Irati Miguel

Morphological and genetic characterization of benthic dinoflagellates of the genera Coolia, Ostreopsis and Prorocentrum from the south-eastern Bay of Biscay

Benthic dinoflagellates of the genera Coolia, Ostreopsis and Prorocentrum isolated from coastal waters of the south-eastern Bay of Biscay were identified morphologically by means of light microscopy (LM) including epifluorescence microscopy, and scanning electron microscopy (SEM). To identify the strains to species level, molecular phylogenetic analyses using the nuclear large subunit rDNA (LSU) were performed for 16 strains of the three genera. These morphological and phylogenetic analyses revealed the presence of the following species: Coolia canariensis S. Fraga, Coolia monotis Meunier, Ostreopsis cf. siamensis Schmidt, Prorocentrum emarginatun Fukuyo, P. lima (Ehrenberg) Dodge, P. rhathymum Loeblich III, Sherley & Schmidt, and two as yet unidentified species, which in the phylogenetic tree were grouped with different strains of Prorocentrum emarginatun and P. fukuyoi Murray et Nagahama from GenBank. A strain from Minorca (Balearic Islands, western Mediterranean Sea) analysed in this study for comparative purposes and fitting morphologically into the P. emarginatum/P. fukuyoi group also appeared in this cluster, which seems to include morphologically cryptic or semicryptic species. The most common taxa were Coolia monotis, Ostreopsis cf. siamensis and Prorocentrum lima, which appeared at most sampling sites. Only the strains corresponding to Ostreopsis cf. siamensis and Prorocentrum lima were toxic to Artemia franciscana.

Gene flow within the M evolutionary lineage of Apis mellifera: role of the Pyrenees, isolation by distance and post-glacial re-colonization routes in the western Europe

We present a population genetic study focused on the two subspecies of the M evolutionary lineage, A. m. mellifera and A. m. iberiensis. Nuclear and mtDNA variation was analysed in 27 bee populations from the Iberian Peninsula, France and Belgium. Microsatellite data provides compelling evidence of a barrier to neutral gene flow at the Pyrenees. In addition, they suggest isolation by distance between populations of the M lineage. Mitochondrial data support the hypothesis that the Iberian Peninsula served as glacial refugia for the honeybees of western Europe. They show two paths of post-glacial re-colonization in the extremes of the Pyrenees and suggest that the western path was more significant in the post-glacial re-colonization process. Thus, we report here on three main factors for mellifera and iberiensis subspecies differentiation: the Pyrenean barrier, isolation by distance and the post-glacial re-colonization process.ZusammenfassungDie Unterarten der Honigbiene (Apis mellifera) werden in die 5 evolutionäre Linien A (African), C (northern Mediterranean), M (western Europe), O (Oriental) and Y (Yemenitica) gruppiert. Die in dieser Studie untersuchte evolutionäre Linie M enthält zwei Unterarten: A. m. mellifera wird von Frankreich bis zu den Bergen des Ural gefunden und A. m. iberiensis ist auf der iberischen Halbinsel verbreitet. Obwohl allgemein angenommen wird, dass die Pyrenäen ein bedeutendes Hindernis für den Genfluss zwischen A. m. mellifera und A. m. iberiensis darstellt, wurde ein solcher Barriereeffekt bislang nicht nachgewiesen. Die Existenz genetischer Gefälle vom Süden der Iberischen Halbinsel bis zum nördlichen Europa und eine ungewisse taxonomische Zuordnung einiger Populationen in den Pyrenäen tragen zu der Unsicherheit bezüglich der Rolle der Pyrenäen als genetische Barriere bei. Andererseits wird seit längerem angenommen, dass die Iberische Halbinsel während der Eiszeit als Refugium für die westliche Honigbiene diente (Ruttner, 1952, 1988) und eine nacheiszeitliche Wiederbesiedlung von Nordeuropa wird von mehreren Autoren unterstützt (Garnery et al., 1998a,b; Franck et al., 1998, 2000b). Gegenstand dieser Untersuchung war es, einen potentiell isolierenden Effekt der Pyrenäen nachzuweisen, neue Daten zu dem Differenzierungsprozess der zwei Unterarten beizutragen und den Ablauf der Wiederbesiedlung zu untersuchen. Wir untersuchten 1398 Völker aus 27 Populationen der Iberischen Halbinsel sowie aus Frankreich und Belgien auf Variation an 10 Mikrosatellitenloci und der COI-COII intergenischen Region der mtDNA. Wir verwendeten verschiedene Arten statistischer Analysen wie die DA genetische Distanzmatrix, neighbor-joining trees, Korrelationen erster Ordnung und partielle Korrelationen, AMOVA, Analyse räumlicher Autokorrelationen und COCOPAN für Mikrosatellitendaten. Die Ergebnisse zeigten eine Isolation zwischen den verschiedenen Populationen der M Linie durch die Entfernung auf und lieferten sehr deutliche Hinweise auf eine Barriere für den neutralen Genfluß bei den Pyrenäen. Die Verteilung der mtDNA Haplotypen bestätigte das Iberische Refugium der Westeuropäischen Honigbiene in der Eiszeit. Wir konnten zwei verschiedene Wege der nacheiszeitlichen Wiederbesiedlung von der Iberischen Halbinsel aus an den beiden Enden der Pyrenäen ableiten. Es gab deutliche Unterschiede in der Verteilung der Mitotypen zwischen den westlichen und östlichen Enden der Pyrenäen, diese legten nahe, dass der westliche Weg für den nacheiszeitlichen Widerbesiedlungsprozess wichtiger war. Nach der in dieser Untersuchung beobachteten hohen Variabilität der M Mitotypen südlich der Pyrenäen könnten diese eine nützliche genetische Ressource für die Konservation der Westeuropäischen Honigbienen darstellen. Der nacheiszeitliche Wiederbesiedlungsverlauf, die Isolation durch die Entfernung und die von den Pyrenäen gebildete Verbreitungsbarriere sind Einflüsse, die zu der Ausbildung der Unterarten A. m. mellifera und A. m. iberiensis beigetragen haben.

