The Nero di Parma is an Italian pig breed with a peculiar breed history. It originates from a native breed called “Nera Parmigiana”, which, in the beginning of the 20th century, was crossed with highly productive breeds, causing the extinction of the original type in the 1970s. During the 1990s a growing interest for organic products and outdoor farming brought the attention back to the local type and a breed recovery project started to reestablish the original breed. The aim of the study was to investigate the genetic diversity of the Nero di Parma breed to provide further insights for breed conservation and to propose breeding strategies.Nero di Parma is an endangered swine breed reared in the North of Italy which nowadays counts 1603 alive pigs. The aims of this study were (i) to explore the genetic diversity of the breed at pedigree level to determine the actual genetic structure, (ii) to evaluate the effectiveness of the breeding recovery project and (iii) to potentially propose breeding strategies for the coming generations. The pedigree dataset contained 14,485 animals and was used to estimate demographic and genetic parameters. The mean equivalent complete generations was equal to 6.47 in the whole population, and it reached a mean value of 7.94 in the live animals, highlighting the quality of the available data. Average inbreeding was 0.28 in the total population, whereas it reached 0.31 in the alive animals and it decreased to 0.27 if only breeding animals were considered. The rate of inbreeding based on the individual increase in inbreeding was equal to 7%. This study showed the effectiveness of the recovery project of the breed. Nevertheless, we found that inbreeding and genetic diversity have reached alarming levels, therefore novel breeding strategies must be applied to ensure long-term survival of this breed. The profound changes in the production systems occurred in agriculture in the past 50 years have caused severe and undesirable impacts on the worldwide genetic variability. After the second World War, the call for animal-based products steeply increased, and rural systems were not any longer able to sustain food demand. In this context, the agricultural system has gone through a period of great changes which led to the development of intensive and commercially oriented systems. These systems necessarily promoted the use of high-yielding breeds at the expenses of the local ones. The changings, driven by the needs at that time, did not consider unfavorable side effects in the long terms. High-yielding breeds increasingly replaced traditional breeds, causing a threatening loss of genetic diversity. Swine system exactly resembles this scenario. Indeed, in the last decades, cosmopolitan lean breeds such as Landrace, Large White, or Duroc have been selected worldwide for their high productivity, at the expense of local breeds which have been crossed to improve their performance. Local breeds generally show strong adaptation to their local environment, even in the presence of harsh conditions, supporting their potential role to address the future climate changes and potential disease outbreaks. They represent a significant economic resource especially in rural areas as they have been mainly used in the context of niche markets. Lastly, conservation of small local breeds relies also on their socio-cultural value and their contribution to the development of local communities . TherefoThe Nero di Parma is a swine breed reared in the North of Italy, mostly in the province of Parma. Its ancestor was the “Nera Parmigiana” breed whose main characteristics were a slate colored coat, presence of wattles , ears haNowadays, the live population counts 1603 pigs. In small populations the risk of permanent loss in genetic material is dramatically high, and the maintenance of genetic diversity represents one of the most important breeding goals . The conSince the beginning of the 2000s, ARAER  in collaboration with University of Parma routinely collects identification data of Nero di Parma animals. The quality control (QC) of the data was performed checking the correctness of sows’ and boars’ registration numbers, birth date and sex identification. After the QC, the pedigree database contained 14,485 pigs  of which 1603 currently alive (RP: reference population): 641 males (39.99%) and 962 females (60.01%). Within RP the number of males includes the castrated males. Reference population is defined as the total number of pigs currently alive, whose pedigrees were traced back to the earliest recorded ancestors. In addition to the RP, the current breeding population (BP) was built considering alive sows and boars used for reproduction purpose. To build the BP two filters were applied: Sows and boars alive with at least one offspring . The farn is the number of generations between individuals and each known ancestor [The quality of pedigree information was investigated using the equivalent complete generations (CGE), which is computed as follow:ancestor . In addie) and the effective number of ancestors (fa) were calculated according to Lacy [a and fe was calculated. The ratio between fe and f was computed to evaluate the presence of bottlenecks after breed foundation [g) was also computed which is defined as the number of founders that would be expected to produce the same genetic diversity if the founders were equally represented and no loss of alleles occurred [g was obtained by the inverse of twice the average coancestry of the individuals present in the reference population. The effective number of non-founders (nfe) was computed following Caballero and Toro [g and fe was calculated to evaluate if the population is affected by genetic drift [Fi) was calculated according to the approach proposed by Gutiérrez et al. [e. The realized effective population size (Ne) was thus estimated as proposed by Cervantes et al. [e is a useful estimator of the inbreeding state in a determined population [IS, FST, FIT) were calculated to detect the reduction of heterozygosity among subpopulations based on farm ID and individuals to measure the total population differentiation. The FST values were interpreted using the qualitative guidelines proposed by Wright [ST value of 0.15–0.25 indicates large differentiation, 0.05–0.15 indicates moderate differentiation, and FST < 0.05 indicates little differentiation among populations. To evaluate the hierarchical relationship among farms in the RP, a clustering algorithm based on the FST results was used in R [ST value among farms.The actual number of founders (f) and ancestors were calculated in the TP, RP, and BP. The effective number of founders  and it reached a mean value above 5 in 2009. Throughout the studied period a gradual increase in the CGE was found and no differences among sex were shown . The avee was equal to 3.42. The actual number of ancestors was 16 and the fa was equal to 3.0. The number of ancestors explaining the 50% of the observed genetic diversity was equal to 2 animals. The ratio between fa and fe was equal to 0.88 whereas the ratio between fe and f was equal to 0.11. The fg was equal to 2.01 and the ratio between fg and fe was equal to 0.59. The actual number of founders identified was 19 and 11 for the RP and BP, respectively. The number of fa and fe were similar for the RP and BP if compared to the TP. The average inbreeding coefficient (F) in the TP was 0.28 (SD = 0.10) with a maximum value of 0.66. In the RP the average inbreeding was equal to 0.32 (SD = 0.11) while, when considering alive breeding animals (BP) (N = 336), the inbreeding decreased reaching a mean value equal to 0.27 (SD = 0.09). The average AR in the TP, RP, and BP was equal to 0.50 (SD = 0.06), 0.51 (SD = 0.048) and 0.50 (SD = 0.09), respectively. In addition we evaluated AR in the BP for boars and sows separately and we found a mean value equal to 0.51 (SD = 0.09) in boars (N = 21) and equal to 0.50 (SD = 0.09) in sows (N = 315). The individual increase in inbreeding (ΔFi) was equal to 0.07, 0.06 and 0.05 in the TP, RP, and BP, respectively. In the case of realized Ne, we found an increase in Ne from the TP to the BP  (The main parameters that characterize the Nero di Parma genetic variability in the TP and in the subgroups RP and BP are given in  = 9.66) .In N = 15) had a percentage of internal father equal to 9.31%, whereas in the fourth percentile class (average N = 1124) this value reached 66.70%. The major difference in terms of average inbreeding was found from the comparison of the first and last percentile, with an average F of 0.32 and 0.28, respectively.A total of 44 farms were identified in the database, of which 25 had a total of registered animals lower than 100 throughout the studied period. In IS, FST and FIT were calculated based on the aforementioned farms and were equal to −0.07, 0.12, and 0.06, respectively. In ST pairwise comparison. In the case of Farm E, the FST showed moderate to high differentiation to all the remaining farms. In contrast, seven farms showed no genetic differentiations based on the FST values.The analysis of the RP in terms of farms available showed that only 10 farms are currently operating. The Wright F parameters FNowadays, preservation of genetic diversity is crucial to overcome the dramatic loss of worldwide animal biodiversity. Biodiversity has a widely recognized role in food security and nutrition; nevertheless, our food systems is under severe threat due to the ongoing loss of genetic diversity . In thisThis study can be considered as the first step in deepening our knowledge on the genetic diversity of the Nero di Parma and to establish the effectiveness of the recovery project. Since the beginning of the project, the number of piglets born per year generally increased, especially after the officially recognition of the breed. This outcome suggests that the breed recovery project was effective to increase the population size, regardless the absence of economic grant from the government. Although Nero di Parma is no longer classified as at risk of extinction, the females breeding population consists of 315 females, thus it still has to be considered as endangered. Indeed, FAO  stated tThe pedigree depth is of main importance in the study of genetic diversity based on pedigree data as it affects the estimation of all the genetic parameters . Thus, we/f was found in the TP compared to RP and BP. In particular, the fe/f in the TP suggested that only 11% of the initial genetic variability was kept throughout the generations. Thus, we can hypothesize that the breed experienced a loss of genetic diversity as side effect of the breeding candidates’ selection during the initial steps of the recovery project. This is a quite common scenario during a recovery project. It is known that most losses of genetic variability occur just straight after the setting up of a conservation program as likely a substantial number of founders do not give progeny to the following generations [a and fe close to 1 ensures the continuity of genetic origin [g and fe, was equal to 0.59 in TP showing a clear loss of genetic diversity. This ratio was higher than in the Bunte Bentheimer (0.35) [g being roughly 40% lower than fa might be related to the partition of genetic variability among farms. We indeed found that the use of own boar within farms was high especially in the biggest farms (66.7% in the fourth percentile). Likewise, the results obtained for the FIS and FIT and FST highlighted the need to implement breeding strategies to assist the exchange of genetic materials among farms. Particularly, from the heatmap, seven farms showed clear genetic homogeneity, whereas in the remaining pairwise comparisons moderate to high level of differentiations were found. It is noteworthy that the largest farms were also on average the ones with the lowest inbreeding. This result is explanatory of the different breeding strategies adopted by the breeders depending on their herd size. Especially in one farm, a remarkable degree of differentiation based on FST value was found being above 0.09 in six pairwise comparisons. Interestingly, this latter farm is one of the biggest farms counting 332 currently alive animals  of which 55 being used as breeding animals . This farm exhibited rather low inbreeding and coancestry compared to the remaining ones. We thus believe that, even though the genetic material exchange with other farms is not commonly practiced in this farm, yet an effective mating scheme is used within the farm. On the other hand, smaller farms showed low differentiation highlighting that the exchange of genetic material is regularly performed. Nevertheless, the remarkable high inbreeding and average relationship found in the smallest farms suggested a reduction in genetic diversity due to the extensively use of a boar family among them. Therefore, the adoption of optimal contribution selection (OCS), a method that maximizes the selection objective while constraining progeny inbreeding, might be an effective tool in this breed. The OCS might help to increase the genetic material exchange among farms in the Nero di Parma. We therefore suggest the use OCS to optimize the contribution of breeding animals and homogenized coancestry via a better exchange of genetic materials among farms. The ΔFi found in the TP was equal to 7.0% which was higher than the ΔFi in most commercial and local pig breeds. The ΔFi was twice as high as in the Bísaro Pig [e based on ΔFi was equal to 7.68, 8.72, and 9.66 in TP, RP, and BP which is similar to what was found in the Gochu Asturcelta pig breed [The ultimate objective of a recovery project is the preservation of breed genetic variability from which founders were drawn. A remarkable difference between ferations ,31,32. Terations  and to cerations , in Czecerations  or Canaderations . A ratioc origin . In the r 0.35)  but lowesaro Pig  and conssaro Pig . In thisig breed . Similar  but lowig breed . The effig breed ,38,39. TTo conclude, in this study we unraveled the genetic diversity of the Nero di Parma at pedigree level and we highlighted few main future strategies that might be implemented for its conservation. The results of our study showed that the recovery project of the Nero di Parma was effective despite the absence of economic national support. To increase the effective population size and lower the rate of inbreeding, we suggest the use of OCS together with AI which can in turn effectively help to improve the exchange of genetic materials among farms. A genomic implementation of this work is needed to completely disclose the state of genetic diversity in Nero di Parma breed and to be able to protect its high-quality production.


