Rangifer colonization and propose that the High Arctic archipelagos of Svalbard, Franz Josef Land, and Novaia Zemlia were colonized by reindeer from the Eurasian mainland after the last glacial maximum. Comparing mtDNA control region sequences from the three Arctic archipelagos showed a strong genetic connection between the populations, supporting a common origin in the past. A genetic connection between the three archipelagos and two Russian mainland populations was also found, suggesting colonization of the Eurasian high Arctic archipelagos from the Eurasian mainland. The age of the Franz Josef Land material (>2000 years before present) implies that Arctic indigenous reindeer colonized the Eurasian Arctic archipelagos through natural dispersal, before humans approached this region.In light of current debates on global climate change it has become important to know more on how large, roaming species have responded to environmental change in the past. Using the highly variable mitochondrial control region, we revisit theories of  Climatic oscillations over the Quaternary (2.4 million years ago–present) have had a major impact on the geographic distribution and genetic structure of species through population extinctions and range shifts . The demRangifer tarandus) is a keystone species in the circumpolar North, not only ecologically through the way they impact upon the plant cover [Rangifer was distributed south of the ice sheet in both Eurasia and in North America, and in the Beringia refugium encompassing the Bering land bridge, Alaska, as well as large parts of Siberia [Rangifer exhibit distinct morphological adaptations to different environments, and populations have been subdivided into various ecotypes according to their life-history strategies and ecological conditions such as the woodland or boreal forest or sedentary form, the barren-ground or tundra or migratory form, the mountain form, and the Arctic form [R.t platyrhynchus), distributed on the Svalbard archipelago, the North American Peary caribou (R.t pearyi) primarily distributed on the Canadian Arctic archipelagos, and the recently extinct R.t eogroenlandicus, formerly distributed on Eastern parts of Greenland [R.t. pearsoni) with a view to restoration [Reindeer and caribou  [Rangifer tarandus) [Alopex lagopus) [Gulo gulo) [R.t eogroenlandicus have been shown to comprise of mtDNA haplotypes signalling a common origin in an ancient Beringian and/or Eurasian pre-glacial population [R.t eogroenlandicus shared certain mtDNA haplotypes, morphological similarities, as well as have been observed to migrate from Ellesmere Island to North Greenland, provides convincing evidence for a North American colonization route for these two subspecies [There has been a wide-ranging debate on the colonization routes and dispersal of Arctic reindeer inhabiting the islands in the western Eurasian and North American Arctic , 18, 19.andicus) , reindeelagopus)  and the lo gulo) . Svalbarpulation . The facSvalbard reindeer are characterized by low genetic variability indicating isolation, possible bottlenecks, and subsequent genetic drift as important population processes , 25. TheThe idea that the wild reindeer on some of the Arctic archipelagos might have been genetically linked to the Eurasian mainland was first raised by Gravlund et al.  who founth century onwards [th century were recorded as living without domestic reindeer [A third account of the colonization of some of the High Arctic islands focusses on human-instigated translocations. Resolving the question of possible human induced translocation is important for the issue of introgression from domestic herds, a key parameter for classifying protection status of populations. There is a relatively broad Russian language literature documenting the human interest in wild reindeer, and the movement of reindeer herds between islands in the Eastern Barents Sea in historic times , 37, 38. onwards . There i onwards , althougreindeer . There areindeer , 37, 38.reindeer . HoweverRangifer populations in both North America and Eurasia, there has been a conspicuous lack of studies comparing the reindeer populations on each of these three neighbouring archipelagos to each other–the clearest approach to discussing possible common colonization routes. A genetic link between Svalbard, Novaia Zemlia and Franz Josef Land would tell us if these archipelagic populations have a common origin and also help to answer the question of whether or not the current distribution of the Arctic type is caused by natural dispersal or recent human induced dispersal of domestic reindeer. We also included sequence data from reindeer on the nearest population of domestic reindeer situated on Kolguev Island to test for introgression from domestic reindeer. Two wild mainland- and one wild island population from Russia were included to help answer the question of a possible biographical link between the Arctic archipelagos and the Eurasian mainland.Here, we use the highly variable CR to compare sequence data from contemporary reindeer populations on Svalbard and Novaia Zemlia, with ancient samples from the now extinct population on the Franz Josef Land archipelago. Despite the work done comparing Svalbard reindeer to the mainland 14C dated  and the headwaters of the Pechora River in the Pechro-Ilychskii Nature Reserve, Komi Republic (n = 10), were downloaded from GenBank and included in the analyses in order to test for possible gene flow between the mainland and the archipelagos , Novaia Zemlia (n = 20) and Franz Josef Land (n = 15.) The Svalbard population was sampled at Nordenskiöld Land on Spitsbergen and on Nordaustlandet. Wild reindeer samples from Novaia Zemlia were collected on the South Island . Archaic4C dated . Skin- aipelagos . Additioipelagos . The PezBlood and muscle samples from Svalbard were collected as part of the Man and the Biosphere (MAB) project which started in 1978. The Svalbard MAB field project was coordinated by the Norwegian Polar Institute . Hunting permits of reindeer within the Svalbard MAB field project was approved by the Governor of Svalbard. Sampling of muscle-, skin- and velvet samples from Novaia Zemlia, Belyi Island and Kolguev required no specific permits and was done under an ethics review for the ERC Arctic Domus and performed under the ERC Arctic Domus ethics annex.Muscle and skin samples of wild reindeer from Novaia Zemlia and Belyi Island were collected from dead animals via subsistence hunting. The sampling was conducted by the authorized managers of these populations, according to the regulations stated by the Ministry of Nature Protection of the Russian Federation. Velvet samples from domestic reindeer on Kolguev Island was collected from dead animals during industrial slaughter. No animals were sacrificed for this study and the field work did not involve endangered or protected species.Tissue samples were stored in ethanol (≥ 80%) or kept frozen until analysed, blood samples were stored in EDTA. DNA extraction of muscle-, velvet- and skin samples was performed using DNeasy Blood & Tissue Kit (Qiagen) following the manufactures protocol. DNA extraction from EDTA blood was carried out using DNeasy Blood & Tissue Kit (Qiagen) or by using a boiling method for DNA extraction (SI).°C for 2 min, 95°C for 30 sec, 55°C for 30 sec and 72 oC for 1 min (step 2–4 cycled 30 times) and finally 72 oC for 10 min. PCR reactions were performed in 20 μl total volume using 1–2 μl DNA template, and with the following final concentrations; 1X buffer, 1.5 mM MgCl2, 0.8 mM dNTPs, 5 pmol of each primer, 0.5 μg/μl Bovine Serum Albumin (BSA), 0.5 U/μl AmpliTaq DNA polymerase (Applied Biosystems), and dH2O to make up the remaining volume.A 503 base pair (bp) long fragment from the mitochondrial control region was amplified using the forward primer RtCRF (5`-AAT AGC CCC ACT ATG AGC ACCC-3`)  and the The samples were cleaned for unincorporated primers and nucleotides using Illustra ExoProStar  diluted 10 times. Cycle sequencing was performed in a 10 μl reaction volume, using BigDye v3.1 sequencing kit (Applied Biosystems) following manufacturer`s recommendations. Purification was carried out using standard EDTA/EtOH precipitation. Capillary electrophoresis and data analysis were performed with an ABI 3130xL- or 3500xL instrument (Applied Biosystems). All sequences were sequenced in both directions and the consensus sequences were aligned by ClustalW  and editDNA was extracted from antler powder using DNeasy Blood & Tissue kit (Qiagen) following Bjørnstad and Røed . StandarFrom the ancient material a 266 bp fragment of the mtDNA control region was amplified using the primer pair 259F/524R (5’–TGCCCCATGCTTATAAGCAAG–3’/ 5’–GTGAGATGGCCCTGAAGAAA–’3), or by amplifying two overlapping amplicons of respectively 140 bp with primers 259F and 398R (5’- CCTTTCTTGTCAACATGCGTA– 3’) and 178 bp with primers 347 F (5’–TGCCCCATGCTTATAAGCAAG–3’) and 524R. PCR amplification and sequencing were performed as in Bjørnstad and Røed . The seq14C dated 4 antler samples from Franz Josef Land which also amplified successful DNA  were calculated in DnaSP  for the A total of 137 samples from seven populations were analyzed for the mitochondrial control region, including 122 contemporary- and 15 ancient samples. The 400 bp long alignment, comprising the contemporary populations, varied from low to relatively high levels of genetic diversity with a total of 30 haplotypes, overall haplotype diversity (Hd) = 0.910, and nucleotide diversity (π) = 0.019 . The SvaIc, Id, Ie and cluster II [Ic showing an intermediate level of support (posterior probability = 71). In the present study, we found sub-cluster Ic to comprise the 3 haplotypes previously found in Svalbard reindeer (n = 27), but also to include haplotypes found on Novaia Zemlia (n = 12), the Pechora River (n = 3), the Peza River (n = 1) and Franz Josef Land (n = 13) , Belyi Island (n = 1), Peza River (n = 2), Pechora River (n = 1), Novaia Zemlia (n = 2) and Franz Josef Land (n = 1) , Novaia Zemlia (n = 6) and on Belyi Island (n = 6)  Figs  and 2. Hm Taimyr . In the and Figs  and 2. Ireindeer . However 1) Figs  and 2. Freindeer . We foun 6) Figs  and 2. Ondinavia , 69.Ic appear to have experienced a recent population expansion as the mismatch distribution analysis showed no significant deviation from the expected distribution under the sudden expansion model (see 2 were highly significant with Fs = 5.948 (p = 0.000) and R2 = 0.103 (p = 0.002) adding support for a demographic expansion for this sub-cluster. Tajima`s D was negative (-1.018), but not significant (p = 0.173). Date since expansion for sub-cluster Ic was calculated to 5862 years before present (YBP) (95% CI: 535–10375) based on the mean number of pairwise differences, τ (τ = 2.762).Sub-cluster odel see . An expaIe, 13 in sub-cluster Ic, and one sample with a haplotype unique for Franz Josef Land  and 24 haplotypes were identified. However, low levels of genetic variation was found in the ancient Franz Josef Land population  . Three hand Figs  and 2.Ic haplotypes in the two mainland populations could be the result of random haplotype survival from a common refugial population. More extensive sampling of Russian wild reindeer, with a special emphasis on islands in northern Siberia, would be necessary to clarify this further.Our results show that the most common haplotype found on Svalbard is also the most common haplotype found among the contemporary wild population on the South Islands of Novaia Zemlia and the extinct population on Franz Josef Land–suggesting that the population history of wild reindeer on these High Arctic islands was tightly linked. The genetic similarity between these archipelagic reindeer populations indicate gene flow and dispersal from a common source population, and supports the theory that all of these islands were likely colonized from the Eurasian mainland. The hypothesis of an eastern colonization of the Arctic archipelagoes is further supported by a genetic connection between the three archipelagic