MOLECULAR TAXONOMY AND PHYLOGENOMICS OF CERAMIALES (RHODOPHYTA) HIGHLIGHT CHALLENGES AND ADVANCES IN UNDERSTANDING THE DIVERSITY AND SYSTEMATICS OF ALGAL TURFS

Algal turfs are ecosystem engineers receiving growing attention in relation to their expansion on temperate reefs as a consequence of global change. However, their species diversity and taxonomy are still poorly understood. Turfs are composed of densely entangled small seaweeds, in which the red algal order Ceramiales is commonly a major component. The advent of classical molecular taxonomy and -omic approaches has revolutionized investigations of seaweed diversity and systematics. These approaches have been applied to the study of the Ceramiales, facilitating the discovery of new species and resolving classification issues. A molecular diversity survey using the rbcL gene and involving the study of ca. 400 specimens of turf-forming rhodomelacean species in Macaronesia detected impressive amounts of newly discovered diversity. A total of 67 species were identified, of which half corresponded to undescribed species presumably endemic to this bioregion. Likewise, the application of molecular species delimitation methods to widely distributed records of Polysiphonia scopulorum (>150 samples collected in Australia, South Africa, southern Europe and Macaronesia) revealed that it is a complex in which 13 species were resolved. These 13 cryptic species range from endemics with narrow known distributions to a species found in all studied regions. At taxonomic levels above species, the use of plastid phylogenomics has produced well-resolved phylogenies that have been applied to the resolution of classification issues in the Ceramiales. These studies illustrate how classical and newer evolving molecular techniques facilitate the understanding of seaweed diversity and systematics, and provide new insights into the complexity of turf assemblages

Phylogenetic analyses reveal a new old introduced red algal species in Europe

The detection of invasive species is commonly challenging in marine environments, often related with the insufficient taxonomic knowledge of the local diversity. Using DNA sequences in diversity surveys and their phylogenetic analysis can assist us to clarify the native or introduced origin of some species. We applied this approach to study the red algae Aphanocladia stichidiosa. It was originally described in the Mediterranean (1955), later recorded in Portugal (1960), the Canary Islands (1986), the northwestern Spain (1990) and the Azores (2012). Unexpectedly, our diversity surveys in Australia revealed its presence in Victoria in 2015, finding that European and Australian specimens had identical DNA sequences (rbcL gene) and suggesting that it is introduced in one of the regions. Phylogenetic analysis resolved A. stichidiosa in a highly supported clade with species restricted to the southern hemisphere, mainly from Australia. Accordingly, we conclude that A. stichidiosa is native from Australia, although it has never been recorded there before, and that it represents a relatively old introduction in Europe. In both native and introduced regions, it is frequent in algal turfs, a type of assemblage composed by a carpet of small, morphologically similar species whose identification is often difficult. The type of growth of this species explains the absence of previous records in Australia, where the number of taxonomists studying this assemblage is lower than in Europe. Sequential reports of A. stichidiosa in different European regions suggest that it is expanding its distribution, as well as its abundance is increasing. The invasive character of this species needs to be determined yet, and its small size should not lead to underestimate its potential harmfulness. In the current context, in which kelp forests are globally declining and algal turfs are expanding, this species might play a relevant role in the transformation of the European marine ecosystems

