Genetic Basis and Evolutionary Forces of Sexually Dimorphic Color Variation in a Toad-Headed Agamid Lizard.

In the animal kingdom, sexually dimorphic color variation is a widespread phenomenon that significantly influences survival and reproductive success. However, the genetic underpinnings of this variation remain inadequately understood. Our investigation into sexually dimorphic color variation in the desert-dwelling Guinan population of the toad-headed agamid lizard (Phrynocephalus putjatai) utilized a multidisciplinary approach, encompassing phenotypic, ultrastructural, biochemical, genomic analyses, and behavioral experiments. Our findings unveil the association between distinct skin colorations and varying levels of carotenoid and pteridine pigments. The red coloration in males is determined by a genomic region on chromosome 14, housing four pigmentation genes: BCO2 and three 6-pyruvoyltetrahydropterin synthases. A Guinan population-specific nonsynonymous single nucleotide polymorphism in BCO2 is predicted to alter the electrostatic potential within the binding domain of the BCO2-ß-carotene complex, influencing their interaction. Additionally, the gene MAP7 on chromosome 2 emerges as a potential contributor to the blue coloration in subadults and adult females. Sex-specific expression patterns point to steroid hormone-associated genes (SULT2B1 and SRD5A2) as potential upstream regulators influencing sexually dimorphic coloration. Visual modeling and field experiments support the potential selective advantages of vibrant coloration in desert environments. This implies that natural selection, potentially coupled with assortative mating, might have played a role in fixing color alleles, contributing to prevalence in the local desert habitat. This study provides novel insights into the genetic basis of carotenoid and pteridine-based color variation, shedding light on the evolution of sexually dimorphic coloration in animals. Moreover, it advances our understanding of the driving forces behind such intricate coloration patterns.

Population Genomics of Wall Lizards Reflects the Dynamic History of the Mediterranean Basin.

The Mediterranean Basin has experienced extensive change in geology and climate over the past six million years. Yet, the relative importance of key geological events for the distribution and genetic structure of the Mediterranean fauna remains poorly understood. Here, we use population genomic and phylogenomic analyses to establish the evolutionary history and genetic structure of common wall lizards (Podarcis muralis). This species is particularly informative because, in contrast to other Mediterranean lizards, it is widespread across the Iberian, Italian, and Balkan Peninsulas, and in extra-Mediterranean regions. We found strong support for six major lineages within P. muralis, which were largely discordant with the phylogenetic relationship of mitochondrial DNA. The most recent common ancestor of extant P. muralis was likely distributed in the Italian Peninsula, and experienced an "Out-of-Italy" expansion following the Messinian salinity crisis (∼5 Mya), resulting in the differentiation into the extant lineages on the Iberian, Italian, and Balkan Peninsulas. Introgression analysis revealed that both inter- and intraspecific gene flows have been pervasive throughout the evolutionary history of P. muralis. For example, the Southern Italy lineage has a hybrid origin, formed through admixture between the Central Italy lineage and an ancient lineage that was the sister to all other P. muralis. More recent genetic differentiation is associated with the onset of the Quaternary glaciations, which influenced population dynamics and genetic diversity of contemporary lineages. These results demonstrate the pervasive role of Mediterranean geology and climate for the evolutionary history and population genetic structure of extant species.

Population genetic differentiation and genomic signatures of adaptation to climate in an abundant lizard.

Species distributed across climatic gradients will typically experience spatial variation in selection, but gene flow can prevent such selection from causing population genetic differentiation and local adaptation. Here, we studied genomic variation of 415 individuals across 34 populations of the common wall lizard (Podarcis muralis) in central Italy. This species is highly abundant throughout this region and populations belong to a single genetic lineage, yet there is extensive phenotypic variation across climatic regimes. We used redundancy analysis to, first, quantify the effect of climate and geography on population genomic variation in this region and, second, to test if climate consistently sorts specific alleles across the landscape. Climate explained 5% of the population genomic variation across the landscape, about half of which was collinear with geography. Linear models and redundancy analyses identified loci that were significantly differentiated across climatic regimes. These loci were distributed across the genome and physically associated with genes putatively involved in thermal tolerance, regulation of temperature-dependent metabolism and reproductive activity, and body colouration. Together, these findings suggest that climate can exercise sufficient selection in lizards to promote genetic differentiation across the landscape in spite of high gene flow.