populations and one individual from Pechora River (Komi Republic), who shared the most common haplotype found on Svalbard, Novaia Zemlia and Franz Josef Land. Also, three similar haplotypes were observed in single samples from wild reindeer in both Pechora river basin and in the Peza river basin. However, the presence of Published radiocarbon dates of archaic bones collected on Franz Josef Land suggest that wild reindeer populated the archipelago 6400–1300 YBP . These rIe). This finding can be explained by the recent translocation of a small number of domestic reindeer from Kolguev to Novaia Zemlia, between 1928 and 1931 [We did find one individual on Novaia Zemlia with a haplotype also found in reindeer on Kolguev Island (sub-cluster and 1931 . The weaRangifer [Lemmus) questioning the centrality of the traditional Beringian refuge for the post-glacial re-colonization of the Arctic. They demonstrate how four different mtDNA linages of the circumpolar lemming (Lemmus) indicate separation by glacial barriers, followed by post glacial colonization from refugia other than Beringia. Shaefer et al. [Ic as recently as 5000–6000 YBP. This implies that the unique genetic composition of these Arctic reindeer populations may have resulted from bottlenecking, isolation, and then subsequent expansion in the High Arctic well after the retreat of the ice, rather than isolation in alternative refugia during the last glacial maximum (LGM). Further studies of the genetic structure in Russian reindeer populations would help to answer this problem.Our results indicate an eastern colonization route of the Eurasian arctic archipelagos. The Bering land refuge has traditionally been most widely discussed as a single origin point for various continental distributions of Rangifer , 75 as wRangifer . HoweverRangifer  performer et al.  in theirr et al.  in theirPicea) and birch (Betula) tree lines in northern Eurasia were located at least 150 km further North from their present location, and in the case of birch, may have reached the seacoast [Post-glacial range shifts and the expansion of reindeer populations, as well as those of other cold-adapted species, is probably connected to the major environmental changes taking place in northern Eurasia during the Holocene . Sea surseacoast , 79. Theseacoast . As mentseacoast , suggestRangifer are in many ways a classic circumpolar species providing an important anchor to the environmental history of the High Arctic, and also to the lives of local people. Contemporary climate change would be expected to alter the distribution and demography of Rangifer today as has been the case in the past. Mammals living in the High Arctic have limited opportunities to migrate further north. To survive, they will have to depend on their ability to adapt where they are. Therefore they are under particular risk. This necessitates having proper management plans with an emphasis on conserving genetic variability for indigenous archipelagic reindeer.This study has established certain strong genetic similarities found between wild reindeer populations on Svalbard, Novaia Zemlia and Franz Josef Land implying that the maternal genetic structure of these archipelagic reindeer populations is indigenous and unique. The study lends considerable weight to the hypothesis that these islands may have colonized from the Eurasian mainland via an eastern route. Moreover the study strongly disproves the suggestion that populations for example on Novaia Zemlia are feral populations of introduced domestic reindeer. It is our hope that these important results will help clarify existing conservation plans for wild reindeer on Novaia Zemlia in the Russian Arctic Strict Nature Reserve and its aim at conserving this important and unique population.S1 FigIc, Id, Ie and II .The Bayesian phylogeny shows 30 control region haplotypes and support for sub-cluster (TIF)Click here for additional data file.S2 FigIc. The mismatch analyses show a unimodal distribution, which is characteristic for a recently expanded population [The observed pairwise difference (blue bars) and the expected mismatch distribution (red bars) under the sudden expansion model among individuals in sub-cluster pulation .(TIF)Click here for additional data file.S1 Table14C dates were calibrated using CALIB 6.1.1 [The IB 6.1.1 , based oIB 6.1.1  with 2σ (PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Genetic counselling is not routinely offered for psychiatric disorders in the United Kingdom through NHS regional clinical genetics departments. However, recent genomic advances, confirming a genetic contribution to mental illness, are anticipated to increase demand for psychiatric genetic counselling. This is the first study of its kind to employ qualitative methods of research to explore accounts of psychiatric health professionals regarding the prospects for genetic counselling services within clinical psychiatry in the UK. Data were collected from 32 questionnaire participants, and 9 subsequent interviewees. Data analysis revealed that although participants had not encountered patients explicitly demanding psychiatric genetic counselling, psychiatric health professionals believe that such a service would be useful and desirable. Genomic advances may have significant implications for genetic counselling in clinical psychiatry even if these discoveries do not lead to genetic testing. Psychiatric health professionals describe clinical genetics as a skilled profession capable of combining complex risk communication with much needed psychosocial support. However, participants noted barriers to the implementation of psychiatric genetic counselling services including, but not limited to, the complexities of uncertainty in psychiatric diagnoses, patient engagement and ethical concerns regarding limited capacity. Genetic counselling is currently available in the United Kingdom (UK) for a variety of disorders and diseases with a substantial genetic component. Concerned with the cause, course, diagnosis and treatment of genetic disorders, genetic counselling is a medical specialty offering information-giving and psychosocial support to affected individuals and their families. Mirroring the broader purpose of genetic counselling  have identified an increasing number of ‘susceptibility’ loci – risk alleles that confer small non-additive genetic effects for a whole range of psychiatric disorders. For instance, the Schizophrenia Working Group for the Psychiatric Genomics Consortium  identifiToday, psychiatric disorders are considered heritable conditions that affect individuals and their families’ worldwide , community psychiatric nurses (n = 7) and consultant psychiatrists (n = 9). Of the participants who returned the initial recruitment questionnaire, 6 were male and 26 were female. All participants were mental health professionals working for Cardiff & Vale University Health Board Trust aged between 18 and 54.Thirty two questionnaires were returned. The study gatekeeper estimated that email links were sent to over 100 eligible members of staff, however, due to the method of dissemination this figure cannot be confirmed. The sample included psychiatric nurses (n = 2), a community psychiatric nurse (n = 1), consultants psychiatrists (n = 2) and consultants psychiatrists with an academic background in psychiatric genetics (n = 4). Eight interviews were conducted face-to-face and one by telephone. Informed consent was obtained prior to each interview. All interviews were audio-recorded and transcribed verbatim.Of the 12 participants who expressed interest in taking part in the qualitative interviews, two did not respond to further correspondence and one was unavailable for interview until after the study deadline. Nine semi-structured interviews were conducted with psychiatric staff nurses  and ‘latent’ (implied) meanings of participants’ knowledge and experience. In this sense, analysis is concerned with how participants account for their knowledge and experience as a discursive practice Whilst many participants indicated that psychiatry required more training in genetics, the demand for genetic counselling had not yet come from the patients. In the following extract, a senior academic psychiatrist gives a historical account of previous clinical referrals for psychiatric disorders in the 1990s.The high demand for cancer genetics had shaped expectations that similar demand for psychiatric genetics would follow. This prompted an informal collaboration between medical and psychiatric genetics. The respondent alludes to the fragility of this arrangement, implying that interest from “genetics” resided mainly with the collaborator rather than the “department”. However, the unmet expectations of a “tidal wave of psychiatric genetics” implies a stronger reason for why the clinic had few referrals..Actually further down the list are other issues that women want to find out and actually quite low down the list is what’s the risk of me passing this illness on to my kid […] because in some respects they are kind of much more proximal problems, aren’t they, like you know, what do I do about my medication now, you know, is this going to make me really ill, these are much more immediate problems Many participants speculated that this low demand related to a priority of environmental rather than genetic factors of psychiatric disorders: “It’s rare for people to think about genetics, issues of genetic risk aren’t top of the list usually” . Many affected individuals and their families seek advice and reassurance about more pressing concerns relating to medication and symptoms rather than genetic risk. Even in the context of prenatal counselling for women, who have a higher risk of postpartum psychosis, genetics was a distal concern:The low demand for genetic counselling is attributed to other priorities, such as giving advice and reassurance about the management of medication and symptoms. In the extract above, the participant is suggesting that issues ‘proximal’ to illness tend to dominate the consultation, which implies that clinical practice is more client-led rather than information-driven..Some of them come to me, parents usually, blaming themselves, worrying about the siblings, the brothers, the sisters. I think that’s a key point for your services to our patients Future demand for genetic counselling was often discussed in the context of what genetic counselling can provide for psychiatric patients and their families. Participants frequently cited methods of managing uncertainty and reducing guilt and stigma as key benefits:…the relatives of our patients often have a number of anxieties which stem from lack of understanding, being scared of the unknown you know. I think genetic counselling may be just what they need, somebody to talk them through the ins and out of the disorder, we don’t tend to spend enough time covering it .Here, genetic counselling provides psychological adaptation and reassurance to unaffected family members. The participant implies that psychosocial support mitigates self-blame by presumably foregrounding the complex aetiology of psychiatric conditions. One participant suggested that genetic counselling provides “time” to explain the disorder and reduce anxiety for families:could perform this role but lack the resources to do it properly.There is a sense in which genetic counselling offers aetiological information to relieve family anxiety, which positions the service in the role of providing additional family support. The statement “we don’t tend to spend enough time covering it” implies that psychiatric nurses .If you’ve got a spouse of somebody that’s had a really severe psychosis, I’d imagine they would want that information. You can imagine that genetic counselling would be really useful in that, which is something that isn’t offered widely but there probably would be uptake for Similarly, there was a notion that genetic counselling could be utilized as an additional “therapeutic tool” for both patients and their families. The availability