Understanding the molecular evolution of tiger diversity through DNA barcoding marker ND4 and NADH dehydrogenase complex using computational biology.

BACKGROUND: Currently, Tigers (the top predator of an ecosystem) are on the list of endangered species. Thus the need is to understand the tiger's population genomics to design their conservation strategies. OBJECTIVE: We analyzed the molecular evolution of tiger diversity using NADH dehydrogenase subunit 4 (ND4), a significant electron transport chain component. METHODS: We have analyzed nucleotide composition and distribution pattern of ND genes, molecular evolution, evolutionary conservation pattern and conserved blocks of NADH, phylogenomics of ND4, and estimating species divergence, etc., using different bioinformatics tools and software, and MATLAB programming and computing environment. RESULTS: The nucleotide composition and distribution pattern of ND genes in the tiger genome demonstrated an increase in the number of adenine (A) and a lower trend of A+T content in some place of the distribution analysis. However, the observed distributions were not significant (P > 0.05). Evolutionary conservation analysis showed three highly align blocks (186 to 198, 406 to 416, and 527 to 545). On mapping the molecular evolution of ND4 among model species (n = 30), we observed its presence in a broader range of species. ND4 based molecular evolution of tiger diversity and time divergence for a tiger (20 different other species) shows that genus Panthera originated more or less at a similar time. CONCLUSIONS: The nucleotide composition and nucleotide distribution pattern of tiger ND genes showed the evolutionary pattern and origin of tiger and Panthera lineage concerning the molecular clock, which will help to understand their adaptive evolution.

Sorting Out the Genetic Background of the Last Surviving South China Tigers.

The South China tiger (Panthera tigris amoyensis) is endemic to China and also the most critically endangered subspecies of living tigers. It is considered extinct in the wild and only about 150 individuals survive in captivity to date, whose genetic heritage, however, is ambiguous and controversial. Here, we conducted an explicit genetic assessment of 92 studbook-registered South China tigers from 14 captive facilities using a subspecies-diagnostic system in the context of comparison with other voucher specimens to evaluate the genetic ancestry and level of distinctiveness of the last surviving P. t. amoyensis. Three mtDNA haplotypes were identified from South China tigers sampled in this study, including a unique P. t. amoyensis AMO1 haplotype not found in other subspecies, a COR1 haplotype that is widespread in Indochinese tigers (P. t. corbetti), and an ALT haplotype that is characteristic of Amur tigers (P. t. altaica). Bayesian STRUCTURE analysis and parentage verification confirmed the verified subspecies ancestry (VSA) as the South China tiger in 74 individuals. Genetic introgression from other tigers was detected in 18 tigers, and subsequent exclusion of these and their offspring from the breeding program is recommended. Both STRUCTURE clustering and microsatellite-based phylogenetic analyses demonstrated a close genetic association of the VSA South China tigers to Indochinese tigers, an issue that could only be elucidated by analysis of historical South China tiger specimens with wild origin. Our results also indicated a moderate level of genetic diversity in the captive South China tiger population, suggesting a potential for genetic restoration.

Tigers of the World: Genomics and Conservation.

Of all the big cats, or perhaps of all the endangered wildlife, the tiger may be both the most charismatic and most well-recognized flagship species in the world. The rapidly changing field of molecular genetics, particularly advances in genome sequencing technologies, has provided new tools to reconstruct what characterizes a tiger. Here we review how applications of molecular genomic tools have been used to depict the tiger's ancestral roots, phylogenetic hierarchy, demographic history, morphological diversity, and genetic patterns of diversification on both temporal and geographical scales. Tiger conservation, stabilization, and management are important areas that benefit from use of these genome resources for developing survival strategies for this charismatic megafauna both in situ and ex situ.

Conservation priorities for endangered Indian tigers through a genomic lens.

Tigers have lost 93% of their historical range worldwide. India plays a vital role in the conservation of tigers since nearly 60% of all wild tigers are currently found here. However, as protected areas are small (<300 km2 on average), with only a few individuals in each, many of them may not be independently viable. It is thus important to identify and conserve genetically connected populations, as well as to maintain connectivity within them. We collected samples from wild tigers (Panthera tigris tigris) across India and used genome-wide SNPs to infer genetic connectivity. We genotyped 10,184 SNPs from 38 individuals across 17 protected areas and identified three genetically distinct clusters (corresponding to northwest, southern and central India). The northwest cluster was isolated with low variation and high relatedness. The geographically large central cluster included tigers from central, northeastern and northern India, and had the highest variation. Most genetic diversity (62%) was shared among clusters, while unique variation was highest in the central cluster (8.5%) and lowest in the northwestern one (2%). We did not detect signatures of differential selection or local adaptation. We highlight that the northwest population requires conservation attention to ensure persistence of these tigers.

