicates, live larvae were counted on day 10 posthatch by gently scooping small groups into replacement containers.To quantify DS ranging from 1 to 15 dph) was extracted using a more cost-effective ‘salting out’ protocol  following the animal tissue protocol of Qiagen DNeasy® kits. Larval DNA  to calculate summary statistics  and assess the suitability of loci for parentage analysis , observed heterozygosity, number of dams, and number of sires. Linear regression was used to relate the four proxies to survival. Subsequently, CERVUS was used to assign individual offspring to candidate mothers and fathers. For each offspring tested, candidate parents were assigned with at least 95% confidence using exclusion probability first and the maximum likelihood score second  software ASReml v3.0.5 . An ‘animal model’ is a mixed model  that has been found advantageous for estimating phenotypic variance components in wild populations , which corresponds to the experimental vessel, given that age  and sex  were not relevant factors. Replicate ID significantly influenced DS  therefore all subsequent analyses included replicate ID as a fixed effect to remove its effect prior to the estimation of genetic parameters. We then estimated the heritability of DS by testing three distinct models: (i) the base model including only the fixed effect, (ii) a model including the fixed effect and an additive genetic random effect (αi with variance VA), and (iii) a model including the fixed effect, additive genetic random effect, and dam identity (mi with variance VM) as a random effect. The components of total phenotypic variance in DS (VDS = VA + VM + VR) were then used to calculate narrow sense heritability h2 of DS as the ratio of additive genetic variance to total phenotypic variance (h2 = VA/VDS) and maternal effects m2 of DS as the ratio of maternal variance to total phenotypic variance (m2 = VM/VDS). Statistical significance was assessed with likelihood-ratio tests [LRT = −2 * (likelihood base model–likelihood model with random effects)] tested against the chi-square distribution (df = 1).Last, we ran a hierarchical set of univariate ‘animal models’ for the trait 2 conditions in the experimental replicates significantly lowered average offspring survival by 34% compared to control replicates  survival of 46% (15%). Survival tended to increase with all four measures of genetic diversity ; however, the linear relationship was only significant for observed heterozygosity  produced from that pair. The maximum number of sires to fertilize a single dam's eggs was 31, resulting in 115 offspring, of which 83 survived and 32 died . Adding maternal identity as a second random effect resulted in a small and nonsignificant model improvement . Hence, in the final model without maternal identity, the estimated total phenotypic variance (±SE) VDS was 29.15 ± 1.69, with an additive genetic variance VA of 5.72 ± 2.10 and thus a heritability of larval survival at high CO2 of 0.20 ± 0.07.The estimated genetic variance components for 2 conditions. Our approach had three priorities, that is, using a (i) single-generation experiment with (ii) enough parents to approximate a wild population's genetic diversity, while (iii) allowing all sire × dam crosses to be reared in a (replicated) common garden environment. We found that genetically related larvae were indeed phenotypically more similar  than unrelated larvae, which translated into a significant component of additive genetic variance and a small but nonzero heritability of 0.20 ± 0.07. Compared to average larval survival at the controls (70%), elevated CO2 conditions significantly reduced survival  in addition to CO2-unrelated mortality agents. Hence, our heritability estimate for survival under elevated CO2 conditions should be considered a maximum, because it assumed that all mortality resulted from these conditions.We used a quantitative genetic approach to estimate the heritability of fish survival during the early developmental stages at elevated CO2 levels. Assuming that neutral genetic variation is a proxy for adaptive genetic variation, this finding highlights the importance of standing genetic variation for a population's capacity to withstand environmental stress.The small contribution of maternal effects was unexpected, given that early life history traits in marine organisms often reflect maternal traits and investment Green . A low l2-related mortality occurs during the earliest and thus smallest life stages  should be weighed against feasibility, cost, and time considerations. In most OA-sensitive organisms, CO2 levels was 0.20 ± 0.07, which is well within the range of narrow sense heritability reported for survival in fish in the literature (Mousseau and Roff n = 79), in contrast to generally higher heritability values associated with morphological traits like length or weight . The current understanding of the many sources of genetic variation in natural populations predicts this difference, because life history traits are more closely associated with fitness than more distantly related morphological traits  were higher than at elevated CO2 . Assuming that h2size > h2survival, our