of a service which deals with at risk relatives as well as affected individuals was identified by participants as a key part of the service:.That a relative can go to see a counsellor independently, not as part of their relatives care, sometimes they think they can ask about themselves and the impact on them, we don’t get around to it, they like to concentrate on the treatment of their kid or parent or whoever it may be. They don’t want to eat into their time The participant offers a hypothetical account of the benefits of genetic counselling for unaffected families, though it is unclear what kind of “information” this entails. Markers such as “would imagine”, “can imagine” and “probably would” suggest a vague understanding of genetic counselling, though, in a positive sense, it provides an alternative space for family support: “somewhere else to discuss and express their concerns”. This was supported by another participant who expressed that families often do not like to raise concerns about their own well-being in the affected individuals’ consultations with the psychiatric team.Genetic counselling is value-adding in terms of providing families an additional space of support. Participants recognized the unique aspect of clinical genetics in its care of families rather than individuals; it provides a private and supportive space to minimize the psychological “impact” of mental illness. Again, the reference to “time” implies that genetic counselling is perceived as an extension of therapeutic support in the context of limited professional resources.We have got to the stage now where we have much more knowledge, and we should start to think about it in a way, setting up a clinic, a clinical application for it .Most participants demonstrated an awareness of genomic advances within the field of psychiatry, though some discordance was observed regarding their implications for future clinical practice. Those involved in psychiatric genetics expressed willingness that genetic research should: “inform clinical psychiatry, part of that would be genetic counselling”:.I don’t think people have really got their heads around the predictive abilities of the genetic knowledge that we have yet, I think it’s still too, too early to say and… I think the position is still… there’s a long way to go before we can use genetics to predict risk in the population and everybody’s really cautious about doing it Though transferring genetic research to the clinical setting is desirable, there is also a sense in which “the next stage” of clinical translation is unclear. Ethical issues regarding early diagnosis and prevention or unnecessary medicalization warrant caution about translating genetic discoveries from the laboratory to the clinic:expectation that affected individuals and their families would benefit from such a service. While all the participants attested to the benefits of a patient-centred approach of genetic counselling, non-genetics professionals conveyed a sometimes vague understanding of its specialization, implying that it provided “additional” care and support for families of affected individuals. Lastly, a sense of caution was raised in relation to explicitly linking genetic services to testing.Although they had not experienced explicit requests for genetic counselling, participants reported an Many participants deliberated over professional responsibility for psychiatric genetic counselling provision. Some concluded that the best arrangement would be a joint collaboration of expertise rather than allocating sole responsibility to either clinical genetics or psychiatry. Several issues were raised regarding the suitability of both services to provide genetic counselling for psychiatric disorders which form the basis of further subthemes..We run our own clinic, tertiary referral clinic, we get some patients who wish to have genetic information, we don’t call it genetic counselling, we don’t believe we’re doing it formally in that sense, but we offer genetics advice. So we do occasionally have people coming [with a] family history of dementia, family history of psychotic disorders and sometimes families with high densities of a number of different things. So, you know, it is very much a question of seeing the person and trying to get the records, but without… to do it properly, it’s very difficult without I think a nurse who goes and takes the time to do the family history For those working in psychiatric genetics, genetic counselling was perceived as a service specific to medical genetics, and thus separate from academic psychiatry. In the extract below, the senior academic psychiatrist is contrasting genetic counselling with their own genetics clinic:.I guess it’s a professional specialism, you know… particularly, medical genetics is, but also the nurses are trained in ways that we’re not. I guess we don’t have the precision… most of the cases we see are much more murky. I think, there is a need to have a more formal set up. And we have thought about that […] So we may well try and develop a formal clinic with a trained nurse, because… I think we are identifying now these cases and families where they have quite highly penetrant mutations, which confusingly predispose to a range of different psychiatric disorders... but mainly neurodevelopmental so, you know, these are quite complicated families to advise and I think there is going to be a need to develop the expertise to deal with that The participant makes a distinction between genetic counselling as a “formal” service and the kind of clinic they offer, which centres on “genetic advice”. Genetic advice-giving is oriented to the collection of information about family history, the gathering of records that may assist in making risk assessments. However, gathering information from families is a specialized skill requiring both “time” and interactional expertise “to do it properly”. From an academic perspective, genetic counselling is perceived as a formal specialism that needs to be developed within psychiatric genetics:The perception of genetic counselling as a “professional specialism” highlights a unique form of training not offered in the psychiatric genetics clinic. The participant attributes this to psychiatric disorders lacking the “precision” of phenotypes seen in medical genetics. The formality of genetic counselling is seen as offering a more rigorous approach to collecting complex case histories and advising families in ways that may support the mutual interests of academic psychiatry and genetic counselling. Interestingly, this expertise is not imagined as a single professional role, but a collaborative relationship between psychiatrists and “trained nurses”.Many participants recognized that genetic counselling embodied a unique set of skills suited to dealing with the genetics of psychiatric disorders. Only one participant expressed concern that genetic counsellors do not feel equipped to deal with psychiatric disorders in their current practice, however, all participants were in agreement that genetic counselling is a “skilled profession”, and better equipped to communicating genetic risk than psychiatric services.I don’t think psychiatrists are trained in that necessarily. No less than any other doctors I’m sure… so you’d imagine that clinical geneticists and people who regularly do genetic counselling are far more skilled in that sense, obviously trained for it and certainly that’s not the case for… people who have gone through sort of generic medical training or even speciality training .A common observation was that psychiatry did not receive training in the provision of genetic risk information; many saw this as a skilled practiced belonging to “clinical genetics”:Genetic counselling involves a “far more” specific set of skills than what is available in the “generic” training of psychiatrists. In this sense, there is very little difference between the training of doctors and psychiatrists. These contrasts serve to distinguish genetic counselling as an established sub-discipline from which psychiatry can benefit.I think the giving of information is a fairly generic skill that any doctor or nurse should be able to master… it’s trying to formulate what information to give, I think that may require more training than the average consultant psychiatrist can do… So for example, the psychiatrist should know the recurrence risks in different classes of relative but then you start to say, well actually I’ve got two, my uncle Fred had bipolar disorder, what does that mean, are my risks different and most people will be thinking, this is beyond my level of expertise, and actually they are very difficult questions to answer. So that’s where I think you might need a more specialised service .In a similar vein, the senior academic psychiatrist also highlighted the generic nature of communicating recurrence risk to families, which implies that genetic counselling is more than merely information-giving:Where communicating recurrence risk is a transferrable skill that “any doctor or nurse should be able to master”, genetic counselling is a “specialised service” more suited to dealing with complex information-giving, especially when multiple family members are affected. However, it is interesting to note the absence of emotion in this description as an important component of risk communication.Combining the expertise and knowledge of psychiatry and genetic counselling will provide our patients with genetic risk information that will allow personal empowerment, that’s our goal, to empower our patients to make informed decisions .Whilst many participants acknowledged that genetic counselling was a skilled practice of communication, only one participant emphasized the distinct benefits this may have for patients:This emphasis on patient empowerment was not articulated by other participants, though it was implied that communicating risk and uncertainty had benefits for individuals and their families. Here, the psychiatric nurse suggests that combining the expertise of psychiatry and genetic counselling is value-adding: that communicating complex risk information is not merely edifying or reassuring, but it enables patients to make difficult decisions.From the perspective of psychiatry, then, we see a glimpse of how psychiatric genetic counselling may be organized in the future. Genetic counselling implies a collaborative arrangement in which emotional work and family history-taking is combined with the diagnostic and medical expertise of psychiatry. This suggests that the provision of psychiatric genetic counselling would be more suited to a separate sub-discipline, with trained genetic counsellors developing expertise in psychiatry.As a result of the service not being currently available, barriers for referral of genetic counselling services were discussed by participants. Four main barriers were highlighted..It’s often quite difficult to make a precise diagnosis in the relative which then alters the precision of what you can do Uncertainty was discussed in relation to two contexts of service provision. Firstly, the difficulties in providing a firm diagnosis for those affected by psychiatric disorders would be a barrier for referrals to the service. Without a definite diagnosis, information and risk communication lacks precision.Arguably, precision of diagnosis is necessary for information to be useful to families, though the literature does not necessarily support this assumption Secondly, factors predisposing to psychiatric disorders are essentially probabilistic rather than deterministic. Uncertainty surrounding the risks of susceptibility loci and pleiotropy of genes (where one gene can influence multiple phenotypic traits) reduces the clinical utility of information provided in a genetic counselling consultation.The complex aetiology of psychiatric disorders encompasses both non-genetic factors as well as genetic factors associated with several disorders. However, the uncertainty arising from probabilistic risk can have positive consequences