Estudio ficológico de los hábitats rocoso-arenosos del Atlántico peninsular

[Resumen] Esta tesis mejora el conocimiento de las especies y comunidades de de algas de hábitats rocoso-arenosos del Atlántico Peninsular, abordando diferentes perspectivas: florística, taxonómica, ecológica y conservacionista. El estudio ha sido principalmente llevado a cabo analizando 2525 muestras de diferentes tipos de comunidades algales recogidas en el intermareal e infralitoral superior de 138 localidades, desde 2002 hasta 2011. En la primera parte de esta tesis se presenta un catálogo de 257 especies. Además se incluyen descripciones morfológicas y, en algunos casos, datos moleculares de las 35 especies más características de hábitats rocoso-arenosos, estudiando 13 de ellas en detalle que en su mayoría pertenecen a la familia Rhodomelaceae. Como resultado, se describen dos nuevas especies y las estructuras sexuales de otras cuatro, se llevan a cabo varias propuestas taxonómicas y se proporcionan nuevos datos corológicos para las costas Ibéricas. La segunda parte se centra en el estudio de las comunidades de algas de hábitats rocoso-arenosos: i) se proporciona la descripición de la vegetación de una localidad gallega, ii) se estudia comparativamente la estructura, variabilidad espacial y diversidad de las comunidades de hábitats rocoso-arenosos y hábitats rocosos, iii) se lleva a cabo la monitorización de la estructura, dinámica temporal y fenología de Erythroglossum lusitanicum a lo largo de un año, iv) se analizan las comunidades cespitosas de hábitats rocoso arenosos, describiendo 29 tipos de céspedes algales. Los datos generados en este estudio fueron analizados desde una perspectiva conservacionista (distribución, biodiversidad, endemismos, especies no nativas, amenazas, etc.) en la última parte de esta tesis, y se proponen 20 localidades de interés para la conservación de las comunidades de hábitats rocoso-arenosos.[Abstract] This thesis improves the knowledge on algal assemblages and their species from sandcovered rocks along the Atlantic Iberian Peninsula, addressing several perspectives: floristic, taxonomic, ecological and conservational. The study was mainly performed through the analysis of 2525 samples of different types of algal assemblages collected in the intertidal and upper subtidal of 138 sites, from 2002 to 2011. A catalogue of 257 species was provided in the first part of this thesis. Morphological descriptions and, in some cases, molecular data of the 35 most common species from sand-covered rocks were included, studying in detail 13 of them which mostly belong to the family Rhodomelaceae. As a result, two new species were described, as well as the sexual structures of four ones, several taxonomic proposals were performed for other taxa, and new chorollogical data were added to the Iberian coast. The second part was focused on the study of the algal assemblages from sand-covered rocks: i) providing a description of the vegetation from a Galician locality, ii) comparing the structure, spatial variability and diversity of the assemblages from sand-covered rocks and rocky shores, iii) monitoring the structure, temporal dynamics and phenology of Erythroglossum lusitanicum through a one-year study, and iv) analzying the turf assemblages from sand-covered rocks, with the description of 29 types of turfs. In the last part of this thesis, the data generated in this study were analyzed from a conservational perspective (distribution, biodiversity, endemisms, nonnative species, threats, etc.) and 20 sites of interest for the conservation of assemblages from sand-covered rocks were proposed.[Resumo] Esta tese mellora o coñecemento das especies e comunidades de de algas de hábitats rochoso-areosos do Atlántico Peninsular, abordando diferentes perspectivas: florística, taxonómica, ecolóxica e conservacionista. O estudo foi realizado principalmente analizando 2525 mostras de diferentes tipos de comunidades algais recollidas no intermareal e infralitoral superior de 138 localidades, dende 2002 ata 2011. Na primera parte desta tese preséntase un catálogo de 257 especies. Ademáis inclúense descripcións morfolóxicas e, nalgúns casos, datos moleculares das 35 especies máis características dos hábitats rochoso-areosos, estudando 13 delas en detalle que na sua maioría pertencen á familia Rhodomelacea. Como resultado, se describen duas especies novas e as estructuras sexuais doutras catro, se levan a cabo varias propostas taxonómicas e se proporcionan novos datos corolóxicos para as costas Ibéricas. A segunda parte céntrase no estudo das comunidades de algas de hábitats rochosoareosos: i) se proporciona a descripición da vexetación dunha localidade galega, ii) se estuda comparativamente a estructura, variabilidade espacial e diversidade das comunidades de hábitats rochoso-areosos e hábitats rochosos, iii) se leva a cabo a monitorización da estructura, dinámica temporal e fenoloxía de Erythroglossum lusitanicum ó longo dun ano, iv) se analizan as comunidades cespitosas de hábitats rochoso-areosos, describindo 29 tipos de céspedes algais. Os datos xerados neste estudo foron analizados dende unha perspectiva conservacionista (distribución, biodiversidade, endemismos, especies non nativas, ameazas, etc.) na última parte desta tesis, e se propoñen 20 localidades de interés para a conservación das comunidades de hábitats rochoso-areosos

Biodiversity of epiphytic macroalgae (Chlorophyta, Ochrophyta, and Rhodophyta) on leaves of Zostera marina in the northwestern Iberian Peninsula

[Abstract] The composition, abundance, and distribution of epiphytic macroalgae living in meadows of Zostera marina L. in the northwestern Iberian Peninsula are here analyzed. We identified 63 species: 40 red algae, 16 brown algae, and 7 green algae. Most of them are classified as filamentous or filiform functional forms, while Pneophyllum fragile Kütz. was the only encrusting species. In general, the surface covered by epiphytes on the leaves of Zostera marina was low and a 43% of species were only found in juvenile stages. Regarding their frequency, 10 species were collected in the majority of the areas, while others were rare. Most species were found both epiphytic and in other substrata of the meadows, but 9 were exclusively epiphytic. We detected 9 introduced species.[Resumen] Se han analizado la composición, la abundancia y la distribución de macroalgas epifitas que viven en praderas de Zostera marina L. del noroeste de la península ibérica. Se han identificado 63 especies: 40 algas rojas, 16 pardas y 7 verdes. La mayoría pertenecen a los grupos funcionales filamentosos o filiformes, excepto Pneophyllum fragile Kütz., la única especie costrosa. En general, la cobertura de epifitos en las hojas de Zostera marina fue baja y un 43% de las especies solo se encontraron como estadios juveniles. En cuanto a su frecuencia, 10 de ellas se recolectaron en la mayoría de las áreas, mientras que el resto fueron más raras. La mayor parte de las especies se encontraron tanto epifitas como en otros substratos de las praderas, si bien 9 resultaron exclusivamente epifitas. Detectamos 9 especies introducidas.Xunta de Galicia; GPC2015/02