study's heritability estimates for early survival in a fish (0.10–0.20) were notably above the two invertebrates studied thus far.In our final model (fixed + additive genetic random effect), the heritability estimate (±SE) for early life survival at high COFalconer . Given tFalconer , where eM. menidia will adapt relatively rapidly to OA? We believe that this conclusion is not warranted yet, given the current lack of understanding regarding the strength and form of OA-induced selection in the wild  resulted in a significant divergence between up- and down-selected lines within only four generations. Incidentally, the heritability of growth rate in this experiment was also 0.20. More recently, Brown et al.  might mitigate or even reverse OA-induced selection  parts of the spawning season, while becoming CO2 tolerant later in the season, when elevated respiration rates increase CO2 levels in tidal marsh spawning habitats , relative allelic richness (0.56–0.72), and observed heterozygosity (0.66–0.72) all varied substantially between replicates. The latter was significantly positively correlated to survival and explained almost 50% of the variation. All proxies for genetic variability were calculated across all individuals per replicate (regardless of dead and surviving), hence assuming that neutral (microsatellites) and adaptive genetic variability are correlated, our finding corroborates the notion that more genetic variability and a higher overall allelic richness among progeny positively affect survival (Ruzzante et al. In addition to heritability, our quantitative genetic approach revealed links between phenotypic and genotypic differences among the 10 ‘replicates’ of this study. The strong statistical effect of replicate ID was most likely due to a nonrandom distribution of genotypes among experimental vessels, despite our meticulous efforts to randomize offspring at the beginning of the trial Fig.. Small bOur work has emphasized the suitability of using a quantitative genetic approach to evaluate species evolutionary potential in the face of continuing OA. We believe that such estimates are of increasing interest to a research field that needs to address the questions of short- and long-term adaptability in marine organisms to allow a more realistic assessment of the OA threat.


Previous studies documented the problem of inbreeding among Italian Greyhounds (IG) from the USA and its possible role in a multiple autoimmune disease syndrome. The present study is an extension of these earlier experiments and had two objectives: 1) to identify pockets of additional genetic diversity that might still exist among IG from the USA and Continental Europe, and 2) to determine how loss of genetic diversity within the genome and in the dog leukocyte antigen (DLA) complex relates to the problem of autoimmune disease in IG from the USA. Genetic testing was conducted using 33 short tandem repeat (STR) loci across 25 chromosomes and 7 STR loci that associated with specific dog leukocyte antigen (DLA) class I and II haplotypes. Standard genetic assessment tests based on allele frequencies and internal relatedness (IR) were used as measures of breed-wide and individual heterozygosity.The results of these tests demonstrated that IG from the USA and Continental Europe belonged to a single breed but were genetically distinguishable by genomic allele frequencies, DLA class I and II haplotypes, and principal coordinate analysis (PCoA). In the second part of the study, 85 IG from the USA that had suffered various autoimmune disorders (case) and 104 healthy dogs (control) of comparable age were studied for genetic associations with disease. Case dogs were found to be significantly more homozygous in the DLA regions than control dogs. Principal coordinate analysis did not differentiate case from control populations. No specific STR-associated DLA-class I or II haplotype was associated with increased autoimmune disease risks. Reasons for the loss of genetic diversity and increased homozygosity among IG from the USA were studied using registration data and deep pedigrees. The breed in the USA started from a small number of founders from Europe and has remained relatively isolated and small in numbers, limiting breeding choices especially in the period before modern transportation and artificial insemination. An additional cause of lost diversity and increased homozygosity has been the influence of famous sires and their show-winning progeny. The most influential of these sires was Ch. Dasa’s King of the Mountain (King) born in 1978. Virtually all contemporary IG from the USA have King at least once in 10 generation pedigrees and 18 % of the genome of contemporary IG from the USA is shared with King.It was concluded that artificial genetic bottlenecks have concentrated numerous genetic polymorphisms responsible for autoimmune disease and that these risk factors did not originate in a specific individual or bloodline of the breed. Rather, they were of ancestral origin in both purebred and random bred dogs and inherited by descent. Italian Greyhound breeders in the USA have several options to improve breed health: 1) breed against