of avoiding a fatalistic impression of disease. Thus, while the inherent uncertainty of genetic risk is a barrier for the service, it also presents important opportunities to reassure patients.I think I agree one needs to be an adult to make those decisions and there’s the question of capacity, people who are already ill, do they understand what they are asking for and what it would mean .Many participants cited the ethical concern of limited capacity or autonomy of clients affected by a psychiatric condition. Acting upon genetic information relies on the ability of patients to give informed consent or engage in shared decision-making:.They come to us on average a standard deviation, at least sort of 15 IQ points below the healthy population, so straight away you’ve got to gear your consultation and communication to somebody in that sense Adapting information to patients with cognitive impairment is both a challenge and a skill which seems to be a more prevalent concern for clinical psychiatry.From this description of patient population, capacity to provide consent is evidently variable. In such cases of reduced autonomy, communicating complex risk information may be neither desirable nor achievable in the consultation, in which case a genetic counselling service would need to balance the “best interests” of clients with issues of capacity..Most of my patients aren’t aware of the genetic implications of their disorder. They don’t think their families are at risk. It could open up a can of worms Among the non-genetic professionals, there were concerns that knowing one’s genetic risk may intensify stigma and blame, which is contrary to the view that genetic explanations may actually reduce stigma and blame .The expression “can of worms” implies that the personal and familial consequences of knowing one’s risk may generate more harm than good. Again, balancing the patient’s autonomy and their best interests would seem to be particularly relevant to clinical psychiatry. The psychiatric nurse alludes to the scenario that attempting to solve one problem might inadvertently create more problems.The challenge posed by this health professional is balancing a paternalistic approach of withholding information with psychosocial support. Genetic information has significant implications for the individual and their family, however, it should not hinder the individual’s right to be aware of his/her genetic risk.People often tend to feel more ashamed in psychiatry for carrying something that causes [a] psychiatric disorder, so a stigma. They might not want to come because they might be told “you’ve got something” .Patients’ unwillingness to cooperate in genetic counselling services is also indicative of potential barriers for provision. For instance, the stigma related to psychiatric disorders may reduce uptake for psychiatric genetic counselling:Psychiatric patients often have a vendetta against our health service, they’ve been let down by it many times in the past. They don’t engage. We have trouble getting patients to attend appointments here, it’s not the same as having a physical complaint, they’re used to it, and they think there’s nothing we can do to help, so why bother .And one psychiatrist stated that patients do not always attend appointments because the service itself is stigmatized:In this candid account, the participant highlights several barriers owing to the poor relationship between patients and mental health services. Patients with chronic histories of illness may not see the benefit of genetic counselling by virtue of its association to existing services.Patient engagement is clearly a factor that may affect the success of provision. As this study has highlighted, respondents perceived that issues of genetic risk are not a major priority for many affected individuals; this, in addition to the barriers identified here, may continue to hinder the implementation of a genetic counselling service.This is the first study to explore accounts of mental health professionals with respect to future service provision of psychiatric genetic counselling in the UK. Participants noted that there was no current demand for psychiatric genetic counselling from the patient group and their families as the aetiology of these disorders are not well understood. From the initial recruitment survey, only 25 % of participants were aware that a genetic counselling service was available for non-psychiatric disorders, highlighting that awareness of the genetic counselling service is limited among other healthcare providers. However, DeLisi and Bertisch  found thProvision of psychiatric genetic counselling services was advocated by the participants of this study as a unique “specialism”. Communicating genetic risk is a “skilled profession” capable of reducing feelings of anxiety, guilt and stigma. Only one participant identified such a service as “empowering” patients to make informed decisions. These findings are consistent with the reported aims and outcomes of genetic counselling more broadly, and fit the model of empowerment proposed by McAllister et al. .These outcomes have been reported by previous studies into the provision of genetic counselling for mental health in Canada . Martin et al.  expresseBarriers to referring individuals and their families to the service were described in detail by participants, highlighting areas of concern over future service-delivery. Over 90 % of the initial survey participants felt they would refer to an available genetic counselling service in future, over 90 % felt the service had some relevance and over half of participants reported the service to be useful and informative to their patient group. This suggests that these barriers would not hinder referrals, or the validity of the counselling provided. However, issues with patient engagement were raised that would affect the success of the service despite patient referrals.The concept of ‘uncertainty’ played two roles in this study. Diagnostic uncertainty was presented as a barrier for referral to the genetic counselling service due to the difficulty in providing accurate psychiatric diagnoses in a field where many conditions have an overlapping phenotype. Concern was expressed over the validity of information genetic counselling could provide in light of this diagnostic uncertainty. However, uncertainty was also described as a tool for reassurance, which may have positive consequences for avoiding a fatalistic impression of psychiatric disorders. Uncertainty is not unique to psychiatric genetics. Genetic counsellors are well-versed in these discussions in the field of cancer genetics and the increasing identification of variants of unknown significance (VUS). The impact of uncertainty in the context of psychiatric diagnoses and psychiatric genetics should be explored further.Ethical considerations’ regarding the provision of psychiatric genetic counselling is an area that requires further consideration. Participants were especially concerned with the limited or variable capacity of affected individuals and their ability to understand complex information on which subsequent decisions are made. A challenge presented by the participants for the service was the ability to adapt communication to enhance informed consent without increasing psychosocial burden. However, many genetic conditions currently seen in clinical genetics also have a significant effect on cognitive ability, often associated with learning disabilities, and therefore should not present a new challenge to genetic counselling.Four participants in this study have a background in psychiatric genetics and therefore have a greater knowledge of genetics than is typically found among clinical psychiatrists. The range of expertise in the sample reflects the variablity of knowledge observed in this study.Due to external constraints of completing the research within the framework of a supervised Masters Dissertation, data collection did not achieve thematic saturation, and therefore provides a limited insight into the accounts of psychiatric health professionals. Further exploration of individuals working in clinical psychiatry is vital to reveal a broader range of perspectives.This study is the first of its kind to explore healthcare providers’ accounts of psychiatric genetic counselling in the UK. Further exploratory research is needed with different and larger cohorts to examine the interface between healthcare professionals and patients in order to provide recommendations for the future of psychiatric genetic counselling. Further research should include exploration of patient perspectives on the value of psychiatric genetic counselling to identify the compatibility of patient and professional perspectives.Although demand for psychiatric genetic counselling has not been formally voiced in the UK at present, the accounts of psychiatric health professionals indicate that such a service would be useful and desirable. Advances in the identification of susceptibility loci for psychiatric disorders may have significant implications for genetic counselling in clinical psychiatry, whether or not these discoveries lead to genetic testing.Psychiatric health professionals describe clinical genetics as a skilled profession capable of combining complex risk communication with much needed psychosocial support. However, they envisage that the possibility of such a service is confronted with a range of barriers and challenges including, but not limited to, the complexities of uncertainty in psychiatric diagnoses, patient engagement and ethical concerns regarding reduced patient autonomy.


Donax trunculus in Europe, relatively little information is known about its population genetic structure and connectivity and their consequences on conservation policies. We employed 16 microsatellite loci to characterise the genetic diversity and population structure of D. trunculus. A total of 514 samples from seven different localities along the Atlantic-Mediterranean transition, from the Atlantic (Gulf of Cádiz) to the north-western Mediterranean were genotyped. The analysis of the population genetic structure displayed a clear distinction along the Atlantic-Mediterranean transition with different clusters in the Atlantic Ocean, the Alboran Sea and the northwestern Mediterranean. Consequently, we recommend that these three areas should be considered as different management units. We showed that all populations seem to be at high long-term risk of extinction with the exception of the protected Doñana National Park population which still seems to have evolutionary potential. Therefore, our results emphasized the necessity of protection of this economic resource and the validity of molecular tools to evaluate the population dynamics.In a resource management perspective, the understanding of the relative influence of the physical factors on species connectivity remains a major challenge and is also of great ecological and conservation biology interest. Despite the overfishing threat on the wedge clam  Many soft and hard faunal barriers to dispersal have been identified in marine environments throughout the world, including, for instance, the Isthmus of Panama1246Genetic studies have demonstrated that gene flow between conspecifics either side of barrier is often limited for marine910121311151017Donax trunculus , is an Atlanto-Mediterranean warm-temperate species found in the Black Sea, the Mediterranean SeaD. trunculus constitutes a very important fishing resource due to its high economic value. Only in Europe, the recorded landings over the last 12 years equal 11,202 tons, with a maximum yield of 1,355 tons in 2005 followed by a steady decline reaching only 516 tons in 2011The wedge clam, D. trunculus has not been studied from a conservation biology or genetics perspective. The few studies that have paid any attention to its genetic makeup have focused on its genome structure25et al.et al. (GenBank Accession Numbers HG792255 to HG792275) recently characterised respectively the first nineteen