PTEROSIPHONIA PENNATA VERSUS P. PINNULATA (CERAMIALES, RHODOPHYTA) EN EL NOROESTE DE LA PENÍNSULA IBÉRICA

In the floristic studies that we are carrying out in roky-sandy marine environments, we have detected populations with individuals having intermediate characters between Pterosiphonia pennata and P. pinnulata. As the second species have not previously recorded in Galicia, we have carried out a comparative study between P. pennata and P. pinnulata. In order to value what species are present in Galicia, distinctive diagnostic characters for both species were considered. Individuals collected in populations of nine Galician localities and herbarium specimens from Atlantic and Cantabrian coasts of the Iberian Peninsula were studied. Regarding, the number of periaxial cells, the axe diameter, as well as the diameter/length ratio of segments in postrate axes and erect axes, the Galician material shows intermedial between P. pennata and P. pinnulata; whereas the erect axe segment in wich the first branch is originated, the coalesced between erect axe and branch as well as the development of tetrasporangia in spread lateral branches show resemblance to P. pinnulata. Adding that in literature a large variability and taxonomic confusion for the diagnostic character values of both species were detected, we suggest the necessity of carrying out a widely study to check the existence of a single species, which will cover the variability detected for P. pennata and P. pinnulata in the present study. In the meantime, we identify the Galician material asEn los estudios florísticos que estamos realizando en ambientes marinos rocoso-arenosos de Galicia hemos detectado poblaciones con individuos que muestran caracteres intermedios entre Pterosiphonia pennata y P. pinnulata; como la segunda especie no ha sido citada en Galicia hemos realizado un estudio comparado entre ambas especies. Se consideraron los caracteres diagnóstico discriminantes en individuos recolectados en poblaciones de nueve localidades de Galicia con el fin de determinar cuál de las dos especies está presente en nuestras costas, confirmándose que el material gallego presenta valores intermedios entre P. pennata y P. pinnulata. Teniendo en cuenta que en la bibliografía de otras regiones geográficas existe gran variación y confusión taxonómica en los valores de los caracteres diagnóstico aportados por diversos autores para las especies objeto de estudio, planteamos la necesidad de realizar un estudio más amplio con objeto de verificar la existencia de una sola especie, la cual incluiría la variabilidad detectada para P. pennata y P. pinnulata en este trabajo. Mientras tanto, proponemos considerar el material gallego como P. pennata en sentido amplio

DIATOMS AND DINOFLAGELLATES DIVERSITY INHABITING A COASTAL UPWELLING SYSTEM: A METABARCODING APPROACH

Small eukaryotic plankton has been traditionally characterized using conventional microscopy techniques. Current advances in sequencing technologies allow the cost-effective study the diversity within microbial plankton based on DNA sequences. This technique has been implemented in the last 15 years for investigating prokaryotic diversity, but its application to unravel the diversity and ecology of eukaryotic organisms is still incipient. In this study we analyze the diversity of the diatoms and dinoflagellates in the marine communities using rDNA sequencing techniques, as well as the taxonomic resolution provided by the V4 region of the 18S rRNA gene. Twenty monthly 6L seawater samples for DNA metabarcoding were collected and filtered through 3 μm polycarbonate filter in a station off the Ría de A Coruña (NW Iberian Peninsula). DNA was extracted, the V4 region of the 18S rRNA was PCR amplified and subsequently sequenced using the High Throughput Sequencing (HTS) platform Illumina. Amplicon sequence variants (ASVs) were differentiated using DADA2 implemented in R. Sequences were aligned against PR2 v4.12.0 and SILVA 132 18S rRNA databases as references, as well as studied using BLAST and phylogenetic trees. Sequence-based taxonomic approach found 128 and 416 metabarcodes corresponding to diatoms and dinoflagellates, respectively. Sequences had limited resolution at species or even genus level, and overall the taxonomic resolution of diatoms was substantially higher than those of dinoflagellates

Nano- and microplankton diversity inhabiting a coastal upwelling system: a metabarcoding approach

