TítuloAnalyses of molecular markers and gene expression in crustacean species

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(1)DOCTORAL THESIS 2018. ANALYSES OF MOLECULAR MARKERS AND GENE EXPRESSION IN CRUSTACEAN SPECIES. ALEJANDRA PERINA CEDRÓN Department of Biology.

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(3) Analyses of molecular markers and gene expression in crustacean species. Alejandra Perina Cedrón Doctoral Thesis 2018. Supervisors: Dr. Andrés Martínez-Lage & Dra. Ana M. González-Tizón. PhD program: Cell and Molecular Biology.

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(5) Analyses of molecular markers and gene expression in crustacean species Análisis de marcadores moleculares y expresión génica en especies de crustáceos PhD Thesis / Tesis de doctorado. Alejandra Perina Cedrón. Supervisors / Directores Dr. Andrés Martínez-Lage & Dra. Ana M. González-Tizón. Review by / Revisado por Alberto Pallavicini – Università di Trieste (Italia) Gerhard Pohle – University of New Brunswick (Canadá). Departamento de Biología. Facultad de Ciencias.

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(7) Los Dres. ANA MARÍA GONZÁLEZ TIZÓN profesora contratada doctora y ANDRÉS MARTINEZ LAGE profesor titular de universidad del área de genética del Departamento de Biología de la Universidade da Coruña.. INFORMAN: que la licenciada Alejandra Perina Cedrón del programa de doctorado de Biología Celular y Molecular, ha realizado en el Departamento de Biología de la Facultad de Ciencias el trabajo con el título Analyses of molecular markers and gene expression in crustacean species bajo nuestra supervisión. Considerándolo finalizado permitimos su presentación bajo la modalidad de mención internacional y compendio de artículos.. Y para que conste firmamos la presente en A Coruña a 25 de enero de 2018.. VºBª de los Directores. Fdo. Ana Mª González Tizón. La doctoranda. Fdo. Alejandra Perina Cedrón. Andrés Martínez Lage.

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(9) To my F1 In loving memory of my Daddy.

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(11) Acknowledgements.

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(13) The pack is the wolf 's strength ! To start I would like to underline that although this PhD thesis bears my signature, it actually has only been feasible thanks to unagging support of numerous people. And as there are many ways to skin a cat... My rst thanks go to my supervisors, Andrés Martínez Lage and Ana González Tizón for their trust in me and for the opportunity to work together. Thank you for your guidance, friendship, and for all your encouragement. Given the chance, I would happily work with you again! Many thanks to Enrique González Ortegón for collecting and spending time under the stereo microscope to identify Palaemon specimens. I am very grateful to all the people who generously hosted me during my internship abroad: Raquel Chaves, Daniela Ferreira and Susana Meles. This research internship was an exciting learning experience. Thanks also to Nani and Raquel from the University Research Support Services, for their willing assistance with lab issues. Many thanks to my colleagues at the Evolutionary Biology Group whom I am so thrilled to have worked with:. Joaquín, Vero, Zeltia, David, Inés, Rosa, Neus, Elba,. Horacio, Manuel, Marta, Cosme Damián, Antón Vila... (I hope I have not left anyone out) all of whom are a part of my past, present and future. Clearly, my special thanks to Vero for the calamitous and good memories we share when begining in the lab. Zeltia my coach, my doula, to whom I owe the rst sentence of these acknowledgements, and Inés who has been a breath of fresh air for me in these last few months. I would also like to thank the AllGenetics Dream team, and especially Joaquín and Antón. Your support, willingness to wait for so long and the shared experience have been very important to me. To all my wonderful friends, especially Javi, Eva, César, Claudia my warmest thanks for putting up with me all this time. Alba and Diego, thank you for sharing a bit of you talent with me and designing the beautiful cover of this thesis. And last but by no means least my most heartfelt thanks to my incredible family! Iago, my love, thank you for your innite patience, you know that this work is as much yours as mine. I love you! Xoel, my baby, you encourage me every day, and Lucas whose purr made the analysis of the microsatellites easier. I would like to thank my parents, Julio and Mary for their love and all the support they have provided me over the last few years, and to my granny Carmen and my dear Lulo for taking care of me. THANKS. Finally, I was endowed with a FPI (Formación de Personal Investigador) grant (BES2012-053288) from the Spanish Goverment and by a Resarch Internship grant from Universidade da Coruña.. This work was nancially supported by grants from the Span-. ish Goverment (CTM2007-62034, and CTM2014-53838-R), and from Xunta de Galicia (10MMA103008PR and GRC2014/050). MUCHAS GRACIAS. v.

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(15) Contents SHORT ABSTRACTS. 1. Resumen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3. Resumo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4. EXTENDED ABSTRACT. 5. Resumen extendido . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. I.. INTRODUCTION. 7. 13. Organisation of the thesis. 15. Introduction to crustacean species. 17. Pollicipes species: Pollicipes pollicipes. . . . . . . . . . . . . . . . . . . . . . . .. State-of-the-art of genetic studies in Pollicipes species. Palaemon species: Palaemon serratus. . . . . . . . . . . .. 18. . . . . . . . . . . . . . . . . . . . . . . .. 19. State-of-the-art of genetic studies in Palaemon species. II.. 17. . . . . . . . . . . .. AIMS AND OBJECTIVES. 20. 21. III. RESEARCH ARTICLES. 25. 1.. Analyses of 5S rDNA. 27. 2.. Accessing transcriptomic data. 61. 3.. Transcriptome assembly of P. pollicipes. 69. 4.. Transcriptome assembly of P. serratus. 83. 5.. Isolation and characterization of microsatellites in P. serratus. 87. 6.. Genetic diversity and population structure of P. serratus. 97. vii.

(16) Alejandra Perina Cedrón. IV. GENERAL DISCUSSION. 113. Contributions of this thesis to the understanding of the evolution of 5S rDNA. 115. Contributions of this thesis to improve access to transcriptomic data. 119. Contributions to assess the population structure of P. serratus. 123. V.. 127. CONCLUSIONS. Bibliography. viii. 131.

(17) SHORT ABSTRACTS.

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(19) Short abstracts. Resumen. Los crustáceos son uno de los grupos más diversos del planeta. Tanto el percebe Pollicipes. pollicipes como el camarón Palaemon serratus representan dos recursos pesqueros importantes en numerosas regiones. Esta tesis pretende ampliar el conocimiento de estas especies analizando la variabilidad genética e identicando genes de interés. En primer lugar, el análisis de la organización molecular del ADN ribosomal 5S (5S rDNA) mostró que el género Pollicipes tiene diferentes variantes de 5S rDNA en su genoma. Las relaciones logenéticas revelaron un patrón de agrupamiento entre especies. Estos resultados conrmaron que el 5S rDNA evoluciona según el proceso de birth and death. En segundo lugar, los ensamblajes de los transcriptomas fueron el paso previo a la identicación génica y a la cuanticación de niveles de expresión. En este sentido, se identicaron y evaluaron mediante RT-qPCR varios transcritos con similaridad a proteínas ralacionadas con el asentamiento larvario en percebes. Finalmente, a través del análisis de la diversidad y estructura genética de P. serratus se situó una barrera biogeográca al oeste del estrecho de Gibraltar.. Se recomendó la delimitación de dos áreas prioritarias de. gestión (Atlántico vs Gibraltar-Meditarráneo) así como que la población del estuario del Guadalquivir sea considerada como un stock separado.. Resumo. Os crustáceos son un dos grupos máis diversos do planeta. Tanto o percebe Pollicipes. pollicipes como o camarón Palaemon serratus son dous recursos pesqueiros importantes en numerosas rexións. Esta tese pretende ampliar o coñecemento destas dúas especies analizando a variabilidade xenética e identicando xenes de interese. En primeiro lugar, a análise da organización molecular do ADN ribosomal 5S (5S rDNA) mostrou que o xénero Pollicipes amosa diferentes variantes do 5S rDNA no seu xenoma. As relacións loxenéticas revelaron un patrón de agrupamento entre especies. Estes resultados conrmaron que o 5S rDNA evoluciona segundo un proceso de birth and death. No segundo lugar, as ensamblaxes dos transcritomas foron o paso previo para a identicación de xenes e a cuanticación da súa expresión.. Neste senso, foron identicados e evalua-. dos mediante RT-qPCR varios transcritos con similaridade a proteínas relacionadas co asentamento larvario nos percebes. Finalmente, a través dunha análise de diversidade e estrutura xenética de P. serratus foi posible localizar unha barreira bioxeográca ao oeste do estreito de Xibraltar. Recomendouse a delimitación de dúas áreas prioritarias para a xestión (Atlántico vs Xibraltar-Mediterráneo) e tamén que a poboación do estuario do Guadalquivir sexa considerada como un stock separado.. 3.

