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Typical avian eyes are phenotypically engineered for photopic vision (daylight). In contrast, the highly derived eyes of the barn owl (Tyto alba) are adapted for scotopic vision (dim light). The dramatic modifications distinguishing barn owl eyes from other birds include: 1) shifts in frontal orientation to improve binocularity, 2) rod-dominated retina, and 3) enlarged corneas and lenses. Some of these features parallel mammalian eye patterns, which are hypothesized to have initially evolved in nocturnal environments. Here, we used an integrative approach combining phylogenomics and functional phenotypes of 211 eye-development genes across 48 avian genomes representing most avian orders, including the stem lineage of the scotopic-adapted barn owl. Overall, we identified 25 eye-development genes that coevolved under intensified or relaxed selection in the retina, lens, cornea, and optic nerves of the barn owl. The agtpbp1 gene, which is associated with the survival of photoreceptor populations, was pseudogenized in the barn owl genome. Our results further revealed that barn owl retinal genes responsible for the maintenance, proliferation, and differentiation of photoreceptors experienced an evolutionary relaxation. Signatures of relaxed selection were also observed in the lens and cornea morphology-associated genes, suggesting that adaptive evolution in these structures was essentially structural. Four eye-development genes (ephb1, phactr4, prph2, and rs1) evolved in positive association with the orbit convergence in birds and under relaxed selection in the barn owl lineage, likely contributing to an increased reliance on binocular vision in the barn owl. Moreover, we found evidence of coevolutionary interactions among genes that are expressed in the retina, lens, and optic nerve, suggesting synergetic adaptive events. Our study disentangles the genomic changes governing the binocularity and low-light perception adaptations of barn owls to nocturnal environments while revealing the molecular mechanisms contributing to the shift from the typical avian photopic vision to the more-novel scotopic-adapted eye.

Sex chromosomes recombine restrictly in their homologous area, the pseudoautosomal region (PAR), represented by PAR1 and PAR2, which behave like an autosome in both pairing and recombination. The PAR1, common to most of the eutherian mammals, is located at the terminus of the sex chromosomes short arm and exhibit recombination rates ~20 times higher than the autosomes. Here, we assessed the interspecific evolutionary genomic dynamics of 15 genes of the PAR1 across 41 mammalian genera (representing six orders). The strong negative selection detected in most of the assessed groups reinforces the presence of evolutionary constraints, imposed by the important function of the PAR1 genes. Indeed, mutations in these genes are associated with various diseases in humans, including stature problems (Klinefelter Syndrome), leukemia and mental diseases. Yet, a few genes exhibiting positive selection (ω-value1) were depicted in Rodentia (ASMT and ZBED1) and Primates (CRLF2 and CSF2RA). Rodents have the smallest described PAR1, while that of simian primates/humans underwent a 3 to 5 fold size reduction. The assessment of the PAR1 genes synteny revealed differences among the mammalian species, especially in the Rodentia order where chromosomic translocations from the sex chromosomes to the autosomes were observed. Such syntenic changes may be an evidence of the rapid evolution in rodents, as previous referred in other papers, also depicted by their increased branch lengths in the phylogenetic analyses. Concluding, we suggest that genome migration is an important factor influencing the evolution of mammals and may result in changes of the selective pressures operating on the genome.

Associations between different organisms have been extensively described in terrestrial and marine environments. These associations are involved in roles as diverse as nutrient exchanges, shelter or adaptation to adverse conditions. Ascidians are widely dispersed marine invertebrates associated to invasive behaviours. Studying their microbiomes has interested the scientific community, mainly due to its potential for bioactive compounds production—e.g., ET-73 (trabectedin, Yondelis), an anticancer drug. However, these symbiotic interactions embrace several environmental and biological functions with high ecological relevance, inspiring diverse biotechnological applications. We thoroughly reviewed microbiome studies (microscopic to metagenomic approaches) of around 171 hosts, worldwide dispersed, occurring at different domains of life (Archaea, Bacteria, Eukarya), to illuminate the functions and bioactive potential of associated organisms in ascidians. Associations with Bacteria are the most prevalent, namely with Cyanobacteria, Proteobacteria, Bacteroidetes, Actinobacteria and Planctomycetes phyla. The microbiomes of ascidians belonging to Aplousobranchia order have been the most studied. The integration of worldwide studies characterizing ascidians’ microbiome composition revealed several functions including UV protection, bioaccumulation of heavy metals and defense against fouling or predators through production of natural products, chemical signals or competition. The critical assessment and characterization of these communities is extremely valuable to comprehend their biological/ecological role and biotechnological potential.

Sexual reproduction is a diverse and widespread process. In gonochoristic species, the differentiation of sexes occurs through diverse mechanisms, influenced by environmental and genetic factors. In most vertebrates, a master-switch gene is responsible for triggering a sex determination network. However, only a few genes have acquired master-switch functions, and this process is associated with the evolution of sex-chromosomes, which have a significant influence in evolution. Additionally, their highly repetitive regions impose challenges for high-quality sequencing, even using high-throughput, state-of-the-art techniques. Here, we review the mechanisms involved in sex determination and their role in the evolution of species, particularly vertebrates, focusing on sex chromosomes and the challenges involved in sequencing these genomic elements. We also address the improvements provided by the growth of sequencing projects, by generating a massive number of near-gapless, telomere-to-telomere, chromosome-level, phased assemblies, increasing the number and quality of sex-chromosome sequences available for further studies.

We focus on the stalked goose barnacle L. anatifera adhesive system, an opportunistic less selective species for the substrate, found attached to a variety of floating objects at seas. Adhesion is an adaptative character in barnacles, ensuring adequate positioning in the habitat for feeding and reproduction. The protein composition of the cement multicomplex and adhesive gland was quantitatively studied using shotgun proteomic analysis. Overall, 11,795 peptide sequences were identified in the gland and 2206 in the cement, clustered in 1689 and 217 proteinGroups, respectively. Cement specific adhesive proteins (CPs), proteases, protease inhibitors, cuticular and structural proteins, chemical cues, and many unannotated proteins were found, among others. In the cement, CPs were the most abundant (80.5%), being the bulk proteins CP100k and -52k the most expressed of all, and CP43k-like the most expressed interfacial protein. Unannotated proteins comprised 4.7% of the cement proteome, ranking several of them among the most highly expressed. Eight of these proteins showed similar physicochemical properties and amino acid composition to known CPs and classified through Principal Components Analysis (PCA) as new CPs. The importance of PCA on the identification of unannotated non-conserved adhesive proteins, whose selective pressure is on their relative amino acid abundance, was demonstrated.

We report here the genome sequences of six Vibrio strains isolated from an Atlantic intertidal marine sponge, Ophlitaspongia papilla. Genome mining and comparative genomics will assist in deciphering the bioactive potential of the symbiotic microbes and molecular mechanisms of sponge-microbial symbioses.

We report here the genome sequences of six Vibrio strains isolated from an Atlantic intertidal marine sponge, Ophlitaspongia papilla . Genome mining and comparative genomics will assist in deciphering the bioactive potential of the symbiotic microbes and molecular mechanisms of sponge-microbial symbioses.

