Hot springs are some of the most special environments on Earth. Many prokaryotic and eukaryotic microbes have been found to live in this environment. The Himalayan geothermal belt (HGB) has numerous hot springs spread across the area. Comprehensive research using molecular techniques to investigate eukaryotic microorganisms is still lacking; investigating the composition and diversity of eukaryotic microorganisms such as protists in the hot spring ecosystems will not only provide critical information on the adaptations of protists to extreme conditions, but could also give valuable contributions to the global knowledge of biogeographic diversity. In this study, we used high-throughput sequencing to illuminate the diversity and composition pattern of protist communities in 41 geothermal springs across the HGB on the Tibetan Plateau. A total of 1238 amplicon sequence variants (ASVs) of protists were identified in the hot springs of the HGB. In general, Cercozoa was the phylum with the highest richness, and Bacillariophyta was the phylum with the highest relative abundance in protists. Based on the occurrence of protist ASVs, most of them are rare. A high variation in protist diversity was found in the hot springs of the HGB. The high variation in protist diversity may be due to the different in environmental conditions of these hot springs. Temperature, salinity, and pH are the most important environmental factors that affect the protist communities in the surface sediments of the hot springs in the HGB. In summary, this study provides the first comprehensive study of the composition and diversity of protists in the hot springs of the HGB and facilitates our understanding of the adaptation of protists in these extreme habitats.

Pelagic cephalopods have evolved a series of fascinating traits, such as excellent visual acuity, high-speed agility, and photophores for adaptation to open pelagic oceans. However, the genetic mechanisms underpinning these traits are not well understood. Thus, in this study, we obtained high-quality genomes of two purpleback flying squid species (Sthenoteuthis oualaniensis and Sthenoteuthis sp.), with sizes of 5450 Mb and 5651 Mb, respectively. Comparative genomic analyses revealed that the S-crystallin subfamily SL20-1 associated with visual acuity in the purpleback flying squid lineage was significantly expanded, and the evolution of high-speed agility for the species was accompanied by significant positive selection pressure on genes related to energy metabolism. These molecular signals might have contributed to the evolution of their adaptative predatory and anti-predatory traits. In addition, the transcriptomic analysis provided clear indications of the evolution of the photophores of purpleback flying squids, especially the recruitment of new genes and energy metabolism-related genes which may have played key functional roles in the process.

Morels (Morchella spp.) are iconic edible mushrooms with a long history of human consumption. Some microbial taxa are hypothesized to be important in triggering the formation of morel primordia and development of fruiting bodies, thus, there is interest in the microbial ecology of these fungi. To identify and compare fungal and prokaryotic communities in soils where Morchella sextelata is cultivated in outdoor greenhouses, ITS and 16S rDNA high throughput amplicon sequencing and microbiome analyses were performed. Pedobacter, Pseudomonas, Stenotrophomonas, and Flavobacterium were found to comprise the core microbiome of M. sextelata ascocarps. These bacterial taxa were also abundant in the soil beneath growing fruiting bodies. A total of 29 bacterial taxa were found to be statistically associated to Morchella fruiting bodies. Bacterial community network analysis revealed high modularity with some 16S rDNA operational taxonomic unit clusters living in specialized fungal niches (e.g., pileus, stipe). Other fungi dominating the soil mycobiome beneath morels included Morchella, Phialophora, and Mortierella. This research informs understanding of microbial indicators and potential facilitators of Morchella ecology and fruiting body production.

Despite the contribution of DNA barcoding towards understanding the biodiversity and distribution of species, the success of the mitochondrial cytochrome c oxidase subunit I gene (COI) amplification has been quite variable when it comes to Cephalopoda. Some species in this class such as Sthenoteuthis oualaniensis seem to be more difficult to amplify COI than others due to failed amplifications with universal primer and lack of specific set of primers. In this study, we developed new Sthenoteuthis - specific primer set, which significantly increased average amplification success. The new primer set will aid the recovery of barcodes from this difficult group and facilitate further studies in phylogeny and cryptic diversity of Sthenoteuthis oualaniensis.

Target sequence capture is an efficient technique to enrich specific genomic regions for high-throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP-based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome-scale data (hundreds or thousands of loci).

Beetles (Coleoptera) are the most diverse and species-rich group of insects, and a robust, time-calibrated phylogeny is fundamental to understanding macroevolutionary processes that underlie their diversity. Here we infer the phylogeny and divergence times of all major lineages of Coleoptera by analyzing 95 protein-coding genes in 373 beetle species, including ~67% of the currently recognized families. The subordinal relationships are strongly supported as Polyphaga (Adephaga (Archostemata, Myxophaga)). The series and superfamilies of Polyphaga are mostly monophyletic. The species-poor Nosodendridae is robustly recovered in a novel position sister to Staphyliniformia, Bostrichiformia, and Cucujiformia. Our divergence time analyses suggest that the crown group of extant beetles occurred ~297 million years ago (Mya) and that ~64% of families originated in the Cretaceous. Most of the herbivorous families experienced a significant increase in diversification rate during the Cretaceous, thus suggesting that the rise of angiosperms in the Cretaceous may have been an 'evolutionary impetus' driving the hyperdiversity of herbivorous beetles.

Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia) by targeting the cell wall components chitin and β-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.

Relative to the commonly used mitochondrial and nuclear protein-coding genes, the noncoding intron sequences are a promising source of informative markers that have the potential to resolve difficult phylogenetic nodes such as rapid radiations and recent divergences. Yet many issues exist in the use of intron markers, which prevent their extensive application as conventional markers. We used the diverse group of snakes as an example to try paving the way for massive identification and application of intron markers. We performed a series of bioinformatics screenings which identified appropriate introns between single-copy and conserved exons from two snake genomes, adding particular constraints on sequence length variability and sequence variability. A total of 1,273 candidate intron loci were retrieved. Primers for nested polymerase chain reaction (PCR) were designed for over a hundred candidates and tested in 16 snake representatives. 96 intron markers were developed that could be amplified across a broad range of snake taxa with high PCR successful rates. The markers were then applied to 49 snake samples. The large number of amplicons was subjected to next-generation sequencing (NGS). An analytic strategy was developed to accurately recover the amplicon sequences, and approximately, 76% of the marker sequences were recovered. The average p-distances of the intron markers at interfamily, intergenus, interspecies, and intraspecies levels were .168, .052, .015, and .004, respectively, suggesting that they were useful to study snake relationships of different evolutionary depths. A snake phylogeny was constructed with the intron markers, which produced concordant results with robust support at both interfamily and intragenus levels. The intron markers provide a convenient way to explore the signals in the noncoding regions to address the controversies on the snake tree. Our improved strategy of genome screening is effective and can be applied to other animal groups. NGS coupled with appropriate sequence processing can greatly facilitate the extensive application of molecular markers.

MicroRNA (miRNA) plays an important role in the control of gene expression. HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE (SE) to process primary miRNA (pri-miRNA) into mature miRNA. Although HYL1 has been shown to partner with DCL1 to enhance miRNA accuracy, the mechanism by which HYL1 selects the DCL1-targeted cleavage sites in pri-miRNA has remained unknown. By mutagenesis of HYL1 and analysis of in vivo pri-miRNA processing, we investigated the role of HYL1 in pri-miRNA cleavage. HYL1 forms homodimers in which the residues Gly147 and Leu165 in the dsRBD2 domain are shown to be critical. Disruption of HYL1 homodimerization causes incorrect cleavage at sites in pri-miRNA without interrupting the interaction of HYL1 with DCL1 and accumulation of pri-miRNAs in HYL1/pri-miRNA complexes, leading to a reduction in the efficiency and accuracy of miRNAs that results in strong mutant phenotypes of the plants. HYL1 homodimers may function as a molecular anchor for DCL1 to cleave at a distance from the ssRNA-dsRNA junction in pri-miRNA. These results suggest that HYL1 ensures the correct selection of pri-miRNA cleavage sites through homodimerization and thus contributes to gene silencing and plant development.

The interordinal relationships of Laurasiatherian mammals are currently one of the most controversial questions in mammalian phylogenetics. Previous studies mainly relied on coding sequences (CDS) and seldom used noncoding sequences. Here, by data mining public genome data, we compiled an intron data set of 3,638 genes (all introns from a protein-coding gene are considered as a gene) (19,055,073 bp) and a CDS data set of 10,259 genes (20,994,285 bp), covering all major lineages of Laurasiatheria (except Pholidota). We found that the intron data contained stronger and more congruent phylogenetic signals than the CDS data. In agreement with this observation, concatenation and species-tree analyses of the intron data set yielded well-resolved and identical phylogenies, whereas the CDS data set produced weakly supported and incongruent results. Further analyses showed that the phylogeny inferred from the intron data is highly robust to data subsampling and change in outgroup, but the CDS data produced unstable results under the same conditions. Interestingly, gene tree statistical results showed that the most frequently observed gene tree topologies for the CDS and intron data are identical, suggesting that the major phylogenetic signal within the CDS data is actually congruent with that within the intron data. Our final result of Laurasiatheria phylogeny is (Eulipotyphla,((Chiroptera, Perissodactyla),(Carnivora, Cetartiodactyla))), favoring a close relationship between Chiroptera and Perissodactyla. Our study 1) provides a well-supported phylogenetic framework for Laurasiatheria, representing a step towards ending the long-standing "hard" polytomy and 2) argues that intron within genome data is a promising data resource for resolving rapid radiation events across the tree of life.

