Population genomics of a marine gastropod with limited dispersal capabilities

A dispersão de organismos é um processo essencial que promove o fluxo genético e contraria a diferenciação das populações. A falta de barreiras aparentes no ambiente marinho significa que os organismls podem alcançar a panmixia em escalas espaciais muito menores do que as que se verificam no meio terrestre. Até recentemente, a genética de populações apoiava-se num conjunto relativamente pequeno de marcadores genéticos para fazer inferências sobre a conectividade de populações em grandes escalas espaciais. No entanto, recentes avanços nas técnicas moleculares produziram um conjunto de novos métodos de sequenciação de DNA, comumente referidos como Sequenciação de Nova Geração (NGS), que permitem a obtenção de milhares de marcadores do genoma, aumentando potencialmente a resolução dos dados. RADseq é um tipo de técnica NGS que emprega enzimas de restrição para cortar o genoma nos locais de restrição, produzindo milhares polimorfismos de nucleotídeo único (SNP, single-nucleotide polymorphism). Buccinum undatum é um neogastropode amplamente distribuído em ambos os lados do Atlântico Norte, desde o Canadá até ao Golfo da Biscaia podendo atingir profundidades de 1000 m. Esta espécie é o maior gastrópode marinho comestível do Atlântico Norte, com um comprimento máximo de concha de 150 mm. Alimenta-se principalmente de bivalves e de pequenss crustáceos. B. undatum constitui uma pescaria relevante no Reino Unido desde 1922, só em 2017 foram desembarcadas 20.800 toneladas, correspondendo à sexta maior pesca de marisco no valor de 22,7 milhões de libras. Apesar de sua importância comercial, apenas três estudos se concentraram no fluxo genético e estrutura populacional de B. undatum. Todos esses estudos concordaram que o B. undatum pode ser geneticamente diferenciado ao longo de dezenas de quilómetros e que as populações são mais divergentes em baías e enseadas do que aquelas mais afastadas da costa. Esses estudos sugeriram um modelo de stepping-stone para uma grande população semi-contínua. No total, 195 indivíduos foram coletados em 13 locais de amostragem do Mar do Norte, Canal da Mancha e Mar da Irlanda de Dezembro de 2018 a Fevereiro de 2019. Destes 191 indivíduos foram escolhidos para prosseguir com a preparação da biblioteca de DNA de dupla digestão (DdRAD) usando ApeKI e BamHI-HF. A sequenciação foi realizada no Illumina HiSeq X Ten. No total, obtiveram-se 1.427.813.991 reads. O controle de qualidade e a desmultiplexação foram realizados com o software STACKS e os parâmetros: m = 3, n = 3 e M = 3. 4. Do total de reads, 19% não tinham código de barras, 0.08% foram removidos devido à baixa qualidade 4.4% não possuíam local de corte RAD, resultando em 1.303.931.081 reads. Depois da remoção dos indivíduos com menos de 20x de cobertura, permaneceram 141 indivíduos, o que corresponde a 6 a 15 indivíduos por local amostrado, representando 92.8% de todas as reads retidas (1.210.090.726). A cobertura obtida variou de 21.41x a 71.52x (média = 48.2x, S.D. = 13.2x). Os SNPS retidos correspondem ao que estavam simultaneamente presentes em 70% dos locais de amostragem, em 80% dos indivíduos, tinham uma frequência alélica menor maior que 5% e não excederam uma heterozigosidade máxima de 50%. Removeram-se ainda todos os SNPS que não estavam em Hardy Weinberg Equilibrium (HWE) e estavem em disequilibrio de ligação (linkage disequilibrium). No final da filtragem foram retidos 885 SNPs. Foram detectados 4 loci outliers sob seleção positiva putativa. No final foram utilizados para análise dois conjuntos de dados; (i) somente loci neutros, correspondendo a 881 loci e (ii) loci outlier - quatro loci outlier identificados como estando sob selecção positiva. Os resultados baseados somente nos loci neutros (i) encontraram evidências de três grupos genéticos distintos com frequências genéticas semelhantes nos locais de amostragem. A comparação entre pares de locais foi significativa em 22% dos pares, que decresceu para 12% após correção para múltiplos testes. A subestrutura detectada foi de amplitude menor do que noutros estudos. No entanto, isso pode ser resultado do esforço de escala na amostragem, pois o presente estudo é de relativamente pequena escala geográfica. Os valores de Fst variaram de 0.0052 a 0.0133, indicando que subestrutura significativa estava presente em todo o âmbito geográfico do estudo. Detectou-se uma significativa tendência de isolamento por distância nos locais de amostragem do Mar do Norte (r = 0.51, p = 0.001) e uma correlação significativa entre a distância genética e geográfica ao usar todos os locais de amostragem (r = 0,54, p = 0,002). Com base na análise de agrupamentos bayesianos, detectam-se três agrupamentos genéticos distintos, um dos quais numa frequência um pouco maior nos locais de amostragem do Canal da Mancha e outro em frequências mais altas no Mar do Norte. O grupo restante tem uma mistura em proporções quase iguais em todos os locais de amostragem. Estes resultados concordam com estudos anteriores de que B. undatum consiste numa grande população isolada com subestrutura significativa. No entanto, a divergência populacional geral é suprimida pelo alto fluxo genético e um grande tamanho efetivo da população. Foi relatado anteriormente que B. undatum é mais diferenciado em baías e enseadas. No entanto, este estudo não encontrou nenhuma evidência nesse sentido, embora apenas um único local de amostragem se localizasse numa uma baía. Será necessário um esforço de amostragem mais intenso nas baías e enseadas para testar a robustez dessa tendência. Os loci outliers não revelaram nenhuma estrutura populacional e, em vez disso, apoiam a existência de uma única população. Outliers sumetidos a blast do National Center for Biotechnology Information não revelaram qualquer correspondência significativa com genes anotados de outras espécies. Assim, na ausência de um genoma de B. undatum anotado ou dados ambientais à escala geográfica da amostragem, não é possível explicar os fatores subjacentes à selecção positiva detectada. Este estudo contribui para a crescente literatura que usa RADseq para delinear a estrutura populacional de escala geográfica fina. Buccinum undatum tem baixo potencial de dispersão e os valores de Fst no presente estudo são muito inferiores ao que seria esperado. Submetemos a explicação possível que um grande tamanho populacional efectivo e uma população semi-contínua suprimem a divergência genómica desta espécie.Population genomics is important for understanding the degree of genetic connectivity and effective dispersal over geographic distances. Connectivity, or the constraint of it, influences both local and regional biodiversity and thus is of primary interest to both evolutionary and ecological studies. In recent years, previous assumptions regarding dispersal capabilities, and their function as a primary driver of expected genetic structure of populations have been challenged by Next-Generation Sequencing techniques. This study investigated the population connectivity of Buccinum undatum with Single Nucleotide Polymorphisms (SNPs) derived from double-digest Restriction associated DNA sequencing. In total, 191 individuals were sequenced from the Southern North Sea, English Channel, and Irish Sea, a geographic scope of 1165 km. After strict quality control and filtering, 885 biallelic SNPs and 141 individuals were retained. Outlier detection revealed 4 loci under putatively positive selection. Two datasets were analysed; a neutral loci dataset which contained 881 SNPS, and an outlier loci dataset that contained the 4 SNPs identified as outliers. Results from the neutral dataset advocated for a single large population with no overall structure but significant sub-structure. However, sub-structure was much less frequent than previously reported for the species. Individuals sampled within a bay were not more genetically differentiated than those outside of bay, a previously reported trait of B. undatum. There was significant isolation by distance observed across the majority of the geographic range. Outlier analysis did not reveal any hidden population structure, nor any isolation by distance. Overall, results presented within fundamentally agreed with previous studies that B. undatum consists of a single population, that is semi-continuous in nature, with sub-structure present. However, high gene flow and a large effective population size supressed overall population divergence

