The Australian dingo is a recent anthropogenic addition to the Australian fauna, which spread rapidly across the continent and has since widely interbred with modern dogs. Genetic studies of dingoes have given rise to speculation about their entry to the continent and subsequent biogeographic effects, but few studies of their contemporary population structure have been conducted. Here we investigated the dingo ancestry and population structure of free-living dogs in western Victoria and contrasted it with a wider southern Australian sample. We wished to determine whether their geographic isolation was mirrored in genetic isolation. To address this question, we analysed 34 microsatellite markers using Bayesian clustering and discriminant analysis of principal components, and summarised genetic diversity at the population and individual level. The broader southern Australia sample (n = 1138) comprised mostly hybrid animals, with 30% considered pure dingoes. All western Victorian individuals (n = 59) appeared to be hybrids with high dingo ancestry. The population showed no evidence of admixture with other populations and low genetic diversity on all measures tested. Based upon our characterisation of this unusual mainland population, we advise against assuming homogeneity of dingoes across the continent. Canis familiaris2 and include the ancient pure-bred Australian dingoes, (very rarely) stray or feral modern domestic dog breeds, and a swarm of hybrids (≡ cross-breeds) with varying dingo and modern domestic dog lineage4. These are collectively called “wild dogs” in much of Australian legislation, policy and management strategies5.Australia has a large and widely distributed population of free-ranging, wild-living dogs. These animals are all members of the species 6), ancient dogs that were most likely derived initially through passive or unconscious domestication from commensal wolves7 and later by active domestication of resultant tamer proto-dogs somewhere in southern east Asia about 30,000 years before present8. The dingo was introduced to Australia by humans via South East Asia at least 3500–8000 years before present, according to archaeological and molecular evidence respectively10, and subsequently feralised11. Corbett6 reported trade between north Australian First Peoples and Malaysian seafarers bearing on-board dogs continuing until the 1920s. Although cultural and trade interactions with New Guinea across the Torres Strait persisted, dingoes remained relatively isolated from other dog populations until British colonisation in 1788 . Being a highly mobile and tameable generalist carnivore, and useful to First Nations peoples, the dingo likely spread from northern Australian introduction points and became established in most Australian environments6.Australian dingoes are medium sized  with pure dingoes being more common in central and western Australia, away from dense human settlement3. As early agriculturalists took their livestock away from the first British settlements on the coast, lethal control of dingoes began through much of the south east to protect the growing sheep and longhorn cattle industry20. The south-eastern mainland state of Victoria was settled early for agricultural pursuits including sheep production, along with New South Wales and south east South Australia21. Subsequently, dingoes were largely extirpated from Victoria except for the rugged north east, Gippsland in the south east, and the semi-arid far western regions . There are few pure or possibly pure dingoes in the north east and eastern populations3 but the status of the extant western Victorian and its contiguous South Australian population extending to the west (hereafter WV) has not been investigated.Due to hybridisation between dingoes and modern domestic dogs since 1788 many populations are now hybrid swarms  and another in north-western and central Australia22. Using single nucleotide polymorphisms (SNPs), analysis of 23 samples indicated that the K’gari population was distinct from the south-eastern Australian sample 4. The region of the proposed distinction between the south-eastern and north-western lineages has not been well sampled in any previous study. Continued sampling of the intermediate zone between the two lineages is required to determine the point of division; if the lineages are to be treated as separate management units, any relationship of the WV population to the Alpine or other divergences requires assessment.Recent studies17. As in other parts of Australia the prevalence of feral domestic dogs appears to be inconsequential3. In this paper we investigated the level of dingo ancestry and population structure of a geographically isolated population of free-living dogs in western Victoria and contiguous South Australia. Our aims were to test for genetic isolation from or continuity with populations identified in previous studies11.Although the free-ranging populations in eastern Victoria exhibit local clusters of high dingo ancestry, the majority have evidence of modern dog introgression, which is also evident in contiguous eastern New South Wales populations18 were used for testing. Details of the reference populations are provided in Stephens et al.3. Dingo ancestry testing identified seven individuals (0.6%) with ≤ 10% dingo DNA present (considered modern domestic dogs), 340 (29.9%) individuals with ≥ 90% dingo DNA (considered likely to be pure dingoes) and the remaining 791 (69.5%) individuals were likely hybrids with 11–89% dingo DNA (Table Dingo ancestry testing was performed using the learning samples approach in Structure (detailed in the Structure v2 documentation), which assigns test individuals to one of two populations (dingoes or domestic dogs) by updating the allele frequencies from assigned reference individuals from each group. Twenty-three microsatellite loci established for allele frequency differences between dingoes and domestic dogsEighty individuals with less than 60% dingo DNA were removed from the wider sample for subsequent analyses to reduce the noise from modern domestic dog DNA  but still retain good coverage of the study area, including all the WV individuals.From the Structure results, inspection of ΔK and lnPr(k) indicated K = 4 as the most likely number of clusters, dividing the sample into clusters from eastern Victoria, eastern New South Wales across to South Australia, western New South Wales and Western Victoria Fig. a, b. The23). As the nine similar replicates contained the runs with the highest log likelihood, these nine were aligned and averaged and the outlier replicate removed.Inspection of the replicates for both K = 4 and K = 6 from the Structure iterations showed nine replicates with very similar results and one replicate with some differences in assignment , separating the dingoes and domestic dogs at the highest level of population structure . The DAPC analysis showed the same pattern as the DAPC in Fig. Two individuals from the north of the WV area clustered with the 57 WV specimens, bringing the size of the WV population analysed for population summary statistics to 59. The WV population showed lower genetic diversity than the other populations on all population summary statistics tested, including allelic richness, which accounts for the difference in sample size, but did not have a higher number of private alleles Table . Mean reST, also confirmed strong genetic differentiation between the WV and all other populations, with the lowest value from the 95% confidence intervals of the WV comparisons (0.23 between WV and NNS) greater than the highest value from the comparisons between the other populations (0.15 between WN and EV) .Pairwise differentiation, as measured by FsGD analyses support the results of the population-level, non-spatial analyses with respect to the WV population . It is unlikely that such direct dingo conservation processes will be introduced into the region because of extant livestock protection imperatives and the legal and animal welfare obligations of livestock producers. However, the current Victorian policy of limiting control activities on public lands to within 3 km of the edge  could slow the rate of hybridisation by reducing the probability of interbreeding between extant high-dingo hybrids and lower dingo hybrids from elsewhere. This suggestion is contrary to the findings of Bohling and Waits25 that lethal control increased hybridisation between red wolves (Canis rufus) and coyotes (C. latrans) and the suggestions of Cairns et al.17 that lethal control increases hybridisation of dingoes and other dogs. Data on the impact of lethal control on dingo hybridisation is lacking, so there is still much to be learned about these processes.Our southern Australian sample showed a similar pattern of dingo hybridisation to other studies, with higher numbers of hybrids near the more densely human-occupied coastal areas and higher dingo ancestry in the sparsely populated inland26. Conversion of ephemeral water supplies to permanency is only recent in the Mallee and Wimmera regions of Victoria. There is no permanent surface water drainage in the Mallee and most water availability was ephemeral prior to the introduction of a channel system over the 50 years from 185627. Indigenous land use practices could also have affected dingo population density and distribution before British colonisation, but permanent water availability would have had influence on their distribution and that of their prey (dogs need between 26.2 and 122.6 mL kg-1 day-1 of water)29. It is likely that pre-British dingo population densities were low in WV because it is semi-arid  and has low primary productivity due to poor soils. Any expansion of dingoes’ range due to a sudden increase in resources, coupled with the rapid spread of modern domestic dogs and associated mating opportunities is likely to have had conflicting effects on the genetic composition of dingoes: a decrease in genetic diversity in areas where rapid expansion has occurred due to small groups colonising newly hospitable regions, and an increase in genetic diversity where domestic dog genes were entering the population.Hybridisation proportions might also correlate with expansion of livestock production and associated water infrastructure11, which makes it easier for the reference population to capture the allelic variation in most dingoes, but unusually isolated populations such as WV and K'gari may show decreased accuracy with any reference sample approach. The DAPC analysis with the dingo and domestic dog references included  confirms that the WV population allele frequencies are distinct from the dingo and modern domestic dog reference populations, and that WV individuals are unlikely to be accurately represented in any method of ancestry analysis which requires such references. Although the number of private alleles was not high in the WV population, which would mean there is no analogue in the reference animals, the low genetic diversity and skewness from the references mean the comparisons are more susceptible to stochastic allele changes from genetic drift and the impact of data scoring errors.Although the population-wide proportion of dingo ancestry was high in the WV population, we are cautious about the finding that there were no ‘pure’ dingoes in the sample. The method of testing relies on updating of the model priors from reference populations, and sufficient deviation in allele frequencies from those represented in the reference populations is likely to decrease the accuracy of dingo ancestry estimation. The genetic diversity of dingoes across Australia is low overall31). A major advantage of microsatellite genotyping currently is the ability to add new samples to a dataset containing many thousands of already typed individuals3, and it will be some time before the availability of genomic data sets can provide similar baseline data. Expanding the genomic coverage of markers can also provide opportunities to clarify the history of the WV population. The increased resolution afforded by resampling a sub-set of the data with single nucleotide polymorphisms (SNPs) should allow much greater insight into the timings of population separations32. Our study also did not provide a clear indication of which of the surrounding areas was most closely related to the WV population, and higher-resolution markers may help to address this question.In the short term, an assessment of the effect of reference population selection and quantification of the genetic variation of dingoes across their range is required to fully understand these impacts. In the longer term, expanding genome coverage should better characterise isolated and distinctive populations such as WV , but all values between the other populations and the WV population were 0.240–0.321, indicating strong genetic discontinuity between WV and the surrounding populations, even over larger geographic distance than between the other