Individuals now have access to an increasing number of internet resources offering personal genomics services. As the direct-to-consumer genetic testing (DTC GT) industry expands, critics have called for pre- and post-test genetic counseling to be included with the product. Several genetic testing companies offer genetic counseling. There has been no examination to date of this service provision, whether it meets critics’ concerns and implications it may have for the genetic counseling profession. Considering the increasing relevance of genetics in healthcare, the complexity of genetic information provided by DTC GT, the mediating role of the internet in counseling, and potential conflicts of interest, this is a topic which deserves further attention. In this paper we offer a discourse analysis of ways in which genetic counseling is represented on DTC GT websites, blogs and other online material. This analysis identified four types of genetic counseling represented on the websites: the integrated counseling product; discretionary counseling; independent counseling; and product advice. Genetic counselors are represented as having the following roles: genetics educator; mediator; lifestyle advisor; risk interpreter; and entrepreneur. We conclude that genetic counseling as represented on DTC GT websites demonstrates shifting professional roles and forms of expertise in genetic counseling. Genetic counselors are also playing an important part in how the genetic testing market is taking shape. Our analysis offers important and timely insights into recent developments in the genetic counseling profession, which have relevance for practitioners, researchers and policy makers concerned with the evolving field of personal genomics. While genetic testing has been an established clinical practice for some time de Vries , it has The prevalence of genetic testing products being advertised and sold on the internet has been a source of concern for healthcare providers and professional organizations, governments and other regulatory and consumer groups. One common point of concern is how individuals may (mis)understand, (mis)interpret and cope with the genetic information that they are provided from genetic tests not ordered through a healthcare professional  data for common conditions which provide relatively small contributions to predictions of disease risk O’Daniel . Health It has also been suggested that preventive health advice in this context sits problematically with non-directive genetic counseling   and the Although we recognize that the distinction between “traditional” and “non-traditional” genetic counseling is now blurred Finucane , p3, cenProfessional roles and boundaries are often disrupted by technological change, as Petrakaki et al.  highlighThe history of genetic counseling has been continually shaped by technological advances Kenen , and theConsidering the emphasis of many European and North American medical organizations on the need for genetic counseling in DTC GT, there has been surprisingly little empirical investigation of the representations of genetic counseling in the DTC GT industry. Hennen et al. , and autrepresented by the DTC GT websites, examining the types of genetic testing products with which counseling is offered, the forms of counseling offered and the roles attributed to counselors. Our study does not profess to make any claims about how genetic counseling sessions are conducted in the DTC GT setting, but rather examines the representation of genetic counseling online, which is important when considering how this market is taking shape and the potential directions in practice. We analyze how the critiques of the lack of involvement of genetic counselors in DTC GT are being played out online.In this paper we examine the role genetic counseling is coming to play in the DTC GT industry. In particular we are interested in how genetic counseling is More specifically, we critically explore how genetic counseling expertise is represented on these websites, as well as in other online material such as blogs. We argue that the DTC GT industry is potentially resulting in a change in how genetic counselors’ professional roles are being represented and consider how these emerging roles sit with more “traditional” aspects of genetic counseling. We also argue that genetic counselors have an important part to play in how the DTC GT market is taking shape, particularly in terms of the mediation of testing, counselors’ engagement with physicians, and the entrepreneurial activities of some genetic counselors, as many of these practices are moving DTC GT more firmly toward medical products and information.In December 2010, a list of websites for companies offering genetic testing services to consumers was compiled by the first author after consulting recently published literature  or by a third party; three blogs administered by DTC GT companies which discussed genetic counseling services; four blogs discussing genetic counseling and DTC GT, which were not administered by DTC GT companies; and one interview with a genetic counselor which discussed DTC GT.We archived all web material using WebCite and transferred written text to tables for ease of analysis. The first author performed discourse analysis of all material. Discourses were considered online texts in the form of the words, images and hyperlinks on company websites, whose combined meaning extends beyond the text itself , five did provide counseling  and one company offered what we have referred to as “independent” counseling (23andMe)  consumer about genetics and the genetic testing product, through the provision of web-based information or pre-test counseling. Genetic counselors are also represented as having an important role in the education of healthcare providers about genetic testing. As examples of the latter, Lineagen offers genetic counseling services primarily as an informational service for physicians. Navigenics genetic counselors are defined as “trained healthcare professionals that specialize in personal genomics,” who are “dedicated to providing you with the most accurate information to help you, your family, and even your doctor.” Pathway Genomics genetic counselors are there for when “a physician or staff member is in need of assistance with clinical information, report interpretation or general customer service.” One genetic counselor regards this as a shift into physician education, asking on the DNA Exchange blog, “will we move from being educators of patients to being educators of health professionals?”The mediator: in many ways part of their educational role, the genetic counselor on some of the analyzed sites also acts as a mediator between the customer and the physician. A good example can be found in the genetic counseling service offered by Navigenics, where their counselors can help the customer “determine what to focus on with your doctor—or even speak to your doctor directly if he or she has questions.” The genetic counselor can also be viewed as a mediator between the customer and the DTC GT company, through their online or phone interactions.The lifestyle/health advisor: the genetic counselor offers advice about lifestyle and health behavior change about a range of complex and rare illnesses as a result of the DTC GT results. Lineagen claims that they offer “counseling to promote informed choices and adaptation to the risk or condition,” and that their counselors help families to understand “what may happen in the future and what treatment or management options are available … (and make) adjustments to the condition and choose courses of action that are best for the children and their families.” Navigenics helps the consumers to integrate information from their test results into their lives, and DeCODEme offers “counseling to help patients understand what their test results mean for their future health.”The risk interpreter: the genetic counselor is represented on websites as a specialist, an expert in risk interpretation. In general, risk interpretation is based principally on the consumers’ genomic information obtained from the genetic test. There was no mention of the potentially unspecific or uncertain nature of information provided by DTC GT on the websites we analyzed or of the difficulties interpreting genetic information without the context of a detailed family history.The entrepreneur: a number of genetic counselors have founded websites which provide telegenetics services, and have been advocates for the important role of genetic counseling in DTC GT. For example, Jordanna Joaquina is a genetic counselor and founder of AccessDNA , then “a leading online consumer resource for genetics,” which educated and directed people towards testing services and counseling services. In a guest post for the DNA Exchange blog, Joaquina states that “in this brave new world of personalized medicine, I imagine that every person will have their own personal genetic counselor.”Interrelated with various types of genetic counseling provided by the DTC GT companies is the representation of the roles of genetic counselors. In general, genetic counselors are represented on the DTC GT websites as personal genetics experts. They are represented as being constantly “on-call,” with websites advertising “unlimited access,” counselors’ availability “7 days a week,” where they are ready “to help you at any time.” The counselors’ professional knowledge is emphasized through markers such as board certification, ethical guidelines, hyperlinks to well-respected websites such as the National Society of Genetic Counselors and scientific research. The genetic counselors’ expertise is evident in various, overlapping roles as: genetics educator; mediator; lifestyle/health advisor; risk interpreter; and entrepreneur.Genetic counselors were represented on the websites and blogs we analyzed as practicing in some or a combination of these roles. The kinds of roles for genetic counselors on each website generally aligned with the company’s targeted consumer, whether this was a proactive customer, patient, carrierIn our analysis, we have identified various forms of genetic counseling represented on the websites, many of which are emerging as a result of genetic counselors’ involvement in the direct-to-consumer genetic testing industry. Changes in professional roles are taking place within a broader context of ongoing changes in genetic testing processes, genetic research and genetic result interpretation. The commercialization of counseling on the internet and the increasing complexity of the genetic information these internet counselors are dealing with, are contributing to shifts in the profession.Whilst the DTC GT industry is extremely heterogeneous, we can nonetheless make some general claims about how genetic counseling is represented in the direct-to-consumer genetic testing field. First, the internet provides various forms of mediation that may be shifting boundaries of the profession, its practice and expertise. The genetic counselors on the sites we analyzed contacted individual consumers either through email or phone. Unlike the use of the telephone and internet to reach remote or hard to reach patients—how telegenetics is most commonly used (Abrams and Geier While genetic counselors have traditionally educated patients and families about genetics, the