Extreme events are increasing globally with devastating ecological consequences, but the impacts on underlying genetic diversity and structure are often cryptic and poorly understood, hindering assessment of adaptive capacity and ecosystem vulnerability to future change. Using very rare “before” data we empirically demonstrate that an extreme marine heatwave caused a significant poleward shift in genetic clusters of kelp forests whereby alleles characteristic of cool water were replaced by those that predominated in warm water across 200 km of coastline. This “genetic tropicalisation” was facilitated by significant mortality of kelp and other co-occurring seaweeds within the footprint of the heatwave that opened space for rapid local proliferation of surviving kelp genotypes or dispersal and recruitment of spores from warmer waters. Genetic diversity declined and inbreeding increased in the newly tropicalised site, but these metrics were relative stable elsewhere within the footprint of the heatwave. Thus, extreme events such as marine heatwaves not only lead to significant mortality and population loss but can also drive significant genetic change in natural populations. Because extreme events, by definition, exceed normal environmental conditions, they drive significant mortality, range shifts and the transition to novel ecosystem states6. However, in contrast to the ecological impacts of extreme climatic events which are often obvious and well documented, their impact on underlying patterns of genetic diversity and structure and the implications for adaptability to future change is obscure and often unknown10, particularly for marine systems.Climate change is increasing the intensity and frequency of extreme events11 and are superimposed on a background of ocean warming. Marine heatwaves are increasing in frequency and duration globally12 with significant consequences for coastal marine species, communities and the ecosystem services they provide17. While we have an emerging understanding of how ocean warming may affect the genetics of marine species e.g.19, there are few empirical studies demonstrating the genetic impact of extreme marine heatwaves  to more closely resemble tropical latitudes and transforming them into novel ecosystem configurations6 following the heatwave. Although there is some suggestion that low genetic diversity of Ecklonia populations prior to the heatwave may have mediated its ecological impact10, the consequence of the heatwave for subsequent genetic diversity and structure of Ecklonia forests is currently unknown. Given that co-occurring kelp species suffered cryptic loss of genetic diversity and potential directional selection following the heatwave20 it is possible that Ecklonia populations were similarly affected. Unravelling whether the heatwave caused genetic changes in Ecklonia forests will help predict vulnerability to future warming and extreme events.Marine forests, comprised of foundation species of kelps and seaweeds that underpin biodiversity throughout temperate regions3. We were fortunate to have genetic samples (DNA) of Ecklonia forests prior to the heatwave, allowing us to provide a rare empirical demonstration of the impacts of an extreme marine heatwave on subsequent genetic properties of kelp forests. We predicted that the heatwave would precipitate loss of genetic diversity given the large mortality of Ecklonia at many sites and a poleward shift in warm-affinity alleles driven by an increased flow of prevailing currents during the reproductive period of Ecklonia immediately following the heatwave31.Because extreme events are largely unpredictable in time and space, robust empirical assessment of their impacts are often hindered by a lack of “before data”24. The MHW engulfed ~ 2,000 km coastline and on the southwest coast, where this study was focused, the coastal region experienced temperature anomalies of 2.5–5.5 °C above the long term maxima  was also stronger during the autumn reproductive period of Ecklonia31 immediately following the heatwave.In the Austral summer of 2010/11 the west coast of Australia experienced an unprecedented marine heatwave (MHW) with temperature anomalies exceeding anything seen in at least 140 years of recorded history Fig. 1Ecklonia radiata is the dominant kelp throughout Australasia30 where it underpins immense biodiversity33. It is a perennial species with an alternation of generations life cycle with macroscopic sporophytes (sampled here) alternating with microscopic gametophytes30. E. radiata does not reproduce asexually, except for the distinctive E. brevipes morphotype in Hamelin Bay34 which was not sampled here. Samples of E. radiata sporophytes for genetics were collected across 800 km of coastline that experienced the highest thermal anomalies during the heatwave 35 and after (2018) the 2011 heatwave from Kalbarri , Geraldton , Jurien Bay, Marmion and Hamelin. There were no large thermal anomalies in the intervening years except a mildly cooler period from 2015 to 2017. We recognise that our sampling represents only 2 points in time and without more temporal replication within before and after periods we cannot definitively attribute any genetic change to the heatwave. However, we believe that the rarity of “before” genetic data in studies that seek to examine impacts of unpredictable extreme events warrants this sampling design (see37 for examples of similar designs with 2 time points for genetic data). Moreover, given that temporal replication of genetic data in any study is rare, our data is a critical first step in assessing genetic impacts of climatic change on natural systems. Both before and after the heatwave, all samples were haphazardly collected (but at least 1 m apart) on scuba or snorkel between two and ten meters depth in an area approximately 20 × 20 m32. Collected samples (n = 30–40 kelp plants per site) were free from epiphytes and were preserved in silica gel desiccant (2006) or snap frozen in liquid nitrogen (2018).39 and in 2018 using Qiagen DNeasy plant kits and cleaned with Qiagen Powerplant Pro kits using a modified manufacturer protocol. Specifically, raw material was soaked in lysis buffer for 24–48 h at 65 °C to aid lysis and DNA was washed on the spin filter 3 times. DNA quality and quantity were checked using picogreen and gel electrophoresis (2006) or on a Qubit and Nanodrop the run on a LabChip nucleic acid analyser (Perkin Elmer) for quality control. Even DNA samples from 2006  that had a lower concentration of DNA amplified well. All DNA samples were genotyped at the same time (in 2019) using n = 8 new polymorphic microsatellite markers developed for E. cava40 and E. radicosa41 and tested on E. radiata (supplementary Table 2O). PCRs conditions were composed of an initial denaturation at 95 °C for 1 min followed by 30 cycles each with denaturing at 95 °C for 15 s, annealing at 59 °C for 15 s and extension at 72 °C for 10 s. Fragment sizes were genotyped by capillary separation at the Australian Genome Research Facility using one individual with known genotype for each loci to avoid reading errors on all sequenced plates.DNA from 2006 was extracted as in42. No evidence of null alleles or scoring errors due to stuttering or large allele dropout was found in the whole data set. Overall, no more than 4% of missing data was allowed within a year with a maximum of 10% of missing data for one population across loci and 8.75% for one locus across populations. We tested for linkage disequilibrium between pairs of loci of the populations in ARLEQUIN 3.543 with a permutation procedure of 10,000 iterations. We found no consistent linkage disequilibrium between loci across populations, instead the standardised index of association  which may be a result of the significant population declines in this site, however, no data was available from prior to the MHW. Departure from panmixia was tested at each locus for each population using the function hw.test implemented in the package Pegas with the conventional Chi-square test. Significance of the deviation from Hardy–Weinberg equilibrium was addressed by a Markov Chain (MC) algorithm in ARLEQUIN  but departure was never found for more than one locus in each population. The total number of alleles or allelic richness (Na) per locus, the observed (Ho) and expected (He) heterozygosity and the number of private alleles (unique to a particular site) were estimated for each population before and after the MHW using GENALEX 6.546 and GENETIX 4.0547 also used to estimate pairwise Weir and Cockerham’s FST. MLH  was calculated using the R package InbreedR at each site before and after the heatwave as this metric has previously been shown to be a sensitive metric of thermal change48. FST and FIS were computed per population in FSTAT 3.249. Significance of FIS values were estimated by running 800 permutations of alleles among individuals for each population and a 5% indicative adjusted nominal level of P = 0.00125. Patterns of genetic structure were inferred by the Bayesian clustering method implemented in STRUCTURE v2.3.4 (https://web.stanford.edu/group/pritchardlab/structure) allowing admixture and independent allele frequencies enabling individuals to be assigned to different genetic groups if admixed50. This method is based on a Markov chain Monte Carlo (MCMC) approach to optimise genotypic equilibrium (linkage equilibrium and HWE) within each cluster. For a K (likely number of genetic clusters) ranging from 1 to 4, with no geographic prior, 10 independent runs were conducted with a burn-in period of 100,000 steps and 500,000 MCMC replications. Both mean log likelihood L(K) and ΔK were used to estimate the likely number of clusters and obtained on Structure Harvester51. Final bar plots were generated with the POPHELPER Structure Web App52 with the alignment of the assignment scores obtained over the ten runs for each K. Discriminant analyses of principal components (DAPCs) were computed using the R package adegenet53. This technique is used to maximise the inter-group component of variation of a principal component analysis and visually explore major genetic patterns among populations before and after the MHW. We retained 25 principal components out of the 69  and 7 linear discriminants. The Bayesian informative criterion (BIC) was minimal for K = 8 which corresponds to the number of sampled populations when differentiating before from after. For every DAPC, loci contributions have allowed us to better identify the loci and alleles driving the observed changes in allelic frequencies between populations. Global analysis of molecular variance (AMOVA) weighted over loci was performed in ARLEQUIN using 1,000 permutations to statistically assess the differences amongst clusters. Given our unbalanced design with extirpation of kelp populations at Kalbarri and genetic change largely attributable to one site, we chose to do AMOVAs separately on data before and after the heatwave. Locus-by-locus outputs were investigated to look for main loci contributions. Pairwise FST tests were used to examine site differences before and after the heatwave.Allele sizes were manually scored using the microsatellite plugin on the Geneious Prime software (v11.1). Ambiguous loci were classed as missing data. Prior to conducting statistical analyses, we checked for genotyping errors  using MICRO-CHECKER54. Conversely, heterozygote deficiency is a sign of expansion. Comparisons between expected heterozygosity under HWE and the expected heterozygosity computed from the actual observed number of alleles under the assumption of mutation-drift equilibrium were performed using the two-phase model (TPM) on the BOTTLENECK v1.2.02 software55 (https://www1.montpellier.inra.fr/CBGP/software/Bottleneck/bottleneck) recommended for