(20) Alejandra Perina Cedrón. Abstract Crustaceans are one of the most diverse groups on the planet. The gooseneck barnacle. Pollicipes pollicipes and the common littoral shrimp Palaemon serratus are both vital shery resources in numerous regions. This PhD thesis aims to contribute to the understanding of these species by analysing the genetic variability and identifying interesting genes. Firstly, the analysis of molecular organisation of the 5S ribosomal DNA (5S rDNA) showed dierent size variants of 5S rDNA arrays in the genome of the genus Pollicipes. Phylogenetic analyses have shown a pattern of gene clustering among species. results suggested that the 5S rDNA evolves in the birth and death process.. These. Secondly,. transcriptome assemblies were a step prior to gene identication and the quantication of gene expression. In this regard, several transcripts related to larval settlement in barnacles were identied and further evaluated using RT-qPCR. Finally, the analysis of the diversity and the genetic structure of P. serratus revealed that a biogegraphical break was located to the west of the Strait of Gibraltar. For future management of P. serratus we suggested delimiting two dierent priority areas (Atlántico vs Gibraltar-Meditarráneo) and considering the population of the Estuary of Guadalquivir as a separate stock.. 4.

(21) EXTENDED ABSTRACT.

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(23) Extended abstract. Resumen extendido. En esta tesis doctoral por compendio de publicaciones se han realizado diversos estudios genéticos en dos especies de crustáceos marinos: en el percebe Pollicipes pollicipes (Gmelin, 1789) y en el camarón común Palaemon serratus (Pennant, 1777). Ambos organismos representan recursos pesqueros de gran importancia económica en diversas regiones europeas, entre las que destaca principalmente Galicia (España). La elevada demanda comercial de estos dos crustáceos provoca de manera directa una notable explotación pesquera de estos dos recursos naturales. Los objetivos concretos sobre los que se ha centrado la presente tesis fueron: 1) el análisis de la variabilidad genética intra e interespecíca utilizando marcadores moleculares nucleares, tanto el ADN ribosomal 5S (5S rDNA) en el caso del percebe, como marcadores de tipo microsatélite para el estudio poblacional del camarón, y 2) la identicación de genes relacionados con funciones biológicas de interés, tales como el reclutamiento y desarrollo larvario, la jación al sustrato o la metamorfosis. Estos objetivos se desarrollaron dentro de la genética evolutiva, la genética poblacional, la transcriptómica y la expresión génica. La metodología aplicada varía en cierto modo a lo largo de cada uno de los capítulos que conforman los resultados de la tesis, sin embargo puede resumirse como sigue: en el capítulo 1 titulado Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection , la familia multigénica del 5S rDNA se estudió mediante un procedimiento de amplicación por reacción en cadena de la polimerasa (PCR), clonación y secuenciación. La totalidad de las secuencias obtenidas experimentalmente en el laboratorio se analizaron posteriormente empleando herramientas bioinformáticas y se depositaron en bases de datos públicas. En el capítulo 2 cuyo título es Accessing transcriptomic data for ecologically important genes in the goose barnacle (Pollicipes pollicipes ), with particular focus on cement proteins , se llevó a cabo el ensamblaje y la anotación de un conjunto de secuencias tipo EST (Expressed. Sequence Tag ) de P. pollicipes, obtenidas tanto experimentalmente como a través de bases de datos públicas. En los capítulos 3 y 4 que llevan por título De novo transcriptome assembly of Pollicipes pollicipes, towards an understanding of larval settlement y De. novo transcriptome assembly of shrimp Palaemon serratus respectivamente, se utilizó la tecnología de secuenciación masiva de Illumina para secuenciar, ensamblar de novo y anotar los transcriptomas del percebe P. pollicipes y del camarón P. serratus en dos etapas diferentes de su ciclo biológico (adulto y larvas). Además, en el caso de P. pollicipes, se aplicó la técnica de RT-qPCR (reverse transcriptase quantitative PCR ) para comparar los niveles de expresión génica de ciertos genes, identicados como resultado de los análisis de los transcriptomas. Por último en los capítulos 5 y 6 Isolation and characterization of 20 polymorphic microsatellite loci in Palaemon serratus and cross-amplication in. Palaemon species by 454 pyrosequencing y Assessment of genetic diversity and population structure of Palaemon serratus, towards a sustainable management , se desarrollaron. 7.

(24) Alejandra Perina Cedrón. veinte marcadores polimórcos de tipo microsatélite en el camarón P. serratus, a partir de una genoteca enriquecida en motivos microstátelite. Seguidamente estos marcadores se utilizaron para evaluar la diversidad genética y la estructura poblacional de esta especie de camarón en aguas europeas. A continuación se detalla cada uno de estos estudios: En el capítulo 1 Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection se estudió la organización molecular y la secuencia de nucleótidos de la familia multigénica del 5S rDNA en el conjunto de 116 secuencias procedentes de tres especies del género Pollicipes, P. pollicipes, P. elegans y P. polymerus en un contexto evolutivo. El 5S rDNA está formado por una región conservada que se transcribe de 120 pares de bases de longitud (5S), y por una región espaciadora intergénica variable en longitud comúnmente conocida como el espaciador no transcrito (NTS). En este trabajo, se predijo la estructura secundaria de las regiones 5S y se generaron dos posibles tipos de estructura para el género Pollicipes, además de la estructura consenso para crustáceos. Se caracterizaron los elementos conservados de las regiones aguas arriba y aguas abajo de las mismas, encontrándose al menos tres regiones conservadas: una región rica en 'AT' aproximadamente a -25 nucleótidos del sitio de inicio de la transcripción; la región 'CGGCCACCGGC' localizada entre los -24 y -14 nucleótidos, y el tramo 'TTC' a -7 nucleótidos del inicio de la transcripción. Se compararon también las regiones reguladoras internas con las descritas previamente en especies modelo. De esta manera, se pudieron identicar las cuatro regiones de control interno (ICR) características de la región 5S. Se demostró que el 5S rDNA del género Pollicipes está organizado en repeticiones en tándem de diferentes tamaños, aunque unidades dispersas también se pueden localizar en el genoma. Se encontraron hasta siete tipos diferentes de 5S rDNA y dos posibles pseudogenes. El análisis de las regiones NTS del 5S rDNA mostró que algunos de ellos estaban evolutivamente más relacionados con NTS de otras especies que con aquellos de la misma especie, sugiriendo la existencia de polimorsmo ancestral y evolución a largo plazo mediante el proceso de birth-and-death. La conservación de la secuencia nucleotídica dentro de las regiones 5S sugirió que la selección puricadora, además de los entrecruzamientos desiguales y las conversiones génicas, estuvieron implicados en la evolución de esta familia multigénica. En el capítulo 2 Accessing transcriptomic data for ecologically important genes in the goose barnacle (Pollicipes pollicipes ), with particular focus on cement proteins se llevó a cabo el desarrollo y la caracterización de una genoteca de 119 secuencias de tipo EST (expressed sequence tags ) en el percebe P. pollicipes mediante secuenciación Sanger. Hasta hace relativamente poco tiempo, las secuencias de tipo EST eran fundamentales para el descubrimiento de nuevos genes y se obtenían comúnmente mediante la secuenciación de un ARN mensajero clonado. Siguiendo una aproximación de ensamblaje híbrido, estas secuencias de tipo EST fueron combinadas con aquellas previamente depositadas en la base de datos correspondiente del Centro Nacional para Información Biotecnológica. 8.