Zooplankton, a key component of marine communities, has essential functions in marine ecosystems partly indorsed by extremely diversified communities. Several efforts have been made to reach a better characterization and identification of those marine organisms. Until recently, morphological taxonomy was the most applied tool, but the rise of molecular techniques and, recently, varied omics approaches have pushed forward the standard of zooplankton study, proving successful even in difficult samples. Omics techniques led for instance to a refinement of species delimitations and characterization of zooplankton diversity, enlightening the ecology and evolution of zooplankton, and providing the baseline data for evaluating community structure shifts due to environmental changes. Despite inherent limitations (e.g. database-dependent), molecular and omics technologies provide a whole new level of accuracy and specificity for assessing and deciphering worldwide zooplankton biodiversity. This chapter provides a concise overview on how omics techniques are currently fostering the research in zooplankton, contributing to characterize their ecology, life cycles, and spatial and temporal patterns. Moreover, the chapter discusses the usefulness of omics techniques for promoting the development of extensive genetic databases, and how these methods can be used for evaluating zooplankton responses to climate variability and change.

Here we provide all datasets and details applied in the construction of a composite protein database required for the proteogenomic analyses of the article “Putative Antimicrobial Peptides of the Posterior Salivary Glands from the Cephalopod Octopus vulgaris Revealed by Exploring a Composite Protein Database”. All data, subdivided into six datasets, are deposited at the Mendeley Data repository as follows. Dataset_1 provides our composite database “All_Databases_5950827_sequences.fasta” derived from six smaller databases composed of (i) protein sequences retrieved from public databases related to cephalopods’ salivary glands, (ii) proteins identified with Proteome Discoverer software using our original data obtained by shotgun proteomic analyses of posterior salivary glands (PSGs) from three Octopus vulgaris specimens (provided as Dataset_2) and (iii) a non-redundant antimicrobial peptide (AMP) database. Dataset_3 includes the transcripts obtained by de novo assembly of 16 transcriptomes from cephalopods’ PSGs using CLC Genomics Workbench. Dataset_4 provides the proteins predicted by the TransDecoder tool from the de novo assembly of 16 transcriptomes of cephalopods’ PSGs. Further details about database construction, as well as the scripts and command lines used to construct them, are deposited within Dataset_5 and Dataset_6. The data provided in this article will assist in unravelling the role of cephalopods’ PSGs in feeding strategies, toxins and AMP production, This study was supported by the Strategic Funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by the Foundation for Science and Technology (FCT) and the European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through the FCT under the project PTDC/CTA-AMB/31774/2017 (POCI-01-0145-FEDER/031774/2017). We are grateful to Robert Carcasses (Full Stack developer at Kenkou GmbH, Germany) for language programming advice and assistance.

Enzyme subunit interfaces have remarkable potential in drug design as both target and scaffold for their own inhibitors. We show an evolution-driven strategy for the de novo design of peptide inhibitors targeting interfaces of the Escherichia coli FoF1-ATP synthase as a case study. The evolutionary algorithm ROSE was applied to generate diversity-oriented peptide libraries by engineering peptide fragments from ATP synthase interfaces. The resulting peptides were scored with PPI-Detect, a sequence-based predictor of protein-protein interactions. Two selected peptides were confirmed by in vitro inhibition and binding tests. The proposed methodology can be widely applied to design peptides targeting relevant interfaces of enzymatic complexes.

Cyanobacteria are a group of microorganisms that can be found in a diverse range of biogeographical areas and produce potent and damaging cyanotoxins, which reveal importance for continuous studies and surveillance efforts. In this study, we analyzed worldwide two-month culture-enriched water samples collected from 12 distinct countries (Costa Rica, Cuba, Fiji, France, Indonesia, Mali, Portugal, South Africa, Spain, Thailand, USA, Vietnam) including two undisclosed areas (Fiji and Mali). We performed a PCR-based molecular multi-step scheme that consisted in the detection of the main cyanobacterial species, genera, and cyanotoxins biosynthesis genes. Results from this study indicate that Microcystis aeruginosa followed by Planktothrix agardhii were the most prevalent species of all the 12 countries analyzed. Cylindrospermospis raciborskii was detected in Costa Rica, while P. agardhii was detected in Fiji and South Africa. M. aeruginosa was detected in Fiji and Mali. Regarding the main cyanotoxins biosynthesis genes, a cyrC gene fragment (cylindrospermopsins) was amplified in the African continent (South Africa), while anaC (anatoxin-a) was detected in two distinct locations, Mali and Vietnam. Saxitoxins biosynthesis gene was also detected in Fiji and Vietnam. Microcystins biosynthesis gene (mcyA) was co-detected with anatoxin-a biosynthesis gene in Mali and with saxitoxins biosynthesis gene (sxtI) in Portugal. This study therefore constitutes a major contribution to the global biogeography of cyanobacteria and its cyanotoxins and recommends continuous vigilance of toxic cyanobacteria particularly in the more undisclosed areas of the world. The PCR analysis data obtained in our 2-month culture-enriched water samples supports molecular methods as a preliminary tool in the environmental surveillance of cyanobacteria and cyanotoxins in undisclosed locations, particularly since the several positive amplifications detected may indicate that though samples were collected under non-bloom conditions, if environmental conditions change in the ecosystem, there is a risk that bloom-forming species may arose along with their detected cyanotoxicity.

The COVID-19 outbreak has infected over 6 million people across the world. The origin of COVID-19 coronavirus (CoV) remains unknown, although pangolins have been suggested as potential hosts. We investigated two pangolins seized in Guangdong Province, China. Molecular screening revealed CoV in one pangolin (“Dahu”), while another (“Meidong”) was infected by Ehrlichia ruminantium. Dahu exhibited difficulty breathing, infections of lung, intestines, and nostrils, as revealed by computed tomography imaging and necropsy. Previous phylogenetic analyses showed bat coronavirus RaTG13 is closer to COVID-19 CoV compared to pangolin coronavirus. Over 20 caregivers have had close physical contact with CoV-positive Dahu, but none became infected with CoV. Our data suggest that pangolins are unlikely the natural reservoir or secondary hosts of COVID-19 CoV. Pangolins seems to be victims infected by CoV carried by a not yet unidentified natural reservoir host species, perhaps due to their weakened immune system.

Horizontal gene transfer (HGT) plays an important role for evolutionary innovations within prokaryotic communities and is a crucial event for their survival. Several computational approaches have arisen to identify HGT events in recipient genomes. However, this has been proven to be a complex task due to the generation of a great number of false positives and the prediction disagreement among the existing methods. Phylogenetic reconstruction methods turned out to be the most reliable ones, but they are not extensible to all genes/species and are computationally demanding when dealing with large datasets. In contrast, the so-called surrogate methods that use heuristic solutions either based on nucleotide composition patterns or phyletic distribution of BLAST hits can be applied easily to the genomic scale, but they fail in identifying common HGT events. Here, we present ShadowCaster, a hybrid approach that sequentially combines nucleotide composition-based predictions by support vector machines (SVMs) under the shadow of phylogenetic models independent of tree reconstruction, to improve the detection of HGT events in prokaryotes. ShadowCaster successfully predicted close and distant HGT events in both artificial and bacterial genomes. ShadowCaster detected HGT related to heavy metal resistance in the genome of Rhodanobacter denitrificans with higher accuracy than the most popular state-of-the-art computational approaches, encompassing most of the predicted cases made by other methods. ShadowCaster is released at the GitHub platform as an open-source software under the GPLv3 license.