representation of genetic counselors employed by the DTC GT industry as genetic educators for physicians appears to be changing as a result of the DTC GT product. A precursor to this may be the emphasis that earlier genetic testing companies, such as Myriad, placed on physician education. Myriad Genetics has been offering a commercial BRACAnalysis genetic susceptibility test for hereditary breast cancer since 1996 and is often seen as a forerunner to the large number of DTC GT companies now in operation (Matloff and Caplan Closely related to their role in physician education is the genetic counselor’s role in providing product advice to healthcare providers and other consumers. The limited training that doctors receive in genetics (Powell et al. The type of genetic information that is provided by DTC GT differs from the monogenetic information for which genetic counselors have traditionally been trained. This means that more traditional roles for genetic counselors, such as risk interpretation and prevention strategies, are, we argue, taking on new forms, as genetic counselors may be called upon to interpret an ambiguous product that provides information about a large number of genetic associations for complex diseases, based on controversial studies of specific populations, which provide probabilities that are very similar to population risk. One result of this shift, as we observed on the websites, and as O’Daniel  has alsoSome genetic counselors writing in blogs are recognizing these roles are “non-traditional,” with a GeneDx counselor commenting that “having genetic counselors work outside their traditional roles, ensures we will have well informed professionals in these new areas of growth, that benefits not only doctors and counselors, but also patients and families” Waltho . AnotherThe emerging roles for genetic counselors represented on the websites that we have discussed are implicitly aligned with representations of more clinically acceptable, or “traditional” forms of genetic counseling on the websites. This occurs predominantly through the use of images that show face-to-face counseling sessions, with many counselors emphasizing their clinical experience in their online personal profiles. This is a crucial aspect of the genetic counseling product provided by DTC GT companies, as these more “traditional” images reinforce the test as a test with clinical significance, thus potentially strengthening consumer faith in the product. We need more research in this area to understand this further. DTC GT includes ambiguous products situated in a gray area between recreational/informational tests and medical tests. By providing genetic counseling services, DTC GT companies are drawing on associations of genetic counseling with clinical services and monogenetic testing. This image includes the professional attributes of autonomy, expertise, and non-directiveness.In summary, our analysis demonstrates that direct-to-consumer genetic testing is leading to important and controversial changes in how the roles of genetic counselors are represented, as well as more broadly through: the utilization of telegenetics for a range of consumers, not only those who are specifically rurally or culturally isolated; the genetic counselors’ entrepreneurial involvement with commercial enterprise; and the new kinds of genetic information being provided by GWAS tests, differing from the monogenetic results that genetic counselors have traditionally dealt with. The participation of genetic counselors in the DTC GT industry raises ethical questions about professional boundaries and moral obligations, highlighting this as an area of professional concern.The involvement of genetic counselors in the genetic testing industry also has a role to play in shaping the direct-to-consumer genetic testing product. Not only do genetic counselors potentially act as markers of clinical trust on the website, as discussed above, but the presence of the genetic counselor also raises interesting questions in regard to the “direct-ness” of genetic tests sold to the consumer, a common point of concern for those critical of DTC GT. As discussed earlier, much research about DTC GT considers “direct-to-consumer” to mean the selling of genetic tests to the public unmediated by a physician. Our analysis suggests that genetic counselors introduce a mediating aspect into the service, which has previously been underexplored in the literature. In dealing directly with physicians, either through education or product advice, genetic counselors working within the DTC GT industry also highlight that we need to consider carefully not only what we mean by “direct,” but also what we mean by “consumer.”Our analysis of the online DTC GT landscape is limited by our focus on testing for mental disorders and by our use of English language search terms. Nonetheless the sample our search generated does seem to be representative of the range of websites available in this field, at least for websites registered in English-speaking parts of the world, where DTC GT is taking flight. Another limitation of the study is our focus on analyzing the online representations of genetic counseling, rather than empirically examining the actual work and practices performed by those employed in the DTC GT industry. More research is needed to investigate this area, including interviews with genetic counselors employed by DTC GT companies and participant-observer fieldwork. However, our research has shed light on an understudied aspect of DTC GT, and generated many questions upon which such research could be based, some of which we explore in the conclusion.Genetic counseling in the DTC GT industry is a controversial area of genomic medicine. DTC GT is resulting in the representation of emerging forms of expertise for genetic counselors. We find, as Kenen , p1384 oRather than take sides in this debate, in this paper we have critically explored the roles that genetic counselors are represented to be assuming as part of the DTC GT industry. As mentioned, our research has raised many questions for further research. First, the normative aspect of genetic counseling and DTC GT needs closer examination. As Wade and Wilfond , p289 wrIn general, little is known about the genetic counselors working for these companies, and what they think about what DTC GT means for their profession and their professional identity. Is there any room left for uncertainty in their sessions and how are they dealing with an ambiguous product that absolves itself from diagnosis, yet is sold on the basis of providing health information? We also know little about how users are engaging with these genetic counseling services, although recent research has shown a low-uptake of the free genetic counseling offered by one of these sites (Bloss et al. How genetic counselors are involved in the DTC GT industry needs serious consideration. In this paper we have shown that genetic counselors are represented as assuming shifting roles, and promoting emerging forms of expertise in the DTC GT industry. The potential diversification of roles for genetic counselors will help them to maintain a particular position in the healthcare marketplace (Nancarrow and Borthwick


Phaseolus vulgaris was recently suggested through analysis of nucleotide polymorphism at the nuclear level. Here, we have used chloroplast microsatellites to investigate the origin of the common bean, on the basis of the specific characteristics of these markers , to validate these recent findings. Indeed, comparisons of the results obtained through analysis of nuclear and cytoplasmic DNA should allow the resolution of some of the contrasting information available on the evolutionary processes. The main outcomes of the present study are: (i) confirmation at the chloroplast level of the results obtained through nuclear data, further supporting the Mesoamerican origin of P. vulgaris, with central Mexico representing the cradle of its diversity; (ii) identification of a putative ancestral plastidial genome, which is characteristic of a group of accessions distributed from central Mexico to Peru, but which have not been highlighted beforehand through analyses at the nuclear level. Finally, the present study suggests that when a single species is analyzed, there is the need to take into account the complexity of the relationships between P. vulgaris and its closely related and partially intercrossable species P. coccineus and P. dumosus. Thus, the present study stresses the importance for the investigation of the speciation processes of these taxa through comparisons of both plastidial and nuclear variability. This knowledge will be fundamental not only from an evolutionary point of view, but also to put P. coccineus and P. dumosus germplasm to better use as a source of useful diversity for P. vulgaris breeding.Evolutionary studies that are aimed at defining the processes behind the present level and organization of crop genetic diversity represent the fundamental bases for biodiversity conservation and use. A Mesoamerican origin of the common bean  Phaseolus vulgaris grow across a wide geographic area of the Americas, from northern Mexico to northwestern Argentina  markers are widely used in population genetics and evolutionary studies of plants  were analyzed previously by Angioi et al. (The seeds were provided by the United States Department of Agriculture (USDA) Western Regional Plant Introduction Station in the USA, the International Center of Tropical Agriculture (CIAT) in Colombia, and the Laboratory of Plant Genetics (D3A) at the Polytechnic University of Marche (UNIVPM) in Italy. Most of these accessions had already been characterized using different types of molecular markers, such as amplified fragment length polymorphism . The PCR conditions were as reported in Table Genomic DNA was extracted from each accession from young leaf tissue of a single, greenhouse-grown plant, using the miniprep extraction method , Mesoamerican wild (MW), and northern Peru and Ecuador (PhI) populations.The percentage of polymorphic loci, the average number of observed alleles per locus (Na), the effective number of alleles per locus  were tested using Wilcoxon signed-ranks non-parametric test for two groups, arranged for paired observations  was used to compare the diversity between the two main gene pools ; this estimate measures the loss of diversity of one population compared to another, and it was originally proposed by Vigouroux et al. (H = 1 − (HePOP1/HePOP2), where POP1 refers to the population that shows the lower level of genetic diversity (He) compared to the other population (POP2).An x et al. : ΔH = 1 P. vulgaris accessions.Using the JMP software, version 8 , principal component analysis (PCA) was performed from allele frequencies. The same analysis was carried out also to investigate the genetic relationships among the P. vulgaris and 10 P. coccineus accessions), and thus to assign the genotypes into genetically structured groups/populations (K). A spatial genetic mixture analysis was conducted  software, version 5.3 . The differences between the clusters identified according to the genetic diversity estimates  were tested using the Wilcoxon signed-ranks non-parametric test for two groups, arranged for paired observations  were estimated as FST  gene, which is involved in the control