1.Genetic basis of morphology and adaptation in Felidae and other mammalian species;

Free-ranging wild species adapt to the varying environment via their phenotypic persity as determined by genomic persity. Understanding the genetic basis of phenotypic variation is of tremendous importance for uncovering the mechanisms of animals’ adaptation and persity. In particular, coat color and pattern are prominent morphological features in mammals and play an essential role in adaptation and survival.

Tigers have intrigued human society for thousands of years, so as the elusive white tiger, a rare variant characterized by dark stripes on a white fur background. Less well known are two other pelage color variants: the golden tiger with a blonde color tone with pale golden fur and red-brown rather than black stripes, and the snow white tiger that is almost completely white, with faint to nearly nonexistent narrow stripes on the trunk and diluted sepia brown rings on the tail. White tigers were occasionally observed in wilderness on the Indian subcontinent, with the oldest record dating back to the 1500s. In 1951, a male white tiger named Mohan was captured, from which numerous white tigers were bred for captivity around the world. Captive breeding records have suggested that the white and golden tiger coat colors are determined by two independent autosomal recessive loci while the dual-recessive homozygote result in the snow white pelage, however, the precise genetic mechanisms remain elusive until now.

In collaboration with captive breeding facilities, we used the tiger as a model to decode natural genetic polymorphisms. We employed a pedigree-based genome-wide association study (GWAS) approach based on restriction site-associated DNA sequencing (RADSeq), followed by whole-genome sequencing (WGS), to map the Mendelian trait. Our results revealed the causative mutation of white tiger to be a single amino acid change (A477V) in transporter protein SLC45A2, the polymorphisms of which are associated with skin and hair pigmentation in several species, including human. Protein structural homology modeling further suggested a functional effect of the A477V substitution on SLC45A2 and the melanin synthesis process. Since the SLC45A2 A477V substitution only affects the white tiger’s pigmentation and does not cause severe physiological defects, we argue that the white tiger morph is a naturally occurring and viable feature of the genetic persity in tigers that is worth conserving. The paper was published and featured as the cover story in Current Biology (Xu et al. 2013).

Subsequently we reached the final product in a series stemming from the work on tiger coat color genomics and published in Cell Research (Xu et al. 2017) to illuminate the phenotypic persity and genetic basis of all coat color morphs currently know in the tiger. We determined the genetic basis of the golden tabby tiger to be an amino acid change (H587Y) in the transmembrane serine protease CORIN. Together with the mutation responsible for the white tiger (SLC45A2 A477V), two single nucleotide substitutions have resulted in the nearly complete loss of melanin in the snow white tiger. The results have been validated in samples from 200 unrelated tigers worldwide. We further conducted immunochemistry study to show that the H587Y mutation impairs the function of CORIN in degrading agouti signaling protein, strengthening the inhibitory effect of agouti signaling protein on eumelanogenesis during hair growth cycles and resulting in a prolonged pheomelanin band in the golden tiger’s hair.

These findings not only advanced our understanding of mammalian coat color genetics, but also established a working pipeline of non-model species genomics from mapping genes underlying phenotypic variation to functional validation of the genetic mutations. Our work on tiger pelage polymorphism has stirred a wide-range public attention by immediate media coverage from Science, National Geographic, the Times, BBC News and Cell etc.. We were also invited to contribute in-depth view on this issue in Science China (Xu and Luo, 2014) and Scientific American (Luo and Xu, 2014).

Besides the tiger, we also explored the genetic basis of coat color variation in domestic yaks with candidate gene approach (Zhang et al., 2014) and elucidated the genetics of tail-length polymorphism in domestic cats (Xu et al., 2016).  Multiple projects are going on with regard to various genomic mechanisms underlying animal morphological persity.

2.Phylogeography, molecular ecology, and conservation genomics of endangered wildlife.

Conservation genomics is an interdisciplinary science that aims to apply molecular methods to the conservation and restoration of biopersity. This research line of our team is at the interface of ecology and evolution, employing computational approaches to reconstruct population-level patterns and to study basic and applied questions associated with adaptation, behavior, demographic history, and taxonomy. Research programs feature species of conservation concern, utilize population genetic information to assess status, define conservation units and guide management programs both in the wild and captivity.