High levels of population differentiation are a common demographic pattern in syngnathids, even at small geographical scales. This is probably the end result of the common life history traits observed within the family, involving limited dispersal capabilities and strong habitat dependency. The worm pipefish, Nerophis lumbriciformis, which displays all these characteristics, also presents an additional variable potentially able to promote population differentiation: high sexual selection intensity, especially at the extremes of its distribution. Nevertheless, an early life pelagic stage, which presumably allows for admixture, could prevent population structuring. Here, we assessed the phylogeography of N. lumbriciformis through the amplification of the cytochrome b, 12S, and 16S rDNA mitochondrial markers as well as the rhodopsin nuclear marker, performed upon 119 individuals. We observed a genetically homogeneous population with indications of extensive gene flow. We tentatively attribute this finding to the dispersal potential of the species’ pelagic larvae, supported by marine currents acting as major dispersal vectors. We also detected a signal of expansion towards the poles, consistent with the current climate change scenario. Despite the marked latitudinal differences in the phenotype of reproducing worm pipefish, the absence of clear population structuring suggests that phenotypic plasticity can have a significant role in the expression of sexual selection-related traits.

Adhesive secretion has a fundamental role in barnacles&rsquo, survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.

Adhesive secretion has a fundamental role in barnacles’ survival, keeping them in an adequate position on the substrate under a variety of hydrologic regimes. It arouses special interest for industrial applications, such as antifouling strategies, underwater industrial and surgical glues, and dental composites. This study was focused on the goose barnacle Pollicipes pollicipes adhesion system, a species that lives in the Eastern Atlantic strongly exposed intertidal rocky shores and cliffs. The protein composition of P. pollicipes cement multicomplex and cement gland was quantitatively studied using a label-free LC-MS high-throughput proteomic analysis, searched against a custom transcriptome-derived database. Overall, 11,755 peptide sequences were identified in the gland while 2880 peptide sequences were detected in the cement, clustered in 1616 and 1568 protein groups, respectively. The gland proteome was dominated by proteins of the muscle, cytoskeleton, and some uncharacterized proteins, while the cement was, for the first time, reported to be composed by nearly 50% of proteins that are not canonical cement proteins, mainly unannotated proteins, chemical cues, and protease inhibitors, among others. Bulk adhesive proteins accounted for one-third of the cement proteome, with CP52k being the most abundant. Some unannotated proteins highly expressed in the proteomes, as well as at the transcriptomic level, showed similar physicochemical properties to the known surface-coupling barnacle adhesive proteins while the function of the others remains to be discovered. New quantitative and qualitative clues are provided to understand the diversity and function of proteins in the cement of stalked barnacles, contributing to the whole adhesion model in Cirripedia.

Historical reports show that in Portugal, cyanotoxins reports were mainly in the Center (cylindrospermopsins) and South (cylindrospermopsins, saxitoxins) regions of the country apart from the well distributed microcystins. Therefore, in our study, seven freshwater ecosystems located in the North and Center Regions of Portugal were screened between April and September of 2017 for the main cyanotoxins (microcystins, cylindrospermopsins, anatoxin-a, and saxitoxins) by a two methods approach that combined the application of molecular (PCR) and immunological (ELISA) assays. Results from our survey reveal that both methods revealed the presence of all main cyanotoxins. ELISA results showed that 48% of the samples were above (1.6&ndash, 18.8 &mu, g/L) the guideline value established for microcystins (1 &mu, g/L), while in the remaining cyanotoxins, 33% of the samples were above (1.1&ndash, 6.8 &mu, g/L) the guideline value established for anatoxin&ndash, a (1 &mu, g/L). Further, for saxitoxins, only one sample gave a value above (4.3 &mu, g/L) the guideline (3 &mu, g/L) and this corresponded to a North Region ecosystem. In the cytotoxin cylindrospermopsins, none of the samples were above the guideline established value (1 &mu, g/L). This study will improve the risk assessment strategy in Portugal, as well as advance water quality and water management.

The human β-coronavirus SARS-CoV-2 epidemic started in late December 2019 in Wuhan, China. It causes Covid-19 disease which has become pandemic. Each of the five-known human β-coronaviruses has four major structural proteins (E, M, N and S) and 16 non-structural proteins encoded by ORF1a and ORF1b together (ORF1ab) that are involved in virus pathogenicity and infectivity. Here, we performed detailed positive selection analyses for those six genes among the four previously known human β-coronaviruses and within 38 SARS-CoV-2 genomes to assess signatures of adaptive evolution using maximum likelihood approaches. Our results suggest that three genes (E, S and ORF1ab genes) are under strong signatures of positive selection among human β-coronavirus, influencing codons that are located in functional important protein domains. The E protein-coding gene showed signatures of positive selection in two sites, Asp 66 and Ser 68, located inside a putative transmembrane α-helical domain C-terminal part, which is preferentially composed by hydrophilic residues. Such Asp and Ser sites substitutions (hydrophilic residues) increase the stability of the transmembrane domain in SARS-CoV-2. Moreover, substitutions in the spike (S) protein S1 N-terminal domain have been found, all of them were located on the S protein surface, suggesting their importance in viral transmissibility and survival. Furthermore, evidence of strong positive selection was detected in three of the SARS-CoV-2 nonstructural proteins (NSP1, NSP3, NSP16), which are encoded by ORF1ab and play vital roles in suppressing host translation machinery, viral replication and transcription and inhibiting the host immune response. These results are insightful to assess the role of positive selection in the SARS-CoV-2 encoded proteins, which will allow to better understand the virulent pathogenicity of the virus and potentially identifying targets for drug or vaccine strategy design.

Cylindrospermopsin (CYN) was found to occur in Portugal for the first time. In this study CYN values varied from a minimum of 1.4 μg L−1 to a maximum of 12 μg L−1 detected through HPLC technique and confirmed by LC-MS method. Amplification of the cyrC gene was done and was confirmed to be from the genera Aphanizomenon. This study is therefore an important contribution to the knowledge on the dispersal and biogeography of CYN. © 2017 This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by Fundação para a Ciência e Tecnologia (FCT) and European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program - COMPETE 2020 and by National Funds through the FCT under the projects PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845) and PTDC/AAG-GLO/2317/2014, and by the Structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Lines NOVELMAR and ECOSERVICES), funded by the Northern Regional Operational Program (NORTE2020) through the ERDF.

