Beyond RuBisCO: Convergent molecular evolution of multiple chloroplast genes in C4 plants

BackgroundThe recurrent evolution of the C4 photosynthetic pathway in angiosperms represents one of the most extraordinary examples of convergent evolution of a complex trait. Comparative genomic analyses have unveiled some of the molecular changes associated with the C4 pathway. For instance, several key enzymes involved in the transition from C3 to C4 photosynthesis have been found to share convergent amino acid replacements along C4 lineages. However, the extent of convergent replacements potentially associated with the emergence of C4 plants remains to be fully assessed. Here, we introduced a robust empirical approach to test molecular convergence along a phylogeny including multiple C3 and C4 taxa. By analyzing proteins encoded by chloroplast genes, we tested if convergent replacements occurred more frequently than expected in C4 lineages compared to C3 lineages. Furthermore, we sought to determine if convergent evolution occurred in multiple chloroplast proteins beside the well-known case of the large RuBisCO subunit encoded by the chloroplast gene rbcL.MethodsOur study was based on the comparative analysis of 43 C4 and 21 C3 grass species belonging to the PACMAD clade, a focal taxonomic group in many investigations of C4 evolution. We first used protein sequences of 67 orthologous chloroplast genes to build an accurate phylogeny of these species. Then, we inferred amino acid replacements along 13 C4 lineages and 9 C3 lineages using reconstructed protein sequences of their ancestral branches, corresponding to the most recent common ancestor of each lineage. Pairwise comparisons between ancestral branches allowed us to identify both convergent and divergent amino acid replacements between C4-C4, C3-C3 and C3-C4 lineages.ResultsThe reconstructed phylogenetic tree of 64 PACMAD grasses was characterized by strong supports in all nodes used for analyses of convergence. We identified 217 convergent replacements and 201 divergent replacements in 45/67 chloroplast proteins in both C4 and C3 ancestral branches. Pairs of C4-C4 ancestral branches showed higher levels of convergent replacements than C3-C3 and C3-C4 pairs. Furthermore, we found that more proteins shared unique convergent replacements in C4 lineages, with both RbcL and RpoC1 (the RNA polymerase beta’ subunit 1) showing a significantly higher convergent/divergent replacements ratio in C4 branches. Notably, significantly more C4-C4 pairs of ancestral branches showed higher numbers of convergent vs. divergent replacements than C3-C3 and C3-C4 pairs. Our results demonstrated that, in the PACMAD clade, C4 grasses experienced higher levels of molecular convergence than C3 species across multiple chloroplast genes. These findings have important implications for both our understanding of the evolution of photosynthesis and the goal of engineering improved crop varieties that integrates components of the C4 pathway.

Evolutionary rates of testes-expressed genes differ between monogamous and promiscuous Peromyscus species

Reproductive proteins, including those expressed in the testes, are among the fastest evolving proteins across the tree of life. Sexual selection on traits involved in sperm competition is thought to be a primary driver of testes gene evolution and is expected to differ between promiscuous and monogamous species due to intense competition between males to fertilize females in promiscuous lineages and lack thereof in monogamous ones. Here, we employ the rodent genus Peromyscus as a model to explore differences in evolutionary rates between testis-expressed genes of monogamous and promiscuous species. We find candidate genes that may be associated with increased sperm production in promiscuous species and gene ontology categories that show patterns of molecular convergence associated with phenotypic convergence in independently evolved monogamous species. Overall, our results highlight possible molecular correlates of differences in mating system, which can be contextualized in light of expected selective pressures.

Single Cell Transcriptomics Trajectory and Molecular Convergence of Clinically Relevant Mutations in Brugada Syndrome

Brugada syndrome (BrS) is a rare, inherited arrhythmia with high risk of sudden cardiac death. To evaluate the molecular convergence of clinically relevant mutations and to identify developmental cardiac cell types that are associated with BrS etiology, we collected 733 mutations represented by 16 sodium, calcium, potassium channels, regulatory and structural genes related to BrS. Among the clinically relevant mutations, 266 are unique singletons and 88 mutations are recurrent. We observed an over representation of clinically relevant mutations (~80%) in SCN5A gene, and also identified several candidate genes, including GPD1L, TRPM4 and SCN10A. Furthermore, protein domain enrichment analysis revealed that a large proportion of the mutations impacted ion-transport domains in multiple genes, including SCN5A, TRPM4 and SCN10A. A comparative protein domain analysis of SCN5A further established a significant (p=0.04) enrichment of clinically relevant mutations within ion-transport domain, including a significant (p=0.02) mutation hotspot within 1321-1380 residue. The enrichment of clinically relevant mutations within SCN5A ion transport domain is stronger (p=0.00003) among early onset of BrS. Our spatiotemporal cellular heart developmental (prenatal to adult) trajectory analysis applying single cell transcriptome identified the most frequently BrS mutated genes (SCN5A and GPD1L) are significantly upregulated in the prenatal cardiomyocytes. A more restrictive cellular expression trajectory is prominent in the adult heart ventricular cardiomyocytes compared to prenatal. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS associated cell type that provides insight into the complex genetic etiology of BrS.

