Habitat-associated Genomic Variation in a Wall Lizard from an Oceanic Island.

The lizard Teira dugesii exhibits morphological divergence between beach and inland habitats in the face of gene flow, within the volcanic island of Madeira, Portugal. Here, we analyzed genomic data obtained by genotyping-by-sequencing, which provided 16,378 single nucleotide polymorphisms (SNPs) from 94 individuals sampled from 15 sites across Madeira. Ancient within-island divergence in allopatry appears to have mediated divergence in similar species within other Atlantic islands, but this hypothesis was not supported for T. dugesii. Across all samples, a total of 168 SNPs were classified as statistical outliers using pcadapt and OutFLANK. Redundancy analysis (RDA) revealed that 17 of these outliers were associated with beach/inland habitats. The SNPs were located within 16 sequence tags and 15 of these were homologous with sequences in a 31 Mb region on chromosome 3 of a reference wall lizard genome (the remaining tag could not be associated with any chromosome). We further investigated outliers through contingency analyses of allele frequencies at each of four pairs of adjacent beach-inland sites. The majority of the outliers detected by the RDA were confirmed at two pairs of these matched sites. These analyses also suggested some parallel divergence at different localities. Six other outliers were associated with site elevation, four of which were located on chromosome 5 of the reference genome. Our study lends support to a previous hypothesis that divergent selection between gray shingle beaches and inland regions overcomes gene flow and leads to the observed morphological divergence between populations in these adjacent habitats.

Association of a single amino acid replacement with dorsal pigmentation in a lizard from the Qinghai-Tibetan Plateau.

Reptiles can evolve adaptive colors in different environments, but relatively little is known about the genetic mechanisms. Here, we identified the MC1R gene and its association with intraspecific color variation in the lizard Phrynocephalus erythrurus. Analysis of the MC1R sequence in 143 individuals from dark South Qiangtang Plateau (SQP) and light North Qiangtang plateau (NQP) populations, revealed two amino acid sites that showed significant differences in frequency between two areas. One SNP, corresponding to Glu183Lys residue, was found to be a highly significant outlier and differentially fixed for SQP and NQP populations. This residue is located in an extracellular area in the second small extracellular loop within the secondary structure of MC1R, which represents an "attachment pocket" part of the 3D structure. Cytological expression of MC1R alleles with the Glu183Lys replacement showed a 39 % increase in intracellular agonist-induced cyclic AMP levels and a 23.18 % greater cell surface expression of MC1R protein in the SQP relative to the NQP allele. Further in silico 3D modeling and in vitro binding experiments indicated a higher MC1R-α-MSH binding for the SQP allele, and elevated melanin synthesis. We provide an overview of how a single amino acid replacement leads to fundamental changes in MC1R function, and hence shapes variation in dorsal pigmentation in lizards from different environments.

Life on a beach leads to phenotypic divergence despite gene flow for an island lizard.

Limited spatial separation within small islands suggests that observed population divergence may occur due to habitat differences without interruption to gene flow but strong evidence of this is scarce. The wall lizard Teira dugesii lives in starkly contrasting shingle beach and inland habitats on the island of Madeira. We used a matched pairs sampling design to examine morphological and genomic divergence between four beach and adjacent (<1 km) inland areas. Beach populations are significantly darker than corresponding inland populations. Geometric morphometric analyses reveal divergence in head morphology: beach lizards have generally wider snouts. Genotyping-by-sequencing allows the rejection of the hypothesis that beach populations form a distinct lineage. Bayesian analyses provide strong support for models that incorporate gene flow, relative to those that do not, replicated at all pairs of matched sites. Madeiran lizards show morphological divergence between habitats in the face of gene flow, revealing how divergence may originate within small islands.

Population Genomics of Variegated Toad-Headed Lizard Phrynocephalus versicolor and Its Adaptation to the Colorful Sand of the Gobi Desert.