and sixteen polymorphic microsatellite markers in D. trunculus, providing a first battery of genetic tools for the study of the genetic structure of the species to allow the delineation of conservation units essential in fisheries managementUntil recently25Donax trunculus. For that, 514 individuals from 7 locations along the Atlantic and Mediterranean coasts of Spain were genotyped using 16 microsatellite markers. After evaluating the genetic diversity, the importance of the two well-known characterised biogeographic boundaries  on the population genetic structure was assessed. Finally, we discuss these findings in a perspective of biodiversity conservation and resource management.Understanding connectivity among populations through genetic structure has important conservation implications because genetic assessment is a central criterion in determining the appropriate units and spatial scale for conservation and management30P < 0.05 after Bonferroni correction). Genotyping was consistent across the 514 samples as revealed by mis-scoring rates from two independent scorers below 2%. The number of alleles (NA) per locus ranged from 12 to 33 O) and expected (HE) heterozygosity and polymorphic information content ranged from 0.207 to 0.768 A total of 514 individuals from seven different localities were genotyped at 16 microsatellite loci. The genetic diversity found in each locus across samples is summarised in bove 10% . Table 2FST analysis revealed a significant genetic differentiation (P < 0.05) between the Gulf of Cádiz (Southern Atlantic Spanish coast), the Alboran Sea (Southwestern Mediterranean coast) and the Northwestern Mediterranean coast. This corroborates the presence of two well characterised biogeographical barriers, the Strait of GibraltarP > 0.05; The analysis of molecular variance (AMOVA) revealed that the majority (98.6%) of the observed variation was explained by differences within the sampling sites, however a small but significant portion of the variation (1.1%) occurred among sampling sites between groups . Resultsq-value for each pooled sampling site were of 0.806 ± 0.080 (range from 0.462 to 0.920) for the Gulf of Cadiz, 0.542 ± 0.084 (range from 0.357 to 0.736) for the Alboran Sea, and 0.845 ± 0.100 (range from 0.524 to 0.959) for the Northwestern Mediterranean Coast. Some individuals were not included into the mean individual admixture proportions of each pooled sampling site because they clustered into a different group: one individual from Isla Canela  clustered into the Gulf of Cadiz and, two individuals from Caleta de Vélez  and two from Cabo de Gata  into the Northwestern Mediterranean Coast.STRUCTURE analyses using the RECESSIVEALLELES and LOCPRIOR functions further substantiated the existence of three genetically differentiated clusters: one in the Gulf of Cádiz; a second one for the samples collected east of the Strait of Gibraltar, in the Alboran Sea, but west of AOF in the Mediterranean Sea; and a third one for the samples collected northwest of the AOF in the Northwestern Mediterranean coast. A clear discrimination was obtained between these three clusters . The assAnalyses using BayesAss suggest a consistent gene flow among the groups of populations . The aveDicentrarchus labrax confirmed that the Atlantic and Mediterranean basins harbour two distinct lineageset al.Crangon crangon) populations from the Atlantic Ocean and the Mediterranean Sea. Very high levels of genetic differentiation between Atlantic and Mediterranean populations  were also found in the Lusitanian sea star (Asterina gibbosa)Palinurus elephas) showed small but significant differences (FST for pairwise comparisons within basins = 0.003 ± 0.004 and FST among basins = 0.011 ± 0.005) indicating the presence of two partially-overlapping groups Pelagia noctiluca, across the Atlantic Ocean and Mediterranean Sea indicated a high degree of connectivity with little evidence of population subdivision between basinsFST = 0.005) among Atlantic and Mediterranean stocks was detected when using microsatellite allele frequencies but no evidence of differentiation was observed using mtDNA haplotype diversity. The magnitude of genetic differentiation observed between the different pooled sampling sites in the wedge clam can be considered as large values for marine taxa (from 2 to 3%).Several recent studies have clearly shown that the degree of connectivity among marine taxa separated by biogeographic barriers varies from extensive isolation to complete panmixia and thus the actual influence of these marine transitions remains controversial11D. trunculus populations as supported by our Bayesass analysis and also as reported in many other taxa151111In this study, the level of genetic differentiation observed between the main oceanographic discontinuities along the Gulf of Cádiz to the Western Mediterranean Sea  confirms that these biogeographic barriers to dispersal effectively restrict gene flow between D. trunculus, Nanton et al.Ruditapes philippinarum) populations in Korea also reported the occurrence of null alleles in 7 loci out of 10Mercenaria mercenaria) in Florida. Null alleles were present in all loci (range from 0.015 to 0.296), but for 4 loci out of 7, the frequency of null alleles was particularly high (>0.146)The high incidence of null alleles found in this study is consistent with many reports in natural populations of bivalve species analysed with DNA markers such as microsatellites2644et al.Pleuronectes platessa). They found that populations examined between 1924 and 1960 were in Hardy-Weinberg equilibrium, whereas populations examined after approximately 1970 were not. They performed extensive testing to rule out that these deviations for HWE were not due to genotyping artefacts and Wahlund effects. Subsequently, the authors were able to attribute the significant heterozygote deficiencies found from 1970 onward to inbreeding and overexploitation. Unfortunately, we do not have access to archived samples to verify if the populations where in HWE in the past. However, we attempted to rule out genotyping errors (see above) and PCR artefacts using the following approaches. First, we redesigned primers whenever the flanking sequences where adequate . Secondly, we re-amplified 10 loci including the loci for which we redesigned primers and 5 more which produced codominant amplicons for a subsample of individuals (approx. 200) under relaxed annealing temperatures (4 °C lower) to verify that additional alleles had not been missed. None of these strategies resulted in a reduction of the HWE deviations and thus, it was concluded that PCR artefacts were minimal and insufficient to explain the large heterozygous deficits observed. While it is impossible with the current dataset and with the funding that as available for the project to demonstrate that overexploitation may play and important part in the lack of conformance with HWE, this explanation remains plausible and interesting.Alternatively and certainly a far more interesting result would be that the deviations from HWE are due to the known severe exploitation that the species has been subjected over the last decades. For example, Hoarau D. trunculus showed variable levels of genetic variation than those previously foundD. trunculusSinonovacula constrictaMactra chinensiset al.D. trunculus and other clam speciesIn this study, the microsatellite loci characterized for et al.D. trunculus populations have been intensively exploited over the last decades, and particularly in Europecrit value recommended for microsatellite markers  is an important parameter of numerically small populations, which is positively correlated with their ability to persist in a changing environment and to evolve. The 50/500 rule often cited by conservation practitionersDonax trunculus fishery. The Doñana beach belongs to the Doñana National Park, and since 1996 has a regulation of the D. trunculus fishery: only 160 shellfish collectors are allowed (have a license). These shellfish collectors have to work using only an artisanal rake with a long net attached to the end of the drag (fishing from boats is forbidden) on the intertidal zone. During five days a week, they can collect a maximum of 25 kg of D. trunculus individuals per day and these must be larger than 25 mm. Furthermore, they have to respect the breeding season (two months a year) without fishing, and they cannot collect when toxic algae are detected in the wedge clams. In the case of Isla Canela, in addition to the shellfish collectors with license that work in the D. trunculus fishery, there are numerous illegal collectors who operate in the area throughout the year and sell their catches in the streets of Huelva and Seville and other smaller villages in both Provinces of Andalusia and, in general, visitors to the beach also harvest clams mostly during the summer months . Fishing from boats is also allowed, so the area of collection is wider, covering both the subtidal and intertidal zones. Moreover, there is neither a strict and respected reproductive period without fishery, nor a strict control over the D. trunculus minimum sizes. All these differences in the exploitation of D. trunculus fishery are reflected in the size structure of both populations. For example, in July of 2011, 2012 and 2014, although significant differences in D. trunculus density between the two beaches were only observed in 2014, individuals from Doñana were always significantly larger than those from Isla Canela  and share a similar habitat structure: long sandy beaches close to a river mouth  . Howevera Canela  and 2, pDonax trunculus is described along the Atlantic-Mediterranean transition. Using 16 microsatellite markers, we identified the presence of a large proportion of null alleles in this species. However, the presence of null alleles in the loci appears to have no effect on the population genetic structure uncovered. Our results showed that all populations seem to be at high long-term risk of extinction with the exception of the Doñana National Park population which still seems to have evolutionary potential. Interestingly, this population is rigorously managed by the authorities of the National Park with restrictions in the number of fishing licences, the amount of allowed harvest and the use of hand operated dredges. This work represents a contribution for the understanding of the genetic diversity and population structure of this species, and de facto in terms of biodiversity conservation and resource management.In conclusion, for the first time, the genetic diversity and population structure of Donax trunculus individuals were obtained in seven different locations in the Month of July 2011 in Spain . The PCR amplifications were done on an Applied Biosystems 9700 DNA thermal cycler. The PCR products were then separated on a capillary sequencer  using GeneScan™ 500 LIZ® Size Standard. Allele sizes were determined with the Gene Mapper®V4.0 program .A total of 514 of MicroCheckerDeviation from Hardy-Weinberg equilibrium and linkage disequilibrium were tested using Genepop (version 4.0A), the observed (HO) and expected (HE) heterozygosity, the polymorphism information contentThe total number of alleles  was implemented in Arlequin (version 3.5.2.1)q-values) for each individual in each sampling site, considering each one like a population, was performed using STRUCTURE v.2.2KThe individual admixture proportions .