Fusarium graminearum is the principal etiological agent of FHB in wheat, producing mainly the trichothecene mycotoxin, deoxynivalenol (DON) and its acetyl derivatives  and 3-acetyl deoxynivalenol (3ADON)). Understanding the population biology of F. graminearum such as the genetic variability, as well as mycotoxin chemotype diversity among isolates is important in developing sustainable disease management tools. In this study, 570 F. graminearum isolates collected from commercial wheat crops in five geographic regions in three provinces in Canada in 2018 and 2019 were analyzed for population diversity and structure using 10 variable number of tandem repeats (VNTR) markers. A subset of isolates collected from the north-eastern United States was also included for comparative analysis. About 75% of the isolates collected in the Canadian provinces of Saskatchewan and Manitoba were 3ADON indicating a 6-fold increase in Saskatchewan and a 2.5-fold increase in Manitoba within the past 15 years. All isolates from Ontario and those collected from the United States were 15ADON and isolates had a similar population structure. There was high gene diversity (H = 0.803–0.893) in the F. graminearum populations in all regions. Gene flow was high between Saskatchewan and Manitoba (Nm = 4.971–21.750), indicating no genetic differentiation between these regions. In contrast, less gene flow was observed among the western provinces and Ontario (Nm = 3.829–9.756) and USA isolates ((Nm = 2.803–6.150). However, Bayesian clustering model analyses of trichothecene chemotype subpopulations divided the populations into two clusters, which was correlated with trichothecene types. Additionally, population cluster analysis revealed there was more admixture of isolates among isolates of the 3ADON chemotypes than among the 15ADON chemotype, an observation that could play a role in the increased virulence of F. graminearum. Understanding the population genetic structure and mycotoxin chemotype variations of the pathogen will assist in developing FHB resistant wheat cultivars and in mycotoxin risk assessment in Canada.Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada,  Fusarium species of which Fusarium graminearum Schwabe is the primary etiological agent in wheat crops in Canada based on the frequency of isolation and impact on grain yield and quality F. graminposition ,47,48. IF. graminearum populations  from western Canada were genetically similar. This is mainly supported by the high gene flow  and low genetic differentiation  among regions. Lower gene flow and relatively high genetic differentiation  between the populations in the western provinces and that in Ontario and PA indicated a genetic structure between eastern and western regions. A similar correlation between genetic distance/identity and geographic location was reported for F. graminearum isolates collected from Alberta, Saskatchewan and Manitoba , where ΦPT was calculated as the proportion of the variance among populations relative to the total. We also determined the genetic variation between and within populations with AMOVA, by partitioning covariance components and their levels of significance (p < 0.05 or p < 0.01) using 1000 permutations. Genetic structure and admixture among populations were carried out with Bayesian clustering analyses of VNTR data using STRUCTURE v 2.3.4 [K) ranged from one to five after performing 100,000 Monte Carlo Markov Chain (MCMC) iterations following a 25,000-iteration burn-in for each run. Ten replicate runs were performed for each K value and STRUCTURE HARVESTER V. A.2 [K) based on the Evanno et al. (2005) method. The proportion of membership (q) in each of the K clusters was determined and isolates were assigned to the cluster that had the highest proportion of the membership.Population genetic analysis of Ex v 6.5 ,32 to es v 2.3.4 . We used v 2.3.4 . The numR V. A.2  was used


Lutjanus griseus) has a tropical and subtropical distribution. In much of its range this species represents one of the most important fishery resources because of its high quality meat and market value. Due to this, this species is vulnerable to overfishing, and population declines have been observed in parts of its range. In recent decades, it has been established that knowing the level of genetic connectivity is useful for establishing appropriate management and conservation strategies given that genetic isolation can drive towards genetic loss. Presently the level of genetic connectivity between subpopulations of L. griseus of the southern region of the Gulf of Mexico and the Caribbean Sea remains unknown.The gray snapper  suggesting ongoing gene flow among the subpopulations. Gene flow within the southern Gulf of Mexico appears to be stronger going from east-to-west.Total heterozygosity was 0.628 and 0.647 in the southern Gulf of Mexico and the Mexican Caribbean Sea, however, results obtained from AMOVA and Migration rates tended to be higher between subpopulations within the same basin compared to those across basins indicating some regionalization. High levels of genetic diversity and genetic flow suggest that the population is quite large; apparently, the fishing pressure has not caused a bottleneck effect. Establishing effective fisheries regulations is a complex and multidisciplinary task . In receLutjanus griseus) is an economic and ecologically important fishery species and can be highly abundant throughout its range, yet there is a paucity of information for its fishery management  have been declining since the 1950’s and populations in the Florida Keys are thought to be potentially over-fished . In CubaCarcharhinus limbatus), a low dispersal species, show strong genetic differentiation between the Mesoamerican Barrier Reef System and the southern Gulf of Mexico (Stegastes partitus), a high dispersal reef fish (Pterois volitans), the most studied invasive species, has been reported as having significant genetic differentiation between both regions, which suggests a phylogeographic break , Puerto de Veracruz (PV) and Tuxpan (TX); (2) Mexican Caribbean Sea, with the subpopulations Bahia de Chetumal (BC), Xahuayxol (X), Punta Herrero (PH) and Chiquilá (CH) (Two regions were studied: (1) Gulf of Mexico, with the subpopulations Campeche (ilá (CH) . Our sevilá (CH) . The Mexilá (CH) .X site, with 48 samples; SEMARNAT approved the field study with the number: 23/K4-0002/05/18). The sampling was carried out during November and December 2016 for the Mexican Caribbean Sea region, and during the same months 2017 for the Southern Gulf of Mexico region. Such periods were chosen in order to only catch resident individuals so as not to bias the estimates of gene flow, given that the migration of the gray snapper occurs from May to October .Microsatellites are commonly used DNA markers in genetic diversity studies because they present greater number of polymorphs  per locus than other markers. This feature makes them more sensitive to changes in size, structure and dispersion rate of populations . AdditioL. griseus, which were selected for their high levels of polymorphism and their ability to amplify in L. griseus. Seven loci were developed from the red snapper (Lutjanus campechanus) while one locus (Ra1) was developed from the vermillion snapper (Rhombloplites aurorubens)  .2O, 2.0 µL of green buffer, 0.8 µL of MgCl2, 0.125 µL dNTP, 0.25 µL of primer forward, 0.30 µL of primer reverse, 0.04 of Taq-polymerase and 1 µL (5 ng) of DNA extract. The Eppendorf thermocycler was programed with an initial denaturing cycle at 95 °C for 2 min, followed by 45 cycles of a denaturing step at 95 °C for 30 s, followed by an annealing step .Allele sizes were estimated using a DNA fragment analyzer (ABI 3730xl DNA). We used ABI DS-33 dye set with G5 filter set. Forward primers were dye labeled with either 6-FAM, VIC, NED or PET dye labels; GS-600 standard set was used with LIZ dye. This protocol allowed the detection of several PCR products at the same time by fluorescence emission. Fragment sizes were estimated using GeneMarkerNa) and effective number of alleles (AE); the latter defined as the alleles with the capacity of passing to the next generation (Ho), expected heterozygosity (He) and total heterozygosity (Ht) were also calculated. Heterozygosity is a measure used to know the diversity of a locus and is defined as the probability that upon selecting two loci, both will be different (Fa) was also estimated, considering that markers exceeding a 0.2 value should be excluded from further analyses  was used to test if the allelic frequencies conformed to the HWE   . Given tis large . The RSTtellites . This insoftware .Ne) and the number of effective migrants per generation (Nm) were estimated using the population parameter theta (θ) and the migration parameter (M) was calculated between subpopulation pairs. The analyses were carried out using the Maximum Likelihood Estimation with ten short