Schrodinger's scat: a critical review of the currently available tiger (Panthera Tigris) and leopard (Panthera pardus) specific primers in India, and a novel leopard specific primer.

BACKGROUND: Non-invasive sampling has opened avenues for the genetic study of elusive species, which has contributed significantly to their conservation. Where field based identity of non-invasive sample is ambiguous (e.g. carnivore scats), it is essential to establish identity of the species through molecular approaches. A cost effective procedure to ascertain species identity is to use species specific primers (SSP) for PCR amplification and subsequent resolution through agarose gel electrophoresis. However, SSPs if ill designed can often cross amplify non-target sympatric species. Herein we report the problem of cross amplification with currently published SSPs, which have been used in several recent scientific articles on tigers (Panthera tigris) and leopards (Panthera pardus) in India. Since these papers form pioneering research on which future work will be based, an early rectification is required so as to not propagate this error further. RESULTS: We conclusively show cross amplification of three of the four SSPs, in sympatric non-target species like tiger SSP amplifying leopard and striped hyena (Hyaena hyaena), and leopard SSP amplifying tiger, lion (Panthera leo persica) and clouded leopard (Neofelis nebulosa), with the same product size. We develop and test a non-cross-amplifying leopard specific primer pair within the mitochondrial cytochrome b region. We also standardize a duplex PCR method to screen tiger and leopard samples simultaneously in one PCR reaction to reduce cost and time. CONCLUSIONS: These findings suggest the importance of an often overlooked preliminary protocol of conclusive identification of species from non-invasive samples. The cross amplification of published primers in conspecifics suggests the need to revisit inferences drawn by earlier work.

A call for tiger management using "reserves" of genetic diversity.

Tigers (Panthera tigris), like many large carnivores, are threatened by anthropogenic impacts, primarily habitat loss and poaching. Current conservation plans for tigers focus on population expansion, with the goal of doubling census size in the next 10 years. Previous studies have shown that because the demographic decline was recent, tiger populations still retain a large amount of genetic diversity. Although maintaining this diversity is extremely important to avoid deleterious effects of inbreeding, management plans have yet to consider predictive genetic models. We used coalescent simulations based on previously sequenced mitochondrial fragments (n = 125) from 5 of 6 extant subspecies to predict the population growth needed to maintain current genetic diversity over the next 150 years. We found that the level of gene flow between populations has a large effect on the local population growth necessary to maintain genetic diversity, without which tigers may face decreases in fitness. In the absence of gene flow, we demonstrate that maintaining genetic diversity is impossible based on known demographic parameters for the species. Thus, managing for the genetic diversity of the species should be prioritized over the riskier preservation of distinct subspecies. These predictive simulations provide unique management insights, hitherto not possible using existing analytical methods.

Tigers of Sundarbans in India: is the population a separate conservation unit?

The Sundarbans tiger inhabits a unique mangrove habitat and are morphologically distinct from the recognized tiger subspecies in terms of skull morphometrics and body size. Thus, there is an urgent need to assess their ecological and genetic distinctiveness and determine if Sundarbans tigers should be defined and managed as separate conservation unit. We utilized nine microsatellites and 3 kb from four mitochondrial DNA (mtDNA) genes to estimate genetic variability, population structure, demographic parameters and visualize historic and contemporary connectivity among tiger populations from Sundarbans and mainland India. We also evaluated the traits that determine exchangeability or adaptive differences among tiger populations. Data from both markers suggest that Sundarbans tiger is not a separate tiger subspecies and should be regarded as Bengal tiger (P. t. tigris) subspecies. Maximum likelihood phylogenetic analyses of the mtDNA data revealed reciprocal monophyly. Genetic differentiation was found stronger for mtDNA than nuclear DNA. Microsatellite markers indicated low genetic variation in Sundarbans tigers (He= 0.58) as compared to other mainland populations, such as northern and Peninsular (Hebetween 0.67- 0.70). Molecular data supports migration between mainland and Sundarbans populations until very recent times. We attribute this reduction in gene flow to accelerated fragmentation and habitat alteration in the landscape over the past few centuries. Demographic analyses suggest that Sundarbans tigers have diverged recently from peninsular tiger population within last 2000 years. Sundarbans tigers are the most divergent group of Bengal tigers, and ecologically non-exchangeable with other tiger populations, and thus should be managed as a separate "evolutionarily significant unit" (ESU) following the adaptive evolutionary conservation (AEC) concept.