Climate Shapes the Geographic Distribution and Introgressive Spread of Color Ornamentation in Common Wall Lizards.

AbstractClimate can exert an effect on the strength of sexual selection, but empirical evidence is limited. Here, we tested whether climate predicts the geographic distribution and introgressive spread of sexually selected male color ornamentation across 114 populations of the common wall lizard, Podarcis muralis. Coloration was highly structured across the landscape and did not reflect genetic differentiation. Instead, color ornamentation was consistently exaggerated in hot and dry environments, suggesting that climate-driven selection maintains geographic variation in spite of gene flow. Introgression of color ornamentation into a distantly related lineage appears to be ongoing and was particularly pronounced in warm climates with wet winters and dry summers. Combined, these results suggest that sexual ornamentation is consistently favored in climates that allow a prolonged reproductive season and high and reliable opportunities for lizard activity. This pattern corroborates theoretical predictions that such climatic conditions reduce the temporal clustering of receptive females and increase male-male competition, resulting in strong sexual selection. In summary, we provide compelling evidence for the importance of climate for the evolution of color ornamentation, and we demonstrate that geographic variation in the strength of sexual selection influences introgression of this phenotype.

Genomic evidence for asymmetric introgression by sexual selection in the common wall lizard.

Strongly selected characters can be transferred from one lineage to another with limited genetic exchange, resulting in asymmetric introgression and a mosaic genome in the receiving population. However, systems are rarely sufficiently well studied to link the pattern of introgression to its underlying process. Male common wall lizards in western Italy exhibit exaggeration of a suite of sexually selected characters that make them outcompete males from a distantly related lineage that lack these characters. This results in asymmetric hybridization and adaptive introgression of the suite of characters following secondary contact. We developed genomewide markers to infer the demographic history of gene flow between different genetic lineages, identify the spread of the sexually selected syndrome, and test the prediction that introgression should be asymmetric and heterogeneous across the genome. Our results show that secondary contact was accompanied by gene flow in both directions across most of the genome, but with approximately 3% of the genome showing highly asymmetric introgression in the predicted direction. Demographic simulations reveal that this asymmetric gene flow is more recent than the initial secondary contact, and the data suggest that the exaggerated male sexual characters originated within the Italian lineage and subsequently spread throughout this lineage before eventually reaching the contact zone. These results demonstrate that sexual selection can cause a suite of characters to spread throughout both closely and distantly related lineages with limited gene flow across the genome at large.

Molecular Convergent Evolution of the MYBPC2 Gene Among Three High-Elevation Amphibian Species.

We report a strong pattern of molecular-level convergent/parallel evolution of the MYBPC2 gene. Three high-elevation amphibian species, Bufo gargarizans minshanicus, Nanorana pleskei, Rana kukunoris, revealed remarkable numbers of convergent and parallel amino acid substitutions. On the MYBPC2 gene tree of eleven anurans, the three distantly related species formed a strongly supported clade that was away from their respective relatives. Furthermore, we generated both model-based and empirical data-based null distributions for neutral convergent evolution. All three pairwise comparisons among the three species showed significantly more convergent and parallel substitutions than the null distributions. This study adds to the very small roster of clear cases of non-neutral molecular convergent evolution (e.g. prestin, rhodopsin). Molecular convergent evolution has significant implications in biology and detailed case studies will likely provide more insight into its genetic mechanisms.

Gene expression variations in high-altitude adaptation: a case study of the Asiatic toad (Bufo gargarizans).

BACKGROUND: Genome-wide investigation of molecular mechanisms for high-altitude adaptation has attracted great attention in the last few years. In order to understand the contribution of gene expression level variations to high-altitude adaptation in Asiatic toads (Bufo gargarizans), we implemented a reciprocal transplant experiment between low- and high-altitude sites and sequenced 12 transcriptomes from brain, heart, and liver tissues. RESULTS: A large number of genes with expression differences (DEGs) between high- and low-altitude individuals (193 fixed and 844 plastic) were identified, and the majority of them were tissue specific. Heart displayed the largest number of DEGs, both plastic and fixed. Fixed DEGs were particularly concentrated in functions associated with muscle contraction, and the majority of them were down-regulated in high-altitude individuals. Plastic DEGs were highly concentrated in several energy metabolism related functional categories, and the majority of them were also down-regulated at high-altitude environments. In liver samples, genes associated with nutrient metabolism experienced a broad-scale expression down-regulation in high-altitude toads. CONCLUSIONS: These broadly suppressed expression patterns at high altitudes are in strong contrast to those of endothermic homeotherms, suggesting poikilothermic vertebrates may have adopted different strategies at high altitudes. Our results strongly support that both genotypic specialization and phenotypic plasticity play crucial role in adaptation to high altitude for Asiatic toads. Poikilothermic vertebrates are among the most hypoxia-tolerant animals known, and many molecular mechanisms remain elusive. We hope that our results will provide useful directions for future research.

