Signal transducers and activators of transcription 3 (STAT3) is a transcription factor that plays a key role in many cellular processes such as cell growth and cancer. However, the functions and mechanisms by which STAT3 regulates cellular processes are not fully understood.Here we describe a novel function of STAT3. We demonstrated that STAT3 plays an important role in DNA replication. Specifically, knockdown of STAT3 reduced DNA replication while activation and ectopic expression of STAT3 promoted DNA replication. We further identified the WD repeat and HMG-box DNA-binding protein 1 (WDHD1), which plays an important role in DNA replication initiation, as a novel STAT3 target gene that mediated the DNA replication function of STAT3. We showed that STAT3 bind the promoter/up regulatory region of WDHD1 gene.These studies identified a novel function of STAT3 that is mediated by its newly identified target gene WDHD1 and have important implications. Signal transducers and activators of transcription (STAT) family members are transcription factors that mediate many cellular processes and involved in the pathogenesis of various human diseases . STAT3 iSTAT3 activation is triggered primarily by interleukin 6 (IL-6) family cytokine receptor-associated Janus kinases (JAKs) , 8 as weActivated STAT3 can up-regulate the transcription of numerous genes, many of these genes are its direct target , 22. STAIn this report we described a novel biological function of STAT3. We showed that STAT3 played an important role in DNA replication. In addition, we identified WDHD1 as a STAT3 regulated target gene that mediated the DNA replication function of STAT3.Our recent studies demonstrated that both STAT3 and WDHD1 were up-regulated in oncogene overexpressing cells . STAT faNext we examined the extent to which ectopic expression of STAT3 would facilitate DNA replication. For this, we transfected MCF-7 cells with plasmid encoding STAT3. To avoid the potential effect of STAT3 on G1/S phase transition, cells were thymidine blocked before transfection. After transfection and releasing from the block, DNA replication was measured. As shown in Fig. Having demonstrated a role for STAT3 in DNA replication, we wanted to explore the mechanism by which STAT3 facilitates replication. Since STAT3 is a transcription factor, it is likely that STAT3 facilitates DNA replication by regulating a gene whose product involves in DNA replication. WDHD1 is certainly a potential candidate to mediate DNA replication function for STAT3. We therefore investigated the possibility that WDHD1 is a transcriptional target gene for STAT3. As mentioned and shown, both STAT3 and WDHD1 were up-regulated in HPV oncogene E7 expressing cells Fig. a. Upon tNext we examined whether the steady-state levels of WDHD1 protein is subjected to STAT3 regulation. As shown in Fig. To further establish the role of STAT3 in regulating WDHD1 expression, we examined the association of STAT3 at the promoter/up regulatory region of WDHD1. STAT family proteins recognize a consensus DNA binding motif of TTCCC/GGGAA . Three pAs a STAT3 target functioning in DNA replication, WDHD1 should be able to rescue the DNA replication defect caused by STAT3 knockdown. We therefore overexpressed WDHD1 in cells STAT3 has been knocked down. For this, MCF-7 cells were synchronized at the late G1/early S-phase of the cell cycle with thymidine, transfected with STAT3 targeting siRNAs, then transfected with WDHD1 plasmid, DNA replication was examined. Significantly, expression of WDHD1 rescued DNA replication reduction caused by STAT3 knockdown , HTB-22) and cervical cancer HeLa cells  were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in a humidified atmosphere of 5% COTotal RNA was isolated with an RNeasy kit (Qiagen) from MCF-7 and HeLa cells and their correspondent targeting siRNA expressing cells according to the manufacturer’s instruction. cDNA was synthesized with a Superscript VILO cDNA synthesis kit (Invitrogen). The iTaq Universal SYBR Green Supermix (Bio-Rad) was used for real-time PCR (qRT-PCR) in the Bio-Rad CFX96 Touch Real-Time PCR Detection system. Data were analyzed using the 2 − ∆∆Ct method.Chromatin immunoprecipitation (ChIP) assay was performed using the ChIP assay kit from Millipore following the manufacturer’s protocol. Immunoprecipitations were performed using anti-STAT3 or control IgG antibodies. PCR was performed with the Simple ChIP Human c-Fos Promoter Primers  that has been shown to interact directly with STAT3 , 43 and Cells were transfected with a final concentration of 20 nM siRNA per target gene Table  using LiTo obtain total protein, cell extraction was prepared in lysis buffer . The protein concentration was measured by the bicinchoninic acid (BCA) protein assay reagent (Pierce) and confirmed by Coomassie blue staining of membranes after blotting. Equal amounts of protein from each cell lysate were separated in an SDS polyacrylamide gel (PAGE) and transferred onto a nitrocellulose filter membrane (NC) membrane. Membranes were blotted with antibodies against WDHD1 , STAT3 , Phospho-STAT3 (Tyr705) , and tubulin .For the bromodeoxyuridine (BrdU) labeling experiment, BrdU  was added to the medium 2 h before collection of cells. Cells were then harvested and fixed in 70% ethanol. The cells were permeabilized with 2 N HCl–0.5% Triton X-100, neutralized with 0.1 M sodium tetraborate, stained with monoclonal anti-BrdU (BD Biosciences), and then with anti-mouse IgG F(ab)2-FITC (Sigma), and counterstained with PBS-7-AAD-RNase A. Flow cytometric analysis was performed on a BD FACSAria™ III sorter instrument equipped with BD FACSDiva™ 7.0 software . FITC 490 nm fluorescence was acquired in logarithmic amplification in FL1 and 7-AAD 650 nm fluorescence was acquired in linear amplification in FL3. Cell cycle analysis was done using Cytomics™ FC500 Flow Cytometry CXP 2.0.t test was used to compare the differences between means. P < 0.05 was considered significant.Data were presented as means and standard deviations (SDs). The Student’s Additional file 1: Figure 1. Time selection of thymidine block and release of cell cycle. a MCF-7 cells were blocked with thymidine for 0h, 20h and 24h. b After thymidine block for 20h, MCF-7 cells were released for 1h, 2h, 3h, 4h, 5h, 6h, 8h, 10h.Additional file 2: Figure 2. STAT3 plays a role in DNA replication in HeLa cells. After thymidine block, HeLa cells were transfected with siRNAs targeting STAT3. After releasing, cells were stained with BrdU and analyzed by flow cytometry. Data from a representative experiment of 3 were shown (Upper panel) and summarized (Lower panel). Western blots were performed using transfected cell extracts without thymidine treatment.Additional file 3: Figure 3. WDHD1 mRNA expression is regulated by STAT3 in HeLa cells. STAT3 and WDHD1 mRNA levels in IL-6 or EGF treated HeLa cells determined by real-time-PCR analysis. Data from a representative experiment of 3 were shown. Error bars reflect the standard deviations of the mean. *p < 0.05 **p < 0.01. NC negative control.Additional file 4: Figure 4. The steady-state levels of WDHD1 is regulated by STAT3 in HeLa cells. a STAT3 and WDHD1 protein levels in HeLa cells were examined by Western blotting after siRNA transfection (Left panel). Data were summarized (Right panel). Data from a representative experiment of 3 were shown. Error bars reflect the standard deviations of the mean. *p < 0.05 **p < 0.01. NC negative control.Additional file 5: Figure 5. STAT3 binds to WDHD1 promoter/up regulatory region in HeLa cells. Immunoprecipitations were performed using anti-STAT3 or control IgG antibodies. PCR was performed with c-Fos or WDHD1 primers. Data from a representative experiment of 3 were shown (Left panel) and summarized (Right panel).