microsatellite data54 and was run for 1,000 iterations with a variance among multiple steps in TPM equal to 1255. The Wilcoxon test was used to depict significant heterozygosity deficiency and only the resulting P values were presented in Table 56 to test for an association between linearised pairwise population genetic differences (FST/(1 − FST)) and coastal distance.Detections of recent mass mortality due to drastic effective reductions in response to extreme events such as MHW can be inferred by a heterozygosity excess compared to the expected heterozygosity based on the observed number of allelesFIS) became significantly positive at Marmion and Jurien Bay after the heatwave indicating that these sites became inbred  and this temporal difference was ~ 8 times greater than the same temporal comparison between any other pair of sites  and Geraldton after the heatwave. Moreover, Jurien Bay appears to have become slightly more similar to Kalbarri/Geraldton in the DAPC plot after the heatwave  cluster prior to the heatwave, became the dominant alleles at Marmion after the heatwave, concurrent with a decline in alleles which were overwhelmingly dominant in the higher latitude (cool) cluster. Thus, we define these changes as “genetic tropicalisation”. This phenomenon is a specific case of a hybrid zone, whereby populations at the interface of species or genetic clusters periodically mix to various degrees62 which can also be climate mediated62.Tropicalisation is traditionally defined as an increase in relative abundance of warm-water species6. Kelp recruitment is often low within dense canopies and turnover largely reliant on the change in physical conditions created as kelp density thins or gaps are created following mortality or loss of adult plants64. Indeed, Marmion initially suffered ~ 25% decline in kelp cover due to the direct physiological effects of the heatwave6 which likely exceeded or approached the thermal tolerance of kelp and co-occurring seaweed species66. This opened up space for rapid recruitment by either (1) locally surviving ‘warm’ genotypes20 or (2) propagules transported from lower latitudes assisted by the increased poleward flow of prevailing currents67 at the time of the heatwave31. These scenarios of recolonization are not mutually exclusive and likely occurred together, although our microsatellite data can only provide definitive evidence for the latter. If genetic tropicalisation was a result of proliferation of surviving warm genotypes at Marmion it would imply there was selection against ‘cool’ genotypes. While this may indeed have occurred, selection cannot be inferred from neutral microsatellite data. Nevertheless, Marmion suffered the greatest loss of genetic diversity of any site (24–31% of expected and multi-locus heterozygosity), suggesting that there could have been some selection. Moreover, Marmion became inbred after the heatwave suggesting that breeding was among a smaller pool of surviving genotypes rather than a large influx of new ones. Regardless of the mechanism driving the change, the timing of the heatwave during the peak reproductive season for Ecklonia68, meant that the genetic tropicalisation signal was likely rapidly entrenched. The rapid recovery of kelp canopy cover at Marmion would have prevented any further major genetic turnover because the ecological driver of such turnover  was removed. Indeed, this genetic tropicalisation signal was evident even 7 years after the heatwave.Genetic tropicalisation at Marmion was likely facilitated by mortality of kelp that occurred as a direct result of the heatwave24 nor has it shown any evidence of ecological tropicalisation24, implying that no space was opened up for significant genetic turnover to occur. Indeed, the relatively greater stability of private alleles at Hamelin and presence of the lowest pairwise FST estimates between times of sampling indicate little genetic turnover at this site.For other sites for which we had before and after data (Hamelin and Jurien Bay) there was only a small genetic effect of the heatwave. Genetic diversity at Hamelin slightly increased, gaining a number of low frequency alleles characteristic of low latitude (warm) sites. Despite experiencing similarly large thermal anomalies, we contend that Hamelin only experienced mild genetic change because there was largely no mechanism for this process to occur. Hamelin did not experience significant loss of kelp during the heatwave presumably because its thermal threshold was not crossed24. While this loss provides a mechanism for genetic turnover, a large shift in neutral alleles could not take place as Jurien Bay was already part of the low latitude cluster and dispersal along this coast is predominately poleward67 with few remaining populations further north. Thus, recolonization following loss of kelp would not leave a genetic signature because recruits would most likely be sourced from within the same genetic cluster. Nevertheless, FIS became significant at Jurien Bay after the heatwave indicating inbreeding and Jurien Bay became slightly more similar to Kalbarri/Geraldton on the DAPC plot after the heatwave concurrent with a drop in the pairwise FST estimate between these sites indicating that the kelp population at Jurien Bay became slightly more genetically similar to its low latitude (warm) trailing edge neighbours, possibly a mild tropicalisation signal.Interestingly, there was only a small genetic effect of the heatwave at Jurien Bay, despite great loss of kelp cover (50–60%) and strong ecological tropicalisation20. Two co-occurring forest forming species Scytothalia dorycarpa and Sargassum fallax29, both showed strong declines in haplotype diversity following the heatwave20 with Scytothalia being completely extirpated from Jurien Bay25. Moreover, there were some signs of possible directional selection for Sargassum with specific genotypes dominating after the heatwave20. Nevertheless, genetic change from before to after the heatwave could also be simply due to one of many temporally stochastic processes that influence genetic turnover in populations. For example, temporal variation in oceanography69, reproductive success70, ecological processes (e.g. herbivory) or post-recruitment survival of different genotypes may result in significant temporal genetic change. Unfortunately there exist very few temporally replicated genetic datasets, particularly for seaweeds, to tease apart the role of such stochastic processes in driving genetic change71. The few existing temporally replicated seaweed studies show contrasting results. For example, Fucus serratus populations sampled 10 years apart across Europe, showed temporal genetic stability48 except in warm range edge populations that were thermally stressed. These populations experienced a 90% decline in abundance and greater homozygosity (decline in MLH), possibly as a result of selection favouring thermal tolerance48. Conversely, an extreme event (earthquake) in Chile resulted in a decline in Agarophyton chilense genetic diversity  immediately following the event, but recovery occurred after 2 years72. Given the weight of evidence we present here and across multiple species and genetic markers20, we find it likely the heatwave was responsible for the observed genetic changes. However, proper understanding of the genetic impacts of climate change and extreme events for marine organisms will require temporally replicated studies and efforts to collect baseline data from which to measure change.Although the correlative nature of this study and lack of more temporal replication means that we cannot definitively demonstrate that the heatwave caused the genetic changes seen here, genetic change seen in other seaweed species and markers following the heatwave is strong evidence to suggest that the heatwave did mediate genetic tropicalisation73. Genetic studies have shown that these banks can also be of mixed origin and age which maximises genetic diversity of recolonising populations74. Given that gametophytes of E. radiata appear to have broad thermal tolerances with lethal upper limits that are generally in excess of temperatures experienced in the field30, it is possible that recovery following heatwave-induced mortality of macroscopic sporophytes was partly from such a bank of microscopic forms. Recovery from a genetically diverse, mixed age or origin bank could explain the general maintenance of genetic diversity across most sites, even where there was large loss of canopy cover (Jurien). Moreover, recovery from an existing bank at Marmion may have dampened the impact of increased gene flow from lower latitudes. However, the difficulties in finding and sampling gametophytes in natural settings mean that large knowledge gaps remain regarding their role in population recovery following extreme events and kelp forest population genetics more generally.Kelps possess a microscopic gametophyte stage which may be able to tolerate different environmental conditions than sporophytes and potentially act as a short term ‘bank of microscopic forms’ for recolonisation following stressors76 and is outside the scope of this study. Nonetheless, while our study used neutral microsatellite markers and can only definitively infer change in gene flow, it is plausible that, because lower latitude populations had a greater thermal tolerance than higher latitude populations prior to the heatwave77, this greater tolerance would now be seen in Marmion due to an increase in low latitude “warm” genotypes. Given that reduction in temperature sensitivity of metabolic processes from cool- to warm-adapted populations has previously been demonstrated for several seaweed species along this coastline77 , we find this scenario likely. Unravelling whether the changes seen here in neutral microsatellite markers are reflected in those under selection (adaptive markers), along with manipulative experiments to assess thermal tolerance of different genotypes will be key for predicting future vulnerability of these kelp forests to ongoing climate change.It is interesting to speculate whether populations at Marmion are now more thermally tolerant than before the heatwave. Properly identifying adaptation in complex natural systems is a challenging task78, genetic signs of population expansion at Geraldton and Marmion (Table 12 and warm low latitude populations remain somewhat vulnerable due to inbreeding coupled with genetic erosion79, factors which may undermine long term adaptive capacity.It has been 9 years since the marine heatwave and while the lowest latitude kelp forests in Kalbarri have not yet recoveredon Table  suggest 81. In the case of Australian kelp forests, the use of interventionist measures may be one of the few routes left to address the alarming predicted range contraction of Ecklonia and extinction of other seaweeds83. These strategies include assisted adaptation and evolution, which are founded on the concept that incorporating stress tolerant genes or genotypes into extant populations can boost their resilience to future change83. This highlights a pressing need to identify where and when one may find such tolerant individuals and populations to apply such approaches. We suggest that extreme events may provide a natural laboratory for identifying tolerant populations or genotypes that could be targeted and utilised in assisted adaptation approaches. Recognising these potentially positive benefits of extreme events may present new opportunities to “future-proof” ecosystems against climate change.Global kelp loss has prompted calls for more interventionist management approaches to actively ensure long-term persistence against climate changeSupplementary file1


Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.Johne’s disease (JD) is an infectious wasting condition of ruminants caused by  Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of chronic wasting disease in domestic livestock species commonly known as paratuberculosis or Johne’s disease (JD) , awarded by Indian Council of Medical Research (ICMR), New Delhi, India. IAEC vide reference number IAEC/CIRG/16-17 dated: 12.05.2016, confirmed that this project do not have any ethical issue. Samples were collected only for laboratory analyses and avoided unnecessary pain and suffering to animals and were not from any endangered or protected species.900-PCR before screening through i-ELISA and Mce-ELISA.Serum samples were collected from four domestic livestock species  suspected for MAP infection. Animals sampled were both from farm and farmer’s herds and flocks located in two states {(Uttar Pradesh (UP) and Madhya Pradesh (MP)} of country. A total of 251 goats , 53 sheep , 117 buffaloes , and 33 cattle  were included in this study. Animals were in variable age mainly from females and they have mixed physical conditions and 30–35% of the animals were suffering from clinical to advance clinical JD. All these animals were ear-tagged for identification.For the collection of blood samples goats and sheep by restraining manually by side of chest and gently holding the the head towards one side. Cattle and buffaloes were properly restrained in cattle crush and blood (2–3 ml) was collected in vacutainers, after properly sterilizing the jugular vein with 70.0% ethyl alcohol. Institute Animal Ethics committee (IAEC) of the CIRG, Mathura, UP, India has approved the technical program of the project. Samples were stored at -20°C until screened. Status of the animals with respect to MAP infection was determined using ‘Gold Standard’fecal culture, fecal microscopy and fecal IS900, IS1311 PCR and IS1311 PCR-REA  Tween-20). Uncoated surfaces were blocked (100 μl/well) with blocking buffer (3.0% skimmed milk) for one hour at 37°C. Following three washes with washing buffer, 100 μl of diluted serum (1:50) in serum dilution buffer (1.0% BSA) were added in duplicate to each well. Plates were incubated at 37°C for one hour, emptied, and washed three times with washing buffer. Secondary antibodies used in this assay were peroxidase-labelled anti-species whole IgG antibody produced in rabbits , at 1:8000 dilution for goats and sheep and 1:6000 for cattle and buffaloes in 1xPBS. 100 μl of secondary anti-species antibody was added to each well and incubated for 50 minutes at 37°C. After washing four times, 100 μl of chromogenic substrate solution of O-Phenylenediamine dihydrochloride (OPD)  was prepared as per manufacturer’s recommendation and added to each well. Plates were incubated for 10–15 minutes in the dark at 37°C. Extent of the color development  was measured at the absorbance of 450 nm using Bio-RAD A robust and extensive validated indigenous ELISA kit based on semi-purified Protoplasmic antigen (sPPA) prepared from the characterized strain (S 5) 'Indian Bison Type' bio-type of MAP of goat origin (provided by CIRG) was used for the comparative studies. PPA used in this kit was harvested as whole cell sonicate. Lysate was centrifuged and supernatant was used as antigen after protein estimation. Initially, i-ELISA was developed for the screening of goats and sheep , 37, 38 S/P ratio for Mce: Optical densities (OD) were expressed as sample-to-positive (S/P) ratios as per Collins (2002) by the following formula .Sensitivity and Specificity:Sensitivity = True Positive X 100 / True Positive + False NegativeSpecificity = True Negative X 100 / True Negative + False PositiveStatistical differences between the results of Mce-ELISA and i-ELISA were analyzed by McNemar’s and kappa agreement was applied to measure the statistical significance between the results of the two tests .900 PCR, confirmed the presence of MAP (Colonies of the MAP reference strain (S 5) appeared on the slants of HEY medium. Colonies were small, straw color, translucent, hemispherical, raised with nipple, smooth, and glistening . Stainine of MAP .MAP2189-MAP2194 cluster organization was also conserved in both the strains. Based on Bioinformatics analysis, MAP2191 gene from MAP was of 1065bp and encodes for a typical linear Mce protein that has 354 amino acids with an estimated molecular weight of 37.510 kDa. Possible secondary structure of the MAP2191 protein was predicted using SOPMA website and analyzed results revealed that this protein consisted of 41.53% α-Helix, 16.95% extended strand, 11.58% β-turn and 29.94% random coils. C-terminal portion of MAP2191 protein was successfully expressed and purified in this study. Partial Mce-truncated protein sequence (amino acids 154 to 354) was chosen based on the predicted sequence to contain most dominant B- and T-cell epitopes. CLC software analysis also revealed that selected region of MAP2191 protein had high antigenic index and was more hydrophilic.Alignment and comparison of MAP K-10 and MAP (S 5) strain sequences illustrated highly conserved sequences, likely to be cross-reactive, as most epitopes were shared. Moreover, MAP2191 gene was successfully amplified by PCR. Agarose gel electrophoresis of PCR products showed a single band of 621bp amplicon (603bp mce gene and extra 18bp were used to restrict sites and bring the coding sequence in-frame) as positive for the truncated gene  expression vector between the restriction sites BamHI and HindIII and transformed into XL10-Gold ultra-competent cells for the high percentage of recombinant plasmid yields. Presence of the insert was approved by colony PCR and double digestion using restriction enzymes (mce-truncated gene in the pET28a vector.Whole DNA was isolated from MAP (S 5) strain, and target region of ted gene . Mce-truanalysis . Gene se enzymes . Gene semce-truncated plasmid was transformed into Rosetta/BL-21 cells. Transformed cells were induced for expression with optimized concentration of 1.5 mM IPTG  . Sample Of the 251 serum screened by Mce-ELISA and i-ELISA, 124 (49.4%) and 139 (55.4%) were positive, and 127 (50.6%) and 112 (44.6%) were negative, respectively . ComparaOf the 53 serum screened by Mce-ELISA and i-ELISA, 27 (51.0%) and 31 (58.5%) were positive and 26 (49.0%) and 22 (41.5%) were negative, respectively . I-ELISAOf 117 buffaloes serum samples screened by Mce-ELISA and i-ELISA, 81 (69.2%) and 84 (71.8%) were positive and 36 (30.8) and 33 (28.2%) were negative, respectively . ResultsOf 33 cattle serum screened, 18 (54.5%) and 21 (63.6%) were positive, and 15 (45.5%) and 12 (36.4%) were negative by Mce-ELISA and i-ELISA, respectively . The 3 . The res(a) Comparison of Mce-ELISA with i-ELISA. Sensitivity of Mce-ELISA was lower 84.8, 87.1, 92.8 and 85.7%, but specificity was higher 94.6%, 100.0%, 90.0 and 100.0% in goats, sheep, buffaloes, and cattle, respectively (ectively .(b) Comparison of Mce-ELISA with i-ELISA. Sensitivity of i-ELISA was higher 95.1, 100.0, 96.3 and 100.0%, but specificity was lower 83.4%, 84.6%, 83.3 and 80.0% in goats, sheep, buffaloes and cattle, respectively , respectively .MAP, the cause of JD in domestic livestock , has alsE. coli, invading non-phagocytic HeLa cells [mce operons of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium smegmatis under different growth conditions and found the expression of mce1, mce3, and mce4 operons present in tissues collected from infected guinea pigs and rabbits. Hou et al. [mce genes were expressed by Mycobacterium avium during growth in macrophages. Another previous study also revealed presence of mce and PE/ PPE family genes in MAP as virulence factors [M. avium subsp. avium (MAC) and MAP which encode for different mce genes that were analogous in all the mycobacteria, indicating their functional importance.Proteins of Mce-family of pathogenic mycobacteria have been associated with virulence by enhancing invasive power and survival in macrophages and various mammalian cell lines , 43–46. La cells , 24, 47.La cells  analyzedu et al.  found th factors . In a st factors , showed MAP2191 gene is one of the mce5 operons (MAP2189 to MAP2194) of the MAP; mce5 cluster which has previously been identified as important for invasion and survival of bacilli within macrophages and also virulence of MAP [MAP2191 gene was based on the identification of putative B- and T-cell epitopes in MAP proteins encoded by the mce5 gene cluster and asses their antigenicity during infection. Using Bioinformatics analyses, we identified B- and T-cell epitopes in the C-terminal region of the MAP2191 putative Mce protein. The selected region of MAP2191 protein had a high antigenic index and was more hydrophilic as crevealed by the CLC software analysis. In this study, we generated and cloned a truncated MAP2121 DNA sequence, and subsequently generated the His-tagged recombinant protein in an E. coli host. Our purification method with Ni-NTA agarose resin was very effective and we obtained a large amount of soluble and purified Mce-truncated peptide.e of MAP , 26. Eige of MAP , 52. TarCurrently, antigen-based ELISA tests for detecting MAP with a mixture of proteins are available including avian or johnin, a MAP-PPD, whole-cell sonicated extract, protoplasmic antigens etc., . BecauseThis in house i-ELISA kit has been validated with other commercial ELISA kits , recombinant secretory proteins based cocktail ELISA (c_ELISA) and natural secretory proteins-based ELISA test , 56, 57 et al. (2015) compared i-ELISA (Indigenous g-ELISA) with commercial antigen-based b-ELISA and commercial sr-ELISA and found 100% sensitivity. In another report, 91.4% sensitivity of i-ELISA was observed when compared with EV-ELISA kit [Our earlier studies reported enhanced sensitivity and specificity of the i-ELISA as compared to the other commercially available ELISA kits for the diagnosis of Johne’s disease in domestic livestock , 39. ForLISA kit . These aLISA kit , 38.M.bovis antibodies. Therefore, this test could be a better choice for screening of paratuberculosis especially in the region where bovine-TB is endemic. Overall the Mce-based ELISA was comparable to the the highly sensitive and robust ‘i-ELISA test’, indigenously developed for the screening of livestock population.In our study, Mce-truncated protein yielded higher absorbance values against the confirmed positive pooled serum samples, and low-level (but above background) antibody reactivity was also observed with confirmed negative pooled serum samples. The ELISA test using Mce-truncated protein was able to discriminate between the most positive and negative serum samples used in this study. However, the Mce-based ELISA showed a relatively higher specificity than i-ELISA but suffered from slight reduction in sensitivity. These results highlighted the potential capability of Mce-truncated ELISA in preventing false-positive reactions that occur when a milieu of antigens is used in i-ELISA tests. Mce-ELISA has shown perfect agreement with i-ELISA and there is no cross-reactivity with anti-In conclusion, this is the first report of a Mce surface protein-based ELISA as new modified ELISA test, as diagnostic test against MAP infection. Affinity of purified Mce-truncated protein was detected by serum antibodies from animals infected with JD and supported the hypothesis that MAP Mce protein was immunogenic and highly expressed protein during MAP infection. True achievement of this study was the isolation and expreseeion of the Mce protein, ‘a new antigen’ with the huge potential to be further exploited not only as a diagnostic tool but also a new target antigen for the development of a highly effective vaccine for the control and eradication of the incurable Johne’s disease in domestic livestock.S1 Appendix(DOCX)Click here for additional data file.S1 Raw Images(DOCX)Click here for additional data file.