(25) Extended abstract. (NCBI) y que habían sido obtenidas mediante secuenciación Roche 454. Así, a partir de un total de 4310 EST se consiguieron ensamblar 1805 unigenes que posteriormente fueron anotados contra la información biológica disponible en bases de datos públicas. El análisis Blastp mostró una gran similaridad con diversos organismos pertenecientes al phylum Arthropoda. La anotación ontológica reveló que el 49 % de los términos GO (Gene Ontology ) se encuentran en la categoría Proceso Biológico, el 30 % corresponden a Componente Celular y el 21 % restante a la categoría Función Molecular. Si bien, solo una pequeña parte de estos unigenes ensamblados se han anotado, estos resultados contribuyen a mejorar el entendimiento de las características biológicas de esta especie. La identicación, en este capítulo, de dos proteínas cementantes, la de 100 kDa y la de 52 kDa, resultó especialmente atractiva. En el siguiente capítulo 3 De novo transcriptome assembly of Pollicipes pollicipes, towards an understanding of larval settlement, dado que el estudio y el análisis del transcriptoma es esencial para el entendimiento de la función de los genes, se utilizó la tecnología de secuenciación de Illumina para secuenciar, ensamblar de novo y anotar el transcriptoma del percebe P. pollicipes. El ARN se aisló a partir de una mezcla de tejidos en dos etapas del ciclo biológico: adulto y larvas en estadio nauplio. Las lecturas de secuenciación se depositaron en la base de datos SRA (Sequence Read Archive ) del NCBI. Estas lecturas fueron ensambladas de novo, traducidas y posteriormente anotadas. Se obtuvieron 121.061 transcritos a partir de tejido de percebe adulto, con una longitud media de contig de 683,39 nucleótidos (nt) y un total de 105.585 genes. En el caso de las larvas, se consiguieron 98.488 transcritos, la longitud media de contig fue de 603,16 nt y 88.182 genes. Los valores del parámetro N50 fueron 1.105 y 848 para cada ensamblaje respectivamente y el total de bases ensambladas fue 82.731.878 para el transcriptoma adulto y 59.403.826 para el de las larvas. Los resultados de la anotación contra la base de datos de secuencias proteicas no redundantes (nr) mostraron 30.960 y 28.182 secuencias (adulto y larva, respectivamente) con al menos un resultado de alineamiento local (Blastp). La anotación funcional produjo 9.254 y 8.288 secuencias anotadas para adulto y larva, respectivamente. Se generó una lista de genes candidatos a ser especícos de adulto y larva donde la mayoría de resultados no se anotaron. Sin embargo, a partir de los resultados del conjunto de la anotación, se han podido identicar diversos transcritos implicados en el asentamiento larvario y la jación al sustrato. Entre ellos se encuentran transcritos con elevada similitud a proteínas relacionadas con el cemento, crustinas, la feromona de asentamiento en el agua (waterborne settlement pheromone ), receptores tirosina quinasa y el complejo proteico inductor del asentamiento, (SIPC, settlement inducing protein. complex ). Estos transcritos junto con la proteína cementante de 100 KDa identicada en el capítulo anterior, se evaluaron mediante experimentos de RT-qPCR. Mientras que las proteínas cementantes no mostraron expresión en larvas, los niveles de expresión del resto de transcritos evaluados fueron más altos en dicha etapa que en la etapa adulta. En el capítulo 4 De novo transcriptome assembly of shrimp Palaemon serratus , uti-. 9.

(26) Alejandra Perina Cedrón. lizando la misma metodología que en el capítulo anterior se generó el transcriptoma del camarón P. serratus en adulto y en larvas en estadio zoea. Las lecturas de secuenciación se depositaron en la base de datos SRA (Sequence Read Archive ) del NCBI. Estas lecturas fueron ensambladas de novo, traducidas y posteriormente anotadas. Se obtuvieron 112.716 transcritos a partir de tejido de camarón adulto, con una longitud media de contig de 996,97 nt, y 95.601 genes. En el caso de las larvas, se recuperaron 152.110 transcritos, la longitud media de contig fue de 1.047,88 nt y 124.389 genes. Los valores del parámetro N50 fueron 2.311 y 2.596 para cada ensamblaje respectivamente y el total de bases ensambladas fue de 112.374.970 para el transcriptoma adulto y 159.393.572 bases para el de larvas. Los resultados de las anotaciones están disponibles como material suplementario. En el capítulo 5 Isolation and characterization of 20 polymorphic microsatellite loci in. Palaemon serratus and cross-amplication in Palaemon species by 454 pyrosequencing , se aislaron marcadores microsátelite polimórcos especícos para el camarón P. serratus. Siguiendo una aproximación basada en el desarrollo de genotecas enriquecidas en motivos microsatélite, se caracterizaron y optimizaron 20 loci microsatélite en veinte individuos procedentes del golfo Ártabro (Galicia, España). El número de alelos por locus osciló de dos a 17. Las heterocigosidades observada y esperada variaron de 0,050 a 0,950 y de 0,049 a 0,916, respectivamente. Además, dieciséis de los 20 marcadores, amplicaron en tres especies más del género Palaemon, en P. elegans, P. adspersus y P. longirostris. A continuación y para nalizar, en el último capítulo de esta tesis, 6 Assessment of genetic diversity and population structure of Palaemon serratus, towards a sustainable management , utilizando estos marcadores microsatélite desarrollados en el capítulo previo se analizaron la diversidad genética y la estructura poblacional del camarón P.. serratus. Diecisiete loci fueron nalmente utilizados en el análisis de diez localidades situadas a lo largo de la costa Atlántica y Mediterránea cubriendo una gran parte de su distribución. Los tres restantes marcadores microsatélite se eliminaron del estudio por presentar más de un 25 % de datos perdidos. El número medio de alelos por locus y localidad fue 5,347, mientras que las heterocigosidades observada y esperada medias fueron 0,408 y 0,516 respectivamente. El análisis bayesiano reveló una clara pertenencia de los individuos analizados a dos clústers. Los especímenes de las localidades Atlánticas (Anglesey, Duinbergen, Calais, Isla de Ré, Golfo Ártabro y Mondego) se agruparon en un clúster y aquellos que pertencen al área del estrecho de Gibraltar y Mediterráneo (Guadalquivir, Conil, Gibraltar, Mallorca) en otro clúster. El nivel de diversidad genética fue mucho más elevada dentro del clúster Atlántico. Se detectaron valores de FST signicativos entre todos los pares de localidades. Asimismo, la localidad del Estuario del Guadalquivir resultó especialmente interesante por ser signicativamente diferente al resto de los sitios de muestreo. Se encontraron evidencias de aislamiento por distancia, a través de la correlación positiva entre la distancia genética y la distancia geográca. Finalmente, se detectó una barrera biogeográca situada al oeste del Estrecho de Gibral-. 10.