The variegated toad-headed agama, Phrynocephalus versicolor, lives in the arid landscape of the Chinese Gobi Desert. We analyzed populations from three different locations which vary in substrate color and altitude: Heishankou (HSK), Guazhou County (GZ), and Ejin Banner (EJN). The substrate color is either light-yellow (GZ-y), yellow (EJN-y), or black (HSK-b); the corresponding lizard population colors largely match their substrate in the degree of melanism. We assembled the P. versicolor genome and sequenced over 90 individuals from the three different populations. Genetic divergence between populations corresponds to their geographic distribution. We inferred the genetic relationships among these populations and used selection scans and differential expression to identify genes that show signatures of selection. Slc2a11 and akap12, among other genes, are highly differentiated and may be responsible for pigment adaptation to substrate color in P. versicolor.

Camouflage versus running performance as strategies against predation in a lizard inhabiting different habitats.

Running speed and camouflage are associated with the foraging and anti-predator abilities of animals. The toad-headed lizard, Phrynocephalus versicolor, has evolved a darker dorsal color in melanistic habitats and maintained a lighter color in adjacent, non-melanistic habitats. We test the hypothesis that lizards have weaker running speed on well-matching backgrounds than on less matching backgrounds. We used lizard models to compare the predation pressure, while the running speed of dark and light lizards were compared in field tunnels using a video recording method. Our results indicated that both the dark lizards in melanistic Heishankou (HSK) and the light lizards in non-melanistic Guazhou (GZ) face lower predation pressure than potential color-background unmatched lizards. The light lizards have a potentially higher running speed than darker lizards in melanistic habitats, which implies that substrate color matching populations with benefits of camouflage might have lower anti-predation pressure, and the costs of investment in melanin production may reduce running capacity.

Elevation as a selective force on mitochondrial respiratory chain complexes of the Phrynocephalus lizards in the Tibetan plateau.

Animals living in extremely high elevations have to adapt to low temperatures and low oxygen availability (hypoxia), but the underlying genetic mechanisms associated with these adaptations are still unclear. The mitochondrial respiratory chain can provide >95% of the ATP in animal cells, and its efficiency is influenced by temperature and oxygen availability. Therefore, the respiratory chain complexes (RCCs) could be important molecular targets for positive selection associated with respiratory adaptation in high-altitude environments. Here, we investigated positive selection in 5 RCCs and their assembly factors by analyzing sequences of 106 genes obtained through RNA-seq of all 15 Chinese Phrynocephalus lizard species, which are distributed from lowlands to the Tibetan plateau (average elevation >4,500 m). Our results indicate that evidence of positive selection on RCC genes is not significantly different from assembly factors, and we found no difference in selective pressures among the 5 complexes. We specifically looked for positive selection in lineages where changes in habitat elevation happened. The group of lineages evolving from low to high altitude show stronger signals of positive selection than lineages evolving from high to low elevations. Lineages evolving from low to high elevation also have more shared codons under positive selection, though the changes are not equivalent at the amino acid level. This study advances our understanding of the genetic basis of animal respiratory metabolism evolution in extreme high environments and provides candidate genes for further confirmation with functional analyses.

Dorsal Pigmentation and Its Association with Functional Variation in MC1R in a Lizard from Different Elevations on the Qinghai-Tibetan Plateau.

Identification of the role of the MC1R gene has provided major insights into variation in skin pigmentation in several organisms, including humans, but the evolutionary genetics of this variation is less well established. Variation in this gene and its relationship with degree of melanism was analyzed in one of the world's highest-elevation lizards, Phrynocephalus theobaldi from the Qinghai-Tibetan Plateau. Individuals from the low-elevation group were shown to have darker dorsal pigmentation than individuals from a high-elevation group. The existence of climatic variation across these elevations was quantified, with lower elevations exhibiting higher air pressure, temperatures, and humidity, but less wind and insolation. Analysis of the MC1R gene in 214 individuals revealed amino acid differences at five sites between intraspecific sister lineages from different elevations, with two sites showing distinct fixed residues at low elevations. Three of the four single-nucleotide polymorphisms that underpinned these amino acid differences were highly significant outliers, relative to the generalized MC1R population structuring, suggestive of selection. Transfection of cells with an MC1R allele from a lighter high-elevation population caused a 43% reduction in agonist-induced cyclic AMP accumulation, and hence lowered melanin synthesis, relative to transfection with an allele from a darker low-elevation population. The high-elevation allele led to less efficient integration of the MC1R protein into melanocyte membranes. Our study identifies variation in the degree of melanism that can be explained by four or fewer MC1R substitutions. We establish a functional link between these substitutions and melanin synthesis and demonstrate elevation-associated shifts in their frequencies.