Ficedula albicollis. Repeatability was found for two social cues: previous year conspecific breeding success and previous year heterospecific abundance. Yet, additive genetic variances for these two social cues, and thus heritabilities, were low. This suggests that most of the phenotypic variation in the use of social cues and resulting conspecific and heterospecific social environment experienced by individuals in this population stems from phenotypic plasticity. Given the important role of social information use on ecological and evolutionary processes, more studies on genetic versus environmental determinism of social information use are needed.Social information use for decision‐making is common and affects ecological and evolutionary processes, including social aggregation, species coexistence, and cultural evolution. Despite increasing ecological knowledge on social information use, very little is known about its genetic basis and therefore its evolutionary potential. Genetic variation in a trait affecting an individual's social and nonsocial environment may have important implications for population dynamics, interspecific interactions, and, for expression of other, environmentally plastic traits. We estimated repeatability, additive genetic variance, and heritability of the use of conspecific and heterospecific social cues (abundance and breeding success) for breeding site choice in a population of wild collared flycatchers  Animals face multiple fitness‐related decisions that they must make adaptively to maximize fitness. To reduce uncertainty in decision‐making, animals need information about environmental quality  that an individual experiences. For example, the use of the presence and/or success of breeding conspecifics as social cues leads individuals to be attracted to areas of higher conspecific breeding density and success  , which uses the presence and success of both conspecifics and ecologically close heterospecific competitors  as social information sources for making breeding decisions at different temporal and spatial scales  for breeding site choice. Our model species was a migratory passerine, the collared flycatcher  and consisted of 15 discrete deciduous forest patches interspersed with agricultural areas and/or roads. In each forest patch, nest boxes have been provided in excess since 1980 , and therefore compete with collared flycatchers  abundance and breeding success of conspecifics and heterospecifics (great tits) by collared flycatcher for breeding site (nest box) choice. The use of these social cues was assessed previously in the same population  that was used for calculating the weights assigned to the social cues (abundance or breeding success of conspecific or heterospecific neighbors) for each nest box j depending on its distance to the focal nest box i. Only the nest boxes j that were within the same forest patch as the focal nest box i were considered, thus restricting the calculation of social cues to intra‐patch level. The weights were calculated as e−(dij/α), where ijd is the distance between nest boxes i and j in meters . These weights thus exponentially decrease with increasing distance between the nest boxes, the rate of decrease being negatively correlated with the value of α. Hence, larger values of α mean that nest boxes over a larger neighborhood get a nonnegligible weight in the calculation of the social cue, or, in other words, that the social environment considered extends farther away from the focal nest box. A large value of α corresponds to a neighborhood including all or most nest boxes in the forest patch, and the maximum investigated value of α, 83 m, indeed leads to nonnegligible weight for nest boxes up to 248 m from the focal nest box. Conversely, with a small value of α, the neighborhood becomes essentially restricted to the few closest nest boxes , and the minimum investigated value of α, 1 m, includes only the focal nest box. To facilitate comparisons of the variance component estimates across social cues and to maintain consistency with the earlier phenotypic analysis  excluded the variation explained by the fixed effects, thus our estimates of repeatability and heritability are conditional on the fixed effects. However, in addition to affecting the access to social cues, age, dispersal status, and timing of breeding may also influence the use of the cues. For example, yearlings lack breeding experience and philopatric individuals benefit from familiarity with their forest patch that may influence their propensity to use social cues, and earlier settling breeders may use already available heterospecific social cues more often than later settling breeders that also have conspecific cues available . We note that the results were very similar irrespective of the fixed effects structure, thus the above issue in defining the VP unlikely affects the accuracy of the repeatability and heritability estimates. The analyses were restricted to the pairs for which the “status” variable could be defined for both female and male. In addition, some response variables had missing values, resulting in varying sample sizes  and, for older adults, breeding dispersal status  may affect access to information sources and thus the possibilities of using social information  was 0.00086. Further details on the pedigree are given in the Supporting Information, Section D.The full collared flycatcher pedigree was pruned to keep all individuals that had a phenotypic value for at least one response variable and their ancestors. This pruned pedigree contained 3557 individuals of which 2019 were phenotyped. Out of these phenotyped individuals, for 685 individuals both parents were known, for 61 only the mother was known, for nine only the father was known, and for 1264 both parents were unknown. These 1264 individuals were either parents of other phenotyped individuals, thus providing information for estimating the additive genetic and dominance genetic parameters, or individuals whose relatedness to other individuals was not known. In the latter case, although they did not provide information for the estimation of the additive genetic and dominance genetic parameters, they are included in the estimation of the other model components. Mean female‐male relatedness across breeding pairs  are design matrices relating individual observations to fixed and random effects, respectively. β is a vector of fixed effects, and ♀PI, ♂PI, Patch, Box, and e are vectors of random permanent individual , breeding patch, nest box effects, and residuals, respectively. The random effects and residuals were assumed to follow normal distributions with zero mean and variances VPI♀, VPI♂, VPATCH, VBOX, and VR, respectively. Less than 2% of adult collared flycatchers in our population mate with the same partner over years . Thus, we assume the covariance to be zero, but also note that, given the overall low VPI♀ and VPI♂ estimates (see Results), the covariances are also likely to be low and ignoring them should not affect the conclusions of this study. The random effects Patch and Box were included to account for potential consistent spatial variation between forest patches and nest boxes, respectively. They were fitted as random effects instead of fixed effects due to the high number of levels in both variables (15 forest patches and 846 nest boxes). Fixed effects β included the female and male “status” variables, date of initiation of nest building, and all their two‐ and three‐way interactions.We estimated individual repeatability of the use of social cues using univariate linear mixed models. Phenotypic variation within each response variable was partitioned into the following components:The total phenotypic variance for the use of social cues for breeding site choice, conditioned on the fixed effects, was estimated asFemale and male repeatabilities were estimated asThe total permanent individual variance in the population was estimated as the sum of female and male permanent individual variances:and the corresponding total repeatability in the populationRtotal were distributed away from zero.As repeatability estimates and their 95% confidence intervals are always positive, we cannot, for example, use the criterion “95% CI excludes zero” to define which response variables are repeatable. We instead include in the following analyses those response variables for which the posterior estimates of total repeatability For repeatable response variables (see Results), we estimated additive genetic variances (and heritabilities) using univariate “animal models”  are design matrices relating individual observations to female and male additive genetic (♀a and a♂) and dominance genetic (Dom♀ and Dom♂) random effects. The female and male additive genetic random effects were assumed to be jointly distributed following a multivariate normal distribution, MVN, wherePhenotypic variation for each response variable was partitioned into the following components:A is the additive genetic relationship matrix between all individuals calculated from the pruned pedigree, and ⊗ denotes the Kronecker product of the two matrices. VA♀ and VA♂ are the female and male additive genetic variances and CovA♀♂ is the cross‐sex additive genetic covariance. Information for estimating female additive genetic variance comes from the covariance between y‐values and relatedness of females, information for estimating male additive genetic variance comes from the covariance between y‐values and relatedness of males, and information for female‐male additive genetic covariance comes from the covariance between y‐values and relatedness between females and males. The female‐male additive genetic covariance thus is related to covariance between related females and males at the population level, not necessarily between a female and a male in a breeding pair . ♀Dom and ♂Dom were assumed to follow normal distributions N and N, where D is the dominance genetic relationship matrix between individuals, calculated from the pruned pedigree. We explicitly accounted for the dominance genetic variance because it may have a relatively important effect in natural populations . Inclusion of •PI is imperative in “animal models” if the dataset includes repeated measures of individuals  between pair members , the population‐level estimate of additive genetic variance, which determines the potential of the trait to respond to selection, needs to include the genetic effects of both individuals Bijma . This isce Bijma . Hence, and the corresponding total heritability asAlthough our approach accounts for the effects of female and male pair members, we note that the indirect genetic effects relevant for an individual's use of social cues for breeding site choice may not be limited to within the breeding pairs but may also include, for example, the other conspecifics (pairs) breeding in the neighborhood. If such indirect genetic effects exist, our estimates of “total” additive genetic variance and heritability are somewhat biased low compared to the real total additive genetic variance and heritability  previous year conspecific success (median [95% CI]: 0.27 0.16 to 0.37]) and (ii) previous year great tit abundance  for conspecific success in the previous year and (ii) –4.6 × 10−5 [–0.016 to 0.012] for great tit abundance in the previous year . Thus, we estimated heritabilities using models excluding the cross‐sex additive genetic covariance. These models suggested low heritabilities  –3.0 × 10We assessed the individual repeatability, additive genetic variance, and heritability of the use of con‐ and heterospecific social cues for breeding site choice in a population of wild collared flycatchers. Social information from both con‐ and heterospecifics was found to influence breeding site choice of collared flycatchers , confirming previous results based on different datasets .The data used in this article have been uploaded to Dryad  conspecific abundance in the current year, (b) conspecific abundance in the previous year, (c) conspecific success in the previous year , (d) great tit abundance in the current year, and (e) great tit abundance in the previous year as social cues for breeding site choice by collared flycatchers.Table S2. Comparison of the variance component estimates in the repeatability models with varying prior specifications.Table S3. Comparison of the variance component estimates in the quantitative genetic models (full models including the cross‐sex additive genetic covariance) with varying prior specifications.Table S4. Parameter estimates (posterior medians) and their 95% credibility intervals in the univariate GLMM estimating repeatability for the use of five social cues for breeding site choice in collared flycatcherTable S5. Parameter estimates (posterior medians) and their 95% credibility intervals in the univariate GLMM estimating repeatability for the use of five social cues for breeding site choice in collared flycatcherFigure S3. Estimates of female (red circles), male (blue triangles) and total (black squares) additive genetic variances (median ± 95% CI) in the use of (c) conspecific success in the previous year and (e) great tit abundance in the previous year as social cues for breeding site choice by collared flycatchers, based on the models with the full fixed effects structure, but excluding the cross‐sex additive genetic covariance (see Table S7).Table S6. Parameter estimates (posterior medians) and their 95% credibility intervals in the univariate GLMM estimating additive genetic variance and heritability for the use of two social cues for breeding site choice in collared flycatcher.Table S7. Parameter estimates (posterior medians) and their 95% credibility intervals in the univariate GLMM estimating additive genetic variance and heritability for the use of two social cues for breeding site choice in collared flycatcher.Table S8. Parameter estimates (posterior medians) and their 95% credibility intervals in the univariate GLMM estimating additive genetic variance and heritability for the use of two social cues for breeding site choice in collared flycatcher.Table S9. Parameter estimates (posterior medians) and their 95% credibility intervals in the univariate GLMM estimating additive genetic variance and heritability for the use of two social cues for breeding site choice in collared flycatcher.Table S10. Comparison of the repeatability estimates derived from the repeatability and the quantitative genetic models.Click here for additional data file.


