Prnp) gene influences host susceptibility to many pathogenic prion diseases. Understanding the distribution of susceptible Prnp variants and determining factors influencing spatial genetic patterns are important components of many chronic wasting disease mitigation strategies. Here, we describe Prnp variability in white-tailed deer (Odocoileus virginianus) from the Mid-Atlantic region of the United States of America, an area with a recent history of infection and low disease incidence. This population is characterized by lower rates of polymorphism and significantly higher frequencies of the more susceptible 96GG genotype compared to previously surveyed populations. The prevalence of the most susceptible genotypes at disease-associated loci did vary among subregions, indicating that populations have innate differences in genotype-dictated susceptibility.Genetic variability in the prion protein ( Infectious diseases are important population stressors that can have significant impacts on the health of natural populations as well as their demographic and genetic trajectories ,2. EmergPrnp) gene influences susceptibility to transmissible spongiform encephalopathies [Ovis aries) and cows (Bos taurus) [Genetic variation in the prion protein  . One not taurus) ,17. Thes taurus) .Prnp gene have been consistently linked to differences in relative susceptibility for many species affected by prion diseases [Odocoileus virginianus) with a rare serine substitution at codon 96 compared to those deer that were homozygous for the wild-type protein (glycine at codon 96). Similar polymorphisms have been observed to affect susceptibility in other species where infection has occurred [Prnp gene likely influences the trajectory and outcome of disease outbreaks and likely influences predictions on population-level responses.Nonsynonymous polymorphisms in the diseases ,19,20. Adiseases  observedoccurred . Understoccurred , prevaleoccurred . Thus, gPrnp variability among cervid populations [Prnp genotypes may prove useful in understanding the relative susceptibility, progression, and outcome of chronic wasting disease epidemics in relation to other populations. Previous studies have largely been non-spatial and have focused on comparing patterns of Prnp variation among infected and uninfected individuals or characterizing the genotype frequencies of a single region [Prnp variation is important for identifying local populations that may be more susceptible to disease establishment. Given that chronic wasting disease outbreaks tend to be locally constrained, at least initially [Prnp variability may also prove more useful in characterizing and predicting local selection dynamics. Thus, a more thorough evaluation of levels of spatial heterogeneity is desirable, especially in regions where the disease is constrained in distribution and where prevalence rates within and among populations are low.Genetic variation is rarely homogeneous across landscapes. This is a trend that also seems to be true of ulations –28. Mease region ,28,29. Te region , so undenitially , measuriPrnp genotypes in an area of relatively recent infection in the Mid-Atlantic region of United States of America. We specifically focused on determining patterns of population-level susceptibility, defined as the frequency of the most susceptible Prnp genotypes, for the main species impacted by chronic wasting disease in the region (white-tailed deer). Levels of susceptibility were compared to studies conducted in regions that have a longer history of chronic wasting disease in order to determine the relative susceptibility of this region to emerging infection. Genotype frequencies were then compared among four subregions and 20 sampling localities to assess how geographic patterns of susceptibility may change across landscapes  ,33 or noPrnp gene  [F = 0.175, P’ < 0.0001) [F = 0.247, P’ = 0.0080; F = 2.468, P’ = 0.0006) [F = 2.733, P’ < 0.0001) [F = 3.566, P’ = 0.0018) [F = 6.172, P’ < 0.0001; Genotype frequencies observed in this study were compared to other regions in order to assess differences among distinct areas within the range of white-tailed deer. The proportion of the most susceptible genotype at codon 95 (95QQ) was 91.6% for the Mid-Atlantic region and was significantly lower than the frequencies of this genotype in populations from New Jersey  , Wiscons 0.0001) ,28, and  0.0006) , Wiscons 0.0001) ,28, Wyom 0.0018) , and wesF = 0.258, P’ = 0.0012; subregion two: F = 0.278, P’ = 0.0002; subregion four: F = 0.279, P’ = 0.0010). These differences were at least 20% lower than those observed in other regions . Only one individual exhibited polymorphisms at both loci (QG/HS genotype), indicating the rarity of individuals with less susceptible alleles at multiple loci. The proportion of individuals that were doubly homozygous ranged from 62.8% (n = 49) in subregion three to 84.4% (n = 146) in subregion four. Spatial interpolation still exhibited a northwest-to-southeast cline in patterns of susceptibility, although the proportion of susceptible individuals was generally lower within sampling units when loci were considered jointly .Prnp gene was less in the Mid-Atlantic region than reported in previous studies. We observed five non-silent and seven silent polymorphisms. For comparison, populations from Midwestern states and western Canada reported 14 and 15 polymorphisms, respectively [Prnp variation within this population when compared to other regions.The observed variability within the ectively ,29. ManyWe did detect novel polymorphisms at nucleotide positions 110 and 499. The change at position 110 led to a change in the amino acid sequence. This novel variant was identified in two individuals from subregion one. Given the frequency of this polymorphism within our sample and the fact that this polymorphism has not been observed in previous studies, we expect that the presence of this polymorphism in free-ranging populations is rare and is unlikely to contribute to susceptibility at the population-scale. Additionally, neither of these deer tested positive for chronic wasting disease, indicating that this polymorphism does not seem to cause a novel variant of the disease.Variability was detected at two loci consistently linked to lower susceptibility to chronic wasting disease in previous studies ,26,28,34Prnp variation is an important goal of many chronic wasting disease management strategies. One factor that has been suggested to drive underlying genetic variation is selection in favor of less susceptible genotypes [Cervus elaphus) [Prnp variability among closely-associated populations.Understanding the factors leading to enotypes ,21. Undeenotypes . This inenotypes , selectielaphus) . BecausePrnp variability. This is a trend that has previously been postulated to affect the Prnp genotype frequencies of white-tailed deer and other cervids [Prnp variability. Landscape features influence genetic connectivity in white-tailed deer and may explain regional variation in genotype frequencies [Prnp frequencies as well. Additionally, previous studies suggest that white-tailed deer also conform to a clinal pattern of genetic structure, which may be consistent with the north-to-south gradient of increasing genetic susceptibility observed in this study [Prnp genotype frequencies was somewhat unexpected, especially given the widespread nature of white-tailed deer gene flow and relative permeability of previously reported dispersal barriers [Prnp heterogeneity. Estimating gene flow and population structure in this region using neutral genetic markers is an area of ongoing research and is likely elucidate the trends observed here.Demographic factors, such as dispersal and population structure, may also affect the spatial distribution of  cervids ,26,30,37quencies . This isquencies . Thus, tis study ,41. The barriers ,42. ThisPrnp variability at disease-associated loci than previously documented for white-tailed deer populations infected with chronic wasting disease. Genotype frequencies varied among subregions and even among counties within subregions. Therefore, spatial variability in Prnp genotype frequencies seems to occur at finer scales than previously reported. Focusing on describing genotype frequencies at a regional-scale may under- or over-represent the genotype frequencies of disease-associated loci within distinct subpopulations. These results highlight the importance of measuring patterns of spatial Prnp variation, particularly in recently-infected populations. Further studies focused on understanding spatial patterns of genetic connectivity may help to resolve whether observed Prnp heterogeneity is reflective of stochastic genetic variation or is a consequence of spatial genetic structure.Attempts to manage chronic wasting disease in areas of recent emergence require an understanding of the factors influencing the susceptibility of populations. Here, we have reported lower Prnp variability. While selection may occur, it will have to be strong enough to overcome migration rates from disparate populations with potentially different disease and genetic dynamics [Prnp variability as a management tool [Continued and long-term genotyping efforts in this region and others recently infected by chronic wasting disease may provide novel insights into the factors that influence prion-disease susceptibility in cervids and other species. Selection dynamics for prion diseases are complex and there is no consensus on the relative importance of balancing versus purifying selection for these or similar loci . There adynamics . Finallyent tool ,28. Trac2 in three Mid-Atlantic states between 2013 and 2017  [Prnp open-reading frame and do not amplify the Prnp pseudogene [2O, 2.00 µL of 20 ng/µL DNA template. All PCRs were carried out using the following protocol: 95°C for 15 minutes, 35x cycles , with a final extension at 72°C for 10 minutes. Successful PCR amplifications were purified using an Exonuclease 1/Shrimp Alkaline Phosphatase cleaning procedure. Purification mixes consisted of 0.58 µL of Exonuclease 1 , 0.58 µL of Shrimp Alkaline Phosphatase , 8.84 µL deionized H2O, and 10 µL of PCR amplicon. The following thermocycler protocol was applied to Exonuclease 1/Shrimp Alkaline Phosphatase purification: 37°C for 30 minutes and 80°C for 15 minutes. Sanger sequencing was performed on an Applied Biosystems 3730 sequencing platform (Applied Biosystems) at the Penn State Genomics Core Facility.Genomic DNA was extracted using the QIAGEN DNeasy blood and tissue extraction protocol (QIAGEN). We used a previously described primer set that encompasses the open reading frame of the on (PCR) . The foreudogene . PolymerPrnp sequence reads were scored and analyzed using the CWDPRNP R package [All  package . The CWD package . All seq package . We filt package ,29,38,37Prnp gene and estimated genotype and allele frequencies of SNP loci. We translated nucleotide sequences into their respective amino acid sequence as disease-associated loci have been typically linked to nonsynonymous base substitutions and are more commonly discussed by their amino acid designation [Prnp variability. Confirmation to Hardy-Weinberg expectations was evaluated for codons 95 and 96. Hardy-Weinberg tests were carried out in Genepop (version 4.6) [We identified all variable sites within the ignation ,26,28,37ion 4.6) ,48 with ion 4.6) ,50. BecaP value designated P’). Genotype frequencies were also compared among subregions using the same procedure to determine whether specific subpopulations existed that were more susceptible to chronic wasting disease invasion and establishment.We compared sample-wide genotype frequencies to those reported from previous studies to determine whether genotype frequencies differed significantly from those previously observed. Results from six previous studies were used for comparisons ,29,35,36Prion gene variation was mapped throughout the region to visualize patterns of population susceptibility. Genotype frequencies were mapped to the centroid of each sampled county. Inverse-distance weighting interpolation was used to create a two-dimensional surface of the expected frequency of the most susceptible genotype across the region. The power parameter was set to three and seemed to appropriately balance weighting of more distant points (avoided concentric ‘bulls-eye’ pattern) and close-by points (appropriately smooths the surface and avoids tessellation).


Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and the evolutionary history of admixture that produced these hybrid populations remain unclear. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterized as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids based on whole-genome sequences. We propose that these hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of data that shows how recombination, and gene flow, shape natural populations of Leishmania.Parasites of the genus  Leishmania. It is transmitted by sand fly insect vectors and is responsible for an enormous burden of human suffering. In this manuscript we examine Leishmania isolates from Ethiopia that cause the most serious form of the disease, namely visceral leishmaniasis, which is usually fatal without treatment. Historically the general view was that such parasites reproduce clonally, so that their progeny are genetically identical to the founding cells. This view has changed over time and it is increasingly clear that recombination between genetically different Leishmania parasites occurs. The implication is that new biological traits such as virulence, resistance to drug treatments or the ability to infect new species of sand fly could emerge. The frequency and underlying mechanism of such recombination in natural isolates is poorly understood. Here we perform a detailed whole genome analysis on a cohort of hybrid isolates from Ethiopia together with their potential parents to assess the genetic nature of hybrids in more detail. Results reveal a complex pattern of mating and inbreeding indicative of multiple mating events that has likely shaped the epidemiology of the disease agent. We also show that some hybrids have very different relative amounts of DNA (polysomy) the implications of which are discussed. Together the results contribute to a fuller understanding of the nature of genetic recombination in natural populations of Leishmania.Leishmaniasis is a spectrum of diseases caused by the protozoan parasite  Leishmania is a diverse genus of kinetoplastid protozoan parasites from the family Trypanosomatidae. These parasites are best known as the cause of human and animal leishmaniasis, which is a clinically important neglected tropical disease affecting millions of people and causing a tremendous burden of mortality and morbidity [Leishmania have a digenetic (two host) life cycle involving a vertebrate host and 166 different species of phlebotomine sand fly that have been implicated as vectors [Leishmania have been reported to infect humans [orbidity ,2. Leishorbidity . Leishma vectors , althoug vectors . Vertebrt humans .Leishmania, other trypanosomatids and indeed most protozoan parasites was considered to be largely clonal [Leishmania species [Leishmania isolates have surprisingly little heterozygosity, which has been widely ascribed to extensive selfing [Leishmania species.Historically, the population structure of y clonal : the prey clonal . Subsequ species –10. Most selfing –13, alth selfing . In cont selfing –17 and e selfing ,19, suggLeishmania species have been achieved in the sand fly vectors [L. major [L. major/L. infantum crosses have also been performed with segregation of cutaneous and visceral traits [Leishmania have not been described, although haploid stages of Trypanosoma brucei have recently been discovered in tsetse flies [Laboratory genetic crosses between at least two  vectors ,21, and L. major . Here mal traits . Howeverse flies .Leishmania can undergo genetic exchange is of profound epidemiological importance. Genetic exchange may facilitate adaptation to new vectors, mammalian hosts or other ecological niches. For example, Leishmania infantum/major hybrids infect Phlebotomus papatasi, a non-permissive vector for L. infantum that is widespread in the Indian subcontinent [L. braziliensis and L. peruviana have also been implicated as agents of destructive forms of mucocutaneous leishmaniasis [The fact that ontinent . Hybridsmaniasis . GeneticLeishmania species [Leishmania from Turkey [L. donovani species complex. One of the parents appeared to be L. infantum, but the precise parentage of this population remains unclear as no putative parental genotypes were isolated in the same region. While genomic patterns were consistent with meiosis they do not formally exclude the possibility of a parasexual process.Genome-wide sequence data are crucial to explore fully the extent of hybridisation and to identify the mechanisms by which hybrids are formed . Data fr species , where mm Turkey . This poL. donovani originating from Ethiopia. East African strains of L. donovani are particularly diverse, consisting of two main populations: one comprising strains from northern Ethiopia and Sudan, the other strains from southern Ethiopia and Kenya [Phlebotomus orientalis is the main vector in northern Ethiopia and Sudan and Phlebotomus martini in the South, although other vectors have also been implicated [L. donovani in Ethiopia also differ in clinical phenotypes [L. donovani in isolates from the northern population. Here, we apply whole-genome sequencing data to characterise more fully these Ethiopian L. donovani to confirm that isolates are true hybrids that originate from recombination between two different sympatric lineages. We reveal a complex pattern of inheritance implying multiple independent origins from similar parents, and backcrossing with parental types. Extensive polysomy, at the level of chromosomes, is apparent in some hybrids, the significance of which is discussed.Here we present a detailed genomic analysis of a natural hybrid population of nd Kenya  and corrplicated . These tenotypes . High inenotypes  and MLSTenotypes  have conL. donovani  these data produced at least 40-fold median coverage across the isolates. Generally, coverage was consistent across the genome, with more than 30 reads per sample covering at least 90% of the genome   were gene genome . SNP cale genome . In conte genome , with moe genome , S1 Fig.L. donovani isolate from an Ethiopian VL patient; JPCM5, a Spanish canine L. infantum, and BPK282 from a Nepalese VL case) revealed the diversity present in this Ethiopian cohort and their distant relationship to both L. donovani in the Indian subcontinent and L. infantum , while DM299 was strikingly polysomic. For this isolate, allele frequency data suggested a minimum of tetrasomy across chromosomes, with half the chromosomes inferred at even higher dosage .Chromosome copy number for each isolate was inferred from read depth and allele frequencies at heterozygous sites see , Fig 2. in vitro passages was analysed. DNA content of these cells was suggestive of diploidy . The difference in allele frequency distribution between DM62 and DM299 is clear: for example, chromosomes 11, 12 and 33 all show a clear peak in allele frequencies close to 0.5 in DM62 suggesting disomy (or at least an even chromosome dosage), but peaks at 0.33 and 0.67 in DM299, suggesting a higher dosage of at least 3 copies . The verdiploidy , leavingLeishmania populations. Indeed, when we applied this approach to our data, neither the recommended cross-validation nor examination of how the likelihood of the data changed with different assumptions about the number of ancestral populations (K) revealed a clear optimum value for the number of ancestral populations for our data  and used these as markers to identify the likely origin of variants identified in the hybrids, effectively ‘painting’ the hybrid isolate chromosomes by their likely ancestry under the hypothesis that these isolates originated as hybrids between the parental groups or close relatives. We identified a total of 49,835 such ‘parent-distinguishing SNP’ sites at which the two parental populations were completely fixed for different alleles. These sites are distributed across all chromosomes. For the vast majority of sites, across all putative hybrid isolates, genotypes consisted of a combination of these parental alleles , supportTo get a more quantitative understanding of the distribution of these parent distinguishing SNP sites in blocks inherited from each parent we developed a hidden Markov model (HMM) to assign putative ancestry for sites without parent-distinguishing SNPs. The HMM assigns every 100 bp window of the genome to three states: either homozygous ‘parent A’, homozygous ‘parent B’ or heterozygous . The advWe used reads and read pairs to phase locally heterozygous sites that were physically close to each other in the genome into sets of variants known to be present on a single haplotype. We subsequently identified regions at which all heterozygous positions were phased in all 15 isolates, so that we have unambiguous information about the haplotypes present in these regions. We inferred haplotype phylogenies for nine such regions that were at least 3 kb long and had an average of at least 4 heterozygous sites per isolate; this included 9 of the 16 ‘fully phased’ regions of 3 kb or longer . All 9 bIn most blocks  possess hybrid genotypes that are phylogenetically intermediate between these two parental groups. On both the tree and PCA, these are not equidistant from the two parental groups, suggesting a greater contribution of the second group of parental isolates (parent B) to these putative hybrid genotypes. Admixture analysis supports the interpretation of these intermediate genotypes as hybrids. Most blocks of variants in these genomes can be interpreted as being inherited from one, or both of the parental populations. The large-scale structure of these genomes as blocks of variants of particular ancestry suggests that these are relatively recent events, at least in terms of the number of crossing-over events that have occurred since. There are however substantial differences between the hybrids in that the number of variants shared by individual hybrid isolates and the parentals, and also their distribution across the genomes differs substantially. The structural variation we identify does not appear to support a hybridisation scenario, but the many shared variants probably represent differences between both Ethiopian populations and the reference genome assembly rather than genuine variation within these populations. It is