Effects of substrate color on intraspecific body color variation in the toad-headed lizard, Phrynocephalus versicolor.

Diversity in animal coloration is generally associated with adaptation to their living habitats, ranging from territorial display and sexual selection to predation or predation avoidance, and thermoregulation. However, the mechanism underlying color variation in toad-headed Phrynocephalus lizards remains poorly understood. In this study, we investigated the population color variation of Phrynocephalus versicolor. We found that lizards distributed in dark substrate have darker dorsal coloration (melanic lizards) than populations living in light substrates. This characteristic may improve their camouflage effectiveness. A reciprocal substrate translocation experiment was conducted to clarify the potential role of morphological adaptation and physiological plasticity of this variation. Spectrometry technology and digital photography were used to quantify the color variation of the above-mentioned melanic and nonmelanic P. versicolor populations and their native substrate. Additionally, substrate color preference in both populations was investigated with choice experiments. Our results indicate that the melanic and nonmelanic populations with remarkable habitat color difference were significantly different on measured reflectance, luminance, and RGB values. Twenty-four hours, 30 days, and 60 days of substrate translocation treatment had little effects on dorsal color change. We also found that melanic lizards choose to live in dark substrate, while nonmelanic lizards have no preference for substrate color. In conclusion, our results support that the dorsal coloration of P. versicolor, associated with substrate color, is likely a morphological adaptation rather than phenotypic plasticity.

Morphological species and discordant mtDNA: A genomic analysis of Phrynocephalus lizard lineages on the Qinghai-Tibetan Plateau.

Many species have been established on the basis of morphology, with markers such as mtDNA used to confirm the existence of independent historical lineages. Discordance between morphology and gene trees makes this less straightforward. Genotyping by sequencing (GBS) was used to analyse general genomic divergence across two recognized high altitude lizard species found in the eastern Qinghai-Tibetan Plateau. One of the species (Phyrnocephalus guinanensis) is found on a large area of sand dune habitat and distinguished from the other (P. putjatia) by morphology. We found that the primary pattern of genomic divergence is discordant with these morphological species: northern P. putjatia populations from around the large saline Qinghai lake are genomically distinct from P. putjatia and P. guinanensis populations located south of the Qinghai South and Riyue Mountains. Two competing historical scenarios were assessed using approximate Bayesian computation which unequivocally favoured a split between populations separated by the Qinghai South and Riyue mountains over a split between morphological species. The findings indicate that historical vicariance due to geographical features underpins the phylogenetic split rather than ecology-mediated divergence between sand dune and non-sand areas which i) is predicted by the mtDNA tree (showing the utility of this marker in species delimitation) and ii) demonstrates the unsuitability of the morphology-based taxonomy (indicating that large morphological differences do not always reflect historical lineages). In addition, we found a clear signal of isolation-by-distance around the periphery of Qinghai lake which suggests: i) a high level of resolution by GBS for detecting local divergence and ii) restricted gene flow over relatively short geographic distances. Overall, we show how morphological variation can mislead taxonomic conclusions and the utility of GBS for resolving these issues.

Advancements of Annexin A1 in inflammation and tumorigenesis.

Annexin A1 is a Ca2+-dependent phospholipid binding protein involved in a variety of pathophysiological processes. Accumulated evidence has indicated that Annexin A1 has important functions in cell proliferation, apoptosis, differentiation, metastasis, and inflammatory response. Moreover, the abnormal expression of Annexin A1 is closely related to the occurrence and development of tumors. In this review article, we focus on the structure and function of Annexin A1 protein, especially the recent evidence of Annexin A1 in the pathophysiological role of inflammatory and cancer. This summary will be very important for further investigation of the pathophysiological role of Annexin A1 and for the development of novel therapeutics of inflammatory and cancer based on targeting Annexin A1 protein.

Evolutionary analysis of mitochondrially encoded proteins of toad-headed lizards, Phrynocephalus, along an altitudinal gradient.