Ne) was 84.7, and mean inbreeding (F) was 0.052 (SD 0.083). For 227 genotyped Border Collies that had available pedigree information (GenoPed), molecular and pedigree estimates of diversity were compared. A reference population  and their ancestors  were used to evaluate the diversity of the population that are contributing to the current generation. The reference population had a Ne of 123.5, a mean F of 0.095 (SD 0.082), 2276 founders (f), 205.5 effective founders (fe), 28 effective ancestors (fa) and 10.65 (SD 2.82) founder genomes (Ng). Removing TNS and NCL carriers from the genotyped population had a small impact on diversity measures , however, there was a loss of > 10% minor allele frequency for 89 SNPs around the TNS mutation (maximum loss of 12.7%), and a loss of > 5% for 5 SNPs around the NCL mutation (maximum 5.18%). A common ancestor was identified for 38 TNS-affected dogs and 64 TNS carriers, and a different common ancestor was identified for 33 NCL-affected dogs and 28 carriers, with some overlap of prominent individuals between both pedigrees. Overall, Border Collies have a high level of genetic diversity compared to other breeds.Maintaining genetic diversity in dog breeds is an important consideration for the management of inherited diseases. We evaluated genetic diversity in Border Collies using molecular and genealogical methods, and examined changes to genetic diversity when carriers for Trapped Neutrophil Syndrome (TNS) and Neuronal Ceroid Lipofuscinosis (NCL) are removed from the genotyped population. Genotype data for 255 Border Collies and a pedigree database of 83,996 Border Collies were used for analysis. Molecular estimates revealed a mean multi-locus heterozygosity (MLH) of 0.311 (SD 0.027), 20.79% of the genome consisted of runs of homozygosity (ROH ) > 1 Mb, effective population size ( As more inherited disorders and causative mutations are identified, a major concern is the level of genetic diversity available in the population and the impact of removing carriers from the population through breeding management. One common example of an inherited disorder in the breed is the autosomal recessive Trapped Neutrophil Syndrome (TNS) (https://omia.org/OMIA001428/9615/), characterised by static neutropenia and hyperplasia of myeloid cells in the bone marrow. Affected dogs present with abnormal craniofacial development, are usually smaller than their littermates, and fail to thrive at a young age3. The cause of the disease is a 4-bp deletion in exon 19 of VPS13B on chromosome 13 3. The mutation was found to be widespread in the working and show dog groups in several countries around the world, occurring at an allele frequency of around 5.9–8%4. Another example is the autosomal recessive Neuronal Ceroid Lipofuscinosis (NCL) (https://omia.org/OMIA001482/9615/), a neurodegenerative lysosomal storage disease characterised by an accumulation of intracellular lipopigments in neurons and other cells, which causes progressive neurological symptoms such as loss of vision, behavioural changes, and motor disturbances and ultimately leads to premature death8. The disease can arise through several genetic mechanisms, and so far 13 mutations across eight genes have been characterised in a variety of dog breeds10. The causative mutation for NCL in Border Collies is a nonsense mutation (Q206X) in exon 4 of CLN5 on chromosome 22 11 and the same mutation has also been found in Australian Cattle Dogs, in a mixed breed dog with German Shepherd Dog and Australian Cattle Dog ancestry, and a mixed breed dog with uncertain breed background13. The frequency of the mutant allele in Border Collies is around 3.5%11. A study of NCL carriers and affected Border Collies in Japan revealed a common ancestor originating from Australia in 19445. Since the discovery of the causative mutations and the development of polymerase chain reaction (PCR) tests for these diseases, registered Border Collie breeders have avoided breeding of carriers in an attempt to reduce the incidence of these diseases.Border Collies are one of the most popular breeds in the world with at least 25 known inherited disorders18, which have not been able to utilise comprehensive genetic data across a large sample size for a single breed. Other studies have also examined genetic diversity of traditional dog breeds in a conservation context21. For example, the Sapsaree is a native Korean dog breed with around 4000 individuals which are protected by the Korean government and bred with strict regulations, as it was close to extinction during Japanese colonisation (1910–1945) and the Korean War (1950–1953)20. Genetic diversity in a breed can be estimated through genealogical data, or more accurately through DNA markers. Few studies have conducted in-depth analyses of a single dog breed using both pedigree and molecular data23. Genealogical data have previously been used to estimate genetic diversity in Border Collies in France18, Australia24, Belgium23, and Hungary25 and are summarised in Table 16. However, pedigree estimates of inbreeding may not reflect true values due to incomplete or inaccurate pedigree information. All published molecular estimates of genetic diversity in Border Collies have been based on small panels of microsatellite markers26. The results from these studies are summarised in Table 30. However, only a few studies have used high-density SNP arrays to evaluate genetic diversity in dog breeds32.Many studies have primarily examined within-breed or between-breed genetic diversity of popular dog breedsThe aims of this study were to assess genetic diversity in the Border Collie breed using SNP markers and compare these values to genealogical estimates of diversity, and to examine the impact of removing TNS- and NCL-affected and carriers dogs on genetic diversity.33 was used for analysis and visualisation.Unless otherwise stated, R version3.6.134. Due to an update in the bead chip, an additional 225 samples were genotyped on a more comprehensive array covering over 220,000 SNPs (220 k array).This study was conducted in accordance with guidelines from the Animal Research Act, NSW, Australia, approved under the Animal Ethics Committee of the University of Sydney under ethics numbers 37/634/6013. Blood from 286 Border Collies  was collected in EDTA coated vacutainers by licensed veterinarians from private practices and were voluntarily submitted from owners with written informed consent. Dogs were considered Border Collies if the dog was registered with the Australian National Kennel Club (ANKC) or based on owner identification and confirmation by visual inspection by veterinarians. Genomic DNA was isolated using the DNeasy Blood and Tissue Kit  following the manufacturer’s protocol. Genotyping of the DNA samples were performed by Geneseek, Lincoln, NE, USA using the CanineHD BeadChip . A total of 61 samples were genotyped on an array covering over 170,000 evenly spaced, genome-wide SNPs (170 k array)Ne, predicted number of animals equally contributing to breeding, see 35 for formula), generation interval , kinship coefficients  and inbreeding coefficients 38. The overall rate of inbreeding (ΔF) was calculated according to the formula 39. Individual increases in inbreeding (ΔFi) was calculated using the formula originally from Falconer and Mackay39 and modified by González-Recio et al.40, F and EqG are the inbreeding coefficient and equivalent generations respectively for individual i. Inbreeding for each individual adjusted for pedigree depth (FEqG), was also calculated using the mean EqG . A total of 12,478 ancestors were available for these dogs. Genealogical analyses of diversity for 227 of the genotyped dogs that passed genotype quality control and had pedigree information (known parents) was conducted separately (henceforth known as the GenoPed population), as the population included 68 dogs born outside of 2005 to 2015 and 5 with unknown year of birth. A total of 5470 ancestors of these 227 dogs were available. The GenoPed population would allow a direct comparison of genetic estimates of diversity to genealogical estimates for the same 227 dogs, while RefPop provided a wider range of animals that could be separately assessed for genetic diversity and would allow a more accurate estimate of diversity in the dogs contributing to the present generation of Border Collies. The R package ‘optiSel’ was used to calculate equivalent complete generations , index of pedigree completeness , effective population size (f), effective number of founders (fe), effective number of ancestors (fa), and effective number of founder genomes (Ng) (after 500 iterations)42. For the calculation of fe, 20 generations were traced (ped_util program) and used to calculate fe (prob_origin program) for RefPop. The prob_origin program was also used to identify the marginal contributions of each ancestors, which is the genetic contribution that has not yet been accounted for by other ancestors43. A placeholder year of birth of 1950 was used for 6270 RefPop ancestors that had missing year of births.The software PEDIG was used to calculate number of founders , refer to the animals that have the greatest expected genetic contribution to the reference population. The effective number of founders is the number of equally contributing founders that would give the same genetic diversity as the studied population, and accounts for selection rate (probability of being a parent) and variation in family size, while the effective number of ancestors is the minimum number of ancestors  that explains the same genetic diversity as the studied population and accounts for bottlenecks in the pedigree44 was used to impute SNP genotypes of the 170 k panel up to the higher density 220 k SNP array. First, both the 170 k dataset and the 220 k dataset were filtered separately using PLINK v1.945 to keep individuals with no more than 20% missing genotype calls (–mind 0.2), and additionally the 220 k dataset was filtered for a minor allele frequency (MAF) of 0.02 (–maf 0.02). The datasets were then merged and converted into vcf files, filtered to remove SNPs with a genotype call rate of < 25% (–geno 0.25), then imputed on BEAGLE v5.144 using default parameters for model states (imp-states = 1600) and minimum cM length of haplotype segments (imp-segment = 6.0), while ‘cluster’ was set to 0.05 and effective population (Ne) was set to 100. A Ne value of 100 was selected since previous studies estimate Ne in Border Collies to be 99 to 12924, and the default value of 1,000,000 is for large outbred populations, whereas purebred dogs are small inbred populations. After imputation, the merged dataset was further filtered in PLINK v1.945 for a minor allele frequency of 0.02. Runs of homozygosity (ROH) for lengths of > 1 Mb, > 2 Mb, > 4 Mb, and > 8 Mb were calculated for autosomes using PLINK v1.945 as previously described22; in particular, the parameters used for computing ROH were: –dog –allow-no-sex –chr 1–38 –homozyg –homozyg-density 50 (ROH has at least one SNP per 50 kb) –homozyg-gap 100 (two SNPs in ROH no more than 100 kb apart) –homozyg-kb 1000/2000/4000/8000 (minimum length in kb for defining ROH) –homozyg-snp 50 (ROH has at least 50 SNPs) –homozyg-window-het 1  –homozyg-window-missing 5  –homozyg-window-snp 50 (scanning window of 50 SNPs) –homozyg-window-threshold 0.05 . Additionally, relationship cutoffs (–rel-cutoff) of 0.3 or 0.45 were also used to define