Fish gut microbial assemblages play a crucial role in the growth rate, metabolism, and immunity of the host. We hypothesized that the gut microbiota of rainbow trout was correlated with breeding program based genetic selection for muscle yield. To test this hypothesis, fecal samples from 19 fish representing an F2 high-muscle genetic line (ARS-FY-H) and 20 fish representing an F1 low-muscle yield genetic line (ARS-FY-L) were chosen for microbiota profiling using the 16S rRNA gene. Significant differences in microbial assemblages between these two genetic lines might represent the effect of host genetic selection in structuring the gut microbiota of the host.p < 0.05). The fecal samples showed statistically distinct structure in microbial assemblages between the genetic lines . Functional profiling of bacterial operational taxonomic units predicted characteristic functional capabilities of the microbial communities in the high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic line samples.Tukey’s transformed inverse Simpson indices indicated that high muscle yield genetic line (ARS-FY-H) samples have higher microbial diversity compared to those of the low muscle yield genetic line (ARS-FY-L)  =14.11, The significant differences of the microbial assemblages between high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic lines indicate a possible effect of genetic selection on the microbial diversity of the host. The functional composition of taxa demonstrates a correlation between bacteria and improving the muscle accretion in the host, probably, by producing various metabolites and enzymes that might aid in digestion. Further research is required to elucidate the mechanisms involved in shaping the microbial community through host genetic selection.The online version contains supplementary material available at 10.1186/s12864-020-07204-7. Aquaculture is one of the fastest-growing industries and plays a vital role in fulfilling the global requirements for human protein consumption . Growth The gastrointestinal compartments of fish contain large microbial communities that play an essential role in homeostasis, physiology, and gut development –7. MicroMicrobiota transmit nutrient signals to their hosts, which might shape the gut microbiome in every stage of life based on diet intake, behavioral change, and environmental influence . Researcp < 0.05; Fig. Divergent selection was practiced for fillet yield to develop high (ARS-FY-H) and low (ARS-FY-L) yield genetic lines of rainbow trout. The two fish groups used in this study were collected after two generations of selection and were statistically different in their average muscle yield as indicated by a one-way Mann-Whitney U test  but not low-muscle yield genetic lines . Therefore, harvest day was treated as a random effect in all models to test for the main effect of genetic line. Using a linear mixed model, we tested for differences in gut alpha diversity between fish genetic lines and found that diversity was greater in high (ARS-FY-H) genetic line  = 14.11, p < 0.05, Fig. 1,36 = 4.7, p < 0.05, R2 = 11.9%) indicated that the muscle-yield genetic line was predictive of gut microbial assemblages in rainbow trout  muscle yield genetic line.A total of eight phyla, 12 classes, 36 families, and 64 genera had significant differences in abundance between the two genetic lines  muscle yield genetic line, whereas, the genus Mycoplasma , and unclassified Firmicutes  have higher abundance in the low (ARS-FY-L) muscle yield genetic line  muscle yield genetic lines and are associated with digestion, fatty acid metabolism, pathogen inhibition, which, are linked to host health and digestion. Family Burkholderiaceae had a higher abundance in high (ARS-FY-H), and genus Burkholderia belonging to this family were reported as the most abundant genus in the fish gut [The genus g humans , 42, micg humans , and tilg humans . These bg humans . A recenty acids . Both thfish gut . HoweverMycoplasma (16.8%) and the unclassified phylum Firmicutes (7.9%). A previous study showed that the genus Mycoplasma is the most abundant taxon in adult Atlantic salmon [Tinca tinca [Taxa with significantly higher abundance in the low (ARS-FY-L) muscle yield genetic line were c salmon . Bacterica tinca . The lesca tinca  and thisca tinca .Bacteroides belonging to phylum Bacteroidetes showed higher abundance in the high (ARS-FY-H) genetic line, and is associated with fatty acid metabolism thus producing SCFAs [Tax4Fun analyses were used in this study to enumerate differential functional capabilities of microbial communities in high (ARS-FY-H) and low (ARS-FY-L) genetic lines Fig. . Bactering SCFAs . A studyng SCFAs .Fig. 5HFusobacterium was more abundant in the high (ARS-FY-H) muscle yield genetic line and known to be involved in lysine degradation and production of SCFAs [Similarly, microbe mediated lysine degradation increased production of SCFAs (butyrate and acetate) in the human gut . The genof SCFAs . Microbiof SCFAs , 57. Ketof SCFAs . Arachidof SCFAs , 59. Theof SCFAs . Bacteriof SCFAs . Fish with low (ARS-FY-L) muscle yield had unique functional profiles that differed from high (ARS-FY-H) muscle yield samples. For example, pyruvate metabolism, amino acid metabolism, folate biosynthesis, glycosphingolipid biosynthesis, glyoxylate and dicarboxylate metabolism, adipocytokine signaling pathway, and two-complement system were enriched in most of the low (ARS-FY-L) genetic line samples Fig. . GlycospIn this study, the gut microbial assemblage  diversity correlated with selectively-bred muscle yield genetic lines. Microbial differences between the two genetic lines could be observed as a host genetic selection signature on the gut microbiota. Both differences in taxonomic groups of microbes and their functional predictions correlated with muscle yield. Our results suggest a role of specific microbial taxa in improving host muscle growth and metabolism. n = 118) from the ARS-FY-C line. The tank received identical water from a partial re-use system and water temperature was maintained at approximately 13 °C for the entirety of the grow-out period. Fish were split at random into a total of two 1800-L and two 3800-L tanks as they grew to maintain biomass densities below 100 kg/cubic meter. At approximately 13 months of age, fish used in the current study were split into three replicate 800-L tanks (n = 46 fish per tank). One week before harvest, the fish were split at random into four 800-L tanks (34–35 fish per tank) to allow the harvest of two complete tanks on each of two successive days and thus minimize netting-associated stress associated with harvesting of a partial tank. Fish were fed a commercial diet  using automatic feeders  that provided feed at a daily ration that was considered as slightly below satiation.Fish were produced at the NCCCWA as a part of our ongoing selective breeding program. Briefly, full-sibling families  were produced from single-sire × single-dam mating events. Each family was reared separately from hatch through approximately 30 g (4 months post-hatch) when 8 fish per family were anesthetized  and uniquely tagged by inserting a passive integrated transponder  into the peritoneal cavity. After tagging, fish were comingled and reared in a single, 1800-L tank that also housed contemporary fish (n = 1 fish per family) for fecal collection at harvest; selection was based on divergent mid-parent breeding values and to maximize genetic diversity within each line. Due to a mortality of a high (ARS-FY-H) genetic line fish, 39 fecal samples were collected for this study, 19 from the high (ARS-FY-H) muscle yield genetic line  and 20 representing the low (ARS-FY-L) genetic line . Fecal samples were stripped manually into sterile Eppendorf tubes , then stored in a − 80 °C freezer until analysis. Fish were eviscerated and the carcasses were placed on ice and held in a 4 °C refrigerator overnight for analysis of muscle yield the following day.At approximately 15 months post-hatch, fish were euthanized using an overdose of anesthetic (300 mg/L MS-222) and processed for analysis of the muscle yield trait. Fish were not fed the day prior to and the day of harvest. Twenty families were pre-selected from the high (ARS-FY-H) and low (ARS-FY-L) genetic lines  and 20 low (ARS-FY-L) genetic line fish were subjected to DNA isolation using a Promega Maxwell DNA Isolation Kit , as we previously described  with a mLibrary preparations and sequencing were done based on 16S rRNA sequencing strategy using the Illumina 16S Metagenomic Sequencing Library Preparation Guide. Briefly, 10 μM of 515F and 10 μM of 806R primers amplifying V4 regions were used to target 16S rRNA gene using McLAB HiFi master mix using polymerase chain reaction (PCR). The final PCR reaction consisted of 12.5 μL 2x HiFi, 1 μL of 10 μM 515F primer and 1 μL of 10 μM 806R primer, 5 μL DNA and 5.5 μL sterile nuclease-free water. The PCR product was then subjected to size selection using a magnetic bead capture kit . After the first PCR clean up, dual indexed primers were used to amplify the V4 region as described by Kozich et al. . After iwww.mothur.org) according to the Mothur Illumina Miseq standard operating procedure (SOP) [pcr.seqs command was used to trim primers and adaptors to the V4 region. The median length of the sequences was determined as 253 by using the summary.seqs command [Screen.seqs command was used to remove sequences with length > 254 bp and < 251 bp containing homopolymers of > 8, and with ambiguous base calls. The split.abund command was used to keep Operational Taxonomic Units (OTUs) with more than two reads [chimera.vsearch and removed from the analysis. The remaining sequences were clustered using cluster.split [remove.rare command [A total of 28,518,046 paired-end raw sequences were obtained during the Miseq run. Sequencing data were analyzed using Mothur  , 68 with command . Screen.wo reads . The SILwo reads  was useder.split  at a thr command , 74. Thevegan [plyr [dplyr [ggplot2 [lmerTest [pheatmap, MuMIn [lme4 [Tax4Fun [DEseq2 [rcompanion [grid [TidyVerse [To test for the statistically significant differences of the muscle yield between the two groups, a one-way Mann-Whitney U test  was performed. Statistical analyses  were performed in R version 3.5.2 using the packages vegan , plyr [7an [plyr , dplyr [r [dplyr , ggplot2[ggplot2 , lmerTeslmerTest , pheatmap, MuMIn , lme4 [8In [lme4 , Tax4Fun[Tax4Fun , DEseq2  [DEseq2 , rcompanompanion , grid [8on [grid , and TididyVerse .lme4) [Sixteen fecal samples  from the high (ARS-FY-H) and 18 fecal samples (that passed QC) from the low (ARS-FY-L) muscle yield line were used for this analysis. A Tukey’s ladder of power transformation was performed to fit inverse Simpson values to a Gaussian distribution. Alpha diversity between the genetic lines was compared using a linear mixed-effects model (LMMs) with the genetic line as a fixed effect and harvest day set as a random effect (package lme4) .vegdist function in the Vegan package [betadisper function was used to test for the homogeneity of multivariate dispersion between gut assemblages from high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic lines. The metaMDS function in Vegan was used to generate a non-metric multidimensional scaling ordination (nMDS), which was then plotted using ggplot2 [adonis function in Vegan was used to perform PERMANOVA on Bray-Curtis dissimilarity values to determine if the genetic line was predictive of gut assemblages, while controlling for harvest day effect . An indicator species analysis was performed in Mothur to determine the microbial assemblages that were characteristic of muscle-yield genetic lines [ggplot2. PERMANOVA was used to test for differences in microbial assemblages with genetic line set as a fixed effect.Beta diversity was calculated to test if muscle yield genetic line was predictive of the gut microbiota. To do this, a Bray-Curtis dissimilarity matrix was generated using the  package . The bet ggplot2 . The adoic lines . A Kruskvegdist function, and non-metric multidimensional scaling (nMDS) ordination was used for visualization using the metaMDS function and plotting in ggplot2. The adonis function was performed to determine if sample type , set as a fixed effect, was descriptive of microbial assemblages. The detailed methodology and results for this experiment have been included in Additional file Feed and water samples were sequenced to determine whether the fecal microbial assemblages of trout differed from the environment. Beta diversity was calculated based on a Bray-Curtis dissimilarity matrix representing sample-to-sample pairwise distances using the phylotype command in Mothur to investigate microbial functional and metabolic capacities of OTUs. The shared file was then converted to the biome format using the make.biom command in Mothur. The Tax4FUN package in R was used to predict the microbial functional and metabolic capacities by linking 16S rRNA gene-based taxonomic profiles to KEGG reference profiles [p-value less than 0.001 were selected for heatmap visualization using the pheatmap R package [Phylotype based OTU clustering and classification was performed using the profiles . The nor package . The R cAdditional file 1. Differentially abundant taxa. Table including taxa having significant differences in abundance between the two genetic lines.Additional file 2. Mothur Standard Operating procedure for microbiota profiling.Additional file 3. Mothur Taxonomy file containing taxa information of all the OTUs.Additional file 4. Mothur Shared file used to calculate alpha and beta diversity metrics.Additional file 5. Comparison of gut and environmental samples. Additional file 6. R code. Additional file 7. Statistical analysis results from alpha and beta diversity analysis.