BACKGROUND: Animals living at high altitude must adapt to environments with hypoxia and low temperatures, but relatively little is known about underlying genetic changes. Toad-headed lizards of the genus Phrynocephalus cover a broad altitudinal gradient of over 4000 m and are useful models for studies of such adaptive responses. In one of the first studies to have considered selection on mitochondrial protein-coding regions in an ectothermic group distributed over such a wide range of environments, we analysed nineteen complete mitochondrial genomes from all Chinese Phrynocephalus (including eight genomes sequenced for the first time). Initial analyses used site and branch-site model (program: PAML) approaches to examine nonsynonymous: synonymous substitution rates across the mtDNA tree. RESULTS: Ten positively selected sites were discovered, nine of which corresponded to subunits ND2, ND3, ND4, ND5, and ND6 within the respiratory chain enzyme mitochondrial Complex I (NADH Coenzyme Q oxidoreductase). Four of these sites showed evidence of general long-term selection across the group while the remainder showed evidence of episodic selection across different branches of the tree. Some of these branches corresponded to increases in altitude and/or latitude. Analyses of physicochemical changes in protein structures revealed that residue changes at sites that were under selection corresponded to major functional differences. Analyses of coevolution point to coevolution of selected sites within the ND4 subunit, with key sites associated with proton translocation across the mitochondrial membrane. CONCLUSIONS: Our results identify mitochondrial Complex I as a target for environment-mediated selection in this group of lizards, a complex that frequently appears to be under selection in other organisms. This makes these lizards good candidates for more detailed future studies of molecular evolution.

Proximate causes of altitudinal differences in body size in an agamid lizard.

Body size is directly linked to key life history traits such as growth, fecundity, and survivorship. Identifying the causes of body size variation is a critical task in ecological and evolutionary research. Body size variation along altitudinal gradients has received considerable attention; however, the underlying mechanisms are poorly understood. Here, we compared the growth rate and age structure of toad-headed lizards (Phrynocephalus vlangalii) from two populations found at different elevations in the Qinghai-Tibetan Plateau. We used mark-recapture and skeletochronological analysis to identify the potential proximate causes of altitudinal variation in body size. Lizards from the high-elevation site had higher growth rates and attained slightly larger adult body sizes than lizards from the low-elevation site. However, newborns produced by high-elevation females were smaller than those by low-elevation females. Von Bertalanffy growth estimates predicted high-elevation individuals would reach sexual maturity at an earlier age and have a lower mean age than low-elevation individuals. Relatively lower mean age for the high-elevation population was confirmed using the skeletochronological analysis. These results support the prediction that a larger adult body size of high-elevation P. vlangalii results from higher growth rates, associated with higher resource availability.

The complete mitochondrial genome of the lizard subspecies, Phrynocephalus vlangalii vlangalii (Reptilia, Squamata, Agamidae).

The complete mitochondrial genome was sequenced from the toad-headed lizard, Phrynocephalus vlangalii vlangalii. The overall length of mitogenome is 16,319 bp, including 22 tRNA, 13 protein coding genes, 2 rRNA genes, and 2 control regions. The gene order and content were same with the published congeneric mitogenomes, besides the small portion between tRNA-Pro and tRNA-Phe.

Partition number, rate priors and unreliable divergence times in Bayesian phylogenetic dating.

More loci/partitions should improve Bayesian estimation of divergence times on phylogenies but it has recently been shown that this can lead to surprisingly poor estimation due to the way it affects the prior on mean substitution rate. Here we consider the likely impact of partition number on divergence time analyses carried out using the program BEAST. Mitochondrial genome data from toad-headed lizards (genus Phrynocephalus) from the Qinghai-Tibetan Plateau were used to examine this effect. Under increased partitioning of the sequences, BEAST posterior divergence times became unreasonably narrow and downwardly biased due to misspecification of the mean substitution rate prior. This effect was detectable when relatively few partitions were used (i.e. between four and eight), but became very acute for 27-86 partitions. Fortunately, a correction that adjusts the standard deviation of the mean of locus rates led to results that were equivalent to those obtained using the latest version of the program MCMCtree, which implements a new gamma-Dirichlet prior to overcome this problem. A review of the literature shows that a substantial number of BEAST dating studies are likely to have been affected by this misspecification of the rate prior.

The geography and timing of genetic divergence in the lizard Phrynocephalus theobaldi on the Qinghai-Tibetan plateau.