(27) Extended abstract. tar que divide los dos clústers anterioremente descritos. A partir de los datos obtenidos con estos marcadores microsatélite, se ha propuesto delimitar dos áreas prioritarias de gestión de este recurso marino y además, que al menos la población muestreada en el Estuario del Guadalquivir sea considerada como un stock genético separado. Por tanto, las principales conclusiones de esta tesis pueden resumirse en los siguientes puntos:. . El análisis sobre la organización molecular del ADN ribosomal 5S (5S rDNA) en especies que pertenecen al género Pollicipes reveló que este grupo de crustáceos puede tener variantes diferentes de 5S rDNA atendiendo principalmente a las diferencias en la región espaciadora intergénica, NTS. En este trabajo, se caracterizaron siete tipos de 5S rDNA en base al análisis de la secuencia de dicha región. Cinco unidades se encontraron en P. pollicipes, dos unidades en P. elegans y otras dos en P.. polymerus. Algunos NTS mostraron una mayor similaridad con NTS de diferentes especies que con NTS de la propia especie donde se encontraron. Las relaciones logenéticas revelaron un patrón de agrupamiento génico entre especies donde variantes de 5S rDNA idénticas se comparten entre especies y variantes únicas son especícas de especies. Los análisis de las regiones aguas arriba y aguas abajo del sitio de inicio de la transcripción, las regiones reguladoras internas, y las señales de terminación muestran los rasgos comunes a especies modelo como Drosophila. melanogaster o Xenopus laevis. La predicción de la estructura secundaria para el 5S ha sido posible en todas las secuencias analizadas, generando dos posibles tipos para el género Pollicipes. Además, se han descrito dos pseudogenes. Los resultados obtenidos sugieren que el 5S rDNA del género Pollicipes se encuentra sometido a un proceso de evolución birth-and-death, con una fuerte selección puricadora que actúa sobre la región que se transcribe.. . El ensamblaje híbrido sobre secuencias de tipo EST del percebe P. pollicipes produjó un total de 1.805 unigenes, de los cuales solo un pequeño porcentaje pudo ser anotado. Entre estas secuencias, la anotación basada en ontología mostró que el 49 % de los términos ontológicos pertenecían a la categoría de Proceso Biológico, el 30 % a la categoría de Componente Celular y el 21 % restante se encontraron en la categoría de Función Molecular. La identicación de dos proteínas relacionadas con la jación al sustrato rocoso (100 kDa y 52 kDa cement proteins ) resultó particularmente interesante para estudios futuros.. . A partir de lecturas procedentes de la tecnología de secuenciación masiva de Ilumi-. na se ensamblaron de novo dos transcriptomas para P. pollicipes, un transcriptoma para adultos y uno para larvas en estadio nauplio. El análisis Blastp contra la base de datos no redundante de proteínas mostró que más del 80 % de las secuencias presentaban al menos un resultado de similaridad mientras que, el 26 % se anotaron funcionalmente contra la base de datos Uniprot en ambos transcriptomas.. 11.

(28) Alejandra Perina Cedrón. Se generaron dos listas de posibles genes especícos de cada etapa biológica, sin embargo la mayoría de ellos no se anotaron. Los transcritos relacionados con el proceso de asentamiento larvario que han sido identicados entre el conjunto de secuencias se evaluaron por RT-qPCR. Mientras que la cuanticación de los niveles de expresión de las tres proteínas cementantes (19 kDa, 52 kDa y 100 kDa) no produjo resultados en larvas, la crustina, la feromona de asentamiento en el agua, el receptor tirosina-quinasa y la proteína SIPC mostraron niveles de expresión génica más elevados en las muestras de larvas en comparación con las de tejido adulto.. . Se ensamblaron de novo dos transcriptomas para el camarón P. serratus a partir de lecturas obtenidas a través de la misma tecnología de secuenciación masiva que se utilizó en el caso del percebe, ensamblándose un transcriptoma a partir de tejido adulto y otro transcriptoma a partir de un conjunto de larvas en estadio zoea. En este caso se obtuvieron resultados de anotación funcional en aproximadamente un 18 % de las secuencias en ambos transcriptomas.. . Se desarrollaron 20 loci microsatélite polimórcos especícos del camarón P. serratus. La amplicación de estos marcadores funcionó en tres congéneres: P. elegans, P.. adspersus y P. longirostris, demostrando su posible utilización en estudios entre especies.. . Después de comprobar la idoneidad de estos marcadores microsatálite en veinte individuos de Galicia, se extendió el análisis a un total de diez localidades que cubren una gran parte del rango de expansión natural de este crustáceo. El análisis bayesiano mostró dos clusters de individuos. Camarones de las localidades de la costa Atlántica (Anglesey, Duinbergen, Calais, Isla de Ré, Golfo Ártabro y Mondego) se agruparon en un clúster, mientras que los camarones del área del Estrecho de Gibraltar y la costa Mediterránea (Guadalquivir, Conil, Gibraltar y Mallorca) se agruparon en otro cluster. La localidad del Estuario del Guadalquivir resultó signicativamente diferente al resto de localidades de muestreo. Los resultados del análisis de aislamiento por distancia mostraron una correlación positiva entre las distancias genéticas y geográcas. A raíz de los datos obtenidos con estos marcadores moleculares se postuló que la barrera biogeográca que limita el ujo génico se encuentra localizada al oeste del estrecho de Gibraltar y se propone delimitar dos áreas prioritarias de gestión de este recurso natural además de considerar a la población muestreada en el Estrecho de Gibraltar como un stock genético diferente.. 12.

(29) Part I.. INTRODUCTION.

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(31) Organisation of the thesis In this PhD thesis two species of marine crustaceans were studied, consisting of the gooseneck barnacle Pollicipes pollicipes (Gmelin, 1789) (Crustacea: Pedunculata) and the common littoral shrimp Palaemon serratus (Pennant, 1777) (Crustacea: Decapoda). In terms of methodology, both molecular genetic and bioinformatic approaches were followed in order to answer questions mainly related to evolutionary genetics, ecology or transcriptomic proling. As the thesis was written as a compendium of research articles, each of them has its own introductory section. Therefore, in this general introduction, I will give an overview on the organisation of the thesis and will provide some additional relevant information that, due to editorial reasons, was not included in the articles themselves. The two species under study were selected, mainly, because of their commercial value in various European regions. As commercialisation in turn, may lead to overexploitation of such natural resources the research and objectives of the present study were supported by dierent programs funded by government institutions.. In the case of P. pollicipes,. the general aim of the rst project was the study of the genetic variability using DNA molecular markers.. Here, the 5S ribosomal DNA was characterised in order to come. to understand the Galician shores, to what extent these shores were genetically related to one another as well as to those of the Portuguese and Moroccan shores, and to two congeneric species, Pollicipes elegans and Pollicipes polymerus. The second project examined how the exploitation of P. pollicipes may be managed with two basic purposes: on one hand the study the environmental factors that aect the recruitment and growth of these animals, and on the other, the generation of molecular markers and gene sequences to identify genes involved in specic biological functions, such as larval recruitment and substrate adhesion. The latter, strictly genetic, was the basis of this PhD thesis. The research project on the shrimp P. serratus proposed to characterise dierent populations of this species.. Particularly, the research delved into the knowledge of the investigate. the genetic status through the development of microsatellite loci and the analysis of population variability. For the sake of clarity, articles have been organised by publishing date and topic.. 15.