Mustela lutreola L., 1761, which is one of the most endangered mammalian species in the world. This article provides a comprehensive description of the studies undertaken over the last 50 years in terms of cytogenetics, molecular genetics, genomics (including mitogenomics), population genetics of wild populations and captive stocks, phylogenetics, phylogeography, and applied genetics . An extensive and up-to-date review and critical analysis of the available specialist literature on the topic is provided, with special reference to conservation genetics. Unresolved issues are also described, such as the standard karyotype, systematic position, and whole-genome sequencing, and hotly debated issues are addressed, like the origin of the Southwestern population of the European mink and management approaches of the most distinct populations of the species. Finally, the most urgent directions of future research, based on the research questions arising from completed studies and the implementation of conservation measures to save and restore M. lutreola populations, are outlined. The importance of the popularization of research topics related to European mink genetics among scientists is highlighted.The purpose of this review is to present the current state of knowledge about the genetics of European mink  Mustela lutreola L., 1761 is considered one of the most endangered mammalian species in the world [Neovison vison [Due to ongoing population depletion, both in terms of the actual number of individuals and area occupied, European mink he world . The spehe world . About 5he world . Reintrohe world . The maion vison . The alaon vison ,5.Ailuropoda melanoleuca [M. lutreola are limited and urgently need to be completed, especially in the context of the progressing extinction process and the disappearance of its numerous populations in France, Belarus, and Russia, among others [Despite this, the number of studies on European mink, reflected in the number of scientific papers devoted to the species, is relatively low. The digital repository of the National Centre for Biotechnology Information (NCBI) records less than 60 scientific articles devoted to various aspects of the biology of the species. In comparison, the same repository lists as many as 2000 articles related to the giant panda g others ,8. The mg others ,10,11,12Notably, only (conservation) genetics can provide tools to rescue species affected by the extinction vortex , which, M. lutreola karyotype. Further research on the genetics of European mink relates primarily to genetic markers [The pioneer genetic studies on European mink concerning cytogenetics were conducted in the former USSR. To date, works by Volobuev and Ternovsky , Volobue markers ,21, the  markers ,24,25,26 markers , assessm markers ,28,29,30 markers ,32. Wort markers ,34,35,36M. lutreola provides a good example of the possibility of achieving the goal of conservation genetics, i.e., applying genetic knowledge and methods to preserve endangered species, as well as to support evidence-informed conservation activities and strategies based on knowledge about the current state of the species’ genetic resources [The purpose of this review was to present the current state of knowledge about the genetics of European mink, with particular emphasis on the possibility of its practical application in species conservation. The case of esources ,37.The added value of this review article is an extensive and up-to-date review of the available specialist literature on the genetics of European mink, including an update detailing in terms of genetics, the species bibliography published by Youngman . The revAdditionally, a definition of conservation (captive) breeding genetics is formulated.n = 38 [Mustela L., 1758 genus, the diploid number of chromosomes ranges from 38 to 44 [n = 30, which, on a cytogenetic basis, additionally indicates a relatively low degree of its evolutionary relationship with European mink [The diploid number of the chromosomes of European mink is 38, which is typical for many species of the Mustelidae Fischer, 1817 family; for more than 60% of species of this group, 2n = 38 ,40. Amon38 to 44 ,42. Signean mink .M. lutreola consists of five pairs of metacentric chromosomes of different sizes, two pairs of subtelocentric chromosomes, five pairs of submetacentric chromosomes, and seven pairs of telocentric chromosomes [FN, number of chromosomal arms) is 62, while the fundamental autosomal number  is 58 [The chromosomal set of omosomes . The X comosomes . As withomosomes . The funs) is 58 . Nucleols) is 58 . The stas) is 58 .Mustela nivalis, mountain weasel Mustela altaica, Japanese marten Martes melampus, European badger Meles meles, and striped polecat Ictonyx striatus was presented by Graphodatsky et al. [Mustela sibirica [The characteristics of the G and C bands of chromosomes of European mink were presented by Graphodatsky et al. ,19,44. Tsibirica ,23.M. lutreola × Mustela putorius hybrids [Mlut”. Cabria et al. [M. lutreola, which are listed in The best-known genetic markers of European mink are the microsatellite nuclear sequences (short tandem repeats (STRs) and simple sequence repeats (SSRs)), which are sequential patterns of DNA consisting of several nucleotides and tandem repeats . They ar hybrids ,45,46,47a et al.  identifiMustela eversmanii, Mustela putorius furo, M. sibirica, M. nivalis, N. vison, Mustela erminea, Martes martes, and Martes foina, among others [M. lutreola in studies on genomes of other mustelids. In turn, Peltier and Lodé [Microsatellite markers of European mink were successfully amplified in other species of the Mustelidae family, including in g others ,48, demoand Lodé , Michauxand Lodé , and Caband Lodé  positivedrb gene from the family of genes of major histocompatibility complex (MHC) class II were analyzed in M. lutreola [est-2; the gene for NADP-dependent cytosolic malate dehydrogenase/EC 1.1.1.40, me−1; the gene for malate dehydrogenase (MDH)/EC 1.1.1.37, mdh−1; and the gene for nonspecific protein). All other loci were monomorphic, whereas for samples analyzed in parallel from the European polecat, nine polymorphic loci were found [drb gene, Becker et al. [drb gene, demonstrating the evolutionary closeness of this sequence in M. sibirica, Mustela itasi, and M. lutreola and its trans-species polymorphism (TSP), indirectly evidencing selection balance.Besides the polymorphism of neutral genetic markers, variants of allozymes and the lutreola ,50,51,52lutreola , two allre found . For theThe genome of European mink has not yet been sequenced. However, Mouton et al.  reportedcytb gene (26 for fragments of 337 to 504 bp and four for the complete gene sequence of 1140 bp), 23 haplotypes for the control region (357 to 990 bp), and the whole mitogenome sequence. In comparison, 357,611 and 19,698 records of the nucleotide sequences of M. putorius and M. putorius furo [N. vison were deposited in GenBank, respectively.For the species, 160 nucleotide sequences (DNA and RNA) were deposited in GenBank . This nuius furo  and N. vM. lutreola was estimated at 6.4 pg, and the shares of rapidly (representing highly repetitive DNA sequences), intermediately (representing moderately repetitive sequences), and slowly-  renaturing fractions were shown to be 17%, 13%, and 70%, respectively [Vormela peregusna [Lushnikova et al.’s  work canectively . In the eregusna . The moseregusna .EcoRI and RFLP-BamHI polymorphisms in M. lutreola, M. putorius, N. vision, M. erminea, M. sibirica, and V. peregusna [EcoRI repeats were dispersed over karyotype, the 1.9 kb BamHI repeats were concentrated in the heterochromatic pericentromeric regions and additional chromosome arms, while the 1.35 kb BamHI repeats were only located in the centromeric regions [BamHI repeats (interspecific variability of DNA–DNA hybridization patterns) were also applied to consider the phylogenetic relationships of the abovementioned species, indicating existence of a common evolutionary group including M. lutreola, M. sibirica, and M. putorius [Pioneering studies on the genome scale included comparative studies concerning RFLP-eregusna . The obt regions . The Bamputorius .M. putorius  as being about 2.411–2.474 million bp, the content of GC pairs as about 42%, and the number of genes as about 27,300 [Due to its high cytogenetic similarity and proven close phylogenetic relationships ,24,34,42t 27,300 .M. lutreola with the complete sequences of mitochondrial genomes of 24 Mustelidae species deposited in GenBank and conducted in BLAST [M. lutreola mtDNA indicated its high affinity with European polecat (and ferret) and its explicit presence in the clade including M. eversmanni, Mustela nigripes, M. sibirica, and M. itatsi [The complete reference mitochondrial genome of European mink was sequenced de novo in 2017 (GenBank accession code: MT304869), with the length of nucleotide sequence being 16,523 bp . The comin BLAST  showed a. itatsi . Compari. itatsi .N. vison and M. putorius, occurring in the same areas, enables the effective control of the population of the former abovementioned invasive alien species that are dangerous for European mink, and allows the assessment of the possible occurrence and scale of hybridization with the latter species [Identification of European mink from environmental and noninvasively obtained samples  is important for species in situ conservation efforts ,61,62,63 species ,65,66,67RsaI and MspI [M. lutreola, two other haplotypes characteristic of M. putorius, and one haplotype characteristic of N. vison [An example is the genetic test for the distinction of European mink, European polecat, and American mink, developed by Gómez-Moliner et al. . The proand MspI . The obtN. vison . The advN. vison .Mel08, according to the procedure described by Domingo-Roura [M. lutreola and M. putorius (221 bp product for both species) can be distinguished from N. vison (436 bp), whereas the use of digestion with restriction enzyme AciI can distinguish European mink (no digestion occurs) from European polecat  [N. vison vs. urgent conservation efforts toward M. lutreola) [Another noninvasive method for the identification of European mink, also differentiating it from European polecat and American mink, was developed by López-Giráldez et al. . This mego-Roura . At the  136 bp) . The advutreola) ,46. Collirbp; 221 bp fragment of exon 1), arguing that identification methods based on mtDNA are subject to risks from nuclear insert copies, high intraspecific diversity, and heteroplasmy. The distinction between European mink, European polecat, and American mink is based on the differentiation of species-specific single-strand conformation polymorphism (SSCP) electrophoretic patterns [M. lutreola and M. putorius [Oliveira et al.  developepatterns . The PCRpatterns . Compariputorius ,69.M. lutreola were detected by the DNA barcoding method following the protocol developed by Fernandes et al. [b gene (cytb) sequence .Kiseleva and Sorokin  performes et al. , which iM. lutreola focused on defining genetic diversity between preserved populations of the species. Analyses of the intraspecies genetic structure of European mink showed its relatively high genetic diversity, especially in comparison with other Mustelidae taxa [Studies on the population genetics of dae taxa ,77,78,79dae taxa .cytb gene in 43 individuals: 14 from Russia and Belarus, 2 from Romania, and 27 from France and Spain, with 11, 2, and 1 haplotypes identified for these populations, respectively [π) and haplotype diversity (h) [The studies of Michaux et al.  on the isity (h)  were higEH) and allelic richness (SR) were calculated as 0.539 and 3.76 for Northeastern Europe, 0.458 and 2.89 for Southeastern Europe, and 0.379 and 2.12 for Western Europe, respectively [ISF (inbreeding coefficient) values reached 0.084 in the French–Spanish population, 0.085 in the Romanian population, and 0.182 in the Russian–Belarusian population [ijF) in the population from West Europe was 0.08 (for distances of <10 km), suggesting mating occurred locally [In a study conducted in 2005, 15 haplotypes for the complete D-loop were identified in the Russian–Belarussian population , four in the Romanian population , and only one in the French–Spanish population  [STF , STG , and STΦ  [An important outcome of the research of Michaux and colleagues ,8, as weπ = 0.0092 ± 0.0055 and h = 0.95 ± 0.054 [π = 0.0134 ± 0.0074 and h = 0.98 ± 0.027 [π = 0.0026 ± 0.0019 and h = 0.74 ± 0.052) [Relatively high genetic diversity in the Eastern European population was confirmed by Korablev et al. , who exa ± 0.054 . Analysi ± 0.054  (Table 4 ± 0.027 . Europea± 0.052) . The dif± 0.052) . An impo± 0.052) .cytb gene and the control region of 344 individuals. The established parameter values for evaluating the genetic diversity for microsatellite loci are summarized in π = 0.004 ± 0.003, h = 0.862 ± 0.016, 13 haplotypes, 92.3% of private haplotypes) and moderate for the Southeastern population , to the lowest for the Western population (only a single haplotype detected). The nucleotide and haplotype diversities calculated for the whole examined group were 0.005 ± 0.003 and 0.857 ± 