Genetic signals of high-altitude adaptation in amphibians: a comparative transcriptome analysis.

BACKGROUND: High-altitude adaptation provides an excellent system for studying how organisms cope with multiple environmental stressors and interacting genetic modifications. To explore the genetic basis of high-altitude adaptation in poikilothermic animals, we acquired transcriptome sequences from a high-altitude population and a low-altitude population of the Asiatic toad (Bufo gargarizans). Transcriptome data from another high-altitude amphibian, Rana kukunoris and its low-altitude relative R. chensiensis, which are from a previous study, were also incorporated into our comparative analysis. RESULTS: More than 40,000 transcripts were obtained from each transcriptome, and 5107 one-to-one orthologs were identified among the four taxa for comparative analysis. A total of 29 (Bufo) and 33 (Rana) putative positively selected genes were identified for the two high-altitude species, which were mainly concentrated in nutrient metabolism related functions. Using SNP-tagging and FST outlier analysis, we further tested 89 other nutrient metabolism related genes for signatures of natural selection, and found that two genes, CAPN2 and ITPR1, were likely under balancing selection. We did not detect any positively selected genes associated with response to hypoxia. CONCLUSIONS: Amphibians clearly employ different genetic mechanisms for high-altitude adaptation compared to endotherms. Modifications of genes associated with nutrient metabolism feature prominently while genes related to hypoxia tolerance appear to be insignificant. Poikilotherms represent the majority of animal diversity, and we hope that our results will provide useful directions for future studies of amphibians as well as other poikilotherms.

Population Genetic Structure and Species Status of Asiatic Toads (Bufo gargarizans) in Western China.

We investigated the population genetic structure of Asiatic toads (Bufo gargarizans) from the mountains of western China to determine their species status, using genotypic data of ten microsatellite DNA loci and DNA sequences from one mitochondrial gene. A total of 197 samples from eight sites were examined, which cover a large range of elevations (559-3457 m), as well as all three traditionally defined species (or subspecies). AMOVA did not reveal any particularly large among-groups structure, whether the sites were grouped by drainage, elevation, region, or species (subspecies). Individual assignment tests placed all samples into two genetic clusters, which largely corresponded to their geographic locations. An isolation-by-distance pattern was also detected when an outlier population (site 3) was excluded. Furthermore, a mitochondrial gene tree revealed deep divergence among haplotypes, sometimes within the same site. The clade patterns were partially associated with geographic distribution but had no resemblance to the traditional 2- or 3-species classification. Overall, these toad populations harbor a large amount of genetic diversity and have very high population differentiation, but taken together the evidence suggests that all populations belong to a single species. Our results are consistent with most previous molecular studies, and we recommend using Bufo gargarizans to represent all Asiatic toad populations from western China without subspecies division.

Adaptive introgression reveals the genetic basis of a sexually selected syndrome in wall lizards.

The joint expression of particular colors, morphologies, and behaviors is a common feature of adaptation, but the genetic basis for such "phenotypic syndromes" remains poorly understood. Here, we identified a complex genetic architecture associated with a sexually selected syndrome in common wall lizards, by capitalizing on the adaptive introgression of coloration and morphology into a distantly related lineage. Consistent with the hypothesis that the evolution of phenotypic syndromes in vertebrates is facilitated by developmental linkage through neural crest cells, most of the genes associated with the syndrome are involved in neural crest cell regulation. A major locus was a ~400-kb region, characterized by standing structural genetic variation and previously implied in the evolutionary innovation of coloration and beak size in birds. We conclude that features of the developmental and genetic architecture contribute to maintaining trait integration, facilitating the extensive and rapid introgressive spread of suites of sexually selected characters.

A wide hybrid zone mediated by precipitation contributed to confused geographical structure of Scutiger boulengeri.

