Search

Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life, but the evolutionary mechanisms that operate on regulatory interactions remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyse genomes, epigenomes and transcriptomes during early development in two Heliocidaris sea urchin species that exhibit highly divergent life histories and in an outgroup species. Positive selection and chromatin accessibility modifications within putative regulatory elements are enriched on the branch leading to the derived life history, particularly near dGRN genes. Single-cell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near conserved cell fate specification genes. Experimentally perturbing key transcription factors reveals profound evolutionary changes to early embryonic patterning events, disrupting regulatory interactions previously conserved for ~225 million years. These results demonstrate that natural selection can rapidly reshape developmental gene expression on a broad scale when selective regimes abruptly change. More broadly, even highly conserved dGRNs and patterning mechanisms in the early embryo remain evolvable under appropriate ecological circumstances.

Carya, in the Juglandiodeae subfamily, is to a typical temperate-subtropical forest-tree genus for studying the phylogenetic evolution and intercontinental disjunction between eastern Asia (EA) and North America (NA). Species of the genus have high economic values worldwide for their high-quality wood and the rich healthy factors of their nuts. Although previous efforts based on multiple molecular markers or genome-wide SNPs supported the monophyly of Carya and its two EA and NA major subclades, the maternal phylogeny of Carya still need to be comprehensively evaluated. The variation of Carya plastome has never been thoroughly characterized. Here, we novelly present 19 newly generated plastomes of congeneric Carya species, including the recently rediscovered critically endangered C. poilanei. The overall assessment of plastomes revealed highly conservative in the general structures. Our results indicated that remarkable differences in several plastome features are highly consistent with the EA-NA disjunction and showed the relatively diverse matrilineal sources among EA Carya compared to NA Carya. The maternal phylogenies were conducted with different plastome regions and full-length plastome datasets from 30 plastomes, representing 26 species in six genera of Juglandoideae and Myrica rubra (as root). Six out of seven phylogenetic topologies strongly supported the previously reported relationships among genera of Juglandoideae and the two subclades of EA and NA Carya, but displayed significant incongruencies between species within the EA and NA subclades. The phylogenetic tree generated from full-length plastomes demonstrated the optimal topology and revealed significant geographical maternal relationships among Carya species, especially for EA Carya within overlapping distribution areas. The full-length plastome-based phylogenetic topology also strongly supported the taxonomic status of five controversial species as separate species of Carya. Historical and recent introgressive hybridization and plastid captures might contribute to plastome geographic patterns and inconsistencies between topologies built from different datasets, while incomplete lineage sorting could account for the discordance between maternal topology and the previous nuclear genome data-based phylogeny. Our findings highlight full-length plastomes as an ideal tool for exploring maternal relationships among the subclades of Carya, and potentially in other outcrossing perennial woody plants, for resolving plastome phylogenetic relationships.

Lytechinus variegatus is a camarodont sea urchin found widely throughout the western Atlantic Ocean in a variety of shallow-water marine habitats. Its distribution, abundance, and amenability to developmental perturbation make it a popular model for ecologists and developmental biologists. Here, we present a chromosomal-level genome assembly of L. variegatus generated from a combination of PacBio long reads, 10× Genomics sequencing, and HiC chromatin interaction sequencing. We show L. variegatus has 19 chromosomes with an assembly size of 870.4 Mb. The contiguity and completeness of this assembly are reflected by a scaffold length N50 of 45.5 Mb and BUSCO completeness score of 95.5%. Ab initio and transcript-informed gene modeling and annotation identified 27,232 genes with an average gene length of 12.6 kb, comprising an estimated 39.5% of the genome. Repetitive regions, on the other hand, make up 45.4% of the genome. Physical mapping of well-studied developmental genes onto each chromosome reveals nonrandom spatial distribution of distinct genes and gene families, which provides insight into how certain gene families may have evolved and are transcriptionally regulated in this species. Lastly, aligning RNA-seq and ATAC-seq data onto this assembly demonstrates the value of highly contiguous, complete genome assemblies for functional genomics analyses that is unattainable with fragmented, incomplete assemblies. This genome will be of great value to the scientific community as a resource for genome evolution, developmental, and ecological studies of this species and the Echinodermata.