Rapid evolution and molecular convergence in cryptorchidism-related genes associated with inherently undescended testes in mammals

Background The mammalian testis is an important male exocrine gland and spermatozoa-producing organ that usually lies in extra-abdominal scrotums to provide a cooler environment for spermatogenesis and sperm storage. Testicles sometimes fail to descend, leading to cryptorchidism. However, certain groups of mammals possess inherently ascrotal testes (i.e. testes that do not descend completely or at all) that have the same physiological functions as completely descended scrotal testes. Although several anatomical and hormonal factors involved in testicular descent have been studied, there is still a paucity of comprehensive research on the genetic mechanisms underlying the evolution of testicular descent in mammals and how mammals with ascrotal testes maintain their reproductive health. Results We performed integrative phenotypic and comparative genomic analyses of 380 cryptorchidism-related genes and found that the mammalian ascrotal testes trait is derived from an ancestral scrotal state. Rapidly evolving genes in ascrotal mammals were enriched in the Hedgehog pathway—which regulates Leydig cell differentiation and testosterone secretion—and muscle development. Moreover, some cryptorchidism-related genes in ascrotal mammals had undergone positive selection and contained specific mutations and indels. Genes harboring convergent/parallel amino acid substitutions between ascrotal mammals were enriched in GTPase functions. Conclusions Our results suggest that the scrotal testis is an ancestral state in mammals, and the ascrotal phenotype was derived multiple times in independent lineages. In addition, the adaptive evolution of genes involved in testicular descent and the development of the gubernaculum contributed to the evolution of ascrotal testes. Accurate DNA replication, the proper segregation of genetic material, and appropriate autophagy are the potential mechanisms for maintaining physiological normality during spermatogenesis in ascrotal mammals. Furthermore, the molecular convergence of GTPases is probably a mechanism in the ascrotal testes of different mammals. This study provides novel insights into the evolution of the testis and scrotum in mammals and contributes to a better understanding of the pathogenesis of cryptorchidism in humans.

Convergent Evolution and Structural Adaptation to the Deep Ocean in the Protein-Folding Chaperonin CCTα

The deep ocean is the largest biome on Earth and yet it is among the least studied environments of our planet. Life at great depths requires several specific adaptations; however, their molecular mechanisms remain understudied. We examined patterns of positive selection in 416 genes from four brittle star (Ophiuroidea) families displaying replicated events of deep-sea colonization (288 individuals from 216 species). We found consistent signatures of molecular convergence in functions related to protein biogenesis, including protein folding and translation. Five genes were recurrently positively selected, including chaperonin-containing TCP-1 subunit α (CCTα), which is essential for protein folding. Molecular convergence was detected at the functional and gene levels but not at the amino-acid level. Pressure-adapted proteins are expected to display higher stability to counteract the effects of denaturation. We thus examined in silico local protein stability of CCTα across the ophiuroid tree of life (967 individuals from 725 species) in a phylogenetically corrected context and found that deep-sea-adapted proteins display higher stability within and next to the substrate-binding region, which was confirmed by in silico global protein stability analyses. This suggests that CCTα displays not only structural but also functional adaptations to deep-water conditions. The CCT complex is involved in the folding of ∼10% of newly synthesized proteins and has previously been categorized as a “cold-shock” protein in numerous eukaryotes. We thus propose that adaptation mechanisms to cold and deep-sea environments may be linked and highlight that efficient protein biogenesis, including protein folding and translation, is a key metabolic deep-sea adaptation.