The head and neck muscles of the serval and tiger: homologies, evolution, and proposal of a mammalian and a veterinary muscle ontology.

Here we describe the head and neck muscles of members of the two extant felid subfamilies (Leptailurus serval: Felinae; Panthera tigris: Pantherinae) and compare these muscles with those of other felids, other carnivorans (e.g., domestic dogs), other eutherian mammals (e.g., rats, tree-shrews and modern humans), and noneutherian mammals including monotremes. Another major goal of the article is to discuss and help clarify nomenclatural discrepancies found in the Nomina Anatomica Veterinaria and in veterinary atlases and textbooks that use cats and dogs as models to understand the anatomy of domestic mammals and to stress differences with modern humans. We propose a unifying nomenclature that is expanded to all the head and neck muscles and to all mammalian taxa in order to help build veterinary and mammalian muscle ontologies. Our observations and comparisons and the specific use of this nomenclature point out that felids such as tigers and servals and other carnivorans such as dogs have more facial muscle structures related to the mobility of both the auricular and orbital regions than numerous other mammals, including modern humans, which might be the result of an ancient adaptation related to the remarkable predatory capacities of carnivorans. Interestingly, the skeletal differences, mainly concerning the hyoid apparatus, pharynx, and larynx, that are likely associated with the different types of vocalizations seen in the Felinae (mainly purring) and Pantherinae (mainly roaring) are not accompanied by clear differences in the musculature connected to these structures in the feline L. serval and the pantherine P. tigris.

Limited genetic diversity preceded extinction of the Tasmanian tiger.

The Tasmanian tiger or thylacine was the largest carnivorous marsupial when Europeans first reached Australia. Sadly, the last known thylacine died in captivity in 1936. A recent analysis of the genome of the closely related and extant Tasmanian devil demonstrated limited genetic diversity between individuals. While a similar lack of diversity has been reported for the thylacine, this analysis was based on just two individuals. Here we report the sequencing of an additional 12 museum-archived specimens collected between 102 and 159 years ago. We examined a portion of the mitochondrial DNA hyper-variable control region and determined that all sequences were on average 99.5% identical at the nucleotide level. As a measure of accuracy we also sequenced mitochondrial DNA from a mother and two offspring. As expected, these samples were found to be 100% identical, validating our methods. We also used 454 sequencing to reconstruct 2.1 kilobases of the mitochondrial genome, which shared 99.91% identity with the two complete thylacine mitochondrial genomes published previously. Our thylacine genomic data also contained three highly divergent putative nuclear mitochondrial sequences, which grouped phylogenetically with the published thylacine mitochondrial homologs but contained 100-fold more polymorphisms than the conserved fragments. Together, our data suggest that the thylacine population in Tasmania had limited genetic diversity prior to its extinction, possibly as a result of their geographic isolation from mainland Australia approximately 10,000 years ago.

Oldest known pantherine skull and evolution of the tiger.

The tiger is one of the most iconic extant animals, and its origin and evolution have been intensely debated. Fossils attributable to extant pantherine species-lineages are less than 2 MYA and the earliest tiger fossils are from the Calabrian, Lower Pleistocene. Molecular studies predict a much younger age for the divergence of modern tiger subspecies at <100 KYA, although their cranial morphology is readily distinguishable, indicating that early Pleistocene tigers would likely have differed markedly anatomically from extant tigers. Such inferences are hampered by the fact that well-known fossil tiger material is middle to late Pleistocene in age. Here we describe a new species of pantherine cat from Longdan, Gansu Province, China, Panthera zdanskyi sp. nov. With an estimated age of 2.55-2.16 MYA it represents the oldest complete skull of a pantherine cat hitherto found. Although smaller, it appears morphologically to be surprisingly similar to modern tigers considering its age. Morphological, morphometric, and cladistic analyses are congruent in confirming its very close affinity to the tiger, and it may be regarded as the most primitive species of the tiger lineage, demonstrating the first unequivocal presence of a modern pantherine species-lineage in the basal stage of the Pleistocene (Gelasian; traditionally considered to be Late Pliocene). This find supports a north-central Chinese origin of the tiger lineage, and demonstrates that various parts of the cranium, mandible, and dentition evolved at different rates. An increase in size and a reduction in the relative size of parts of the dentition appear to have been prominent features of tiger evolution, whereas the distinctive cranial morphology of modern tigers was established very early in their evolutionary history. The evolutionary trend of increasing size in the tiger lineage is likely coupled to the evolution of its primary prey species.

Sumatran tiger (Panthera tigris sumatrae): a review of conservation status.