clear that structural variation appears rapidly during in vitro culture of Leishmania, so we cannot be sure that all of the variants we detect are present in natural populations rather than being an artefact of promastigote culture [In more detail, our results are indicative of complex  culture –41. In tLeishmania seems challenging, given the facultative nature of sexuality in this genus, and our current lack of understanding of recombination in Leishmania. In particular, we do not possess quantitative information on the mutation rate, recombination rate or even likely generation time of Leishmania in vivo. In all cases a greater proportion of the genomic variation was shared with the ‘parent A’ population, suggesting a more recent common ancestry with this population or backcrossing with this population during evolutionary history. The close relatedness between the LV9 isolate, isolated in the Humera district of Ethiopia in 1967, and one of the parental groups indicates that parental like genotypes have been present in this region for extended periods of time. While the age of the hybridisation events we have described are unclear, the presence of genotypically stable “parental donors” over time may have facilitated the emergence of multiple hybrid populations. In summary, while we cannot reconstruct the precise history of this population, our data confirm that the population of L. donovani in Ethiopia has undergone multiple rounds of hybridisation, including more complex patterns of crossing than simple F1 hybridisation between parents or subsequent crossing within a hybrid population.Results are indicative of at least three different histories of crossing between parental types, and probably within the hybrid population itself. Here with DM299 and DM62 (isolated from the same patient) showing very similar patterns probably arising from the same isolate, while both DM19 and DM295 are distinct with different histories. Using a hidden Markov model, we estimated the lengths of blocks of sequence inferred to originate from each parental population. The distribution of sizes of contiguous sequence fragments derived from each parent was not consistent between the hybrids, probably reflecting differences in the timing of hybridization events; we expect that continued interbreeding and the accompanying crossing-over gradually broke down these blocks of ancestry. Hence, blocks inherited from parents in older crossing events have smaller stretches of continuity. Our HMM results thus gives qualitative insight into the relative age of the different events that gave rise to these hybrid isolates. Using this approach, we infer that DM62 and DM299 have crossed onto a parent B like ancestor more recently than to parent A, while the ancestry of DM19 and DM295 from both parents is approximately equally old. This approach of assessing length of inherited haplotypes has been previously used to gain quantitative results into the history of human populations , but parL. donovani that has previously been identified within this region [Isolate DM481 emerged as a consistent outlier that forms a distinct lineage, intermediate between the two parental groups but also distinct from the other four putative hybrid isolates. The presence of haplotypes at a number of loci in this isolate which are not present in either parental population makes it seem unlikely that this is a very recent hybrid between the two parental groups in the current cohort. One explanation is that DM481 is simply a representative of a distinct, divergent population, which appears plausible considering the genetic diversity within s region ,33,35. AL. donovani populations transmitted by P. orientalis, and that resulting hybrid progeny may be widely disseminated. This complex evolution also implies that co-infection of P. orientalis with different L. donovani isolates occurs frequently, at least in this region. It is currently unknown if hybrids are most likely to emerge when a sand fly is co-infected with different Leishmania strains ingested in the same blood meal or subsequent feeds. For T. brucei there is evidence that the production of hybrid genotypes is most successful when both parental types are taken up in a single meal by the tsetse vector [The multiplicity of hybrid and parental genotypes within this small sample of isolates from northern Ethiopia suggests that genetic exchange is commonplace among e vector .Leishmania promastigotes described both experimentally [L. donovani in the Indian subcontinent. Indeed, aneuploidy patterns do not seem to strongly segregate between Ethiopian L. donovani populations [Saccharomyces and Candida, to rapidly generate adaptive diversity [Leishmania [Leishmania promastigotes [Leishmania lacks classical regulation of transcription at initiation through promoters, this could contribute to parasite adaptation to at least some conditions [in vitro or in vivo: while it is clear at least some variation does occur in both [in vitro culture.The pattern of polysomy we observe across the cohort did not reflect the phylogenetic relationship between isolates or their assignment to hybrid or parental classes; this is consistent with a highly dynamic chromosome complement in mentally  and in fulations  despite iversity , and likishmania ,47. Aneustigotes ,49. As Lnditions ,46. Howe in both ,50 it apLeishmania can vary dramatically and rapidly in culture [in vitro culture is known to systematically reduce ploidy in experimentally derived T. cruzi [Leishmania [A striking result is that the sequence data suggests that isolate DM299, a recrudescent infection (from DM62) taken from the same patient was remarkably polysomic across all chromosomes relative to other isolates. Previous flow cytometry measurements of DNA content were suggestive of diploidy for both strains DM62 and DM299 and all other strains in the cohort  and are  culture ,51 and pT. cruzi . Here, rT. cruzi . HoweverT. cruzi ,48,54. Tishmania  and concishmania ,56.T. cruzi [Broadly, mitochondrial phylogenies corresponded to the expected nuclear genotypes  was isolated from a human VL case in Nepal in 2003. Illumina whole-genome data for these isolates were obtained from the ENA database, with parasite material, sequencing approach and analysis of these data detailed in [DNA library preparation was performed by shearing genomic DNA into 400–600 base pair fragments by focused ultrasonication , standard multiplex Illumina libraries were prepared using the NEBNext DNA Library Kit. The libraries were amplified with 8 cycles of PCR using Kapa HiFi DNA polymerase’ and were then pooled. 100bp paired-end reads were generated on the Illumina HiSeq 2000 v3 according to the manufacturer’s standard sequencing protocol. All sequencing data for these isolates are available from the European Nucleotide Archive under project ERP106107. The LV9 strain  was originally isolated from a VL case in the Humera district in the far North of Ethiopia in 1967 . The JPCailed in  for BPK2L. donovani LV9 reference assembly using SMALT v0.7.0.1 [-x flag). Variants were called using the HaplotypeCaller algorithm of Genome Analysis Toolkit v3.4 [Reads for each isolate were mapped to the v0.7.0.1 , indexinv0.7.0.1  and mappkit v3.4 , followikit v3.4  except akit v3.4  to identkit v3.4 , and valde novo from read mapping data rather than using the variant calls, and reconstructs at most two haplotypes at any locus. Phylogenies for the inferred haplotypes were generated using raxmlHPC v8.2.8 [2 > 0.25.The whole-genome phylogeny and principal components analysis presented here were generated by using VCFtools v0.1.15  to conveC v8.2.8  under a C v8.2.8  with a sC v8.2.8 ,74 so thParent-distinguishing sites were identified as those for which both parent A isolates shared an identical homozygous genotype and all four parent B isolates were homozygous for a different allele. These sites could be unambiguously assigned as being derived from one or other parent in the putative hybrid isolates, assuming these other isolates were hybrids of these parents. To extend this analysis to other sites across the genome, a Hidden Markov model (HMM) was used to classify every 100bp window along the genome of the 5 suspected hybrid isolates by likely ancestry. Three hidden ancestry states  were used to explain the pattern across the genome of 4 observed parent-distinguishing SNP “symbols” . The 100bp window size was chosen to make the HMM computationally tractable and so that almost every window  was unambiguous for the observed symbol. All transitions between hidden states were allowed, but each hidden state could emit only the corresponding observation or the non-determinate symbol. Initial transition and emission probabilities and trained parameters are shown in L. tarentolae [L. tarentolae maxicircle is 20,992bp), including the entire transcribed region: tests using less strict criteria for assembly produced longer but less reliable assemblies. The assembled contigs were then rotated using CSA [kDNA maxicircle genome sequences were generated by mapping illumina sequence data against the available maxicircle sequence assembly for rentolae  and usinrentolae  to perfosing CSA  before asing CSA  with autsing CSA ,80. A masing CSA  with 10 S1 Table(DOCX)Click here for additional data file.S2 TableInitial transition (a) and emission (b) probability matrix and trained transition (c) and emission (d) probabilities for HMM. NA represents “Not Allowed” emissions from that state.(DOCX)Click here for additional data file.S1 FigWhite bars represent homozygous reference genotypes, dark grey homozygous non-reference genotypes and the intermediate shade heterozygous positions. Purple bars represent low-quality calls where genotype is uncertain.(EPS)Click here for additional data file.S2 Figk). (C) Maximum-likelihood ancestry proportions for each isolate under a model with k = 2. Colors of bars represent two different hypothetical ancestral populations, colors of isolate labels on X-axis represent parental and proposed hybrid populations of isolate, as shown in (A) Five-fold cross-validation error and (B) Likelihood of the data for models with different numbers of ancestral populations ((EPS)Click here for additional data file.