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Moreover, microRNA-126a-5p overexpression in H9C2 cells inhibited cell viability but increased LDH release and caspase 3 activity. Cardiac function analysis based on the measurements of hemodynamic parameters showed that microRNA-126a-5p expression ablation in M-I/R injured mice led to the reversal of the symptoms caused by M-I/R injury. Transesophageal echocardiography also revealed that the values of LVIDd and LVIDs were decreased while the values of LVFS% and LVEF% were increased in M-I/R injured mice after treatment with microRNA-126a-5p inhibitor, compared with the M-I/R injured mice treated with the control. Bioinformatic analysis demonstrated that Hspb8, a protective protein in myocardium, was the target of microRNA-126a-5p. Thus, these findings indicated that microRNA-126a-5p was up-regulated in mouse M-I/R model and promoted M-I/R injury in vivo through suppressing the expression of Hspb8, which may shed light on the development of potential therapeutic target for M-I/R injury.Previously, we found several genes are involved in myocardial ischemia-reperfusion (M-I/R) injury. In this report, we first developed a mouse model of M-I/R injury and demonstrated microRNA-126a-5p was associated with the M-I/R injury by using high-throughput microRNA expression analysis. We further investigated the expression and function of microRNA-126a-5p during mouse M-I/R injury. We observed high expression of microRNA-126a-5p in the M-I/R mice and increased levels of LDH and CK-MB (damage markers) in the serum. H Ischemic heart disease (IHD) contributes to the development of cardiovascular disease, which in turn leads to patient death. The current IHD treatment involves reperfusion of the blocked artery, but reperfusion may lead to myocardial ischemia-reperfusion (M-I/R) injury, including myocardial stunning, cardiomyocyte death, microvascular obstruction and arrhythmias . TherefoMicroRNAs are a kind of endogenous, single-stranded RNAs about 18-20 nucleotides, which can regulate gene expression by targeting 3′-end untranslated region (UTR) of mRNAs at posttranscriptional level. It has been speculated that more than 60% of human genes are regulated by microRNAs . Number in vivo through suppressing the expression of Hspb8, a protective protein in cardiomyocytes. These findings provide strong insights for developing a new therapeutic strategy for M-I/R injury.Among various microRNAs, microRNA-126a-5p is abundantly expressed in the endothelial cells and tissues with lots of vessels, such as lung, liver, and heart . Its assP < 0.01)  Figure . These r) Figure . To veri) Figure . These din vitro expression of microRNA-126a-5p, H9C2 cells were treated with various concentrations of H2O2 for different time points to induce cell damage, then the expression of microRNA-126a-5p was assessed by RT-QPCR. As shown in Figure 2O2 treatment increased the expression of microRNA-126a-5p in H9C2 cells and 0.5 mmol/L of H2O2 treatment for 24 hrs showed the maximum effect. Moreover, the increase in microRNA-126a-5p expression by H2O2 treatment was time-dependent  to induce hypoxia/reoxygenation (H/R) injury. As shown in Figure To further examine the t Figure . Further2O2 or H/R, we used its mimic and inhibitor to regulate its expression. MicroRNA-126a-5p mimic enhanced its expression in H9C2 cells compared with the control and mimic-negative transfected cells    . Compared to the model group, pretreatment with microRNA-126a-5p mimic, cell viability was further decreased. While pretreatment with microRNA-126a-5p inhibitor significantly attenuate hypoxia/reoxygenation-induced decrease in cell viability in H9C2 cells (P<0.05). These data demonstrate that microRNA-126a-5p enhance H9C2 cells injury induced by H2O2 or H/R.To further study the role of elevated microRNA-126a-5p in H9C2 cell injury induced by Hs Figure . Convers) Figure . We furt) Figure . LDH levr Figure  and 3D. ) Figure . Also, tP < 0.01) , while injection of control inhibitor (LNA-Scr) did not show any effect  Figure . We alsot Figure . Moreovet Figure  and caspt Figure  were inhmax (the maximum rate of left ventricular pressure rise) was significantly higher in I/R injury mice, but -dp/dtmax (the maximum rate of left ventricular pressure decline) was lower than that in the sham group. Interestingly, inhibition of microRNA-126a-5p by its inhibitor showed significant reversal of these parameters (i.e. higher LVSP and markedly lower LVEDP compared with I/R group). The +dp/dtmax and -dp/dtmax values were also reversed by the microRNA-126a-5p inhibitor  but significantly decreased the LVSP (left ventricular systolic pressure) compared to the mice in the sham group. Moreover, the +dp/dtFurther, we tested the effect of microRNA-126a-5p ablation on the left ventricular cardiac function by echocardiography measurements. To verify this effect, transthoracic echocardiography and M-mode tracings were used to evaluate LVIDd, LVIDs, LVFS% and LVEF%. As expected, I/R injury significantly increased the LVIDd and LVIDs Figure  and 6B, http://pictar.mdc-berlin.de/), TargetScan (http://www.targetscan.org/) and miRBase (http://www.mirbase.org/) were used to predict the targets of microRNA-126a-5p. Among the candidates predicted by bioinformatics analysis, we found that Hspb8 is a potential target of microRNA-126a-5p because the seed sequences of microRNA-126a-5p align perfectly with the 3`-UTR of Hspb8 mRNA  Hspb8 3`-UTR or mutated Hspb8 3`-UTR (MT) at the predicted microRNA-126a-5p target sequences was transfected into 293T cells and then co-transfected with mimic-NC, microRNA-126a-5p mimic, microRNA-126a-5p inhibitor or inhibitor-NC. Luciferase reporter assays showed that microRNA-126a-5p mimic significantly decreased the luciferase activity of the WT Hspb8 3`-UTR by approximately 37.3% in 293T cells relative to the control  inhibited the down-regulation of Hsbp8 o Figure . Similarr Figure .2O2 treatment. Treatment with microRNA-126a-5p inhibitor significantly rescued H2O2-induced decrease in cell viability in H9C2 cells (p < 0.05), however, treatment with Hspb8-specific siRNA (#1) relieved the increase in cell viability mediated by microRNA-126a-5p inhibitor  inhibited the changes of caspase 3 activity mediated by microRNA-126a-5p inhibitor  and ∼67% (#2) depletion of Hspb8, respectively Figure . Then thr Figure . Moreover Figure .Myocardial ischemia and reperfusion (I/R) injury is a phenomenon where blood supply to the heart is restricted momentarily and then restored back, but the damages to the tissues and organs may not recover completely. This is a complex pathophysiological event involving various mechanisms. The myocardial I/R injury induces the myocardial damage including cell death, cardiomyocyte hypertrophy and angiogenesis. There are many factors that can lead to cell death, such as pH abnormality , cellulaMicroRNAs function as gene silencers and are emerging as important regulators for gene expression and biological processes. Recently, several studies have suggested that miRNAs are involved in cardiac events such as muscle contraction, heart growth and conductance of electric signal . They plin vivo and in vitro studies have indicated that microRNA-126a-5p plays crucial roles in maintaining the integrity of blood vessels, the formation of new blood vessels and repairing of the damages /(LVIDd)3 × 100. All echocardio-graphic measurements were averaged from at least three separate cardiac cycles.Hemodynamic measurements were performed in mice after I/R. Mice were kept under isoflurane (2%) anesthesia. A Millar catheter (SPR-1000) was inserted through the right carotid artery into the LV. The LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximum rate of LVSP increase (+dp/dt max) and decrease (−dp/dt max) were recorded and analyzed using a PowerLab data acquisition system .http://www.targetscan.org) miRBase (http://www.mirbase.org/) and TargetScan (http://www.Targetscan.org/) were used.To predict the target genes of miR-126a-5p, bioinformatics algorithms TargetScan . The differences between two groups were analyzed using unpaired Student’s t-test, while the differences among three or more groups were analyzed using one-way ANOVA  followed by Student-Newman-Keuls.


Steindachneridion scriptum is an important species as a resource for fisheries and aquaculture; it is currently threatened and has a reduced occurrence in South America. The damming of rivers, overfishing, and contamination of freshwater environments are the main impacts on the maintenance of this species. We accessed the genetic diversity and structure of S. scriptum using the DNA barcode and control region (D-loop) sequences of 43 individuals from the Upper Uruguay River Basin (UUR) and 10 sequences from the Upper Paraná River Basin (UPR), which were obtained from GenBank. S. scriptum from the UUR and the UPR were assigned in two distinct molecular operational taxonomic units (MOTUs) with higher inter-specific K2P distance than the optimum threshold (OT = 0.0079). The COI Intra-MOTU distances of S. scriptum specimens from the UUR ranged from 0.0000 to 0.0100. The control region indicated a high number of haplotypes and low nucleotide diversity, compatible with a new population in recent expansion process. Genetic structure was observed, with high differentiation between UUR and UPR basins, identified by BAPS, haplotype network, AMOVA  and Mantel test. S. scriptum from the UUR showed a slight differentiation , but not isolation-by-distance. Negative values of Tajima’s D and Fu’s Fs suggest recent demographic oscillations. The Bayesian skyline plot analysis indicated possible population expansion from beginning 2,500 years ago and a recent reduction in the population size. Low nucleotide diversity, spatial population structure, and the reduction of effective population size should be considered for the planning of strategies aimed at the conservation and rehabilitation of this important fisheries resource. Freshwater ecosystems are among the most endangered ecosystems . HabitatSteindachneridion scriptum  and adequate fisheries control.The correct management of fish stocks depends on the precise identification of the target species, which may present very similar morphological characteristics to other species. The latest revision of the genera  species . Using m the UUR , 2013. IS. scriptum from UUR represent a single molecular operational taxonomic unit (MOTU). Posteriorly, we investigate the genetic diversity and population structure of S. scriptum from the UUR and UPR basins using the mitochondrial control region.Taxonomic uncertainties are common in fishes  and can S. scriptum were collected by scientific fishing and local fishermen between 2006 and 2015, with authorization of the Brazilian Institute of the Environment and Renewable Natural Resources . A total of 19 individuals from the Uru River and 24 from the Canoas River (Can), UUR basin, were sampled . Tissues were preserved in 95% ethanol until extraction. This research was conducted under Animal Care Protocol PP00788 of the Federal University of Santa Catarina (UFSC).The Uruguay River (Uru) originates in Brazilian territory  in Southern Brazil, together with the Paraná and Paraguay Rivers form the La Plata Basin . SamplesTotal DNA was obtained with fin clips following a salt extraction method . For theS. scriptum  from the UPR basin, S. parahybae (access FPSR293-10–FPSR297-10) from the Paraíba do Sul Basin and one specimen of Pseudoplatystoma corruscans and one specimen of Zungaro jahu to root our phylogenetic analyses. All sequences available in Barcode of Life Data System (BOLD). Intra- and inter-specific genetic distances based on the Kimura 2-parameter (K2P) evolution model were calculated using Mega 6  approach based on single-locus data that is a relative robust tool for species delimitation . The ultlocal minima function in the R package SPIDER  and specimens from Tibaji River (UPR) (access EU930039.1–EU930044.1). The overall genetic diversity was estimated using the following DnaSP software (S). Genetic diversity within and between sample sites was hierarchically tested by Analysis of Molecular Variance (AMOVA) (FST) using Arlequin 3.1 (k (1–6) without origin information  and subsequently using “clustering of groups of individuals.” We used Mantel tests as implemented in the Alleles In Space (AIS) 1.0   with 10,quin 3.1 . Spatialquin 3.1 . First, AIS) 1.0  to test th DnaSP . A mediath DnaSP . Demograth DnaSP  with theth DnaSP .S. scriptum from the UUR basin was deposited in the Zoology Museum of the Universidade Estadual de Londrina MZUEL 15569 .The COI sequences were deposited in BOLD