Changes in developmental gene regulatory networks (dGRNs) underlie much of the diversity of life1, but the evolutionary mechanisms that operate on interactions with these networks remain poorly understood. Closely related species with extreme phenotypic divergence provide a valuable window into the genetic and molecular basis for changes in dGRNs and their relationship to adaptive changes in organismal traits. Here we analyze genomes, epigenomes, and transcriptomes during early development in two sea urchin species in the genus Heliocidaris that exhibit highly divergent life histories and in an outgroup species. Signatures of positive selection and changes in chromatin status within putative gene regulatory elements are both enriched on the branch leading to the derived life history, and particularly so near dGRN genes; in contrast, positive selection within protein-coding regions have at most a modest enrichment in branch and function. Single-cell transcriptomes reveal a dramatic delay in cell fate specification in the derived state, which also has far fewer open chromatin regions, especially near dGRN genes with conserved roles in cell fate specification. Experimentally perturbing the function of three key transcription factors reveals profound evolutionary changes in the earliest events that pattern the embryo, disrupting regulatory interactions previously conserved for ∼225 million years. Together, these results demonstrate that natural selection can rapidly reshape developmental gene expression on a broad scale when selective regimes abruptly change and that even highly conserved dGRNs and patterning mechanisms in the early embryo remain evolvable under appropriate ecological circumstances.

Aureococcus anophagefferens caused brown tides for three consecutive years from2009 to2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dynamics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spatial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July,2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from2011 to2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other stations for its more selective feeding.

Brown tides caused by Aureococcus anophagefferens have occurred frequently in the coastal waters of Qinhuangdao since 2009. The Changli coast is the area most strongly affected by the brown tides, which result in significant economic losses and serious ecological harm. To understand the effects of brown tides on nanoplankton diversity and biomass, samples were collected from May to July in 2012 at station XKK, which is approximately 0.2 nautical miles off the Changli coast. The V9 region of the 18S rDNA gene was used as a marker and was analyzed by Illumina sequencing to assess the nanoplankton diversity and biomass, and real-time fluorescence quantitative PCR was performed to analyze the temporal variations in the copy concentrations of the 18S rDNA of nanoplankton. The results showed that nanoplankton diversity, as determined by the Shannon index, decreased from May to June and increased in July. Illumina sequencing analysis revealed that the plankton community differed among the samples from May to July, and over this period, the dominant phytoplankton groups changed from pelagophytes to chlorophytes. Sufficient nutrients and low grazing pressure by zooplankton and scallops accelerated the growth of small-celled phytoplankton. A. anophagefferens was the most dominant phytoplankton species, with an 18S rDNA maximum copy concentration detected on 26 June, followed by Micromonas sp. and Bathycoccus prasinos, with maximum copy concentrations observed on 3 July. Ciliophora, Arthropoda and Cercozoa were the dominant taxa of the local zooplankton community. The Ciliophora and Arthropoda biomasses decreased, and the Cercozoa biomass increased when the copy concentration of A. anophagefferens peaked on 26 June. We suspect that A. anophagefferens had a significantly negative effect on the growth of Ciliophora and Arthropoda and could be ingested by Cercozoa to promote its proliferation. The small and low-abundance plankton that are likely missed in microscopic and clone library analyses could be detected using Illumina sequencing and qPCR, which provide an effective method for a comprehensive analysis of the plankton community.