Population genomics reveals a single semi-continuous population of a commercially exploited marine gastropod

Buccinum undatum is a commercially important marine gastropod with limited dispersal capabilities. Previous genetic studies utilising microsatellites and Double-digest Restriction-site Associated DNA sequencing (ddRADseq) provided evidence that B. undatum exhibits fine-scale genetic structure. Using ddRADseq, 128 individuals from the southern North Sea, English Channel, and the Irish Sea were genotyped using 7015 filtered single nucleotide polymorphisms (SNPs), 19 of which were identified as being under putative selection. Multiple genetic clustering methods – Discriminant analysis of principal components, Principal component analysis, and Sparse non-negative matrix factorisation,– were used to investigate population structure. Spatially explicit genetic structure was investigated using an Estimated Effective Migration Surface analysis and a Mantel correlogram. A single genetic population was found using neutral SNPs, with weak within-population structuring. Global FST was low (0.0046, p < 0.001), and pairwise FST estimates between sampling locations were between 0.0004 and 0.0224. There was a significant trend of isolation-by-distance across all sampling locations (r = 0.743, p < 0.001). Positive spatial autocorrelation indicated whelks located ≤ 50 km of one another were significantly more related than by chance (r = 0.12, p = 0.003), further emphasising the low dispersal capabilities of B. undatum. Finally, two barriers of lower-than-average dispersal were discovered; the Thames estuary and across the English Channel. Management implications are discussed for the sustainability of whelks from inshore fisheries