The majority of wild Sumatran tigers are believed to live in 12 Tiger Conservation Landscapes covering approximately 88,000 km(2) . However, the actual distribution of tigers across Sumatra has never been accurately mapped. Over the past 20 years, conservation efforts focused on the Sumatran tigers have increased, but the population continues to decline as a result of several key threats. To identify the status of the Sumatran tiger distribution across the island, an island-wide questionnaire survey comprised of 35 respondents from various backgrounds was conducted between May and June 2010. The survey found that Sumatran tigers are positively present in 27 habitat patches larger than 250 km(2) and possibly present in another 2. In addition, a review on major published studies on the Sumatran tiger was conducted to identify the current conservation status of the Sumatran tiger. Collectively, these studies have identified several key factors that have contributed to the decline of Sumatran tiger populations, including: forest habitat fragmentation and loss, direct killing of tigers and their prey, and the retaliatory killing of tigers due to conflict with villagers. The present paper provides management authorities and the international community with a recent assessment and a base map of the actual distribution of Sumatran tigers as well as a general overview on the current status and possible future conservation challenges of Sumatran tiger management.

Mitochondrial phylogeography illuminates the origin of the extinct caspian tiger and its relationship to the amur tiger.

The Caspian tiger (Panthera tigris virgata) flourished in Central Asian riverine forest systems in a range disjunct from that of other tigers, but was driven to extinction in 1970 prior to a modern molecular evaluation. For over a century naturalists puzzled over the taxonomic validity, placement, and biogeographic origin of this enigmatic animal. Using ancient-DNA (aDNA) methodology, we generated composite mtDNA haplotypes from twenty wild Caspian tigers from throughout their historic range sampled from museum collections. We found that Caspian tigers carry a major mtDNA haplotype differing by only a single nucleotide from the monomorphic haplotype found across all contemporary Amur tigers (P. t. altaica). Phylogeographic analysis with extant tiger subspecies suggests that less than 10,000 years ago the Caspian/Amur tiger ancestor colonized Central Asia via the Gansu Corridor (Silk Road) from eastern China then subsequently traversed Siberia eastward to establish the Amur tiger in the Russian Far East. The conservation implications of these findings are far reaching, as the observed genetic depletion characteristic of modern Amur tigers likely reflects these founder migrations and therefore predates human influence. Also, due to their evolutionary propinquity, living Amur tigers offer an appropriate genetic source should reintroductions to the former range of the Caspian tiger be implemented.

Subspecies genetic assignments of worldwide captive tigers increase conservation value of captive populations.

Tigers (Panthera tigris) are disappearing rapidly from the wild, from over 100,000 in the 1900s to as few as 3000. Javan (P.t. sondaica), Bali (P.t. balica), and Caspian (P.t. virgata) subspecies are extinct, whereas the South China tiger (P.t. amoyensis) persists only in zoos. By contrast, captive tigers are flourishing, with 15,000-20,000 individuals worldwide, outnumbering their wild relatives five to seven times. We assessed subspecies genetic ancestry of 105 captive tigers from 14 countries and regions by using Bayesian analysis and diagnostic genetic markers defined by a prior analysis of 134 voucher tigers of significant genetic distinctiveness. We assigned 49 tigers to one of five subspecies (Bengal P.t. tigris, Sumatran P.t. sumatrae, Indochinese P.t. corbetti, Amur P.t. altaica, and Malayan P.t. jacksoni tigers) and determined 52 had admixed subspecies origins. The tested captive tigers retain appreciable genomic diversity unobserved in their wild counterparts, perhaps a consequence of large population size, century-long introduction of new founders, and managed-breeding strategies to retain genetic variability. Assessment of verified subspecies ancestry offers a powerful tool that, if applied to tigers of uncertain background, may considerably increase the number of purebred tigers suitable for conservation management.

In defense of species.

In this paper, I address the charge that the category species should be abandoned in biological work. The widespread appeal to species in scientific discourse provides a presumption in favor of the category's usefulness, but a defeasible presumption. Widely acknowledged troubles attend species: these troubles might render the concept unusable by showing that 'species' is equivocal or meaningless or in some similar way fatally flawed. Further, there might be better alternatives to species. I argue that the presumption in favor of species is not defeated on these scores. Troubles attending species, which arise on account of contextual variation attending the use of 'species', do not indicate that the concept is unusable. And alternatives to the use of 'species', which have been proposed in connection with rank-free systematics and in connection with conservation efforts, fail to provide a proper replacement for species.

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris).

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) of Panthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8-17 million years ago in the tiger and 4.6-16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular 'fossils' that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.