populations . The sGD results also show lower individual values for observed heterozygosity and allelic richness in the WV than any other area, which supports the results of the partitioned summary statistics and provides further evidence for the isolation of the WV population.The individuals across most of the study area displayed a pattern consistent with weak or clinal population structure: low FThe effective population size, by contrast, is not uniquely low within the WV, with individuals in western New South Wales and in South Australia showing similar values. Broader comparisons of effective population size in high dingo-ancestry populations, particularly in inland, north and western Australia where high numbers of ‘pure’ dingoes are available, could indicate the typical effective population size for Australian dingoes, and their capacity to persist with low genetic diversity when dog introgression is minimal.35.The presence of low genetic diversity, high mean relatedness and, in particular, the low incidence of private alleles is consistent with a population that is a reduced version of a larger regional gene pool and subject to drift and inbreeding—remnant after ~ 150 years of intermittent control in the region—rather than a population translocated from a different ancestral source36. Allelic richness, observed heterozygosity and effective population size values were similar , with the K’gari study using a reduced version of the same microsatellite marker set employed in this study. Although low genetic diversity due to founder effects, inbreeding and genetic drift are common in island populations38, finding comparable levels of isolation in a mainland dingo population was unexpected.The WV population of free-ranging dogs shows measures of genetic diversity close to those found in the dingo population on K’gari11 or a more limited area with more intensive sampling36. The Australia-wide studies identified a split between the lineages of dingoes in the south east and north west. The population structure analysis in this study, which is the first to characterise the west of NSW and Victoria, did not identify a contemporary boundary of the south east-north west split11. At the highest level of population structure (K = 4) the north of the study area showed a continuous population from eastern NSW to South Australia, with the exception of the WN population, which appears to exist within this continuum and may be the result of local environmental factors or a migration event. A discontinuity may still be present to the west or north of the area studied here, but this requires further investigation. Although the northern population is not the focus of this study, it further highlights the need for adequate sampling of dingo populations to disentangle their complex history of migration, colonisation, hybridisation, persecution and translocation which has occurred in the very short time, in evolutionary timescales, since their arrival in Australia.The reasons for genetic isolation in a large mobile apex predator in the face of an increasingly hybridised swarm require investigation. Prior to British colonisation and settlement, indigenous population density and their trading and cultural movements likely influenced the spread and continental distribution of dingoes. Biogeographic barriers to human and animal movements possibly affected colonisation by dingoes and subsequently impeded gene flow through biogeographic semi-isolation. Previous analyses of genetic subdivision in dingoes focussed on either a broad geographic range with few samplesSampling in inner-eastern Australia is necessary to understand the contemporary and finer-scale processes at work in this separation, but the current study highlights an important caveat; sampling of individuals in the WV as representative of this area would provide an inaccurate genetic profile of the broader area. The high mean relatedness and lack of admixture with surrounding individuals could appear as a distinct lineage under some analyses, and care should be taken to examine demographic parameters for inbreeding and outbreeding that may be caused by isolation or introgression, respectively.4, subsequent analyses suggest the processes that caused them differed. The K’gari (and WV) populations appear to have arisen from isolation after the colonisation of the continent by dingoes36, while the mitochondrial south east/north west lineages are more likely to have occurred during their initial or early migration into Australia, with unknown contemporary ecological significance. We should therefore consider more subtle effects across the landscape given complex but temporally shallow demographic history of dingoes, to then relay this information to local managers to enable more precise recommendations in the context of regional conditions and pressures.Although the south east/north west split and the K’gari subdivision have previously been combined under ‘ESUs’40. Many questions remain about their introduction, ecology and behaviour, and the use of molecular data will continue to elucidate aspects of dingo distribution, subpopulation structure, and lineages nationally. This information will assist in the management of dingoes so that their genetic diversity can be maintained in the context of necessary control of livestock predation15 and conservation of predation-susceptible fauna . Here our objective was to add to that general body of work by detailing the genetics of one isolated population of dingoes. We hope it will fit into a broader context of studies into free-ranging dog genetic diversity across the continent and significant island populations of dingoes such as that on K’gari, utilising different genetic markers, analysis techniques and spatial scales of interest43.The fate of the Australian dingo is a concern for many stakeholders across its broad and varied rangeEucalyptus spp.) woodland as national park (gazetted in 1909) and forestry lands. The region is part of the Murray-Darling Depression bioregion44, is semi-arid with mean annual rainfall of 300–320 mm45 and characterised by sandy soils of low fertility and productivity46. Agricultural enterprises, primarily cropping with some livestock production (mostly sheep at low carrying capacity), surround the mallee woodland. This geographic arrangement contributes to conflict between sheep graziers and free-ranging dogs, and the region is subject to ongoing free-ranging dog management by lethal control through trapping and baiting. The Wyperfeld/ Big Desert Wild Dog Management Zone employed five registered private trappers during the study, and contractors in contiguous nearby South Australia were responsible for controlling errant dogs. Together, these areas comprised the WV sampling region.The Mallee region of north-western Victoria is sparsely populated by people and contains a large area of unoccupied mallee (https://doi.org/10.5061/dryad.2rd32) were also included  with the recommended protocol. Extracts were amplified at 34 microsatellite markers in seven multiplexed PCRs48. PCRs were performed in 10 µl reactions, containing 5 μl Qiagen Multiplex PCR solution , 1 μl Qiagen Q-Solution, 1 μl DNA, 0.2 μM of each primer and 2 μl DNAase/RNAase-free water. PCR conditions were: 15 min at 95 °C, 35 cycles of 30 s at 94 °C, 90 s at 55 °C or 58 °C and 60 s at 72 °C, then 30 min at 60 °C. All fragments were visualised on the same equipment and peaks called by the same individual for consistency.Dog ear tissue specimens were stored in ~ 3 mL Longmire buffer3 23 microsatellite loci18 were analysed in STRUCTURE v2.3.449 along with 322 reference dingoes and 109 domestic dogs3. Reference dingoes were collected from across Australia, and although higher numbers from the central and west were used, repeating the tests with different references showed mean differences between results of < 1%3. Reference animals were coded with USEPOPINFO = 1 and test animals with USEPOPINFO = 0 (the ‘learning samples’ approach49). The sample was run for 300,000 iterations and 30,000 burn-in replicates, with allele frequencies initialised and updated from the reference individuals. Ten replicates were performed and the results combined using the R package pophelper v2.3.150. The assignment of each individual’s genotype to the dingo reference population was then reported as a percentage. Individuals with less than 60% dingo ancestry were excluded from further analyses, to maximise the geographically relevant information in the genotype.Dingo purity testing was carried out on all samples using the method outlined in Stephens et al.51. Runs were repeated from K = 1–15 for 10 replicates. STRUCTURE output was assessed for optimal number of clusters (K) by the ΔK method52, by inspection of lnPr(K) using Structure Harvester v0.6.9453 and using the thermodynamic integration approach with the admixture model implemented in rMaverick v1.0.554. The latter method provides an alternative estimate of K from multiple MCMC chains without assuming normal distribution of the likelihood. Replicate runs of the optimal K were then aligned, averaged and visualised using pophelper. Individuals were assigned to the cluster to which they had the highest ancestry coefficient .The remaining samples were then assessed for population differentiation using all 34 loci. The additional 11 loci were added to the 23 loci used for dingo ancestry testing to mitigate potential bias introduced by the selection of the 23 loci for allele frequency differences between dogs and dingoes. Each sample was analysed using STRUCTURE using the admixture model and correlated allele frequencies for 500,000 iterations and 50,000 burn-ins. The sample was run with initial Alpha = 0.25 and allowed to vary among populationsadegenet 2.1.356 which provides an assumption-free method for comparison with the Bayesian clustering approach. The number of clusters was inferred using the find.clusters function56 for K = 1–20 and inspection of the BIC graph for the minimum values. Optimal K value was also informed by the Ward’s clustering algorithm implemented in adegenet (criterion “diffNgroup”). The number of principal components to retain was chosen using the cross-validation method in adegenet, with missing data replaced with either zeroes or the mean of allele frequencies and the results compared.Populations were also assessed using Discriminant Analysis of Principal Components (DAPC) in R using the package diveRsity v1.9.9057. The number of private alleles, unbiased expected heterozygosity, observed homozygosity and bootstrapped mean relatedness were calculated using GenAlEx v6.51b259. Mean relatedness values were calculated from the matrix of Lynch & Ritland estimators60 between individuals then averaged per population. These data were bootstrapped and permuted for 999 replicates to obtain 95% confidence intervals (CI) and to test for significant deviation from the null hypothesis of average relatedness, respectively. Pairwise Weir & Cockerham’s FST61 and DEST62 were also calculated using diveRsity and bootstrapped for 1,000 replicates to obtain 95% CI values. Effective population size was calculated using NeEstimator V2.163 and the results are reported for the single-sample linkage disequilibrium method with random mating and alleles with frequency below 0.02 excluded, to reduce bias introduced by the inclusion of rare alleles.Population summary statistics were calculated for each of the STRUCTURE populations to characterise the relative genetic diversity within and between each population. The per-population allelic richness was calculated using sGD v2.1164. This method does not assume pre-defined clusters to assess population statistics, but instead uses a user-defined radius around each individual to calculate properties such as observed heterozygosity and allelic richness. This method may reveal subtle genetic variations within populations, as well as being less reliant on assumptions of population boundaries which may be unclear in continuously distributed taxa such as free-ranging dogs. Based on previous extents of spatial autocorrelation in dingoes16 we selected a distance of 200 km across the study area of approximately 1500 km × 1200 km. Geographic coordinates were converted to UTM using PBSmapping v2.73.0 in R65. Although the study area covered three UTM zones, the majority of the individuals were collected in Zone 55, so this zone was selected by PBSmapping and the results inspected to ensure the individual locations were reasonably accurate. Observed heterozygosity, allelic richness and effective population size using NeEstimator V2.1 were calculated against a Euclidean distance matrix for individuals with a minimum of 10 other individuals within the radius. Individuals which did not have a minimum of 10 other individuals within 200 km were recorded as missing data.Summary statistics were additionally analysed across the study area using the R package Supplementary Information.