We set up two in-house resource and technical platforms for this line of research: (1) a biological sample repository and (2) an ancient DNA laboratory. The sample repository involved transferring part of one of the world’s largest mammalian frozen sample banks from the former Laboratory of Genomic Diversity at the US National Cancer Institute (Chief: Dr. Stephen O’Brien) to China. This provides an invaluable and sustainable resource platform for a broad array of research projects in genomic persity, comparative genomics, evolution, and phylogeography. Our ancient DNA lab was dedicated for DNA extraction, PCR setups, or next-generation sequencing library preparation from biological materials that are old, degraded, or intrinsically contain trace quantities of DNA. For contamination control, the aDNA lab is under positive and equipped with UV lamps and HEPA filters both for the source of air into the room and within the laminar fume hood. The entire room and its equipment can be sterilized with UV light and bleached, destroying unwanted DNA fragments before each experiment.

As with many endangered species, tigers have been classified into subspecies - natural geographically separate populations - for purposes of recognition and conservation. The subspecies concept is controversial, but the recognition of subspecies has particular relevance in tigers because conservation strategies for this charismatic flagship species are inextricably tied to subspecies taxonomic pisions. Establishment of subspecies definitions and classifications and a comprehensive understanding of the implications of tiger subspecies are critically important.

In 2004, my colleagues and I published our work on tiger genetic ancestry and phylogeography (Luo et al. 2004), in which we recognized six extant subspecies and split the Malayan tiger from its Indochinese counterpart as a distinct, new living subspecies Panthera tigris jacksoni. This paper in PLoS Biology has become a landmark for big cat conservation and established the present taxonomic system in the tiger that is recognized worldwide. Subsequently we further elucidated that the extinct Caspian tiger was not significantly different from the extant Amur tiger (Driscoll et al. 2009; Driscoll et al. 2012) and revealed a close molecular genetic affinity of the extinct Javan and Bali tigers and living Sumatran tiger, with ancient DNA approaches (Xue et al. 2015). This was the first time the complete evolutionary history by mtDNA of all nine tiger subspecies was revealed. In addition, some other publications on genetics of tigers including review of conservation genetic applications in tiger (Luo et al. 2010) and optimization of a multiplex microsatellite panel for identification of inpidual, subspecies and sex in tigers (Zou et al. 2015). More recently, we are now able to present the strongest-ever population genomic evidence for tiger subspecies differentiation, validating six living named subspecies with whole genome sequencing data from voucher tiger specimens (Liu et al. in revision). Genome-wide analysis identified regions bearing strong signals of positive selection in the Sumatran tiger, likely associated with local adaptation and pergence.

Ernst Mayr (formulator of the Biological Species Concept), Stephen O’Brien, and many others suggested, subspecies are rather useful when validated because of their evolutionary potential to develop into new species. Understanding the tiger’s natural history from a genomic perspective now provides a data-driven foundation for conservation strategy and management, which aims to reverse species’ decline by maximizing the effort to preserve the genetic persity and evolutionary uniqueness of the species.

In addition, this line of research has also encompassed a wide range of endangered species with evolutionary or conservation significance. We completed comparative phylogeography of six sympatric Asian felids based on 445 specimens and revealed several novel aspects about the biogeographical history of Southeast Asia, including a major pergence between the Indochinese and Sundaic felids on the Thai-Malay Peninsula (Luo et al. 2014). The first complete phylogeny of pangolins was generated, providing a solid scientific basis for molecular tracing of the world’s most trafficked mammals (Gaubert et al. 2017). Within the landscape of China, we illustrated for the first time the very recent fragementation effect of the Qinghai-Tibet railway on the landscape genetics of the endangered Przewalski’s gazelle (Yu et al. 2017).

Xiao Xu, Yan Zhuang, Ke Liu, yizhou Fu Ye, Zhengyan Sui, Sicheng Han, Daowei Lu, Lanhui Peng, Duchen Wang, Yuchen Han, Zhenbo Wu, Mengluan Jiang, Yazhi Zhang, Xiaofan Niu