Confused geographical structure of a population and mitonuclear discordance are shaped by a combination of rapid changes in population demographics and shifts in ecology. In this study, we generated a time-calibrated phylogeny of Scutiger boulengeri, an endemic Xizang alpine toad occurring in mountain streams on the Qinghai-Xizang (Tibet) Plateau (QTP). Based on three mitochondrial DNA (mtDNA) genes, eight clades were assigned to three deeply divergent lineages. Analysis of nuclear DNA (nuDNA) genes revealed three distinct clusters without geographic structure, indicating significantly high rates of gene flow. Coalescent theory framework analysis (approximate Bayesian computation model DIYABC and Migrate-N) suggested that divergence of the main intraspecific clusters was the result of hybridization after secondary contact in the Holocene around 0.59 million years ago (Ma). The ratio of mtDNA F ST (fixation index) to nuDNA F ST was 2.3, thus failing to show male-biased dispersal. Geographic cline analysis showed that a wide hybrid zone was initially established in southwestern China, without significant reproductive isolation but with strong introgression in S. boulengeri, suggesting high hybrid fitness. Furthermore, mtDNA genes exhibited isolation by distance (IBD) while nuDNA genes exhibited significant isolation by environment (IBE). Results suggested that mitonuclear discordance may have initially been caused by geographic isolation, followed by precipitation-mediated hybridization, producing a wide hybrid zone and geographic structure confusion of nuDNA genes in S. boulengeri. This study indicated that complicated historical processes may have led to specific genetic patterns, with a specific climate factor facilitating gene flow in the system.

Toward understanding the genetic basis of adaptation to high-elevation life in poikilothermic species: a comparative transcriptomic analysis of two ranid frogs, Rana chensinensis and R. kukunoris.

BACKGROUND: Understanding how organisms adapt to high-elevation environments at a genome scale provides novel insights into the process of adaptive evolution. Previous studies have mainly focused on endothermic organisms, while poikilothermic species may have evolved different mechanisms to cope with high-elevation environments. In this context, we sequenced transcriptomes of a pair of closely related anuran species, Rana chensinensis and R. kukunoris, which inhabit respective low- and high-elevation habitats. By comparing the two transcriptomes, we identified candidate genes that may be involved in high-elevation adaption in poikilothermic species. RESULTS: Over 66 million sequence reads from each transcriptome were generated. A total of 41,858 and 39,293 transcripts for each species were obtained by de novo assembly. By comparing the orthologous transcripts, we identified 125 protein-coding genes that have likely experienced strong positive selection (Ka/Ks>1). In addition, 335 genes that may bear a signature of positive selection (1≥Ka/Ks>0.5) were also recognized. By considering their functions, fourteen candidate genes were determined to be likely involved in high-elevation adaptation, including two CYP genes, USP-1, and several others. CONCLUSIONS: We identified a set of candidate genes that may have promoted adaptation of R. kukunoris to its high-elevation environment. These include several genes that have previously been associated with oxygen binding, response to UV radiation, and repair of free radical injury. Detailed molecular, physiological, and phenotypic studies are required to better understand the roles of these genes in improving the performance of R. kukunoris in a high-elevation environment. We have evidence for both convergent and divergent evolution between endothermic and poikilothemic species, but additional research across a wide range of organisms will be necessary to comprehend the complexity of high-elevation adaptation.

Extensive introgression and mosaic genomes of Mediterranean endemic lizards.

The Mediterranean basin is a hotspot of biodiversity, fuelled by climatic oscillation and geological change over the past 20 million years. Wall lizards of the genus Podarcis are among the most abundant, diverse, and conspicuous Mediterranean fauna. Here, we unravel the remarkably entangled evolutionary history of wall lizards by sequencing genomes of 34 major lineages covering 26 species. We demonstrate an early (>11 MYA) separation into two clades centred on the Iberian and Balkan Peninsulas, and two clades of Mediterranean island endemics. Diversification within these clades was pronounced between 6.5-4.0 MYA, a period spanning the Messinian Salinity Crisis, during which the Mediterranean Sea nearly dried up before rapidly refilling. However, genetic exchange between lineages has been a pervasive feature throughout the entire history of wall lizards. This has resulted in a highly reticulated pattern of evolution across the group, characterised by mosaic genomes with major contributions from two or more parental taxa. These hybrid lineages gave rise to several of the extant species that are endemic to Mediterranean islands. The mosaic genomes of island endemics may have promoted their extraordinary adaptability and striking diversity in body size, shape and colouration, which have puzzled biologists for centuries.