Ostrinia furnacalis (ACB) and Ostrinia nubilalis (ECB), which are sympatric in the Yili River Valley in Xinjiang, China.Tianshan Mountains provide a model for studying biological evolution and speciation. Here we assess the evolutionary history of COI, COII, Cytb) and four nuclear DNA  markers obtained from representatives of HC (Huocheng), YN (Yining), XY (Xinyuan), and MNS (Manasi).Our study is based on the historical gene flow analyses of two species by using three mitochondrial DNA , and mismatch distribution test results suggest that population expansion events may not have occurred in the recent past. The demographic history and gene flow pattern between ACB and ECB may follow the “mountain isolation” hypothesis. The ML and BI trees of the mtDNA haplotype dataset show that ECB haplotypes are grouped together in a distinct clade and are clearly separate from ACB haplotypes. However, the geographical pattern of haplotype distribution is less clear for both ACB and ECB, supporting that there has been frequent gene flow among the geographic populations in the Tianshan Mountains.Our results reveal that there is an asymmetrical gene flow pattern between the four populations. The population migratory pathways between these different populations show inflow into HC and YN, outflow from XY, and that MNS maintained a flow balance. Bayesian divergence time dating based on the These findings indicate that the Tianshan Mountains are less likely a barrier to gene flow of the two species. Ostrinia furnacalis and Ostrinia nubilalis, which are sympatric in the Yili River Valley in Xinjiang, China. Our study is based on the historical gene flow analyses of two species by using three mitochondrial DNA  and four nuclear DNA  markers obtained from representatives of HC (Huocheng), YN (Yining), XY (Xinyuan) and MNS (Manasi).Tianshan Mountain provides a model for studying biological evolution and speciation. Here we assess the evolutionary history of the  SeveralPrimula nutans and Primula fasciculata  and the Holocene (present to 0.0117 Mya) are located in the northwestern China and provide an excellent model for inferring biogeographical processes associated with species divergence according to proposed biogeographic hypotheses , and the ECB (European corn borer), O. nubilalis (Hübner) (Crambidae: Pyraustinae), are worldwide maize pests that cause substantial yield losses in corn production , Ostrinia showed that assortative mating is evident among sympatric ECB and Ostrinia scapulalis (the Adzuki bean borer) in Europe based on pheromone race‐specific gene marker genetic analysis  to reveal gene flow direction of ACB and ECB in sympatric regions within the Yili area and on both sides of the Tianshan Mountains; (ii) to determine the demographic history and diffusion pathways between the different populations of ACB and ECB; and (iii) to explore whether any possible barriers in the Tianshan Mountains are expected to impact their gene flow and biogeographical patterns on both sides, which may follow the “mountain isolation” hypothesis.However, any assumption about biogeographic patterns of genetic variation and gene flow patterns within and between 22.1Adult specimens were collected from four regions in Xinjiang, China during July and August 2017, including three regions in the Yili Kazak Autonomous Prefecture Area where ACB and ECB co‐occur. We defined these four regions, namely Huocheng, Yining, Xinyuan, and Manasi, as the four geographical populations HC, YN, XY, and MNS, respectively. The former three populations, namely HC, YN, XY, are located in the Yili River Valley, and MNS population is situated on the other side of the Tianshan Mountains. Specimens were collected using both sweeping net and light trap methods and have been stored as pinned dry collection at the Entomological Museum, Northwest A&F University, Yangling, Shaanxi Province, China (NWAFU). Some of the adult specimens were preserved in 95% ethanol at −20°C at the NWAFU. We selected 15–19 samples from each population for DNA extraction Appendix .2.2COI (cytochrome coxidase subunit I), COII (cytochrome coxidase subunit II), Cytb (cytochrome b), EF‐1α (elongation factor‐1α), Wingless (wingless), RPS5 , and CAD (carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase). PCR primers used in this study are listed in Appendix COI), 52°C (COII), 45 °C (Cytb), 57°C (EF‐1α), 60°C (Wingless), 58°C (RPS5), 45°C (CAD), and an extension for 1 min at 72°C; a final extension step of 72°C for 10 min. PCR products were detected by 1.0% agarose gel electrophoresis, stained with ethidium bromide, and visualized by ultraviolet light. DNA products were subsequently bidirectionally sequenced by AuGCT Biotech.Genomic DNA was extracted from one or two legs of adult specimens using the DNeasy DNA Extraction Kit (TransGen Biotech) following the manufacturer's protocols. Three mitochondrial and four nuclear gene markers were obtained including: COI: MK286109‐MK286173; COII: MK286174‐MK286237; Cytb: MK286307‐MK286374; EF‐1α: MK303832‐MK303897; Wingless: MK292217‐MK292285; RpS5: MK286238‐MK286306; CAD: MK292148‐MK292216.Sequence chromatograms were checked by using Chromas Pro v 2.6.5 (Technelysium Pty Ltd.) and edited through MAFFT , haplotype diversity (Hd), and nucleotide diversity (π) for each population of different species using DnaSP v 6.12.03  was used to investigate the genetic variation between and within populations in the Arlequin 3.5 software  of different geographical populations for each species was calculated in the DnaSP v 6.12.03 software , 0.05 < Fst < 0.15 (moderate differentiation), 0.15 < Fst < 0.25 (relatively large differentiation), and Fst > 0.25  (H27) was selected as the outgroup in the phylogenetic analyses. ML and BI analyses were executed in the PhyloSuite v 1.2.2 software  in Tracer v 1.7.2  between geographical populations of the two species and within each species was calculated in the DnaSP v 6.12.03 software , 0.25 < Nm < 0.99 (Moderate frequency), Nm > 1 (high frequency)  = 2.4. In the concatenated nuDNA alignment, 80 nucleotide positions were variable sites, and 46 were parsimony informative. The analysis of nucleotide composition showed a low A + T content (47.5%). The estimated ratio of transition to transversion was (R) = 3.8.Variation of the seven gene markers of ACB and ECB was shown in Appendix Hd) of the four geographical populations  ranged from 0.923 to 1.000 based on MTD while the values of Hd were ca. 1.000 based on NUD. The nucleotide diversity (π) ranged from 0.01046 to 0.01810 and from 0.00347 to 0.00451 based on MTD and NUD, respectively. For ECB, haplotype diversity (Hd) of the three geographical populations  ranged from 0.345 to 0.733 based on MTD while the values of Hd were ca. 1.000 based on NUD. The nucleotide diversity (π) ranged from 0.00030 to 0.00095 and from 0.00357 to 0.00535 based on MTD and NUD, respectively. In general, the total sampling individuals of ACB (0.934 and 0.01366) exhibited a higher Hd and π values than ECB (0.505 and 0.00051) based on MTD, while ACB (1.000 and 0.0041) and ECB (1.000 and 0.00492) showed an approximate Hd and π values based on NUD.Genetic diversity indices were summarized separately for each population and species Appendix . For ACBFst was applied to detect the genetic differentiation within the two species (ACB and ECB) across their different geographical populations . Our results showed that the Fst values between the ACB and ECB ranged from 0.58109 to 0.66714 and from 0.19175 to 0.37037 based on MTD and NUD, respectively. Moreover, for the three sympatric regions , the values of Fst between the ACB and ECB ranged from 0.58109 to 0.66139 and from 0.22095 to 0.33358 based on MTD and NUD, respectively  was high; however, genetic differentiation among different geographical populations was low for both ACB and ECB.The AMOVA analyses revealed a different pattern of genetic variation based on MTD and NUD. The total variation based on MTD was mainly explained by groups of both species (55.83% of the variation) and within populations (45.65% of the variation), while the total variation based on NUD was mainly accounted for by within populations (73.97% of the variation), compared with that among groups (25.60%) or among populations within groups  for MTD and R = −.23 (p = .2986 > .05) for NUD, respectively. These results indicated that there was no significant correlation between genetic differences and geographical distances in either dataset.The mean genetic distance (K2P) values between different geographical populations ranged from 0.014 to 0.019 and from 0.004 to 0.006 based on MTD and NUD, respectively Appendix . The cor3.2We identified 27 haplotypes based on MTHD Figure . A totalp < .5, Iss (0.0301) ≪ (0.7851)). For the MTHD dataset, phylogenetic analyses inferred with BI and ML methods were slightly different  values were all positive based on MTD, while Tajima's D and Fu's Fs values (−0.90279 and −55.649) were all negative based on NUD. For ECB, Tajima's D and Fu's Fs values were −1.42151/−0.55939 and −1.282/−9.594 based on MTD and NUD, respectively  and four geographical populations . For the total sampling individuals of ACB, Tajima's Appendix . In gene3.4O. latipennis was estimated at 0.022 Mya , the age of most recent common ancestor (MRCA) of ACB individuals was 0.003 Mya (95% HPD: 0.002–0.006 Mya), and the age of MRCA of ECB individuals was 0.002 Mya  based on MTD and NUD , the values of Nm between the ACB and ECB ranged from 0.13 to 0.18 and from 0.25 to 0.575 based on MTD and NUD, respectively. For ACB, the Nm values between the different populations ranged from −97.15 to 2.17 and from −20.31 to 13.79 based on MTD and NUD, respectively  based on MTD were low; however, gene flow among different geographical populations was high for both ACB and ECB.The UD Table . At the BSSVS analyses Table  showed t44.1Our findings indicate that there is high genetic differentiation between the two species (ACB and ECB) in Xinjiang. However, the degree of genetic differentiation among different geographical populations within each species is low. These results are consistent with previous studies reported that a low level of genetic differentiation is present among Chinese populations of ACB , not in the Yili River Valley where the overall topography of the middle vein of the Tianshan Mountains opens westward. However, gene flows were found between MNS of ACB and the other three populations of ACB and ECB in the Yili River Valley, suggesting that the Tianshan Mountains might have many refuges according to the “glacial refugia” hypothesis Avise, . In this4.3Given the divergence time between ACB and ECB is approximately 0.008 Mya (0.003–0.0127 Mya), this implies that the interspecific divergence occurred in Holocene in the Yili area. Meanwhile, the second intraspecific divergence time of ACB was estimated at 0.003 Mya (0.002–0.006 Mya) and observed at 0.002 Mya (0.0045–0.001) for ECB. Our findings indicate that large‐scale climate changes associated with tectonic events during early Holocene may have caused considerable fragmentation of habitats for sympatric separation of ACB and ECB in the Yili area ; writing – original draft (lead); writing – review and editing (lead). Zhaofu Yang: Project administration (lead).The authors declare no competing interest.Appendix S1Click here for additional data file.