subsets of more distantly related dogs for ROH analyses. The R package ‘ggplot2’ was used to plot the frequency in which each SNP was in a ROH > 1 Mb across all genotyped dogs46. Inbreeding coefficients were calculated as a frequency of ROH (FROH) by summing the ROH across all individuals and dividing by the autosomal genome length covered by the SNPs (difference in base pairs (bp) between the first and last SNP for each chromosome, summed across all chromosomes). Inbreeding was also additionally calculated with the –het flag in PLINK v1.945 (Fgeno), which was also used to calculate multi-locus heterozygosity (MLH) using the formula 45. Pearson’s correlation was used to compare FROH with Fped and Fgeno. To calculate effective population size, a subset of 10,000 SNPs were selected as previously described22. Briefly, the filtered SNP data was cut into 12,001 segments and 10,000 segments were randomly sampled, then one SNP was selected at random in each segment. Effective population size (Ne) was then calculated using NeEstimator v2.147 based on the subset of SNPs.As there were differences in the number of SNPs in the two SNP arrays used, BEAGLE v5.148.The copy number variation (CNV) analysis was detected using the algorithm implemented by PennCNV using the 225 dogs genotyped on the 220 k SNP array. The algorithm for CNV analysis from SNP array data was developed and described previouslyThe script of PennCNV “detect_cnv.pl” was used with the Hidden Markov Model parameter file and the GC Model to reduce waviness. The quality of the final dataset was assessed with the following thresholds: a logR ratio standard deviation (LRR_SD) > 0.20, BAF drift > 0.002, and waviness factor  > 0.04 or <  − 0.04 for each sample. To reduce the possible false CNV calls, only CNV consisting of at least three consecutive SNPs were considered.49. The output was refined into discrete, non-overlapping common regions.The identification of CNV regions was performed by using ‘cnvpack’ package in R with the Cumulative Overlap Using Very Reliable Regions (COVER) methodTo assess the impact of the introduction of testing for TNS and NCL in 2007, the proportions of affected, carrier and normal dogs per year were examined from our database containing the status for 6439 dogs tested for TNS  and 5956 dogs tested for NCL . These dogs were tested for TNS and NCL upon request by the owner or breeder. Following exclusions based on missing information (year of birth), 5626 dogs  remained for proportions per year analysis for TNS, while 5173 dogs  remained for analysis for NCL.50 using a mixed linear model association (MLMA) analysis incorporating a genetic relationship matrix (GRM), where carriers were also coded as cases since there were so few affected animals genotyped. The R package ‘qvalue’ v2.16.0 was used to calculate chromosome-wise q-values, with a false discovery rate of 0.05, based on p-values from the MLMA output51. The most significant SNP from the MLMA for each disease were used as a proxy for the causal mutation and were used to calculate r2 values with PLINK v1.9 to examine linkage disequilibrium around the respective mutations – VPS13B on chromosome 13 for TNS and CLN5 on chromosome 22 for NCL. Regional association plots were constructed using these r2 values, recombination rates from Campbell et al.52, and protein-coding genes from publicly available data from the National Center for Biotechnology Information (NCBI) (GCF_000002285.3_CanFam3.1). Additionally, for each disease, carriers/affected dogs were removed to examine the impact on genetic diversity, changes to MAF (plotted with ‘ggplot2’46 in R), ROH, and MLH across the genome in the remaining genotyped samples.Of the genotyped dogs remaining after genotype quality control filtering, 206 dogs were tested for TNS  and 205 were tested for NCL . Three dogs were carriers for both mutations. Genome-wide association analyses were conducted for each disease through GCTA v1.2653 at a default k-value of 1055, to visualise relationships between normal, carrier and affected dogs for both diseases. Pedigrees were constructed using a combination of the R packages ‘kinship2’, ‘pedigree’, and ‘FamAgg’58.A distance matrix (based on IBS) was created in PLINK v1.9 (--distance-matrix) and was used to generate a NetView plot with the R package ‘netview’There were 28 dogs that passed genotype quality control and were included in genetic analyses but were not included in GenoPed due to missing pedigree information. Of the GenoPed population , 79.7% were from Australia, 10.6% were of unknown origin, 2.6% were from UK, 2.2% were from Japan, 1.8% were from USA, while the remaining 3.1% were from Belgium, Romania, New Zealand, Italy, Germany, and Finland. For each dog used in this study, the details on sex, year of birth, country of origin, parentage, TNS status, NCL status, and inclusion in RefPop (and ancestors) or GenoPed (and ancestors) are available in Supplementary Data Ne estimate for GenoPed was 65.4. There were 11 dogs in GenoPed where inbreeding could not be calculated due to incomplete pedigree information. Of these 11 dogs, eight dogs only had one fully traceable generation, while the other three only had two to three fully traceable generations. Average inbreeding from pedigree estimates (Fped), excluding these 11 dogs, was 0.139 (SD 0.063).A summary of average values for index of pedigree completeness, equivalent generations, inbreeding, generation interval, and kinship coefficient are in Table ped) for each group is available in supplementary data  were plotted over time, excluding dogs where inbreeding could not be calculated due to incomplete pedigree, dogs that had only one complete generation, and years where only one dog was available  has remained constant since 1980 at approximately 0.1, while adjusting for pedigree depth (FEqG) showed inbreeding has declined between 1960 and 1985 and has not deviated much beyond 1985 .Distribution of inbreeding  and mean kinship was higher than previous studies . Generation interval was similar at I = 4.41. Ne was within range of previous studies at 123.5. Number of observed founders (f), effective founders (fe), effective ancestors (fa),effective founder genomes (Ng), indications of uneven founder contribution (fe/f), indications of genetic bottle necks (fa/fe), and indications of genetic drift (Ng/fe) were as follows:f = 2276, fe = 205.5, fa = 28, Ng = 10.65 (SD 2.82), fe/f = 0.09, fa/fe = 0.14, Ng/fe = 0.05. Although fa/fe value indicated a genetic bottleneck in the population, we were unable to identify a year where there was a substantial drop in number of dogs born per year  was 0.052 , and for allele frequencies < 0.02 the estimated Ne was 86.0 (95% CI 86.0–86.1). Average observed heterozygosity across all dogs was 0.309 (SD 0.147), while average expected heterozygosity was 0.328 (SD 0.146)  for allele frequency of < 0.05, and was 79.3 (95% CI 79.2–79.4) for allele frequency of < 0.02. The Fgeno for this subset of dogs was 0.042 (SD 0.076). A comparison of inbreeding coefficients calculated through pedigree analysis (Fped) against genotype data (Fgeno), as well as pedigree analysis (Fped) against FROH (> 1 Mb), both revealed moderate correlations at R = 0.62  and R = 0.63 , respectively  compared to duplications . When CNVs were assigned to clusters, there were 112 unique regions that met the criteria for a CNV regions. CNV regions ranged in size from 1.16 kbp to 1.01 Mbp, with an average length of 697,562.9 bp. These CNV regions were distributed between 29 autosomes. Eight chromosomes had no evidence for CNV regions, including chromosomes 11, 19, 23, 26, 29, 32, 33, and 35  we would expect the population to have 0.59% affected, 14.21% carriers, and 85.19% normal dogs. Across the entire database, we observed higher frequencies of TNS with 58 affected (0.9%), 1448 carriers (22.49%), and 4933 normal (76.61%) at an allele frequency of 0.121. After filtering out individuals with missing year of births and excluding years where there were fewer than ten dogs tested, there was a maximum of 160 carriers born in 2007 (27.58% of dogs tested that year) for TNS, while the highest proportion of TNS carriers were born in 2010  11, which would be an expected 0.12% affected dogs, 6.76% carriers, and 93.12% normal dogs under HWE. Across the entire database, we observed slightly higher frequencies of NCL-affected dogs , lower frequencies of carriers , and slightly higher frequency of normal dogs , at a similar allele frequency of 0.029. After exclusions stated above, there were considerably fewer carriers for NCL across the years analysed compared to TNS, with a maximum of 19 carriers (25.33% of dogs tested that year) born in 1995 for NCL , 837,026 bases away from the TNS mutation  and was not in VPS13B   and were strongly linked to the top SNP (r2 = 0.96). A total of 36 SNPs were strongly linked (r2 ≥ 0.8) to the top SNP and 12 SNPs were moderately linked (0.5 ≤ r2 < 0.8) . For NCL, the filtered genotyped population consisted of 184 dogs clear for the disease risk, and 21 carriers (50 dogs were not tested). In the GWAS for TNS, there were 840 significant SNPs (chromosome q < 0.05) on chromosome 13 . The most significant SNP was at 22:30,143,975 , which was 430,662 bases upstream from the NCL mutation   on chromosome 22  when carriers and affected dogs were removed (0.037 to 0.034), a slight increase in the average proportion of genome in a ROH from 21.03% to 21.39%, and MLH dropped slightly from 0.31 to 0.309 (Table ROH (> 1 Mb) slightly from 0.036 to 0.033, while average proportion of genome in a run slightly increased from 19.82% to 22.04%. The number of total runs on chromosome 13 reduced from 745 to 600 and MLH also had a slight reduction from 0.316 to 0.31. Across the genome, gains and losses of MAF were evenly distributed except a pronounced loss of MAF was identified on chromosome 13 around the TNS mutation, with 89 SNPs losing ≥ 10% MAF (maximum loss of 12.7%), while 67 SNPs in this region also gained ≥ 5% MAF , MLH (0.31), or average proportion of genome in a run (21.04%) (Table ROH (0.038) and a small increase in average proportion of genome in a run from 21.43% to 21.64%. The number of total runs decreased from 805 to 746 and MLH increased slightly from 0.311 to 0.312. Gains and losses of MAF were mostly evenly distributed across the genome, and at chromosome 22 around the NCL mutation, there were just four SNPs losing > 5% MAF (maximum loss of 5.18%)  Table . Examina run 21.0% 25.Based on EqG values of RefPop, the depth of pedigree data was more extensive than previous studies59. Mean molecular estimates for inbreeding in the present study  = 0.037) were comparable to previous estimates in Border Collies 18. The genealogical estimate of inbreeding in the reference population (0.095) was consistent with previous estimates (0.008 to 0.099)25. The mean genealogical inbreeding estimates for GenoPed was higher  compared to molecular estimates for these animals . These values are not directly comparable due to differences in calculation for the different data types, but pedigree estimates of inbreeding has been shown in other studies to correlate well with several different inbreeding estimates from SNP chip data and sequence data61. Importantly, the standard deviation for both estimates were large, indicating a high degree of variation in inbreeding across the population. This trend was the opposite to the study