of fruit shattering in Arabidopsis thaliana. These data allowed a comparison of the data from the population structure analyses carried out using cpSSRs and nuclear sequences. Thus, for the 71 accessions shared between this study and that of Bitocchi et al. (The sequences of five gene regions (from 500 to 900 bp) for 71 accessions were available from Bitocchi et al. . These fi et al. ; PvSHP1 i et al. , a populi et al. , while fq) was performed according to the kriging method, with each of the clusters identified by population structure analysis, which was implemented in the R packages spatial (http://www.r-project.org/). In the case of the cpSSRs, due to the non-recombinant nature of these markers, which does not allow admixture, the membership coefficients were represented by one or zero ; thus, the interpolation for plastidial data represents an approximation.To compare the geographical distributions of the clusters identified through the cpSSR and nucleotide data, spatial interpolation of membership coefficients (2) test, which was performed using the JMP 8.0 software .The association between the results obtained by the BAPS analyses carried out with the cpSSR and nucleotide data was tested by analysis of contingency tables with the likelihood ratio chisquared  to 378 bp (ccSSR19). Overall, the number of alleles per locus (Na) ranged from two (cp2) to 12 (ccSSR20); in parallel the same two markers showed the lowest and the highest genetic diversity, He = 0.13 and He = 0.85, respectively  to six (ccSSR20). One locus (cp2) was monomorphic in the P. vulgaris sample. For the remaining 16 loci, Na ranged from two (cp3 and ccSSR12) to 11 (ccSSR20). The highest level of genetic diversity was detected for the ccSSR20 locus, as an He of 0.84 for both P. vulgaris and P. coccineus  and within the P. vulgaris Andean (AW), Mesoamerican (MW), and northern Peru and Ecuador accession (PhI) populations.Genetic diversity estimates were computed considering the whole sample and the following major subdivisions: different species . There was a 26% reduction in genetic diversity (ΔH) of the AW population compared to the MW population.Among the three rs Table . In partrs Table . This diP. vulgaris and P. coccineus accessions, were investigated by PCA  as the best partition of the whole sample  and 13 MW accessions from Central Mexico. Cluster C2 included 21 MW and three PhI accessions, along with two P. coccineus genotypes. There were accessions from all of the three common bean populations in cluster C3 , while cluster C4 was exclusive to the remaining eight P. coccineus accessions. The geographical distribution of the P. vulgaris accessions based on the BAPS cluster membership is showed in Figure The population structure analysis identified four different clusters C1, C, C3, C4 P. vulgaris accessions  showed similar levels of genetic diversity . Cluster C4 showed the lowest Ne (1.6) and He (0.29) estimates. However, there were no significant differences in the levels of genetic diversity between these four clusters .The genetic diversity estimates for the BAPS clusters are showed in Table P. vulgaris populations  and the P. coccineus accessions was estimated as FST, D, and RST. The FST and D estimates were very similar, as expected for populations that have a very low number of unique alleles  was greater than that between AW and MW . The highest values of FST were those in the comparisons with the P. coccineus population; however, the MW population showed the lowest levels of differentiation with P. coccineus  compared to the other P. vulgaris populations [FP. coccineus)STST, and PhI [RP. coccineus)STST identified by the BAPS analysis (Table FST and RST) were significantly different from zero (P ≤ 0.001). We observed less differentiation (lower FST and RST) among the three clusters predominated by the P. vulgaris accessions , than between any of these and C4, which was comprised exclusively of P. coccineus accessions. When considering these comparisons with the P. coccineus cluster (C4), the lowest FST was with the C2 cluster [FC2–C4)ST. Three clusters were identified by the analysis carried out with cpSSRs. They corresponded to clusters  determined previously using all the accessions, while the Cluster C4 was not determined due to the exclusion, in this comparative analysis, of the P. coccineus accessions. Six clusters , as in Bitocchi et al.  between the genetic clusters obtained with these different markers , the Andes (B6), and northern Peru and Ecuador (B5).The availability of sequence data for five gene fragments for 71 out of the 109 i et al.  were ideP. vulgaris through an analysis of its genetic diversity at the plastidial DNA level, in comparison with the study of Bitocchi et al. . Moreover, the absence of phaseolin type I in the Mesoamerican gene pool might be due to its extinction in Mesoamerica, or it might still be present, but just not included in the samples analyzed in the literature.The data obtained here are in agreement with the Mesoamerican origin of i et al. , where t−3–10−5 mutations per generation; Provan et al., −3–10−4 mutations per generation; Estoup and Angers, −6–10−5 mutations per generation; Mariette et al., The first outcome was the reduction in the genetic diversity (26%) in the Andean gene pool, compared to that of Mesoamerica. This has already been shown, even if to different extents, by analyses carried out with different nuclear molecular markers . The Andean accessions are almost all included in cluster C1, with the only exception being an accession from southern Peru that belongs to cluster C3. Considering the nuclear data, cluster C1 is significantly associated with clusters B3, B6, and B4. This supports the close relationship between the Andean (B6) and the MW accessions from central Mexico . These data provide further confirmation of the evidence highlighted by the nuclear data  and are not significantly associated with any genetic cluster identified with the nuclear data. In particular, almost all of the MW in cluster C3 are from Central Mexico, with the only exception being one Colombian genotype; moreover, cluster C3 comprised four PhI accessions and one AW accession. The wide distribution in cluster C3 can be interpreted as evidence that these accessions carry the ancestral plastidial genome that spread over the entire distribution that is now covered by P. vulgaris. This pattern is also confirmed by the RST divergence estimations, where cluster C3 shows the lowest values compared to all of the other clusters, including most of the various alleles, when the size of the alleles is considered as a measure of the evolutionary distance among alleles. However, the same does not hold when the infinite allele model is considered: FST. Indeed, for FST, C2 shows the lowest divergence. This appears to be determined by the higher diversity (He) of C2 compared to C3, but not as alleles number (richness), with C2 showing the more uniform distribution of allele frequencies. Thus, we can speculate that the different results obtained for RST and FST might be the result of the more precise estimation of allele divergence using RST and because C3 has more skewed allele frequencies due to the drift .However, an interesting and novel outcome is revealed by the cpSSRs, which is probably due to the different characteristics of the nuclear and plastidial genome (and in particular to the presence of recombination for the nuclear genome): the identification of cluster P. coccineus genotypes to cluster C2 suggests that this cluster can be considered as having been derived from an ancestral lineage from which P. vulgaris separated from P. coccineus. Alternatively, this might result from post speciation introgression from P. vulgaris . This putative introgression of plastidial DNA from P. vulgaris to P. coccineus is consistent with the hypothesis that the P. dumosus species originated from a cross of P. vulgaris as maternal and P. coccineus as paternal parent, followed by successive backcrosses from P. coccineus as paternal donor , P. vulgaris , and P. dumosus (intermediate characteristics between P. coccineus and P. vulgaris) are partially intercrossable, although only when P. vulgaris is the female parent  with the aim of obtaining a more detailed picture of the history of the common bean. These cpSSR data strongly support the nuclear data of Bitocchi et al. , that inC3) that includes accessions distributed from northern Mexico to Peru that appear to carry a putative ancestral plastidial genome.A novel outcome was also provided by these analyses based on the polymorphism at the chloroplast DNA level: the identification of a genetic group  for the introgression of important traits; e.g., resistance to biotic and abiotic stress in P. vulgaris elite germplasm (Singh et al., Finally, the present study highlights the potential to evaluate the evolutionary history of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ovis canadensis) were used to re-establish a population that had been extirpated in the San Andres Mountains in New Mexico, USA.A key challenge for conservation biologists is to determine the most appropriate demographic and genetic management strategies for wildlife populations threatened by disease. We explored this topic by examining whether genetic background and previous pathogen exposure influenced survival of translocated animals when captive-bred and free-ranging bighorn sheep  among translocated animals. While heterosis or genetic rescue effects may occur in F1 and later generations as the two source populations interbreed, we conclude that previous pathogen exposure was a more important marker than genetic heterozygosity for predicting survival of translocated animals. Every wildlife translocation is an experiment, and whenever possible, translocations should be designed and evaluated to test hypotheses that will further improve our understanding of how pathogen exposure and genetic variability influence fitness. Innate and adaptive immune responses evolved in vertebrates as a first and secondary line of defense, respectively, against a diverse and changing array of pathogenic organisms. The effectiveness of these immunologic responses, and hence the fitness of individuals, populations, and species, is driven by pathogen exposure history and the immunogenetic repertoire of major histocompatibility complex (MHC) genes and non-MHC genes ,2. NovelOvis canadensis) are a useful model for examining this interplay between disease, demography, and genetics. They are a polygynous, highly philopatric species found in small, fragmented populations in the mountainous regions of western North America [Ovis aries). Pneumonia epizootics appear to be driven by density-dependence, serving to constrain population size [Bighorn sheep  predation, and drought had reduced this population to the point of extinction, and translocation from captive and/or free-ranging herds was necessary to reestablish a self-sustaining population in the SAM. The two founder populations chosen for reintroduction were a genetically diverse free-ranging herd in the Kofa National Wildlife Refuge (KNWR), Arizona, and a less diverse captive herd in the Red Rock Wildlife Area (RRWA) that was originally derived from the native SAM population. In November 2002, 51 bighorn sheep were translocated into the SAM from the KNWR (n = 20) and the