The Anthropocene has witnessed catastrophic amphibian declines across the globe. A multitude of new, primarily human‐induced drivers of decline may lead to extinction, but can also push species onto novel evolutionary trajectories. If these are recognized by amphibian biologists, they can be engaged in conservation actions. Here, we summarize how principles stemming from evolutionary concepts have been applied for conservation purposes, and address emerging ideas at the vanguard of amphibian conservation science. In particular, we examine the consequences of increased drift and inbreeding in small populations and their implications for practical conservation. We then review studies of connectivity between populations at the landscape level, which have emphasized the limiting influence of anthropogenic structures and degraded habitat on genetic cohesion. The rapid pace of environmental changes leads to the central question of whether amphibian populations can cope either by adapting to new conditions or by shifting their ranges. We gloomily conclude that extinction seems far more likely than adaptation or range shifts for most species. That said, conservation strategies employing evolutionary principles, such as selective breeding, introduction of adaptive variants through translocations, ecosystem interventions aimed at decreasing phenotype–environment mismatch, or genetic engineering, may effectively counter amphibian decline in some areas or for some species. The spread of invasive species and infectious diseases has often had disastrous consequences, but has also provided some premier examples of rapid evolution with conservation implications. Much can be done in terms of setting aside valuable amphibian habitat that should encompass both natural and agricultural areas, as well as designing protected areas to maximize the phylogenetic and functional diversity of the amphibian community. We conclude that an explicit consideration and application of evolutionary principles, although certainly not a silver bullet, should increase effectiveness of amphibian conservation in both the short and long term. ApproxiExtant amphibians live in environments exposed to a multitude of primarily human‐induced drivers of decline. These drivers have been exhaustively reviewed elsewhere  into the world's air, soil, and particularly water supply alters development and physiology of amphibians and increases mortality. Furthermore, pollution can compromise host immune response or its protective microbiome, increasing pathogen virulence  involving reduced susceptibility to the frog‐killing chytrid fungus  is usually lower than census size and depends on sex ratio among breeders, variance in reproductive success between individuals, and, crucially, population size fluctuations across generations. Probably most relevant for conservation is contemporary, current, or recent  Ne of individual populations. These Ne estimates in amphibians usually go in tens, and only rarely in hundreds or thousands of individuals  effective sizes of a number of populations connected by migration can be inferred from DNA sequences sampled from a single population. The distinction between different aspects and timescales of Ne is well understood in the population genetic literature, but not sufficiently appreciated in the conservation context. Thus, paradoxically, a highly subdivided species harboring as a whole substantial genetic variation and characterized by large species‐wide Ne can actually consist of very small populations on the verge of extinction.Modest contemporary Hyla arborea), larval fitness in small fragmented populations suffered from fixation load but HFCs were undetectable  in the field but not in the laboratory  , with contemporary Ne of several tens of individuals, still retains substantial microsatellite variation  occurrence in Oviedo, Spain, showed that despite isolation for hundreds of generations, populations in the city center still harbor substantial genetic variation   population in the UK due to the inadvertent release of tadpoles from another population.A potentially highly effective conservation intervention alleviating the adverse genetic effects of reduced Lithobates sevosus). The implementation of intense conservation efforts, including a headstarting program (raising hatchlings or juveniles in captivity and then releasing them into the wild) in 2002, resulted in an increase in Ne, as assessed using microsatellite markers, over a 17‐year period  in amphibians is quantitative PCR (qPCR) which has eliminated the need for laborious and unreliable histological examinations of amphibian skin  populations in France  exemplifies the power of genome‐wide data in detecting effects of anthropogenic fragmentation  into similar habitat (grassland) does not seem to decrease population connectivity or genetic diversity (measured as microsatellite variation) in the new populations; in contrast, expansion into novel habitat (desert) resulted in strong population differentiation, likely the result of bottlenecks and lack of connectivity  from nearby donor populations consistently failed to establish at a receptor site, whereas a single translocation from a distant site with different environmental characteristics became established and eventually thrived. The authors suggest that success was determined by the large size of the distant donor population, and by its higher mean fitness or more adaptive variation than the local but smaller donor sites. After 10 years, the receptor population retained allele frequency distributions at microsatellite loci similar to the donor site, but intriguingly MHC allele frequencies shifted to resemble local ones. This study implies that the risks of losing local adaptation can be offset if the introduced individuals originate from large populations because of a strong correlation between population size and fitness or adaptive variation in amphibians. Moreover, local selection acting on some loci of the genome can be very rapid (~3 toad generations), and these loci may often be involved in disease resistance. At a practical level, this study adds weight to the suggestion that the release of many individuals  increases the chances of success of a translocation project . This, A leading amphibian example for rapid adaptation to temperature change is provided by studies on the wood frog demonstrating shifts in thermal tolerance and preference as well as temperature‐specific developmental rate  and B. salamandrivorans . The adaptive potential to overcome the pathogen has been demonstrated for some amphibian populations , even though captive assurance programs outnumber programs aimed at reintroductions  found that more than eight generations in captivity negatively affected not only genetic variation, but also a trait clearly related to fitness—induced antipredator defense  is likely to increase  consequences of hybridization and we focus on them here.5.1Incilius nebulifer) and Fowler's toad (Anaxyrus fowleri) has contributed to a decline of the latter due to strong postzygotic isolation  and the green frog (L. clamitans) puts into question the distinctiveness of the former   may have facilitated the expansion of plains spadefoot toads into novel habitat in the southwestern United States  and southern chorus frog (P. nigrita), reinforcement has led to the evolution of both female preferences and male signals, resulting in increased prezygotic isolation countering maladaptive hybridization  have undisputed and significant ecological impacts and have generated the most scientific interest  are also subclinical carriers of deadly amphibian pathogens and aid in their spread and maintenance in the environment , the natural range of which encompasses Central and tropical South America. This species has been intentionally introduced into other tropical regions, most notably into northern Australia and many Pacific islands. All life stages of this toad produce toxic substances, and lethal ingestion is the primary pathway of direct impact on native species. In Australia, the range of this species has expanded rapidly over the last eight decades  in France  in South Africa has evolved a suite of behavioral, phenotypic, and reproductive traits that allow it to better cope with the drier conditions in its new range. However, it is not yet possible to distinguish whether these shifts represent local adaptation or phenotypic plasticity and thus should be viewed as responses lowering phenotypic mismatches between invasive species and their novel environments.In another toad example  have detrimental effects on native amphibian species  in southern South Africa, finding that all new populations were most likely the result of human transport and thus should be considered alien and potentially invasive.Identifying the provenance of extralimital amphibian populations is important for prioritizing conservation actions (do we eradicate or protect?) but can be tricky. Importantly, the contrasting population genetic hallmarks of expansions in human‐mediated versus landscape‐ or climate‐mediated range expansions can be put to work for this purpose. For instance, Tolley, Davies, and Chown  pitted jResearch on several species showed that the phenotypes of invasive amphibians may undergo rapid optimization in their new habitats which, coupled with increased dispersal ability, may render invasive populations unmanageable. Unfortunately, after establishment, alien‐species naturalizations are usually irreversible. Novel evolutionary measures against invasive amphibian populations have been proposed. One as yet untested idea to counter the hyperdispersive toads leading the cane toad expansion in northwestern Australia is to introduce individuals of lower dispersal capacity (from established populations) to the invasion front. It is thought that this “genetic backburning” strategy  are tolerant to trematode infection during larval development and resistant after metamorphosis  from CBP into suitable habitat in which Bd is present with the expectation that natural selection will eventually generate a tolerant or resistant variant , Craugastor (Craugastoridae), Pristimantis (Strabomantidae), and the Neotropical salamanders