systems (accession UUR001-17–UUR052-17), and DNA D-loop sequences were uploaded in GenBank (accession MF045370–MF045412). The voucher corresponding to S. scriptum, resulting in a total length of 611 bp, with 7 polymorphic sites and 6 haplotypes defined. The inter-specific nucleotide frequencies were 26.02% of Cytosine, 28.97% of Thymine, 27.19% of Adenine, and 17.82% of Guanine. No stop codons, insertions, or deletions were observed in the COI sequences, indicating that they represent fragments of functional mitochondrial genes and not nuclear mitochondrial pseudo-genes (Numts). Considering P. corruscans and Z. jahu as the outgroup, the maximum likelihood for the GMYC model was significantly superior (L = 628.3242) to the likelihood of the null model . The single-threshold GMYC model suggested the presence two clusters  of four ML entities  with a confidence interval of 4–17 .The consensus alignment of 43 COI sequences were obtained from samples identified morphologically as S. scriptum sequences used in this study was OT = 0.0079 (0.79%) of divergence. From the set of the genus Steindachneridion sequences available on BOLD, three MOTUs were identified using the OT value and software jMOTU: S. scriptum from the UUR, S. scriptum from the UPR, and S. parahybae. The COI inter-MOTU between S. scriptum from the UUR and S. scriptum from the UPR showed values larger than the OT , while intra-MOTU values for fishes from both basins were lower than the average OT  (Table 1). The minimum inter-specific distances between S. scriptum MOTUs (UUR and UPR) and S. parahybae were 0.100 (10%) and 0.090 (9%), respectively.The optimal threshold calculated for all Figure 2) were clustered in two principal clusters, corresponding to S. scriptum and S. parahybae species. The S. scriptum clade appeared divided into two well-supported sub-clades formed by UUR and UPR individuals, in both NJ and BI phylogenetic trees. Based on these results, all specimens from Uru were composed of a single MOTU, named here as S. scriptum.The NJ-K2P  was 865 bp. A total of 56 variable sites were found in the region defining a total of 36 haplotypes. Overall, 30 haplotypes identified in the analyses were unique and exclusive. Of these, 20 were from the Uru, 4 from Can, and 6 from UPR basin (Tibagi River). The most common haplotype was H8, which was recorded 12 times and was shared by samples from the Uru and Can rivers, and haplotypes H4 and H27, both with three records, were exclusive to the Uru and Can rivers, respectively. The haplotype network  revealed a high degree of similarity between the specimens from the Uru and Can rivers, even though with slight differences in the haplotype frequencies and exclusive haplotypes from each river. These specimens were also differentiated from the samples of UPR by 12 mutations.The final alignment size of the 59 D-loop consensus sequences of S. scriptum from the Uru (Hd = 0.959/π = 0.007) in comparison with the Can River (Hd = 0.837/π = 0.004) (Table 2). In addition, the samples from UPR have high diversity indices, in comparison with samples from UUR .The average nucleotide frequencies were 34.38% of Adenine, 33.17% of Thymine, 12.19% of Guanine, and 20.26% of Cytosine. Genetic variability, expressed as Hd) and nucleotide diversity (π), was higher in Table 3). When the populations were considered as two basin groups, the AMOVA among groups was 78.17% and FST value was highly significant . Genetic divergence between individuals from the Uruguay Basin (Uru vs. Can rivers) was low but significant . The population groupings generated by the BAPS, without origin information of the samples, revealed the existence of three clusters , corresponding to Uruguay and Paraná Basins. The IBD analysis showed a significant positive correlation  between the geographical distance and corresponding FST for S. scriptum from UUR and UPR .The patterns of genetic variability found within and between populations in the AMOVA were based on the two principal clusters: Uruguay vs. Paraná Basins and Uru vs. Can rivers . Non-significant negative values were estimated for D and Fs indexes for specimens from the Can River and UPR, whereas fish sampled in the Uru showed significant negative values estimated for D and Fs indexes. The BSP analysis  (S. scriptum from the UUR and S. scriptum from the UPR could be explained by geographic isolation between watersheds that occurred during the Miocene epoch (between 5 and 24 million years ago) when the UUR and the UPR became isolated  . The MOTh (0.3%)  but can h (0.3%) . The twoisolated . Based o factors . Congenecode gap , 2004 thhoming . The haplotype network indicated a greater genetic similarity between the specimens from the Can and Uru, whereas is possible to observe differences in the haplotype frequencies, and exclusive haplotypes for each river. Recent studies with potamodromous fish species reported genetic structure in hydrographic systems without apparent physical barriers, resulting from behaviors related to IBD , homing homing , and isohoming .S. scriptum from the UUR was low in comparison with the values found in neotropical freshwater fish (π = 1.5%) (Nucleotide diversity of  = 1.5%) . The hig = 1.5%) .Steindachneridion species, and the knowledge about genetic characteristics of the genus is incipient. RAPD markers indicated low genetic diversity for S. scriptum from the UUR, as reported by S. melanodermatum from the Iguaçu River Basin (S. scriptum from the UUR can be attributed to the following: (1) population history of this species: a Bayesian skyline plot analysis revealed a subtle increase in effective population size over time and demographic swings in the recent past, with a notable increase in effective population size between 1,000 and 2,500 years ago and a subsequent reduction in the effective size of females; and (2) evolutionary history of the species: according to S. scriptum notably maintain conserved morphological and cytogenetic patterns.Individuals from the Uru River were genetically more diverse than fishes from the Can River, indicating that the main channel of the Uru River allows the meeting of individuals of different areas (or tributaries), favoring the maintenance of the highest level of genetic diversity. Although it is a relevant fishing resource, useful molecular markers such as microsatellites, extensively employed in fish genetic studies, are still not developed for any er Basin . Low gener Basin . In thisS. scriptum in some stretches of the Itá and Machadinho reservoirs (S. scriptum.In general, concerns and actions of conservation are more related to the perception of the disappearance of a given species than of genetic diversity reduction . Therefoservoirs , 2013, tRP made substantial contributions to the design of the work and acquisition, analysis, and interpretation of data for the work, and drafted it until the approval for publication of the content. JR made substantial contributions to the conception and design of the work, analysis and interpretation of the data, and drafted and revised it critically for important intellectual content. EZ-F made substantial contributions to the conception of the work, revised it critically for important intellectual content, and provided approval for publication of the content. The authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Arenaria balearica (Caryophyllaceae) could be a relict Tertiary plant species, this has never been experimentally tested. Nor have the palaeohistorical reasons underlying the highly fragmented distribution of the species in the Western Mediterranean region been investigated. We have analysed AFLP data (213) and plastid DNA sequences (226) from a total of 250 plants from 29 populations sampled throughout the entire distribution range of the species in Majorca, Corsica, Sardinia, and the Tuscan Archipelago. The AFLP data analyses indicate very low geographic structure and population differentiation. Based on plastid DNA data, six alternative phylogeographic hypotheses were tested using Approximate Bayesian Computation (ABC). These analyses revealed ancient area fragmentation as the most probable scenario, which is in accordance with the star-like topology of the parsimony network that suggests a pattern of long term survival and subsequent in situ differentiation. Overall low levels of genetic diversity and plastid DNA variation were found, reflecting evolutionary stasis of a species preserved in locally long-term stable habitats.Although it has been traditionally accepted that  Arenaria balearica L. from the family Caryophyllaceae is a good example.Within the Mediterranean global biodiversity hotspot, the Tyrrhenian Islands represent ca. 22% of the total surface, and lodge a high percentage of endemic taxa  Greuter in Aquilegia L. in With the closure of the Strait of Gibraltar  promoted the expansion of xerophytic elements and typically Mediterranean taxa . Later, Thymus herba-barona Loisel.  nor in Sardinia as A balearica and that it is absent from the islets of the Tuscan Archipelago.Previous studies on phylogeopraphic patterns of Mediterranean disjunct endemic species have focused on examples from the Eastern Mediterranean region e.g., , as wellA. balearica. More specifically our objectives are: (1) to test to which extent the observed distribution of A. balearica is concordant with the geological history of the continental fragment islands from the Western Mediterranean region; (2) to give a satisfactory answer to the question on how the colonization of the different islands and islets took place; and (3) to evaluate whether the low morphological variation observed among populations of A. balearica located in different islands is in correspondence with overall low levels of genetic diversity.Using both sequencing of plastid DNA regions and amplified fragment length polymorphism (AFLP) fingerprinting, this study aims to reconstruct the phylogeographic patterns and differentiation of intraspecific lineages within the disjunct endemic plant During the Last Glacial Maximum (LGM), ice sheets covered large areas in northern latitudes, and global temperatures were significantly lower than today . At the To map the past and current shorelines in detail, the present-day topographic and bathymetric data covering the area were taken from the ETOPO1, which is a 1 arc-minute global relief model of the Earth’s surface that integrates land topography and ocean bathymetry. This model was built from numerous global and regional data sets, and is available in “Bedrock” (base of the ice sheets) versions . EstimatArenaria balearica is an herbaceous perennial delicate plant whose filiform, branched stems and small leaves form low, compact ever-green moss-like dense mats, preferentially on cool, moist soils in shaded rocky places (comophyte), although it can be secondarily found also on shady moist slopes, between 0 and 1,800 m a.s.l. .m a.s.l. . Althoug , Corsica (8), Sardinia (9), Tavolara (1), and Montecristo (2), representing the entire distribution range of lica gel . Each saThe intent was to include a minimum of 10–12 plants per population in the analysis, but sometimes the population sizes were small and it was not possible to collect such a quantity of well separated (>5–10 m) individuals. Also, further problems were encountered in some cases in the DNA extraction and amplification processes (the leaves are only 2–4 mm and it was many times difficult to get an adequate quantity of DNA). In this situation, a variable number of 1–16 plants per sampling site were finally used .A. bertolonii were selected to be used as outgroup in the plastid DNA haplotype analyses. Given the uncertain phylogenetic position of A. balearica within the genus according to the most recent data  Fior & P.O. Karis, A. tejedensis (Willk.) Fior & P.O. Karis and A. suffruticosa Fior & P.O. Karis. These data further support the sister group relationship between A. balearica and A. bertolonii already proposed by Three additional samples from ent data , the selTotal genomic DNA was isolated from