12 pages, 5 figures, 6 tables, supplemental material https://doi.org/10.25387/g3.12640691, The octocoral, Paramuricea clavata, is a habitat-forming anthozoan with a key ecological role in rocky benthic and biodiversity-rich communities in the Mediterranean and Eastern Atlantic. Shallow populations of P. clavata in the North-Western Mediterranean are severely affected by warming-induced mass mortality events (MMEs). These MMEs have differentially impacted individuals and populations of P. clavata (i.e., varied levels of tissue necrosis and mortality rates) over thousands of kilometers of coastal areas. The eco-evolutionary processes, including genetic factors, contributing to these differential responses remain to be characterized. Here, we sequenced a P. clavata individual with short and long read technologies, producing 169.98 Gb of Illumina paired-end and 3.55 Gb of Oxford Nanopore Technologies (ONT) reads. We obtained a de novo genome assembly accounting for 607 Mb in 64,145 scaffolds. The contig and scaffold N50s are 19.15 Kb and 23.92 Kb, respectively. Despite of the low contiguity of the assembly, its gene completeness is relatively high, including 75.8% complete and 9.4% fragmented genes out of the 978 metazoan genes contained in the metazoa_odb9 database. A total of 62,652 protein-coding genes have been annotated. This assembly is one of the few octocoral genomes currently available. This is undoubtedly a valuable resource for characterizing the genetic bases of the differential responses to thermal stress and for the identification of thermo-resistant individuals and populations. Overall, having the genome of P. clavata will facilitate studies of various aspects of its evolutionary ecology and elaboration of effective conservation plans such as active restoration to overcome the threats of global change, We acknowledge the funding support of the European Union’s Horizon 2020 research and innovation program under grant agreement No 689518 (MERCES) and the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. JBL is funded by an assistant researcher contract framework of the RD Unit - UID/Multi/04423/2019 - Interdisciplinary Centre of Marine and Environmental Research – financed by the European Regional Development Fund (ERDF) through COMPETE2020 - Operational Program for Competitiveness and Internationalisation (POCI) and national funds through FCT/MCTES (PIDDAC). AA was partially supported by the FCT project PTDC/CTA-AMB/31774/2017 (POCI-01-0145-FEDER/031774/2017). DGG was supported by an FPU grant (FPU15/05457) from the Spanish Ministry of Education. We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme / Generalitat de Catalunya, the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III, the Generalitat de Catalunya through Departament de Salut and Departament d'Empresa i Coneixement, and the Co-financing by the Spanish Ministry of Science and Innovation with funds from the European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth Operating Program. JBL and JG planned and granted the funding to start the project, With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI)

Indexación: Scopus. Alignment-free (AF) methodologies have increased in popularity in the last decades as alternative tools to alignment-based (AB) algorithms for performing comparative sequence analyses. They have been especially useful to detect remote homologs within the twilight zone of highly diverse gene/protein families and superfamilies. The most popular alignment-free methodologies, as well as their applications to classification problems, have been described in previous reviews. Despite a new set of graph theory-derived sequence/structural descriptors that have been gaining relevance in the detection of remote homology, they have been omitted as AF predictors when the topic is addressed. Here, we first go over the most popular AF approaches used for detecting homology signals within the twilight zone and then bring out the state-of-the-art tools encoding graph theory-derived sequence/structure descriptors and their success for identifying remote homologs. We also highlight the tendency of integrating AF features/measures with the AB ones, either into the same prediction model or by assembling the predictions from different algorithms using voting/weighting strategies, for improving the detection of remote signals. Lastly, we briefly discuss the efforts made to scale up AB and AF features/measures for the comparison of multiple genomes and proteomes. Alongside the achieved experiences in remote homology detection by both the most popular AF tools and other less known ones, we provide our own using the graphical–numerical methodologies, MARCH-INSIDE, TI2BioP, and ProtDCal. We also present a new Python-based tool (SeqDivA) with a friendly graphical user interface (GUI) for delimiting the twilight zone by using several similar criteria. https://www.mdpi.com/2218-273X/10/1/26

The octocoral, Paramuricea clavata, is a habitat-forming anthozoan with a key ecological role in rocky benthic and biodiversity-rich communities in the Mediterranean and Eastern Atlantic. Shallow populations of P. clavata in the North-Western Mediterranean are severely affected by warming-induced mass mortality events (MMEs). These MMEs have differentially impacted individuals and populations of P. clavata (i.e. varied levels of tissue necrosis and mortality rates) over thousands of kilometers of coastal areas. The eco-evolutionary processes and genetic factors contributing to these differential responses remain to be characterized. Here, we sequenced a P. clavata individual with short and long read technologies, producing 169.98 Gb of Illumina paired-end and 3.55 Gb of Oxford Nanopore Technologies (ONT) reads. We obtained a de novo hybrid assembly accounting for 712.4 Mb and 107,682 scaffolds. The contig and scaffold N50 are 15.85 Kb and 17.01 Kb, respectively. Despite of the low contiguity of the assembly, the gene completeness was relatively high, including 86% of the 978 metazoan genes contained in the metazoa_odb9 database. A total of 76,508 protein-coding genes and 85,763 transcripts have been annotated. This assembly is one of the few octocoral genomes currently available. This is undoubtedly a valuable resource for characterizing the genetic bases of the differential responses to thermal stress and for the identification of thermo-resistant individuals and populations. Overall, the genome of P. clavata will help to understand various aspects of its evolutionary ecology and to elaborate effective conservation plans such as active restoration actions to overcome the threats of global change.

The most recently described bacterial members of the genus Endozoicomonas have been found in association with a wide variety of marine invertebrates. Despite their ubiquity in the host holobiont, limited information is available on the molecular genomic signatures of the symbiotic association of Endozoicomonas with marine sponges. Here, we generated a draft genome of Endozoicomonas sp. OPT23 isolated from the intertidal marine sponge Ophlitaspongia papilla and performed comprehensive comparative genomics analyses. Genome-specific analysis and metabolic pathway comparison of the members of the genus Endozoicomonas revealed the presence of gene clusters encoding for unique metabolic features, such as the utilization of carbon sources through lactate, L-rhamnose metabolism, and a phenylacetic acid degradation pathway in Endozoicomonas sp. OPT23. Moreover, the genome harbors genes encoding for eukaryotic-like proteins, such as ankyrin repeats, tetratricopeptide repeats, and Sel1 repeats, which likely facilitate sponge-bacterium attachment. The genome also encodes major secretion systems and homologs of effector molecules that seem to enable the sponge-associated bacterium to interact with the sponge and deliver the virulence factors for successful colonization. In conclusion, the genome analysis of Endozoicomonas sp. OPT23 revealed the presence of adaptive genomic signatures that might favor their symbiotic lifestyle within the sponge host.

Horizontal gene transfer (HGT) plays an important role in the evolution of many organisms, especially in prokaryotes where commonly occurs. Microbial communities can improve survival due to the evolutionary innovations induced by HGT events. Thus, several computational approaches have arisen to identify such events in recipient genomes. However, this has been proven to be a complex task due to the generation of a great number of false positives and the prediction disagreement among the existing methods. Phylogenetic reconstruction methods turned out to be the most reliable but they are not extensible to all genes/species and are computationally demanding when dealing with large datasets. On the other hand, the so-called surrogate methods that use heuristic solutions either based on nucleotide composition patterns or phyletic distribution of BLAST hits can be applied easily to genomic scale, however, they fail in identifying common HGT events. Here, we present ShadowCaster, a hybrid approach that sequentially combines compositional features under the shadow of phylogenetic models independent of tree reconstruction to improve the detection of HTG events in prokaryotes. ShadowCaster predicted successfully close and distant HTG events in both artificial and bacterial genomes. It detected HGT related to heavy metal resistance in the genome of Rhodanobacter denitrificans with higher accuracy than the most popular state-of-the-art computational approaches. ShadowCaster’s predictions showed the highest agreement among those obtained with other assayed methods. ShadowCaster is released as an open-source software under the GPLv3 license. Source code is hosted at https://github.com/dani2s/ShadowCaster and documentation at https://shadowcaster.readthedocs.io/en/latest/.