by Wijnrocx et al.23, which reported a lower inbreeding coefficient of 0.019 using genealogical data from 13,142 dogs compared to an inbreeding value (FIS) of 0.031 using microsatellite markers in 11,66 dogs. In a study of diversity in Bullmastiffs, the estimates for inbreeding using molecular and genealogical data were similar (0.047 and 0.035 respectively). Although our estimates using molecular markers (FROH) are similar to Wijnrocx et al.23 (FIS), higher estimates for inbreeding estimated from genealogical data may be due to the greater depth of pedigree in the present study, which allowed for a more accurate estimation. A comparison of inbreeding coefficients for GenoPed calculated from genealogical data to the molecular calculations from FROH over 1 Mb and to Fgeno  showed moderate correlations. Higher values in the molecular estimates may be due to popular sire effects or linebreeding in older generations that were not captured in the pedigree data. Higher values in the genealogical estimates could reflect more recent inbreeding or be due to the large variation in the estimates. Our reference population showed higher mean kinship compared to Leroy et al.18 and Wijnrocx et al.23, which is in part explained by higher mean inbreeding values compared to these studies.Inbreeding in dogs not only has an effect on the occurrence of disease variants in the population but may also reduce longevity and litter size23 and Shariflou et al.24. The differences compared to Shariflou et al.24 may be attributed to a greater depth of pedigree captured in the current study (EqG = 7.6 compared to 10.38 in RefPop). The effective number of founders for RefPop was close to the value reported by Wijnrocx et al.23. Wijnrocx et al.23 had a more limited depth of pedigree (EqG = 4.7) compared to the present study (EqG = 10.38), and has a much larger number of effective ancestors (115) compared to the present study (28).The number of observed founders in RefPop was much higher than previous studies, however, the number of effective ancestors and the number of effective founder genomes were lower compared to previous studies, indicating the presence of genetic bottlenecks and genetic drift in the population. Effective number of founder genomes was also a lot lower in RefPop compared to that reported by Wijnrocx et al.fe/f indicates the founder contribution, with smaller values indicating more uneven founder contribution. The fe/f value calculated for RefPop (0.09) was close to previous reports by others25, but was less than half that of Wijnrocx et al.23, which again may be due to differences in depth of available pedigree. The Border Collie breed has a large registry and so our findings of uneven founder contribution are consistent with previous reports that uneven founder contribution is more pronounced in breeds with larger registries24. The value of fa/fe is an indication of genetic bottlenecks in the population43 and differed greatly to estimates by others24 . It is possible that genetic bottlenecks occurred further back in the pedigree that were not captured by these studies, however, it remains unclear why the fa/fe values of RefPop and by Shariflou et al.24 differed extensively despite having similar EqG. The number of effective founder genomes is the smallest estimate for founder contribution43 and was a great deal smaller in the current study (Ng = 10.65 (SD 2.82)) compared to previous estimates (Ng = 25.2 to 55.5)24.The value of 24. The Ne of RefPop (123.5) was within the bounds of previous estimates (99.1 to 129)24. The genealogical Ne estimates for GenoPed (65.4) and from SNP data (78.1) were comparable to each other, indicating that between 65 and 78 dogs captured the available genetic variation in a sample of 227 dogs, and the pedigree estimate is suitable to use when genetic data is not available to evaluate genetic diversity. Genetic estimates for effective population size has only previously been measured for 13 generations (Ne13 = 50) based on 16 Border Collies32, hence these estimates are not directly comparable. Two other studies have reported Ne estimates in other breeds using SNP data, and reported an Ne of 29.1 in Bullmastiffs22, and an Ne of 27 in Braque Français type Pyrénées31. These two studies are from breeds that have much smaller registries compared to Border Collies and it is not surprising that they have much lower Ne compared to the present study’s estimates.Effective population size is a measure of the number of equally contributing animals that explain the available genetic variation in a population, with higher numbers representing a greater genetic diversityROH was approximately 0.1 lower than previously reported for 16 Border Collies by Mastrangelo et al.32, implying there was more genetic diversity in the Australian population in our study, or our measures of genetic diversity were more precise owing to a larger sample size. Compared to other breeds previously reported32, the estimates from the present study are much lower, potentially due to the Border Collie’s large registry. One study, which also imputed data from the 173 k SNP array up to the higher density SNP 220 k array, examined ROH in 102 Border Collies and showed small clusters with moderate density of ROH on chromosomes 3, 21, 22, 23, and several others62. Compared to other breeds examined in that same study, the ROH density across the genome in the Border Collie was one of the lowest. The study also showed Border Collies had a mean FROH of approximately 0.2 and ranged from 0.1 to 0.5, however their criteria for detecting a run of homozygosity was less stringent (500 kb) than the present study and hence cannot be directly compared62. In the present study, chromosomes 3, 21, 22 and 23 did not show greater density in ROH, and instead chromosomes 2, 5, 14, 24 and 26 displayed the highest frequency of SNPs in a ROH.Our estimates for F26. The difference may be explained by the large number of alleles in microsatellites as well as higher mutation rates in microsatellites, and extrapolation of a small number of loci to a generalised estimate of heterozygosity in those studies. The estimates from the present study were comparable to a study that used the 170 k CanineHD Beadchip for the Braque Français type Pyrénées dog breed with a reported expected and observed heterozygosity of 0.371 (± 0.142) and 0.359 (± 0.124), respectively31. Multi-locus heterozygosity (MLH) was higher in the present study (0.311) and towards the upper end of the MLH range (0.1 to 0.36) measured in other breeds22.The findings for expected and observed heterozygosity were lower than previous estimates derived from a small number of microsatellitesAnother source of variation that contributes to diversity is CNV. In other studies, CNV have been associated with phenotypic variation and in some cases, inherited disorders. Here the identified CNV regions were mostly described in other breeds but some appeared to be novel to Border Collies. On a chromosome-by-chromosome basis the number of dogs carrying an individual CNV region varied between 1 and 13, indicating that CNV contribute to diversity within the breed. Many CNV overlap genes, and may contribute to disorders in the breed, although there was no indication of this in the present cohort.11. However, these observations should be taken with caution as it may not be representative of the population since there may be some sampling bias. Breeders that identify a carrier among their dogs may choose to test all related animals, so sampling is not completely random. Upon inspection of the pedigrees of affected dogs for both diseases, there were several prominent ancestors that were bred repeatedly and were likely carriers, which would have contributed disproportionately to the disease allele frequency in the population. A previous study found a common ancestor (dog_50624 in the current study) for seven TNS-affected dogs63. In this study, we were able to build a much larger pedigree including 38 TNS-affected dogs and 71 carriers. The common ancestor identified previously, dog_50624, could be traced to a total of 47 TNS carriers and 22 affected dogs. The paternal grandfather of dog_50624 was also identified to be a common ancestor for 20 other TNS carriers and another 10 affected dogs (including four shared with dog_50624). A total of 106 dogs in the database were sired by dog_50624. Importantly, there was a common ancestor identified (dog_811) for both TNS- and NCL-affected dogs, which sired a total of 34 dogs in the database and was the fourth top contributing ancestor to RefPop. This exemplifies how a single dog can affect the population decades later, and these genetic impacts can be further exacerbated through inbreeding or breeding of descendants, in a manner that may reduce the effective population size. Although all NCL-affected dogs and carriers could be traced to a common ancestor, the origin of the disease in Border Collies likely predates the common ancestor (born in 1966) found in this study, since the study of NCL in Border Collies in Japan found a common ancestor originating from Australia in 19445. It is possible that the mutation originated before the formation of the breed, as the same mutation has also been identified in Australian Cattle Dogs, and two mixed breed dogs13. As suggested previously12, there may have been interbreeding between Border Collies and Australian Cattle dogs which possibly introduced the mutation from one breed to the other64. The possibility of the mutation originating in an ancestor of multiple breeds is supported by the study of Villaini et al.13, as the mixed breed dog in the study had little to no Border Collie or Australian Cattle Dog ancestry, and the other mixed breed dog in the study by Katz et al.9 that had a German Shepherd Dog sire and Australian Cattle Dog dam shared the rare, 87 kb haplotype around CLN5 with five Border Collies. The German Shepherd Dog breed is from a more ancestral clade to the Border Collie and the Australian Cattle Dog breeds, and the breeds suggested by Villani et al.13 to harbour the same mutation are from various other clades64. Since the mutation has not yet been characterised in any other breeds it is difficult to determine the breed or ancestor from which it originated, or when the mutation arose, but does suggest that this mutation should be investigated in all breeds exhibiting the disease. Similarly, the origin of the TNS mutation is also likely older than dog_811, since it could not account for 7 carriers in the pedigree. This disease has been exclusively reported in the Border Collie breed so it may be possible to identify the dog in which the mutant allele originated, however, the uncertainty of identifying animals that are carriers in older generations makes it difficult to pinpoint the origin of the mutation.There was a greater number of TNS carriers identified for each year compared to NCL, with over 100 TNS carriers identified per year between 2006 and 2010, compared to less than 20 NCL carriers found per year across 1986 to 2014. However, the number and proportion of carriers for both diseases declined between 2011–2014. We also observed a greater allele frequency for the mutations than previously reportedThe NetView visualisation of the population showed the distribution and prevalence of TNS carriers in the genotyped population of dogs. They were distributed throughout the network and were not