chains and one long chain with 50,000 and 500,000 genealogies respectively at a constant mutation rate using the Migrate 3.6 software  an Analysis of Molecular Variance (AMOVA) was carried out using Arlequin 3.5 software .FST) and the geographic distance can indicate restricted gene flow. We tested for this association with the Mantel test . Based on the PIC results, six microsatellites were considered to be highly informative given that they exceed the 0.5 value, locus Prs320 (0.469) was medium level and locus Lca43 (0.239) was poorly informative  varied among subpopulations, between 3.115 (TX) and 3.384 (X). Higher values were observed in the Mexican Caribbean Sea subpopulations. He mean values for the Gulf of Mexico varied between 0.626 and 0.631 (C and PV (He = 0.631 both). He mean values observed in the Mexican Caribbean Sea region ranged between 0.641 and 0.655 . Ht values were similar for the Gulf of Mexico and the Mexican Caribbean Sea, with mean values of 0.628 and 0.647 respectively.Mean number of effective alleles . In subpopulation pairwise comparison, the FST and RST  ranged between 1.3 and 11.2. Highest values were observed in the pairs CH–C (8.5), C–PV (10.0), and PV–TX (11.2) (ectively . Per genX (11.2) .PV% = 98.22). The FIS value was 0.017, which suggests that the subpopulations present in the studied regions constitute a panmictic population. The FCT and FSC values indicated no genetic structure , therefore, we found no relationship between the genetic and geographic distances.The coefficient of determination from the Mantel test was 0.0221 and the coefficient of correlations was 0.149 (L. griseus) in two regions of the Mexican Atlantic: the Gulf of Mexico and the Mexican Caribbean Sea. We found no significant genetic differences between basins or among subpopulations (AMOVA: between regions and among subpopulations). Similarly, estimations of FST and RST between subpopulation pairs were all not significant. This lack of genetic difference indicates high genetic connectivity among all subpopulations of L. griseus along this ~1,950 km stretch of coastline. This result is also supported by the Mantel Test (p > 0.05), which didn’t show an association between genetic differences and geographic distances. In all cases, the number of migrants per generation (Nm) entering any given subpopulation was greater than four, this indicates that there is unrestricted gene flow and that the populations behave like a panmictic population, as theoretically with the migration of a single organism (Nm = 1) allele fixation is avoided  are known to show strong genetic differentiation between the Mesoamerican Barrier Reef System (Caribbean) and the southern Gulf of Mexico (S. partitus) also shows evidence of a weak restriction in gene flow between the Mexican Caribbean and southern Gulf of Mexico , which later becomes the Loop Current . This could be due to displacing reproductive aggregations that occur between the months of June and August around the new moon on the shelf border with a duration period of 8–10 days  were large, however, they must be considered with some caution, especially when interpreting for management and conservation issues. The mutation rate that is, considered for the estimate .Ht general average = 0.640; PIC general average = 0.597). According to the PIC, the microsatellites used are highly informative and polymorphic (n = 8); previous studies of L. griseus have reported means of 5.43 (n = 14) (n = 14) (Genetic diversity in these populations was high (ymorphic . The num(n = 14)  and 7.0 (n = 14) , similarFIS varying from 0.066 (X) to 0.188 (TX), which suggests a heterozygote deficit not previously reported for L. griseus. MicroChecker indicated that the most possible cause for the significant values in the FIS index could be the presence of null alleles. Null alleles are produced when there is a mutation in one of the primer binding sites of the microsatellites; this prevents annealing with the designed primer preventing the amplification of one of the alleles, resulting in a false homozygote . The next priority for understanding gray snapper populations in the Western Atlantic is to determine the degree of connectivity between Mexican populations and the northern Gulf of Mexico and between Mexico and Cuba.The high levels of genetic diversity, similar to those observed in other gray snapper populations from the northern Gulf of Mexico, as well as the high levels of gene flow in general, suggest that 10.7717/peerj.8485/supp-1Supplemental Information 1C), Puerto de Veracruz (PV), Tuxpan (TX), Bahia de Chetumal (BC), Chiquilá (CH), Punta Herrero (PH) and Xahuaxol (X).Abbreviations: Campeche , Puerto de Veracruz (PV), Tuxpan (TX), Bahia de Chetumal (BC), Chiquilá (CH), Punta Herrero (PH) and Xahuaxol (X).Abbreviations: Campeche (Click here for additional data file.


Prochilodus magdalenae is a freshwater fish endemic to the Colombian Magdalena-Cauca and Caribbean hydrographic basins. The genetic structure patterns of populations of different members of Prochilodus and the historic restocking of its depleted natural populations suggest that P. magdalenae exhibits genetic stocks that coexist and co-migrate throughout the rivers Magdalena, Cauca, Cesar, Sinú and Atrato. To test this hypothesis and explore the levels of genetic diversity and population demography of 725 samples of P. magdalenae from the studied rivers, we developed a set of 11 species-specific microsatellite loci using next-generation sequencing, bioinformatics, and experimental tests of the levels of diversity of the microsatellite loci. The results evidenced that P. magdalenae exhibits high genetic diversity, significant inbreeding coefficient ranging from 0.162 to 0.202, and signs of erosion of the genetic pool. Additionally, the population genetic structure constitutes a mixture of genetic stocks heterogeneously distributed along the studied rivers, and moreover, a highly divergent genetic stock was detected in Chucurí, Puerto Berrío and Palagua that may result from restocking practices. This study provides molecular tools and a wide framework regarding the genetic diversity and structure of P. magdalenae, which is crucial to complement its baseline information, diagnosis and monitoring of populations, and to support the implementation of adequate regulation, management, and conservation policies. Prochilodus, Semaprochilodus, and Ichthyoelephas, and encompasses 21 Neotropical freshwater fish species in the main river basins of South America (The family Prochilodontidae (Teleostei: Characiformes) comprises the genera  America . Most of America . Further America .In addition to the economic importance, Prochilodontidae plays an important trophic role in aquatic ecosystems. These detritivorous and migratory fishes contribute to the nutrient cycling, distribution, equilibrium, and maintenance of energetic flows and support a wide trophic network for a great number of predators . Hence, Prochilodus magdalenae Steindachner 1878 is the most representative endemic species of the Colombian ichthyofauna, considered the emblematic fishery resource of the Magdalena-Cauca Basin, with an estimated unload for the Magdalena Basin of 2,182.67 metric tons in 2013 . However, between 1978 and 2012, this species experienced drastic decreases in its population densities, catches (approx. 85%), and mean catch sizes. These effects resulted from overfishing during migratory periods, violations of legislation related to mean catch sizes, and habitat disturbances including deforesting, floodplain lake desiccations, agrochemical or chemical contamination derived from farming and mining activities, sedimentation, and dam/hydropower construction, among others   , it is rlineatus  and Procrgenteus  show fidP. magdalenae since similar patterns of genetic population structure have been found in other congeners such as P. reticulatus  previously studied by This study tests the hypothesis that P. magdalenae from the river mainstream and floodplain lakes along the different Colombian Magdalena-Cauca and Caribbean hydrographic areas , the “whole genome shotgun” strategy and the Nextera library preparation kits  for the sequence reads. All steps regarding the read cleaning, contig assemblage, identification of microsatellite loci, primer design, in silico alignment of primers using primer-BLAST (available in https://ncbiinsights.ncbi.nlm.nih.gov/2017/06/28/e-pcr-is-retiring-use-primer-blast/), PCR optimization, and polymorphism analysis of 52 microsatellites were performed following the methodology described by Low-coverage sequencing of the genomic library of one specimen of ™ Taq DNA Polymerase , 2.5 mM MgCl2, 2% formamide , 0.35 pmoles/μl labeled forward primer , and 0.5 pmoles/μl reverse primer . The PCRs were performed on a T100 thermocycler  with an initial denaturation step of 95 °C for 3 min followed by 32 cycles consisting of a denaturation step of 90 °C for 22 