The white shark ( Carcharodon carcharias ; Chondrichthyes, Elasmobranchii) is one of the most publicly recognized marine animals. Here we report the genome sequence of the white shark and comparative evolutionary genomic analyses to the chondrichthyans, whale shark (Elasmobranchii) and elephant shark (Holocephali), as well as various vertebrates. The 4.63-Gbp white shark genome contains 24,520 predicted genes, and has a repeat content of 58.5%. We provide evidence for a history of positive selection and gene-content enrichments regarding important genome stability-related genes and functional categories, particularly so for the two elasmobranchs. We hypothesize that the molecular adaptive emphasis on genome stability in white and whale sharks may reflect the combined selective pressure of large genome sizes, high repeat content, high long-interspersed element retrotransposon representation, large body size, and long lifespans, represented across these two species. Molecular adaptation for wound healing was also evident, with positive selection in key genes involved in the wound-healing process, as well as Gene Ontology enrichments in fundamental wound-healing pathways. Sharks, particularly apex predators such as the white shark, are believed to have an acute sense of smell. However, we found very few olfactory receptor genes, very few trace amine-associated receptors, and extremely low numbers of G protein-coupled receptors. We did however, identify 13 copies of vomeronasal type 2 (V2R) genes in white shark and 10 in whale shark; this, combined with the over 30 V2Rs reported previously for elephant shark, suggests this gene family may underlie the keen odorant reception of chondrichthyans.

Background: The Ribonuclease III (RNase III) enzymatic class is involved in many important biological processes from bacteria to higher eukaryotes. Consequently, they have been useful as drug-target candidates for drug development. Despite their high molecular diversity, RNases III share common structural and catalytic features and some degree of enzymatic activity. However, the role of accessory domains as key determinants of substrate selectivity and over the biological function of each RNase III type is still under study. Results: The in vitro enzymatic activity of three RNase III members from class I (Escherichia coli RNase III, Schizosaccharomyces pombe Pac1 and Saccharomyces cerevisiae Rntp1) and the human Drosha placed in class II was revisited against four different substrates. These two RNase III classes comprise members showing different domain organization. Enzymatic activity differences were found among members of the class I, which were even higher when the human Drosha (class II) was tested. The substrate promiscuity of the E. coli RNase III was corroborated in vivo through its expression in S. cerevisiae, as reported previously, but was extended here to Pichia pastoris. The putative molecular mechanisms contributing for the lethal effect of the heterologous RNase III on the yeast lives were deeply discussed. Conclusion: The new generated biochemical data integrated with previous available information affirmed that RNases III substrate specificity as well as their cellular biological role is highly influenced by its protein structure architecture. This fact also allowed drawing evolutionary links between RNase III members from their structural and substrate specificity differences.

A diverse group of genes are involved in the tooth development of mammals. Several studies, focused mainly on mice and rats, have provided a detailed depiction of the processes coordinating tooth formation and shape. Here we surveyed 236 tooth-associated genes in 39 mammalian genomes and tested for signatures of selection to assess patterns of molecular adaptation in genes regulating mammalian dentition. Of the 236 genes, 31 (∼13.1%) showed strong signatures of positive selection that may be responsible for the phenotypic diversity observed in mammalian dentition. Mammalian-specific tooth-associated genes had accelerated mutation rates compared with older genes found across all vertebrates. More recently evolved genes had fewer interactions (either genetic or physical), were associated with fewer Gene Ontology terms and had faster evolutionary rates compared with older genes. The introns of these positively selected genes also exhibited accelerated evolutionary rates, which may reflect additional adaptive pressure in the intronic regions that are associated with regulatory processes that influence tooth-gene networks. The positively selected genes were mainly involved in processes like mineralization and structural organization of tooth specific tissues such as enamel and dentin. Of the 236 analyzed genes, 12 mammalian-specific genes (younger genes) provided insights on diversification of mammalian teeth as they have higher evolutionary rates and exhibit different expression profiles compared with older genes. Our results suggest that the evolution and development of mammalian dentition occurred in part through positive selection acting on genes that previously had other functions.

Cephalopods, successful predators, can use a mixture of substances to subdue their prey, becoming interesting sources of bioactive compounds. In addition to neurotoxins and enzymes, the presence of antimicrobial compounds has been reported. Recently, the transcriptome and the whole proteome of the Octopus vulgaris salivary apparatus were released, but the role of some compounds&mdash, e.g., histones, antimicrobial peptides (AMPs), and toxins&mdash, remains unclear. Herein, we profiled the proteome of the posterior salivary glands (PSGs) of O. vulgaris using two sample preparation protocols combined with a shotgun-proteomics approach. Protein identification was performed against a composite database comprising data from the UniProtKB, all transcriptomes available from the cephalopods&rsquo, PSGs, and a comprehensive non-redundant AMPs database. Out of the 10,075 proteins clustered in 1868 protein groups, 90 clusters corresponded to venom protein toxin families. Additionally, we detected putative AMPs clustered with histones previously found as abundant proteins in the saliva of O. vulgaris. Some of these histones, such as H2A and H2B, are involved in systemic inflammatory responses and their antimicrobial effects have been demonstrated. These results not only confirm the production of enzymes and toxins by the O. vulgaris PSGs but also suggest their involvement in the first line of defense against microbes.

Studying the diet of crustaceans based on the direct visual inspection of the gut contents can be difficult because they can ingest only a portion of the prey and further crush it into very small pieces, making a very few food items identifiable. Here, the usefulness of molecular methods to identify the species present in the gut contents of two crustaceans, the shore crab Carcinus maenas and the brown shrimp Crangon crangon, was tested. Stomach contents were removed, and their total genomic DNA extracted followed by amplification (PCR) with four specific prey mtDNA primers. Since both crustaceans are opportunistic predators, preying upon around 40 different species, the study was restricted to common potential preys in the sampled area, the Minho Estuary (Portugal): the common goby Pomatoschistus microps, the ragworm Hediste diversicolor, the flounder Platichthys flesus and the peppery furrow shell Scrobicularia plana. After testing the most appropriate methodology for DNA extraction and PCR amplification to detect the presence of each potential prey with positive control samples, the same methodology was applied to the stomach content samples. The molecular methods allowed for detection of the presence of P. microps, P. flesus and H. diversicolor in the stomachs of both crustaceans. Therefore, once potential prey and their availability in the system are known, PCR technique can be an alternative tool to overcome some of the limitations of traditional methods in the study of the crustaceans’ diet, even when stomach contents include a great part of ‘detritus’. This study reflects also the need to develop more tools (primers) to improve the assessment of predator–prey interactions in aquatic ecosytems.

