Species conservation can be improved by knowledge of evolutionary and genetic history. Tigers are among the most charismatic of endangered species and garner significant conservation attention. However, their evolutionary history and genomic variation remains poorly known, especially for Indian tigers. With 70% of the worlds wild tigers living in India, such knowledge is critical. We re-sequenced 65 individual tiger genomes representing most extant subspecies with a specific focus on tigers from India. As suggested by earlier studies, we found strong genetic differentiation between the putative tiger subspecies. Despite high total genomic diversity in India, individual tigers host longer runs of homozygosity, potentially suggesting recent inbreeding or founding events, possibly due to small and fragmented protected areas. We suggest the impacts of ongoing connectivity loss on inbreeding and persistence of Indian tigers be closely monitored. Surprisingly, demographic models suggest recent divergence (within the last 20,000 years) between subspecies, and strong population bottlenecks. Amur tiger genomes revealed the strongest signals of selection related to metabolic adaptation to cold, while Sumatran tigers show evidence of weak selection for genes involved in body size regulation. We recommend detailed investigation of local adaptation in Amur and Sumatran tigers prior to initiating genetic rescue.

Increasing habitat fragmentation leads to wild populations becoming small, isolated, and threatened by inbreeding depression. However, small populations may be able to purge recessive deleterious alleles as they become expressed in homozygotes, thus reducing inbreeding depression and increasing population viability. We used whole-genome sequences from 57 tigers to estimate individual inbreeding and mutation load in a small–isolated and two large–connected populations in India. As expected, the small–isolated population had substantially higher average genomic inbreeding (FROH = 0.57) than the large–connected (FROH = 0.35 and FROH = 0.46) populations. The small–isolated population had the lowest loss-of-function mutation load, likely due to purging of highly deleterious recessive mutations. The large populations had lower missense mutation loads than the small–isolated population, but were not identical, possibly due to different demographic histories. While the number of the loss-of-function alleles in the small–isolated population was lower, these alleles were at higher frequencies and homozygosity than in the large populations. Together, our data and analyses provide evidence of 1) high mutation load, 2) purging, and 3) the highest predicted inbreeding depression, despite purging, in the small–isolated population. Frequency distributions of damaging and neutral alleles uncover genomic evidence that purifying selection has removed part of the mutation load across Indian tiger populations. These results provide genomic evidence for purifying selection in both small and large populations, but also suggest that the remaining deleterious alleles may have inbreeding-associated fitness costs. We suggest that genetic rescue from sources selected based on genome-wide differentiation could offset any possible impacts of inbreeding depression.

Knowledge of relationships in wild populations is critical for better understanding mating systems and inbreeding scenarios to inform conservation strategies for endangered species. To delineate pedigrees in wild populations, study genetic connectivity, study genotype‐phenotype associations, trace individuals, or track wildlife trade, many identified individuals need to be genotyped at thousands of loci, mostly from noninvasive samples. This requires us to (a) identify the most common noninvasive sample available from identified individuals, (b) assess the ability to acquire genome‐wide data from such samples, and (c) evaluate the quality of such genome‐wide data, and its ability to reconstruct relationships between animals within a population. We followed identified individuals from a wild endangered tiger population and found that shed hair samples were the most common compared to scat samples, opportunistically found carcasses, and opportunistic invasive samples. We extracted DNA from these samples, prepared whole genome sequencing libraries, and sequenced genomes from these. Whole genome sequencing methods resulted in between 25%–98% of the genome sequenced for five such samples. Exploratory population genetic analyses revealed that these data were free of holistic biases and could recover expected population structure and relatedness. Mitochondrial genomes recovered matrilineages in accordance with long‐term monitoring data. Even with just five samples, we were able to uncover the matrilineage for three individuals with unknown ancestry. In summary, we demonstrated that noninvasive shed hair samples yield adequate quality and quantity of DNA in conjunction with sensitive library preparation methods, and provide reliable data from hundreds of thousands of SNPs across the genome. This makes shed hair an ideal noninvasive resource for studying individual‐based genetics of elusive endangered species in the wild.