s and an annealing step for 18 s using the annealing temperatures described for each primer in The PCRs were conducted in a volume of 10 µl, which contained 2–4 ng/µl of template DNA isolated with the GeneJET Genomic DNA Purification kit  following the manufacturer´s instructions, 1 × buffer , 0.2 mM dNTPs , 0.05 U/μl PlatinumO) and expected (HE) heterozygosities and inbreeding coefficient (FIS) were estimated using Arlequin v3.5.2.2 software . For a best estimation of genetic stocks (K), the web-based software STRUCTURESELECTOR  were amplified in a subset of samples using primers and PCR conditions previously described by Prochilodus magdalenae, Prochilodus reticulatus, Prochilodus mariae, Prochilodus nigricans and using Ichthyoelephas longirostris as outgroup. For this purpose, we performed two independent runs of 20 million generations sampled each 1,000 generations using 25% as burn-in. The remaining values were left as default. The convergence of each parameter was checked based on a potential scale reduction factor nearing 1, an average standard deviation of the split frequencies lower than 0.010, and the visualization of the resulting trees was performed with FigTree v1.4.3 software  generated 277,133 reads and 14,124 of 50,404 that contained microsatellite loci, were flanked by suitable PCR priming sites. The dinucleotides (47.758%) were the most abundant repeat motifs, followed by tetranucleotide (28.353%), trinucleotide (16.193%), pentanucleotide (5.146%), and hexanucleotide (2.549%) repeats. The most common motifs found were AC (29.661%), TC (18.905%), ATT (4.811%) and AAAT (4.794%). The sequences of contigs containing the microsatellite loci obtained in the present study are provided in Genomic sequencing of the Illumina shotgun library of O = 0.589 and HE = 0.876, and the PIC values ranged from 0.399 to 0.949 (average 0.867)  . A totalO: 0.809; HE: 0.884) and Cesar , followed by Sinú , Magdalena , and Cauca  and were lowest in Atrato , the fish hatchery , and Nare   .E: 0.895; HO: 0.792), whereas the lowest was observed in Beté, a site of the Atrato River . In addition, all sites exhibited a highly significant deficit of observed heterozygosity (FIS) per site in main rivers of the different Colombian hydrographic areas were significant and ranged from 0.120 to 0.255  exhibit substantial evidence of selection in the Magdalena River .K = 2; MEDMEDK = 2; MEDMEANK = 2), one in the Magdalena River  and the other one in the remaining evaluated rivers  = 0.009; P = 0.000). Together with Chucurí + Puerto Berrio + Palagua, a predominant genetic stock with different levels of genetic admixture in Sinú and Atrato rivers was revealed in the clusters suggested by the MAXMEANK and MAXMEDK statistics (K = 3). The additional clustering patterns  homogenously distributed in Magdalena River and its tributaries  = 0.033; P = 0.000; F’ST = 0.027; P = 0.004; D’est = 0.149; P = 0.005). In Atrato River, the Bayesian analysis showed a single genetic stock  = 0.045; P = 0.000; F’ST = 0.047; P = 0.000; D’est = 0.330; P = 0.000).However, excluding samples that exhibit loci putatively under selection , comparisons among sites within each river revealed a genetic admixture of two stocks (Δbutaries  and 7. Acox1 gene clustered our samples (GenBank accession numbers MK330430–MK330494) with sequences of P. magdalenae and P. reticulatus deposited in public databases and in a different group, P. mariae and P. nigricans  than among P. magdalenae and P. reticulatus haplotypes (0.000–0.005).Finally, the Bayesian tree using the igricans . Moreoveigricans  were larP. magdalenae, the consistency in the amplification in a larger sample, allelic size class distribution, and high definition peaks allowed the selection of only 11 microsatellite loci for further population genetic analysis. Most of the loci showed departures from Hardy–Weinberg equilibrium and significant observed heterozygosity deficit in the random sample. The observed heterozygosity deficit may be related to technical problems such as silent alleles; however, it remains to explore the potential variations in the primer alignment sequences, since this study sequenced the genome of a single specimen of P. magdalenae. Two non-excluding explanations may be related to the significant levels of inbreeding and the genetic structure of the samples by the coexistence of two genetic stocks (see below).This work developed species-specific microsatellite loci using next-generation sequencing and bioinformatic analysis. Although a total of 21 of 52 microsatellite loci with tri- and tetra-nucleotide motifs were polymorphic in P. magdalenae considering that the levels of heterozygosity constitute a parameter used to estimate the genetic diversity of the populations. In addition to the applications in harvest management, stocking programs, definition of conservation units, recovery of threatened species, and management of invasive species, these tools may be useful in forensic genetics since partially degraded DNA samples are often found in this area  among the highest values found in other Prochilodontidae species, only surpassed by those reported for P. costatus  as this was evidenced by the genetic structure analysis (see below). Another biological cause of observed heterozygosity deficit, assortative mating, does not seem to explain the results found in this study because spawning  as descrcostatus  and P. llineatus . Even molineatus .P. magdalenae shows signs of erosion of the genetic pool, likely by the constant pressure from fishing and other anthropogenic activities exerted on its populations. Although paradoxical to the observed heterozygosity deficit found in all populations evaluated, this outcome is plausible considering that the Bottleneck algorithm tests not for an excess of heterozygotes (HO > HE) but rather for an excess of heterozygosity (He > He at mutation-drift equilibrium)  . Besideslibrium) . Consided Tobago .P. magdalenae exhibits genetic stocks that coexist and co-migrate along sections of the main channel and some tributaries of the Magdalena River , Sinú, and Atrato rivers. Before testing this hypothesis, we compared the genetic structure at regional scale, finding two spatially structured populations: one in the Magdalena River  and the other in the remaining rivers evaluated.This study tested the hypothesis that P. magdalenae. The phylogenetic analysis using partial sequences of cox1 gene indicates that samples do not correspond to species such as P. mariae or P. nigricans because this genetic stock is clustered with previously published sequences of P. magdalenae , Sinú, and Atrato. Since similar patterns of genetic structure are found in P. reticulatus  as well as geographical differences among the rivers analyzed . This genetic structure also explains the significant heterozygosity deficit observed in all sites analyzed (Wahlund effect) as discussed above. Similar evidence of the Wahlund effect has been documented in the congener solation . AlthougP. magdalenae exhibits high genetic diversity, significant inbreeding levels per genetic stock, and signs of erosion of the genetic pool and conforms a mixture of genetic stocks heterogeneously distributed along the rivers studied. Additionally, this study developed a set of 11 microsatellite loci that allows the reliable detection of levels of genetic diversity, providing a tool for monitoring changes in the genetic diversity of the species, brood stocks and juveniles used for supportive breeding and for measuring the efficacy of current population restocking activities. Management and conservation strategies need to be implemented at the level of the basins Magdalena-Cauca, Sinú and Atrato concordantly with their genetic population structure.This study provides evidence that 10.7717/peerj.10327/supp-1Supplemental Information 11: main channel of the river; 2: Floodplain LakeClick here for additional data file.10.7717/peerj.10327/supp-2Supplemental Information 2First row indicates respectively: Number of loci, Number of individuals, Number of sampling sites, Sample size for eight sites. Third row indicates respectively: River, Sampling site, Sampling site ID, Sample ID, Locus name for 11 loci.Click here for additional data file.10.7717/peerj.10327/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.10327/supp-4Supplemental Information 4Forward and reverse primers are indicated in gray. Microsatellite motifs are highlighted in green.Click here for additional data file.10.7717/peerj.10327/supp-5Supplemental Information 5LnLHood_LD: Likelihood of linkage disequilibrium; LnLHood_LE: Likelihood of linkage equilibrium; Alpha-t: Alpha per test; K: Number of comparisons. Loci highlighted in blue were included in the analysis. Values in bold denote statistical significance after Sequential Bonferroni correction.Click here for additional data file.10.7717/peerj.10327/supp-6Supplemental Information 6Click here for additional data file.