0.014, respectively [M. lutreola revealed significant geographic structuring . The presented results indicated higher overall genetic polymorphism and structure in the Northeastern population, slightly lower values in the Romanian population, and significantly lower values in the Western population [Cabria et al.  focused ectively . Analysipulation .b gene in the Eastern European population  and two in the Spanish population . The same study revealed the existence of four different haplotypes in both the Eastern European and Spanish populations of M. putorius.Studies conducted by Davison et al.  and CabrThe cause of high genetic homogeneity of populations living in France and Spain is attributed to the bottleneck and founder effects that might have occurred  in the relatively recent past, as well as to the limited gene flow, thereby altering reproductive exchanges ,14,30. AEst−2, Mdh−1, Me−1, and Pnp) were found to be polymorphic , with observed heterozygosity averaging 0.02, an expected heterozygosity of 0.038, and an ISF of 0.48 [N = 49), the polymorphism reached 25.8% and the observed heterozygosity levels averaged 0.057 [These conclusions were also supported by results of genetic polymorphism analysis of 38 genes’ loci  conducte of 0.48 . In Euroed 0.057 .M. lutreola from the Poitou-Charentes region (Western France), conducted by Peltier and Lodé [ISF = 0.1907). The authors suggested that a significant heterozygote deficit in the Southwestern population of European mink was associated with depletion of genetic diversity, which is characteristic of species near extinction.Eight microsatellite loci  in 12 inand Lodé  were genEst−2, Mdh−1, Me−1, and Pnp) and four (36.4%) microsatellite loci  were found to be polymorphic. The effective number of microsatellite alleles per locus was estimated at 1.45 and mean observed heterozygosity was 0.095 [OH = 0.246, N = 114), according to parallel analysis [The level of genetic polymorphism in the French population (Southwestern France) was also studied by Lodé et al. . Geneticas 0.095 . In thisMustela lutreola lutreola L., 1761, M. l. biedermanni Matschie, 1912, M. l. binominata Ellerman and Morrison-Scott, 1951, M. l. cylipena Matschie, 1912, M. l. novikovi Ellerman and Morrison-Scott, 1951, M. l. transsylvanica Éhik, 1932, and M. l. turovi Kuznetsov and Novikov, 1939), was not confirmed by intraspecies genetic diversity studies. In the view postulated by Cabria et al. [High phenotypic diversity, expressed in the postulated distinction of six  to sevena et al. , populata et al. . Currenta et al. .Notably, knowledge about the origin of certain populations derived from genetic data affects their management and conservation. An example illustrating this is the lively debate on the controversial natural (colonization) or human-induced (introduction) origin  were reported by Davison et al. [M. lutreola and polecats , and its more distant relationships with M. sibirica and M. itatsi [cytb gene and the D-loop sequence confirmed these results [M. putorius and M. eversmanii) may be evidence of relatively recent speciation or gene flow through hybridization (reticulate evolution) occurring between these species [The results of pioneering molecular research on European mink phylogeny based on mitochondrial sequence  were also used in a multispecies analysis of diversification timing of taxonomic groups within the Mustelidae family [The phylogenetic analysis based on the nucleotide sequence of the nuclear o et al. , proved e weasel A,B. The 12S rRNA ,42,95 anNA [cytb ,25,31, ae family .12S RNA, cytb, and ND2) and three nuclear  genes were particularly interesting. The results of these studies indicated that the European polecat is phylogenetically the closest species to European mink, whereas other closely related species include the Siberian weasel, the least weasel, and the stoat .According to phylogenetic analyses of Davison et al.  M. putorM. lutreola and M. putorius, M. eversmanii, M. nigripes, M. sibirica, or M. itatsi [Mustela genus, including M. lutreola, and instead classified into the separate genus Neovison by Baryshnikov and Abramov in 1997 [The phylogenetic distance between the European mink and the American mink is much greater than between . itatsi ,95,97,98 in 1997 .Amdovirus, Dependovirus, Parvovirus, Astrovirus, and Picobirnavirus [Research concerning European mink in the field of molecular ecology, understood as the application of molecular genetic methods to address ecological questions , is mainrnavirus .M. lutreola was investigated to examine the potential of its protein product to be used as a receptor by the SARS-CoV−2 virus, and thus to verify if European mink could potentially be an intermediate host species for this pathogen [M. lutreola to SARS-CoV−2 [Recently, the nucleotide sequence of the angiotensin I converting enzyme 2 gene in pathogen . The obtRS-CoV−2 .VP2 gene of the Aleutian mink disease virus (AMDV) using a PCR method in European mink from Spain. Leimann et al. [M. lutreola from Hiiumaa Island (Estonia) for the presence of the AMDV DNA by PCR amplification of the NS1 and VP2 gene fragments. AMDV DNA was not detected in the examined animals [Mañas et al.  detectedn et al.  tested f animals .Borrelia burgdorferi, which was the first report in this regard concerning animals originating in Romania [M. lutreola (Southwestern France) for the presence of pathogenic Leptospira interrogans DNA by PCR amplification. Of the 34 examined individuals, 8 were found to be infected with this pathogen, however, the role of European mink as a reservoir of L. interrogans was not confirmed [An example of a bacterial pathogen identified in European mink using a PCR-Restriction Fragment Length Polymorphism (RFLP) method is  Romania . Moinet  Romania  tested tonfirmed .In 2019, a pilot study on European mink detection and population monitoring using the environmental DNA (eDNA) metabarcoding method was performed in Northeastern Spain (La Roja and Basque Country) ,63. Due Although the number of studies on the genetics of European mink is relatively low, the important application aspect of almost every study in this field should be emphasized. Such studies directly contribute to obtaining valuable and practical knowledge about planning effective protective measures, both ex situ (conservation captive breeding) and in situ , conducted both ad hoc and as long-term strategies ,106,107.M. lutreola conservation to be addressed by conservation genetics are: (1) Determining the optimal species conservation scenario for management of the existing populations, including translocations and reintroductions ; (2) captive breeding genetics; and (3) identification and assessment of possible threats to interspecies genetic nature, i.e., hybridization and introgression [The key issues for gression ,8,66,108Since the publication of the first study results showing significant differences in genetic diversity between the Northeastern, Western, and Southeastern populations of European mink, the ESU concept  and defiM. lutreola, including reintroductions and translocations, experience in the conservation of closely related endangered species, e.g., the black-footed ferret, can be used [The consequence of such reasoning is the acceptance of supplying the Western European ex situ stock with individuals from the Eastern European ex situ stock, exhibiting “better” values of the genetic diversity indicators . Additio be used ,116,117.The case of inbreeding depression is not simple and obvious—even if a loss of genetic variability is regarded as one of the major threats to European mink conservation , the lowM. lutreola originates from the Black Sea area, was formulated by Lodé [Considering the historical range extent of European mink, an interesting colonization scenario hypothesis, according to which the French population of  by Lodé . Accordi by Lodé .The potential risk of outbreeding depression (which is connected with translocations between populations) as noted previously, needs to be further investigated and assessed ,30,122. M. lutreola in the study area, preceded by a period of population insularization, led to a gradual reduction in its phenetic polymorphism, albeit without major loss of genetic diversity [Historical material, although on a limited scale covering the period 1983–2006, was included in the analysis of genetic variation conducted by Korablev et al. [M. lutreola. Nevertheless, the results indicated highly significant differences in parameters describing the genetic diversity between all three populations of European mink [For management units, since the results of the conducted research did not allow for their definition, the concept of a distinct population segment (DPS) was proposed to apply to European mink ,8,125. Aean mink ,45,49,85eN), which determines heterozygosity decreases due to genetic drift at a rate of 1/2eN per generation, and genetic neighborhood area (aN), which represents the area within which adults can randomly mate [aN was a diameter of 31.7 km, allowing for population size within the neighborhood area to range from 16.1 to 22.8 individuals [eN/N ratio, where N is the adult population size, ranged from 0.089 to 0.197 for the studied population [Important aspects for in situ conservation of wild populations are the demographic indicators that describe the conditions needed to retain genetic diversity in the population and determine the potential survival of endangered species, i.e., effective population size , preventing inbreeding and genetic erosion in ex situ stocks, and studying the genetic basis of traits that are important for reproduction in captivity [Although the abovementioned achievements of population genetics, phylogenetics, phylogeography, studies on genetic markers, identification by genetic methods, and molecular ecology of European mink focused on wild populations and their conservation in situ, captive breeding genetics addresses the issues of maintaining and developing captive stocks to preserve the species’ genetic pool. Captive (or conservation) breeding genetics can be defined as a subfield of conservation genetics that aims to provide genetic theory and techniques for conservation breeding of captive wildlife stocks before the reintroduction (repopulation) phase can be achieved. The key issues covered by captive breeding genetics for aptivity .The first conservation breeding program for European mink was launched in the Tallinn zoo (Estonia) in 1984; the European Endangered Species Program (EEP) for the species was launched by the European Association of Zoos and Aquaria (EAZA) in 1992 . The capM. lutreola is to preserve the genetic diversity of the species in captivity, as well as supply individuals for reintroduction [M. lutreola aims to maintain 97.5% of the original genetic diversity of the founders in an ex situ population for as long as possible [The conservation goal of the captive breeding programs for oduction . They aloduction ,134. Capoduction ,135. Spepossible .FGE; the number of wild-caught individuals/founders that would produce the same amount of gene diversity as does the population under study), (retained) genetic diversity , population mean kinship , mean inbreeding , and effective population size/census size ratio (eN/N). The values of the indicators for the Eastern (EEP) and Western (Spanish) European captive populations were: FGE, 7.3 and 1.18; GD, 93.2% and 57.5%; MK, 0.068 and 0.425; I, 0.079 and 0.364; and the eN/N ratios were 0.283 and 0.073, respectively [Effective population size for the Eastern ex situ population was calculated as 65.6 individuals . Maran eectively .M. lutreola individuals in captivity [Conservation breeding programs are characterized by the selection of the genetically most suitable pairs for reproduction, identified according to their genetic representation among the population to avoid inbreeding . A commoaptivity .M. lutreola in captivity may be enhanced by implementation of assisted reproductive techniques (ARTs), including semen and embryo cryopreservation, artificial insemination, and embryo transfer [Conservation breeding of transfer ,140,141.transfer ), and thtransfer . Biobanktransfer .M. lutreola in situ cannot be overestimated. Conservation genetics can provide knowledge that could be applied to reorient conservation strategies to prioritize the survival of threatened distinctive populations [M. nigripes, M. eversmanni, and M. putorius conservation breeding and reintroduction program [The role of captive breeding of European mink is of critical importance for the survival of the species. However, per Ehrlich , “the loulations . Appropr program ,148,149. program .It was claimed that genetically managed, long-term breeding programs within zoological gardens can be a source of individuals for reintroduction . HoweverM. putorius males mate with M. lutreola females [2n) in hybrids can be 39 or 40 [Two important issues addressed by conservation genetics include whether the risks of hybridization and introgression exist for European mink and, if so, the size of these risks. The close phylogenetic relationships between European mink and European polecat were proven by the lack of a complete interspecies reproductive barrier between these taxa, expressed by spontaneous hybridization in nature ,49,152. 