Spatial variation in gene flow across a hybrid zone reveals causes of reproductive isolation and asymmetric introgression in wall lizards.

Hybrid zones provide insights into the evolution of reproductive isolation. Sexual selection can contribute to the evolution of reproductive barriers, but it remains poorly understood how sexual traits impact gene flow in secondary contact. Here, we show that a recently evolved suite of sexual traits that function in male-male competition mediates gene flow between two lineages of wall lizards (Podarcis muralis). Gene flow was relatively low and asymmetric in the presence of exaggerated male morphology and coloration compared to when the lineages share the ancestral phenotype. Putative barrier loci were enriched in genomic regions that were highly differentiated between the two lineages and showed low concordance between the transects. The exception was a consistently low genetic exchange around ATXN1, a gene that modulates social behavior. We suggest that this gene may contribute to the male mate preferences that are known to cause lineage-assortative mating in this species. Although female choice modulates the degree of reproductive isolation in a variety of taxa, wall lizards demonstrate that both male-male competition and male mate choice can contribute to the extent of gene flow between lineages.

An ultrasensitive electrochemical immunosensor for apolipoprotein E4 based on fractal nanostructures and enzyme amplification.

Human apolipoprotein E4 (APOE4) is a major risk factor for Alzheimer's disease (AD) and can greatly increase the morbidity. In this work, an ultrasensitive sandwich-type electrochemical immunosensor for the quantitative detection of APOE4 was designed based on fractal gold (FracAu) nanostructures and enzyme amplification. The FracAu nanostructures were directly electrodeposited by hydrogen tetrachloroaurate (HAuCl4) on polyelectrolytes modified indium tin oxide (ITO) electrode. The sensing performances of the modified interface were investigated by cyclic voltammetry (CV). After functionalization with HRP-labeled APOE4 antibody, the human APOE4 could be detected quantitatively by current response. The current response has a linear relationship with the logarithm of human APOE4 concentrations in a range from 1.0 to 10,000.0 ng/mL, with a detection limit of 0.3 ng/mL. The fabricated APOE4 electrochemical immunosensor exhibits strong specificity, high sensitivity, low detection limit and wide linear range. The detection of human APOE4 provides a strong support for the prevention of AD and early-stage warning for those susceptible populations.

Exploring the genetic basis of adaptation to high elevations in reptiles: a comparative transcriptome analysis of two toad-headed agamas (genus Phrynocephalus).

High elevation adaptation offers an excellent study system to understand the genetic basis of adaptive evolution. We acquired transcriptome sequences of two closely related lizards, Phrynocephalus przewalskii from low elevations and P. vlangalii from high elevations. Within a phylogenetic framework, we compared their genomic data along with green anole, chicken and Chinese softshell turtle, and identified candidate genes and functional categories that are potentially linked to adaptation to high elevation environments. More than 100 million sequence reads were generated for each species via Illumina sequencing. A de novo assembly produced 70,919 and 62,118 transcripts for P. przewalskii and P. vlangalii, respectively. Based on a well-established reptile phylogeny, we detected 143 positively selected genes (PSGs) along the P. vlangalii lineage from the 7,012 putative orthologs using a branch-site model. Furthermore, ten GO categories and one KEGG pathway that are over-represented by PSGs were recognized. In addition, 58 GO categories were revealed to have elevated evolutionary rates along the P. vlangalii lineage relative to P. przewalskii. These functional analyses further filter out PSGs that are most likely involved in the adaptation process to high elevations. Among them, ADAM17, MD, and HSP90B1 likely contributed to response to hypoxia, and POLK likely contributed to DNA repair. Many other candidate genes involved in gene expression and metabolism were also identified. Genome-wide scan for candidate genes may serve as the first step to explore the genetic basis of high elevation adaptation. Detailed comparative study and functional verification are needed to solidify any conclusions. High elevation adaptation requires coordinated changes in multiple genes that involve various physiological and biochemical pathways; we hope that our genetic studies will provide useful directions for future physiological or molecular studies in reptiles as well as other poikilothermic species.

Transcriptome profile of the green odorous frog (Odorrana margaretae).