Both geometric morphometric and microsatellite data consistently support the differentiation of the Apis mellifera M evolutionary branch

Traditional morphometrics, allozymes, and mitochondrial data have supported a close relationship between the M branch subspecies A. m. iberiensis and the North African subspecies (A branch). However, studies using nuclear DNA markers have revealed a clear distinction between the latter and the two European M branch subspecies. In help resolve this paradox, we analyzed 663 colonies from six European and African subspecies. A geometric morphometrics approach was applied to the analysis of wing shape, and the results were compared with data of six microsatellite loci. Both data sets were found to be highly consistent and corroborated a marked divergence of West European subspecies from North African ones. This supports the hypothesis that the presence of the African lineage mitotype in Iberian honey bee populations is likely the consequence of secondary introductions, with a minimal African influence within the current Iberian genetic background. Wing geometric morphometrics appears more appropriate than mitochondrial DNA analysis or traditional morphometrics in the screening and identification of the Africanization process.

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo‐nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo‐nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo‐nitzschia species containing only chlorophyll c2 and c3.

DIVERSITY OF PSEUDO-NITZSCHIA IN THE SOUTHEASTERN BAY OF BISCAY

Ten species of the genus Pseudo-nitzschia were identified in the Nervion River estuary from preserved net tow samples and 31 strains isolated from the estuary. Species identification was performed by means of ultrastructural analysis of valve ornamentation and genetic analysis of selected strains of the P. delicatissima complex. Identified species include: P. australis, P. fraudulenta, P. pungens and P. subpacifica from the P. seriata complex, and P. arenysensis, P. galaxiae, P. multistriata, P. pseudodelicatissima and two uncertain P. pseudodelicatissima-like genotypes from the delicatissima complex. Most clonal strains corresponded to P. pseudodelicatissima-like taxa, P. fraudulenta and P. galaxiae. We were unable to identify the strains of the P. delicatissima-like complex solely on the basis of morphology. A comparison of sequences of the rDNA ribosomal Internal Transcribed Spacers (ITS), including the regions ITS1, 5.8S and ITS2, of four strains of the P. delicatissima complex plus 23 sequences of taxonomically related strains available in GenBank, revealed the presence of P. arenysensis among strains of the P. delicatissima-morphologies and three different ITS types among the three P. pseudodelicatissima-like morphologies analysed. One corresponded to P. pseudodelicatissima sensu stricto (strain Ner-D5) whereas the other two (Ner-D6 and Ner-D8) constituted genetically distinct entities, which appeared in the phylogenetic tree as sister taxa to either P. cuspidata, the former, or to P. calliantha and P. mannii, the latter. The genetic differences among these three strains of P. pseudodelicatissima-like morphologies were corroborated by analysing their ITS2 secondary structure and comparing them with the ITS2 secondary structure of phylogenetically related species from GenBank.