39 or 40 ,49 and 039 or 40 , respect39 or 40 ,49,153.Identification of hybrids using genetic methods is important for captive breeding, as using hybrids in the ex situ breeding programs is not advisable . PotentiAnalyses conducted by Cabria et al.  involvedDDX3Y gene located on the Y chromosome [F1) hybrids of M. lutreola to M. putorius [Hybridization direction  was detected by direct nucleotide comparison of a sequenced 614 bp mitochondrial control region and partial sequences of introns 5 and 7 of the romosome . The obtputorius . An impoputorius . Additioputorius ,49.M. putorius furo [The possibility of crossbreeding between European mink and the ferret ius furo ,157 was ius furo  or Siberius furo  was provius furo ,159.The final reflection on the European mink conservation genetics may be a reference to the definition of this subdiscipline of genetics, namely, a reminder that the imperative, apart from preserving the natural intraspecies genetic diversity, is to ensure the continuity of evolutionary and ecological processes responsible for the formation and maintenance of this diversity .M. lutreola, advanced genomics (whole-genome and reduced-representation approaches) can help to describe and explain adaptive genetic variations, resolving phylogenetic and phylogeographic questions, identifying adaptive alleles, as well as identifying and quantifying inbreeding, hybridization and introgression in a more accurate way than genetics [The overall conclusion from the analysis of the history and scope of genetic research on European mink is a gradual increase in the genomic representativeness of the nucleotide sequences used. This trend is in line with the widespread transition from genetics to genomics, which is also  observed in conservation genetics ,161,162.genetics ,166,167.genetics ,166. Forgenetics ,169.Lycaon pictus to detect inbreeding and population-specific selection [Canis lupus lycaon [Sarcophilus harrisii. From the M. lutreola population genetics perspective, the same research demonstrated that individual heterozygosity was not associated with breeding success in captivity but was negatively associated with litter sizes of breeding females [Genomic methods considerably improved conservation efforts toward some mammalian endangered species. The whole-genome approach was previously applied to the conservation of African wild dog election . Genomicelection ,172,173.s lycaon . Wright s lycaon  identifi females .M. lutreola also needs to be further investigated, as sampling size may be a significant limitation of genomics in conservation [The correct selection of an appropriate sample size for population genetics research seems to be a neglected issue. Determination of the optimal level of sampling effort required for adequate characterization of the intraspecies genetic variation is of fundamental importance . Too manervation .In future research on the genetic diversity of the surviving European mink populations, much more attention should be paid to optimization of the sample size, as sampling many individuals per population but using a small number of genetic markers does not contribute to accurate and reliable results ,182. GenM. lutreola genetics, the following research issues remaining to be addressed and resolved are as follows:Based on the analysis of the research work completed to-date in the field of Establishing a karyotype reference standard;M. putorius GenBank assembly accession GCA_902207235.1, and a platinum quality genome available for M. erminea, RefSeq assembly accession GCF_009829155.1, could be used for reference-based assembly or designing primers for any genomic location for targeted sequencing);Initiation and completion of the whole-genome sequencing project  SNPs panel, optimal for the study of inter- and intrapopulation genetic diversity, and the species phylogeny (possible revision of taxonomic status at the genus level) and phylogeography.Research Issues Per Se:4.Resolving the issue of undertaking conservation actions (including translocations) of wild persisting populations ;5.Assessment of the impact of the breeding process on the development of traits essential for survival in the wild (adaptation to captivity) and the role of (re)introduced individuals in shaping the gene pools of wild populations ;6.Phylogeographic reconstruction in terms of origin of the French–Spanish population.Conservation Issues:7.Encouraging the scientific community to undertake research related to European mink genetics, as well as its promotion and popularization );8.Scientific cooperation and networking ;9.Development of protocols for the preservation and biobanking of the species’ genetic resources, which could be based on the extensive achievements and experience in this field with the Black-footed ferret ,117,143.Promotional and Organizational Issues:Maran et al.  listed tM. lutreola species viability in a global change scenario [The question of the potential influence of reduced genetic diversity  on the scenario ,164.M. lutreola, thereby indicating the application value of this knowledge.Just as important as experimental research are reviews and (meta-)analytical works that indicate how gained genetic knowledge can be used to solve specific problems facing the conservation of www.europeanminkcentre.org) is worth mentioning; an internet platform, acting as a digital repository, information hub, and think tank dedicated to conservation biology, including conservation genetics, of the species [M. lutreola conservation genetics [M. lutreola and implementation of genetic research results in its conservation [The initiative to establish the European Mink Centre ( species . The Cengenetics . The proervation .


Candida albicans, can display extensive ploidy variation, assessing how ploidy impacts virulence has important clinical relevance. As an opportunistic pathogen, C. albicans causes nonlethal, superficial infections in healthy individuals, but life‐threatening bloodstream infections in individuals with compromised immune function. Here, we determined how both ploidy and genetic background of C. albicans impacts virulence phenotypes in healthy and immunocompromised nematode hosts by characterizing virulence phenotypes in four near‐isogenic diploid and tetraploid pairs of strains, which included both laboratory and clinical genetic backgrounds. We found that C. albicans infections decreased host survival and negatively impacted host reproduction, and we leveraged these two measures to survey both lethal and nonlethal virulence phenotypes across the multiple C. albicans strains. In this study, we found that regardless of pathogen ploidy or genetic background, immunocompromised hosts were susceptible to fungal infection compared to healthy hosts. Furthermore, for each host context, we found a significant interaction between C. albicans genetic background and ploidy on virulence phenotypes, but no global differences between diploid and tetraploid pathogens were observed.Studying fungal virulence is often challenging and frequently depends on many contexts, including host immune status and pathogen genetic background. However, the role of ploidy has often been overlooked when studying virulence in eukaryotic pathogens. Since fungal pathogens, including the human opportunistic pathogen  Candida albicans, in healthy and immunocompromised nematode hosts. We found a significant interaction between ploidy and pathogen genetic background, regardless of host immune status.Virulence phenotypes depend on the genotype of both the host and the pathogen, but ploidy is an important, but understudied component of an organism's genotype. Therefore, we measured lethal and nonlethal virulence phenotypes of diploid and tetraploid pathogen, the yeast  The pellet was resuspended in a 5.25% bleach solution to dissolve adult hosts and select for eggs, transferred to a microcentrifuge tube and inverted for 90–120 s and subsequently centrifuged for 30 s at 440 RCF. The pellet was washed thrice with 1 ml M9 buffer and resuspended in 500 μl M9 buffer. ~100 eggs were added to uninfected or C. albicans treatment plates. 48 hr later, a single L4 nematode (×10 per treatment) was randomly selected and transferred to an uninfected or C. albicans treatment plate and incubated at 20°C. Each founder nematode was transferred to new treatment plates every 24 hr for seven consecutive days. For survival analysis, we documented whether the founder was alive, dead, or censored . For fecundity analysis, any eggs laid for each 24‐hr interval remained undisturbed on the plate and incubated at 20°C for an additional 24 hr and the number of viable progeny produced per day was scored. Total brood size is the sum of viable progeny produced over seven days. Delayed reproduction is calculated by the number of progeny produced on Day 4 or later divided by the total progeny produced for each founder nematode. All experiments were performed in triplicate or more.To synchronize host populations, 2.3C. albicans and/or E. coli and incubated at 20°C for five days in which the founder produces F1 and F2 progeny. The entire population derived from the single founder were washed with M9 buffer and transferred to 15‐ml conical tubes and brought to a final volume of 10 ml. Tubes were placed at 4°C for 1 hr to allow the nematodes to settle at the bottom. 20 μl samples were taken from each population and counted six independent times to determine the final population size for each founder nematode. All experiments were performed at least in duplicate.Lineage growth assays were performed as previously described Feistel, . In brie2.4C. albicans genetic background and ploidy or between C. albicans ploidy and host context.All statistical analyses were performed with GraphPad Prism. Survival curves were tested for significant differences using log‐rank (Mantel–Cox) tests. Lineage growth, total brood size, and delayed reproduction data sets were tested for normality using the D'Agostino & Pearson omnibus normality test. For comparisons across genetic backgrounds, Kruskal–Wallis and Dunn's multiple comparison tests were performed. For pairwise comparisons between ploidy or host context, Mann–Whitney tests were performed. Two‐way ANOVAs were performed to test for interactions between 33.1C. albicans genetic background differentially impacted host fitness. To do this, we infected C. elegans hosts with four genetically distinct strain backgrounds of C. albicans. Two of these genetic backgrounds are laboratory strains: a “laboratory heterozygous,” which consists of the SC5314 reference strain and a “laboratory homozygous,” a derivative of SC5314 in which the genome is completely homozygous. The other two genetic backgrounds are clinical strains: a “bloodstream,” isolated from a candidemia infection of a male immunosuppressed patient and an “oral/vaginal,” a pair of clinical strains isolated from a single immune competent female patient with vulvovaginal candidiasis. For each C. albicans genetic background, we assessed four measures of host fitness: host survival  whereas the other two C. albicans genetic backgrounds were not. The laboratory heterozygous strain impacted host reproductive timing significantly more than the three other pathogen genetic backgrounds. Together, these results suggest that while pathogen genetic background may not obviously contribute to host survival phenotypes, it may be important for other measures of fungal infection that are less lethal.First, we wanted to investigate whether l Figure , host lil Figure , host brl Figure , and hosl Figure . For surC. albicans genetic backgrounds is ploidy. For each genetic background, we used a related diploid strain and a tetraploid strain , brood size .Finally, we were curious if host immune status impacted the virulence relationship between each diploid‐tetraploid pair of strains. Since there are significant differences for most host fitness measures between healthy and immunocompromised hosts even when uninfected Table , we normr Figure . We alsosek‐1) host backgrounds for all four C. albicans genetic backgrounds  and tetraploid (dotted lines) in healthy (N2) and immunocompromised  is due to these allelic differences. By measuring virulence phenotypes in laboratory‐derived genomes that only differ in the number of chromosome sets they contain, we can directly assess the impact ploidy has on virulence. Here, we still observe different patterns of ploidy‐specific virulence, depending on genetic background. In the laboratory heterozygous genome, we consistently observe the tetraploid as more virulent than its diploid in both healthy  declare no competing interests.Dorian J Feistel: Conceptualization ; Data curation ; Formal analysis ; Methodology . Rema Elmostafa: Data curation ; Formal analysis . Meleah Hickman: Conceptualization ; Formal analysis ; Funding acquisition ; Supervision ; Visualization ; Writing‐original draft ; Writing‐review & editing .Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Table S6Click here for additional data file.