RRWA (n = 31), and 30 more bighorn were translocated from KNWR in November 2005. We examined genetic variation at 33 microsatellite loci to compare genetic variability, and we conducted population health analyses at the time of capture to assess infectious disease exposure. Radiocollared sheep released into the SAM were then monitored through February 2007 to determine survivorship and cause specific mortality.We approached this problem by testing whether genetic background and previous pathogen exposure influenced survival when animals from two different founder populations were simultaneously translocated into the San Andres Mountains (SAM) in New Mexico, USA. The SAM once supported the largest population of native bighorn sheep in the state. However, by the late 1990's, a combination of disease, mountain lion  and microsatellite (0.359) loci . ImmediaThe RRWA is a 500-hectare enclosure for bighorn sheep located in central New Mexico. The RRWA population was initiated in 1972 with founders from the SAM, and has served as the source for >260 bighorn sheep translocated within New Mexico since 1979 . This poThe KNWR is located in southwestern Arizona and contains a free-ranging population of bighorn sheep that has significantly higher heterozygosity (0.60) than the RRWA captive population . Like thGenomic DNA was extracted from blood and genetic variability was assessed by examining 33 microsatellite loci , DRB3 (MHC-linked loci), BMC1009, CELJP23, BM203, OARCP026, TGLA94, FCB193, IRBP, CELB9, BM6506, CELJP15, BM4107, CSRD247, HSC, INRA023, INRA063, INRA105, MAF214, McM527, OarAE129, OarCP49, OarFCB20, SPS113). Many of these loci have been examined in previous publications [Multi-locus heterozygosity (MLH) values were calculated for each animal as the preferred metric of genetic variability. Chapman et al.  showed tAntibodies are a marker of previous exposure and priming of the adaptive immune response to specific pathogens. Sera from blood collected at the time of capture were tested for the presence of antibodies to three viruses known to cause respiratory disease and pneumonia in bighorn sheep: bovine respiratory syncytial virus (BRSV), bluetongue virus (BTV), and parainfluenza-3 virus (PI3). Assays were performed at the California Animal Health and Food Safety laboratory in Davis, California, and results were classified as positive or negative. Results for BTV were reported as positive or negative; titers ≥ 1:20 were considered positive for BRSV, and titers > 1:16 were considered positive for PI3.A VHF radiocollar  with mortality sensor was placed on animals to be translocated at the time of capture to facilitate monitoring after their release in the SAM. Animals were transported to the San Andres National Wildlife Refuge in the SAM and released within 48 hrs of capture. Resight of individual animals by radio-signal was conducted by state and federal wildlife biologists through February 2007 to detect mortalities and determine specific causes of death. Mortalities were investigated immediately and a field investigation and necropsy typically performed within 72 hrs of death. Mortalities were classified as lion predation based on criteria of Hayes et al. , or as pWe used independent univariate and multivariate approaches to explore correlation within our dataset, and to identify factors most influencing pathogen exposure, number of days until death (or end of study period if survived) and death due to suspected pneumonia. Independent variables evaluated for their impact on these three outcomes included sex, age-class , source population (RRWA vs KNWR), year released , exposure status to each pathogen  at time of release, and genetic variability (MLH). Significant associations with outcomes variables, confounding, and effect modification were evaluated by stratified univariate analyses using exact categorical tests and the student's t test. The relationship between MLH (as a continuous variable) and age and sex class were evaluated by the two-way ANOVA in order to adjust for source population. Independent categorical variables were also examined for their relationship to loss to follow-up by the two-sided Fisher exact test to determine whether variables were related to censorship, and therefore could not be included in the survival analyses.Binary logistic regression was used to evaluate whether independent variables were related to death due to pneumonia, which was the most common cause of death detected during the study period. Only sheep that died of suspected pneumonia or were confirmed alive at the end of the study were included in this analysis (n = 50). Sheep that died from predation or unknown causes were excluded from this analysis. Variables were selected by backward stepwise elimination (likelihood ratio test P < 0.1) and confidence intervals for the logistic model evaluating risk factors for pneumonia were calculated using conditional exact inference due to low sample size. Hosmer-Lemeshow goodness of fit test was used to estimate overall fit of the final logistic model.For the survival analyses, median survival time was calculated for all risk factors of interest measured at time of translocation. The Kaplan-Meier product-limit estimator was used to estimate the survivor function, and the log-rank test  was usedBighorn sheep translocated from RRWA were predominantly male (18/20), while 72% (34/47) of the sheep translocated from KNWR were female (Fisher exact P < 0.001). Age class was not significantly associated with source population (Fisher exact test P = 0.068), but twice as many sheep in the 3-6 yr old range were captured in KNWR (23/36) compared to RRWA (11/31). All sheep relocated to SAM in 2005 were from KNWR, but these newly introduced sheep were demographically similar and did not differ with regard to pathogen exposure or genetic diversity from sheep introduced from KNWR in 2002.Serologic evidence of previous pathogen exposure varied substantially by source population for all pathogens, except PI3 Table . Sheep fThree of the 33 microsatellite loci tested were monomorphic and were excluded from further analyses . Complete genotypes were determined for all animals at each of the remaining 30 loci with the exception of a single animal at a single locus. Allele frequencies did not significantly differ from HWE expectations across loci, and null alleles were not detected. The genetic diversity parameter, MLH . Most deaths due to pneumonia  occurred in the fall from September thru November, and seven of these occurred in a cluster in the fall of 2006. Pneumonia impacted all age classes with 27% (4/15) of 1-3 year old sheep, 35% (9/26) of 3-6 year old sheep and 44% (4/9) of > 6 year old sheep dying of pneumonia. Mean MLH was not significantly different among the 33 sheep that survived  compared to the 17 sheep that died of pneumonia , but this analyses lacked power due to low sample size . Similarly, risk factors were not significantly associated with death due to pneumonia in the multivariate analyses using exact conditional logistic regression.Year of release was not associated with overall survival in univariate or multivariate analyses and survival time did not differ significantly by source population. However, age class at time of release was marginally associated with survivorship (log rank test P = 0.079) in the univariate analyses. More than half of the sheep released at 1 to < 3 years old survived the entire study period , while median survival time was 1,234 days for sheep 3 years to < 6 years old and only 475 days for sheep released when 6 years or older. Genetic diversity and pathogen exposure were not related to survival time, even after stratifying by source population.Risk factors found to be significantly associated with time to death in the Cox proportional hazards multivariate framework were the oldest age class and previous exposure to BRSV Table . Based oA key challenge for conservation biologists is to determine the most appropriate demographic and genetic management strategies for wildlife populations threatened by disease -19.Bighorn sheep in North America provide a useful model for examining this issue because they are highly susceptible to infectious diseases, and they are frequently translocated to re-establish or augment populations. In this study, we took advantage of a management effort - the restoration of bighorn sheep to the SAM - to test whether genetic background and previous pathogen exposure influenced the survival of translocated bighorn sheep.Variability and local adaptation are considered to be key genetic factors influencing the persistence of small populations of bighorn sheep ,20,21, aWhile the genetic management goal of achieving increased heterozygosity in the new SAM population was accomplished, it was not associated with enhanced survivorship of the individual sheep that were translocated. Bighorn sheep from the KNWR had significantly higher genetic diversity than those from the RRWA, but neither source population nor genetic background (MLH) influenced their survival in the SAM. Our failure to detect a significant relationship between genetic diversity and adult survival is consistent with the Chapman et al.  meta-anaPrevious examination of genetic variability among bighorn sheep suggests that microsatellite diversity has been influenced primarily by neutral factors, and MHC diversity by balancing selection ,9,22,23.Pasteurella and Manheimia spp.) and viruses  have been implicated as primary or secondary pathogens in enzootic and epizootic pneumonia, and we associate these organisms with pneumonic disease because they can be detected in sick and dead animals. However, we know far less about causal relationships. The multifactorial etiology of pneumonia, coupled with the complex interplay of host, pathogen, and environment, has made it very difficult to identify a single, specific cause of pneumonia.Pneumonia was the most frequent cause of death, accounting for 50% 17/34) of the documented mortalities. This mirrors what has been reported for many other populations, illustrating the ongoing importance of pneumonia as a cause of morbidity and mortality across the range of bighorn sheep  of the d,27,28. SInstead of focusing on identifying pathogens after death, we looked at how previous pathogen exposure influenced the future survival of healthy bighorn sheep. The presence of antibodies was used as a marker of pathogen exposure, indicating that viral transmission and infection had occurred at some point in the past. Animals translocated into the SAM showed evidence of previous exposure to BTV (RRWA only) as well as BRSV and PI3 (both RRWA and KNWR; Table Age class at the time of release was significantly related to survival, and younger animals lived longer than those > 6 yrs of age Table . This inWe conclude that increased heterozygosity did not increase survival following translocation, nor did it reduce individual susceptibility to dying specifically from pneumonia. Instead, we found that previous pathogen exposure was more important than genetic heterozygosity as a marker for predicting survival of translocated animals. In their review of > 600 published HFC effects, Chapman et al.  reportedWMB and MEW designed and implemented the study. MCTP and CKJ conducted genetic and statistical analyses, respectively. All authors have approved the final manuscript.