Small eukaryotic plankton has been traditionally characterized using conventional microscopy techniques. Current advances in sequencing technologies allow the costeffective study the diversity within microbial plankton based on DNA sequences. This technique has been implemented in the last 15 years for investigating prokaryotic diversity, but its application to unravel the diversity and distribution of eukaryotic organisms is still incipient. Alleged advantages of this approach include the ability of detecting the smaller fraction of the community that trend to be overlooked in microscopy studies, as well as improving the resolution of taxonomic identification for groups whose morphological study is challenging due the paucity of morphological characters. In this study we characterize the diversity of the nano- and microplankton marine communities using microscopy and rDNA sequencing techniques. The aim of this work is to compare results of both techniques, assessing whether rDNA data can provide new insights into the study of the diversity of eukaryotic planktonic communities. Twenty monthly samples were collected in a station off the Ría de A Coruña (NW Iberian Peninsula). Planktonic samples for DNA studies were collected by filtering seawater samples through a 3 μm pore size polycarbonate filter. DNA was extracted, the V4 region of the 18S rRNA was PCR amplified and subsequently sequenced using the High Throughput Sequencing (HTS) platform Illumina. Amplicon sequence variants (ASVs) were differentiated using DADA2 implemented in R. Sequences were aligned against PR2 v4.12.0 and SILVA 132 18S rRNA databases as references. Microscopic identification of plankton taxa (phytoplankton and protozoa) was made using the Uthermöhl technique. In total 1182 ASVs were identified based on rDNA data, which contrasts with only 65 species identified by microscopy. Morphological identifications of eukaryotic plankton was restricted mainly to diatoms, dinoflagellates and ciliates, while rDNA data allowed the detection of additional taxonomic groups (i.e. protist). Some of them were very abundant, such as the algae Cryptophyceae and Mamiellophyceae or the heterotrophs Ascomycota or parasitic Stramenopiles. Therefore, our study further evidenced that DNA metabarcoding is a valuable tool to uncover part of the great diversity within microbial plankton, improving the detection of small eukaryotes

Biodiversidad de macroalgas epifitas (Chlorophyta, Ochrophyta y Rhodophyta) en hojas de Zostera marina en el noroeste de la península ibérica

The composition, abundance, and distribution of epiphytic macroalgae living in meadows of Zostera marina L. in the northwestern Iberian Peninsula are here analyzed. We identified 63 species: 40 red algae, 16 brown algae, and 7 green algae. Most of them are classified as filamentous or filiform functional forms, while Pneophyllum fragile Kütz. was the only encrusting species. In general, the surface covered by epiphytes on the leaves of Zostera marina was low and a 43% of species were only found in juvenile stages. Regarding their frequency, 10 species were collected in the majority of the areas, while others were rare. Most species were found both epiphytic and in other substrata of the meadows, but 9 were exclusively epiphytic. We detected 9 introduced species.Se han analizado la composición, la abundancia y la distribución de macroalgas epifitas que viven en praderas de Zostera marina L. del noroeste de la península ibérica. Se han identificado 63 especies: 40 algas rojas, 16 pardas y 7 verdes. La mayoría pertenecen a los grupos funcionales filamentosos o filiformes, excepto Pneophyllum fragile Kütz., la única especie costrosa. En general, la cobertura de epifitos en las hojas de Zostera marina fue baja y un 43% de las especies solo se encontraron como estadios juveniles. En cuanto a su frecuencia, 10 de ellas se recolectaron en la mayoría de las áreas, mientras que el resto fueron más raras. La mayor parte de las especies se encontraron tanto epifitas como en otros substratos de las praderas, si bien 9 resultaron exclusivamente epifitas. Detectamos 9 especies introducidas

"Alsidium oliveiranum" sp. nov. (Rhodomelaceae, Rhodophyta), an overlooked species from the southwestern Atlantic based on morphology and DNA sequence data

[Abstract] The exploration of seaweed diversity in poorly studied habitats has often led to the discovery of new species. Sand-covered rocks are an example, as they received less attention than sand-free rocky intertidal habitats during seaweed diversity surveys in Brazil. In sand-covered rocks from Espírito Santo and Rio de Janeiro we found an alga whose morphology was unique among rhodomelacean species previously reported in Brazil. With the aim to clarify the taxonomic identity of this species we studied its morphology, as well as its phylogenetic relationships. Molecular analyses resolved this species in the genus Alsidium (tribe Alsidieae) and differed from sequenced congeners with divergences ≥2.5 and 4.2% in the rbcL and cox1 genes, respectively. Morphological characters were in agreement with the genus Alsidium, and differed from other species currently recognized in the genus. The species consisted of a basal crust and scarcely branched erect axes with seven pericentral cells covered by a continuous layer of cortical cells. Reproductive structures were formed on clusters of short determinate branches. Therefore, the new species A. oliveiranum is proposed based on morphological and molecular evidence. Our findings contribute to better understand the diversity of the tribe Alsidieae, which is particularly diverse in the Americas.Xunta de Galicia; GPC2015/025Brasil. Conselho Nacional de Desenvolvimento Científico e Tecnológico; 304899/2017–8Fundação de Amparo à Pesquisa do Estado de São Paulo; FAPESP 2016/50370-