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(33) Introduction to crustacean species The gooseneck barnacle Pollicipes pollicipes (Gmelin, 1789) and the common littoral shrimp Palaemon serratus (Pennant, 1777) are marine crustaceans that belong to phylum Arthropoda, that is comprised of 3 subphyla of living organisms: Chelicerata, Uniramia, and Crustacea. The latter forms a large and diverse taxon. In fact, according to Martin and Davis (2001) no group of plants or animals on the planet exhibits the range of morphological diversity seen among the extant Crustacea.. In addition to boundless. diversity of form, crustaceans exhibit a great range of sizes, from minute interstitial and parasitic forms (e.g. Tantulocarida) measuring as little as a tenth of a millimetre to giant crabs, lobsters, and isopods with a body size of up to half a metre in length or width and weighing up to 20 kilograms (Webber et al. 2010). Crustaceans are an ancient group, dating from at least the Early Cambrian (Chen et al. 2011) and have diversied abundantly since then. Calculations of the number of named living species of Crustacea range from approximately 50,000 to 67,000 (Webber et al. 2010). Nowadays, it is noteworthy that by virtue of their edibility, many crustaceans are prized items on restaurant menus around the world (Webber et al. 2010). Below, each of the studied species is introduced separately.. Pollicipes species: Pollicipes pollicipes The gooseneck barnacle Pollicipes pollicipes (Gmelin, 1789) belongs to the order Pedunculata and family Pollicipedidae within the phylum Arthropoda. The genus Pollicipes includes three other living species: P. elegans Lesson, 1831, P. polymerus Sowerby, 1833, and the recently discovered P. caboverdensis Fernandes, Cruz and Van Syoc, 2010. These species have a restricted geographic distribution. Whereas P. elegans is found on the west coast of South America, from Mexico to Peru, P. polymerus is common in the intertidal region of more exposed parts of the west coast of North America (Barnes 1996).. P. polymerus overlaps with P. elegans at its southern distributional limit.. P.. caboverdensis (Fernandes et al. 2010) appears restricted to the tropical region o the Cape Verde Islands, whereas P. pollicipes is found along the northeastern Atlantic coast, from Dakar in Senegal to the north coast of Brittany in France (Barnes 1996, Quinteiro et al. 2007). The most remarkable characteristic presented by these barnacles is a muscular and. 17.

(34) Alejandra Perina Cedrón. exible stalk or peduncle. This peduncle shows a cuticle constituted by calcied scales disposed alternately and symmetrically (Barnes 1996) which protect the internal tissues and maintain the integrity of peduncle. The peduncle also contains feminine gonad and cement glands, which produce a proteinaceous adhesive secretion or cement (Barnes 1996). Above the peduncle, a capitulum protects the main organs (Molares Vila 1993).. P. pollicipes is a hermaphroditic organism with internal cross-fertilisation through copulation, breeding mainly from March to September on the rocky areas of Galician shores. This reproductive period includes two larval release periods, one at the end of the winter (March-April) and another in the summer (July-October), which is the most important in terms of adult population (Molares Vila 1993). Self-fertilisation does not occur because sperm and ovaries mature alternately (Cruz & Hawkins 1998). At a given moment, one specimen is either a functional male or a functional female (Klepal 1995). The life cycle of P. pollicipes comprises six naupliar stages, in which the larvae live freely in the water, followed by a cypris stage. Throughout larval development, each stage change is associated with metamorphosis, involving skin shedding and changes in size and shape (Molares et al. 1994). Cypris larvae, nally, attaches to an adult individual. The duration of larval life has been estimated to be one month (Molares Vila 1993). These barnacles represent an important economic resource.. They are considered to. be a delicacy mainly in the Spanish market. P. pollicipes is harvested by a specialised group of local shermen, named percebeiros. Generally, the captures of P. pollicipes are maintained at constant levels and have ranged in the last ten years, from 306,147 to 416,139 Kg per year, with a net value of 7,000,000. ¿/year. on the sh market. However,. there are large temporal oscillations depending on market demand and the state of the sea. Thus, in 2016, the price ranged between 0.50 and 207.00. ¿/kg.. The shery totaled. 3,499,371.93 kg of barnacle captured from 2007 to 2017, representing an economic prot of 91,446,127.63 euros (data obtained from pescadegalicia.gal, Xunta de Galicia).. State-of-the-art of genetic studies in. Pollicipes. species. Evolutionary and population genetic studies on barnacles are relatively scarce. However, the commercial importance of P. pollicipes has probably fuelled them.. For example,. Van Syoc (1995) studied the phylogenetic relationships of three species of the genus. Pollicipes, analysing the mitochondrial gene cytochrome c oxidase subunit 1 (COI). Quinteiro et al. (2007) investigated the inuence of Atlantic coastal currents and mesoscale hydrographic structures on the population genetic structure of P. pollicipes. Subsequently, Van Syoc et al. (2010) produced a phylogeny for the four species of edible goose barnacles in the genus Pollicipes, using concatenated DNA sequence data from mitochondrial genes, COI and ribosomal subunit 16s (16s), and nuclear gene histone subunit 3 (H3) fragment. These results supported the hypothesis that eastern Atlantic species, P. pollicipes and an, at that moment, undescribed P. caboverdensis, and P. elegans, were the most recently diverged of the four species in the genus.. 18. According to the results obtained.

(35) Introduction. from COI, Campo et al. (2010) concluded that the extreme climatic conditions during the Pleistocene had a strong inuence on the current distribution of the genetic variation of P. pollicipes. Fernandes et al. (2010) described P. caboverdensis sp. nov. from the Cape Verde Islands. Simultaneously, Quinteiro et al. (2011) characterised the Cape Verde population of stalked barnacles and studied the phylogenetic relationships among the species included in the Pollicipes genus (P. pollicipes, P.elegans, P. polymerus, and this new species from the Cape Verde Islands).. The phylogenetic trees were obtained. from the analyses of mitochondrial COI and nuclear ITS1, 5.8S rDNA, and ITS2. Recently, Seoane-Miraz et al. (2015) characterised 15 polymorphic microsatellite loci in P.. pollicipes. These microsatellite markers have not been used so far. Genomic and protein databases are largely unavailable for P. pollicipes.. To date. (March, 2017), the number of sequence entries available for P. pollicipes in the National Center for Biotechnology Information (NCBI) is limited. Meusemann et al. (2010) carried out a phylogenomic approach to resolve the arthropod tree of life using extensive sequence data from genome and expressed sequence tag (EST) projects. In that study 4,191 EST sequences were obtained from P. pollicipes. However, for example, there are no entries available for high-throughput DNA and RNA in the sequence read archive (SRA) database.. The only genome sequencing project is for microsatellite loci development. from P. elegans (PRJNA216107).. Palaemon species: Palaemon serratus The common littoral shrimp Palaemon serratus (Pennant, 1777) belongs to the order Decapoda and family Palaemonidae. Until recently, the genus Palaemon was composed of 41 species which were distributed throughout ve continents, inhabiting marine shallow waters, marshes, estuaries, and rivers (Cuesta et al. 2012). According to previous classication and González-Ortegón & Cuesta (2006) six species of Palaemon can be found in European waters, namely, P. adspersus Rathke, 1837, P. elegans Rathke, 1837,. P. longirostris H. Milne- Edwards, 1837, P. serratus (Pennant, 1777), P. xiphias Risso, 1816, and the recently introduced P. macrodactylus Rathbun, 1902. These species inhabit with four species of Palaemonetes. The systematic status of the genus has been revised only recently.. Palaemon currently consists of 86 species, taking into account. the recent synonymization of the genera Palaemonetes, Coutierella, and Exopalaemon (De Grave & Ashelby 2013) with Palaemon (Carvalho et al. 2017).. Palaemon serratus inhabits the intertidal and subtidal soft-sediment of estuaries and rocky bottoms covered with seagrass and algae (Figueras 1984). The world distribution covers the Atlantic Ocean, from Scotland and Denmark to Mauritania, and all the Mediterranean Sea, Marmara and the Black Sea (d'Acoz 1999). The life cycle includes four main stages: eggs, larvae, juveniles, and adults. The total length for adult individuals varies between 25 and 90 mm, with females generally larger than the males. The rostrum. 19.