Cantharellus enelensis is a recently described species of the golden chanterelles found in eastern North America. At present, the genetic diversity and population structure of C. enelensis are not known. In this study, we analyzed a total of 230 fruiting bodies of C. enelensis that were collected from three regions of Canada: near the east and west coasts of Newfoundland (NFLD), with 110 fruiting bodies each, and around Hamilton, Ontario (10 fruiting bodies). Among the 110 fruiting bodies from each coast in NFLD, 10 from 2009 were without specific site information, while 100 sampled in 2010 were from each of five patches separated by at least 100 m from each other. Each fruiting body was genotyped at three microsatellite loci. Among the total 28 multilocus genotypes (MLGs) identified, 2 were shared among all three regions, 4 were shared between 2 of the 3 regions, and the remaining 22 were each found in only 1 region. Minimal spanning network analyses revealed several region-specific MLG clusters, consistent with geographic specific mutation and expansion. Though the most frequently observed MLGs were shared among local (patch) and regional populations, population genetic analyses revealed that both local and regional geographic separations contributed significantly to the observed genetic variation in the total sample. All three regional populations showed excess heterozygosity; for the eastern NFLD population, we reject the null hypothesis of Hardy–Weinberg equilibrium (HWE) at all three loci. However, the analyses of clone-corrected samples revealed that most loci were in HWE. Together, our results suggest that the three discrete regional populations of C. enelensis were likely colonized from a common refugium since the last ice age. However, the local and regional populations are diverging from each other through mutation, drift, and selection at least partly due to heterozygous advantage.The wild edible mushroom  Cantharellus enelensis is a popular edible mushroom which is native to eastern North America. This golden chanterelle was historically regarded as part of the Cantharellus cibarius species complex until it was described in 2017 [enelensis” in Latin refers to its presence in NL , where it is the most common species of chanterelle, both on the island of Newfoundland (NFLD) and in mainland Labrador. However, C. enelensis has also been reported in several other parts of eastern North America, from Quebec, Canada to Wisconsin, USA [C. enelensis in NFLD provides an excellent opportunity for studying this group of edible mushrooms.Cantharellus enelensis is an ectomycorrhizal fungus and, consequently, its growth and distribution pattern are closely related to its host plants. The golden chanterelles found in NFLD are associated with Abies balsamea , Picea glauca (white spruce), and Picea mariana (black spruce) [Picea and Abies suggested that they originated in western North America and then spread to eastern North America and through the Bering Strait to Eurasia in the early Miocene [C. enelensis in NFLD, its sources could be similarly from southern parts of North America or somewhere else, or both. At present, C. enelensis has not been reported from outside of eastern North America. Different colonization histories would show different population structures. For example, if there was a single colonization event of NFLD by one individual that subsequently spread across the island, we would see very limited genetic diversity. In contrast, if there were multiple colonization events on the island by different genotypes, genetic differentiations between regional populations may be observed. spruce) . The bio Miocene ,4. The l Miocene ,6. The cC. enelensis. Among these 230, 110 were from five patches at a site near Harbour Grace in eastern NFLD and 110 were from five patches at a site near Stephenville in western NFLD. We also included 10 fruiting bodies from Hamilton, Ontario for comparison. These samples were used to address the following questions: First, how much diversity is there within and between the eastern and western NFLD populations of C. enelensis? Second, is there evidence for genetic differentiation between the two regional populations? Third, how does the Hamilton, Ontario population of C. enelensis compare with the two NFLD populations, and do they have completely different genotypes? Finally, are there signatures of selection and local adaptation?In this study, we collected and analyzed a total of 230 fruiting bodies of Cantharellus formosus, a close relative of C. enelensis [Aspergillus fumigatus, a saprotroph and opportunistic human pathogen, and found that arctic isolates share many alleles with each other and with those collected from other parts of the globe. Similarly, STR markers also helped reveal the genetic diversity and population structure of the wild edible mushroom Leucocalocybe mongolica in Mongolian Plateau, East Asia [C. formosus using a representative set of fruiting bodies of C. enelensis. The markers that showed high amplification success and were polymorphic for our samples were used to genotype all 230 fruiting bodies. Aside from addressing the questions above, using the same markers allows us to compare the allelic profiles of the two closely related species, C. formosus from western North America and C. enelensis from eastern North America.To address these questions, we used short tandem repeat (STR) markers developed previously to analyze nelensis . STR marnelensis ,10,11,12http://www.esri.com/software/arcgis/arcgisfor-desktop (accessed on 15 April 2022)). In NFLD, 10 fruiting bodies of C. enelensis were sampled from each region on 15 August 2009 and 200 fruiting bodies, 20 from each of 10 local populations, five from eastern NFLD and five from western NFLD, were sampled on 18 and 21 August 2010. Voucher specimens from each site were preserved, as reported previously [The samples analyzed here were from three geographic regions. One region is located in eastern NFLD, near Harbour Grace  and another is in western NFLD, at Sandy Point, near Stephenville . The third is in Hamilton, Ontario, Canada . The three discrete collection places are shown in eviously .C. enelensis. In addition, the whole-genome sequence of C. enelensis is not available to help guide the development of potential polymorphic markers that are specific for this species. Instead, in this study, to identify the genotypes of our specimens, we screened the markers described in the study of C. formosus by Dunham et al. [C. enelensis samples, even after four attempts using different annealing temperatures. For another marker (Cf339), while we were able to obtain amplification products from our C. enelensis samples, DNA sequence analyses of amplified products from six representative specimens of C. enelensis (two from each geographic region) revealed no polymorphism within or among these six specimens. However, for the remaining three markers, we successfully obtained amplified products for all specimens and fragment length polymorphisms were observed within and/or among specimens of C. enelensis. Those three markers were Cf113, Cf126, and Cf642, and they were respectively labelled using the fluorescent tags HEX, ATTO565, and 6-FAM (IDT). These primers were used to amplify the three STR regions using the following PCR program: pre-denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 54 °C for 45 s, elongation at 72 °C for 60 s, and a final elongation at 72 °C for 10 min. STR fragments at the three loci were separated using capillary electrophoresis at the MoBix Lab of McMaster University and then analyzed by the STR genotyping software Osiris 2.4  [There is currently no genotyping marker for discriminating strains of m et al. . Among tMD, USA)  to deterNa) and the effective number of alleles (Ne) were calculated for each of the three STR loci in each population using the program GenAlEx 6.5 [uh) were also generated. The unbiased allelic diversity measures the probability that two randomly drawn alleles in the population will be different. Similarly, the unbiased genotypic diversity measures the probability that two randomly drawn individuals will have different multilocus genotypes. Both diversity indices have values that range from 0 to 1. A value of 0 means no variation, where all samples have the same allele at a specific locus or all samples have the same multilocus genotype. In contrast, an uh value of 1 means high genetic variation, where any two randomly drawn alleles in the population will be different or any two randomly drawn specimens will have different multilocus genotypes. To determine whether the populations differ significantly in their mean uh and mean Ne, two-tailed Student’s t-tests were conducted.Allelic diversity including the number of unique alleles  to quantify the contributions of geographic separation to the overall observed genetic variations . Two hieC. enelensis in this study, minimum spanning network (MSN) trees were generated using the R package poppr [To visualize the distributions and genetic relationships among multilocus genotypes (MLGs) within and among the local and regional populations of ge poppr  based onK-values ranging from 1 to 7, with 10 independent runs per K value. For each run, the initial burn-in period was set to 200,000 with 1,200,000 MCMC iterations. To determine the most probable value of K, the ΔK method was used and implemented in Structure Harvester [K-cluster affiliations of individual fruiting bodies and geographic populations based on STRUCTURE results, respectively.The number of potential genetic clusters within our sample was inferred by STRUCTURE v2.3.4 based on an admixture model . Models arvester . In addiarvester  and CLUMarvester  were useC. enelensis is not known. However, the patterns of allelic associations that we observe could provide critical information about its potential mode of reproduction in natural populations. Specifically, in a heterothallic species and in the absence of selection, the genotype frequencies at each locus should approach HWE. In contrast, in homothallic species, we would expect significant deviations of genotype frequencies from HWE, seen as a heterozygote deficit [C. enelensis were in HWE [The Hardy–Weinberg equilibrium (HWE) test was conducted to determine whether the observed genotype frequencies at each locus were different from those expected under the hypothesis of random mating and no selection. At present, the mating system of  deficit . The Gene in HWE ,17.C. enelensis. As in other species in the genus Cantharellus, C. enelensis is an ectomycorrhizal fungus in which most of its biomass is allocated in the soil and tree roots, with fruiting body formation being only a temporary phenomenon induced by environmental cues. In nature, genetic individuals may occupy a range of areas from 5 m2 to upwards of 250 m2 and produce multiple fruiting bodies [C. enelensis populations were conducted for both the total sample data and the clone-corrected data.In this study, we collected and analyzed above-ground fruiting bodies of g bodies . To reduNa, (ii) effective alleles Ne, and (iii) unbiased haploid genetic diversity uh. However, the differences among the three regional populations in each of the three allelic diversity indicators were not statistically significant. Specifically, the p values for the pairwise comparisons in uh between eastern NFLD and western NFLD, between eastern NFLD and Hamilton (Ontario), and between western NFLD and Hamilton were 0.36, 0.37, and 0.19, respectively. For the number of observed alleles and effective number of alleles, the differences were also statistically nonsignificant . Together, the results  observed alleles  results  indicateBased on allelic and genotypic information at the three STR loci, we found a total of 28 multilocus genotypes (MLGs) among the 230 fruiting body specimens collected from the two regions in NFLD, as well as the Hamilton, Ontario population. Each region contained multiple MLGs. The distributions of the MLGs within and among the three regions are shown in Briefly, the numbers of MLGs for the eastern NFLD, western NFLD, and the Hamilton samples were 15, 14, and 7, respectively. The numbers of private MLGs for the 3 regions were 10, 8, and 5, respectively. The frequencies of individual MLGs within each regional population are indicated by the size of the pie diagrams. Among the 28 MLGs detected, 15 were represented by only 1 fruiting body each, with 6 found in the eastern NFLD population, 6 in western NFLD population, and 3 in the Hamilton population. The remaining 13 genotypes were shared by 215 fruiting bodies distributed within and among the populations. Specifically, two MLGs were shared among all three regional populations while four MLGs were shared by two of the three populations. Bruvo’s genetic distances were calculated among the 28 MLGs, and their relationships are shown in C. enelensis in Canada.Aside from analyzing the relationships among specimens from the three regions, we also analyzed the samples separately from within the eastern and western NFLD populations to identify the distributions and relationships among local sites within a region. With only 1 exception (Eastern population 1), all local populations (patches) consisted of more than 1 MLG, ranging from 2 to 8 (mean of 4.2) MLGs per patch. There were 11 MLGs in total in the eastern regional population, of which 3 MLGs were represented by only 1 specimen each while 8 were shared by more than 1 specimen within and among the 5 local populations. Of these eight shared MLGs, three were represented by only one local population and the other five were shared by two or more local populations. The most frequently shared MLG was found in 25 specimens from 4 local populations. For the western NFLD regional population, there were 12 MLGs, among which 5 were represented by only 1 specimen each while 7 were shared by more than 1 specimen within and among the 5 local populations, among which 1 MLG was represented by only 1 population and the other 6 MLGs were represented by fruiting bodies from 2 or more local populations. The most frequently shared MLG was found in 22 specimens from all 5 local populations in western NFLD. Together, the results indicated both unique and shared multilocus genotypes among local and regional populations of Comparisons between the observed and expected genotype frequencies revealed differences among the three loci and the three regional populations. In the nonclone-corrected sample, the null hypothesis of HWE was rejected for all three loci in the eastern NFLD population. However, for the western NFLD population, the null hypothesis of HWE was not rejected at two of the three loci. Similarly, for the Hamilton, Ontario population, the genotype frequencies at two of the three loci did not deviate significantly from HWE. After clone-correction, all three loci in the western NFLD population and two of the three loci in eastern NFLD population were in HWE. Locus Cf126 showed a significant excess of heterozygotes for both the Hamilton, Ontario and the eastern NFLD populations, and as a result, we can reject the null hypothesis of HWE .In all six comparisons between observed and expected heterozygosities in the two regional populations in NFLD, the observed heterozygosities were all higher than those expected under random mating in the nonclone-corrected data. In the clone-corrected data, four of the six comparisons had higher observed than expected heterozygosities . In the The number of potential genetic clusters within the 230 specimens were inferred using STRUCTURE v2.3.4 based on the alleles and genotypes detected at the three marker loci. The admixture model-based simulations indicated that the most suitable ΔK was one, which means the samples collected from the three sites belonged to one inter-breeding metapopulation.Due to the small sample size (10 specimens) of the Hamilton population, we excluded this population from the population genetic differentiation analyses and focused on the two regional populations from NFLD. Specifically, we conducted two-level hierarchical AMOVA analyses for both the nonclone-corrected sample and the clone-corrected sample, excluding the 20 samples collected in 2009. AMOVA results showed that most of the observed genetic variations were found within individual local populations, accounting for 53% and 81% of the total genetic variance for the nonclone-corrected dataset and the clone-corrected dataset, respectively .p < 0.001 in both cases)  . After ch cases) . RegardlC. enelensis. For example, the number of unique and effective alleles were similar among the three regional populations: 3 MLGs were shared between the Hamilton and NFLD populations , and STRUCTURE analyses showed that all 3 regional populations belonged to one inter-breeding metapopulation. However, both unique alleles and unique MLGs were observed in the 2 provinces (NFLD and Ontario), with the NFLD population having 20 MLGs not found in Hamilton while the Hamilton population had 5 MLGs not found in NFLD.Both similarities and differences were observed between the NFLD population and the Hamilton, Ontario population of n = 3), the number of effective alleles , and the unbiased gene diversity  were similar between the two years. Together, these results suggested that even a small number of fruiting bodies (20 fruiting bodies with 10 on each coast) were able to capture most of the gene diversity observed in the 200 specimens in NFLD.We obtained fruiting bodies from two consecutive years (2009 and 2010) from both the east and west coasts of NFLD. Despite the differences in sample sizes between the two years, our comparisons indicated that the observed number of alleles  heterozygosity. Indeed, the three regions differed in the mean and ranges of temperatures, soil physical and chemical conditions, humidity, and flora and fauna [C. enelensis fruiting bodies from eastern NFLD are known to be frequently infested with slugs and maggots (fly larvae), while those in western NFLD are relatively free of slugs and maggots [The observation of broad excess heterozygosity within each of the three regional populations of nd fauna ,33,34,35 maggots . At presC. enelensis has a heterothallic mating system. Microscopic observations of C. enelensis indicated that each basidium typically bears four to eight basidiospores. In basidiomycete mushrooms, basidia with four or more basidiospores each typically can be either homothallic or heterothallic [C. enelensis could also be rejected. In secondarily homothallic species, most basidia typically have two basidiospores on each, with each basidiospore containing two genetically distinct nuclei. Examples of secondary homothallic mushrooms include Amanita exitialis [Agaricus bisporus [C. enelensis is bipolar or tetrapolar. Further research such as whole-genome sequence analyses and crossing experiments using germinated basidiospores are needed to determine whether the mating system in this species is bipolar  or tetrapolar  [C. cibarius have been germinated 6–12 weeks after being spread on modified Fries medium [The frequent finding of HWE in the clone-corrected samples suggests that othallic ,38. Howexitialis  and the bisporus ,41. Howectorial) ,43,44,45Alces alces), the observed low genetic variation in