Marine sediments can reserve many environmental pollutants. Lipophilic marine phycotoxins (LMPs) are natural toxic substances widespread in the marine environment; however, evidence of their existence in sediment is scarce. In the present study, in order to explore the occurrence and distribution characteristics of LMPs in sediment, surface sediment samples collected from a tropical area of Daya Bay (DYB) at different seasons, were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). According to the results, up to six toxin compounds were detected in sediment samples from DYB, OA and DTX1 had the highest levels, followed by PTX2, homo-YTX, AZA2, and GYM. Although AZA2 and GYM were found in most of the sediment, OA, DTX1, homo-YTX, and PTX2 were the predominant toxin compounds, and PTX2 was the most ubiquitous toxin in sediment. The spatial distribution of LMP components in the sediment fluctuated with sampling times, partially according to the physical-chemical parameters of the sediment. There are likely several sources for LMPs existing in surface sediments, but it is difficult to determine contributions of a specific toxin-source in the sediment. Therefore, marine sediments may be a toxin reservoir for LMPs accumulation in benthic organisms via food chains.

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2-C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Friable embryogenic callus (FEC) is considered as the most suitable material for efficient genetic transformation of cassava. Heavy genotype dependence of FEC induction and amenability to somaclonal variation limits the production and maintenance of reliable FEC. Identifying key elements involved in biological processes from somatic embryos (SEs) to FEC at different stages provides critical insights for FEC improvement. Cytological observation showed a dramatic change of subcellular structures among SEs, fresh FEC (FFEC), and old FEC (OFEC). Decrease of sucrose and increase of fructose and glucose were detected in OFEC. A total of 6871 differentially expressed genes (DEGs) were identified from SEs, FFEC, and OFEC by RNA-seq. Analysis of the DEGs showed that FEC induction was accompanied by the process of dedifferentiation, whereas the epigenetics modification occurred during the continuous subculturing process. The cell structure was reconstructed, mainly including the GO terms of "cell periphery" and "external encapsulating structure"; in parallel, the internal mechanisms changed correspondingly, including the biological process of glycolysis and metabolisms of alanine, aspartate, and glutamate. The significant reduction of genomic DNA methylation in OFEC indicated altered gene expression via chromatin modification. These results indicate that the induction and long-term subculture of FEC is a complicated biological process involving changes of genome modification, gene expression, and subcellular reconstruction. The findings will be useful for improving FEC induction and maintenance from farmer-preferred cassava cultivars recalcitrant to genetic transformation, hence improving cassava through genetic engineering.

Phenolamides represent one of the largest classes of plant-specialized secondary metabolites and function in diverse physiological processes, including defense responses and development. The biosynthesis of phenolamides requires the BAHD-family acyltransferases, which transfer acyl-groups from different acyl-donors specifically to amines, the acyl-group acceptors. However, the mechanisms of substrate specificity and multisite-acylation of the BAHD-family acyltransferases remain poorly understood. In this study, we provide a structural and biochemical analysis of AtSHT and AtSDT, two representative BAHD-family members that catalyze the multisite acylation of spermidine but show different product profiles. By determining the structures of AtSHT and AtSDT and using structure-based mutagenesis, we identified the residues important for substrate recognition in AtSHT and AtSDT and hypothesized that the acyl acceptor spermidine might adopt a free-rotating conformation in AtSHT, which can undergo mono-, di-, or tri-acylation; while the spermidine molecule in AtSDT might adopt a linear conformation, which only allows mono- or di-acylation to take place. In addition, through sequence similarity network (SSN) and structural modeling analysis, we successfully predicted and verified the functions of two uncharacterized Arabidopsis BAHD acyltransferases, OAO95042.1 and NP_190301.2, which use putrescine as the main acyl-acceptor. Our work provides not only an excellent starting point for understanding multisite acylation in BAHD-family enzymes, but also a feasible methodology for predicting possible acyl acceptor specificity of uncharacterized BAHD-family acyltransferases.

Pit vipers possess a unique thermal sensory system consisting of facial pits that allow them to detect minute temperature fluctuations within their environments. Biologists have long attempted to elucidate the genetic basis underlying the infrared perception of pit vipers. Early studies have shown that the TRPA1 gene is the thermal sensor associated with infrared detection in pit vipers. However, whether genes other than TRPA1 are also involved in the infrared perception of pit vipers remains unknown. Here, we sequenced the whole exomes of ten snake species and performed genome-wide evolutionary analyses to search for novel candidate genes that might be involved in the infrared perception of pit vipers. We applied both branch-length-comparison and selection-pressure-alteration analyses to identify genes that specifically underwent accelerated evolution in the ancestral lineage of pit vipers. A total of 47 genes were identified. These genes were significantly enriched in the ion transmembrane transporter, stabilization of membrane potential, and temperature gating activity functional categories. The expression levels of these candidate genes in relevant nerve tissues (trigeminal ganglion, dorsal root ganglion, midbrain, and cerebrum) were also investigated in this study. We further chose one of our candidate genes, the potassium channel gene KCNK4, as an example to discuss its possible role in the infrared perception of pit vipers. Our study provides the first genome-wide survey of infrared perception-related genes in pit vipers via comparative evolutionary analyses and reveals valuable candidate genes for future functional studies.