Alexandrium ostenfeldii, from the Baltic Sea, a geologically young and ecologically marginal brackish water estuary which is predicted to support evolution of distinct, genetically impoverished lineages of marine macroorganisms. Analyses of the internal transcribed spacer (ITS) sequences and Amplified Fragment Length Polymorphism (AFLP) of 84 A. ostenfeldii isolates from five different Baltic locations and multiple external sites revealed that Baltic A. ostenfeldii is phylogenetically differentiated from other lineages of the species and micro-geographically fragmented within the Baltic Sea. Significant genetic differentiation (FST) between northern and southern locations was correlated to geographical distance. However, instead of discrete genetic units or continuous genetic differentiation, the analysis of population structure suggests a complex and partially hierarchic pattern of genetic differentiation. The observed pattern suggests that initial colonization was followed by local differentiation and varying degrees of dispersal, most likely depending on local habitat conditions and prevailing current systems separating the Baltic Sea populations. Local subpopulations generally exhibited low levels of overall gene diversity. Association analysis suggests predominately asexual reproduction most likely accompanied by frequency shifts of clonal lineages during planktonic growth. Our results indicate that the general pattern of genetic differentiation and reduced genetic diversity of Baltic populations found in large organisms also applies to microscopic eukaryotic organisms.This study investigates the genetic structure of an eukaryotic microorganism, the toxic dinoflagellate  Due to their small body size, large population sizes, high rates of predominantly asexual reproduction and their open and seemingly homogenous pelagic habitat, aquatic microorganisms have been critically discussed to possess more or less cosmopolitan distributions and do not show biogeographies as found in macroorganisms Recent work on the genetic structure of eukaryotic microalgae revealed that most of the investigated species are highly structured at and below the population level As a historically young aquatic system with steep environmental gradients, the Baltic Sea is a particularly intriguing model system for the study of genetic differentiation and species population structure. Marine organisms most likely colonized this system after the postglacial opening to the North Sea approximately 8000–3000 BP The populations living in the Baltic Sea are considered peripheral to their marine temperate sister populations. They inhabit a geographically and ecologically marginal environment where isolation and exposure to harsh physical conditions, environmental gradients, and anthropogenic impact represent strong selection regimes Given the importance of genetic diversity for ecosystem function and stability as well as for adaptation to environmental change Alexandrium ostenfeldii (Paulsen) Balech et Tangen in the Baltic Sea. This dinoflagellate is part of the A. ostenfeldii/peruvianum species complex with unresolved species boundaries, comprising different morphotypes. Typically a rare background species that co-occurs at relatively low numbers with other phytoplankton A. ostenfeldii forms dense toxic blooms at the Baltic Sea coasts A. ostenfeldii produces dormant resting cysts as a part of its meroplanktonic life cycle To gain insights into genetic diversity patterns of Baltic phytoplankton populations and the relative importance of isolation, local adaptation and specific life history traits as underlying mechanisms, we investigated the genetic structure of the dinoflagellate A. ostenfeldii populations from neighboring North Atlantic and other global populations of this species and 2) the genetic structure and patterns of genetic diversity of this species among and within its populations in the Baltic Sea.By analyzing internal transcribed spacer (ITS) and 5.8S rDNA sequences and amplified fragment length polymorphism (AFLP) data of 84 isolates from 5 geographically separated bloom locations in the Baltic Sea and comparing them with data obtained from global isolates, we here address the following overarching question: Do metapopulations of marine protist species exhibit similar diversity profiles as metazoans in the Baltic Sea and to what extent is this due to Baltic Sea particularities? Specifically we investigate 1) the degree of genetic divergence of Baltic Alexandrium ostenfeldii blooms  a re-isolation step was performed: once germinated cells began to proliferate, single cells possessing one longitudinal flagellum  were randomly picked and placed in a new culture well containing f/8-Si medium. Well established clonal cultures were transferred to vented 50 mL polycarbonate tissue culture flasks and maintained in f/2–Si culture medium at 16°C, 12∶12 light:dark cycle, and 100 µmol photons m–2 s–1.Sediment samples were collected from different locations using a gravity corer , and subsamples of the uppermost (0–3 cm) layer were processed for cyst isolations. Single resting cysts were randomly picked from sonicated, sieved sediment slurries using a micropipette. Cysts were placed in individual wells of 24 well tissue culture plates, each filled with 2 mL of f/8–Si enriched natural sea water of a salinity of 6.5, and incubated at 16°C, 12∶12 light:dark cycle and 100 µmol photons mIsolation success ranged from 35 to 71% in the different populations  and was generally higher in material from northern Baltic bloom sites than in central and southern Baltic material . IsolatiFifteen mL of clonal cultures were harvested from exponentially growing cultures. Strains were washed several times on 10 µm filters  with sterile, filtered seawater  to remove possible bacteria. The cells were then rinsed into 15 mL centrifuge tubes containing 20 ml of sterile seawater and concentrated by centrifugation at 4000 g (15–30 min). The sample was re-centrifuged for 15 min at 14 000 g and the overlying liquid was aspirated to obtain a cell pellet. The cell pellets were disrupted by applying a pestle , and DNA was extracted using a Plant Mini Kit (Qiagen) and purified with a PCR Template Purification Kit (Roche) according to the manufacturer’s instructions. DNA purity and concentration were measured with a NanoDrop ND-1000 (Thermo Scientific) and purified DNA samples were stored at –20°C until further processing.All isolates were sequenced for the internal transcribed spacers , and will be also provided upon request.Prior to the phylogenetic analysis the 84 ITS-sequences obtained for each algal isolate  were assAlexandrium ostenfeldii/peruvianum group we used a reduced set of the generated alignment containing all represented 24 haplotypes/ribotypes within the dataset. For a phylogenetic analyses based on Bayesian inference (BI), the software MrBayes v3.2 6 generations, sampling every 500 trees. In each run, the first 25% of samples were discarded as the burn-in phase. The stability of model parameters and the convergence of the two runs were confirmed using Tracer v1.5 To determine phylogenetic relationships of Baltic isolates within the Additionally, a maximum likelihood (ML) phylogenetic tree based on the reduced alignment was calculated with PhyML Network analyses were done for ITS sequences using median joining in Network 4.6. AFLP fragments were generated using the modified conditions previously described in Results from the capillary electrophoresis of a total of 84 individuals remained un-normalized when detecting size and height of peaks in the software GENEMAPPER 4.0 (Applied Biosystems) employing GENEMAPPER settings from A. ostenfeldii was analysed by Bayesian probabilistic population assignment of AFLP multilocus genotypes in STRUCTURE 2.3.3. K), ranging from one to seven. Convergence of parameter estimates was observed during runs with both settings and assessment of differences in the variation of the Log likelihood showed that parameters could not be estimated with less variation by extending the burn-in or the MCMC chain from which estimates were sampled. Results of STRUCTURE-analyses were evaluated following the ΔK-method of Genetic structure of Baltic Sea FST values . The fixation index, FST, is a measure of population differentiation based on the partitioning of genetic polymorphism, as e.g. represented in biallelic AFLP data. Values of FST range from 0 to 1, where zero implies no genetic differentiation and the value of one indicates that populations are completely differentiated.The scored data set was subsequently divided into putative populations based on their geographical origin to be used in ARLEQUIN 3.5 FST given the set of AFLP markers used in this study was assessed in POWSIM version 4.1 FST = 0.07, p<0.05 after correction). As POWSIM is restricted to 50 loci, we selected those 50 AFLP loci with highest differentiating capacity . In POWSIM the standard settings of 1,000 dememorizations (burn-in iterations), 100 batches with 1,000 iterations per batch were used.The p-values were corrected for multiple pair-wise tests with Bonferroni and Šidák procedures Nei’s unbiased estimate of gene diversity across all loci IsAIsA and their significance were estimated by Monte Carlo simulation  to assess if multilocus linkage disequilibrium was present within the populations of A. ostenfeldii. The calculations were performed in the software LIAN version 3.5 http://adenine.biz.fh-weihenstephan.de/cgi-bin/lian/lian.cgi.pl).Association analyses were applied on the multilocus genotype data to obtain information on the relative