Quercus fabri populations is essential for the conservation and utilization of Q. fabri resources. Here, the genetic diversity and structure of 158 individuals from 13 natural populations of Quercus fabri in China were analyzed using genotyping‐by‐sequencing (GBS). A total of 459,564 high‐quality single nucleotide polymorphisms (SNPs) were obtained after filtration for subsequent analysis. Genetic structure analysis revealed that these individuals can be clustered into two groups and the structure can be explained mainly by the geographic barrier, showed gene introgression from coastal to inland areas and high mountains could significantly hinder the mutual introgression of genes. Genetic diversity analysis indicated that the individual differences within groups are greater than the differences between the two groups. These results will help us better understand the genetic backgrounds of Q. fabri.Analysis of genetic diversity and population structure among  The analysis of genetic diversity and population structure among white oak populations is essential for the conservation and utilization of white oak resources. We present a diversity study of a collection of 158 Q. Fabri individuals from 13 representative natural distribution areas in China using genotyping‐by‐sequencing (GBS). We found that the genetic structure was explained mainly by their geographic location. The genetic structure of adjacent populations was more similar, and infiltration of genetic background from coastal areas to inland areas was observed, where the hindrance of genetic infiltration by mountainous areas was obvious. Quercus plants in China are distributed all over the country, many of which are dominant tree species of zonal vegetation in subtropical evergreen broad‐leaved forests and warm temperate deciduous broad‐leaved forests in China, and they play an important role in China's forest ecosystem.Oak trees (Fagaceae) originated in China during the Eocene period   , the simplified genome sequencing technology genotyping‐by‐sequencing (GBS) has gradually come to be used to analyze the genetic evolution of multiple species . The purity of the extracted DNA was detected using a NanoDrop® spectrophotometer , and DNA electrophoresis was simultaneously performed in a 1% agarose gel to ensure DNA integrity. A Qubit™ 2.0 Fluorometer  was used to accurately measure the DNA concentration. High‐quality DNA was used for GBS library construction and sequencing.Young leaves from 158 individuals of Quercus robur reference genome, and the capture of 10w tags was used as the evaluation standard. Three different enzyme digestion protocols were selected for the evaluation, namely EcoRI and MseI, HaeII and MseI, and MspI and MseI. The results of enzyme digestion showed that the number of tags captured by HaeII and MseI was the largest and the coverage of the reference genome was higher  was greater than 50% in the read in single‐end sequencing. The selected high‐quality clean reads were aligned against the Quercus robur reference genome (https://www.ncbi.nlm.nih.gov/bioproject/PRJEB19898) using the Burrows‐Wheeler Aligner (BWA version 0.7.8‐r455) set to the default settings. The data mapping rate ranged from 84.78% to 96.19%, and the coverage 1X ranged from 9% to 15.62%. The alignment result was good, and the reference sequence was available  using GCTA (version 1.26.0) based on the high‐quality SNPs. PC1 and PC2 as well as PC1 and PC3 were plotted against each other, with individuals assigned to different sample sites.2.5K values) , and the cross‐validation error (CV error) calculated by ADMIXTURE 1.23 with the sum of the values of 10 permutations was used to evaluate the most likely number of clusters.Use ADMIXTURE 1.23 to assign each sample to the most likely cluster using different numbers of clusters (2.6A phylogenetic tree was constructed using the neighbor‐joining method in the MEGA program (version 7.0) based on a pairwise genetic distance matrix of 158 individuals, which was obtained using TreeBest (version 1.9.2) with 1,000 bootstrap replicates  and observed heterozygosity (Ho) analyses, and analysis of molecular variance (AMOVA). Expected heterozygosity could be calculated as described by Harris and DeGiorgio under Hardy–Weinberg equilibrium , and the error was lower than K = 1 and K = 3  among individuals in each group (Table p < .001).A genetic divergence was observed in the two groups and expected heterozygosity  is the probability that the alleles of two randomly selected samples are not the same and are the ratio of the number of heterozygous individuals observed to the total number of individuals in the sample. Expected heterozygosity (He) describes the expected proportion of heterozygous genotypes, which can be calculated according to the method provided by Harris and DeGiorgio  database under accession number PRJNA564867. The authors declare no conflict of interest.Shifa Xiong: Writing‐original draft (lead). Yunxiao Zhao: Data curation ; Formal analysis . Yicun Chen: Formal analysis ; Methodology . Ming Gao: Data curation ; Methodology . Liwen Wu: Funding acquisition ; Methodology ; Project administration ; Supervision ; Validation ; Writing‐review & editing . Yangdong Wang: Supervision (lead); Validation ; Writing‐review & editing .supinfoClick here for additional data file.


Prochilodus costatus in the Três Marias dam in the São Francisco River basin, Brazil, and examined the possible effects of fish stocking programs on P. costatus populations in this region. In total, 1,017 specimens were sampled from 12 natural sites and a fish stocking program, and genotyped for high‐throughput sequencing at 8 microsatellite loci. The populations presented low genetic variability, with evidence of inbreeding and the presence of only four genetic pools; three pools were observed throughout the study region, and the fourth was exclusive to one area in the Paraopeba River. Additionally, we identified high unidirectional gene flow between regions, and a preferred migratory route between the Pará River and the upper portion of the São Francisco River. The fish stocking program succeeded in transposing the genetic pools from downstream to upstream of the Três Marias dam, but, regrettably, promoted genetic homogenization in the upper São Francisco River basin. Moreover, the data show the fragility of this species at the genetic level. This monitoring strategy could be a model for the development of conservation and management measures for migratory fish populations that are consumed by humans.Fish stocking programs have been implemented to mitigate the blockage of original riverbeds by the construction of hydropower dams, which affects the natural migration of fish populations. However, this method raises concerns regarding the genetic rescue of the original populations of migratory fish species. We investigated the spatial distribution of genetic properties, such as genetic diversity, population structure, and gene flow (migration), of the Neotropical migratory fish  We investigated the genetic diversity, population structure, and gene flow (migration) of the Neotropical migratory fish Prochilodus costatus in the Três Marias dam in São Francisco River basin, Brazil. The samples showed low genetic variability, with inbreeding and the presence of only four genetic pools. The fish stocking program succeeded in transposing the genetic pools from downstream to upstream of the Três Marias dam, but, regrettably, promoted genetic homogenization in the upper São Francisco River basin. We used22.1Prochilodus costatus sampling was carried out between 2013 and 2017 upstream and downstream of the TM hydropower dam in the upper São Francisco River basin . All individuals were captured using cast nets and gillnets, and small pieces of their caudal fins were cut and stored in 70% ethanol. All individuals were taxonomically identified based on Britski, Sato, and Rosa (2) of all caudal fin samples were placed into 96‐well plates to start the genomic DNA extraction protocol . All primers used contain overhang adapters compatible with the Illumina sequencing system employed and were incorporated into the target DNA through 30 polymerase chain reaction (PCR) cycles. An index primer containing the Illumina MiSeq adapter  was attached to amplicons through 10 PCR amplification cycles, enabling locus identification for each individual using bioinformatics tools. Genotyping libraries were diluted using the Illumina buffer to attain the final concentration of 10 pMol DNA in a final volume of 1 ml. Sequencing was performed with 600 µl of the final solution using a nano kit  and the MiSeq platform (Illumina®).The NGS approach was chosen in this study due to its high performance with large numbers of samples, low cost–benefit ratio, and rapid data acquisition. NGS genotyping and treatment of data were performed following Pimentel et al. . BrieflyRaw reads with Phred < 30 and length < 75 pb were removed using Prinseq‐Lite  established a minimum of two reads to confirm the less frequent allele. In our work, we set the minimum depth at 10 reads, 20% of which would give at least two reads confirming the less frequent allele. This strategy was developed to avoid false positives. Loci that did not fulfill these criteria were identified as NA. The output of the script was a worksheet (in.xls format) containing the distribution of alleles per locus per individual.The GenotypeMicrosat.pl Perl script was developed to filter the genotyping results from RepeatSeq. We need to establish a minimum number of reads to infer the genotype. As individuals are diploids, only two alleles are possible and we establish whether they are homozygous or heterozygous according to the number of reads. Among the amplicons of a locus, if a given allele has more than 80% of reads, we consider that the individual is homozygous for that allele. If the allele with the largest number of reads does not exceed 80%, we consider the second allele, and if it encompasses ≥20% of the total reads, we consider the individual to be heterozygous. Rosa, Salvador, Bialetzki, & Santos, 2.3P. costatus populations in the upper São Francisco River basin, we estimated genetic indexes and pairwise ϴst, and performed discriminant analysis of principal components (DAPC), comparing the eight study regions. The number of alleles (A), effective number of alleles (Ae), Shannon's index (I), observed heterozygosity (Ho), expected heterozygosity (He), and fixation index (Fi) of P. costatus populations were analyzed using the GenALEx 6.5 software  was applied, and the proper genetic clusters were attributed using the Bayesian information criterion. Then, the membership probability of each individual for the different groups (K) was estimated using the dapc function based on the retained discriminant components. Two distinct combinations of the dataset were analyzed using the compoplot function: (a) an admixture model with correlated allele frequencies, which included optim.a.score information about the sampling regions to infer individuals with mixed genotypes from distinct populations; and (b) a possible hierarchical genetic structure separating regions. This multivariate analysis was implemented because it does not determine loci in Hardy–Weinberg equilibrium and does not require assumptions about the inheritance pattern of each locus . On average, the genotyping produced 109.37 reads per population for Pcos10, 79.75 reads per population for Pcos18, 207.91 reads per population for Pcos22, 347.62 reads per population for Pcos36, 23.83 reads per population for Pcos37, 108.09 reads per population for Pcos44, 92.67 reads per population for Pcos48, and 700.97 reads per population for Pcos49. Only 3% of the produced genotypes for all individuals resulted in no allele amplification [due to nonamplification or low stipulation ] and were designed as NA in the genotype. From the reads, we identified 63 alleles at the 8 loci analyzed for all P. costatus. A values ranged from 2 (ProC10 locus) to 12 (ProC49 locus), and the latter was the most polymorphic locus. Exclusive alleles were found in PAO , PA2 , ABA , and SFD .From the 5,598,163 raw reads generated, 3,294,490 reads remained after quality trimming (p < .05). Overall, the Ho was low and significantly lower than the He, for all analyzed samples and loci. The lowest Ho was found for ABA and the highest was found for PA2 . However, PAO