The most recently described bacterial members of the genus Endozoicomonas have been found in association with a wide variety of marine invertebrates. Despite their ubiquity in the host holobiont, limited information is available on the molecular genomic signatures of the symbiotic association of Endozoicomonas with marine sponges. Here, we generated a draft genome of Endozoicomonas sp. OPT23 isolated from the intertidal marine sponge Ophlitaspongia papilla and performed comprehensive comparative genomics analyses. Genome-specific analysis and metabolic pathway comparison of the members of the genus Endozoicomonas revealed the presence of gene clusters encoding for unique metabolic features, such as the utilization of carbon sources through lactate, L-rhamnose metabolism, and a phenylacetic acid degradation pathway in Endozoicomonas sp. OPT23. Moreover, the genome harbors genes encoding for eukaryotic-like proteins, such as ankyrin repeats, tetratricopeptide repeats, and Sel1 repeats, which likely facilitate sponge-bacterium attachment. The genome also encodes major secretion systems and homologs of effector molecules that seem to enable the sponge-associated bacterium to interact with the sponge and deliver the virulence factors for successful colonization. In conclusion, the genome analysis of Endozoicomonas sp. OPT23 revealed the presence of adaptive genomic signatures that might favor their symbiotic lifestyle within the sponge host. Anoop Alex was supported in part by the project PTDC/BIA-BMA/29985/2017 (POCI-01-0145-FEDER- 029985) from the European Regional Development Fund (ERDF) through COMPETE 2020—Operational Program for Competitiveness and Internationalization (POCI) and National Funds through the Fundação para a Ciência e a Tecnologia (FCT)/MCTES. Agostinho Antunes was funded in part by the Strategic Funding UID/Multi/04423/2019 through National Funds provided by FCT and the ERDF in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program - COMPETE 2020 and by National Funds through the FCT under the project PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845).

Corbicula fluminea and Dreissena polymorpha are two non-indigenous species (NIS), known to provoke biodiversity loss of the existent native communities and alterations in the ecosystem functioning structure. Both of these NIS have successfully founded well established populations in Lakes Maggiore and Garda (Northern Italy). Here, we evaluated the mitochondrial COI genetic diversity of C. fluminea and D. polymorpha populations from the aforementioned lakes. The COI gene analysis revealed one C. fluminea haplotype, belonging to the FW5 androgenetic invasive lineage. Two D. polymorpha haplotypes – LM1 and LM2 – were detected in Lake Maggiore. The D. polymorpha comparative phylogeographical haplotype analysis between Lake Maggiore and the retrieved COI data available from Lake Garda revealed that LM1 is the dominant haplotype in both populations, whereas LM2 a rare haplotype was only detected in Lake Maggiore. These findings contribute for a better understanding of the demographic history of these highly invasive species in these Italian lakes, thus suggesting that C. fluminea and D. polymorpha populations present a similar genetic pattern. The low genetic diversity detected in both of these bivalve populations seems to reflect a pre-existent low genetic pool prevenient from the introductory source(s).

As the only endemic neotropical parrot to have recently lived in the northern hemisphere, the Carolina parakeet (Conuropsis carolinensis) was an iconic North American bird. The last surviving specimen died in the Cincinnati Zoo in 1918 [1]. The cause of its extinction remains contentious: besides excessive mortality associated to habitat destruction and active hunting, their survival could have been negatively affected by its range having become increasingly patchy [2] or by the exposure to poultry pathogens [3, 4]. In addition, the Carolina parakeet showed a predilection for cockleburs, an herbaceous plant that contains a powerful toxin, carboxyatractyloside, or CAT [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To explore the demographic history of this bird, we generated the complete genomic sequence of a preserved specimen held in a private collection in Espinelves (Girona, Spain), as well as of a close extant relative, Aratinga solstitialis. We identified two non-synonymous genetic changes in two highly conserved proteins known to interact with CAT that could underlie a specific dietary adaptation to this toxin. Our genomic analyses did not reveal evidence of a dramatic past demographic decline in the Carolina parakeet; also, its genome did not exhibit the long runs of homozygosity that are signals of recent inbreeding and are typically found in endangered species. As such, our results suggest its extinction was an abrupt process and thus likely solely attributable to human causes. Gelabert et al. report the whole genome of the extinct Carolina parakeet and provide evidence of its phylogeny, adaptation to a toxic cocklebur diet, and demographic history. The lack of signs of recent inbreeding typically found in endangered species suggests its abrupt extinction was human mediated., This work was supported by ERC Consolidator Grant 681396 “Extinction Genomics” to M.T.P.G. and M.S.-V., Obra Social ‘‘La Caixa’’ and Secretaria d’Universitats i Recerca ( GRC2017-SGR880 ) to T.M.-B. and C.L.-F., BFU2017-86471-P and PGC2018-101927-B-I00 from MINECO/FEDER, UE to T.M.-B, and PGC2018-095931-B-100 from MINECO/FEDER, UE to C.L.-F. T.M.-B. is also supported by a U01 MH106874 grant and Howard Hughes International Early Career and CERCA Programme (Generalitat de Catalunya). A.A. is partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by the Portuguese Foundation for Science and Technology (FCT) and the European Regional Development Fund —program PT2020 , by the European Structural and Investment Funds —Competitiveness and Internationalization Operational Program-COMPETE 2020, and by National Funds through the FCT under the project PTDC/CTA-AMB/31774/2017(POCI-01-0145-FEDER/031774/2017 ).

Cnidaria is a phylum of predominantly marine organisms that evolved into two basic body forms: “polyp” characterized by a tubular body; and “medusa” the free-floating stage characterized by a bell-shaped body. A fast increase in the abundance of the free-living stage of cnidarians known as “jellyfish”, can produce huge impacts on fisheries, public health, tourism, and the normal functioning of factories and aquaculture. Those mass occurrence events are influenced by some natural factors such as water temperature, wind and water tides, but also induced by global warming, overfishing, eutrophication and widening of invasive species habitats. These events have also been reported in the Portuguese coast, but the information is still scarce and therefore their impacts are underestimated. Herein, we provide an updated list of medusozoans in mainland Portugal and archipelagos, and their available genetic information. The data gathered showed a total of 272 medusozoans species comprising 254 hydrozoans, 15 scyphozoans, 2 staurozoans, and 1 cubozoan. The data compiled revealed that 26 % of the reported species did not present any genetic information in public databases (May 2019). Moreover, most of these sequences belonged to the mitochondrial markers 16S ribosomal RNA (16S rRNA) and cytochrome c oxidase subunit I (COX1), and to nuclear markers 18S ribosomal RNA (18S rRNA) and 28S ribosomal RNA (28S rRNA). However, the behavior of the medusozoans registered revealed that the hydrozoans Blackfordia virginica, Physalia physalis, Velella velella and the scyphozoans Aurelia aurita, Catostylus tagi, Pelagia noctiluca and Rhizostoma luteum can produce mass occurrence events. Hence, far more than an updated list of medusozoans species in Portugal, and their genetic information, we revealed the species of medusae more capable of provoking events of mass occurrence that could suddenly affect the dynamic of the ecosystems.

Sea anemones are among the most ubiquitous organisms inhabiting the rocky shores of the Atlantic Ocean. We assessed the occurrence of intertidal sea anemones along several beaches on the Portuguese coast, identifying three families: (i) Actiniidae, including Actinia equina, Actinia fragacea, Anemonia viridis, Aulactinia verrucosa, and Anthopleura krebsi species, (ii) Hormathiidae, including Calliactis parasitica, and (iii) Sagartiidae , including Cereus pedunculatus. Analyses of concatenated sequences from one nuclear (18S) and four mitochondrial (16S, COI, COIII and ND6) genes revealed minimal intraspecific phenotypic or genotypic heterogeneity among the sampled locations or relative with species records in NCBI. A comparison among preservation methods across several different tissues demonstrated that tissues preserved with ethanol, and in particular column tissue, yielded higher amounts of DNA compared with samples from other body parts, including tentacles, gastrodermis and ectodermis preserved at −80 °C.