importance of asexual reproduction. The ‘standardized index of association’ Alexandrium ostenfeldii form a monophyletic group. Within the Baltic clade the ITS sequence did not show high differentiation. Minor nucleotide changes were observed among some, but not all, isolates from Kalmar (difference being 0 to 3 nucleotides) , and the other containing groups of strains from the West Atlantic, East Atlantic and the Tasman Sea.The phylogenetic analyses . showed eotides) . While mNetwork analysis  found fiA. peruvianum morphotype from England, Ireland and Spain. The largest genetic distances were between Baltic Sea and global Atlantic and the Tasman Sea in the South Western Pacific A. ostenfeldii strains with a range from 4.4 to 6.1 percent).Uncorrected genetic distance values calculated from ITS sequences  among diK-method of A. ostenfeldii  Following the suggested strategy for inferring the correct number of populations by Pritchard et al. (2010) FST values for all but one pair-wise comparison after multiple test corrections , whereas, the differences between Föglö and Kökar (FST = 0.05) and Föglö and Gotland (FST = 0.05) were the smallest  with geographic distance.The genetic differentiation among the five geographical subpopulations observed in the analysis of STRUCTURE results according to rections . Howeversmallest . The relsmallest , was sigA. ostenfeldii, indicating that genotypic diversity (defined as the proportion of unique genotypes) was high, reaching the value of 1 within each of the five population samples as well as the combined Baltic Sea sample. Values of Nei’s gene diversity, indicative for the distribution of allele frequencies averaged over all AFLP loci, were generally low, ranging from 0.07 to 0.11 at the different Baltic locations  was detected within population samples from Gotland (AOVA), Kalmar (AOKAL) and Puck Bay, Poland (AOPL) with A. ostenfeldii/peruvianum species complex, resembling the complex phylogenetic relationships among global isolates of other Alexandrium species A. ostenfeldii/peruvianum the present phylogenetic analyses resolved six distinct clades. One of these was exclusively comprised by the distinct ITS sequences found in the five investigated Baltic bloom populations. High statistical support for this clade suggests that Baltic Sea A. ostenfeldii form a divergent phylogenetic lineage that is clearly separated from all other known global populations of the A. ostenfeldii/peruvianum species complex.Phylogenetic analyses of the internal transcribed spacer (ITS) sequence data of the rDNA operon revealed a pronounced structure of the A. ostenfeldii description A. ostenfeldii isolates including the ones from the Icelandic type location, and from strains morphologically assigned to A. peruvianum. The apparent mismatch between morpho- and ribotypes emphasizes the present lack of understanding of the evolutionary relationships in the A. ostenfeldii/peruvianum species complex and it indicates the incompatibility of molecular phylogenetic evidence with the hitherto applied morphological species concept. The differentiation of several ribotypes within a single morphotype and the intermingling of morphotypes in phylogenetic clades suggest that cryptic species may also exist in the A. ostenfeldii/peruvianum complex . Though morphologically largely in accordance with the A. ostenfeldii seems to be paradoxical with regard to the timing of colonization of the Baltic and current understanding of the speed of ribosomal sequence evolution in dinoflagellates. The observed differentiation at rDNA level suggests long-standing divergence of the Baltic populations. Even though the rDNA marker used in this study (ITS) is considered to be evolving rapidly compared to 18S rDNA and 28S rDNA, sequence divergence still represents geological rather than ecological time spans according to general expectations. Even neutral mutations resulting from different, local selective regimes are not considered to be expressed rapidly in the rDNA A. ostenfeldii and their geographically neighboring populations from the North Atlantic suggests separation times that by far exceed the postglacial formation of the Baltic Sea ecosystem. Assuming that this degree of genetic divergence represents evolutionary processes on longer time scales, the relatively young Baltic Sea system might have been colonized by dispersing individuals of low salinity adapted populations from other brackish water habitats. Such process would well agree with the idea of large scale dispersal and environmental filtering of microbial populations according to Beijerinck’s hypothesis and its contemporary variants.The divergence among ITS lineages within the Baltic strains of A. ostenfeldii might have accelerated divergence rates, which could have led to the observed pattern. These, in turn are most likely explained by strong effects of neutral and/or adaptive processes governed by the very specific environmental regime in the Baltic Sea. Such conditions may even support the evolution of new species as shown by the case of the brown algae Fucus radicans, which has diverged from Fucus vesiculosus well after the postglacial opening of the Baltic Sea within only a few hundred years However, while the evolution rate of rDNA generally seems to be relatively slow in microbial metapopulations A. ostenfeldii from the Baltic Sea is in contrast to the pattern shown for the congeneric dinoflagellate A. minutum in the Mediterranean Sea, where ITS ribotypes in a large number of isolates were identical despite the presence of clear population differentiation as assessed by polymorphic microsatellite loci It has, furthermore, been suggested that the rise of genetically unique and highly differentiated Baltic populations of widely distributed bivalve species may be a result of postglacial hybridization of long time isolated, geographically distant lineages that were naturally dispersed into the newly opened Baltic Sea. In such lineages processes of genetic differentiation seem to be significantly faster than it would generally be expected from neutral mutation rates A. ostenfeldii may have been selected through environmental filtering from a global pool of brackish-adapted populations not covered by the present strain sampling. More extensive representation of global strains including samples from other brackish-water habitats may reveal that monophyly of the Baltic clade is not provided which would imply that the adaptation to brackish water could have arisen long before the appearance of the Baltic Sea, and the Baltic lineage would have derived from dispersal.Although the present data support the concept of a distinct Baltic lineage that diverged due to adaptation to low salinity conditions of the Baltic Sea after the last glaciations, it cannot be excluded that Baltic A. ostenfeldii clade does not reflect an obvious differentiation pattern. ITS haplotype network analyses revealed that the two major haplotypes were present in northern, central as well as southern bloom sites. The only exception is Kalmar, which includes a unique haplotype represented by one third of the strains analyzed from that population, but lacks another haplotype that is shared by all other populations sampled in this study. This could be interpreted as a indication of weak genetic differentiation. Apparently the level of gene flow among the majority of Baltic bloom populations is too high to generate a clear signature of differentiation at the rDNA level.The distribution of divergent ITS lineages within the Baltic FST, among all studied Baltic subpopulations, except for the pairwise analysis between Gotland and Kalmar where statistical support was considerably lower though still significant at p<0.05. Interestingly, the two methods for inference of the number of genetic population subgroups applied in this study yielded different results. These findings may indicate that the ΔK-method tends to identify the largest genetic break among hypothetical population subgroups, whereas the method proposed by Pritchard et al. 2010 K-method in some cases, especially when relatively weak genetic differentiation among geographically separated populations must be assumed, tends to not reveal the finest underlying population structure As expected, a more obvious differentiation pattern was revealed by the analysis of >500 independent AFLP markers. Here the two hypothetical genetic population subdivisions identified by STRUCTURE according to the ΔK-method are predominantly related to geographic locations in the northern (Föglö and Kökar) and central (Gotland) Baltic or to southern locations  see  A and B)K = 2, becomes more evident in the STRUCTURE assignment pattern with K = 5. Whereas the “southern group” does not show further subdivision, the “northern group” divides into additional subgroups. By displaying a geographical prevalence these subgroups allow for identifying a pattern of more subtle genetic differentiation and gene flow. Whereas the dominant proportion of the subdivided “northern” genetic component A. ostenfeldii seems to be linked to the current regime prevailing in the Baltic Sea and possibly also to life cycle peculiarities of the organism as reasoned in the following. Gene flow between the southern, Puck Bay, Poland and Kalmar) and northern populations can be expected to be limited, since these bloom locations are not immediately connected by the major circulation pattern: From the Polish coast, the major currents move along the southern coasts northeastwards up into the Gulf of Finland. The currents passing Åland, on the other hand, predominantly take northern