Myotis lucifugus), whose populations have experienced declines of over 90% because of the introduced fungal pathogen that causes white-nose syndrome (WNS), survival of the species may ultimately depend upon its capacity for adaptive change. Here, we present evidence of selectively driven change (adaptation), despite dramatic nonadaptive genomic shifts (genetic drift) associated with population declines. We compared the genetic makeups of wild survivors versus non-survivors of WNS, and found significant shifts in allele frequencies of genes associated with regulating arousal from hibernation (GABARB1), breakdown of fats (cGMP-PK1), and vocalizations (FOXP2). Changes at these genes are suggestive of evolutionary adaptation, given that WNS causes bats to arouse with unusual frequency from hibernation, contributing to premature depletion of fat reserves. However, whether these putatively adaptive shifts in allele frequencies translate into sufficient increases in survival for the species to rebound in the face of WNS is unknown.The degree to which species can rapidly adapt is key to survival in the face of climatic and other anthropogenic changes. For little brown bats ( Invasive species are a major trigger of these declines3, including invasive pathogens, against which native species can experience high mortality due to a lack of co-evolutionary defenses6. Introduced fungal pathogens can be particularly dangerous—they can frequently survive in the environment for extended periods, affect a relatively broad range of hosts, and can be highly virulent7, thereby driving mass-mortalities of native species  as well as threatening agricultural crops15 (e.g. rice blast disease16 and Fusarium wilt in bananas17).Events that kill large portions of populations, including naturally and anthropogenically induced disasters, increasingly threaten biodiversity18. A pressing conservation question is whether host populations can evolve resistance or tolerance during such epidemics—a necessary first step towards preventing extinction. Strong selective pressures might theoretically lead to an evolutionary rescue effect if host populations adapt19. However, acute events that kill off most members of a species also reduce the genetic diversity upon which natural selection can act, thereby limiting the capacity for adaptive change20.Although host mortalities may have little impact on fungal pathogens, the pathogens can exert incredibly strong selective pressures on their host populationsPseudogymnoascus destructans21. This highly destructive pathogen has decimated populations of bats, with 12 North American species currently affected22, and some populations experiencing losses of 90–100%23. The fungus was first inadvertently introduced to North America by humans in 2006 (in the northeastern U.S.)24, and is spreading across the continent, largely via infected bats25. The exact mechanism of death is not known, but bats apparently die from secondary physiological complications (e.g. depleted fat reserves) associated with too frequent arousals from hibernation26.White-nose syndrome (WNS) is a disease affecting bats, which is caused by the invasive fungus Myotis lucifugus) in response to WNS. The recent expansion of the fungus into our study area in 2014 combined with the staggering impact of WNS on the local population (roughly 78%)27 provides an opportunity to study the initial evolutionary effects of this pathogen, which continues to spread throughout the continent. Eurasian bats within the genus Myotis—in the native range of the pathogen—tolerate fungal growths with no noticeable mortality29. In contrast, little brown bats were the most common bats in eastern North America prior to WNS, but due to population losses, the species has now been listed as endangered by the IUCN30 and the federal government of Canada31, with a similar decision by the U.S. government pending32. Despite large observed declines, some individuals may have greater genetic-based tolerance or resistance to the disease, raising the potential for adaptive change in little brown bats via selective forces acting on standing genetic variation. However, dramatic population losses may confound the effectiveness of selection or purge potential adaptive variants via genetic drift. Information about these evolutionary processes can help inform the tempo and pace of management efforts for this species, by indicating which, if any, populations are adapting to the pathogen and what traits may be important for survival.Here, we conduct a genome scan to test for evidence of evolutionary changes in little brown bats (33). We detected the effects of stochastic, non-adaptive genomic changes in otherwise neutral portions of the genome (genetic drift) reflective of the large numbers that have died from WNS in this species. Nevertheless, we also identified genetic changes  that may have contributed to survival (as opposed to changes simply due to strong genetic drift), where the signature of selection can be detected by levels of genetic differentiation at a gene that exceeds background levels across the genome35. See methods for more details.In our tests for evolutionary changes in little brown bats, we compared the genetic makeup of “survivors” and “non-survivors” of the disease see Fig.  in a genF = 0.04 ± SE 0.0001) relative to non-survivors (F = 0.006 ± 0.0003), using the F-model in Structure37. This amount of drift-induced genetic change  indicates three mRNA-coding SNPs are located in introns of annotated genes  introduced to the central USA from coastal areas show signs of adaptation to freshwater conditions, despite small founder populations42. Likewise, extremely rapid phenotypic adaptation in Caribbean lizards followed a hurricane, with surviving lizards having larger toe pads which were presumably better at gripping surfaces during strong winds43.The putative selectively driven genetic changes we identify Fig.  have als44. In addition to GABA, these receptors are also sensitive to histamines45, which similarly help regulate hibernation in mammals46 and are released in response to tissue damage from WNS47. The importance of an individual’s sensitivity to histamines is further hinted by PLA2G7, which regulates release of histamines from mast cells48. Because arousals account for 80–90% of bats’ energy budget during hibernation32, genetic variation that contributes to even small changes in arousal frequencies could result in large differences in energy expenditures, making the difference between life and death . We speculate that bats genetically predisposed to release fewer histamines, or be less prone to arousals induced by histamines, are better able to survive WNS through conservation of energy reserves.The putatively adaptive SNPs among the surviving bats in our study are located within or in close proximity to four genes , which when mapped to the annotated reference genome suggest different ways adaptive shifts might contribute to survival. GABARB1 is a receptor for the neurotransmitter GABA, which is a major neural inhibitor in the brains of vertebrates, and has also long been suspected to be involved in regulating hibernation50, which might prove beneficial for WNS-infected bats facing premature depletion of winter fat reserves. In fact, a recent study documented a post-WNS phenotypic shift towards fatter bats of this species51. Although this may be due to a variety of potential mechanisms, including non-evolutionary ones (see discussion in51), our findings suggest a genetic component to this shift.Links between metabolic demands and survival are further suggested by cGMP-PK1, which was implicated by two significant SNPs in our dataset (one within the gene and one nearby). This gene is part of pathways involving cellular metabolism and breakdown of fat, and allelic variants have been linked to obesity in mammals52. Because variation in calls is closely associated with the type of prey and habitat bats must navigate, echolocation is an important functional trait, and potentially adaptive shifts might be related to hunting proficiency, speed of developing foraging abilities in juvenile bats, or subtle differences in prey preferences. These could affect the type and amount of fat that bats store for hibernation. In addition to echolocations for hunting, bats also emit social calls. Sociality may influence the impact of the disease in this species53, and due to the importance of FOXP2 in communication, the gene has been linked to variations in social behavior in other species56. A more detailed study is needed to test these hypotheses, and there are possibly alternative unknown functions of FOXP2 in bats. Interestingly, no individuals in our dataset were heterozygous for this SNP.In contrast to the SNPs linked to physiological mechanisms during winter hibernation, a SNP within FOXP2 suggests behavioral differences might confer a selective advantage. Specifically, FOXP2 is associated with vocalizations in other vertebrates, and echolocation in bats57, we think it is unlikely the mRNA-coding SNPs we detected are statistical artifacts. The four genes we identify had putatively adaptive alleles that were entirely absent from our non-survivors . With the much greater sampling of non-survivors (n = 29) this difference is also not due to limited sampling , potentially confounding the signals of selection and drift. Although we cannot rule out a role for non-selective processes, we note that annotation of the alleles suggests that selection is involved given that the functions are consistent with an adaptive response.Although outlier analyses can contain false positives, potentially inferring selectively driven differentiation when there is noneing Fig. . An alte58 may be important. Specifically, the alleles we identify could assist some bats in “holding out” until spring, when they leave sites in which growth of the pathogen is restricted to. While infected bats do exhibit an immune response to the fungus59, they likely ultimately die due to secondary physiological complications linked to starvation while hibernating60. Such tolerance in little brown bats to WNS may be important for survival in both in intraspecific61 and interspecific62 contexts. However, others argue that resistance is the primary mechanism of survivorship63. Future work is needed to resolve this question.Whether the putative adaptive changes described here reflect host resistance or tolerance to the fungal pathogen has consequences for evolutionary and ecological pressures, as well as management strategies. While our study does not explicitly test whether bats survive WNS via resistance versus tolerance mechanisms, and the genomic approach we used only looked at a small portion of the genome, we found putative selection acting on non-immune genes, which suggests disease tolerance39 it will take time to determine whether surviving remnant populations have sufficient reproductive and recruitment levels to avoid extinction or extirpation. However, the functions of the genes we identify suggest that for this species, and possibly other bats effected by WNS, conservation of summer foraging habitat—not just winter hibernation sites—may promote population recovery, given that the selective advantages underlying shifts in FOXP2 would most likely manifest when