Detection of evolutionary conserved and accelerated genomic regions related to adaptation to thermal niches in Anolis lizards.

Understanding the genetic basis for adapting to thermal environments is important due to serious effects of global warming on ectothermic species. Various genes associated with thermal adaptation in lizards have been identified mainly focusing on changes in gene expression or the detection of positively selected genes using coding regions. Only a few comprehensive genome-wide analyses have included noncoding regions. This study aimed to identify evolutionarily conserved and accelerated genomic regions using whole genomes of eight Anolis lizard species that have repeatedly adapted to similar thermal environments in multiple lineages. Evolutionarily conserved genomic regions were extracted as regions with overall sequence conservation (regions with fewer base substitutions) across all lineages compared with the neutral model. Genomic regions that underwent accelerated evolution in the lineage of interest were identified as those with more base substitutions in the target branch than in the entire background branch. Conserved elements across all branches were relatively abundant in "intergenic" genomic regions among noncoding regions. Accelerated regions (ARs) of each lineage contained a significantly greater proportion of noncoding RNA genes than the entire multiple alignment. Common genes containing ARs within 5 kb of their vicinity in lineages with similar thermal habitats were identified. Many genes associated with circadian rhythms and behavior were found in hot-open and cool-shaded habitat lineages. These genes might play a role in contributing to thermal adaptation and assist future studies examining the function of genes involved in thermal adaptation via genome editing.

Genetic differentiation and local adaptation of the Japanese honeybee, Apis cerana japonica.

We examine the population genetic structure and divergence among the regional populations of the Japanese honeybee, Apis cerana japonica, by re-sequencing the genomes of 105 individuals from the three main Japanese islands with diverse climates. The genetic structure results indicated that these individuals are distinct from the mainland Chinese A. cerana samples. Furthermore, population structure analyses have identified three genetically distinct geographic regions in Japan: Northern (Tohoku-Kanto-Chubu districts), Central (Chugoku district), and Southern (Kyushu district). In some districts, "possible non-native" individuals, likely introduced from other regions in recent years, were discovered. Then, genome-wide scans were conducted to detect candidate genes for adaptation by two different approaches. We performed a population branch statistics (PBS) analysis to identify candidate genes for population-specific divergence. A latent factor mixed model (LFMM) was used to identify genes associated with climatic variables along a geographic gradient. The PBSmax analysis identified 25 candidate genes for population-specific divergence whereas the LFMM analysis identified 73 candidate genes for adaptation to climatic variables along a geographic gradient. However, no common genes were identified by both methods.

Environmental pH signals the release of monosaccharides from cell wall in coral symbiotic alga.

Reef-building corals thrive in oligotrophic environments due to their possession of endosymbiotic algae. Confined to the low pH interior of the symbiosome within the cell, the algal symbiont provides the coral host with photosynthetically fixed carbon. However, it remains unknown how carbon is released from the algal symbiont for uptake by the host. Here we show, using cultured symbiotic dinoflagellate, Breviolum sp., that decreases in pH directly accelerates the release of monosaccharides, that is, glucose and galactose, into the ambient environment. Under low pH conditions, the cell surface structures were deformed and genes related to cellulase were significantly upregulated in Breviolum. Importantly, the release of monosaccharides was suppressed by the cellulase inhibitor, glucopyranoside, linking the release of carbon to degradation of the agal cell wall. Our results suggest that the low pH signals the cellulase-mediated release of monosaccharides from the algal cell wall as an environmental response in coral reef ecosystems.

Coral reefs are known as 'treasure troves of biodiversity' because of the enormous variety of different fish, crustaceans and other marine life they support. Colonies of marine animals, known as corals, which are anchored to rocks on the sea bed, form the main structures of a coral reef. Many corals rely on partnerships with microscopic algae known as dinoflagellates for most of their energy needs. The dinoflagellates use sunlight to make sugars and other carbohydrates and they give some of these to the coral. In exchange, the coral provides a home for the dinoflagellates inside its body. The algae live inside special compartments within coral cells known as symbiosomes. These compartments have a lower pH (that is, they are more acidic) than the rest of the coral cell. Previous studies have shown that the algae release sugars into the symbiosome but it remains unclear what triggers this release and whether it only occurs when the algae are in a partnership. Ishii et al. studied a type of dinoflagellate known as Breviolum sp. that had been grown in sea water-like liquid in a laboratory. The experiments found that the alga released two sugar molecules known as glucose and galactose into its surroundings even in the absence of a host coral. Increasing the acidity of the liquid caused the alga to release more sugars and resulted in changes to some of the structures on the surface of its cells. The alga also produced an enzyme, called cellulase, to degrade the wall that normally surrounds the cell of an alga. Treating the alga with a drug that inhibits the activity of cellulase also suppressed the release of sugars from the cells. These findings suggest that when dinoflagellates enter acidic environments, like the guts of marine animals or symbiosomes inside coral cells, the decrease in pH can activate the algal cellulase enzyme, which in turn triggers the release of sugars for the coral. This research will provide a new viewpoint to those interested in how partnerships between animals and algae are sustained in marine environments. It also highlights the importance of the alga cell wall in establishing partnerships with corals. Further work will seek to clarify the precise biological mechanisms involved.