homozygosity within the genome and in the DLA region, 2) avoid breeding dogs that have suffered an autoimmune disorder, 3) increase diversity by incorporating the genetic differences that exist in IG from Continental Europe, or 4) outcross to other small sighthound breeds. The latter two approaches must be undertaken with care to avoid introduction of new deleterious traits and to maximize retention and dissemination of new genetic diversity. Italian Greyhounds (IG) in the USA began with a small number of founders imported from Europe, with limited introduction of new dogs in the subsequent decades resulting in narrow breeding choices. These genetic bottlenecks were enhanced by a series of popular sires and their progeny, each followed by an extended period of close linebreeding. The resultant inbreeding has reduced genetic diversity, and increased homozygosity, in contemporary dogs. This has resulted in a greater susceptibility to a wide range of autoimmune diseases and inadvertent positive selection of other complex and simple deleterious traits. This loss of genetic diversity prompted a search for dogs currently in Continental Europe, parts of which were once behind the Iron Curtain. However, Italian Greyhounds from Continental Europe also lacked genetic diversity, but were genetically distinguishable from North American IG, both across the genome, and also in the DLA region. The cause of their lost diversity was not determined, although they probably suffered some of the same types of genetic bottlenecks as IG in the USA. Italian Greyhounds from Continental Europe appear to suffer from many of the same common heritable health problems as IG from the USA, but no information was obtained pertaining to the incidence of autoimmune diseases. The incidence of autoimmune diseases in IG from the USA may decrease with a concerted effort to decrease homozygosity across the genome and especially in the DLA region. Dogs that have suffered autoimmune disorders should not be used for breeding. Italian Greyhounds from the USA and Continental Europe, together with other small sight-hound breeds, could also be used to increase genetic diversity in both populations. However, care should be taken to avoid introductions of polygenic or simple recessive genetic disorders that may be unique to one population or the other.A wide range of autoimmune disorders affect dogs, and in particular pure breeds . Some auAlthough it is generally accepted that autoimmune disease in both dogs –13 and hAlthough multiple genetic associations in various regions of the genome have been linked to autoimmune disease in humans, the strongest single genetic contributor of risk for autoimmune disease in people involves the human leukocyte antigen (HLA) complex , 15. TheIt is evident from human studies, that risk for autoimmune disease also involves regions outside of the major histocompatibility complex , 15. GenAllele frequencies for 33 genomic STR markers of 213 IG from the USA and 174 from Continental Europe are listed in Table Internal relatedness based on 33 genomic STRs for IG from the USA and Continental Europe is shown in Fig. Internal relatedness values described above were based on alleles and allele frequencies measured in contemporary IG and did not reflect genetic diversity that has been lost during breed evolution. This lost diversity can be approximated by using allele frequency data from indigenous or village dogs that currently reside in the Middle East, SE Asia, and Pacific nations such as Taiwan, New Guinea, Bali, Australia, Brunei and the Philippines , 19. VilDLA-DRB1, DQA-1 and DQB-1 as shown in Table There were noticeable differences in the actual DLA class I and II haplotypes and their frequencies between IG from the USA and Continental Europe Tables  and 4. AThe control population contained 104 dogs with known gender, 59 female and 45 males (F:M = 1.31). The F:M ratio for case dogs varied greatly depending on the form of autoimmune disease that they manifested (Table The inbreeding coefficient (FIS) calculated from genomic STRs indicated that the control population was nearly in Hardy-Weinberg equilibrium, while the case population as a group was significantly more inbred than the healthy control dogs Table ; Fig. 4.The distribution of dogs with autoimmune disease and healthy controls in the entire population was also examined by PCoA Fig. . Italianp <0.05.) was associated with homozygosity for alleles at 3/4 class I and 2/3 STR-associated DLA class II loci  calculated from pedigrees mirrored what was found with DNA markers, i.e., case dogs had higher COI than control dogs, but significance was at the 93 % probability level Table . The genty Table .Table 11The search for genetic diversity among IG concentrated on the USA and Continental Europe, because IG from these two regions have been long separated by geography, two World Wars and the Iron Curtain. Although there have been imports of European dogs to the USA over the decades, they have been few in numbers and their impact relatively small. A report in a breed publication listed 177 imports from other countries to the USA between 1945 and 1977, but 47 were from