Bamboozled! Resolving deep evolutionary nodes within the phylogeny of bamboo corals (Octocorallia: Scleralcyonacea: Keratoisididae).

Keratoisididae is a globally distributed, and exclusively deep-sea, family of octocorals that contains species and genera that are polyphyletic. An alphanumeric system, based on a three-gene-region phylogeny, is widely used to describe the biodiversity within this family. That phylogeny identified 12 major groups although it did not have enough signal to explore the relationships among groups. Using increased phylogenomic resolution generated from Ultraconserved Elements and exons (i.e. conserved elements), we aim to resolve deeper nodes within the family and investigate the relationships among those predefined groups. In total, 109 libraries of conserved elements were generated from individuals representing both the genetic and morphological diversity of our keratoisidids. In addition, the conserved element data of 12 individuals from previous studies were included. Our taxon sampling included 11 of the 12 keratoisidid groups. We present two phylogenies, constructed from a 75% (231 loci) and 50% (1729 loci) taxon occupancy matrix respectively, using both Maximum Likelihood and Multiple Species Coalescence methods. These trees were congruent at deep nodes. As expected, S1 keratoisidids were recovered as a well-supported sister clade to the rest of the bamboo corals. S1 corals do not share the same mitochondrial gene arrangement found in other members of Keratoisididae. All other bamboo corals were recovered within two major clades. Clade I comprises individuals assigned to alphanumeric groups B1, C1, D1&D2, F1, H1, I4, and J3 while Clade II contains representatives from A1, I1, and M1. By combining genomics with already published morphological data, we provide evidence that group H1 is not monophyletic, and that the division between other groups - D1 and D2, and A1 and M1 - needs to be reconsidered. Overall, there is a lack of robust morphological markers within Keratoisididae, but subtle characters such as sclerite microstructure and ornamentation seem to be shared within groups and warrant further investigation as taxonomically diagnostic characters

The Biodiversity of Calcaxonian Octocorals from the Irish Continental Slope Inferred from Multilocus Mitochondrial Barcoding

Deep-sea corals are important benthic inhabitants that support the biodiversity and function of the wider faunal community; however, their taxonomy is underdeveloped and their accurate identification is often difficult. In our study, we investigated the utility of a superextended (>3000 bp) barcode and explored the effectiveness of various molecular species delimitation techniques with an aim to put upper and lower bounds on the estimated number of calcaxonian species in Irish waters. We collected 112 calcaxonians (70 Keratoisididae, 22 Primnoidae, 20 Chrysogorgiidae) and one chelidonisid from the Irish continental slope and sequenced a 3390 bp DNA barcode comprising four mitochondrial regions (mtMutS, COI + igr1, 16S rRNA-ND2, and igr4), recovering 38 haplotypes. Individuals that shared a haplotype were often morphologically distinct, and we thus undertook detailed morphological work, including SEM of sclerites, on one representative of each morphotype within each haplotype. GMYC, bGMYC, and mPTP returned incongruent estimates of species numbers. In total, there are between 25 and 40 species, although no definitive number could be assigned, primarily due to poorly defined keratoisidid species boundaries. As expected, the superextended barcode provided greater discrimination power than single markers; bGMYC appeared to be the most effective delimiter. Among the identified species were Chelidonisis aurantiaca, collected deeper than previously known at 1507 m, and Calyptrophora clinata, recorded for the second time from the Northeast Atlantic. A full understanding of the diversity and distribution of calcaxonians requires substantial taxonomic work, but we highlight the Irish continental slope as harbouring significant diversity

The Biodiversity of Calcaxonian Octocorals from the Irish Continental Slope Inferred from Multilocus Mitochondrial Barcoding

