Deciphering the origin of developmental stability: The role of intracellular expression variability in evolutionary conservation.

Progress in evolutionary developmental biology (evo-devo) has deepened our understanding of how intrinsic properties of embryogenesis, along with natural selection and population genetics, shape phenotypic diversity. A focal point of recent empirical and theoretical research is the idea that highly developmentally stable phenotypes are more conserved in evolution. Previously, we demonstrated that in Japanese medaka (Oryzias latipes), embryonic stages and genes with high stability, estimated through whole-embryo RNA-seq, are highly conserved in subsequent generations. However, the precise origin of the stability of gene expression levels evaluated at the whole-embryo level remained unclear. Such stability could be attributed to two distinct sources: stable intracellular expression levels or spatially stable expression patterns. Here we demonstrate that stability observed in whole-embryo RNA-seq can be attributed to stability at the cellular level (low variability in gene expression at the cellular levels). We quantified the intercellular variations in expression levels and spatial gene expression patterns for seven key genes involved in patterning dorsoventral and rostrocaudal regions during early development in medaka. We evaluated intracellular variability by counting transcripts and found its significant correlation with variation observed in whole-embryo RNA-seq data. Conversely, variation in spatial gene expression patterns, assessed through intraindividual left-right asymmetry, showed no correlation. Given the previously reported correlation between stability and conservation of expression levels throughout embryogenesis, our findings suggest a potential general trend: the stability or instability of developmental systems-and the consequent evolutionary diversity-may be primarily anchored in intrinsic fundamental elements such as the variability of intracellular states.

Effects of Temperature and Nutrition during the Larval Period on Life History Traits in an Invasive Malaria Vector Anopheles stephensi.

Anopheles stephensi is an Asian and Middle Eastern malaria vector, and it has recently spread to the African continent. It is needed to measure how the malaria parasite infection in A. stephensi is influenced by environmental factors to predict its expansion in a new environment. Effects of temperature and food conditions during larval periods on larval mortality, larval period, female wing size, egg production, egg size, adult longevity, and malaria infection rate were studied using a laboratory strain. Larval survival and female wing size were generally reduced when reared at higher temperatures and with a low food supply during the larval period. Egg production was not significantly affected by temperature during the larval period. Egg size was generally smaller in females reared at higher temperatures during the larval period. The infection rate of mosquitoes that fed on blood from malaria-infected mice was not affected by rearing temperature or food conditions during the larval period. Higher temperatures may reduce infection. A. stephensi; however, larger individuals can still be infective. We suggest that routinely recording the body size of adults in field surveys is effective in finding productive larval breeding sites and in predicting malaria risk.

Stability in gene expression and body-plan development leads to evolutionary conservation.

BACKGROUND: Phenotypic evolution is mainly explained by selection for phenotypic variation arising from factors including mutation and environmental noise. Recent theoretical and experimental studies have suggested that phenotypes with greater developmental stability tend to have a constant phenotype and gene expression level within a particular genetic and environmental condition, and this positively correlates with stronger evolutionary conservation, even after the accumulation of genetic changes. This could reflect a novel mechanism that contributes to evolutionary conservation; however, it remains unclear whether developmental stability is the cause, or whether at least it contributes to their evolutionary conservation. Here, using Japanese medaka lines, we tested experimentally whether developmental stages and gene expression levels with greater stability led to their evolutionary conservation. RESULTS: We first measured the stability of each gene expression level and developmental stage (defined here as the whole embryonic transcriptome) in the inbred F0 medaka population. We then measured their evolutionary conservation in the F3 generation by crossing the F0 line with the distantly related Japanese medaka line (Teradomori), followed by two rounds of intra-generational crossings. The results indicated that the genes and developmental stages that had smaller variations in the F0 generation showed lower diversity in the hybrid F3 generation, which implies a causal relationship between stability and evolutionary conservation. CONCLUSIONS: These findings suggest that the stability in phenotypes, including the developmental stages and gene expression levels, leads to their evolutionary conservation; this most likely occurs due to their low potential to generate phenotypic variation. In addition, since the highly stable developmental stages match with the body-plan-establishment stage, it also implies that the developmental stability potentially contributed to the strict conservation of animal body plan.

Wickerhamiella bidentis sp. nov., a novel yeast species isolated from flowers and insects in Japan.

Two strains were isolated from flowers and insects in Japan, namely NBRC 115686T and NBRC 115687, respectively. Based on sequence analysis of the D1/D2 domain of the 26S large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region and physiological characteristics, these strains were found to represent a novel yeast species of the genus Wickerhamiella. Considering pairwise sequence similarity, NBRC 115686T and NBRC 115687 differ from the type strain of the most closely related species, Wickerhamiella galacta NRRL Y-17645T, by 65-66 nucleotide substitutions with 12 gaps (11.65-11.83 %) in the D1/D2 domain of the LSU rRNA gene. The novel species differ from the closely related Wickerhamiella species in some physiological characteristics. For example, compared with Wickerhamiella galacta JCM 8257T, NBRC 115686T and NBRC 115687 assimilated d-galactose, and could grow at 35 and 37 °C. Hence, the name Wickerhamiella bidentis sp. nov. is proposed to accommodate this species in the genus Wickerhamiella. The holotype is NBRC 115686T (ex-type strain JCM 35540=CBS 18008).