Canada and 66 from the UK [A second and major objective of the study was to compare and contrast health problems between the two populations, and in particular to better understand genetic polymorphisms that might explain the high incidence of autoimmune disease that was documented earlier for IG from the USA . UnfortuThe association between inbreeding and autoimmune disease in IG from the USA involved genetic comparisons between 85 affected (case) and 104 healthy (control) dogs. Although these populations were small compared to human studies of autoimmune disease, previous reports suggest that due to the unique makeup of their genomes, ~100 case and ~100 control dogs from a pure breed of dogs would be sufficient to detect genetic associations even in complex genetic traits such as those involved in autoimmunity . The numIn spite of published relationship of certain DLA class I and II haplotypes with specific autoimmune diseases , 11, 27,The strongest association with autoimmunity in IG from the USA was homozygosity at both genomic and DLA loci. FIS values based on observed and expected heterozygosity in the 33 genomic loci were significantly greater among case than control dogs, but even more so for the DLA region markers. The DLA region is rich in genes controlling immune responses and is in extended linkage disequilibrium. Therefore, it is possible that additional genes in extended linkage to DLA class I and II markers were also involved with autoimmunity in IG, especially when in a homozygous state.nd in popularity by the American Kennel Club. This has made it more difficult to locate the most diverse mates in earlier times. However, the rapid nature of modern transportation and shipment of frozen semen should ease the problem of mate selection. The problem is not only a limited pool of potential mates, but a limited pool of dogs with both a desired genetic backgrounds and sound health. Although the limited founder population, lack of significant introgression with dogs from outside of the country and small population size have been factors favoring inbreeding, it is difficult to deny the effects popular sires and their progeny have had on further narrowing the available pool of genetic diversity. There have been several noteworthy popular sire influences on the breed in the USA during the last 6 decades. Ch. Ulisse di Peltrengo of Winterlea came to the USA via Italy and Scotland as an Italian and English champion [Born to Win, Breed to Succeed [http://www.pedigreedatabase.com/italian_greyhound/dog.html?id=1961170-windriver-ruby-tuesday&p=simplechart&, accessed October 26, 2015).What is the cause of the inbreeding observed in IG from the USA? The breed started with the introduction of a small number of dogs from Europe and expanded from that initial population with limited introgressions from Europe. The breed in the USA has remained relatively small for most of its existence and is currently ranked 72champion . “Ulissechampion .” He bec Succeed  - “Ebonyhttps://www.vgl.ucdavis.edu/services/italiangreyhound.php, accessed October 26, 2015). Other disorders are brought into the breed by deliberate or surreptitious outcrossing with other breeds, such as terriers from England [It is tempting to implicate certain dogs or bloodlines for introducing a novel deleterious mutation into a breed. This may be the case for breed specific recessive mutations such as those responsible for enamel hypoplasia, PRA and glaucoma . Indeed, there are also anecdotal and scientific findings that suggest crossbreeding has occurred to some extent over the last century or less. It has been rumored that other breeds such as Chihuahua, Toy Manchester terrier, Miniature Pinscher, Fox Terrier and Whippet were used at an earlier time to bring in certain coat colors, size, and other desired phenotypic traits. The introduction of terrier blood, especially from England, has been a source of much concern [How do you know when the health of a breed is in trouble? One can argue that it is when undesirable ancestral traits, both complex and Mendelian, reach troublesome levels and when breed specific deleterious simple recessive traits began to appear. How do you respond when a breed becomes overly inbred and health is unfavorably affected? The best solution is to never reach this point, which requires starting a breed with the widest possible genetic base and then diligently breeding to maintain that diversity. Unfortunately, this does not usually occur; one study of 26 breeds registered with the Swedish Kennel Club concluded that 90 % of initial genetic diversity of a breed is rapidly lost . It is o concern . Indeed, concern , 37. ColItalian Greyhounds (IG) in the USA began with a small number of founders imported from Europe and only limited introgression of new dogs from Europe thereafter. This genetic bottleneck was enhanced by both a lack of numbers and a series of popular sires and their progeny, each followed by an extended period of close linebreeding. The resultant inbreeding has reduced genetic diversity and increased homozygosity in contemporary dogs from the USA and has resulted in a greater incidence of a number of polygenic