Background This investigation examined the effects of the microRNA miR-34c-5p on the proliferation, migration, and invasion of oral squamous cell carcinoma (OSCC) and the mechanisms involved. Methods The Gene Expression Omnibus (GEO) database was used to filter the chips, and the GEO2R software (https://www.ncbi.nlm.nih.gov/geo/geo2r/) was used to analyze the microarray data (GSE28100 and GSE45238). Gene set enrichment analysis (GSEA) was used to study the relationship between the expression of miR-34c-5p and the distant metastasis and pathological grade of OSCC. The correlation between TRIM29 (tripartite motif containing 29) expression and the malignant clinical phenotype of OSCC was also examined. The mRNA and protein expression levels of miR-34c-5p and TRIM29 were measured by real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analysis. The proliferation, migration, invasion and apoptosis of the human oral squamous carcinoma cell lines CAL-27 and Tca8113 was assessed by performing cell-counting kit-8 (CCK-8) assays, colony formation assays, transwell tests, wound scratch tests and flow cytometry. Luciferase reporter assays were used to predict the relationship between miR-34c-5p and TRIM29. A xenograft nude model was established and used to evaluate the effect of miR-34c-5p on tumor growth in female BALB/c mice. Results The expression of miR-34c-5p was significantly correlated with the proliferation, migration, and metastasis of OSCC. Overexpression of miR-34c-5p promoted the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and suppressed their apoptosis. Inversely, low expression of miR-34c-5p suppressed the proliferation, migration, and invasion of CAL-27 and Tca8113 cells, and promoted their apoptosis. Overexpression of miR-34c-5p promoted tumor growth in the xenograft nude mice model. The expression of TRIM29 was related to malignant clinical phenotype of OSCC. Overexpression of TRIM29 inhibited the proliferation, migration and invasion of CAL-27 and Tca8113 cell, and induced their apoptosis. TRIM29 knockout had just the opposite effect. Importantly, miR-34c-5p binds to TRIM29 and inhibited TRIM29 expression. Conclusions MiR-34c-5p regulates the proliferation, migration, invasion, and apoptosis of OSCC through targeted binding of TRIM29. This may represent a novel therapeutic target for the treatment of patients with OSCC.

In selective breeding, a central parameter in summarizing the proportion of variance due to genetics for the purpose of predicting gains from selection is realized heritability (h2). We applied the genome-wide complex trait analysis (GCTA) method to genome-wide SNP data obtained by 2b-RAD reduced-representation genotyping and phenotypic data for four traits (shell length, shell height, shell width and whole wet weight) with the aim of establishing the heritability for growth in Zhikong scallop (Chlamys farreri) in a selective breeding program. The GCTA-based heritabilities of 0.42 (S.E. 0.09) for shell length, 0.47 (S.E. 0.07) for shell height, 0.54 (S.E. 0.11) for shell width and 0.28 (S.E. 0.03) for whole wet weight, which were estimated with uncommon SNPs (26,471 SNPs with MAF >2%), were close to and strongly correlated (r = 0.957) with traditional estimates of realized heritability and in the moderate-to-high range, in line with values previously obtained for growth rates in bivalves. hGCTA2 with 20,000 and 10,000 SNPs were very close to estimates with uncommon SNPs and appeared relatively robust to SNP number. Removing causal SNPs had little effect on obtaining reliable estimates of hGCTA2, suggesting that causal SNPs is not necessary for accurate estimates of h2. Chromosome-wise heritability estimates suggested that the genetic contribution to growth complex traits is scattered across the genome and driven by many loci with small effect rather than a few causal loci with a large effect. These results indicate that the genome-wide complex trait analysis method may be useful for estimating realized heritability for growth in Zhikong scallop with SNPs obtained by reduced-representation genotyping approaches, which is less expensive and faster than full-genome sequencing for non-model species growing in natural environments.

Cardiac activity has been widely used in marine molluscs as an indicator for their physiological status in response to environmental changes, which is, however, largely less studied in scallops. Here, we monitored cardiac performance of Zhikong scallop Chlamys farreri using an infrared-based method, and evaluated the effects of several biotic (shell height, total weight, and age) and environmental factors (circadian rhythm and temperature) on scallop heart rate (HR), amplitude (HA), and rate-amplitude product (RAP). Results revealed that size has a significant effect on both HR (negative) and HA (positive), but RAP values are similar in different sized scallops. Age also affects scallop cardiac performance, significantly for HR, but not for HA or RAP. Circadian rhythm affects cardiac activity, with significant elevation of HR, HA and RAP during 1:00–8:00 and 17:00–19:00. With seawater temperature elevation, HR peaks at 30.03 ± 0.23°C, HA at 15.08 ± 0.02°C, and RAP at 15.10 ± 0.19 and 30.12 ± 0.28°C. This suggests HR is a good indicator for thermal limit, whereas HA may indicate optimal growth temperature, and RAP could be an index of myocardial oxygen consumption to indicate myocardium stress. Our study provides basic information on the factors that may affect scallop cardiac performance. It also elucidates the feasibility of HA and RAP as cardiac indices in marine molluscs.