bats are echolocating and hunting, and not in the hibernation sites where the bats are confronted with the fungus , during warmer periods they do not roost in the same sites in which they hibernate, thus the relative geographic isolation is important for assuring that bats sampled during both seasons were from the same population. Winter hibernation sites are concentrated in the northwestern portion of our study area , and primarily consist of abandoned iron and copper mines. As a consequence, bats in our area  were adult bats that had been born the previous year or earlier and thus had survived at least one hibernation period with the WNS pathogen . Most individuals which succumb to the disease are found within the subterranean sites that afflicted species of bats rely upon in winter, and in which the fungus thrives, however some infected bats leave hibernation sites prematurely in winter in search of food or water, but quickly die due to lack of available resources and sub-freezing temperatures. Correspondingly, most non-survivors we sampled were bats found dead in or near hibernation sites during winter . Note that comparing survivors to this more general group of non-survivors makes tests for loci under selection more conservative, in that some of the euthanized bats categorized as non-survivors may not have died from WNS naturally. However, if non-survivors actually carried adaptive alleles, this would not produce a bias  to detect putatively selected alleles—in fact it would make such detection more difficult. In addition, all analyses were repeated excluding the euthanized bats to confirm the robustness the results.All sampled bats Table  were catn = 23) were from bat carcasses found during winter either in or proximal to the caves or mines in which they were hibernating. Prior to the introduction of WNS, it was uncommon to find dead bats at hibernacula, whereas conspicuous numbers of dead individuals are found in and around these sites post-introduction of the disease .Among the survivors, collection methods varied Table . Three sEcoRI and MseI, were used to digest extracted DNA (ddRadSeq33), to which barcodes (unique tags 10 base-pairs long) and adapters for Illumina sequencing were then ligated. Ligation and amplification were done via polymerase chain reaction (PCR), and 350 to 450 bp long fragments were size selected using Pippin Prep . The library of 38 samples was sequenced in one HiSeq. 2500 lane , at the Centre for Applied Genomics .DNA was extracted from membrane of wing tissue using DNeasy Blood and Tissue Kit  and used to prepare a reduced representation genomic library for sequencing. Two restriction enzymes, 67 , and processed in conjunction with supporting programs. The first step, process radtags, allowed up to one mismatch in the adapter sequence and two mismatches in the barcode, with rescue of RAD-Tags allowed. A sliding window of 15% of the read length was used for an initial exclusion of any reads with a Phred score68 below 10 within the window . Of 102,419,857 initial sequences, process radtags removed 1,144,865 reads containing the adapter sequence, 18,775,218 reads with ambiguous barcodes, 156,274 low quality reads, and 2,495,192 reads with ambiguous RAD-Tags.Genomic sequences were demultiplexed using the STACKS bioinformatics pipeline41), and mapped our sequences to the genome using the Burrows-Wheeler Alignment Program (v. 7.17) indexing and MEM algorithms, respectively70. The resulting files were filtered , converted to .bam files, and sorted using SAMtools72 (v. 1.8-27).We then indexed a previously generated reference genome for the species, ftp://ftp.ncbi.nih.gov/genomes/Myotis_lucifugus  was run using the Marukilow model73, minimum Phred68 score of 30, and alpha thresholds (for mean and variance) of 0.05 for discovering single nucleotide polymorphisms (SNPs). This resulted in 59,888,201 BAM records and 581,607 loci . All remaining loci were genotyped, with a mean per-sample coverage of 10.5x ± 7.1x, a mean of 138.5 bps per locus, and consistent phasing for 88.3% of diploid loci.The reference-based method of Populations was then run with default settings and the resulting loci were filtered with a custom script in R74 (v. 3.5.0) to remove loci and SNPs that may be artifacts of sequencing or alignment errors , leaving 273,261 unique loci.ors Fig.  based onpopulations was then run again, retaining loci present in at least 56% of both survivors and non-survivors, ensuring a minimum sample size of at least six survivors; note the actual missing data was typically much lower . This resulted in 40,963 loci (140-bp segments), of which were variable, containing 19,797 SNPs , all of which had a minor allele frequency of >0.01. Minor allele thresholds of 0.01 and 0.05 were evaluated for downstream analyses, and when warranted the higher threshold was used (noted below). Mean genotyped sites per locus was 142.41 bp (SE ± 0.02). Because some loci contained more than one SNP, the robustness of downstream analysis to inclusion of multiple versus a single SNP per 140-bp fragment was evaluated. Main findings did not differ, thus we present analyses based on multiple SNPs per locus in the main text  of survivors were compared with the non-survivors using BBMap75 (v. 38.01). The proportion of GC per individual per locus was averaged across all loci for each individual using a custom script in R. Mean GC content was 43% for survivors (n = 9) and 42% (n = 29) for non-survivors, which confirmed non-survivors were not biased towards higher GC because of decomposition.We also checked that the data were not biased due to different levels of genetic decomposition between the survivors and non-survivors by analyzing the Guanine-Cytosine (GC) content of each sample. Specifically, raw Illumina reads  indicated no related individuals within our dataset. We kept all individuals in downstream analyses, because the presence of a single pair of potential half siblings is not expected to influence estimates of allele frequencies or FST, and removal of putatively related individuals can actually increase the error .In addition, the relatedness of sampled individuals was evaluated in two ways: with see Fig.  caption see Fig. . HoweverStructure37(v. 2.3.4) to evaluate if genome-wide differentiation indicated a single, panmictic population. We selected the ADMIXTURE model with ‘Allele frequencies correlated’ turned on and no prior information about sampling population and explored the best supported model, considering a range of genetic clusters  with 10 repetitions for each k, for 500,000 Markov chain Monte Carlo iterations with a burn in of 50,000. Visual assessment was used to ascertain convergence by examining plots of FST, alpha, and likelihood versus iterations, and to check for consistency among the ten iterations. No evidence of genetic subdivision based on geographic sampling locality was detected  can be particularly great when only a small proportion of the population survives, sometimes causing population bottlenecks. To visualize the drift-induced changes that occurred broadly across the genome, we conducted a principal components analysis (PCA) of the survivors, and projected the non-survivors onto the estimated PC axes, and the degree of drift was quantified using the et al.80). Generating a PCA in this manner is a method of visualizing differences when one group is a subset of the other (in terms of the proportion of variance), for example due to a series of founder events80.The PCA was performed in R74, in conjunction with the packages Adegenet81 (v. 2.1.1) and Plyr82 (v. 1.8.4) using the prcomp function. One survivor and four non-survivors were excluded from this analysis because of missing data , as were loci missing in >50% individuals . The F-model accounts for differences in population sizes, and has been used to quantify differences in drift between groups of contrasting sample sizes that are similar in proportion to our own83. For our parameter of interest, F, we used a prior mean and SD of 0.10, which places similar probabilities on both large and small values of F. To implement this Bayesian approach, we preassigned individuals to one of the two groups (survivor or non-survivor), and used a burn-in of 50,000 followed by 500,000 reps. We fixed lambda at 1, and used a uniform prior from 1 to 10 for alpha, with a standard deviation of 0.025. Three iterations were run, with different random seeds for initiating the Markov Chains.We also directly estimated the amount of genetic drift between survivors and non-survivors in FST-outlier analyses, where the signature of selection can be detected by considering the proportional split of allelic variants between groups relative to background levels across the genome35. We identified candidate loci using three methods of outlier detection—identification of outliers via (i) the number of standard deviations from the mean using an AMOVA-corrected FST84, (ii) by assessing confidence intervals from bootstrap permutation across loci, and (iii) measuring departure from a chi-squared distribution (detailed below). Variable sites which met all three requirements were regarded as candidate loci apparently undergoing positive selection. All tests of selection were conducted with and without the four non-survivors sampled in 2014 (collected prior to the other specimens), to confirm that the results were robust. Note that the low number of sampled survivors reflects the devastating impact of WNS on this species; despite the small sample size, it is not beyond a size in which SNPs under selection can be detected with FST-outlier analyses85.To identify genetic differences among individuals that might have contributed to their survival of WNS, we used FST84 calculated by populations in STACKS35. SNPs with an FST-value of greater than nine standard deviations from the mean (mean = 0.018 ± 1 SD of 0.026) were considered outliers . A threshold of five standard deviations is often used in detection of outlier SNPs under positive selection87. We increased our threshold of significance to nine standard deviations to reduce the potential for false-positives. In the second approach, confidence intervals (95% CI) were estimated using diveRsity88. Using the diffCalc function, Weir and Cockerham’s FST89 was calculated for all loci, with 1,000 bootstraps performed across loci. Only loci for which the lower limit of the CI remained five SD from the mean were considered outliers. In the third approach, outliers were identified with OutFLANK90, which estimates the expected neutral variation of FST-values under a chi-squared distribution. As per the developer guidelines90, we excluded loci with low expected heterozygosity (<0.1), and visually adjusted the trim functions to best fit the observed distribution .In our first approach, we used the AMOVA-corrected 74, often in conjunction with the package ggplot292. A custom script was used to identify SNPs which were identified as candidate loci under all three methods, and putatively selected sites were then cross-referenced with the species’ annotated reference genome41 to infer possible phenotypic function . If the SNP’s position was not within a gene, the nearest annotated areas in each direction were identified.All results were visualized in RSupplemental Information.