the NFLD population was consistent with founder effects due to the small number of founders that were introduced to the island [Pinus strobus), a potential host plant of C. enelensis, population genetic analyses revealed little difference between the NFLD population and those in the other regions in eastern Canada, with limited evidence for genetic isolation by geographic distance [C. enelensis populations is consistent between these two species. Such a pattern likely reflects their common ancestral populations and, in evolutionary terms, the relatively short time  that these northern populations of both species have been established since the last glaciation event.Because of its historical isolation, the Island of Newfoundland has been the subject of biogeographic and population genetic studies for several species, including humans, other animals, and plants ,39,46,47distance . Though C. enelensis. While the three STR markers showed both unique and similar patterns of variations among the local and regional populations, more markers will likely enable us to identify more MLGs for the 230 analyzed fruiting bodies. However, the small number of MLGs identified here was not entirely due to the lack of discriminating power of our markers. With the current allelic diversity , assuming random mating, the presence of 3 alleles at a locus  could generate 6 possible diploid (or dikaryotic) genotypes and the presence of 4 alleles at a locus  could generate 10 possible diploid genotypes each. If the 3 STR marker loci are not tightly linked with each other, a total of 6 × 10 × 10 = 600 MLGs could be produced from these 3 marker loci. Thus, the large discrepancy between the potential number of genotypes  and the observed number of genotypes  suggest that the overrepresented MLGs in the population were not completely due to lack of power in our markers. Instead, selection and/or genetic linkage have likely played significant roles.In this study, we used three STR markers to investigate the patterns of genetic variations within and among geographic populations of C. cibarius. However, sequence analyses based on both the ITS and the TEF1 sequences revealed its distinctiveness, resulting in it being named a different species (C. enelensis) in 2017. We note that though these two gene fragments are commonly used for identifying species of Cantharellus, they often contain one- or two-base insertion or deletion (indel) mutations (as well as base substitutions) between haplotype copies within an individual dikaryotic genome, making it difficult to obtain clean sequences from PCR products for these two genes [C. formosus, in western North America [The golden chanterelle in NFLD had been historically labeled as its European counterpart, C. enelensis, from three separate areas of Canada were analyzed using three STR loci. Most genetic variations were found within local populations. However, statistically significant genetic differentiations were found among regional populations. Our allelic and genotypic analyses revealed that the three regional populations were likely colonized from the same refugium population after the retreat of the last ice age about 10,000 years ago. The region-specific alleles and genotypes were likely due to new mutations accumulated since their colonization events. Tests for HWE revealed evidence for heterozygote advantage within all three regional populations, but to different degrees. Together, our analyses identified evidence for mutation, drift, and selection in the natural populations of this species.In this study, the genetic diversities and population structures of a gourmet edible mushroom, https://sugarhillinn.ca/cuisine/, accessed on 1 June 2022), and there is an active commercial harvest of this species [C. enelensis in other regions in NFLD and how they might be related to these two populations are not known. Similarly, with only 10 fruiting bodies analyzed, the genetic diversity of C. enelensis outside of NFLD remains largely unknown. While the current samples allowed us to identify several differences among the local and regional samples, additional samples from other parts of NFLD, as well as from across the broad distribution range in eastern North America, are needed to identify the potential center(s) of diversity for C. enelensis and its potential routes of dispersal. Furthermore, only three markers are available to analyze our samples, and this number is lower than those in many recent population genetic studies of fungi. Although these three markers allowed us to reveal several population genetic features of this species, developing and applying additional molecular markers, especially those located in different parts of both the nuclear and mitochondrial genomes, will enhance our ability to discriminate the genotypes among fruiting bodies and provide better assessments of both local and regional population structures. Overall, as an ectomycorrhizal mushroom, its productivity in nature depends on many biotic and abiotic factors, including the host species, nutrients, and physical and chemical properties of the soil [C. enelensis need to be collected and analyzed to help identify the key factors influencing their reproduction and fruiting in nature.The golden chanterelle is a popular wild delicacy around the world. In NFLD, aside from the two locations analyzed here, other locations have also reported golden chanterelles, including a restaurant named after this species at the heart of the Gros Morne National Park ( species . At presthe soil ,50. To e


N = 60) and estimated common genetic assessment metrics, predicting minimal hybridization with non‐native trout, low diversity, and declining diversity over time. We further hypothesized genetic metrics would correlate positively with MPVA‐estimated abundance and negatively with extinction probability. We uncovered several instances of hybridization that pointed to immediate management needs. After removing hybridized individuals, cautious interpretation of low effective population sizes (2–63) suggested reduced evolutionary potential for many LCT populations. Other genetic metrics did not decline over time nor correlate with MPVA‐based estimates of harmonic mean abundance or 30‐year extinction probability. Our results demonstrate benefits of genetic monitoring for efficiently detecting hybridization and, though genetic results were disconnected from demographic assessment of conservation status, they suggest reduced evolutionary potential and likely a higher conservation risk than currently recognized for this threatened fish. We emphasize that genetic information provides essential complementary insight, in addition to demographic information, for evaluating species status.The current extinction crisis requires effective assessment and monitoring tools. Genetic approaches are appealing given the relative ease of field sampling required to estimate genetic diversity characteristics assumed related to population size, evolutionary potential, and extinction risk, and to evaluate hybridization with non‐native species simultaneously. However, linkages between population genetic metrics of diversity from survey‐style field collections and demographic estimates of population size and extinction risk are still in need of empirical examples, especially for remotely distributed species of conservation concern where the approach might be most beneficial. We capitalized on an exceptional opportunity to evaluate congruence between genetic diversity metrics and demographic‐based estimates of abundance and extinction risk from a comprehensive Multiple Population Viability Analysis (MPVA) in a threatened fish, the Lahontan cutthroat trout (LCT). We sequenced non‐native trout reference samples and recently collected and archived tissue samples of most remaining populations of LCT ( Importantly, we included reference samples  and Yellowstone cutthroat trout , trout not native to this region but which have been introduced to the LCT range and present a primary threat to the genetic integrity of LCT via introgression. Effective population sizes were interpreted cautiously due to reliance on small and multi‐cohort samples, as we discuss, but provide a standing gage of genetic health and evolutionary potential for LCT. We were also able to test for genetic erosion  attained from available survey‐style field collections, and demographic‐based estimates of abundance and extinction risk from a comprehensive population viability analysis applied across the historical range of a threatened fish in the inland United States. We first applied modern sequencing techniques to recently collected (~2015–2017) tissue samples to estimate common genetic monitoring metrics across most remaining conservation populations  recently applied across LCT stream populations  libraries were prepared with well and plate barcodes  as well an admixture analysis with all samples  to characterize potential rainbow and YCT ancestry. Due to biases associated with highly variable coverage among samples, we did not call genotypes for our PCA or admixture analyses. For the PCA, we instead used the Analysis of Next Generation Sequencing Data (ANGSD) software package  assumed to represent the three parental species or subspecies  to these clusters, where we might most expect to see cluster switching). Accordingly, we used K = 3 for our interpretation of introgression in LCT samples. Since both the IBS PCA method implemented in ANGSD and ngsAdmix are designed to work with low sequencing coverage, these analyses were not restricted to bait‐targeted RAD tags, resulting in 43,263 SNPs contributing to both the PCA and admixture analyses. Although Rapture enriches for bait‐targeted loci, a significant proportion of reads still come from off‐target loci  in any set of samples collected from a given stream in a given year using an exact test , doPost 2 , and postCutoff 0.95 (genotype posterior probability cutoff). Furthermore, since genotype calling requires higher sequencing coverage and off‐target loci are usually sequenced at a much lower depth, this analysis was restricted to sites from the bait‐targeted RAD tags (using the rf flag). These quality filtering criteria resulted in 897 SNPs