(36) Alejandra Perina Cedrón. is toothed with 6-8 dorsal teeth, and 5 teeth on the ventral margin.. The body is not. coloured with red chromatophores on the margins of the abdominal segments. External morphological characters for identication of the species are provided in an illustrated key (González-Ortegón & Cuesta 2006). This decapod has a very crucial traditional activity as well. In the last ten years, the volume of catches was from 47,576 to 90,710 Kg per year, which signies an approximate worth of 2 million euros per year (data obtained from pescadegalicia.gal, Xunta de Galicia).. Moreover being an relevant economic resource, P. serratus is also used as a. biomarker (Oliveira et al. 2012, 2013, Silva et al. 2013).. State-of-the-art of genetic studies in. Palaemon species. The few genetic existing studies on P. serratus are focused on cytogenetics, DNA barcoding, biomonitoring studies and phylogenetic relationships. Cuesta et al. (2012) carried out a molecular study using 16S mtDNA to clarify the phylogeny of a large number representatives of Palaemon and Palaemonetes that inhabit in European waters. Results conrmed the paraphyly of these genera, as already pointed out by Murphy & Austin (2003, 2005) based on Australian representatives. Bilgin et al. (2014) suggested a potential history of isolation between the populations of P. serratus in the Atlantic and eastern Mediterranean, and the Aegean Sea.. Recently, Weiss et al. (2017) using COI. and 16S mtDNA and enolase nuclear gene, detected the ocurrence of two independent evolutionary lineages thereby corroborating dierences between Mediterranean and Atlantic individuals. An uncommon phylogeographic break, located west of the Strait of Gibraltar, was also revealed. González-Tizón et al. (2013) carried out a karyotype and chromosome banding study in P. serratus revealing the diploid chromosome number, 2n=56. Recently, Torrecilla et al. (2017) reported dierences between the karyotypes of. P. elegans and P. serratus, including their sex chromosome systems. The latter showed an absence of heteromorphic sex chromosomes. The chromosome evolution within the genus Palaemon might involve several fusion events giving rise to a reduction on the chromosome number in P. serratus.. Finally, Erraud et al. (2017) adjusted the comet. assay as a marker to assess the contamination impact on the sperm quality.. 20.

(37) Part II.. AIMS AND OBJECTIVES.

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(39) Aims and objectives. Within the broad aim of studying crustacean species, the particular objectives of this thesis are:. . To study the genomic organisation and evolution of the 5S ribosomal DNA (5S rDNA) multigene family in barnacles of the genus Pollicipes, with a focus on the gooseneck barnacle Pollicipes pollicipes.. . To generate an EST (Expressed Sequence Tag ) library in order to nd tissue-specic gene expression in Pollicipes pollicipes.. . To de novo assemble and annotate the transcriptome in dierent growth stages both in Pollicipes pollicipes and in Palaemon serratus.. . To isolate and characterise polymorphic microsatellite loci from Palaemon serratus.. . To infer the genetic variability and the population structure of Palaemon serratus along the European coast.. 23.

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(41) Part III.. RESEARCH ARTICLES.

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(43) 1. Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection Alejandra Perina, David Seoane, Ana M González-Tizón, Fernanda Rodríguez-Fariña, Andrés Martínez-Lage (2011) Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection. BMC Evolutionary Biology, 11:304. BMC EVOLUTIONARY BIOLOGY ISSN: 1471-2148 Bibliometrics 2011 JCR Science Edition Impact factor: 3.521 (3.285-2012) Evolutionary Biology: Q2 Genetics & Heredity: Q2. 27.

(44) Alejandra Perina Cedrón. Perina et al. BMC Evolutionary Biology 2011, 11:304 http://www.biomedcentral.com/1471-2148/11/304. RESEARCH ARTICLE. Open Access. Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection Alejandra Perina, David Seoane, Ana M González-Tizón, Fernanda Rodríguez-Fariña and Andrés Martínez-Lage*. Abstract Background: The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. Results: The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. Conclusions: These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.. Background In higher eukaryotes, nuclear ribosomal DNA (rDNA) genes are usually organized in two multigene families, each composed of hundreds to thousands of copies. A major family encodes for 28S, 5.8S, and 18S rRNA, and a minor family contains only 5S rRNA genes. The 5S rDNA consists of a conserved transcribing region of 120 bp (hereafter 5S) with a variable intergenic spacer usually referred to as the nontranscribed spacer (NTS). The 5S region is highly conserved in length and sequence even among unrelated species, although there is a high rate of heterogeneity within the NTS region * Correspondence: [email protected] Department of Cell and Molecular Biology. Evolutionary Biology Group (GIBE), Universidade da Coruña, A Fraga 10, E-15008 A Coruña, Spain. among closely related species. This variation of NTS is due to insertions, deletions, mini-repeats, base-substitutions and pseudogenes and has been used for evolutionary studies and as a source of species-specific or population specific markers [1-3]. The evolution of ribosomal gene families recently became controversial after it was analyzed in several taxa. The evolution of 5S rDNA units has classically been explained by the concerted evolution model, which suggests that molecular mechanisms such as gene conversion and unequal crossing-over play an important role in the homogenization of repeated units. These mechanisms maintain a high sequence similarity between copies and prevent the independent evolution of each member of a multigenic family [4]. However, several cases have been reported in which sequence. © 2011 Perina et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.. 28.

