Ταυτοποίηση ειδών του γένους Pseudo-nitzschia Peragallo (Βacillariophyceae) από Ελληνικά παράκτια ύδατα

Abstract

Samples were collected from the main shellfish farming and harvesting areas along the Greek coasts during the period March 2007 - April 2010. From these samples, Pseudo-nitzschia cells were isolated and 98 clonal cultures were established, aiming to detect and identify the species Pseudo-nitzschia in Greek coastal waters (by using morphological and molecular characters), investigate the capability of toxin (domoic acid, DA) production by Pseudo-nitzschia strains in culture, estimate the degree of morphological and genetic divergence between Pseudo-nitzschia species and define their phylogenetic relationships. Morphological analysis was conducted in light (LM) and transmission electron microscopes (TEM), and the ITS2 region (internal transcribed spacer 2) was used for genetic analysis. Totally, 161 sequences were analyzed (34 derived from the present study). The majority of the cultures (88) were tested for DA production with the aid of a high-performance liquid chromatography coupled with UV-diode array detection (HPLC-UV-DAD). Furthermore, three P. pseudodelicatissima strains were studied over the course of a batch culture growth experiment in order to investigate the growth phase that DA is produced. Fifteen Pseudo-nitzschia taxa were identified and morphologically described: P. brasiliana, P. delicatissima, P. galaxiae, P. mannii and P. subfraudulenta, reported for the first time from Greek waters, P. subpacifica, recorded for the first time from the Mediterranean basin, and P. calliantha, P. fraudulenta, P. multiseries, P. multistriata, P. pseudodelicatissima and P. pungens var. pungens. Three morphotypes were also found: P. cf. dolorosa, P. cf. delicatissima and P. cf. pseudodelicatissima. Identification was confirmed by phylogenetic analysis. This, additionally, revealed higher than previously estimated levels of genetic diversity. In particular, P. cf. pseudodelicatissima morphotype, along with the P. calliantha and P. mannii species, probably constitutes a case of potential incipient speciation. The morphotype P. cf. delicatissima probably corresponds to a case of semi-cryptic speciation within the P. delicatissima species complex. In addition, intraspecific genetic variation consistent with geographical differentiation was revealed, for the species P. brasiliana, P. delicatissima, P. fraudulenta, P. galaxiae, P. arenysensis and P. cuspidata (geographically distant ITS2 variants of the same species). Domoic acid was detected in cultures of three taxa (P. pseudodelicatissima, P. pungens var. pungens and P. galaxiae), and for species P. pseudodelicatissima and the variety P. pungens var. pungens was referred for the first time. The three examined Pseudo-nitzschia pseudodelicatissima strains indicated low levels of DA production during the mid-exponential phase and highest DA concentrations in late exponential and stationary phase, which are in accordance with existing data for other Pseudo-nitzschia species.%%%%Δeίγματα νeρού συλλέχθηκαν από τις κυριότeρeς πeριοχές καλλιέργeιας και αλιeίας δίθυρων μαλακίων της…