Background: Viral myocarditis (VMC) is a common inflammatory cardiovascular disease with unclear mechanisms, which mainly affects children and adolescents. Apoptosis is the key to CVB3-induced myocarditis, and blocking this process may be beneficial to the therapy of VMC. Hence, this study aimed to explore the protective function of STAT3 on cardiomyocyte apoptosis of VMC and its underlying mechanisms.Methods and Results: In this research, we confirmed that STAT3 was significantly activated in both animal and cell models of VMC. To further clarify what role did STAT3 play in VMC, AG490, an inhibitor of STAT3, was used to suppress p-STAT3. Our results demonstrated that decreased expression of p-STAT3 caused by AG490 significantly aggravated severity of VMC with elevated myocardial inflammation, deteriorative ventricular systolic function and increased mortality. It suggested that STAT3 plays a protective role in VMC. To further identify the anti-apoptosis impact that activated STAT3 made, we constructed lentivirus to regulate the expression of STAT3 in NMCs. We found that up-regulated activated STAT3 attenuated cardiomyocyte apoptosis, but down-regulated one aggravated that, which verified activated STAT3 played an anti-apoptosis role in VMC. Following that, we explored what elements are involved in the anti-apoptotic mechanism of activated STAT3 by using survivin inhibitor YM155. The result showed the anti-apoptotic effect of activated STAT3 does not work in the case of survivin inhibition.Conclusion: Our findings demonstrated STAT3 by targeting survivin alleviated cardiomyocyte apoptosis in CVB3-induced myocarditis. Viral myocarditis (VMC) is a kind of cardiovascular disease characterized by myocardial inflammatory infiltration. It can cause heart failure, cardio-brain syndrome, and even sudden cardiac death . As the STAT3, a member of signal transducer and activator of transcription (STAT) family, is involved in transmission of various signals, including cytokines, apoptosis pathway, and so on . A great6 PFU of purified CVB3, while the control groups were intraperitoneally injected with the sterile PBS. Besides, starting from day 1, the AG490 and AG490 + CVB3 group were intraperitoneally injected with the STAT3 inhibitor AG490 at a dose of 10 mg/kg per day. The NC and CVB3 group were intraperitoneally injected with the same amount of sterile PBS every day. The mice were sacrificed on the seventh day of the experiment. Some mouse heart specimens were fixed with 4% paraformaldehyde, and some were quickly frozen in liquid nitrogen and stored at −80°C for later use.All BALB/c mice were ordered from SLAC Laboratory Animal Center in China and bred at the Animal Laboratory Center of Wenzhou Medical University. All experiments were approved by the Ethics Committee of Wenzhou Medical University and in compliance with the guidelines for experimental animals. 4-week-old male BALB/c mice were randomly divided into four groups: the normal control group (NC), the AG490 treatment group (AG490), the CVB3 infection group (CVB3), the AG490 and CVB3 treatment group (AG490 + CVB3). Mice were infected with the virus on day 0. The CVB3 and AG490 + CVB3 group were injected intraperitoneally with 0.1 ml PBS containing 1 × 102, and 21% O2 for 40 mins. Afterward, the supernatant was transferred to a cell culture dish pre-coated with 1% gelatin (Sigma). After 1 day of cultivation, the planting liquid was replaced with maintenance liquid. After culturing in a cell culture incubator for 1 day, the cells can be used for further experiments. Under serum starvation conditions, neonatal mouse cardiomyocytes (NMC) were cultured in the medium containing CVB3 with an infection multiple of 10 to establish a viral myocarditis cell model.According to the previous protocol, newborn mice within 24 h were killed to isolate cardiomyocytes . The heaUnder 2% isoflurane and 0.5 L/min oxygen gas flow, mice were subjected to transthoracic echocardiography by using the Vevo 2100 imaging system . Related indicators in the echocardiogram measured by supporting software include left ventricular internal diameter at end-systole (LVIDs), left ventricular internal diameter at end-diastole (LVIDd), left ventricular ejection fraction (LVEF).The mouse heart was fixed with formalin and embedded in paraffin, cut into 5 μm thick tissue sections, and stained with hematoxylin and eosin . To assess inflammation and myocardial damage, we use the myocarditis score. As mentioned in previous studies, the severity of myocarditis is assessed by the percentage of infiltrated inflammatory cells and cardiomyocyte necrosis .Cell lysates and heart extracts were separated by 12% SDS-PAGE and transferred to PVDF membrane. After blocked in dissolving in 5% skim milk in TBS for 2 h, incubate in p-STAT3, STAT3, survivin, Cleaved Caspase-3, Caspase-3, Bax  and Bcl-2(Santa Cruz) antibody, and overnight at 4°C. After washed with TBST, the membrane was placed in a box containing goat anti-rabbit IgG for 2 h. After combined with ECL developer, the membrane was developed under the Bio-Rad gel imaging system.The RNA extracted by the TRIZOL method was reverse transcribed into cDNA . Quantitative Real-time PCR was used to detect the expression of STAT3 and survivin. All primers were ordered from Sangon Biotech Co., Ltd. . The following primers were used: survivin, the forward primer 5′- GAGGCTGGCTTCATCCACTG-3′, and reverse primer 5′- CTTTTTGCTTGTTGTTGGTCTCC-3′; GAPDH, the forward primer 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse primer 5′-TGTAGACCATGTAGTTGAGGTCA-3′; STAT3, the forward primer 5′-CAATACCATTGACCTGCCGAT-3′ and 5′-GAGCGACTCAAACTGCCCT-3′.Apoptosis was evaluated by TUNEL Assay (Roche). The cells or tissues were fixed with 4% paraformaldehyde at room temperature for 20 mins and placed in 0.1% Triton X-100 solution on ice for 10 mins. The samples were covered with a 30 μl TUNEL reaction solution and incubated in the humid and dark environment at 37°C for an hour. Then samples were reacted with DAPI fluorescent dye solution for 5 mins. A fluorescence microscope captured the images.LDH-Cytotoxicity Assay Kit (Beyotime) was applied to assess the severity of cardiomyocytes damage. 120 μL/well supernatant of each sample was transferred to a 96-well plate and mixed with 60 µL working solution. After incubated at room temperature for 30 mins in the dark, the absorbance was measured at 490 nm.p-value < 0.05 was considered significant.When using GraphPad Prism seven software for analysis, results were expressed as mean ± standard error. To analyze the statistical significance between multiple groups, one-way analysis of variance was used. To analyze multiple treatment experiments, a two-way analysis of variance was used. The log rank test was used to analyze the survival rate of different groups. The Previous studies have illustrated the protective effect of STAT3 on autoimmunity  and CVB3At the same time, we constructed a viral myocarditis model of NMCs. There were no significant changes in the morphology and pulsation of cardiomyocytes compared with the control group at 12 h post infection (pi); Then some cardiomyocytes showed a wire-drawing phenomenon with the beating frequency slowing down and even irregular meanwhile at 24 h pi; Finally the cardiomyocytes gradually shrank and became round, and even detached and dissolved at 48 h pi . What’s Next, we tried to clarify what role STAT3 played in viral myocarditis. For this purpose, we injected STAT3 inhibitor AG490 into the abdominal cavity of mice with viral myocarditis. We confirmed that AG490 eliminated the upregulation of p-STAT3 induced by CVB3 through western blot . Then weEchocardiography was used to evaluate murine cardiac function by calculating left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). After CVB3 inoculation, the LVFS and LVEF of mice significantly decreased. Compared with CVB3-infected mice, the LVFS and LVEF of AG490-treated myocarditis mice was significantly reduced by 36.38% and 31.01% respectively, indicating that ventricular function worsened after AG490 treatment . What’ mTo further determine the effect of STAT3 in viral myocarditis, we constructed Lv-STAT3 and Lv-sh-STAT3 to regulate the level of p-STAT3 in NMCs. As demonstrated in Since activated STAT3 may play a protective role in viral myocarditis, the effect of activated STAT3 on cardiomyocyte damage caused by CVB3 infection was evaluated through LDH assay. When treated with the Lv-sh-STAT3, the LDH of the CVB3-infected NMCs was sharply increased, while the release of LDH in the Lv-STAT3 group decreased . Given tSurvivin, a member of the inhibitor-of-apoptosis protein (IAP) family, inhibits caspase activation, thereby preventing cell apoptosis among some cardiovascular diseases . Our preTo further clarify whether STAT3 plays an anti-apoptotic role through survivin, we introduced a survivin inhibitor, YM155, which is a novel, small, imidazolium-based compound that suppresses survivin via transcriptional inhibition of the survivin gene promoter. When we added YM155 and Lv-STAT3 simultaneously, the upregulation of survivin induced by Lv-STAT3 was reversed . WhereafViral myocarditis is an acute inflammatory disease of the heart, and is currently a leading cause of sudden death in children and young adults . NeverthPrevious studies found that STAT3 protected the heart, and mutations in STAT3 made the heart more vulnerable . To confApoptosis is programmed cell death, primarily characterized by the formation of apoptotic bodies. To initiate apoptosis, cells activate caspase-3 through exogenous death receptors and endogenous mitochondrial pathways. Apoptosis maintains the body's physiological homeostasis and participates in many pathophysiological processes, including ischemia, hypoxic injury, and viral pathogenesis . A numbeWe next explored those elements that are involved in the anti-apoptotic mechanism of STAT3 activation. As a well-known inhibitor of apoptosis, survivin blocks the activation of caspase three and exerts an effective anti-apoptotic function in cell lines and animal models . Althougin vivo and in vitro. More importantly, we confirmed for the first time that this anti-apoptotic function depends on the up-regulation of survivin (In summary, our study demonstrated that activated STAT3 protects cardiomyocytes from CVB3-induced apoptosis survivin . These n


Erysimum mediohispanicum. To this end, we conducted two independent complementary approaches. First, we empirically estimated temporal migration rates and temporal genetic structure in four populations of E. mediohispanicum during three consecutive years using nuclear microsatellites markers. Second, we developed a demographic genetic simulation model to assess genetic structure for different migration scenarios differing in temporal migration rates and their occurrence probabilities. We hypothesized that genetic structure decreased with increasing temporal migration rates due to the homogenizing effect of migration. Empirical and modelling results were consistent and indicated a U-shape relationship between genetic structure and temporal migration rates. Overall, they indicated the existence of temporal genetic structure and that such genetic structure indeed decreased with increasing temporal migration rates. However, genetic structure increased again at high temporal migration rates. The results shed light into the effects of reproductive asynchrony on important population genetic parameters. Our study contributes to unravel the complexity of some processes that may account for genetic diversity and genetic structure of natural populations.Migration is a process with important implications for the genetic structure of populations. However, there is an aspect of migration seldom investigated in plants: migration between temporally isolated groups of individuals within the same geographic population. The genetic implications of temporal migration can be particularly relevant for semelparous organisms, which are those that reproduce only once in a lifetime after a certain period of growth. In this case, reproductive asynchrony in individuals of the same population generates demes of individuals differing in