Venomous animals are found through a wide taxonomic range including cartilaginous fish such as the freshwater stingray Potamotrygon motoro occurring in South America, which can injure people and cause venom-related symptoms. Ensuring the efficacy of drug development to treat stingray injuries can be assisted by the knowledge of the venom composition. Here we performed a detailed transcriptomic characterization of the venom gland of the South American freshwater stingray Potamotrygon motoro. The transcripts retrieved showed 418 hits to venom components (comparably to 426 and 396 hits in other two Potamotrygon species), with high expression levels of hyaluronidase, cystatin and calglandulin along with hits uniquely found in P. motoro such as DELTA-alicitoxin-Pse1b, Augerpeptide hhe53 and PI-actitoxin-Aeq3a. We also identified undescribed molecules with extremely high expression values with sequence similarity to the SE-cephalotoxin and Rapunzel genes. Comparative analyses showed that despite being closely related, there may be significant variation among the venoms of freshwater stingrays, highlighting the importance of considering elicit care in handling different envenomation cases. Since hyaluronidase represents a major component of fish venom, we have performed phylogenetic and selective pressure analyses of this gene/protein across all fish with the available information. Results indicated an independent recruitment of the hyaluronidase into the stingray venom relative to that of venomous bony fish. The hyaluronidase residues were found to be mostly under negative selection, but 18 sites showed evidence of diversifying positive selection (P &lt, 0.05). Our data provides new insight into stingray venom variation, composition, and selective pressure in hyaluronidase.

Post-print: 41 páginas, 2 tablas, 2 figura. Contiene material suplementario., The Harderian gland is a cephalic structure, widely distributed among vertebrates. In snakes, the Harderian gland is anatomically connected to the vomeronasal organ via the nasolacrimal duct, and in some species can be larger than the eyes. The function of the Harderian gland remains elusive, but it has been proposed to play a role in the production of saliva, pheromones, thermoregulatory lipids and growth factors, among others. Here, we have profiled the transcriptomes of the Harderian glands of three non-front-fanged colubroid snakes from Cuba: Caraiba andreae (Cuban Lesser Racer); Cubophis cantherigerus (Cuban Racer); and Tretanorhinus variabilis (Caribbean Water Snake), using Illumina HiSeq2000 100 bp paired-end. In addition to ribosomal and non-characterized proteins, the most abundant transcripts encode putative transport/binding, lipocalin/lipocalin-like, and bactericidal/permeability-increasing-like proteins. Transcripts coding for putative canonical toxins described in venomous snakes were also identified. This transcriptional profile suggests a more complex function than previously recognized for this enigmatic organ., DDP was supported by a PhD grant (SFRH/BD/80592/2011) from the Portuguese Foundation for Science and Technology (FCT—Fundação para a Ciência e a Tecnologia, Portugal). GACh was also supported by a Postdoctoral grant (SFRH/BPD/92978/2013) from the FCT. This study was funded in part by the projects PTDC/AAG-GLO/6887/2014 and by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT and the European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through the FCT under the project PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845), and by the Structured Programs of R&D&I INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources (NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), and funded by the Northern Regional Operational Program (NORTE2020) through the ERDF. Work performed at the Evolutionary and Translational Venomics Laboratory, Instituto de Biomedicina de Valencia (CSIC) was funded by grant BFU2013-42833-P from the Ministerio de Economía y Competitividad, Madrid, Spain (to JJC).

The bacterial members of the genus Shewanella are widely distributed and inhabit both freshwater and marine environments. Some members of Shewanella have gained considerable attention due to its ability to survive in redox-stratified environments. However, a gap of knowledge exists on the key genomic features of the sponge-associated Shewanella sp. involving the successful host-bacteria interaction, as sponge-symbiotic Shewanella are largely underrepresented in the public repositories. With the aim of identifying the genomic signatures of sponge-Shewanella association, we generated a high-quality genome data of a sponge-associated, Shewanella sp. OPT22, isolated from the intertidal marine sponge Ophlitaspongia papilla and performed comprehensive comparative analyses of 68 genome strains of the genus Shewanella including two previously reported genomes of sponge-associated bacteria, Shewanella spongiae KCTC 22492 and Shewanella sp. Alg231_23. The 16S rRNA-based phylogenetic reconstruction showed the well-supported affiliation of OPT22 and KCTC 22492 with previously reported sponge-associated bacteria, affirming the “sponge-specific” nature of these two bacterial strains isolated from different marine sponge species from the Atlantic and Pacific (East Sea) Oceans, respectively. The genome comparison of the 68 strains of Shewanella inhabiting different habitats revealed the unusual/previously unreported abundance of genes encoding for ankyrin-repeat containing proteins (ANKs) in the genomes of the two sponge-associated strains, OPT22 (ANKs; n = 45) and KCTC 22492 (ANKs; n = 52), which might be involved in sponge-Shewanella interactions. Focused analyses detected the syntenic organization of the gene cluster encoding major secretion system (type III/IV/VI) components and the presence of effector homologs in OPT22 and KCTC 22492 that seem to play a role in the virulence of the sponge bacteria. The genomic island (GI) of Shewanella sp. OPT22 was identified to localize a gene cluster encoding T4SS components and ANK (n = 1), whereas S. spongiae KCTC 22492 harbored a total of seven ANKs within multiple GIs. GIs may play a pivotal role in the dissemination of symbioses-related genes (ANKs) through the horizontal gene transfer, contributing to the diversification and adaptation of sponge-associated Shewanella. Overall, the genome analyses of Shewanella isolates from marine sponges revealed genomic repertoires that might be involved in establishing successful symbiotic relationships with the sponge hosts.

Olfactory receptors (ORs) govern a prime sensory function. Extant birds have distinct olfactory abilities, but the molecular mechanisms underlining diversification and specialization remain mostly unknown. We explored OR diversity in 48 phylogenetic and ecologically diverse birds and 2 reptiles (alligator and green sea turtle). OR subgenomes showed species- and lineage-specific variation related with ecological requirements. Overall 1,953 OR genes were identified in reptiles and 16,503 in birds. The two reptiles had larger OR gene repertoires (989 and 964 genes, respectively) than birds (182-688 genes). Overall, birds had more pseudogenes (7,855) than intact genes (1,944). The alligator had significantly more functional genes than sea turtle, likely because of distinct foraging habits. We found rapid species-specific expansion and positive selection in OR14 (detects hydrophobic compounds) in birds and in OR51 and OR52 (detect hydrophilic compounds) in sea turtle, suggestive of terrestrial and aquatic adaptations, respectively. Ecological partitioning among birds of prey, water birds, land birds, and vocal learners showed that diverse ecological factors determined olfactory ability and influenced corresponding olfactory-receptor subgenome. OR5/8/9 was expanded in predatory birds and alligator, suggesting adaptive specialization for carnivory. OR families 2/13, 51, and 52 were correlated with aquatic adaptations (water birds), OR families 6 and 10 were more pronounced in vocal-learning birds, whereas most specialized land birds had an expanded OR family 14. Olfactory bulb ratio (OBR) and OR gene repertoire were correlated. Birds that forage for prey (carnivores/piscivores) had relatively complex OBR and OR gene repertoires compared with modern birds, including passerines, perhaps due to highly developed cognitive capacities facilitating foraging innovations.