crushed dried leaf material (ca. 25 mg) following the 2× CTAB (cetyl trimethyl ammonium bromide) protocol  with minA. balearica, it was not always possible to extract enough DNA to provide clear and reliable AFLP profiles. Therefore, five populations among the 29 initially sampled had to be excluded from the AFLP analysis EcoRI-AGA/MseI-CTG, (VIC)EcoRI-AAG/MseI-CAT, (VIC)EcoRI-AGG/MseI-CC, were used for the selective polymerase chain reaction. These combinations were selected because they generated a relatively high number of clearly reproducible bands. A relatively high number of alleles per individual is desirable, given that AFLP are dominant markers . As an initial approach to the global genetic relationships among the individuals analysed and possible structure of the data, a Neighbour-Joining (NJ) analysis including 1,000 bootstrap pseudoreplicates based on a matrix of Nei-Li (Paup 4.0b10 (SplitsTree 4.12.3. Raw AFLP data with amplified fragments from 150 to 500 base pairs (bp) were scored and exported as a presence/absence matrix using the software f Nei-Li  distancep 4.0b10 . An unroK genetic clusters and does not assume an a priori assignment of individuals to populations, nor to clusters. Analyses were based on an ancestral admixture model with correlated allele frequencies among populations. The proportion of membership of each individual and population to the K clusters was calculated by performing 20 runs for each K value between 2 and 9 with a run length of the Markov chain Monte Carlo of 1 ×106 iterations after a burn-in period of 1 ×106 iterations, with λ adjusted at 0.4523. The optimal number of K clusters was estimated using the ad hoc parameter (ΔK statistic) of Population genetic structure was additionally investigated using a Bayesian clustering method implemented in STRUCTURE v. 2.3.4  followinAFLPdat , among populations within these groups (FSC), and among populations across the entire study area (FST)  was performed with the software ARLEQUIN 3.5.1.2 . For thiea (FST) . AdditiotrnLUAA-trnFGAA (′ trnK-matKpsbA-3 and rpS16 (trnQ(UUG)-rps16x1, trnL-rpl32F,  atpI-atpH,rpoB-trnC, trnH-psbA,A. balearica. PCR conditions and primers for DNA amplification are detailed in Three regions of the plastid DNA were sequenced and haplotype variation was explored to complement the information given by the mainly nuclear AFLPs. The plastid regions -trnFGAA , psbA-3′nd rpS16  showed tGeneious pro ™ 5.4 . One million data sets were simulated for each scenario , as implemented in DIYABC v2.1 software . DIYABC  basalts . After sscenario . The posscenario . The posscenario . Bias anscenario .A. balearica produced a total of 792 reproducible fragments.The four primer combinations applied to 213 plants representative of the variation of the species Both the NJ and NeighbourNet diagrams conducted on all individuals revealed a relatively weak overall structure of the genetic variation into two main groups: one comprised the samples collected in Majorca  were detected across the whole dataset, five (four substitutions/1 indels), eight (four substitutions/four indels) and four substitutions were detected for the plotypes . The resplotypes  method wA. balearica showed a single major haplotype (present in 24 from the 29 populations studied), probably ancestral (haplotype I), which occurred in all islands (including Tavolara and Montecristo). In addition, there were 15 haplotypes, nine haplotypes  separated one step from the ancestral one, haplotypes VI and XIV derived one step from haplotypes V and XIII respectively and haplotype XV derived two steps from XIV, two haplotypes derived two steps from haplotype I (IV and VIII) and IX derived one step from VIII. The most derived haplotypes were endemic to one individual island and usually were restricted to single populations . Apart from haplotype I, only haplotype V was shared by populations located in different islands (Corsica and Sardinia). Arenaria bertolonii is separated 50 steps from the A. balearica central haplotype. The levels of haplotypic variation found in Corsica and Sardinia seems to be in accordance with the high levels of overall genetic diversity revealed by AFLP markers.The statistical parsimony algorithm implemented in TCS inferred a 95% parsimony network with a maximum limit of four steps and star-like topology . As infeScenario 1  was revealed as the most probable. The posterior probability of the logistic regression was 75%, while the alternative hypotheses  receivedArenaria balearica and the Western Mediterranean region satisfy these prerequisites. One of the most basic questions related with Mediterranean plant populations that still remains open is what part of their present genetic diversity is, as generally assumed, due to isolation in refugia during the Pleistocene glaciations and what part can be traced back to the Tertiary history of taxa  and plastid DNA sequences, which included samples from all the Tyrrhenian islands where the species is represented, indicated that the study group is clearly monophyletic . Additionally, both the careful review of herbarium materials prior to the sampling performed within this study, as well as the field observations, indicate very low morphological variation among populations .Several hypotheses may explain the presence of A. balearica considered as a unique group, which are even lower among the different groups tested with AMOVA. These results contrast with the expectation of high population or geographical group divergence in species that occur in spatially isolated territories, particularly when the species shows limited dispersal abilities . In the case of A. balearica, the moderate levels of divergence found may represent remnants of Messinian contacts among the Tyrrhenian territories and long-term genetic stasis followed by recent differentiation in different stable habitats. Furthermore, the star-like arrangement of plastid DNA haplotypes (in situ differentiation. These results strongly agree with the idea of an ancient haplotype (I) widespread throughout the Tyrrhenian islands where the plant is present today, with different geographically scattered younger in situ derived haplotypes. In most cases, they represent endemic local variants that originated in isolation from each other, probably due to insularity or geography, on the one hand, and to the scattered availability of rupicolous habitats, on the other.Both plastid and nuclear markers show the lack of a phylogeographic break among populations from different islands. Low levels of genetic structure are repeatedly found by the data analyses derived from the anonymous, mostly nuclear, DNA fingerprints  are consistent with some of the criteria that usually characterized palaeoendemic species  was detected for the Tertiary relict Ramonda myconi (L.) Rchb. (Zelkova abelicea (Lam.) Boiss. and Z. sicula Di Pasq., Garfì & Quézel by Quercus suber L. by Cephalaria squamiflora (Sieber) Greuter by A. balearica, independent markers suggest low levels of intraspecific molecular diversity  that covers the whole genome and also with low ITS sequence variation  that covers a small proportion of the nuclear DNA), but an explicit correlation between these data and either long-term morphological constancy or slowed mutation rates cannot be established with the available data..) Rchb. , which wR. myconi  A. Chev.,  Nananthea perpusilla DC., Naufraga balearica Constance & Cannon, Soleirolia soleirolii (Req.) Dandy, etc). Arenaria balearica represents a further example of the importance of rocky sites as conservation habitats and as long-term reservoirs of plant diversity within the Mediterranean region.Tertiary relict species have been forced to survive in refugia for long periods of time and their present genetic structure may therefore reflect the impact of a combination of ancient climatic and geographic changes. The ability to persist and resist overall adverse climatic conditions is probably coupled with the availability of relatively stable habitats, where intrinsic local properties have buffered the impact of historical climatic changes, thus allowing long-time persistence of particular species . The imp. myconi . Further. myconi  have com10.7717/peerj.2618/supp-1Data S1Click here for additional data file.10.7717/peerj.2618/supp-2Data S2MatK.fasta file : Sequences corresponding to psbA-3’trnK-matK ; Rps16.fClick here for additional data file.10.7717/peerj.2618/supp-3Data S3Click here for additional data file.


Torreya yunnanensis (Taxaceae) to investigate the genetic diversity, phylogeography, and population structure of the species.Microsatellite markers were developed in T. fargesii, T. grandis, T. jackii, and T. nucifera.Sixteen primer sets were identified using Illumina 2 × 100‐bp paired‐end sequencing and bioinformatic screening. Most primers also amplified in T. yunnanensis for future studies of molecular ecology as well as their applicability across the genus.These results indicate the utility of these microsatellite markers in  Torreya yunnanensis C. Y. Cheng & L. K. Fu (Taxaceae) is a Chinese endemic evergreen tree that is highly valued for its timber, as well as for its ornamental and ecological benefits. Wild resources of T. yunnanensis have declined sharply as a result of overexploitation and deforestation. It was listed as endangered in the China Species Red List  markers, are a neutral molecular marker widely distributed in the nuclear genome of eukaryotes. Because of advantages such as high polymorphism, ability to facilitate genotyping, and low demand of DNA quality, microsatellites have been widely used as DNA markers in population genetic studies and parentage analysis. In this study, 16 polymorphic microsatellite markers were developed using Illumina 2 × 100‐bp paired‐end sequencing and bioinformatics screening. We believe these markers are useful for investigating genetic diversity and population structure for T. yunnanensis individuals were sampled from five wild populations in this study (Appendix T. yunnanensis is an endangered species and is rare in the field. Young leaves were collected and dry preserved with silica gel. Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method . De novo assembly based on trim reads was performed with Velvet 1.2.10 , observed heterozygosity (Ho), expected heterozygosity (He), and fixation index (F) of each locus for each population were calculated using GENETIX version 4.0 ; these values were higher than those found in T. jackii Chun  Rehder  after Bonferroni correction. Most of the F values were negative, indicating low levels of inbreeding in these populations. There was no evidence of linkage disequilibrium among pairs of loci in the sample. The sequences of these microsatellite loci have been deposited in the GenBank database (Table SRR6428886).According to MICRO‐CHECKER analysis, there is no evidence for scoring error due to stuttering, large allele dropout, or the existence of null alleles. Fourteen loci were polymorphic in five wild populations Table  and two T. fargesii Franch., T. grandis Fortune ex Lindl., T. jackii, and T. nucifera (L.) Siebold & Zucc. Most primers also amplified in these species (Table Cross‐amplification of these 16 primer sets was tested in five individuals in each of the following species: es Table .T. yunnanensis populations was shown to be high. The SSR markers that we developed were polymorphic and will be a useful tool to investigate the genetic diversity, population structure, and levels of gene flow, as well as to optimize breeding for T. yunnanensis and related species.Genetic diversity in wild


Bd or Rv in multiple populations, we used general linear models (GLMs) weighted based on population size to predict pathogen prevalence  and the natural log of intensity based on genetic and environmental variables, as well as location.There is an error in the first sentence of the “Genetic and environmental disease modeling” subsection of the Methods section. The correct sentence is: For species that tested positive for There is an error in