AbstractFormulating strategies for species conservation requires knowledge of evolutionary and genetic history. Tigers are among the most charismatic of endangered species and garner significant conservation attention. However, the evolutionary history and genomic variation of tigers remain poorly known. With 70% of the worlds wild tigers living in India, such knowledge is critical for tiger conservation. We re-sequenced 65 individual tiger genomes across their extant geographic range, representing most extant subspecies with a specific focus on tigers from India. As suggested by earlier studies, we found strong genetic differentiation between the putative tiger subspecies. Despite high total genomic diversity in India, individual tigers host longer runs of homozygosity, potentially suggesting recent inbreeding, possibly because of small and fragmented protected areas. Surprisingly, demographic models suggest recent divergence (within the last 10,000 years) between populations, and strong population bottlenecks. Amur tiger genomes revealed the strongest signals of selection mainly related to metabolic adaptation to cold, while Sumatran tigers show evidence of evolving under weak selection for genes involved in body size regulation. Depending on conservation objectives, our results support the isolation of Amur and Sumatran tigers, while geneflow between Malayan and South Asian tigers may be considered. Further, the impacts of ongoing connectivity loss on the health and persistence of tigers in India should be closely monitored.

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Increasing habitat fragmentation leads to wild populations becoming small, isolated, and threatened by inbreeding depression. However, small populations may be able to purge recessive deleterious alleles as they become expressed in homozygotes, thus reducing inbreeding depression and increasing population viability. We used genome sequencing of 57 tigers to estimate individual inbreeding and mutation loads in a small-isolated, and two large-connected populations in India. As expected, the small-isolated population had substantially higher average genomic inbreeding (FROH=0.57) than the large-connected (FROH=0.35 and FROH=0.46) populations. The small-isolated population had the lowest loss-of-function mutation load, likely due to purging of highly deleterious recessive mutations. The large populations had lower missense mutation loads than the small-isolated population, but were not identical, possibly due to different demographic histories. While the number of the loss-of-function alleles in the small-isolated population was lower, these alleles were at high frequencies and homozygosity than in the large populations. Together, our data and analyses provide evidence of (a) high mutation load; (b) purging and (c) the highest predicted inbreeding depression, despite purging, in the small-isolated population. Frequency distributions of damaging and neutral alleles uncover genomic evidence that purifying selection has removed part of the mutation load across Indian tiger populations. These results provide genomic evidence for purifying selection in both small and large populations, but also suggest that the remaining deleterious alleles may have inbreeding associated fitness costs. We suggest that genetic rescue from sources selected based on genome-wide differentiation should offset any possible impacts of inbreeding depression.

The tiger, a poster child for conservation, remains an endangered apex predator. Continued survival and recovery will require a comprehensive understanding of their genetic diversity and the use of such information for population management. A high-quality tiger genome assembly will be an important tool for conservation genetics, especially for the Indian tiger, the most abundant subspecies in the wild. Here, we present high-quality near-chromosomal genome assemblies of a female and a male wild Indian tiger (Panthera tigris tigris). Our assemblies had a scaffold N50 of >140 Mb, with 19 scaffolds, corresponding to the 19 numbered chromosomes, containing 95% of the genome. Our assemblies also enabled detection of longer stretches of runs of homozygosity compared to previous genome assemblies which will improve estimates of genomic inbreeding. Comprehensive genome annotation identified 26,068 protein-coding genes, including several gene families involved in key morphological features such as the teeth, claws, vision, olfaction, taste and body stripes. We also identified 301 microRNAs, 365 small nucleolar RNAs, 632 tRNAs and other noncoding RNA elements, several of which are predicted to regulate key biological pathways that likely contribute to tiger’s apex predatory traits. We identify signatures of positive selection in the tiger genome that are consistent with the Panthera lineage. Our high-quality genome will enable use of non-invasive samples for comprehensive assessment of genetic diversity, thus supporting effective conservation and management of wild tiger populations.

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