co-located in clusters, further emphasising how widespread the mutant allele is in the population. When the network was visualised for NCL, one cluster of 16 individuals was identified, indicating that in the genotyped population, dogs that have the NCL mutation may be restricted to a few breeding lines or closely related dogs. Removing all carriers that are widespread in a population, such as TNS, is likely to reduce overall genetic diversity compared to removing carriers that are clustered together, such as NCL, where rare variants that exist only in the cluster may be lost, and there will be less of an impact on overall genetic diversity.4. The authors suggested that, although possible, it would be impractical to immediately remove all carriers from the population over concerns about reduction in genetic diversity and increased inbreeding. In the present study, we sought to identify the genetic impact of removing TNS and NCL carriers from the population. Homozygosity analysis of dogs that did not carry the TNS and NCL mutation revealed very little change in number of runs and frequency of ROH, suggesting that removing all TNS and NCL carriers in the population will have little impact on overall genetic diversity. The small impact on diversity from removing carriers is likely in part due to the breed’s large registry in Australia. If the disease was seen in a breed with a small registry, the impact of removing carriers may be more profound and potentially cause a genetic bottleneck in the breed. Although there was little effect of removing carriers and affected dogs from the population on ROH, there was up to 12.7% loss in MAF around VPS13B and up to 5.18% loss around CLN5. The loss of MAF in these regions is an indication of the alleles that may become fixed if carriers of each mutation are removed from the breeding pool. The smaller impact on MAF for NCL may be in part due to fewer carrier dogs (21 carriers and no affected dogs) in the genotyped population for NCL compared to TNS (47 carriers and two affected). Future studies may wish to investigate the loss of MAF or other diversity parameters for NCL with a larger population of carriers/affected dogs. Breeding away from the TNS disease allele in the population may also cause a loss of MAF in the region close to VPS13B, as 36 SNPs were strongly linked to the top SNP and spanned several genes such as RIMS2, and STK3. The gene RIMS2 encodes a presynaptic protein that is essential for neurotransmitter release and also plays a role in insulin secretion66, while STK3  encodes a serine/threonine kinase that plays an important role in apoptosis68. The loss of MAF in these genes could therefore have implications on apoptosis and neurotransmitter release or insulin secretion, but the potential consequences will require further investigation.A study on the prevalence of seven genetic disorders (including TNS and NCL) in 500 Border Collies in Japan found that 56% of the population did not carry any of the disease allelesIn conclusion, we report lower levels of heterozygosity in Border Collies from molecular estimates from SNP data than previous estimates from microsatellites, however, multi-locus heterozygosity in the breed is high. Some genealogical measures of diversity differed from previous studies, particularly in the number of effective ancestors and effective founder genomes. Estimations of effective population size from genetic data and from pedigree data were similar, while inbreeding coefficients calculated from both datasets showed moderate correlations. The number of TNS carriers per year was much higher than the number of NCL carriers. The common ancestor for all TNS-affected dogs, dog_811, also appeared in the pedigrees of all NCL-affected dogs, however the origin of these mutations likely predates this dog. We show that the removal of TNS and NCL carriers from the population has a small impact on overall genetic diversity, but some loss of MAF would result in the regions around the affected genes and may be expected to have a larger impact within smaller sub-populations.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


Puntius sophore, Pethia ticto, and Pethia conchonius) sharing the same kind of habitat using the mitochondrial cytochrome c oxidase subunit 1 (CO1). We used 80 samples of the three species from different river/streams. In which we observed total 4–9 haplotypes in all three species with the intra-species sequenced divergence ranges between 0.002 and 0.019. The nucleotide and haplotype diversity was ranged from 0.002040 to 0.01007 and from 0.251 to 0.822, respectively. Neutrality test values were found to be positive only in the P. ticto but statistically non-significant. The AMOVA variation among the populations was 8.89–84.30% whereas, within the populations, it was ranged from 15.70 to 91.11%. The median-joining haplotype network suggests the stable population size over the time and haplotypes were clustered with respect to their geographic locations except the P. conchonius. Similar pattern observed in the phylogenetic tree.Different pattern of genetic diversity and population genetic structure among the species are reported due to their different ecological requirements, adaptability and the evolutionary histories. Understanding such patterns in a species and between the populations is important to develop the effective conservation plans. Very limited studies are available, how different factors influencing the gene flow of a species especially in fish communities. Therefore, the present study is aimed to document the genetic diversity and population genetic structure of the three species of Cyprinidae fishes ( Puntius/Pethia (Family Cyprinidae) are small size and have the beautiful colorations pattern make them popular as freshwater aquarium fishes and many species are traded internationally  are highly adaptive to different aquatic habitat such as streams, rivers, canals, lakes, reservoirs, and other wetlands. In India, the species of both the genus almost distributed throughout India except higher altitude of the Himalayan ranges , Site-II, Vikasnagar, Asan River (30°43′N 77°72′E) and Site III, Kotdwar, Kho River (29°73′N 78°52′E). Samples were collected using different fishing gears viz., hand net, cast net, and some were collected from fisherman and other indigenous trapping method during 2013–2014. Samples were identified up to species level in the field using the taxonomic key , P. ticto , and P. conchonius .Samples of the P. sophore (30), P. ticto (20), P. conchonius (30)) using DNeasy Blood & Tissue Kit  and the partial fragment of cytochrome c oxidase subunit 1 (CO1) gene , and 40 ng of genomic DNA. PCR thermal cycling parameters included initial denaturation at 94 °C for 5 min followed by 45 cycles of denaturation at 94 °C for 45 s, annealing at 45 °C for 1 min, and extension at 72 °C for 50 s with one cycle of a final extension for 20 min at 72 °C for the CO1 gene. Amplification was verified on the 2% (w/v) agarose gel by loading a mixture of 4 μl PCR product and 1 μl loading dye. The bands of amplified product were observed under the UV light. Amplified PCR products were processed for the cycle sequencing followed by Exo-Sap  treatment to remove the residual primers and dNTPs prior to cycle sequencing PCR. PCR products were than cycle sequenced with their respective primers following the suggested composition of master mixture by manufacture . These products then subjected to DNA sequencing on ABI 3130 genetic analyzer.DNA was extracted from the 80 tissue samples . The sequences generated in this study were validated using reference data through the BLAST tool of GenBank (http://www.ncbi.nlm.nih.gov) in which all the sequences were identified as the respective species. The sequences were aligned with the complete genome of Pethia ticto using Bioedit ; P. sophore (KT957189–KT957196); P. ticto (KT957197–KT957203).Raw sequences of the dit Hall . All theπ) and haplotype (h) diversity values were estimated with ARLEQUIN version 3.0 , maximum-likelihood (ML), and maximum-parsimony (MP) as executed in MEGA version 6  haplotype  were observed in samples of P. sophore collected from the Doiwala whereas in P. ticto only two sites were sampled, among which 8 haplotypes observed. In the P. conchonius only three haplotypes were observed.We sequenced 650 bp of the the ATGC . In the ectively . The seqP. sophore; . A total of five private haplotypes were observed in site A . One of the sequences from the Kotdwar found in the outer clade shows the high genetic distance (0.017) with the other samples of the P. sophore. Whereas for the P. ticto three samples are showing the genetic distance (0.019) and formed the separate clade . These sequences are suggestive presence of cryptic species or species complex for the P. ticto in the same kind of the habitat. However, the diversity indices are observed all the studied fish species were similar as reported in the other studies of the different cyprinid fishes  revealed that positive values were observed only for P. ticto from the Kho river, suggest the presence of hybrid or missing of the alleles in the gene pool. However, further confirmation study is needed using large samples size and loci. In the phylogenetic tree, all the samples from different locations formed separate clades; 3 for P. sophore and 2 for P. ticto (samples from only two location). Whereas for P. conchonius only single clade were formed irrespective to their geographic/river affinity (P. conchonius may be due to occurrence of incomplete lineage shorting in this gene (Ward P. conchonius CO1 gene may also be due to slow evolutionary rate or this species may be highly adaptive to different ecological conditions (Lenski Schistura live in seasonal streams and channel unit with low water. Such organisms have presumably run out of ways to become better adapted to their environment (Lenski P. ticto and P. sophore, that indicates these populations have not gone under recent population expansion and showing long-term stability (Mila et al. P. conchonius, but other demographic test reject the population expansion.The present investigation, a total of 9, 8, and 4 haplotypes were observed in  species . In the te clade  when we affinity . Such paene Ward . On othes Lenski . Anothert Lenski . MJ-haplPethia/Puntius using the CO1 gene. These patterns indicate how the different species are adapted to same kind of the environment through exhibiting the different pattern of population genetic structuring, where two species show a sharp population genetic structuring signals, whereas other rejects. We also observed the private haplotype in the P. sophore in Song river as the water of this sparsely distributed and this river also having the high anthropogenic pressure. Results indicate P. ticto, shows the cryptic diversity and we recommend to use of large samples size and loci to delineate the species boundaries.We have explored the genetic diversity and pattern of the population genetic structure in three species genus BDJ designed the study, carried out the samples collection, DNA extraction, Sequencing and data analysis, write the MS; JAJ provided the reagents and helped in the MS writing; TN has helped in analysis and MS writing; AS has compiled the MS performed data analysis; SPG has co-supervised the work edited MS and provided the Lab support. RKN conceived the study and supervised manuscript.