their developmental stage (non-reproductive and reproductive). These demes are connected by temporal migrants, that is, individuals that become annually asynchronous with respect to the rest of individuals of their same deme. Here, we investigated the extent of temporal migration and its effects on temporal genetic structure in the biennial plant  Many plants require two or more years to produce flowers, but flowering individuals can be observed every year in natural populations. This is due to ‘temporal migrants’, that is, individuals flowering apart (at a different time) from the majority of their contemporaries. In this work, we explore the consequences of temporal migration on the genetic diversity of these populations. For the first time, we have reported a U-shape relationship, meaning that intermediate migration rates maximize genetic differences between flowering and non-flowering individuals. Our study contributes to unravel the complexity of some processes shaping genetic diversity of natural populations. Population is a concept with several definitions that still generates vivid discussions in biological sciences . IntuitiThe mentioned evolutionary and demographic processes structure populations spatially but also temporally. Recent studies on annuals comparing population genetic attributes in different points in time provided insight into how fast and how much plant populations can modify their genetic composition and genetic structure over several generations . For exaTemporal variation in reproductive time will surely be more important in organisms requiring >1 year between reproductive periods. Among them, biennial species are the simplest example. Such organisms are able to show asynchrony at larger time scales (e.g. between years) in addition to the within-year asynchrony described above for isolation by time. Consequences of this reproductive asynchrony will depend on how heritable reproduction times are . Thus, iIn this context of temporal genetic structure, individuals from different demes of the same population co-occur in the same physical site, and developmental variation in reproduction—and probably microhabitat conditions , 2006, bErysimum mediohispanicum to estimate temporal migration rates and temporal genetic structure. We estimated temporal migration rates by monitoring the fate of 1-year-old vegetative rosettes from E. mediohispanicum populations over several years. To assess temporal genetic structure, we genotyped with polymorphic nuclear microsatellites  Do demes within a geographic population show significant genetic differentiation? (ii) Does temporal migration rate reduce between-demes differentiation? Overall, the results highlight the intricacy of ecological and biological factors accounting for the genetic structure of plant populations.Here, we quantified the consequences of temporal migration for genetic structure of plant populations over time. To this end, we conducted two independent complementary approaches using a monocarpic plant with a biennial life history. First, we collected data from natural populations of the biennial plant E. mediohispanicum (Brassicaceae), which is an endemic but common herb of the Iberian Peninsula exhibiting a biennial habit , separated by 0.33–1.45 km and coded as Em01, Em08, Em23 and Em25 . Overall, we tagged and monitored 807 E. mediohispanicum vegetative rosettes.During field campaigns, we haphazardly tagged with numbered metal plates 100–120 vegetative rosettes at each sampling site per year to estimate the proportion of rosettes that completed the life cycle in 2 years. We estimated the proportion of rosettes that required >2 years to become reproductive individuals, representing an estimation of the temporal migration rate between demes. All tagged vegetative rosettes were of similar size (~40–50 mm diameter and 10–15 leaves). We purposely discarded too small or too large rosettes to avoid merging individuals from different demes. Based on previous field observations on E. mediohispanicum and other genetic properties appropriate for population genetic studies (2 (50 mM), 0.2 μL of dNTPs (25 mM), 1 μL of forward and reverse primers (50 ng μL−1), 5 μL of DNA (2 ng μL−1), 0.15 μL of Taq polymerase (5U μL−1) and 15.15 μL of Milli-Q water. Forward primers were fluorescently tagged with 6-FAM™ , NED™  and VIC®  labels . Polymerase chain reactions were performed in a Biometra Gradient Thermal Cycler . Thermocycler conditions consisted of an initial 2-min denaturation at 94 °C followed by 35 cycles of denaturing for 1 min at 94 °C, annealing for 1 min at primer-specific annealing temperatures, extension for 2 min at 72 °C and a final extension step of 10 min at 72 °C. Annealing temperatures were 54 °C for D2 and E5, and 56 °C for the rest of microsatellites. The fluorescently tagged PCR products were sized using GeneScan™ LIZ 500 size standard  on an ABI 3130 Genetic Analyser . Alleles were called by the same person with Peak Scanner v.1.0 .In addition, we collected leaf material from 30 individuals per deme to quantify genetic diversity and differentiation. Because our sampling design includes 3 years, four populations and two demes per population, we collected tissue from 720 individuals. We extracted DNA from all samples using DNeasy Plant Mini Kit  and stored them at −80 °C until genotyping with nine nuclear microsatellite markers . These markers previously showed polymorphism in E. mediohispanicum individuals with more than two alleles at few particular markers, probably because of partial genome duplication. This finding is in accordance with the genome size variation previously described for this species , mean observed heterozygosity (HO) and mean gene diversity (HS). To estimate the proportion of the total genetic variation explained by each of these levels we performed a hierarchical analysis of molecular variance as implemented in the ‘hierfstat’ package in R  were analysed with FSTAT v.2.9.3  and GenAage in R . The sigFST statistics . We also applied the same procedure for every population separately, ranging K value from 1 to 6 in this case.The genetic structure among all 687 Genetic distance between demes was estimated based on the Discriminant Analysis of Principal Components DAPC; , after rtemporal migration rate as the proportion of individuals from a given deme and year reproducing in the other deme 1 year later. Second, we define temporal migration event probability as the frequency at which a migration event occurs over time. Based on this, strict biennials have no temporal migration rates or no temporal migration event probabilities, whereas facultative biennials typically exhibit temporal migration rates and temporal migration event probabilities higher than zero.A time lag of 2 years between seed germination and sexual reproduction characterizes the lifespan of biennial plants. In populations of a biennial plant, both vegetative and reproductive individuals can occur simultaneously. Obviously, vegetative and reproductive individuals cannot interbreed because of their different developmental stages. Reproductive individuals come from seeds germinated 2 years back, whereas vegetative individuals come from seeds germinated just 1 year back. Hence, these populations encompass two distinct demes of reproductive individuals separated temporally by a single year . Some inNT) taking into account the total number of reproductive individuals recruited into a deme, via sexual reproduction (NR) or immigration (NM), and the number of individuals emigrating (NE) from that deme. This basic demographic model has the following structure:We developed a basic demographic model to compute total deme size over time  and λ are deme growth rates for deme d1 in year y and deme d2 in year z, respectively. Such λ values simulated random fluctuations of deme size sampling from a normal distribution with mean 1 and standard deviation 0.01. Although such distribution parameter values are arbitrary, they generated a stable population with slight fluctuations over time. We performed previous simulations with the same mean and different standard deviations to confirm the expected effect of increasing variation on increasing extinction rates (results not shown). Our model only explored the genetic effects of temporal isolation and migration between demes within a demographically stable population.where d1 and d2, the number of emigrating individuals, NE, in years y and z, respectively, was computed as follows:For each of the two demes m12(y) and m21(z) is the migration rate of vegetative individuals from deme d1 to deme d2 in year y, and from deme d2 to deme d1 in year z, respectively.where d1 and d2, the number of recruited individuals via immigration, NM, in years y and z, respectively, was computed as follows:Finally, for each of the two demes d1 and d2  over the 100 generations. The no-migration scenario represented the baseline simulation and showed the demographic behaviour of the simulated population. This simulation schedule was repeated 100 times totalling 11 000 runs (10 migration rates × 11 migration event probabilities × 100 replicates). The model started with 1000 initial individuals from each deme. Simulated individuals can only interbreed either when they are 2 years old or when they are 3 years old if they have become temporal migrants.For each deme, the model ran for 100 generations. A detailed exploration of model performance at each generation indicated that initial conditions did not affect the model, and that 100 generations were enough to accurately estimate all genetic parameters of interest HS) and genetic differentiation between demes and among individuals within demes with a hierarchical analysis of molecular variance (AMOVA). FST statistics  and increasing genetic distances with high temporal migration rates above 50 % .[see]. In contrast, the mean number of individuals per deme reached the minimum values for higher migration event probabilities  but significant in all the scenarios with no migration and the proportion of significant P-values decreased as migration rate and migration event probability increased  in our simulation was obtained for higher migration rates and low migration event probabilities.Temporal genetic differentiation between demes estimated using ncreased . The larInterestingly, model simulations mimicked the results obtained with empirical data. In particular, genetic distances between simulated demes also showed a U-shaped relationship between genetic differences between demes and temporal migration rates, with decreasing genetic distances with increasing temporal migration rates, and increasing genetic distances with higher temporal migration rate .E. mediohispanicum, leads to temporal genetic structure of populations. Examples of the consequences of such temporal genetic structure in biennial plant populations are scarce in the literature . Hence, gene flow and genetic homogenization among sites can rapidly increase in that particular area, erasing population structure at the population level. Interestingly, our analyses only detected interpretable signals of genetic structure between demes within populations when using DAPC rather than the Bayesian clustering method STRUCTURE, probably because the former has the power to unravel complex population subdivisions (The four E. mediohispanicum. We believe that temporal genetic structure could emerge with more intensity in systems with lower effective gene flow. Our study shed light on the complexity of population genetic features of some biological systems, such as biennial plants, in which the extent of temporal migration rates between demes within populations determines temporal genetic structure through migration or demographic stochasticity.In summary, our empirical and modeling approaches showed consistent patterns between genetic differences between demes within populations and temporal migration rates in The following additional information is available in the online version of this article—plaa037_suppl_Supplementary_MaterialClick here for additional data file.plaa037_suppl_Supplementary_DataClick here for additional data file.Data available in the Supplementary Material.