Bolitoglossa (Plethodontidae), are being disproportionately affected by habitat conversion. These lineages are typically species‐rich and share some life‐history and ecological traits; for instance, many species have lost the free‐swimming larval stage, have small body sizes, and inhabit pristine rainforest. Their survival hinges on the protection of large swathes of rainforest in countries that are undergoing human population expansion and rapid development. Nowakowski et al. (Fejervarya (Dicroglossidae) that inhabit rice paddies, Dendropsophus treefrogs (Hylidae) that live in deforested areas of the Neotropics, and some members of the large families Leptodactylidae, Ranidae, and Bufonidae.Another study that used phylogenetic diversity measures as a basis for conservation guidelines , and one is now available for amphibians  revive threatened amphibians, especially the evolutionary unique, specialist species? There is some evidence that this indeed may be the case. In a study examining the effects of habitat restoration in southern Mexico, Díaz‐García, Pineda, López‐Barrera, and Moreno  showed t9It is abundantly clear from our review that amphibian populations can evolve to accommodate new environmental situations. However, the evolutionary response may not be rapid enough to cope with the pace of current global change. Collectively, the field has amassed a substantial amount of information on evolutionary processes in amphibians and we have a reasonable idea of what to protect and how to prioritize protection in the case of limited resources. However, there is a dearth of studies that have attempted to implement evolutionary principles in or that are directly informative for amphibian conservation. Table Ne and minimizing unwanted adaptation, selective breeding, and targeted gene flow for specific traits or adaptive variants , translocations of more fit individuals to populations experiencing fitness declines, and interspecific hybridization as a source of adaptive variation. Few of these tools have actually been implemented in amphibian conservation, and all are in need of evaluation. We also raise awareness of the possibility that the implementation of evolutionary solutions to amphibian conservation may have ecological implications for the local communities. Most obviously, actions that directly modify habitat such as breeding site restoration (aimed at securing population connectivity) or establishment of thermal refugia (to mitigate disease) will likewise affect the local ecology. However, translocations of individuals or genes may also seriously and unpredictably alter local associations. Ideally, the potential conflict between an evolutionary solution for an imperiled amphibian species and the ecological outcome of the action should be acknowledged and factored into a conservation strategy.Can evolutionary principles aid in halting amphibian declines? Yes, by securing the adaptive potential of (declining) amphibian species, by optimizing habitat management to ensure landscape‐scale population dynamics, by reducing phenotype–environment mismatch, and by recognizing and spreading fitness‐increasing variants. A number of conservation tools have been proposed, including evolutionary approaches to captive breeding aimed at increasing Protecting the existing primary habitat patches and increasing their quality or extent is a direct conservation measure of immediate effect and is essential for many nongeneralist species. However, as we watch most pristine ecosystems, especially tropical forests, disappear, secondary habitat in disturbed areas or restored forest is becoming increasingly important for many amphibian species. An evolutionary perspective incorporating phylogenetic and functional diversity is essential in the planning of the secondary habitat, and management approaches manipulating the environment to create climatic refuges from disease could become valid approaches in a more homogenous landscape.None declared.


Studies examining genetic conditions common in Latin America are highly underrepresented in the scientific literature. Understanding of the population structure is limited, particularly Chile, in part due to the lack of available population specific data. An important first-step in elucidating disease mechanisms in Latin America countries is to understand the genetic structure of isolated populations. Robinson Crusoe Island (RCI) is a small land mass off the coast of Chile. The current population of over 900 inhabitants are primarily descended from a small number of founders who colonized the island in the late 1800s. Extensive genealogical records can trace the ancestry of almost the entire population. We perform a comprehensive genetic analysis to investigate the ancestry of the island population, examining ancestral mitochondrial and Y chromosome haplogroups, as well as autosomal admixture. Mitochondrial and Y chromosome haplogroups indicated a substantial European genetic contribution to the current RCI population. Analysis of the mitochondrial haplogroups found in the present-day population revealed that 79.1% of islanders carried European haplogroups, compared to 60.0% of the mainland Chilean controls from Santiago. Both groups showed a substantially lower contribution of indigenous haplogroups than expected. Analysis of the Y chromosome haplogroups also showed predominantly European haplogroups detected in 92.3% of male islanders and 86.7% of mainland Chilean controls. Using the near-complete genealogical data collected from the RCI population, we successfully inferred the ancestral haplogroups of 16/23 founder individuals, revealing genetic ancestry from Northern and Southern Europe. As mitochondrial and Y investigations only provide information for direct maternal and paternal lineages, we expanded this to investigate genetic admixture using the autosomes. Admixture analysis identified substantial indigenous genetic admixture in the RCI population (46.9%), higher than that found in the Santiago mainland Chilean controls (43.4%), but lower than a more representative Chilean population (Chile_GRU) (49.1%). Our study revealed the Robinson Crusoe Island population show a substantial genetic contribution for indigenous Chileans, similar to the level reported in mainland Chileans. However, direct maternal and paternal haplogroup analysis revealed strong European genetic contributions consistent with the history of the Island. ASGR1 gene that are associated with a reduced risk of coronary heart disease in the well-studied Icelandic population  is the only permanently inhabited island within the Juan Fernandez Archipelago, located 670 km due east of San Tiago, Chile. Originally named Más a Tierra (Closer to Land) it is the second largest island of the archipelago, after Más Afuera (Farther Out) . The arcThroughout the 17th to 19th centuries, Robinson Crusoe Island was frequently used as a stopping point by buccaneers seeking refuge on their voyage around the Cape Horn, the southernmost tip of Chile, the Wollaston Islands ancestry . VillanuNFXL1 conferring an increased risk of language disorder and explaining 17% of the trait variance found on RCI . All subjects and/or their parents, where applicable, gave written informed consent, in accordance with the Declaration of Helsinki.Genomic DNA samples were collected from 163 residents from RCI and 30 Chilean controls (referred to as Chilean) consisting of 15 male university students and 15 female adult controls residing in the Santiago area. DNA extractions were performed using a standard chloroform extraction protocol from EDTA whole blood samples .2, supplemented with a custom array designed to cover South American-specific variants. Standard quality control procedures were completed within PLINK v1.90b4.4 1.90b4.4 , during N = 81) were excluded from further analysis . Maternal ancestral haplotypes  were estimated using 180 mitochondrial SNPs using Haplogrep2  from 270 SNPs contained on the Y chromosome  using Y-fitter  (plogrep2 . Y chromY-fitter . Y-fitteN = 40), Iberian (IBS) (N = 14), and African Yoruba (YRI) (N = 40) control population datasets obtained from 1000 Genomes3. An additional Chilean control population (N = 190) containing individuals from across a range of geographic areas were accessed through dbGaP  (N = 32) and those who identified as non-indigenous Spanish-Chilean .European (CEU) (5.v1.p1) . From thK = 3) was performed using the ADMIXTURE software .N = 86) compared to Chilean controls (N = 30). In general, indigenous South American haplogroups were less common in the RCI population than mainland Chilean controls. In particular, mitochondrial haplogroups B2 and D1g3 have been reported to be common to Indigenous America populations  (left) compared to Santiago Chilean controls (N = 15) (right) as reported by Y-Fitter. The common native Indigenous South American Y haplogroup Q (defined by M242) was detected, and was common to both islanders and controls (controls . Interescontrols  and Soutcontrols  populaticontrols  and is ccontrols . R1 (M17The R1b1 (defined by P297) haplogroup was found in both groups. This is the most common haplogroup in Europe and is widespread across Northern and Southern regions. The less frequent group, I2 (M438), was also present in the RCI and Chilean control groups. I2 is found all across Europe, but is considered a predominantly North European haplogroup, particularly in the Balkans region (see text footnote 4) . In contN = 68/86), with some influences from indigenous haplogroups , and common African haplogroups . Overall, the Chilean control groups showed a lower proportion of European haplogroups , and a higher proportion of both indigenous  and African haplogroups . The Y chromosome ancestral groups told a similar story with the proportion of Indigenous South American haplogroups being substantially lower in the islanders  compared to 13.3% (N = 2/15) in the Chilean Controls.Both mitochondrial and Y chromosome analyses , 2B suggPrevious studies, which included genealogical interviews, reported eight founder families were the original island colonizers , 2015a. The mitochondrial and Y haplogroups were combined with family structure data to infer the ancestral haplogroups