out of 60,589 total interrogated genomic sites. Using the snpR package , one of the most important indicators of evolutionary potential , observed heterozygosity (ho), Watterson's θ (θW), Tajima's θ (θT), the proportion of polymorphic SNPs (out of the 893 loci that were polymorphic in any population), and θ skew ([θT − θW]/[(θT + θW)/2])  can be indicative of nonequilibrium processes  to examine how divergence between them changes as overall genetic diversity changes.We then estimated several commonly‐used metrics of population genetic/genomic diversity . We expected populations with higher genetic diversity  to have higher abundance estimates and lower 30‐year extinction probabilities. To test this hypothesis, we fit a series of a linear mixed‐effect models with each genetic statistic above as a fixed effect explaining MPVA extinction and the harmonic mean abundance estimates (see below), with a nested random effect of creek within basin where possible as a covariate. Where these models failed to converge, we modeled the genetic metric as a fixed effect with no random effects. These comparisons included a subset of our samples, i.e., only conservation populations  were used as possible independent variables.Because these models showed little relationship between genetic and MPVA metrics (see Section 2.8Ne) in samples collected from the same population in different years. Additionally, to determine if temporal changes in genetic diversity were associated with among‐population differences in 30‐year extinction risk or changes over time in estimated harmonic mean abundance, we constructed simple linear regressions between these MPVA estimates and the per‐year average change in each of our diversity metrics for populations where we had both MPVA estimates and multiple years of genetic data (n = 12). We did not use any random effects for these models due to the small number of samples with both MVPA estimates and multiple years of genetic data. We again used Holm's . While most individuals were clustered along a continuous distribution of large values of PC1  fell closer to the non‐native trout samples in the PCA and had more than 1% hybrid ancestry in the clustering analysis when combining both RBT and YCT ancestry. The majority of these individuals had mostly RBT hybrid ancestry . Beyond this, however, a handful of individuals had much higher hybridization levels, with a clear break at about 20% LCT‐RBT hybrid ancestry. Strikingly, in addition to the Truckee River and Columbia River reference RBT samples  using Random Forest models yielded no predictive power and an extremely high error rate for out‐of‐bag samples for harmonic mean abundance or 30‐year extinction probability .Lahontan cutthroat trout populations were estimated to have a broad range of abundances and extinction probabilities .Twenty populations had temporal sampling, with the span between sample periods ranging from 2 to 20 and averaging 12 years Table . Further4Our results highlight benefits of broad‐scale genetic assessment for a threatened species distributed across remote, difficult‐to‐sample watersheds, but also demonstrate several complexities of this approach. Drawing from population samples collected largely during standard agency sampling across this fish's range, we were able to characterize multiple aspects of the conservation status of LCT, most of which are concerning. For instance, introgression is a primary threat to the conservation of cutthroat and other trout species  as potentially useful for gaging abundance , particularly in small populations, has warranted caution when assuming a consistent relationship for monitoring changes in demographic attributes , meaning the influence of mixture LD would cause our estimates to fall somewhere from 70% to 90% of the actual Ne . Though genetic metrics did not relate to abundance or extinction risk from our MPVA models (see below), a few notable patterns arose when considering all populations including transplanted or supplemented populations not evaluated in the MPVA comparisons. The very lowest values generally characterized transplanted populations that were established in the 1970s and 1980s outside of the historical range of LCT (in the Steens Mountains in Oregon) using small founder groups, as has been typical in the management of this fish . Though relatively large for LCT in terms of stream miles, these streams have recently been found to be comprised of more marginal/intermittent habitat than previously appreciated  ranked at the lower end for several genetic metrics  and abundance  was significantly associated with abundance and occupancy but explained only a small portion of variation (Grundler et al., Other recent studies have been mixed in finding correlations between genetic/genomic and demographic metrics. Across a suite of desert lizards nucleotide diversity (do capture influences other than time‐bound abundance estimates (Doyle et al., Undoubtedly, however, the established strength of genetic metrics is that they The authors declare no conflict of interest.Tables S1‐S2Click here for additional data file.Appendix S1Click here for additional data file.


Riparia diluta in Central and East Asia consists of three genetically deeply differentiated lineages which vary only gradually in morphology but broadly reflect traditional taxonomy. We detected no signs of gene flow along the eastern edge of the Qinghai‐Tibetan plateau between lowland south‐eastern Chinese R. d. fohkienensis and high‐altitude R. d. tibetana. Largely different breeding and migration timing between these low and high altitude populations as indicated by phenology data suggests that allochrony might act as prezygotic isolation mechanism in the area where their ranges abut. Mongolian populations of R. d. tibetana, however, displayed signs of limited mixed ancestries with Central Asian R. d. diluta. Their ranges meet in the area of a well‐known avian migratory divide, where western lineages take a western migration route around the Qinghai‐Tibetan plateau to winter quarters in South Asia, and eastern lineages take an eastern route to Southeast Asia. This might also be the case between western R. d. diluta and eastern R. d. tibetana as indicated by differing wintering grounds. We hypothesize that hybrids might have nonoptimal intermediate migration routes and selection against them might restrict gene flow. Although further potential isolation mechanisms might exist in the pale sand martin, our study points towards contrasting migration behaviour as an important factor in maintaining evolutionary diversity under morphological stasis.Morphological differentiation associated with evolutionary diversification is often explained with adaptive benefits but the processes and mechanisms maintaining cryptic diversity are still poorly understood. Using genome‐wide data, we show here that the pale sand martin  Studies on the evolution of ecological and phenotypic diversity within rapidly multiplying lineages ‐ often adaptive radiations ‐ have been instrumental for our understanding on how adaptive processes trigger speciation  of Central and East Asia provides such a promising natural system to study diversification under morphological stasis. Its four recognized subspecies overlap in morphometrics and have no diagnostic differences in plumage features  based on a comprehensive geographic sampling of pale sand martin populations around the Qinghai‐Tibetan plateau, we aimed to test (a) if the deep phylogeographic structure indicated by mtDNA is also reflected in genome‐wide variation and (b) if the phylogeographic lineages indeed overlap in morphospace and can thus be considered cryptic. Finally, we (c) investigated, if gene flow between phylogeographic lineages is actually reduced in areas of potential contact.22.1Riparia individuals  on their breeding grounds  for further analyses  was used as outgroup and one sequence of R. d. indica .To investigate whether the deep phylogeographic structure among subspecies in mtDNA can be recovered with a more extensive geographic sampling, we amplified a fragment (~850 bp) of the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene of all 149 individuals using the same protocol as Schweizer et al. . Sanger 2.3R. riparia collected in Zhengzhou, Henan province, China in 2017  using Jellyfish  . We only retained uniquely mapped reads and biallelic SNPs with <10% missing data .Genotyping by sequencing (GBS)  for each K and the number of ancestral populations K set from 2 to 10. The optimal K was evaluated using clumpak  based on individual genotype likelihoods using PCAngsd ) and logarithmic (ln) Euclidean geographic distances between populations using the r package ade4  and computed pairwise  Mantel,  to half R. riparia for the characterization of ancestral states in populations of R. diluta. For populations or R. riparia, the folded SFS was used.Nucleotide diversity (π) of each population was computed using angsd , subspecific identity of the others was based on breeding colony origin. Eight traits were measured following Eck et al.  were available stemming mainly from museum specimens. After log‐transformation of measurements, morphometric differences among subspecies were explored using a principal component analysis (PCA) on the correlation matrix using the function prcomp of the r package stats.To investigate the extent of morphological differences between subspecies of k et al. : length k et al. . In an ak et al.  from whi2.7R. diluta occurs in different climate zones and across a broad altitudinal range, we assessed differences in seasonal occurrence patterns (phenology) between different geographic regions corresponding to the supposed distribution of the three subspecies. To this end, we used data from our own fieldwork and compiled records of R. diluta from the two citizen science databases ebird (https://ebird.org/home) and BirdReport of China (http://www.birdrecord.cn/). Each record was allocated to three periods in each month, that is, before the 10th, between the 10th and the 20th, and after the 20th. The three geographic regions were defined as follows: (a) south and central China east of the Qinghai‐Tibetan plateau below 3000 m above sea level (asl) corresponding to the supposed breeding distribution of R. d. fohkienensis, (b) Qinghai‐Tibetan plateau above 3000 m asl corresponding to the supposed breeding range of R. d. tibetana, (c) the region of China north‐west of the Qinghai‐Tibetan plateau corresponding to the supposed breeding distribution of R. d. diluta.As 33.1R. diluta and 11 haplotypes in R. riparia. Within R. diluta, BI and ML phylogenetic reconstructions recovered well supported clades mostly consistent with the distribution ranges of the morphologically‐defined subspecies  did not cluster with the remaining samples from this region and R. d. tibetana, and was instead found in the R. d. diluta clade. The positions of these different clades as well as that of R. d. indica were not robustly supported throughout. The monophyly of the haplotypes found in R. riparia was only supported in ML analyses, but not with BI.The final ND2 alignment was 851 bp in length with 32 haplotypes in s Figure : One cla3.2R. diluta and R. riparia after aligning trimmed GBS reads to the collared sand martin draft genome. These SNPs were distributed across most autosomes except for the two microchromosomes 16 (1.22 Mb) and 29 (4.21 Mb), and there was a highly significant positive correlation  between the number of SNPs called per chromosome and chromosome size . In a PCA on both species, R. diluta was clearly separated from R. riparia in PC1 (22.12% of the variance), while PC2 (18.34% of the variance) separated R. d. fohkienensis and the remaining samples of R. diluta . PC2 (10.95% of variance) separated north‐western Chinese R. d. diluta from a cluster containing R. tibetana from the Qinghai‐Tibetan plateau and all samples from Mongolia.The heterozygosity of the collared sand martin reference genome was estimated to be 0.1%. In total, we obtained 7640 autosomal SNPs in the data set including a Figure . It is ny Figure , R. d. fK = 4 as the best‐fitting number of ancestral populations with a somewhat lower likelihood for K = 3 .Admixture analyses resulted in 3 Figure . Howevers Figure . For K =R. d. tibetana. Overall differentiation between populations was very high with FST = 0.225 (p < .0001). When the Mongolian population was grouped in an AMOVA with R. d. tibetana, the total variation explained by the subspecies reached FCT = 0.216 with very little differentiation within these groups . When the Mongolian population was alternatively grouped with R. d. diluta, the proportion of the explained variation dropped to FCT = 0.186 and differentiation within the groups of populations increased accordingly . We thus consider the population in Mongolia to belong to R. d. tibetana outside the assumed distribution range of this subspecies. With this assignment, pairwise FST between populations within subspecies ranged from 0 to 0.051 . Nucleotide diversity was overall similar across R. d. diluta and R. d. tibetana populations from the Qinghai‐Tibetan plateau with no detectable relation to longitude or latitude . The highest values of nucleotide diversity were found in the three analysed populations of R. riparia (π ranging from 6.69 × 10−3 to 7.49 × 10−3).Comparison of molecular diversity showed further that the Mongolian birds featured the highest nucleotide diversity among all analysed 3.3R. diluta. Overall, a Mantel test detected a highly significant relationship between genetic differentiation and spatial distance between populations , indicating considerable dispersal among populations over large distances.Given distances of thousands of kilometres between the analysed populations, we tested for the importance of isolation by distance in genetic differentiation between and within subspecies of 3.4R. diluta samples presented here revealed gradual differences between subspecies with some overlap  from Schweizer et al. , while PC2 (30.43% of variance) tended to differentiate subspecies of these two pairs. However, there was gradual overlap overall.The analysis of three morphological traits only, which enabled the inclusion of additional 120 individuals  of pale sand martin from China were compiled for the years 1985–2021. Pale sand martins were found throughout the year but with distinct differences between the regions occupied by the three subspecies. In north‐western China in the breeding area of s Figure . A simila Figure . The fira Figure . Althougc. Table  while R.4Riparia diluta contains multiple deep evolutionary lineages despite extremely subtle and gradual morphological variation among them and little genetic differences within lineages over large geographic areas. Differentiation patterns in nuclear SNPs are largely consistent with phylogeographic structure in mtDNA  ‐ might act as prezygotic isolation mechanisms. Speciation through allochrony, that is, temporal segregation of breeding populations as an important contributor to reproductive isolation, has been invoked in several animal groups, including birds (e.g., Bearhop et al., R. d. fohkienensis and R. d. tibetana remain to be investigated. Moreover, the involvement of additional factors cannot be excluded, especially ecological differences as R. d. fohkienensis and R. d. tibetana might have adapted to different climate regimes. Comparatively fast rates of climate‐niche evolution could be expected in the temporally and spatially heterogeneous climate in the region of the Qinghai‐Tibetan plateau and central and south China (c. Lawson & Weir, We detected no evidence of gene flow in the potential areas of contact between lowland R. d. fohkienensis and R. d. tibetana at the edge of the plateau given that the closest sampled populations were 512 km apart. Hybrid zones between bird species may be considerably narrower, but in bird taxa with recent divergence, relatively wide hybrid zones of >100 km have also been found (Price, R. diluta, rendering it difficult to make predictions about expected potential hybrid zone width. In R. riparia, its sister species, however, 7% of juveniles were found >199 km away from their natal colonies in consecutive years in Britain (Mead, R. diluta by low genetic differentiation and a lack of patterns of isolation by distance within evolutionary lineages. Finer‐scale sampling of the potential contact area would be necessary to specifically test for interbreeding. Given the absence of any traces of admixture in the sampled populations (e.g. Figure R. d. fohkienensis and R. d. tibetana has probably ceased comparatively long ago given that divergence of R. d. fohkienensis has been estimated to have occurred about 1.2 million years ago (Schweizer et al., However, we cannot exclude the existence of an undetected contact zone with hybridization between d Price, . Hybrid d Price, . This hain Mead,  indicatiin Mead, . ConsideR. d. diluta and R. d. tibetana and thus the possibility of hybridization between them may not be too unexpected. It is probably more surprising that the signals of limited autosomal introgression were not detected at the north‐western edge of the Qinghai Tibetan plateau (cf. Figure R. d. tibetana. The comparatively high levels of nucleotide diversity in the Mongolian population of R. d. tibetana might also be a consequence of admixture with R. d. diluta. Further sampling in western Mongolia would be needed to determine the extent and spatial structure of hybridization between the two.Our results clearly show closer relationships between R. d. diluta and Mongolian R. d. tibetana have been identified as potentially limiting extensive hybridization between two subspecies of barn swallow Hirundo rustica which show contrasting migration routes around the Qinghai‐Tibetan plateau (Scordato et al., R. d. diluta and R. d. tibetana might be in contact in Mongolia is indeed located in a migratory divide in different bird species complexes (Irwin & Irwin, R. d. diluta has been recorded to winter in the north‐western part of the Indian Subcontinent and rarely on the Arabian peninsula, and probably takes a western route around the Qinghai‐Tibetan plateau with migration documented in north‐western South Asia (Rasmussen et al., R. d. tibetana might winter in Southeast Asia and circumnavigate the Qinghai‐Tibetan plateau in the east or follow river valleys at its south‐eastern edge and winter in the northern, central and north‐eastern Indian Subcontinent (own data; Rasmussen et al., R. d. diluta and R. d. tibetana in Mongolia. Secondary contact of populations with different migration directions could trigger the evolution of prezygotic isolation mechanisms (Scordato et al., R. diluta, we hypothesize that the secondary contact might be too recent and/or admixture not extensive enough for this process to play a role. However, mating behaviour of the evolutionary lineages remains to be thoroughly investigated to reveal potentially hitherto undocumented differences.The mountains of Central Mongolia between north‐western Chinese populations of 4.3Riparia diluta represents different levels, and we hypothesize that they might differ in their strength of reproductive isolation. While genomic differentiation of R. d. fohkienensis suggests effective reproductive isolation for a comparatively long time period, footprints of introgression were found between R. d. diluta and R. d. tibetana. In the absence of obvious sexually selected traits, seasonal migration behaviour might be an essential factor in maintaining genetic integrity of these morphologically cryptic evolutionary lineages.Genome‐wide differentiation between the morphologically cryptic lineages of the pale sand martin Seasonal migration behaviour has for a long time been considered as playing an important role in generating and maintaining evolutionary divergence (reviewed in Turbek et al., Manuel Schweizer and Gerald Heckel conceived the study; Qindong Tang, Manuel Schweizer and Gombobaatar Sundev collected samples with assistance from Yang Liu. Qindong Tang conducted laboratory work and analysed the data together with Gerald Heckel and Manuel Schweizer and assistance from Reto Burri; Alexander Suh and Reto Burri contributed materials; Qindong Tang, Manuel Schweizer and Gerald Heckel wrote the manuscript with support from all authors.Figure S1‐S5Click here for additional data file.Table S1‐S5Click here for additional data file.