Establishment of a New Model Sea Anemone for Comparative Studies on Cnidarian-Algal Symbiosis.

Frequent coral bleaching has drawn attention to the mechanisms of coral dinoflagellate endosymbiosis. Owing to the difficulty of rearing corals in the laboratory, model symbiosis systems are desired. The sea anemone Exaiptasia diaphana, hosting clade B1 of the genus Breviolum, has long been studied as a model system; however, a single species is insufficient for comparative studies and thus provides only limited resources for symbiosis research, especially regarding the specificity of host-symbiont associations. We established a clonal strain of the sea anemone Anthopleura atodai, whose symbiont was identified as a novel subclade of Symbiodinium (clade A) using a novel feeding method. We also developed a method to efficiently bleach various sea anemone species using a quinoclamine-based herbicide. Bleached A. atodai polyps were vital and able to reproduce asexually, exhibiting no signs of harmful effects of the drug treatment. Pilot studies have suggested that host-symbiont specificity is influenced by multiple steps differently in A. atodai and E. diaphana. RNAseq analyses of A. atodai showed that multiple NPC2 genes were expressed in the symbiotic state, which have been suggested to function in the transport of sterols from symbionts to host cells. These results reveal the usefulness of A. atodai in comparative studies of cnidarian-algal symbiosis.

On the origin of appetite: GLWamide in jellyfish represents an ancestral satiety neuropeptide.

Food intake is regulated by internal state. This function is mediated by hormones and neuropeptides, which are best characterized in popular model species. However, the evolutionary origins of such feeding-regulating neuropeptides are poorly understood. We used the jellyfish Cladonema to address this question. Our combined transcriptomic, behavioral, and anatomical approaches identified GLWamide as a feeding-suppressing peptide that selectively inhibits tentacle contraction in this jellyfish. In the fruit fly Drosophila, myoinhibitory peptide (MIP) is a related satiety peptide. Surprisingly, we found that GLWamide and MIP were fully interchangeable in these evolutionarily distant species for feeding suppression. Our results suggest that the satiety signaling systems of diverse animals share an ancient origin.

Does selection occur at the intermediate zone of two insufficiently isolated populations? A whole-genome analysis along an altitudinal gradient.

Adaptive divergence occurs even between insufficiently isolated populations when there is a great difference in environments between their habitats. Individuals present in an intermediate zone of the two divergent populations are expected to have an admixed genetic structure due to gene flow. A selective pressure that acts on the genetically admixed individuals may limit the gene flow and maintain the adaptive divergence. Here, we addressed a question whether selection occurs in the genetically admixed individuals between two divergent populations. Arabidopsis halleri is a perennial montane plant, which has clear phenotypic dimorphisms between highland and lowland habitats in Mt. Ibuki, central Japan. We obtained the whole-genome sequences of Arabidopsis halleri plants along an altitudinal gradient of 359-1,317 m with a high spatial resolution (mean altitudinal interval of 20 m). We found a zone where the highland and lowland genes were mixing (intermediate subpopulation). In the intermediate subpopulation, we identified 5 and 13 genome regions, which included 3 and 8 genes, that had a high frequency of alleles that are accumulated in highland and lowland subpopulations, respectively. In addition, we also found that the frequency of highland alleles of these selected genome regions was smaller in the lowland subpopulation compared with that of the non-selected regions. These results suggest that the selection in the intermediate subpopulation might limit the gene flow and contribute to the adaptive divergence between altitudes. We also identified 7 genome regions that had low heterozygote frequencies in the intermediate subpopulation. We conclude that different types of selection in addition to gene flow occur at the intermediate altitude and shape the genetic structure across altitudes.