Phenotypic Convergence Is Not Mirrored at the Protein Level in a Lizard Adaptive Radiation

There are many compelling examples of molecular convergence at individual genes. However, the prevalence and the relative importance of adaptive genome-wide convergence remain largely unknown. Many recent works have reported striking examples of excess genome-wide convergence, but some of these studies have been called into question because of the use of inappropriate null models. Here, we sequenced and compared the genomes of 12 species of anole lizards that have independently converged on suites of adaptive behavioral and morphological traits. Despite extensive searches for a genome-wide signature of molecular convergence, we found no evidence supporting molecular convergence at specific amino acids either at individual genes or at genome-wide comparisons; we also uncovered no evidence supporting an excess of adaptive convergence in the rates of amino acid substitutions within genes. Our findings indicate that comprehensive phenotypic convergence is not mirrored at genome-wide protein-coding levels in anoles, and therefore, that adaptive phenotypic convergence is likely not constrained by the evolution of many specific protein sequences or structures.

Convergent genomic signatures of high-altitude adaptation among domestic mammals

Abundant and diverse domestic mammals living on the Tibetan Plateau provide useful materials for investigating adaptive evolution and genetic convergence. Here, we used 327 genomes from horses, sheep, goats, cattle, pigs and dogs living at both high and low altitudes, including 73 genomes generated for this study, to disentangle the genetic mechanisms underlying local adaptation of domestic mammals. Although molecular convergence is comparatively rare at the DNA sequence level, we found convergent signature of positive selection at the gene level, particularly the EPAS1 gene in these Tibetan domestic mammals. We also reported a potential function in response to hypoxia for the gene C10orf67, which underwent positive selection in three of the domestic mammals. Our data provide an insight into adaptive evolution of high-altitude domestic mammals, and should facilitate the search for additional novel genes involved in the hypoxia response pathway.

A functional enrichment test for molecular convergent evolution finds a clear protein-coding signal in echolocating bats and whales

Distantly related species entering similar biological niches often adapt by evolving similar morphological and physiological characters. How much genomic molecular convergence (particularly of highly constrained coding sequence) contributes to convergent phenotypic evolution, such as echolocation in bats and whales, is a long-standing fundamental question. Like others, we find that convergent amino acid substitutions are not more abundant in echolocating mammals compared to their outgroups. However, we also ask a more informative question about the genomic distribution of convergent substitutions by devising a test to determine which, if any, of more than 4,000 tissue-affecting gene sets is most statistically enriched with convergent substitutions. We find that the gene set most overrepresented (q-value = 2.2e-3) with convergent substitutions in echolocators, affecting 18 genes, regulates development of the cochlear ganglion, a structure with empirically supported relevance to echolocation. Conversely, when comparing to nonecholocating outgroups, no significant gene set enrichment exists. For aquatic and high-altitude mammals, our analysis highlights 15 and 16 genes from the gene sets most affected by molecular convergence which regulate skin and lung physiology, respectively. Importantly, our test requires that the most convergence-enriched set cannot also be enriched for divergent substitutions, such as in the pattern produced by inactivated vision genes in subterranean mammals. Showing a clear role for adaptive protein-coding molecular convergence, we discover nearly 2,600 convergent positions, highlight 77 of them in 3 organs, and provide code to investigate other clades across the tree of life.

Convergent evolution and structural adaptation to the deep ocean in the protein folding chaperonin CCTα

The deep ocean is the largest biome on Earth and yet it is among the least studied environments of our planet. Life at great depths requires several specific adaptations, however their molecular mechanisms remain understudied. We examined patterns of positive selection in 416 genes from four brittle star (Ophiuroidea) families displaying replicated events of deep-sea colonization (288 individuals from 216 species). We found consistent signatures of molecular convergence in functions related to protein biogenesis, including protein folding and translation. Five genes were recurrently positively selected, including CCTα (Chaperonin Containing TCP-1 subunit α), which is essential for protein folding. Molecular convergence was detected at the functional and gene levels but not at the amino-acid level. Pressure-adapted proteins are expected to display higher stability to counteract the effects of denaturation. We thus examined in silico local protein stability of CCTα across the ophiuroid tree of life (967 individuals from 725 species) in a phylogenetically-corrected context and found that deep sea-adapted proteins display higher stability within and next to the substrate-binding region, which was confirmed by in silico global protein stability analyses. This suggests that CCTα not only displays structural but also functional adaptations to deep water conditions. The CCT complex is involved in the folding of ∼10% of newly synthesized proteins and has previously been categorized as ‘cold-shock’ protein in numerous eukaryotes. We thus propose that adaptation mechanisms to cold and deep-sea environments may be linked and highlight that efficient protein biogenesis, including protein folding and translation, are key metabolic deep-sea adaptations.