Nuclear Ca2+ oscillations allow symbiosis signaling, facilitating plant recognition of beneficial microsymbionts, nitrogen-fixing rhizobia, and nutrient-capturing arbuscular mycorrhizal fungi. Two classes of channels, DMI1 and CNGC15, in a complex on the nuclear membrane, coordinate symbiotic Ca2+ oscillations. However, the mechanism of Ca2+ signature generation is unknown. Here, we demonstrate spontaneous activation of this channel complex, through gain-of-function mutations in DMI1, leading to spontaneous nuclear Ca2+ oscillations and spontaneous nodulation, in a CNGC15-dependent manner. The mutations destabilize a hydrogen-bond or salt-bridge network between two RCK domains, with the resultant structural changes, alongside DMI1 cation permeability, activating the channel complex. This channel complex was reconstituted in human HEK293T cell lines, with the resultant calcium influx enhanced by autoactivated DMI1 and CNGC15s. Our results demonstrate the mode of activation of this nuclear channel complex, show that DMI1 and CNGC15 are sufficient to create oscillatory Ca2+ signals, and provide insights into its native mode of induction.

Symbiotic microorganisms improve nutrient uptake by plants. To initiate mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, plants perceive Myc factors, including lipochitooligosaccharides (LCOs) and short-chain chitooligosaccharides (CO4/CO5), secreted by AM fungi. However, the molecular mechanism of Myc factor perception remains elusive. In this study, we identified a heteromer of LysM receptor-like kinases consisting of OsMYR1/OsLYK2 and OsCERK1 that mediates the perception of AM fungi in rice. CO4 directly binds to OsMYR1, promoting the dimerization and phosphorylation of this receptor complex. Compared with control plants, Osmyr1 and Oscerk1 mutant rice plants are less sensitive to Myc factors and show decreased AM colonization. We engineered transgenic rice by expressing chimeric receptors that respectively replaced the ectodomains of OsMYR1 and OsCERK1 with those from the homologous Nod factor receptors MtNFP and MtLYK3 of Medicago truncatula. Transgenic plants displayed increased calcium oscillations in response to Nod factors compared with control rice. Our study provides significant mechanistic insights into AM symbiotic signal perception in rice. Expression of chimeric Nod/Myc receptors achieves a potentially important step toward generating cereals that host nitrogen-fixing bacteria.

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1-Mdm35-PA complex and the functional characterization of Ups1-Mdm35 in PA binding and transfer. Ups1 features a barrel-like structure consisting of an antiparallel β-sheet and three α-helices. Mdm35 adopts a three-helical clamp-like structure to wrap around Ups1 to form a stable complex. The β-sheet and α-helices of Ups1 form a long tunnel-like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate-binding site also plays an important role in the function of Ups1-Mdm35. Our study reveals the molecular basis of the function of Ups1-Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins.

Iron (Fe) deficiency is one of the most common micronutrient deficiencies limiting crop production globally, especially in arid regions because of decreased availability of iron in alkaline soils. Sweet potato [Ipomoea batatas (L.) Lam.] grows well in arid regions and is tolerant to Fe deficiency. Here, we report that the transcription of type I H+ -pyrophosphatase (H+ -PPase) gene IbVP1 in sweet potato plants was strongly induced by Fe deficiency and auxin in hydroponics, improving Fe acquisition via increased rhizosphere acidification and auxin regulation. When overexpressed, transgenic plants show higher pyrophosphate hydrolysis and plasma membrane H+ -ATPase activity compared with the wild type, leading to increased rhizosphere acidification. The IbVP1-overexpressing plants showed better growth, including enlarged root systems, under Fe-sufficient or Fe-deficient conditions. Increased ferric precipitation and ferric chelate reductase activity in the roots of transgenic lines indicate improved iron uptake, which is also confirmed by increased Fe content and up-regulation of Fe uptake genes, e.g. FRO2, IRT1 and FIT. Carbohydrate metabolism is significantly affected in the transgenic lines, showing increased sugar and starch content associated with the increased expression of AGPase and SUT1 genes and the decrease in β-amylase gene expression. Improved antioxidant capacities were also detected in the transgenic plants, which showed reduced H2 O2 accumulation associated with up-regulated ROS-scavenging activity. Therefore, H+ -PPase plays a key role in the response to Fe deficiency by sweet potato and effectively improves the Fe acquisition by overexpressing IbVP1 in crops cultivated in micronutrient-deficient soils.