showed low connectivity with the other study regions, despite the detection of few migrants. Furthermore, we identified unidirectional gene flow among the sampled regions, independently of the genetic index inferred  between the PA1 and SFU regions, and moderate gene flow between the SFD and the SFU regions, as well as between the ABA site and the PA1 region. High gene flow (p > .7) was identified between the COD site and the PA1 region, and between the COD site and the SFU region. Notably, we detected high gene flow between the PA2 and SFU regions, suggesting the existence of intensive genetic exchange between these populations. The gene flow between the SFD and PA2 sites was moderate. However, all genetic diversity indices and migration rates indicated low connectivity between PAO and other regions. Indeed, migration analysis detected extremely low gene flow (Nm < 0.3) among sites in the SFU region.The migration and gene flow estimates detected connectivity among all regions  ; data curation ; formal analysis (lead); funding acquisition (supporting); Investigation (lead); Methodology (lead); project administration ; resources ; software (supporting); supervision ; validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Sandra Ludwig: Conceptualization ; data curation (lead); formal analysis (lead); funding acquisition (supporting); investigation (lead); methodology ; project administration (supporting); resources (supporting); software (lead); supervision ; validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Leonardo C. Resende: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; resources ; validation ; visualization ; writing – original draft ; writing – review and editing . Pedro F. P. Brandão‐Dias: Conceptualization (supporting); formal analysis (supporting); investigation ; methodology ; resources (supporting); validation (supporting); writing – original draft (supporting); writing – review and editing (supporting). Adriana H. Pereira: Conceptualization (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting); software (supporting); writing – original draft (supporting). Nazaré L. de Abreu: Data curation (supporting); formal analysis (supporting); methodology (supporting). Izinara C. Rosse: Formal analysis (supporting); methodology (supporting); software (lead); validation ; writing – original draft (supporting); writing – review and editing (supporting). Ana P. V. Martins: Formal analysis (supporting); funding acquisition (supporting); software (supporting); writing – original draft (supporting). Susanne Facchin: Data curation (supporting); formal analysis (supporting); methodology (supporting). João de M. Lopes: Data curation (supporting); funding acquisition (supporting); investigation (supporting); project administration (supporting). Gilmar B. Santos: Data curation (supporting); funding acquisition (supporting); project administration (supporting). Carlos B. M. Alves: Data curation (supporting); investigation (supporting). Evanguedes Kalapothakis: Conceptualization ; data curation ; formal analysis (supporting); funding acquisition (lead); investigation ; methodology ; project administration (lead); resources ; software (supporting); supervision (lead); validation ; visualization ; writing – original draft ; writing – review and editing .Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Oncorhynchus mykiss that exhibits both migratory (steelhead) and non-migratory (resident rainbow) forms. While the negative effects of dams on salmonids have been extensively documented, few studies have had the opportunity to compare population genetic diversity and structure prior to and following dam removal. Here we examine the impacts of the removal of two dams on the Elwha River on the population genetics of O. mykiss. Genetic data were produced from >1200 samples collected prior to dam removal from both life history forms, and post-dam removal from steelhead. We identified three genetic clusters prior to dam removal primarily explained by isolation due to dams and natural barriers. Following dam removal, genetic structure decreased and admixture increased. Despite large O. mykiss population declines after dam construction, we did not detect shifts in population genetic diversity or allele frequencies of loci putatively involved in migratory phenotypic variation. Steelhead descendants from formerly below and above dammed populations recolonized the river rapidly after dam removal, suggesting that dam construction did not significantly reduce genetic diversity underlying O. mykiss life history strategies. These results have significant evolutionary implications for the conservation of migratory adaptive potential in O. mykiss populations above current anthropogenic barriers.Dam construction and longitudinal river habitat fragmentation disrupt important life histories and movement of aquatic species. This is especially true for  Habitat fragmentation caused by anthropogenic disturbance is one of the largest contributors to loss of species and biodiversity globally ,2. DisruFor salmonids, blockages of upstream habitat access for spawning adults and downstream access for juvenile out migration have been especially devastating consequences of dam construction . SalmoniOncorhynchus mykiss is a species that exhibits both an ocean-migrating (anadromous steelhead trout) and freshwater form (resident rainbow trout) [w trout) . While rw trout) ,24. Mechw trout) ,26, but w trout) ,28,29,30w trout) ,19,31,32w trout) ,33 and bw trout) ,35. Genow trout) ,37,38,39w trout) ,41,42.O. mykiss found a major-effect locus for migration return timing in the GREB1L gene on chromosome 28 [GREB1L gene has also been identified as a candidate for adult run timing in Chinook salmon [Steelhead also vary in temporal life history transitions including ocean migration patterns and return timing (run timing) . Steelheosome 28 ,45,46. Vk salmon ,48; howek salmon . For exak salmon . Howeverk salmon ,49,51,52O. mykiss in the Elwha River prior to dam removal identified genetic differentiation among populations separated by anadromous barriers, and natural and hatchery origin steelhead using thirteen microsatellites [O. mykiss population structure, the impacts of dam removal remain unexplored.Dams built without fish passage prevent ocean migration and restrict movement of aquatic organisms throughout the river. The construction of the impassable Elwha River and Glines Canyon dams, completed in 1913 and 1927, respectively, on the Olympic Peninsula north of Port Angeles, WA, present one such example. These dams fragmented the river into three sections: seven river kilometers (rkm) below the dams, 47 rkms in between the dams, and >70 rkms above both dams ,54. Ultitellites . While tO. mykiss before and after removal of dams on the Elwha River. In this study, we genotyped 71,320 SNPs from 567 samples of both steelhead and resident rainbow trout collected prior to dam removal and from 556 steelhead post-dam removal to investigate how steelhead are recolonizing the river. We hypothesized that if dams were the only barrier to gene flow between freshwater and anadromous life history forms of rainbow trout, then admixture should ensue following dam removal. If diverse migration and residency phenotypes existed across the Elwha River prior to dam construction, then we would expect diminished genetic and life history diversity in above-dam populations as a result of selection against the migratory phenotype. We thus expect recolonizing steelhead to be coming from populations previously restricted to below the dams that retained ocean access. Based on these initial hypotheses, we asked: (1) How does population genetic structure and diversity change following dam removal? (2) What was the source for adaptive alleles putatively involved in adult run timing (GREB1L) and anadromy (Omy5) post-dam removal? (3) What do these loci tell us about how steelhead are recolonizing the Elwha River watershed? The results from this study will provide crucial information for future decision making regarding the potential effects of dam removals for the conservation of diverse salmonid life histories.To fill this gap, we compared genetic and ecological life history diversity of Oncorhynchus mykiss before  and after  the removal of the Elwha River and Glines Canyon Dams over a fifteen-year period (2004–2019). Both life history forms (anadromous and resident) of O. mykiss were sampled prior to dam removal (before 2011), across seven sites from above the dams, seven sites from in between the dams, one site above a natural waterfall, and one site from below the dams. We were primarily interested in characterizing and assessing whether returning steelhead post-dam removal were ancestors of pre-dam Elwha River collections of O. mykiss. Post-dam removal samples included only O. mykiss sampled 2015 and onwards when removal of the Glines Canyon dam was completed. Fin clips samples collected both prior to and post-dam removal were stored in 95% ethanol or on chromatographic blotting paper and quickly air dried.Samples were taken from 1,334 Prior to removal of the first dam, the lower Elwha River dam (2012), samples were obtained from sites on the main stem of the Elwha River as well as in tributaries. In future analyses, we divided sampling sites into three main sampling locations that were separated by impassable barriers: above both of the dams (AD), in between the dams (ID), and below the dams (BD) . The SouFollowing the removal of the second dam, the upriver Glines Canyon Dam (2014), the lower (below both former dams) and middle portions (between both former dams) of the Elwha River were mainly sampled, with only three samples collected from the upper portion (formerly above the dams). Juvenile smolts were sampled in rotary screw traps from three sampling sites in 2016 and 2017: Little River (river kilometer (rkm) 0.2), Indian Creek (rkm 0.6), and on the main stem Elwha River (0.3–3.3 rkm) . Adult sO. mykiss fry and juveniles were acquired through electrofishing and smolt traps. These fish were designated as “unknown” life history phenotypes unless recaptured as adults. Juvenile O. mykiss captured in the rotary screw traps could be distinguished as smolts. Smolts were defined by length and coloration at the time of capture. During the early period of outmigration, for example, smolts still had faint parr markings. Fin edges during the early period were starting to darken, but were not fully black. In addition, the average smolt length was 165 mm, but could be as small as 87 mm and as large as 244 mm. During peak and late migration coloration was a fully silver bodied with a solid dark back and completely blackened fin edges and parr markings were very faint to completely gone.Fin clips taken from Adult steelhead sampled post-dam removal included fish that were produced from naturally spawning adults as well as fish that originated from the Lower Elwha Klallam’s Tribe integrated hatchery program . This program used eyed eggs and trout fry from naturally spawning, native steelhead in the Lower portions of the Elwha River prior to dam removal (2005–2011) from below the lower Elwha River Dam (LEKT 2012). Samples from many of these naturally spawning parents were included in the below dam, prior to dam removal, collections included in this study, as well as previous genetic studies . In total, 209 steelhead sampled post-dam removal were identified as hatchery origin, 259 were of natural origin and 88 were of unknown origin. SbfI enzyme and sonication using the QSonica Q800R  to selectively fragment the genome. RAD libraries were sequenced on an Illumina HiSeq 2500 or 3000/4000, pooling 2–3 libraries per lane, and indexed with NEBnext Multiplex i7 indices . Paired-end 100 base pair reads were obtained. After initial sequencing of pooled libraries, read counts were assessed, and in some cases, libraries were re-balanced and re-sequenced to acquire more data.DNA from 1334 fin clip samples was isolated and extracted using the Qiagen DNeasy blood and tissue extraction kit . dsDNA was quantified with Quant-iT picoGreen ds DNA Assay kit  and UV spectrophotometry. All samples were normalized to 12.5 ng/μL, and 125 ng per sample was used for restriction site-associated DNA sequencing (RADseq) library preparation, using 96 samples per library. Briefly, RADseq was performed as described in  using thSbfI site is found in