of the founder lineages, through unbroken paternal and maternal lines . These aInterestingly, all of the inferred founder haplogroups that were identified were of European origin, the majority being common European mitochondrial H2a2a1 or R1b1 Y chromosome haplogroups . None ofMitochondrial and Y chromosome haplotype analyses only tell us about the direct maternal and paternal lineages, and the ability to infer ancestry and structure from these data is highly limited. We therefore further explored the population structure of RCI using autosomal markers and principal component analysis (PCA).To investigate this indigenous contribution in more detail, we repeated the PCA including more data from indigenous South American and Iberian Spanish European reference populations.The Chile_GRU individuals represent non-indigenous participants from the “Evaluation of Ancestry Admixture Among Chileans” controls . The indThe CEU Europeans and IBS Iberian (Spanish and Portuguese) control groups were indistinguishable from each other at this level of resolution .As mitochondrial and Y haplogroup analysis suggested the RC Islanders have a substantial European ancestry, and PCA indicated there was a high degree of individual variability in indigenous ancestry within the RCI group, we performed an admixture analysis . This waN = 68/86) on Robinson Crusoe Island than Chilean controls , although a high degree of European ancestry was evident in both populations. Indigenous South American haplogroups were less common on RCI  than in the Chilean control population from Santiago . Both these rates of indigenous haplogroups are substantially lower than those reported by previous studies of Chilean populations. N = 2/86) and Chilean controls , and may represent either Southern European or African admixture.Mitochondrial analysis revealed that European haplogroups were more common  and Chilean controls . Indigenous haplogroups were present in 7.7% (N = 3/39) of RC Islanders and 16.7% (N = 2/15) in the Chilean controls. These findings are similar to previous population studies who reported predominantly European haplogroups (8.5% indigenous)   in mixedigenous) . The appThe haplogroups reported in this paper were output by Haplogrep2 and Y-Fitter. These methods often assign haplogroups based on the presence or absence of one SNP according to the method and the decision tree they are built upon. To fully resolve and confirm haplogroups it would be necessary to sequence all the defining variants required to assign a subclade.Using extensive, near-complete genealogical records of the island population , 2014, mThe identified founder maternal and paternal haplogroups show an interesting trend. The maternal haplogroups able to be detected through unbroken lineages showed a distinction between Northern European (H2a2a and U4) haplogroups, and the Western European (H1) mitochondrial group. H1 is particularly common on the Iberian Peninsula, although also present at a lower rate across the rest of Europe . The femThe inferred founder Y haplogroups can be divided into Northern European (R1b1 and I2b) likely representing German, Swiss or British ancestry, and Southern European (E1b1b and J2) indicative of Iberian ancestry. These findings are consistent with male founders being of Northern European , Southern European (Spanish) and Chilean ancestry , 2015a. Interestingly, of the reported eight founding families only six unions were able to be accurately discerned from the genealogical records. This may be because the remaining individual founders recorded by the genealogy were partnered to the offspring of founder couples, and therefore considered a separate family unit. Alternatively, it may be because the recorded founders were ascertained from their relationship to the current island population, therefore any founders without living descendants would have been missed. While either scenario could be plausible, it does not impact this study as only founders who genetically contributed to the current island gene pool are of interest.We were unable to find any direct evidence to support the reported eight families founding the current population , 2015a. Both mitochondrial and Y analyses show a predominant contribution of European ancestry in the Founder individuals. Indigenous mitochondrial haplogroups are more common in both Robinson Crusoe and Chilean populations than indigenous Y haplogroups, likely as a result of the colonization of the Americans by Europeans. It should be noted that carrying a European Y or mitochondrial haplogroup does not exclude a high degree of indigenous ancestry, as European haplogroups (particularly Y) are extremely common in modern South American populations.K = 3) showed similar population structure between the current island population, Santiago Chilean controls and the Chile_GRU non-indigenous group. Our findings estimate the genetic contribution from indigenous South Americans to the RC Island population at 46.9%, similar to both Chilean controls (43.4%) and Chile_GRU non-indigenous controls (49.1%). Previous research in outbred Chilean populations identified 40–45% indigenous admixture with European and African estimated at 49–52 and 3% respectively  indicative of a high degree of recent admixture. Within the Mapuche indigenous population, who self-identified as Mapuche, carried a high level of European ancestry. Individually, some Robinson Crusoe islanders showed a predominantly European ancestry, and some a larger proportion of indigenous ancestry (with Mapuche as a proxy for indigenous South American). This reflects the history of how the island was colonized by Europeans, as well as Chileans, who themselves carry a high proportion of European ancestry , p. 205.There are a number of challenges in the investigation of population structure in Chilean populations and this is compounded in a population such as RCI which includes a recent bottleneck and consanguinity. Individuals in the RCI population were selected on the basis of being as distantly related as possible, however, they are still more related to each other than two mainland individuals. A range of methods are therefore required to delineate ancestral contributions. ADMIXTURE is an allele-based approach which is poor at detecting more subtle structure within a population, but is robust for inter-continental and recent admixture . Newer mThe main challenge for studying the genetic contribution of native South American populations is accessing appropriate control populations from which to examine population structure. The long and narrow geography of Chile mean there is a large number of distinct indigenous populations, including Aymara in the north and Mapuche in the south. Similarly, the physical barrier of the Andes separating Chile from neighbors Bolivia and Argentina mean this geographical distribution is constrained . Until vThe Mapuche dataset was used as a proxy for Indigenous South American populations in this study as this was the only dataset that was publicly available at the time of analysis. By utilizing recently available publicly available data from relevant indigenous populations, we have performed the first complete genetic investigation into the population structure of Robinson Crusoe Island. Despite 200 years in isolation, the current island population revealed a predominantly European genetic background, but with a greater than expected Native American genetic component, and showed a similar structure to that seen in mainland Chile. These findings inform genetic studies of the Robinson Crusoe Island population and that of Chile, moving forward and highlight the importance of using appropriate ethnically matched controls in genetic studies.N = 190) are available on request through dbGaP (phs001385.v1.p1). The datasets generated and analyzed during this study are not publicly available to preserve anonymity for the Robinson Crusoe Island population. Requests to access the datasets should be directed to DN, diannenewbury@brookes.ac.uk.European (CEU), Iberian (IBS), and Yoruba (YRI) control populations can be accessed from 1000 Genomes . Chilean population controls (Chile_GRU and Mapuche) . All subjects and/or their parents, where applicable, gave written informed consent, in accordance with the Declaration of Helsinki.HM, LC-C, DN, J-BC, and PV conceived and designed the experiments. MF, ZD, PV, and LJ performed the sample collection and genealogical analysis. HM performed the genetic analyses. HM and DN wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Planorbella trivolvis (Gastropoda: Planorbidae) in the western United States. We amplified two genetic markers  from individuals belonging to western USA populations and downloaded genetic data from GenBank. We utilized minimum spanning networks to assess the genetic patterns and performed Analysis of Molecular Variance and linear regression analyses to determine how geographic distance and watershed identity contributed to the observed genetic structuring. For both markers, we found that the majority of genetic variation was associated within and among populations, rather than among watersheds. Correspondingly, there was no significant effect of geographic distance on genetic distance, suggesting that long-distance dispersal was promoting gene flow between populations. The genetic similarity could reflect avian-mediated dispersal of snails along the Pacific Flyway, a major waterfowl migratory corridor. Further analysis of the population structuring across North America revealed East-West genetic structuring, suggesting that across longitudinal gradients P. trivolvis experiences significant genetic isolation.Despite the important roles of freshwater gastropods in aquatic ecosystems, the taxonomic status of many taxa is unclear, which is compounded by a lack of information on species population genetic structuring, distribution, and dispersal patterns. The objective of this study was to address the biogeography of the freshwater snail  Freshwater snails play important roles in complex ecological interactions, acting as bioindicators of water quality, invasive species, and intermediate hosts for multi-host parasites –5. HowevOne of the major factors that determines