Potential contribution of intrinsic developmental stability toward body plan conservation.

BACKGROUND: Despite the morphological diversity of animals, their basic anatomical patterns-the body plans in each animal phylum-have remained highly conserved over hundreds of millions of evolutionary years. This is attributed to conservation of the body plan-establishing developmental period (the phylotypic period) in each lineage. However, the evolutionary mechanism behind this phylotypic period conservation remains under debate. A variety of hypotheses based on the concept of modern synthesis have been proposed, such as negative selection in the phylotypic period through its vulnerability to embryonic lethality. Here we tested a new hypothesis that the phylotypic period is developmentally stable; it has less potential to produce phenotypic variations than the other stages, and this has most likely led to the evolutionary conservation of body plans. RESULTS: By analyzing the embryos of inbred Japanese medaka embryos raised under the same laboratory conditions and measuring the whole embryonic transcriptome as a phenotype, we found that the phylotypic period has greater developmental stability than other stages. Comparison of phenotypic differences between two wild medaka populations indicated that the phylotypic period and its genes in this period remained less variational, even after environmental and mutational modifications accumulated during intraspecies evolution. Genes with stable expression levels were enriched with those involved in cell-cell signalling and morphological specification such as Wnt and Hox, implying possible involvement in body plan development of these genes. CONCLUSIONS: This study demonstrated the correspondence between the developmental stage with low potential to produce phenotypic variations and that with low diversity in micro- and macroevolution, namely the phylotypic period. Whereas modern synthesis explains evolution as a process of shaping of phenotypic variations caused by mutations, our results highlight the possibility that phenotypic variations are readily limited by the intrinsic nature of organisms, namely developmental stability, thus biasing evolutionary outcomes.

Derivedness Index for Estimating Degree of Phenotypic Evolution of Embryos: A Study of Comparative Transcriptomic Analyses of Chordates and Echinoderms.

Species retaining ancestral features, such as species called living fossils, are often regarded as less derived than their sister groups, but such discussions are usually based on qualitative enumeration of conserved traits. This approach creates a major barrier, especially when quantifying the degree of phenotypic evolution or degree of derivedness, since it focuses only on commonly shared traits, and newly acquired or lost traits are often overlooked. To provide a potential solution to this problem, especially for inter-species comparison of gene expression profiles, we propose a new method named "derivedness index" to quantify the degree of derivedness. In contrast to the conservation-based approach, which deals with expressions of commonly shared genes among species being compared, the derivedness index also considers those that were potentially lost or duplicated during evolution. By applying our method, we found that the gene expression profiles of penta-radial phases in echinoderm tended to be more highly derived than those of the bilateral phase. However, our results suggest that echinoderms may not have experienced much larger modifications to their developmental systems than chordates, at least at the transcriptomic level. In vertebrates, we found that the mid-embryonic and organogenesis stages were generally less derived than the earlier or later stages, indicating that the conserved phylotypic period is also less derived. We also found genes that potentially explain less derivedness, such as Hox genes. Finally, we highlight technical concerns that may influence the measured transcriptomic derivedness, such as read depth and library preparation protocols, for further improvement of our method through future studies. We anticipate that this index will serve as a quantitative guide in the search for constrained developmental phases or processes.

Genomic insights of body plan transitions from bilateral to pentameral symmetry in Echinoderms.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

BACKGROUND: Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. RESULTS: By subjecting zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and chicken (Gallus gallus) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. CONCLUSION: In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the conserved pharyngula period, but rather around the early period in all the vertebrate species tested. These results suggest that negative selection by embryonic lethality could not explain hourglass-like conservation of animal embryos. This highlights the potential contribution of alternative mechanisms such as the diversifying effect of positive selections against earlier and later stages, and developmental constraints which lead to conservation of mid-embryonic stages.

Centromere evolution and CpG methylation during vertebrate speciation.

Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here, we perform de novo long-read genome assembly of three inbred medaka strains that are derived from geographically isolated subpopulations and undergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain three chromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource of twenty-two centromeric regions of length 20-345kbp. Centromeres are positionally conserved among the three strains and even between four pairs of chromosomes that were duplicated by the teleost-specific whole-genome duplication 320-350 million years ago. The centromeres do not all evolve at a similar pace; rather, centromeric monomers in non-acrocentric chromosomes evolve significantly faster than those in acrocentric chromosomes. Using methylation sensitive SMRT reads, we uncover centromeres are mostly hypermethylated but have hypomethylated sub-regions that acquire unique sequence compositions independently. These findings reveal the potential of non-acrocentric centromere evolution to contribute to speciation.