Gene expression alterations from reversible to irreversible stages during coral metamorphosis.

For corals, metamorphosis from planktonic larvae to sedentary polyps is an important life event, as it determines the environment in which they live for a lifetime. Although previous studies on the reef-building coral Acropora have clarified a critical time point during metamorphosis when cells are committed to their fates, as defined by an inability to revert back to their previous states as swimming larvae (here referred to as the "point of no return"), the molecular mechanisms of this commitment to a fate remain unclear. To address this issue, we analyzed the transcriptomic changes before and after the point of no return by inducing metamorphosis of Acropora tenuis with Hym-248, a metamorphosis-inducing neuropeptide. Gene Ontology and pathway enrichment analysis of the 5893 differentially expressed genes revealed that G protein-coupled receptors (GPCRs) were enriched, including GABA receptor and Frizzled gene subfamilies, which showed characteristic temporal expression patterns. The GPCRs were then classified by comparison with those of Homo sapiens, Nematostella vectensis and Platynereis dumerilii. Classification of the differentially expressed genes into modules based on expression patterns showed that some modules with large fluctuations after the point of no return were biased toward functions such as protein metabolism and transport. This result suggests that in precommitted larvae, different types of GPCR genes function to ensure a proper environment, whereas in committed larvae, intracellular protein transport and proteolysis may cause a loss of the reversibility of metamorphosis as a result of cell differentiation.

Genetic basis of orange spot formation in the guppy (Poecilia reticulata).

BACKGROUND: To understand the evolutionary significance of female mate choice for colorful male ornamentation, the underlying regulatory mechanisms of such ornamentation must be understood for examining how the ornaments are associated with "male qualities" that increase the fitness or sexual attractiveness of offspring. In the guppy (Poecilia reticulata), an established model system for research on sexual selection, females prefer males possessing larger and more highly saturated orange spots as potential mates. Although previous studies have identified some chromosome regions and genes associated with orange spot formation, the regulation and involvement of these genetic elements in orange spot formation have not been elucidated. In this study, the expression patterns of genes specific to orange spots and certain color developmental stages were investigated using RNA-seq to reveal the genetic basis of orange spot formation. RESULTS: Comparing the gene expression levels of male guppy skin with orange spots (orange skin) with those without any color spots (dull skin) from the same individuals identified 1102 differentially expressed genes (DEGs), including 630 upregulated genes and 472 downregulated genes in the orange skin. Additionally, the gene expression levels of the whole trunk skin were compared among the three developmental stages and 2247 genes were identified as DEGs according to color development. These analyses indicated that secondary differentiation of xanthophores may affect orange spot formation. CONCLUSIONS: The results suggested that orange spots might be formed by secondary differentiation, rather than de novo generation, of xanthophores, which is induced by Csf1 and thyroid hormone signaling pathways. Furthermore, we suggested candidate genes associated with the areas and saturation levels of orange spots, which are both believed to be important for female mate choice and independently regulated. This study provides insights into the genetic and cellular regulatory mechanisms underlying orange spot formation, which would help to elucidate how these processes are evolutionarily maintained as ornamental traits relevant to sexual selection.

Detection of genes positively selected in Cuban Anolis lizards that naturally inhabit hot and open areas and currently thrive in urban areas.

Species of Anolis lizards of the West Indies that naturally inhabit hot and open areas also tend to thrive in urban areas. In this study, transcriptome was sequenced for nine species of Cuban Anolis lizards that are closely related to each other, but inhabit different thermal microhabitats. Using PAML and HyPhy software, we attempted to identify genes and amino acid sites under positive selection in the common ancestral branch of A. porcatus and A. allisoni, and the branch of A. sagrei, which inhabit hot and open areas, and thrive in urban areas. Although there were no genes where positive selection was commonly detected on both of the tested branches, positive selection was detected in genes involved in the stress response (e.g., DNA damage and oxidative stress) and cardiac function, which could be related to adaptive evolution of tolerance to heat or ultraviolet radiation, on both branches. These findings suggest that adaptive evolution of the response to stress caused by heat or ultraviolet radiation might have occurred in ancestors of Anolis species inhabiting hot and open areas and might be related to the current thriving in urban areas of them.

Human-specific mutations in VMAT1 confer functional changes and multi-directional evolution in the regulation of monoamine circuits.