Targeted genotyping of transcriptome-scale genetic markers is highly attractive for genetic, ecological, and evolutionary studies, but achieving this goal in a cost-effective manner remains a major challenge, especially for laboratories working on nonmodel organisms. Here, we develop a high-throughput, sequencing-based GoldenGate approach (called HD-Marker), which addresses the array-related issues of original GoldenGate methodology and allows for highly multiplexed and flexible targeted genotyping of more than 12,000 loci in a single-tube assay (in contrast to fewer than 3100 in the original GoldenGate assay). We perform extensive analyses to demonstrate the power and performance of HD-Marker on various multiplex levels (296, 795, 1293, and 12,472 genic SNPs) across two sequencing platforms in two nonmodel species (the scallops Chlamys farreri and Patinopecten yessoensis), with extremely high capture rate (98%–99%) and genotyping accuracy (97%–99%). We also demonstrate the potential of HD-Marker for high-throughput targeted genotyping of alternative marker types (e.g., microsatellites and indels). With its remarkable cost-effectiveness (as low as $0.002 per genotype) and high flexibility in choice of multiplex levels and marker types, HD-Marker provides a highly attractive tool over array-based platforms for fulfilling genome/transcriptome-wide targeted genotyping applications, especially in nonmodel organisms.

Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3 h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.

Thermal tolerance has become an active research area in marine poikilotherms due to the influence of increased sea temperature caused by global warming. Previous indicators of thermal tolerance in bivalves are generally laborious and time-consuming and even require killing the specimens. In this study, we demonstrated that heart rate (HR) was a stable and reliable indicator for scallop physiological status by applying an infrared-based cardiac performance monitoring system. The feasibility of HR-based Arrhenius break temperatures (ABTs) as a scallop thermal tolerance indicator was evaluated by investigating ABTs of four species with different thermal limits, including the Yesso scallop (Patinopecten yessoensis), the Zhikong scallop (Chlamys farreri), the bay scallop (Argopecten irradians), and the Catarina scallop (Argopecten ventricosus). In accordance with the thermal limits, ABTs of the Yesso scallop, Zhikong scallop, bay scallop, and Catarina scallop were 22.03 ± 0.19, 29.10 ± 0.25, 32.20 ± 0.25, and 34.09 ± 0.19 °C, respectively, suggesting that the ABT could indicate thermal limits in interspecific scallops. Variations in the ABTs were observed among intraspecific scallops with different sizes, weights, and ages, suggesting that smaller and younger scallops tend to have higher thermal limits. Significant differences in ABT were also observed between pre- and post-spawning individuals, implying that spawning behavior could decrease scallop thermal limits. The above results suggest that HR-based ABT can detect not only interspecific but also intraspecific thermal tolerance in scallops. This study reports the feasibility of infrared-based cardiac performance as a rapid, efficient, and noninvasive indicator for bivalve thermal tolerance.

Reconstructing the genomes of bilaterian ancestors is central to our understanding of animal evolution, where knowledge from ancient and/or slow-evolving bilaterian lineages is critical. Here we report a high-quality, chromosome-anchored reference genome for the scallop Patinopecten yessoensis, a bivalve mollusc that has a slow-evolving genome with many ancestral features. Chromosome-based macrosynteny analysis reveals a striking correspondence between the 19 scallop chromosomes and the 17 presumed ancestral bilaterian linkage groups at a level of conservation previously unseen, suggesting that the scallop may have a karyotype close to that of the bilaterian ancestor. Scallop Hox gene expression follows a new mode of subcluster temporal co-linearity that is possibly ancestral and may provide great potential in supporting diverse bilaterian body plans. Transcriptome analysis of scallop mantle eyes finds unexpected diversity in phototransduction cascades and a potentially ancient Pax2/5/8-dependent pathway for noncephalic eyes. The outstanding preservation of ancestral karyotype and developmental control makes the scallop genome a valuable resource for understanding early bilaterian evolution and biology.