Transcriptome profiles provide a practical and inexpensive alternative to explore genomic data in non-model organisms, particularly in amphibians where the genomes are very large and complex. The odorous frog Odorranamargaretae (Anura: Ranidae) is a dominant species in the mountain stream ecosystem of western China. Limited knowledge of its genetic background has hindered research on this species, despite its importance in the ecosystem and as biological resources. Here we report the transcriptome of O. margaretae in order to establish the foundation for genetic research. Using an Illumina sequencing platform, 62,321,166 raw reads were acquired. After a de novo assembly, 37,906 transcripts were obtained, and 18,933 transcripts were annotated to 14,628 genes. We functionally classified these transcripts by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 11,457 unique transcripts were assigned to 52 GO terms, and 1,438 transcripts were assigned to 128 KEGG pathways. Furthermore, we identified 27 potential antimicrobial peptides (AMPs), 50,351 single nucleotide polymorphism (SNP) sites, and 2,574 microsatellite DNA loci. The transcriptome profile of this species will shed more light on its genetic background and provide useful tools for future studies of this species, as well as other species in the genus Odorrana. It will also contribute to the accumulation of amphibian genomic data.

Genetic evidence for male-biased dispersal in the Qinghai toad-headed agamid Phrynocephalus vlangalii and its potential link to individual social interactions.

Sex-biased dispersal has profound impacts on a species' biology and several factors have been attributed to its evolution, including mating system, inbreeding avoidance, and social complexity. Sex-biased dispersal and its potential link to individual social interactions were examined in the Qinghai toad-headed agamid (Phrynocephalus vlangalii). We first determined the pattern of sex-biased dispersal using population genetic methods. A total of 345 specimens from 32 sites in the Qaidam Basin were collected and genotyped for nine microsatellite DNA loci. Both individual-based assignment tests and allele frequency-based analyses were conducted. Females revealed much more genetic structure than males and all results were consistent with male-biased dispersal. First-generation migrants were also identified by genetic data. We then examined eight social interaction-related morphological traits and explored their potential link to sex-biased dispersal. Female residents had larger heads and longer tails than female migrants. The well-developed signal system among females, coupled with viviparity, might make remaining on natal sites beneficial, and hence promote female philopatry. Dominant females with larger heads were more likely to stay. Contrary to females, male migrants had larger heads and belly patches than residents, suggesting that dispersal might confer selective advantages for males. Such advantages may include opportunities for multiple mating and escaping from crowded sites. Large belly patches and several other morphological traits may assist their success in obtaining mates during dispersal. Furthermore, a relatively high relatedness (R = 0.06) among females suggested that this species might have rudimentary social structure. Case studies in "less" social species may provide important evidence for a better understanding of sex-biased dispersal.

Using genes as characters and a parsimony analysis to explore the phylogenetic position of turtles.

The phylogenetic position of turtles within the vertebrate tree of life remains controversial. Conflicting conclusions from different studies are likely a consequence of systematic error in the tree construction process, rather than random error from small amounts of data. Using genomic data, we evaluate the phylogenetic position of turtles with both conventional concatenated data analysis and a "genes as characters" approach. Two datasets were constructed, one with seven species (human, opossum, zebra finch, chicken, green anole, Chinese pond turtle, and western clawed frog) and 4584 orthologous genes, and the second with four additional species (soft-shelled turtle, Nile crocodile, royal python, and tuatara) but only 1638 genes. Our concatenated data analysis strongly supported turtle as the sister-group to archosaurs (the archosaur hypothesis), similar to several recent genomic data based studies using similar methods. When using genes as characters and gene trees as character-state trees with equal weighting for each gene, however, our parsimony analysis suggested that turtles are possibly sister-group to diapsids, archosaurs, or lepidosaurs. None of these resolutions were strongly supported by bootstraps. Furthermore, our incongruence analysis clearly demonstrated that there is a large amount of inconsistency among genes and most of the conflict relates to the placement of turtles. We conclude that the uncertain placement of turtles is a reflection of the true state of nature. Concatenated data analysis of large and heterogeneous datasets likely suffers from systematic error and over-estimates of confidence as a consequence of a large number of characters. Using genes as characters offers an alternative for phylogenomic analysis. It has potential to reduce systematic error, such as data heterogeneity and long-branch attraction, and it can also avoid problems associated with computation time and model selection. Finally, treating genes as characters provides a convenient method for examining gene and genome evolution.