(45) Research articles. Perina et al. BMC Evolutionary Biology 2011, 11:304 http://www.biomedcentral.com/1471-2148/11/304. divergence levels between ribosomal genes or spacers seem to be much higher than would be expected under a strict concerted evolution scenario [5]. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA in distantly related taxa [3,5-9]. Under the birth-and-death evolution model, new variants are created by gene duplication and can remain as functional genes in the genome or become pseudogenes. In this way, transcribing region conservation could be explained by purifying selection, as suggested by Nei and Rooney [10]. The genus Pollicipes consists of four species: P. pollicipes, P. elegans, P. polymerus and P. caboverdensis. These stalked barnacles are sessile pedunculate cirripede occurring in dense aggregations exposed to heavy swell on rocky intertidal sites. Distribution of P. pollicipes (Gmelin 1789) is on the northeastern Atlantic coast, from Dakar in Senegal to the north coast of Brittany in France [11,12]. P. elegans (Lesson 1831) is found on the west coast of South America from Mexico to Peru, and P. polymerus (Sowerby 1833) is common in the intertidal region of more exposed parts of the west coast of North America [11]. P. polymerus overlaps P. elegans at its southern limit, and P. caboverdensis [13] occurs off the Cape Verde Islands. Studies focused on 5S rDNA have been performed on a small number of crustacean species and, different genomic organization types found. So, in some crustaceans 5S rDNA genes are linked to the major ribosomal genes [14,15] whereas in Artemia salina and Asellus aquaticus they are linked to the tandem repeats of the histone genes [16,17]. 5S rDNA genes are also linked to U1 small nuclear DNA (snDNA) in A. aquaticus [18], whereas they are unlinked to other multigene families in Proasellus coxalis [19]. In the present study, the nucleotide sequences, molecular organization and secondary structure of the 5S rDNA were investigated in three species of the genus Pollicipes (P. pollicipes, P. elegans, and P. polymerus) to know the evolution of these genes in this group of crustaceans.. Results Nucleotide sequence analysis of 5S rDNA. A total of 116 5S rDNA sequences from the genomes of P. pollicipes, P. elegans and P. polymerus was obtained experimentally to study the molecular evolution of 5S rDNA. PCR amplification generated different fragments with different size of 5S rDNA units, as among the different species as within the same species. A different electrophoretic pattern was obtained in each of the three species. Six bands of approximately 200 bp, 280 bp, 350 bp, 400 bp, 440 bp, and 600 bp were observed in P. pollicipes. Four bands were found in. Page 2 of 11. two other species: 350 bp, 550 bp, 700 bp and 900 bp in P. elegans, and 440 bp, 600 bp 870 bp and 1040 bp in P. polymerus. Sequence-similarity searches showed that all sequences matched other 5S rDNA, and BLASTN analysis of the NTS region did not detect any significant similarity with sequences from any other organisms. Primers were designed in such a way that only tandemly arranged 5S rDNA units could yield amplification products. Most of them corresponded to monomers formed by the last portion of the 5S (88 bp), the NTS, and the first portion of the contiguous 5S (32 pb). To maintain the similarity with other 5S rDNA sequences from the international nucleotide sequence databases, the 3’ end of the 5S was transferred to the 5’ end. In all species analyzed here, we obtained several dimer sequences and a trimer in P. elegans (906 bp), formed by two and three contiguous monomers respectively (see Additional File 1, Figure S1). Sequences were named with the letters a, b, and c when they were the first, second, or third unit of the array. The b and c sequences had a complete 5S + NTS units, respectively. From these sequences we designed a more specific primer (5S-Poll-R2) which differs by only 2 nt from 5SPoll-R. By using the 5S-Poll-R2 primer, we amplified 82 out of 116 sequences obtained in this work. Almost all dimers were homogeneous (both monomers were identical or almost identical), but in one clone of P. polymerus we detected a 5S rDNA unit of 1041 bp which consisted of two monomers of 605 and 436 bp. Therefore, we have discovered the linkage of two different units. The trimer was also composed of different monomers. Two of them consisted of the same repeat, but the other had a completely different nucleotide sequence in its NTS. In P. pollicipes only two dimer sequences (407 bp) were formed by divergent monomers, the shortest of which consisted of 123 bp (see Additional File 2, Table S1). These sequences were considered truncated pseudogenes because they lacked seven nucleotides in the 5S region and the spacer. These sequences were not included in the subsequent analyses. The 5S region showed a high GC content (59.2%) and was 120 bp long in all sequences except one, Py02Oly03 (119 bp). It displayed 27 polymorphic sites which were analysed excluding the primer-annealing regions in the a sequences whereas these regions were studied in the b and c sequences because they present a complete 5S + NTS units, respectively. Regarding NTS analyses, the initial alignment showed that the NTSs of genus Pollicipes were highly divergent, revealing the existence of different types (see Additional File 3, Figure S2). The TGI Clustering Tools showed seven types of NTS that we named using letters from A to G. The putative pseudogenes were not classified. A local BLAST allowed us to confirm the types and to. 29.

(46) Alejandra Perina Cedrón. Perina et al. BMC Evolutionary Biology 2011, 11:304 http://www.biomedcentral.com/1471-2148/11/304. classify two doubtful sequences into their corresponding types through the E-value. We also carried out a BLAST among sequences that belonged to different types. In most cases there was no similarity among them when we used megablast except in sequences of F and G types, in which case the E value was 10 -100 or less. F and G types have the same nucleotide sequence, with some fixed nucleotide substitutions and large insertions that increase the length of the sequence from 436-448 (F type) to 604-605 bp (G type). The NTS showed a high degree of variation produced by several insertion-deletion polymorphisms (indels) and nucleotide substitutions. The size of the NTS region was highly variable, ranging from 78 to 489 bp. In P. pollicipes, the lengths of the 5S rDNA units ranged between 605-609 (A type), 203-207 (B type), 284 (C type), 353 (E type) and 436 bp (F type); in P. elegans they were 198 to 351-357 bp (D and E types respectively); and in P. polymerus, they were 436-448 bp for the F type sequences and 604-605 bp for the G type ones. This length disparity in the F type of P. polymerus (436-448 bp) is due to an insertion of 12 bp in the 324 position. The NTS minimum average size was 83 bp (78-87bp). The number of polymorphic sites is given in Table 1. Sequence divergence was examined separately for the 5S and NTS regions. Values of nucleotide diversity for the different 5S rDNA types for each species were higher in the spacer region than in the 5S (Table 1), except for the D and G types and for sequences of P. pollicipes F type. Estimates of evolutionary mean distances within types were relatively small (0.004-0.039) (Table 2), emphasising the low value of the sequences of the A type with respect to those of E and F types. These three types are not as biased by sample size as the others. Phylogenetic analysis. Despite the length variation of the sequences, we were able to perform a blastn among them since there were some regions of similarity (see Additional File 3, Table S2). An MP tree (Figure 1) was calculated implementing the “using all sites” option in order to show the evolution of different variants. On the other hand, the networks created for each of the most frequent types of sequence (A, E and, F-G) (Figure 2) did not show a clear clustering by species. The network for A type sequences did not detect any association between different localities. Similarly, the network for the E type did not reveal any pattern of clustering for the two species that belong to this group of sequences. The E type included 47 sequences: 43 sequences of P. elegans and 4 sequences of P. pollicipes. The F type also included two. 30. Page 3 of 11. Table 1 5S rDNA polymorphism by species within types region A type. 5S rDNA. P. pollicipes. 120 pb. n 35. nts. s. h. π. 69. 34. 0.018. 5. 5. 0.004. 64. 31. 0.021. B type. 5S rDNA. P. pollicipes. 120 pb nts. C type. 5S rDNA. P. pollicipes. 120 pb nts. 3. 6 17 4. 2. 0.020. 2. 4. 2. 0.033 0.000. 3. 11. 2. 0.036. 1. 2. 0.006. 10. 2. 0.080. 23. 2. 0.054. 2 2. 0.033 0.069. D type. 5S rDNA. P. elegans. 120 pb nts. 0. 1. E type. 5S rDNA. 16. 3. 0.025. P. pollicipes. 120 pb. 2. 2. 0.008. 14. 3. 0.034. 66. 37. 0.037. 9. 9. 0.015. 57. 29. 0.048. 4. nts E type. 5S rDNA. P. elegans. 120 pb. 43. nts F type. 5S rDNA. P. pollicipes. 120 pb. 7. 4. 0.009. 2. 2. 0.011. F type. nts. 5. 3. 0.008. 5S rDNA. 35. 1. P. polymerus. 120 pb nts. 0.017. 5 3. 5 14. 0.009 0.020. G type. 5S rDNA. P. polymerus. 120 pb. 2. 2. 0.002. 1. 2. 0.006. 1. 2. 0.001. 4. 17. 3. nts. n: sample size; s: number of segregating (polymorphic) sites; h: number of haplotypes; π: nucleotide diversity. species: 17 sequences of P. polymerus and 4 sequences of P. pollicipes. Sequences of F and G types could be aligned because of their similarity and in this way a network linking both them could be built. The advantage of network methods is that give easy-to grasp representation of the considerable noise in the data. The resulting topology of the maximum likelihood trees using 5S + NTS sequences of the three main. Table 2 Estimates of average evolutionary divergence over sequence pairs within types Type. d. S.E.. A. 0.009. 0.003 0.011. B. 0.031. C. 0.039. 0.013. D. 0.023. 0.012. E F. 0.034 0.015. 0.010 0.005. G. 0.004. 0.004. d: distance within type; S.E: standard error.