biogeographical distribution of freshwater invertebrates is their dispersal mode and ability , 11. TheAquatic snails can move through the environment by both active and passive dispersal mechanisms. Active dispersal, such as crawling, is considered important at a local scale . FreshwaPlegadis chihi) (Vieillot) [Cygnus columbianus) (Ord) [Extra-aquatic dispersal can occur via abiotic vectors such as flooding events or tornadoes; and biotic vectors such as mammals (including humans), insects, and birds . Migratoieillot)  and expes) (Ord) . This sus) (Ord) , 28 and s) (Ord) , 30.Planorbella (formerly Helisoma) [Planorbella is generally considered to be a less effective disperser. Species, such as Physella acuta (Draparnaud) (Family Physidae), are known to have higher fecundity, a shorter hatching time, and significant reproductive plasticity, which all aid stochastic dispersal [Planorbella is hermaphroditic and able to reproduce via self-fertilization, studies indicate this genus has lower viability through self-fertilization and reproduces primarily through outcrossing [Planorbella is less effective at utilizing passive dispersal mechanisms. For instance, Planorbella spp. have been found attached to feathers of waterfowl, but experimental flight simulations have shown they are less likely to remain attached to a feather for any length of time [Planorbella [Planorbella is less likely to disperse long distances compared to other common freshwater snails.Planorbidae is one of the most abundant and widespread freshwater gastropod families in the world , 32. Amoelisoma) . Compareispersal –37. Whilcrossing –42. Low crossing . In addi of time , 24. Whinorbella , 23. CumPlanorbella classification [Planorbella was raised from a subgenus of Helisoma to the genus level by Burch [Helisoma, 16 were moved to Planorbella based on differences in shell coiling, leaving only Helisoma anceps. However, previous work concluded that classification in Planorbella based on shell alone is not sufficient in distinguishing species [Planorbella in North America. To date, studies focus predominantly on Planorbidae species at global scales, with considerably less sampling within the United States [Like many freshwater gastropod families, Planorbidae snails exhibit considerable diversity in shell morphology within species and share extremely homogenous anatomical traits between species –47, oftefication . Planorbby Burch , as was by Burch . Of the  species . Therefo species , 53. Furd States . OverallP. trivolvis in the western United States. Specifically, we opportunistically collected 60 samples from 22 ponds and reservoirs in the western USA and sequenced the 16S and COI mitochondrial loci. We created haplotype networks and calculated molecular diversity indexes and Fst values to describe levels of gene flow between the populations. An AMOVA was conducted to characterize the genetic variance within populations, among populations and across watersheds in the West Coast. By building a statistical model, we tested the relative influence of geographic distance on population genetic structuring. We further compared our findings with samples from GenBank to describe population structuring across North America. These results aim to provide insights into the genetic variation of this ecologically important group of gastropods while concurrently testing the influence of alternative hypothesized explanatory variables on population genetic structure.The objective of the current study was to investigate the biogeographic genetic structuring of Planorbella trivolvis snails were opportunistically collected by hand or by dipnet from 22 ponds and reservoirs in California, Oregon, and Washington States (https://water.usgs.gov/wsc/map_index.html). The localities spanned six different watersheds along the West Coast of the United States (Lower Columbia (HUC 1708), Middle Columbia (HUC 1707), Oregon-Washington Coastal (HUC 1710), Willamette (HUC 1709), Sacramento River (HUC 1802), and San Francisco Bay (HUC 1805)). Detailed locality information can be found in n States . Samplesn States , as deteH. trivolvis, based on diagnostic shell attributes originally described in Hubendick & Rees [Helisoma and Planorbella are genus names used today to refer to P. trivovlis specimens [https://www.itis.gov/), Planorbella trivolvis, as described in Burch [At each of the 22 sites, between one and seven snails were opportunistically collected for a total of 60 specimens. The snails were identified by the collectors as k & Rees . Both Hepecimens , 55. In in Burch . SpeciesGenomic DNA was extracted from the snail foot tissue utilizing the E.Z.N.A Mollusc DNA Kit (Omega Biotek). Two mitochondrial DNA loci were amplified and sequenced (16S and COI) as both have been used successfully to recover population genetic variation in freshwater snails , 53, 56.μL with 6.25 μL GoTaq Green Master Mix (Promega), 3.75 μL of nuclease-free water, 1 μL of each primer and 0.5 μL of the DNA template.The 16S mitochondrial gene was amplified using primer sets 16Sar 5’-CGCCTGTTTATCAAAAACAT-3’ and 16Sbr 5’-CCGGTCTGAACTCAGATCACGT-3’ . The COIThe PCR protocol for 16S included an initial denaturation at 94 °C for 60 s, 35 cycles of 94 °C for 30 s, 55 °C for 50 s and 72 °C for 60 s, and a final extension at 72 °C for 10 minutes . The PCRPlanorbella specimens sampled were confirmed to be P. trivolvis based on BLAST match with existing sequences in GenBank (>97% similarity with known P. trivolvis and H. trivolvis sequences).Amplified products were sequenced by Sanger sequencing at Quintara Biosciences . Raw sequences were processed in CodonCode Aligner 3.1.7 to remove primer sequences and manually correct for low quality read. The final size of the 16S gene segment was 456 bp. The final size of COI gene segment was 606 bp. All P. trivolvis (including the one named H. trivolvis) were downloaded from GenBank, resulting in 38 COI sequences for P. trivolvis. 23 of the 38 COI sequences for P. trivolvis are West Coast samples and the remaining 15 are from other North American samples, including Maryland, New Mexico and Canadian localities. GenBank accession numbers and associated studies can be found in In addition, all available COI sequences for Amplified sequences were aligned using the ClustalW algorithm  implemenhttp://popart.otago.ac.nz) for both gene fragments to view the connections among haplotypes. First, we constructed MSN networks for P. trivolvis in their native range along the West Coast of the United States (Minimum Spanning Networks (MSN) were constructed with PopARt v. 1.7  .To assess the genetic structure in the West Coast, we conducted an Analysis of Molecular Variance (AMOVA) . The AMOGenetic diversity summary statistics were calculated for each gene segment in Arlequin v. 3.5. Summary statistics were estimated for each watershed and across the entire sampling range. The molecular diversity indexes for each watershed included number of haplotypes (h), haplotype diversity (Hd), and nucleotide diversity. The indexes estimated for across the entire sampling range include gene diversity, mean number of pairwise differences and nucleotide diversity.P. trivolvis population structuring, general linear models were built using the lm function in R version 3.4.2 [To further assess whether geographic distance is associated with on 3.4.2  to test P.trivolvis across North America, we aligned the newly amplified sequences and the ones downloaded from GenBank using the ClustalW algorithm [P. trivolvis . A maximum-likelihood reconstruction was conducted using RAxML 8.2.11 [P. trivolvis have been documented globally [P. trivolvis population genetics outside of North America.To assess the structuring of lgorithm  implemenrivolvis . The aveL 8.2.11  with theglobally , there iP. trivolvis individuals were successfully amplified for the 16S gene fragment. The genotyped individuals yielded 16 unique haplotypes, and seven singleton haplotypes are present in the populations (ST) accounts for 71.84% of the total genetic variation (p < 0.05), among populations within a watershed and among watershed account for 12.31% and 15.85% (p < 0.05) respectively.A total of 57 out of 60 ulations . Two hapP. trivolvis individuals were successfully amplified. 20 of the individuals were also analyzed for the 16S gene fragment. The low amplification success for the COI gene fragment results in lower statistical power compared to the 16S gene fragment. Seven unique haplotypes were obtained as a result, and three singletons are present in the populations . Among populations within a watershed accounts for 77.59% of the genetic variation, within population and among watershed account for 1.5% and 20.91% respectively   .P. trivolvis across their entire native range. A lack of available genetic data meant we were only able to access the genetic structure of P. trivolvis within North America, despite its scattered global appearance [GenBank sequences were included in this study in order to assess the overall genetic diversity of pearance . Even wiP. trivolvis highlights phylogenetic structuring and the presence of five well supported clades  [The objective of this study was to address the genetic structuring of the freshwater snail ershler) , 19.Differences in the results between the two genes could be a consequence of smaller sample sizes in the COI dataset or from the differences between the two mitochondrial genes. The COI gene used in this study is considered a universal marker because of its ability to successfully recover gene fragments and rapid rate of evolution in diverse invertebrates , 68, 69.Planorbella spp. in particular are often considered to be dispersal-limited owing to the apparent dependence on hydrological connectivity for direct movement [Another goal of this study was to determine the degree to which watershed identity  or geographic distance contributed to the observed genetic structuring. We found that watersheds were not a significant factor contributing to the observed population structure. Similarly, we discovered that geographic distance was unrelated to genetic distance, at least based on our measurements from 16S and COI. This result corroborates prior research, which shows that geographic distance