Molecular Phylogeny Of Felidae And Conservation Genetics Of Tiger

Living cats(felidae)are important predators in many ecosystems.Due to pressures such as human hunting,habitat loss,lack of prey,and climate change,much of the extant felid diversity is currently threatened.Conservation genetics analyses have provided multiple tools to aid in felid conservation efforts.However,to date,few studies have developed molecular markers that could help elucidate felid evolutionary relationships and aid in conservation efforts.Here,we build a molecular phylogeny to explore the evolutionary history of felids.Specifically,we developed a series of simple sequence repeat(SSR)molecular markers(i.e.,microsatellites)and use parallel tagged amplicon sequencing to help develop conservation strategies for captive tiger populations.We first reconstructed the felid phylogeny,and phylogeny of the feliformia more broadly,using whole mitochondrial genome sequences.the phylogenetic framework is critical for discovering,understanding,and preserving extant felid diversity.By analyzing divergence times among felid species,we found that the felids originated 32-16.8 MYA.Additionally,our analysis suggests that climate cooling after the mid-Miocene likely drove felid diversification.This analysis offers new evolution insight to guide future discovery and conservation of felid diversity.Next,we used published nuclear genome sequences of four felid species to identify SSR loci(i.e.,microsatellites)and potentially amplifiable SSR loci(PALs).We found that each SSR motif(i.e.,nucleotide repeat length)exhibit similar frequencies among felid species.Critically,we identified many PALs for our two focal species,tiger and leopard,(both in pantheriinae),which provide new opportunities for large scale conservation genetics research.This work has provided an enhanced toolset of genetic markers that can be used for ongoing and future felid conservation genetics.In our third study,our goal was to test the utility of PALs as higher resolution molecular markers for Amur tigers.To this aim,we assessed whether 70 PALs exhibited nucleotide repeat sequence-length variation by sequencing these PALs for 37 captive Amur tigers on an Illumina Hiseq.We identified 12 length-variable loci that were less than five repeats,13 loci with lengths between 5-9 repeats,and 13 SSR loci with more than 10 repeats.Thirty-four loci were not usable because the PCR products are too long and the repeat locus is located in the middle of the fragments.Our analyses illustrates the utility of next generation sequencing platforms like Illumina for identifying a diversity of useful PALs.The method described here is simpler,more efficient,and cheaper than traditional length polymorphic SSR microsatellite analyses that require fluorescently-labeled primers and allele length discrimination via capillary electrophoresis instrument.Next,we re-sequenced the 37 captive Amur tigers at three multicopy SSR loci,which hold multicopy and length-variable within tiger genome,to explore their efficacy for identifying individuals and any genetic structiring.Our analyses detected high diversity in fragment length for each multicopy SSR among tiger samples.Using STRUCTURE with the three multicopy SSR markers,we found no significant genetic structuring within the captive Amur tigers samples.Our analyses also suggested each of the three SSR loci were under neutral selection as is expected with microsatellites.The three SSR markers exhibit remarkable discrimination power(DP)with network analyses suggesting complex networks among individual tigers.Additionally,all tigers exhibited unique haplotypes across the three SSR markers.Together,these results suggest these three SSR markers may be useful DNA fingerprints for identifying individual tigers.Finally,we used the Illumina Hiseq platform and parallel tagged amplicon sequencing to sequence over 10,000 bp of the mitochondrial genome for all but one of the 37 captive Amur tigers(excluding tiger STPH03364).We used three phylogenetic methods(NJ,ML,BI)on 94 SNPs to explore genetic structuring among individuals.Based on the mitochondrial genome,all three analyses identified three sub-populations within the captive Amur tigers.Compared with previous results using multicopy SSR makers which found no marked genetic structuring within these captive Amur tigers,we suggest that the genetic structure within the mitochondrial genome only represents variation due to matrilineal inheritance as is expected with mitochondrial DNA.Compare with the previous results by using multicopy SSR,we suggest there may be introduced male Amur tigers as new genetic resources for this small captive population.Combined,these analyses indicate the mitochondrial DNA is likely insufficient as for conservation genetics to understand the endangered status of tiger populations.However,this study confirms that NGS and Parallel tagged amplicon sequencing methods provide larger sequences and with simpler methods and cheaper costs.