or western directions before they join the coastal currents moving southwards between Gotland and the coast of Sweden FST values obtained from AFLP among Kökar, Föglö and Gotland might therefore be explained by relatively high rates of gene flow. The Coastal Swedish current, in turn, is likely to facilitate gene flow from Gotland to Kalmar. The presence of “southern” genotypes in the Gotland bloom location despite the North-South direction of the major current flow may be explained by transport through an internal circulation system of the Central Baltic Proper, which surpasses the loop of eastwards coastal currents into to Gulf of Finland and re-enters the western current after passing the northern tip of Gotland. This internal loop directly connects the southern locations  with central Gotland.The genetic substructure suggested by multilocus genotype assignment data for Baltic K of FST = 0.05, p<0.001 after correction) is evident in the STRUCTURE results from K = 5 with genetic partitions uniquely found in both populations  depend on suitable sites for cyst deposition.Our findings on A. ostenfeldii is not possible due to the lack of sufficient isolates for AFLP analyses from the latter, comparison with adequate data for other dinoflagellate species A. ostenfeldii (ranging from 0.07 to 0.11) are generally lower. For example, the gene diversity reported for an Atlantic A. tamarense population – a species that does not exist in the Baltic Sea – was twice as high as that of Baltic Sea A. ostenfeldiiA. ostenfeldii – have been detected in Polarella glacialis and Scrippsiella aff. hangoei populations from saline Antarctic lakes A. ostenfeldii populations are attributed to the ecologically extreme and geographically marginal habitat conditions, the species experiences in the central and northern Baltic Sea.Although direct comparison between Baltic and North Atlantic populations of A. ostenfeldii, in which life cycle strategies combine sexual and asexual reproduction modes, such phenomena might be especially common. Association analyses, an approach that is commonly used to assess reproduction modes in microbial organisms, where sexuality cannot be observed directly IsA values significantly different from zero – in the southern and central subpopulations , which indicates a strong influence of asexual reproduction.Besides population genetic factors such as bottlenecks and genetic drift, other factors that have been proposed to cause a decrease in gene diversity include the loss of sexual reproduction or shifts towards a higher contribution of asexual reproduction. In organisms such as A. ostenfeldii/peruvianum can produce asexual resting cysts Scrippsiella hangoei, which mainly encysts without prior sexual zygote formation This finding could be the result of an atypical life cycle strategy with prevalence of asexual cysts instead of the typical sexual resting stages IsA as their specific signature of selection. The last of these possible causes of multilocus LD has been proposed to explain findings in the congeneric species A. tamarense in a detailed study on a planktonic population of this species from the North Sea Low incidence of recombination as suggested by significant LD may also be a result of homothallism and high selfing rates A. ostenfeldii.In addition to the above detailed causes for intrinsic generation of multilocus LD within a local population, admixture LD caused by admixture of individuals from other genetically differentiated populations might explain within population multilocus LD especially in the case of the population sample from Gotland, where the most complex pattern of within population genetic structure was observed. Here, similar to the basin wide detected multilocus LD, the observed deviation from an independent distribution of alleles within multilocus genotypes might predominantly result from the simultaneous analysis of multilocus genotypes originating from genetically differentiated populations. The (relative) contribution of one of the possible causes for multilocus LD, however, cannot be specified with the data at hand and more intensive studies of processes at the within population level are certainly needed to unravel the significance of intrinsic population genetic processes in meroplanktonic dinoflagellates such as A. ostenfeldii analyzed in this study. This confirms what has so far been shown by most other studies on marine microalgae using markers with enough differentiating capacity  analysis 84 individual A. ostenfeldii AFLP-genotypes.(TIF)Click here for additional data file.Table S1Sampling area and rate of culture survival.(DOCX)Click here for additional data file.Table S2Information on strains and species used for ITS phylogenetic analysis.(DOCX)Click here for additional data file.Table S3Description of AFLP primer characteristics.(DOCX)Click here for additional data file.Table S4Genetic difference and distance between populations based on ITS regions (572 bp).(DOCX)Click here for additional data file.Text S1AFLP reaction protocol.(DOCX)Click here for additional data file.


We estimated neutral diversity of 21 European cattle breeds with 105 microsatellites. Nine of them resembled unselected Balkan Buša strains with diffuse breeding barriers and the 12 others were strongly differentiated, isolated breeds. Because of the impact of neutral genetic diversity on long-term population adaptive capacity, we discuss the long-term outcome of different conservation priorities in a subdivided metapopulation of the investigated cattle breeds. The optimal contribution to a pool of total genetic diversity allocated more than 95% of long-term relevant neutral diversity to virtually unselected strains of the Balkan Buša, while the maximization of total variance preferred inbred breeds. Current artificial selection methods, such as genomic selection sped up and a recovery of underestimated traits becomes quickly impossible. We emphasize that currently neutral and even deleterious alleles might be required for future genotypes in sustainable and efficient livestock breeding and production systems of a 21st century. We provide cumulative evidences that long-term survival relies on genetic complexity and complexity relies on allelic diversity. Our results suggest that virtually unselected, nonuniform strains harbor a crucial proportion of neutral diversity and should be conserved with high global priority. As one example, we suggest a cooperative maintenance of the nondifferentiated, highly fragmented, and fast vanishing metapopulation of Balkan Buša. Population genetic diversity is the basis of evolutionary potential of species to respond to environmental changes e.g., . In popuMVT; POOLGD; NC;NC maximizes the allelic richness (AR) of the protected pool and thus, according to the discussion in POOLGD approach. Different weighting of within- and between-subpopulation diversity results in diametric conservation concepts (MVT) coupled with inbreeding depression and direct dependence on expected heterozygosity (EH) (POOLGD or NC) prefer subpopulations with high AR  or virtually unselected and hosted in challenging environments  are regarded. We discuss different conservation methods that are compatible with the existing population genetics theory but lead to diametric results. First, we look at the maximization of the total genetic variance of a hypothetical trait MVT; , which iconcepts . One preity (HE) , and othWe used the data of 21 European cattle subpopulations. These subpopulations are characterized as breeds or strains depending on their breeding monitoring level and administrative isolation. While animals of commercial breeds fulfill some phenotypic standards defined by breeding organizations and are subjected to recording (pedigree and phenotypes), assortative mating, and administrative isolation, local strains are more diffuse domestic subpopulations. In general, local strains are formed by many more or less fragmented subpopulations without organized recording, selection, strict breeding isolation, and monitoring by breeding associations see .Another subdivision of the domesticated cattle species follows the geographic distribution into some cosmopolitan and many local subpopulations. Commercial cosmopolitan breeds are isolated from local subpopulations by their administration. Local subpopulations can be divided into two groups: (1) local breeds, that have been managed in the past and are well differentiated but have lost economic importance and remain in small census numbers with a small effective population size and (2) local strains, that have never been intensively managed or differentiated, whose effective population size is still large but whose census number is dramatically decreasing.The core sample set of this study consists of nine virtually unselected autochthonous local strains of the Balkan with more diffuse barriers  and is eA total of 105 microsatellites was used and all 965 samples were genotyped twice in two independent courses. The standard methods of genomic DNA extraction, PCR amplification, microsatellite genotyping, and exclusion of 12 outliers  followedOH) and  that implements the approach described by genepop v.4.0 software package , private alleles (pA) , and rare alleles (rA) that are nonprivate alleles with an arbitrary chosen frequency less than 0.01 over the whole population. This criterion mostly implies a frequency of <5% in the respective subpopulation according to the total sample size of 965, an average sample size of 46 animals, and a distribution of rA (present in <20 gametes) mostly over two to three subpopulations.Estimates