both forward and reverse reads, process_radtags was run independently on these reads from each lane of sequencing, the SbfI site and barcode end identified. Data from the SbfI site were then combined into the R1 file for each sample, and the random fragmented sequence end concatenated into the R2 file for each sample. Following quality filtering, data were filtered to remove PCR clones using the clone_filter script from STACKS. Quality and clone filtered reads from each sample were aligned to the Oncorhynchus mykiss reference genome  and 71,320 SNPs  using d163495.1 . Samtool163495.1 . The res163495.1  to filte163495.1 , we used163495.1 . Post-fi320 SNPs . All furWe conducted a principal component analysis (PCA) using the R package Adegenet  to obtaiWe compared the results of two individual-based population assignment methods to determine whether the population structure that existed prior to dam removal remained after the dams were removed. Discriminant analysis of principal components (DAPC) in the R package Adgenet  uses K mO. mykiss populations temporally to determine whether there were significant changes to genetic variation post-dam removal. All analyses were run separately across all pre-dam removal populations  and post-dam removal life history cohorts separately . We calculated population genetic differentiation , Tajimaon  in VCFton , the inbreeding coefficient (FIS), and effective population size (Ne) within populations prior to dam removal and within life history cohorts post-dam removal. Average relatedness was calculated in VCFtools (Ajk [jk range is between 0 and 1) or less (Ajk range is between 0 and −1) closely related to one another on average [IS) was calculated using the R package Adegenet [e using NeEstimator v2 using the linkage disequilibrium parameterization [e which internally calculates confidence intervals, we performed non-parametric bootstrapping by resampling 10,000 samples from the original data set with replacement and generating 95% confidence intervals [Long-term conservation and management of ols (Ajk ) to dete average . The inbAdegenet  to test rization . Across ntervals .We used genetic stock identification (GSI) analysis to determine whether steelhead recolonizing the Elwha River following dam removal were produced from formerly above-dam populations. The R package RUBIAS  uses a cST estimator [ST. Since we used different individuals in each reference sampling set, we tested different SNPs with each sample set. We compared the accuracy of the two SNP panels by testing the SNPs used in the GSI analysis of the first sample set on the second sample set  and SNP set two (262 SNPs).In our GSI analysis, reference individuals were those sampled prior to dam removal with concordant assignment between population and DAPC group assignment; mixed individuals were all other samples. We additionally tested the inclusion of all individuals sampled prior to dam removal as the reference and all steelhead sampled post-dam removal were included in the mixed sample set. We thus divided individuals into two different reference and mixed sample sets hereafter referred to as sample set one and sample set two  double inversion on Omy5 with high genotyping rates across our samples and were previously shown to be associated with anadromy to look at genetic variation associated with the anadromous phenotype [GREB1L gene on Omy28 to look at genotypic variation associated with migration run timing [Finally, we investigated the distribution of adaptive genetic variation associated with henotype ,39,80. Dn timing ,47.t-test for equal means to first compare the average allele frequencies of our candidate loci between anadromy (Omy5 loci) and adult run timing (GREB1L locus) phenotypes (α = 0.01). Next, we performed generalized linear modelling in the R package lme4 [GREB1L locus allele frequencies explained run timing phenotypes. Only steelhead were included in this analysis with summer-run steelhead recoded with a value of “1” and winter-run steelhead were recoded with a value of “0”. Contingency tables implement in the Genepop package in R were also conducted to test for allelic differentiation in the Omy5 and the GREB1L loci among life history phenotypes, populations, and time periods [We used a two-sample age lme4  to test  periods .GREB1L allele frequencies prior to dam removal were correlated with migratory phenotypes following dam removal. Since we did not necessarily have accurate information regarding natal/spawning population for most steelhead collected post-dam removal, we used population assignments from the GSI to identify inferred, “ancestral” population for steelhead collected post-dam removal. Steelhead were assigned to a single reference population if they had >90% shared genetic composition. All other steelhead were excluded from this analysis. We used these data to explicitly test two hypotheses regarding the source of putatively adaptive loci involved in migration on the Elwha River.We next investigated whether the geographic structuring of Omy5 and Our first hypothesis was that if below-dam populations retained ocean access, then we should detect higher frequencies of the “A” type Omy5 alleles. To test this, we performed a generalized linear model in the R package lme4 to determine whether Omy5 allele frequency variance explained the proportion of recolonizing steelhead from a given inferred population post-dam removal.Our second hypothesis was that summer steelhead that migrate from the ocean earlier would move further upriver closer to the headwaters, therefore primarily coming from above-dam locations. To test this, we conducted an ANOVA in the R aov to determine whether populations location explained the proportion of summer alleles detected across populations.O. mykiss genome  or relatedness (Ajk) that would suggest large differences in the amount of standing genetic variation among populations (e), Tajima’s D and nucleotide diversity compared to the main stem of the Elwha River populations (jk range = 0.0047–0.0090), and effective population size (Ne range = 120.9–150.7)  .e) exhibited statistically significant differences river wide temporally  individuals sampled prior to dam removal in reference sample set one that had concordant DAPC population assignment and sampling location K = 4; . We usedSix individuals out of the 645 tested mixture samples were not assigned to any single reference sampling site with significant confidence (Posterior probability of membership < 0.90). Three of these individuals were sampled prior to dam removal from in-between-dam populations and three were sampled post-dam removal . All anaThe majority of recolonizing steelhead across all life history cohorts appeared to be descendants of collections from below the dams . Over tiOf the 567 individuals collected prior to dam removal, 88 individuals were not included in the reference sample set and tested in the mixture sample set. After removing the three individuals with low log-likelihood values , 86 sampGSI results using sample set and SNP set 1 yielded the greatest power for self-assignment comparison to the other two GSI analyses conducted. We had the higher power to self-assign reference individuals back to their respective population and sampling site using SNP set 1  comparedGREB1L loci among the genotypes of populations sampled prior to dam removal  among life history forms and populations prior to dam removal, and temporally at 11 out of the 12 loci  and at the GREB1L variant. Similarly, we observed global allelic differentiation (p < 0.001) post-dam removal among life history cohorts at 11 out of the 12 loci , and at the GREB1L variant. We also observed allelic differentiation (p < 0.001) among phenotypic forms at the GREB1L variant.We observed substantial geographic variation in the Omy5 and  removal . There w removal . The popt-test: −14.16, df = 1084.5, p < 0.001) and “R” type  Omy5 allele frequencies between steelhead and unknown O. mykiss phenotypes (p < 0.001) and was negatively correlated with the “R” Omy5 allele frequency  . These results provided support for our hypothesis that the below-dam population had the highest frequency of “A” alleles and produced the largest proportion of anadromous individuals (We identified differences in the mean “A” (two sample enotypes a. Simila= 0.024) b. Howeveividuals c. The SoGREB1L summer-run timing allele. We found differences in the mean associations of the GREB1L summer  and winter  alleles between run timing phenotypes in steelhead  . The above-dam population, in particular, had the highest frequencies of the summer-run alleles both prior to and post-dam removal  e. Furthe removal f.Oncorhynchus mykiss populations on the Elwha River within two generations following dam removal. Prior to dam removal, we observed genetic structure consistent with the findings from Winans and colleagues [GREB1L loci were associated with anadromy and adult run timing, respectively. However, the strengths of those genotypic–phenotypic associations were variable and will require further investigation over time as summer-run steelhead increasingly return to the upper watershed. Although dam construction led to declines in anadromous fish abundance, our findings suggest that the dam construction did not diminish genetic variation underlying the steelhead life history form on the Elwha River.In this study, we observed significant shifts in genetic structure in lleagues  illustraLike previous studies, we found less genetic structure within river reaches separated by anadromous barriers compared to among them ,35,83. PO. mykiss in the Russian River basin in California similarly detected increased genetic differentiation among fish separated by natural barriers rather than man-made ones [e and Tajima’s D based on life history forms and cohorts  were primarily produced from the below the dams . We obseO. mykiss upstream of Glines Canyon Dam had historically produced migrants that were seawater tolerant and apparently capable of an anadromous life history as late as the early 1990′s [Although adult steelhead cohorts lacked variation in GSI assignment of inferred population, out-migrating smolt cohorts (2016–2017) had high proportions of above- and in-between-dam genetic ancestries . With thy 1990′s . While sO. mykiss in high-elevation lakes above-dam and in-between-dam high-elevation lakes was recorded on the Elwha River prior to dam removal until 2001 [Oncorhynchus clarkii) subspecies [Across all of our GSI analyses, we did not detect more than five fish that appeared to be of non-Elwha origin . As we htil 2001 . Goldendtil 2001 . Movemenbspecies .O. mykiss and other Pacific salmonids in the Elwha River following dam construction, we did not detect significant spatial or temporal changes to population genetic variation suggestive of significant dam-mediated evolution of O. mykiss populations on the Elwha River. Indeed, recolonizing steelhead appeared to primarily be derived from Elwha populations harboring similar levels of genetic diversity and adaptive genetic variation. One reason for this may have been related to the time frame. Although the dams were in place for over 100 years, selection may not have had the time to remove the genetic variation underlying the migratory phenotypes from above-dam populations [In spite of extensive population declines in ulations ,96.O. mykiss populations in southeastern Alaska, suggesting a latitudinal limit to this genotypic–phenotypic association with anadromy [Another possibility is that the candidate loci identified in previous studies for their involvement in life history ,39,80 weanadromy .GREB1L alleles with run timing phenotypic forms  or remove these obstructions altogether . Dam removal case studies offer the unique opportunity to evaluate the effects of dam construction on species’ population genetics. Previous studies have found that populations of anadromous fish above human-made barriers, such as dams, do not necessarily have reduced genetic diversity. While these populations often experience significant population declines, maintenance of standing levels of genetic variation at loci putatively involved in anadromous phenotypes leaves the potential for rapid reestablishment of formerly dammed populations with anadromous life history forms of salmonid fishes, such as steelhead. This study characterizes the impacts of dam removal on the genetic variation of