(47) Research articles. Perina et al. BMC Evolutionary Biology 2011, 11:304 http://www.biomedcentral.com/1471-2148/11/304. Page 4 of 11. Figure 1 A maximun parsimony (MP) tree. Numbers on the tree correspond to nonparametric bootstrap supports (1000 replicates) and they are reported only for nodes with values ≥50. Outgroup species, Artemia salina, Asellus aquaticus and Lepas anatifera correspond to accession numbers: Y00128; X14815; AJ243001; FR832613; FR832614.. variants showed an among-species gene clustering pattern supported by high bootstraps (see Additional File 4, Figure S3). Phylogenetic analysis of the same data set with the NJ algorithm gave essentially the same topology as that obtained from the ML tree (data not shown).. Upstream and downstream elements. As the spacer regions contain some conserved elements that may be involved in 5S transcription, the 78 nt upstream from the transcription start site of 5S rDNA of Pollicipes species were arranged together. These. Figure 2 Phylogenetic networks of 5S rDNA constructed using the neighbor net algorithm. Pollicipes species are shown in different colors: P. pollicipes in blue, P. elegans in red and P. polymerus in green; (a) Network of A type: sequences of Galician localities are displayed in dark blue and Morocco in light blue; (b) Network of E type; (c) Network of F and G types. 31.

(48) Alejandra Perina Cedrón. Perina et al. BMC Evolutionary Biology 2011, 11:304 http://www.biomedcentral.com/1471-2148/11/304. regions formed the terminal region of each NTS. A search of upstream sequences revealed a conserved AT rich region at about -25 nt from the 5S rDNA transcription start site (see Additional File 4, Figure S4) in three groups of sequences: B, C and F. Another conserved region (CGGCCACCGGC) was identified at positions -24 to -14 nt from the 5S rDNA transcription start site. All sequence types except B, C and F displayed this region. These were the same groups in which the ATrich region was found. Finally, a TTC stretch located at -7 nt was also identified. Another clear disparity between the 5S rDNA types was the number of thymidine residues located in the Trich region, five in the A, B, C, F and G types and four in the D and E types. These repeated sequences corresponded to transcriptional terminators [20,21]. Internal regulatory regions. The 5S internal control regions (ICRs) were compared to those of Drosophila melanogaster described by Sharp and Garcia [22]. As some ICRs coincided with the primer annealing regions, only sequences classified as b or c were included in the Pollicipes ICRs analysis, in addition to other sequences from other crustaceans available from EMBL/GenBank/DDBJ: Parhyale hawaiensis [FN434137]; Proasellus coxalis [Y14281]; Asellus aquaticus [AJ243001] Calanus finmarchicus [X06056] and Artemia sp. [X14815; V00086; M16191; Y00128; X14816; X14817]. The consensus internal regulatory regions of Pollicipes and the other crustaceans are shown in Figure 3. The four ICRs involved in the transcription of 5S rDNA [22] were identified in the 5S sequences. Thus in Pollicipes consensus positions 3-18, 37-44, 48-61, and 78-98 were very similar to their orthologs in D. melanogaster (16/16; 8/8; 13/14 and 18/21 matches respectively). We also identified the sequence elements described in Xenopus laevis [23] that are functionally equivalent to the ICRs: positions 50-61 (box A), 67-72 (intermediate element), and 80-90 (box C) which. Page 5 of 11. also displayed a high degree of similarity (9/12; 5/6; 10/ 11 matches respectively). 5S predicted secondary structures. Sequences included in the secondary structure prediction were those classified as b or c. All sequences were folded (see Additional File 4, Figure S5). We also obtained the consensus secondary structures, two putative types of structures for Pollicipes and another for other crustaceans used in this study (Figure 4). In agreement with Delihas and Andersen [24] the 5’ ends were purines whereas the 3’ ends were pirimidines. Lengths of helix I were 7 nt (type I) and 9 nt (type II and Crustacea). Helix II has a length of eight nt and a looped-out residue at position 63 that is a C (characteristic of metazoans); the two base-pairs that follow this residue are C-G in the general structure, consistent with metazoan and plant 5S rRNA. The two positions 49 and 50 are also flanked by G-C base-pairs on both sides. In the loop bound by helix III there are twelve nucleotides, and the purine at position 37 is a G in metazoans. The alternative structure can be observed in helix IV (as shown in [24]). This helical model includes a C-A mispairing and an increased content of non-canonical basepairs, G-U. The C - loop is formed by 12 bp, the hairpin E - loop displays the AGUA motif and the terminal loop contains the conserved G-U-G-A motif.. Discussion Most of the sequences of Pollicipes analyzed in the present study, except the pseudogenes found in P. pollicipes, might be functional genes because they possess the necessary elements for gene expression, viz the presence of control elements in the NTS, the poly-T tail at the 3’ end of the transcribing region, and conserved ICRs that function as internal promoters of the gene. Until recently it was thought that the NTS had no function, but studies of deletion mutants have shown that upstream control elements are required for the. Figure 3 Schematic comparison of the control elements involved in the transcription of the 5S rDNA. Sequences up and down represent the internal control regions (ICRs) and sequence elements of D. melanogaster (D. m) and X. laevis (X. l), while sequences in the middle represent the consensus crustacea and G. Pollicipes orthologues. Similarities respect to the consensus sequences are denoted by asterisk (*).. 32.

(49) Research articles. Perina et al. BMC Evolutionary Biology 2011, 11:304 http://www.biomedcentral.com/1471-2148/11/304. Page 6 of 11. Figure 4 Predicted consensus secondary structures of the 5S rRNA. Helices are named with roman numbers, and loops with letters, following Barciszenwska et al., (2000). Red indicates one type of base pair, ochre two types of base pairs, and pale colors indicate pairs that cannot be formed by all sequences. (a) Type I Pollicipes. (b) Type II Pollicipes (c) Consensus of crustacea.. expression of 5S rDNA genes [4]. The NTS minimun average size was 83 bp. This size agrees with Martins and Galetti [25], who proposed that an NTS of 60-80 bp can represent the minimum size for the organization of this rDNA in the genome. Although in general 5S displays a high degree of conservation among species and variants, we found some nucleotide substitutions in Pollicipes spp. In the comparisons with D. melanogaster, the ICR I and ICR II regions were the most conserved. Furthermore, the proportion of conserved nucleotide positions in Pollicipes spp. is higher than those obtained for razor clams [8] and mussels [26] which is not surprising since Pollicipes barnacles and D. melanogaster belong to the Arthropoda. The degree of conservation of internal control elements in the 27 crustacean sequences was 10/16 matches within ICR I, 6/8 matches within ICR II, 8/14 matches within ICR III, and 19/21 matches within ICR IV. Many nucleotide substitutions in ICR III were unique for Artemia spp. The highest degree of conservation was in Pollicipes spp. (11/16, 6/8, 12/14 and 21/21 matches respectively) (see Additional File 4, Figure S6). The poly-T tail transcription termination signal of 5S rDNA has been studied in several organisms and seems to be quite conserved. It is part of a transcribed 15-16 nucleotide segment specific to the 5S rRNA precursor. The 135-nucleotide primary transcript was identified in D. melanogaster by in vitro. transcription and 3’-processed to yield the approximately 126-nucleotide pre’ 5S species and the 120nucleotide mature-size 5S rRNA [22]. The analysis of upstream sequences of 5S from genus Pollicipes revealed a putative regulatory region, a TATA - like control element, located around positions -30 to -25 as observed in several fish species [21] and in razor clams [8]. This region, together with RNA pol II-like transcriptional factors, may be involved in RNA pol III transcription [27]. The high degree of conservation of TATA-like sequence positions in all organisms examined to date (e.g.. elasmobranch fishes, [21]) suggests a shared structural pattern. However, in our case, many sequences did not show the TATA-like motif. The fact that certain conserved regions are associated with a specific variant could be related to a differential expression throughout development as seen in Xenopus [28]. Furthermore, we found a TTC sequence, as previously observed in the silkworm Bombyx mori [29]. The predicted secondary structure of all 5S sequences analysed in this work consists of five helices, two hairpin loops, two internal loops and a hinge region. This structure is consistent with the general eukaryotic 5S rRNA structure [24,30] and with that obtained for A. salina [31]. According to Smirnov [32], helix I is potentially important for RNA-protein recognition and helix III seems to be associated with the integration of 5S rRNA. 33.