of genetic variability, observed heterozygosity  by weighted least squares regression, which takes into account the heterogeneity of the LD variances (ldNe does not directly represent the true effective population size (Ne) but it is well related  in the dataset, independent of breed affiliation. Ten independent runs of K = 1 to 11 were carried out, burn-in period 50,000 and 200,000 replicates. To determine the most likely hierarchical structure, we used the log probability of data LnP(D) and estimated the delta K (ΔK) statistics as explained in structure results were plotted with distruct  protected subpopulations or breeds.In total, 105 microsatellites were genotyped in 21 cattle subpopulations. For phylogenetic and conservation studies, 93 neutral markers were chosen see . TherefoGDpoolc), the methods described by metapop 1.0.1  reflect the importance of the breeds for the actual diversity defined by the total genetic variance of a hypothetical quantitative trait, which implicitly gives two times more weight to between- than within-population diversity . First, the nine Buša subpopulations were divided into two metapopulations  each containing one-half of randomly chosen individuals. This subdivision was performed within strains, therefore both MetaB1 and MetaB2 contain one random half of each of the nine Buša subpopulations. Second, the most diverse Buša subpopulation, RMB, was used to derive the simulated subpopulation of small effective population size. In successive steps, RMB was downsized and then reproduced by random mating. We used a random mating of 44 RMB animals to form the next generation of 42 animals. After subsequent 13 non-overlapping generations, a final size of 20 animals was reached. These were reproduced at constant population size for the next 10 generations again by random mating. This led to the depleted population RMBD. Third, following the procedure for RMBD, the most diverse Alpine breed, FV, was also downsized to 20 animals and simulated as the depleted subpopulation FVD. For both RMBD and FVD, the random mating allowed that one random animal could have several offspring while another could have none.To corroborate a decision-making process and to illustrate different outcomes of distinct strategies for conservation in subdivided populations, we added four simulated populations to our samples. These synthetic populations were derived from the real sample data. This relatively simple procedure was carried out with an own Finally, we considered a set of 16 subpopulations including (1) all 12 non-Buša breeds without any changes, (2) two by simulation depleted subpopulations, and (3) two metapopulations each including the half random chosen Buša animals. This set of 16 subpopulations was analyzed by all three conservation strategies.nA), and the EH and OH over all 965 individual animals. The genetic variability of BHB, MNB, and PRB with 0.724, 0.723, and 0.728, respectively, is within the Buša range of 0.723–0.747. tA), pA and E, HOrA, AR, H, and the ISF statistic estimated for 21 breeds for 93 neutral loci. According to previous results  that have been used intensively for upgrading of cattle populations in the Buša sampling region during the past. The assignment test directed 185 (44%) of the 420 individuals to their respective populations. The structure results of the nine Buša strains and two Alpine breeds, TGV and OBV, and the ΔK values are shown in K = 2, K = 6, and K = 11 with highest ΔK values. TGV and OBV were found in distinct clusters, also PRB and, with increasing K, IMB. The remaining seven Buša strains showed a more diffuse clustering with single groups of individuals within a local strain being concise clusters rather than the whole subpopulation.The results of the assignment test and MVTNC, c, and GDpoolc. Apparently, MVTc prioritizes inbred breeds with a known recent bottleneck such as HRP, GLW, MWF, and TGV. Six prioritized breeds with MVTc≥ 10%  have AR, numbers of observed total, private, and rare alleles  contribution to the total genetic pool and account for 94.5%. These five populations have a significantly higher AR, higher numbers of observed total, private, and rare alleles  appear on top of the conservation priority of MVTc. HRP is a Croatian strain of a formerly large podolian cattle population of the Pannonian plain. It is well documented  did not prioritize either FV or FVD. In addition, RMB as a highly diverse subpopulation was listed highest with GDpoolc. After the simulated bottleneck and substantially loss of neutral diversity , only a limited (3.5%) GDpoolc contribution was estimated for RMBD. Remarkably, this contribution is still exceeding the contribution of well-managed modern breeds (0.0–2.6%).The results of the simulated populations follow the trend outlined above. Both FV and RMB do not appear in the ranking of the cumented  that thiNC gave highest priority for these two metapopulations but lowest for two artificially depleted breeds  before the random fusion but none thereafter. By minimizing coancestry in a subdivided metapopulation, we estimated a cumulative GDpoolc = 95.2% of nine Buša strains  given to within- versus between-population diversity. These methods representing different conservation concepts have been compared , and it LDNe estimator of effective population size is 1.84 times larger in five GDpoolc-prioritized Buša strains than in six MVTc-prioritized breeds. The MVT core set approach aims to conserve breeds that show comparatively large differences in the respective population mean of a hypothetical quantitative trait. This seems to make the MVT core set method attractive, because the efficiency of upgrading a breed by introducing alleles from another breed is a function of the difference between the respective population means  still overemphasizes on between-breed variation and gives the by far highest priority to a population with the lowest diversity  that is not unified by systematic selection but simply by chance due to a strong bottleneck (GDpoolc) does not favor the commercially breeds with large census sizes  but the virtually unselected strains of the Buša metapopulation with a large effective population size. Four synthetic populations derived from the sample data corroborate this. Simple depletion by chance of the Alpine breed  or the most diverse Buša strain (RNBD) led to MVTc values comparable with HRP  and non-market  issues are given consideration. Thus, livestock producers will maintain their drive for maximization of output but with diminished reliance on industrialization and therapeutic measures to alleviate environmental stress and rely more on the genetic make-up of their animals to combat stress from heat, parasites, pathogens, and poor nutrition . FitnessThe cattle breeds chosen for this study present a wide spectrum of productivity in current farm animals. On the one hand, there are virtually unselected Buša strains e.g.  with higGDpoolc concept (A) and total possible response (TR). The long-term survival relies on balance between the best possible current adaptation  and a widest open evolutionary window for future developments . Similar as demonstrated for selection response  can be achieved with a lower frequency of currently deleterious or neutral alleles of each single polymorphic locus. But in effectively small populations, the allele with a very low frequency can be often get lost by genetic drift. Likewise, selection can easily drive to fixation of an adaptive locus. Both is directly penalized by the reduction of the evolutionary window (TR) and consequently decreases the long-term survival probability (TR/σA→Max), even if currently not favorable variants are affected by the loss. Therefore, the number of segregating loci (n) should be maintained or even increased by recurrent mutations. The maintenance of new and low frequent alleles is more probable in effectively large populations , only partly by MVTc (λ = 0.5) but neglected by the Weitzman The future environmental changes a species will face cannot be predicted, thus, it is not sensible to prioritize a few actual adaptive alleles that may not be the adaptive alleles of the future . This isstructure results these strains were not very well differentiated except for PRB and IMB and the Alpine breeds, which was to be expected. PRB is found in a geographical isolated position . Second, only animals confirmed as not admixed with foreign breeds  should be chosen for exchange. Third, spatially closer neighbors  are to bWe investigated cattle breeds in Europe on a wide scale by a relatively large marker set and allocated more than 95% of long-term relevant neutral diversity to virtually unselected strains of cattle in the Balkan area. It is a complex and multifaceted decision-making process to prioritize breeds to participate in a conservation program and other factors than the genetic may need to be considered. However, relating to the latter, we recommend by cumulative evidence to emphasize equally on within- and between-breed variations. We suggested controlling tools for cooperative maintenance of the nondifferentiated but highly fragmented and fast vanishing metapopulation of Balkan Buša. The maintenance of the genetic diversity is a global and long-term task and we appeal for global coordinated efforts to step up the long-term conservation infrastructure. As there are obvious parallels to some other strains of domesticated ruminants, we think that the conservation strategy we suggested for cattle might also be relevant for sheep and goat breeds, and in general for other farm animals or subdivided populations.