Deep-sea corals are important benthic inhabitants that support the biodiversity and function of the wider faunal community; however, their taxonomy is underdeveloped and their accurate identification is often difficult. In our study, we investigated the utility of a superextended (&gt;3000 bp) barcode and explored the effectiveness of various molecular species delimitation techniques with an aim to put upper and lower bounds on the estimated number of calcaxonian species in Irish waters. We collected 112 calcaxonians (70 Keratoisididae, 22 Primnoidae, 20 Chrysogorgiidae) and one chelidonisid from the Irish continental slope and sequenced a 3390 bp DNA barcode comprising four mitochondrial regions (mtMutS, COI + igr1, 16S rRNA-ND2, and igr4), recovering 38 haplotypes. Individuals that shared a haplotype were often morphologically distinct, and we thus undertook detailed morphological work, including SEM of sclerites, on one representative of each morphotype within each haplotype. GMYC, bGMYC, and mPTP returned incongruent estimates of species numbers. In total, there are between 25 and 40 species, although no definitive number could be assigned, primarily due to poorly defined keratoisidid species boundaries. As expected, the superextended barcode provided greater discrimination power than single markers; bGMYC appeared to be the most effective delimiter. Among the identified species were Chelidonisis aurantiaca, collected deeper than previously known at 1507 m, and Calyptrophora clinata, recorded for the second time from the Northeast Atlantic. A full understanding of the diversity and distribution of calcaxonians requires substantial taxonomic work, but we highlight the Irish continental slope as harbouring significant diversity

Cochrane Rapid Review Protocol - The Effectiveness of Video Calls for Reducing Social Isolation and Loneliness in Older People

The COVID-19 pandemic has resulted in many countries implementing physical distancing and restriction of movement, particularly for older adults and other vulnerable populations. This requires millions of older people to remain in their homes, many of whom will be cut off from their usual social support network (Armitage &amp; Nellums, 2020). The primary objective of this rapid review is to assess the effectiveness of video calls for reducing social isolation and loneliness in older adults. The review will also seek to address the effectiveness of video calls for mental health outcomes including quality of life and depression

Activated Graphene Oxide-Calcium Alginate Beads for Adsorption of Methylene Blue and Pharmaceuticals

The remarkable adsorption capacity of graphene-derived materials has prompted their examination in composite materials suitable for deployment in treatment of contaminated waters. In this study, crosslinked calcium alginate–graphene oxide beads were prepared and activated by exposure to pH 4 by using 0.1M HCl. The activated beads were investigated as novel adsorbents for the removal of organic pollutants (methylene blue dye and the pharmaceuticals famotidine and diclofenac) with a range of physicochemical properties. The effects of initial pollutant concentration, temperature, pH, and adsorbent dose were investigated, and kinetic models were examined for fit to the data. The maximum adsorption capacities qmax obtained were 1334, 35.50 and 36.35 mg g−1 for the uptake of methylene blue, famotidine and diclofenac, respectively. The equilibrium adsorption had an alignment with Langmuir isotherms, while the kinetics were most accurately modelled using pseudo- first-order and second order models according to the regression analysis. Thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated and the adsorption process was determined to be exothermic and spontaneous

The development of novel flexible electrode arrays for the electrochemotherapy of solid tumour tissue. (potential for endoscopic treatment of inaccessible cancers)

Therapeutic “electroporation” involves application of electric fields to target cells/tissues, thereby rendering their cell membranes transiently porous, thus making feasible the cellular uptake and efficacy of previously impermeant and ineffective therapeutic agents. The objectives of this research are a) the development of flexible electrode arrays for incorporation into microsystem endoscopic devices, and b) the assessment of their efficacy in delivering selected genetic and pharmaceutical anticancer therapies. Gold electrodes were fabricated on flexible polyimide substrates following predictive modeling and simulation of electric fields using FEMLABtm software. Subsequent assessment of electroporation efficiency in-vitro involved 1) enumeration of viable tumour cells after delivery of electric pulses and exposure to low concentrations of bleomycin, otherwise known as electrochemotherapy 2) Efficacy of gene delivery by detection of emitted green fluorescence by cells after electroporation with the pEGFP plasmid and 3) In-vivo efficacy of electrochemotherapy in a variety of human solid tumour masses in nude mouse models (xenografts). The flexible electrode system was found to be successful for electrical delivery of plasmids and drugs in-vitro and invivo. We found in-vivo complete regression of prostate, colon, oesophageal, and renal cancers with reduced growth rates for fibrosarcoma and breast cell lines. These flexible electrodes are suitable for electrochemotherapy or gene therapy to solid tumours masses and may be fabricated for application to the treatment of some cancers in humans by transcutaneous or endoscopic delivery systems