is not strongly correlated to the genetic distance between populations of freshwater organisms , 71, 72.movement .One explanation for the lack of association between watersheds, geographic distance and genetic distance is that dispersal via waterfowl is promoting gene flow across the watersheds. The Pacific Flyway, the westernmost of the four primary bird corridors in North America , is a maP. trivolvis geography, it will likely strengthen the conclusion of this study. There is still a possibility that, by chance, we sampled more common haplotypes than what is actually present in the populations. This can also be evaluated with expanded sampling efforts in the future.Importantly, a lack of geographically isolated populations suggests that despite the limited sampling of this study, most of the shared common haplotypes among populations have been recovered. If the populations were truly isolated, and thus do not share a lot of common haplotypes, the limited sampling would have retrieved a higher number of unique haplotypes. Additionally, if waterfowl were only transporting a small number of snails that successfully establish new populations, genetic bottlenecks are more likely to occur and result in more genetically diverse populations. This suggests that dispersal among populations is relatively frequent. Therefore, although a more comprehensive sampling is needed to further explore P. trivolvis. These results contradict the historical view that Planorbella would be a less effective long-distance disperser when compared to other freshwater gastropods [At the regional scale, watersheds are not major barriers to the distribution of stropods –24. Theistropods . Additiostropods . Second,Future studies are needed to examine the role of waterfowl in freshwater gastropod dispersal. The emphasis should be on smaller, more abundant birds, such as ducks and waders, that are likely important dispersers, but have received less scientific attention relative to larger birds, such as geese and swans . At thisP. trivolvis populations emphasize the degree to which this taxon is grossly under sampled for molecular analyses of population structure. Efforts to understand the population structuring are complicated by lack of available genetic data, unawareness of their global occurrence and the historic methods of identification based on morphology. This study represents the first published effort to characterize the population genetic structure of P. trivolvis.Results of our efforts to compile genetic sequences of P. trivolvis displays strong East-West genetic structuring in their native range, despite their continuous distribution throughout North America (https://www.gbif.org/). These results imply that P. trivolvis experiences a high degree of genetic isolation across North America, even if such isolation was not observed along the West Coast specifically. The structuring observed in the haplotype network is further corroborated by the COI gene tree. The phylogenetic analysis supports the presence of five clades, where the Maryland and Canadian samples form their own clade (A) and exhibits long genetic distance from the western US clades B-E. The East-West genetic structuring coincides with the Rocky Mountains. Specimens collected east of the Rocky Mountains fall within clade A, and samples collected west of the Rocky Mountains fall within clades B-E. This pattern has been seen in other freshwater gastropods, such as P. acuta [Rhagada [Eobania [Iberus [Helixena [Goniobasis species [Pyrgulopsis [Physella [Pleurocera, 2.2-22.6% in Leptoxis and up to 7.9% in Elimia [Planorbella species in their native range.The COI range-wide haplogroup network suggests that P. acuta . The iso[Rhagada , Eobania[Eobania , Iberus  [Iberus , HelixenHelixena , and Gon species . Other sgulopsis  and 5% iPhysella  to distin Elimia . The seqPlanorbella is arguably unknown. Small, scattered populations of P. trivolvis have been identified outside North America, but their general abundance and distribution levels have yet to be elucidated [P. trivolvis existing outside their native range, the species is not considered invasive. One area of study relevant to their global distribution is their dispersal ability. As evidenced by its current proposed distribution, the ability of P. trivolvis to disperse effectively at a regional scale does not translate to the global scale. A possible explanation is that cross-continental dispersal, primarily mediated by humans, may be a less effective dispersal mechanism for P. trivolvis. Anthropogenic transport may occur infrequently, or the snails are not able to survive the journey. Additionally, waterfowl species that effectively disperse P. trivolvis in their native range may not be present in other parts of the globe, limiting P. trivolvis ability to successfully colonize new habitats. Alternatively, the life history traits of Planorbella could play a significant role in their limited global distribution. For example, once P. trivolvis reaches a new environment, it may be less likely to successfully establish a population. This could be a result of the species preferential outcrossing mating system, where more than just one specimen is required to start a population [P. trivolvis from successfully establishing a population. The aquatic environments may lack suitable habitat or resources, support effective predators and competitors, or have physiochemical properties or pollution levels that P. trivolvis cannot tolerate [The current global distribution of ucidated , 85. Despulation . Ecologitolerate .Planorbella is compounded by their phenotypic plasticity. There is a large gap in the understanding of many freshwater snails due to the traditional methods in snail identification, which rely almost exclusively on shell morphology and internal anatomy. The morphological similarity of Planorbella to other planorbid snail species has impeded the overall understanding of their distribution [The lack of knowledge about the global distribution of ribution . ImproviP. trivolvis in their native range has prevented an understanding of their role in freshwater ecological interactions. Our results suggest that long-distance dispersal helps shape and maintain the structure of P. trivolvis, which can influence their ecosystem interactions both in their native and global range. Their effective dispersal has important consequences for their role as intermediate hosts to trematode parasites and conservation. Mechanical barriers, such as dams, are often proposed as a way to stop the spread of parasites in freshwater habitats by preventing the spread of their gastropod hosts [P. trivolvis relies on, in part, the frequency and abundance of contact with waterfowl. These important interactions with waterfowl depend on the availability of suitable habitat. Freshwater habitat modification threatens both aquatic and avian biodiversity by impairing the ability of freshwater snails to disperse locally and impeding contact with waterfowl that may be required for long-distance dispersal [P. trivolvis influence its ecological interactions.A scarcity of information on the population structuring of od hosts . Our resispersal , 19. ImpP. trivolvis in the western United States and across North America. The observed genetic patterns were not explained by watersheds. Additionally, genetic distance was unrelated to geographic distance. The North-South genetic similarity reflects the Pacific Flyway, which suggests that long-distance dispersal vectors, such as waterfowl, are promoting ongoing gene flow over large geographic ranges and that the population structure of P. trivolvis may be strongly influenced by long-distance dispersal in the West Coast. Cumulatively, these results indicate that, at a regional scale, their distribution is not impeded by dispersal.This study addressed the population structuring of the freshwater snail P. trivolvis. This study is the first to examine the population structure of P. trivolvis. Our results showed that P. trivolvis exhibits significant East-West genetic structuring across their native range. Overall, this paper provides an initial framework for continued biogeographical analysis of Planorbella in their native range.The efforts to compare the range-wide structuring of the species was complicated by lack of available information on S1 Appendix(CSV)Click here for additional data file.


Sabellaria chandraae is the most common reef-building polychaete in the intertidal area along the south-eastern Arabian Sea. We used the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene to examine the levels of genetic variation the population. The samples were collected from seven intertidal areas along the south-eastern Arabian Sea. The hierarchical Analysis of Molecular Variance (AMOVA) and conventional population statistics (STF) showed a low level of genetic differentiation , indicating no geographical patterning among seven populations. The present results were able to provide a reference for the single stock population along the south-eastern Arabian Sea.The honeycomb worm  Sabellaria chandraae de Silva, 1961, is broadly distributed along the intertidal zone of the Indian and Sri Lanka coast , and photographed with transmitted illumination. The PCR-amplified DNA fragments were purified using QIAquick gel purification kit according to manufacturer’s instructions . Sequences were produced using the same primers and determined on an Applied Biosystems  3730xl automated DNA sequencer, following the standard cycle sequencing protocol. The sequences were submitted to NCBI GenBank. The mtCOI gene sequences were aligned using CLUSTAL W  and nucleotide (π) diversities were estimated using DnaSPv5  showed no significant differences among seven samples. The AMOVA results of S. chandraae shows within populations  and among populations  between north and south populations (S. chandraae species along the ∼1260 km the west coast of India. Since, the distribution of the species depends the existence of historical factors, reversing WICC, lacking an oceanic barrier to the larval movement among study areas were further confirmed by the low values of STF.The Haplotype (SPv5 Nei . Total hSPv5 Nei . The lowulations . The exi