The Qinghai-Tibetan Plateau (QTP) represents one of the earth's most significant physical features and there is increasing interest in the historical generation of biodiversity within this region. We hypothesized that there should be clear geographically coherent genetic structuring within one of the world's highest altitude lizards, Phrynocephalus theobaldi, due to considerable historical population fragmentation in this environment. This was tested using a major mitochondrial DNA (mtDNA) survey and sequencing of two nuclear markers (AME and RAG-1) from P. theobaldi, from across the southern QTP. A Bayesian method (BPEC) was used to detect four geographically structured mtDNA clusters. A Bayesian phylogenetic tree, together with associated dating analyses, supported four corresponding evolutionary lineages with a timing of 3.74-7.03 Ma for the most basal P. theobaldi split and Pliocene splits of 2.97-5.79 Ma and 2.40-5.39 Ma in the two daughter lineages. Himalayan uplift and changes in the Jilong basin may have contributed to these divergences, but uplift of the Gangdese mountains is rejected due to its timing. The nuclear markers appeared to be sorted between the four mtDNA groups, and species delimitation analyses supported the four phylogeographical groups as candidate species. The study contributes to our understanding of biodiversity on the QTP.

The complete mitochondrial genome of the toad-headed lizard subspecies, Phrynocephalus theobaldi orientalis (Reptilia, Squamata, Agamidae).

The first complete mitochondrial genome sequence of Phrynocephalus viviparity was determined for the toad-headed lizard Phrynocephalus theobaldi orientalis. The 16,608 bp mitogenome contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 2 control regions (CRs). The overall base composition of H-strand is T: 26.7%, C: 24.5%, A: 37.6%, G: 11.2%. The gene arrangement and composition of P. theobaldi orientalis was similar to other published mitochondrial genomes of Phrynocephalus oviparity, except that tRNA-Phe and tRNA-Pro were exchanged. The control region comprised two parts, one between tRNA-Thr and tRNA-Phe and another between tRNA-Pro and 12S RNA. The complete mitogenome sequence of P. theobaldi orientalis provided fundamental data for resolving phylogenetic and genetic problems related to Phrynocephalus viviparity.

The complete mitochondrial genome of the subspecies, Phrynocephalus erythrurus parva (Reptilia, Squamata, Agamidae), a toad-headed lizard dwell at highest elevations of any reptile in the world.

The complete mitochondrial genome was sequenced from the toad-headed viviparous lizard subspecies, Phrynocephalus erythrurus parva, which occupies the highest regions of any reptile on the earth. The mitogenome sequence was 16,431 bp in size, with the overall base composition of H-strand is T: 26.06%, C: 25.14%, A: 36.45%, G: 12.35%. It consists of 13 protein coding, 22 tRNA, 2 rRNA genes and 3 control regions, and its gene order and gene content were identical with the published congeneric mitogenomes of other Phrynocephalus, except for the small protion between tRNA-Pro and tRNA-Phe.

The complete mitochondrial genome of Phrynocephalus guinanensis (Reptilia, Squamata, Agamidae).

Recent phylogenetic researches discovered no reciprocal monophyly between morphological species of Phrynocephalus putjatia and P. guinanensis, P. guinanensis exhibits extensive sexual color dimorphism, whereas P. putjatia does not have. The complete mitochondrial genome of the putative taxa, Phrynocephalus guinanensis, was determined in order to compare the mitogenomes of both ecological forms. The mitogenome sequence was 16,279 bp in size. It consists of 13 protein-coding, 22 tRNA, 2 rRNA genes and 3 control region, and its gene order and gene content were identical with the mitogenome of P. putjatia.

The complete mitochondrial genome of an agama, Phrynocephalus putjatia (Reptilia, Squamata, Agamidae).

The complete mitochondrial genome was sequenced from the toad-headed viviparous lizard, Phrynocephalus putjatia. The mitogenome was 16,283 bp in length; it contained 13 protein coding, 22 tRNA, 2 rRNA genes and 2 control regions. The gene order and compositions were identical with all published congeneric mitogenomes for the fragment between 12 s RNA and tRNA-Thr, but with some differences for the remaining sequences including CR, tRNA-Pro and tRNA-Phe. The characteristics of the mitogenome was analyzed and discussed in detail.