BACKGROUND: Neurochemicals like serotonin and dopamine play crucial roles in human cognitive and emotional functions. Vesicular monoamine transporter 1 (VMAT1) transports monoamine neurotransmitters, and its variant (136Thr) is associated with various psychopathological symptoms and reduced monoamine uptake relative to 136Ile. We previously showed that two human-specific amino acid substitutions (Glu130Gly and Asn136Thr/Ile) of VMAT1 were subject to positive natural selection. However, the potential functional alterations caused by these substitutions (Glu130Gly and Asn136Thr) remain unclear. To assess functional changes in VMAT1 from an evolutionary perspective, we reconstructed ancestral residues and examined the role of these substitutions in monoamine uptake in vitro using fluorescent false neurotransmitters (FFN), which are newly developed substances used to quantitatively assay VMATs. RESULTS: Immunoblotting confirmed that all the transfected YFP-VMAT1 variants are properly expressed in HEK293T cells at comparable levels, and no significant difference was seen in the density and the size of vesicles among them. Our fluorescent assays revealed a significant difference in FFN206 uptake among VMAT1 variants: 130Glu/136Asn, 130Glu/136Thr, and 130Gly/136Ile showed significantly higher levels of FFN206 uptake than 130Gly/136Asn and 130Gly/136Thr, indicating that both 130Glu and 136Ile led to increased neurotransmitter uptake, for which 136Thr and 136Asn were comparable by contrast. CONCLUSIONS: These findings suggest that monoamine uptake by VMAT1 initially declined (from 130Glu/136Asn to 130Gly/136Thr) in human evolution, possibly resulting in higher susceptibility to the external environment of our ancestors.

Global Shifts in Gene Expression Profiles Accompanied with Environmental Changes in Cnidarian-Dinoflagellate Endosymbiosis.

Stable endosymbiotic relationships between cnidarian animals and dinoflagellate algae are vital for sustaining coral reef ecosystems. Recent studies have shown that elevated seawater temperatures can cause the collapse of their endosymbiosis, known as 'bleaching', and result in mass mortality. However, the molecular interplay between temperature responses and symbiotic states still remains unclear. To identify candidate genes relevant to the symbiotic stability, we performed transcriptomic analyses under multiple conditions using the symbiotic and apo-symbiotic (symbiont free) Exaiptasia diaphana, an emerging model sea anemone. Gene expression patterns showed that large parts of differentially expressed genes in response to heat stress were specific to the symbiotic state, suggesting that the host sea anemone could react to environmental changes in a symbiotic state-dependent manner. Comparative analysis of expression profiles under multiple conditions highlighted candidate genes potentially important in the symbiotic state transition under heat-induced bleaching. Many of these genes were functionally associated with carbohydrate and protein metabolisms in lysosomes. Symbiont algal genes differentially expressed in hospite encode proteins related to heat shock response, calcium signaling, organellar protein transport, and sugar metabolism. Our data suggest that heat stress alters gene expression in both the hosts and symbionts. In particular, heat stress may affect the lysosome-mediated degradation and transportation of substrates such as carbohydrates through the symbiosome (phagosome-derived organelle harboring symbiont) membrane, which potentially might attenuate the stability of symbiosis and lead to bleaching-associated symbiotic state transition.

Isolation of uracil auxotroph mutants of coral symbiont alga for symbiosis studies.

Coral reef ecosystems rely on stable symbiotic relationship between the dinoflagellate Symbiodinium spp. and host cnidarian animals. The collapse of such symbiosis could cause coral 'bleaching' and subsequent host death. Despite huge interest on Symbiodinium, lack of mutant strains and readily available genetic tools have hampered molecular research. A major issue was the tolerance to marker antibiotics. Here, we isolated Symbiodinium mutants requiring uracil for growth, and hence, useful in transformation screening. We cultured Symbiodinium spp. cells in the presence of 5-fluoroorotic acid (5FOA), which inhibits the growth of cells expressing URA3 encoding orotidine-5'-monophosphate decarboxylase, and isolated cells that require uracil for growth. Sequence analyses and genetic complementation tests using yeast demonstrated that one of the mutant cell lines had a point mutation in URA3, resulting in a splicing error at an unusual exon-intron junction, and consequently, loss of enzyme activity. This mutant could maintain a symbiotic relationship with the model sea anemone Exaiptasia pallida only in sea water containing uracil. Results show that the URA3 mutant will be a useful tool for screening Symbiodinium transformants, both ex and in hospite, as survival in the absence of uracil is possible only upon successful introduction of URA3.