and simple recessive deleterious traits. This loss of genetic diversity among IG from the USA and its effect on breed health prompted a search for additional genetic diversity, particularly among IG found in Continental Europe, parts of which were sheltered behind the Iron Curtain. Italian Greyhounds from Continental Europe were found to be genetically distinguishable from IG in the USA with evident population substructure even between various countries. Although IG from Europe were genetically distinguishable from American IG, they were also found to lack genetic diversity, although somewhat less so than dogs from the USA. Unfortunately, no information on autoimmune disease problems was provided by breeders from outside the USA, thus negating a comparative study of autoimmune disease in both populations. Loss of genetic diversity among IG from the USA has led to the emergence of a subpopulation of IG that was significantly more inbred than healthy control dogs. The genetics of autoimmune disease were found to be complex and not related to any specific DLA class I or II haplotype. The increased risk for autoimmune disease among this subpopulation could not be attributed to any particular popular sire and was presumed to be ancestral in many pure and random bred dogs and inadvertently enhanced by positive selection for desirable phenotypic traits. The incidence of autoimmune diseases in IG from the USA may decrease with a concerted effort to minimize homozygosity across the genome and especially in the DLA region. Dogs that have suffered autoimmune disorders or are closely related to affected individuals should not be used for breeding. Italian Greyhounds from the USA and Continental Europe and other small sight-hound breeds could also be used to increase genetic diversity between and among their respective populations. However, much more needs to be learned about existing health problems in IG, and in particular IG from Continental Europe. Such information is necessary to avoid introductions of polygenic or simple recessive genetic disorders that may be unique to one population or the other.http://www.vetmed.ucdavis.edu/ccah/research/Italian_greyhound_autoimmune_study.cfm), accessed October 26, 2015. DNA for genetic testing was available from 213 IG from the USA and 175 mainly from Continental Europe including France (n = 33), Germany (n = 16), Belgium (n = 14), Italy (n = 6), Slovenia (n = 7), Romania (n = 4), Ukraine (n = 35), Latvia (n = 4), Russia (n = 4), Finland (n = 11), Poland (n = 28), Great Britain (n = 10), Norway (n = 2), and Sample collection was conducted under UC Davis IACUC protocol #16643.DNA from IG was submitted in the form of buccal swabs or EDTA whole blood. Owners were also asked to complete a form listing details on the dogs’ pedigree, signalment and health  and DLA class II . The STR designations, forward and reverse primers, alleles/locus, and allele size ranges for the four DLA class I and three class II STRs have been previously published [Thirty three STR loci across the canine genome were multiplexed into two panels Table . Amelogeublished . Specifiublished . Furtherublished .STRs rather than single nucleotide polymorphisms (SNPs) were chosen for this study because of their power to discern genetic differences between individual dogs, breeds, and randomly breeding village (indigenous) dog populations in a rapid, reproducible and cost-effective manner using low quality DNA. The power or STRs in discerning genetic relationships has been demonstrated for African village dogs, where 89 STR loci yielded identical results to 300 SNP loci in principal component analysis and STRUCTURE . STR locAllele frequencies from 33 genomic STR loci were used to determine average alleles/locus (Aa), average effective alleles/locus (Ae), observed heterozygosity (Ho), expected heterozygosity (He), inbreeding coefficient FIS and PCoA using GenAIEX 6.5 , 48. AllInternal relatedness (IR) was measured based on the number and frequency of alleles at a locus as described by Amos et al.  and basePedigrees of 45,308 IG were obtained from a database maintained by one of the authors (AL). This includes pedigrees on every IG that was registered and bred with the American Kennel Club (AKC) from 1884 to present and the Kennel Club (UK) and Canadian Kennel club from the 1880s to 1980s. In addition to pedigrees from the AKC, the database incorporates The Irish Kennel Club, stud pages, and show catalogs. Websites from several countries such as Australia and Norway have large lists of pedigrees on their IG pages. Additional pedigrees were obtained from Germany, Russia and Italy. About 80 % of pedigrees after 1950 go back at least 10 generations and cover 70–75 % of the IG that are being bred worldwide. This database is available to any researcher upon request.Coefficients of inbreeding (COI) were calculated using complete pedigrees and CompuPed v.4.0 professional software. This version is no longer available and has upgraded to Millennium Pro . The program can also calculate the total number of times that a particular dog appears in a 10 generation pedigree, the genetic contribution of specific ancestors , and the