Characterization of a Strain of Fukuyoa paulensis (Dinophyceae) from the Western Mediterranean Sea

A single cell of the dinoflagellate genus Fukuyoa was isolated from the island of Formentera (Balearic Islands, west Mediterranean Sea), cultured, and characterized by morphological and molecular methods and toxin analyses. This is the first report of the Gambierdiscus lineage (genera Fukuyoa and Gambierdiscus) from the western Mediterranean Sea, which is cooler than its eastern basin. Molecular analyses revealed that the Mediterranean strain belongs to F. paulensis and that it bears LSU rDNA sequences identical to New Zealand, Australian, and Brazilian strains. It also shared an identical sequence of the more variable ITS‐rDNA with the Brazilian strain. Toxin analyses showed the presence of maitotoxin, 54‐deoxyCTX1B, and gambieric acid A. This is the first observation of the two latter compounds in a Fukuyoa strain. Therefore, both Gambierdiscus and Fukuyoa should be considered when as contributing to ciguatera fish poisoning. Different strains of Fukuyoa form a complex of morphologically cryptic lineages where F. paulensis stands as the most distantly related nominal species. The comparison of the ITS2 secondary structures revealed the absence of CBCs among strains. The study of the morphological and molecular traits depicted an unresolved taxonomic scenario impacted by the low strains sampling.

Broad distribution of Coolia monotis and restricted distribution of Coolia cf. canariensis (Dinophyceae) on the Atlantic coast of the Iberian Peninsula

Abstract: The genus Coolia, which contains potentially toxic epibenthic dinoflagellate species, was studied throughout 17 sampling sites on the Atlantic coast of the Iberian Peninsula during the summers of 2010–2012. Cells from natural samples were isolated into culture, and the strains were observed by light and scanning electron microscopy in order to study and measure the morphological traits. A detailed compilation of previously compiled Coolia measurements was used for comparisons. To gain insight into the taxonomy of the strains, two regions of the nuclear-encoded rDNA were used as markers to infer phylogenetic relationships: the large subunit (LSU) and the internal transcribed spacers ITS1 and ITS2 plus the 5.8S region of the gene (ITS). Two species were found within the study area: Coolia monotis, which was widely distributed throughout the entire area, and Coolia cf. canariensis, which was found at only one sampling site. The observed morphology of the clonal cultures was supported by their accepted taxonomical descriptions in concordance with the phylogenetic analysis of both LSU and ITS regions. This study provided evidence of the limited geographic distribution of Coolia cf. canariensis in the studied area.

Molecular and morphological analyses of solitary forms of brackish Thalassiosiroid diatoms (Coscinodiscophyceae), with emphasis on their phenotypic plasticity

Abstract Blooms of centric diatoms are a common feature in the Bilbao estuary during summer when river flow is at its lowest and water temperature is above 20ºC. To gain insight into the specific composition of these diatom blooms, net samples and cultures of estuarine isolates were analysed under the scanning electron microscope (SEM) and by molecular analyses of the Internal Transcribed Spacers 1 and 2 plus the coding region 5.8S (ITS region) and the 28S rRNA gene. Seven species of solitary centric diatoms belonging to four genera were found in the estuary including: Conticribra weissflogii, Cyclotella atomus var. atomus, Cyclotella cryptica, Cyclotella marina, Cyclotella meneghiniana, Discostella pseudostelligera and Thalassiosira pseudonana. Dominant species during blooms were C. meneghiniana and Co. weissflogii in the upper estuary and D. pseudostelligera and T. pseudonana in the middle estuary. The morphological traits used to differentiate between species pairs of similar morphology (C. meneghiniana/C. cryptica or D. pseudostelligera/D. woltereckii) were observed to vary with environmental conditions, denoting a great deal of phenotypic plasticity which would hinder accurate identification of the species when using morphological approaches alone.

Origin, evolution and conservation of the honey bees from La Palma Island (Canary Islands): molecular and morphological data

Genetic studies have shown a significant hybridization of local populations of Apis mellifera in the Canary Islands, as a consequence of queen importation. Since La Palma Island was a probable exception to this hybridization rule, a local honey bee Conservation Project was developed. As a first step, the genetic characterization of the population was performed with the scope of generating fundamental knowledge to correctly manage honey bee genetic resources. In this sense, the origin of the Canarian honey bee is at issue, since diverse morphological and genetic studies have shown it to be either African or European. In our study, 499 colonies from La Palma were analyzed using mitochondrial DNA, microsatellites and wing geometric morphometric data. Hybridization from the C evolutionary lineage was observed mainly at mitochondrial level, but this hybridization was focused on a restricted area and hybrid colonies were detected for replacement. All genetic and geometric morphometric results showed the Canarian honey bee clustered with Western European M branch, specifically with A. m. iberiensis subspecies, far from African ones. We concluded that the present Canarian honey bee gene pool would have originated most likely from human introductions from Portugal dating back to the conquest of the islands in the sixteenth century, although an ancestral natural colonization from Iberia cannot be ruled out. Results also showed that Canarian honey bees are genetically differentiated from A. m. iberiensis, probably due to micro-evolutionary processes, such as founder effects, bottleneck and local adaptation. Therefore, we propose the classification of these local populations as a differentiated ecotype of A. m. iberiensis. This study highlights the idiosyncrasy of the Canarian honey bee and the need to protect it, as it is a population that extends considerably the genetic diversity of M branch.