Swinholides are 42-carbon ring polyketides with a 2-fold axis of symmetry. They are potent cytotoxins that disrupt the actin cytoskeleton. Swinholides were discovered from the marine sponge Theonella sp. and were long suspected to be produced by symbiotic bacteria. Misakinolide, a structural variant of swinholide, was recently demonstrated to be the product of a symbiotic heterotrophic proteobacterium. Here, we report the production of swinholide A by an axenic strain of the terrestrial cyanobacterium Nostoc sp. strain UHCC 0450. We located the 85-kb trans -AT polyketide synthase (PKS) swinholide biosynthesis gene cluster from a draft genome of Nostoc sp. UHCC 0450. The swinholide and misakinolide biosynthesis gene clusters share an almost identical order of catalytic domains, with 85% nucleotide sequence identity, and they group together in phylogenetic analysis. Our results resolve speculation around the true producer of swinholides and demonstrate that bacteria belonging to two distantly related phyla both produce structural variants of the same natural product. In addition, we described a biosynthesis cluster from Anabaena sp. strain UHCC 0451 for the synthesis of the cytotoxic and antifungal scytophycin. All of these biosynthesis gene clusters were closely related to each other and created a group of cytotoxic macrolide compounds produced by trans -AT PKSs of cyanobacteria and proteobacteria. IMPORTANCE Many of the drugs in use today originate from natural products. New candidate compounds for drug development are needed due to increased drug resistance. An increased knowledge of the biosynthesis of bioactive compounds can be used to aid chemical synthesis to produce novel drugs. Here, we show that a terrestrial axenic culture of Nostoc cyanobacterium produces swinholides, which have been previously found only from marine sponge or samples related to them. Swinholides are polyketides with a 2-fold axis of symmetry, and they are potent cytotoxins that disrupt the actin cytoskeleton. We describe the biosynthesis gene clusters of swinholide from Nostoc cyanobacteria, as well as the related cytotoxic and antifungal scytophycin from Anabaena cyanobacteria, and we study the evolution of their trans -AT polyketide synthases. Interestingly, swinholide is closely related to misakinolide produced by a symbiotic heterotrophic proteobacterium, demonstrating that bacteria belonging to two distantly related phyla and different habitats can produce similar natural products.

Cnidarian toxic products, particularly peptide toxins, constitute a promising target for biomedicine research. Indeed, cnidarians are considered as the largest phylum of generally toxic animals. However, research on peptides and toxins of sea anemones is still limited. Moreover, most of the toxins from sea anemones have been discovered by classical purification approaches. Recently, high-throughput methodologies have been used for this purpose but in other Phyla. Hence, the present work was focused on the proteomic analyses of whole-body extract from the unexplored sea anemone Bunodactis verrucosa. The proteomic analyses applied were based on two methods: two-dimensional gel electrophoresis combined with MALDI-TOF/TOF and shotgun proteomic approach. In total, 413 proteins were identified, but only eight proteins were identified from gel-based analyses. Such proteins are mainly involved in basal metabolism and biosynthesis of antibiotics as the most relevant pathways. In addition, some putative toxins including metalloproteinases and neurotoxins were also identified. These findings reinforce the significance of the production of antimicrobial compounds and toxins by sea anemones, which play a significant role in defense and feeding. In general, the present study provides the first proteome map of the sea anemone B. verrucosa stablishing a reference for future studies in the discovery of new compounds. © 2018 by the authors. Acknowledgments: Dany Domínguez Pérez was supported by a Ph.D. grant (SFRH/BD/80592/2011) from the Portuguese Foundation for Science and Technology (FCT—Fundação para a Ciência e a Tecnologia, Portugal). A. Campos work were supported respectively by Postdoc grants SFRH/BPD/92978/2013 and SFRH/BPD/103683/2014 from the FCT. Armando A Rodríguez was supported by an Alexander von Humboldt postdoctoral fellowship (3.2-KUB/1153731 STP and the Collaborative Research Centre 1279 funded by the German Research Foundation). We are grateful to Isabel Cunha and Daniela Almeida from CIIMAR, FCUP, University of Porto, for the help in the identification, sampling and transporting of B. verrucosa specimens. To Barbara Frazão, from Instituto Português do Mar e da Atmosfera, Lisbon, Portugal for suggestions and by providing information related the species studied. This study was funded in part by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT and the European Regional Development Fund (ERDF) in the framework of the program PT2020, by the European Structural and Investment Funds (ESIF) through the Competitiveness and Internationalization Operational Program—COMPETE 2020 and by National Funds through the FCT under the project PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845), and by the Structured Programs of R&D&I INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources (NORTE-01-0145-FEDER-000035, Research Line NOVELMAR) and CORAL NORTE (NORTE-01-0145-FEDER-000036), and funded by the Northern Regional Operational Program (NORTE2020) through the ERDF.

Bacteria belonging to the genus Pseudovibrio have been frequently found in association with a wide variety of marine eukaryotic invertebrate hosts, indicative of their versatile and symbiotic lifestyle. A recent comparison of the sponge-associated Pseudovibrio genomes has shed light on the mechanisms influencing a successful symbiotic association with sponges. In contrast, the genomic architecture of Pseudovibrio bacteria associated with other marine hosts has received less attention. Here, we performed genus-wide comparative analyses of 18 Pseudovibrio isolated from sponges, coral, tunicates, flatworm, and seawater. The analyses revealed a certain degree of commonality among the majority of sponge- and coral-associated bacteria. Isolates from other marine invertebrate host, tunicates, exhibited a genetic repertoire for cold adaptation and specific metabolic abilities including mucin degradation in the Antarctic tunicate-associated bacterium Pseudovibrio sp. Tun.PHSC04_5.I4. Reductive genome evolution was simultaneously detected in the flatworm-associated bacteria and the sponge-associated bacterium P. axinellae AD2, through the loss of major secretion systems (type III/VI) and virulence/symbioses factors such as proteins involved in adhesion and attachment to the host. Our study also unraveled the presence of a CRISPR-Cas system in P. stylochi UST20140214-052 a flatworm-associated bacterium possibly suggesting the role of CRISPR-based adaptive immune system against the invading virus particles. Detection of mobile elements and genomic islands (GIs) in all bacterial members highlighted the role of horizontal gene transfer for the acquisition of novel genetic features, likely enhancing the bacterial ecological fitness. These findings are insightful to understand the role of genome diversity in Pseudovibrio as an evolutionary strategy to increase their colonizing success across a wide range of marine eukaryotic hosts. © 2018 Alex, Antunes. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Anoop Alex was funded by the Funda??o para a Ci?ncia e a Tecnologia (FCT) SFRH/BPD/ 99251/2013 grant. Agostinho Antunes was supported by the MarInfo ? Integrated Platform for Marine Data Acquisition and Analysis (reference NORTE-01-0145-FEDER-000031), a project supported by the North Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). We thank Emanuel Maldonado and Jo?o Paulo Machado for help with various stages of data analyses and the anonymous reviewers for helpful comments on an earlier version of the manuscript.