Identifying Pseudo-nitzschia species in natural samples using genus-specific PCR primers and clone libraries

Abstract

The diatom genus Pseudo-nitzschia contains a number of toxic and non-toxic species that are difficult to distinguish using light microscopy (LM) and at times even with electron microscopy (EM). In order to investigate the actual diversity and seasonal occurrence of Pseudo-nitzschia species, we developed genus-specific ribosomal DNA LSU primers to be used in PCR reactions with environmental DNA samples. Using this approach, we constructed clone libraries from samples collected in the Gulf of Naples (Mediterranean Sea) on six dates between April and October 2004 and compared molecular results with those obtained from counts using LM on the same dates. Thirteen distinct genotypes could be distinguished by their LSU sequence, against five species discriminated using the light microscope. Despite the limited number of samples, 10 out of 14 LSU genotypes known in the area were recovered. In addition, three new genotypes were retrieved, two of which were from within the P. galaxiae clade and one possibly corresponding to an undescribed P. delicatissima-like morph. Molecular results matched LM findings in the case of P. multistriata, whereas they provided a much higher resolution for morphs such as P. delicatissima- and P. pseudodelicatissima-like, which include several pseudo-cryptic species. Overall, the direct amplification with the primers developed proved to be an effective and useful tool to assess Pseudo-nitzschia diversity in the natural environment.

Introduction

Pseudo-nitzschia is a diatom genus containing several potentially toxic species. Although the morphology of the frustule, as with all diatoms, yields a number of useful taxonomic characters, the identification of species within this genus is often problematic. In recent years, several new species have been circumscribed that are not easily distinguishable using light microscopy (LM) and often also using electron microscopy (EM) (Lundholm et al., 2002, Lundholm et al., 2003, Lundholm et al., 2006). These include P. dolorosa and P. decipiens, which were previously classified as P. delicatissima, and P. calliantha and P. caciantha, formerly included within P. pseudodelicatissima. Pseudo-nitzschia cuspidata and P. inflatula are also quite similar to P. pseudodelicatissima. All these species are referred to as pseudo-cryptic because of the subtle morphological dissimilarities between them, mainly consisting of slight differences in the ultrastructure of the poroids in the valve striae, a character that is only visible using TEM. Identification of pseudo-cryptic species is obviously critical when some of them are toxic, as in the case of Pseudo-nitzschia. In addition, lumping distinct taxa under a single name does not allow the recognition of species-specific patterns of occurrence, nor their relationship to environmental conditions, thus limiting ecological studies and bloom prediction capabilities.

Like other phytoplankton species, Pseudo-nitzschia species tend to exhibit a repeating seasonal occurrence (Fryxell et al., 1997, Fehling et al., 2006). In the Gulf of Naples (Mediterranean Sea), several Pseudo-nitzschia species regularly occur in restricted periods of the year (Zingone et al., 2003, Zingone et al., 2006). However, species identified using LM as P. delicatissima, P. pseudodelicatissima and P. galaxiae seem to be present and bloom several times during the year. Prolonged periods of occurrence and multiple blooms could be due to single ecologically plastic species thriving in different seasons or, rather, to distinct pseudo-cryptic taxa hidden under these morphs.

A high genetic diversity in the Gulf of Naples was first demonstrated by Orsini et al. (2004), who found several distinct genotypes within P. delicatissima-like morphs during the late winter-spring bloom of the species, with a dominance of P. delicatissima sensu stricto (ss) at the peak of the bloom. In a more recent investigation on strains resembling P. delicatissima or P. pseudodelicatissima, eight taxa were identified and circumscribed based on morphological and molecular data and on mating incompatibility, including two new genotypes, P. calliantha2 and P. delicatissima2 (Amato et al., 2007). In this latter study, the presence of P. cuspidata was also revealed using ITS, while the LSU marker fails to separate this taxon from P. pseudodelicatissima ss. The two above-mentioned investigations required an intensive cultivation effort, with almost 200 strains isolated over several months. Pseudo-nitzschia species are frequent in the area in all seasons and often at high concentrations (million cells l⁻¹), which implies that the actual numbers of distinct taxa present in the area may have been so far under-sampled.

Problematic identification of microalgae in natural samples has in some cases been addressed using a molecular approach. PCR using specific primers has been performed on whole community DNA to recognise classes or other supra-generic taxa of marine picoeukaryotic assemblages (e.g. Díez et al., 2001a, Díez et al., 2001b, Zeidner et al., 2003, Fuller et al., 2006), In a few cases, genera or species, mainly harmful, have been targeted with PCR, with the aim of detecting and quantifying known taxa (e.g. Gray et al., 2003, Galluzzi et al., 2004, Galluzzi et al., 2005, Skovhus et al., 2004). Galluzzi et al., 2004, Galluzzi et al., 2005 used genus-specific markers for 5.8S rDNA in real-time PCR to quantify the toxic dinoflagellate Alexandrium minutum in natural samples, whereas a species-specific rbcL primer was used in the case of Karenia brevis (Gray et al., 2003).

The aim of this study was to develop a molecular method to track different pseudo-cryptic or possibly cryptic species within Pseudo-nitzschia, thus overcoming problems posed by microscopy analyses and by the isolation/cultivation method. Genus-specific primers were developed to amplify a fragment from rDNA LSU from community DNA. LSU was chosen as the marker region because, with the exception of the case of P. cuspidata, it is a good marker to distinguish among species of the genus, and also because a high number of sequences were available for both Pseudo-nitzschia and other diatoms, making the design of primers more accurate. Clone libraries were constructed on selected dates in the Gulf of Naples, from samples collected at the long-term station MareChiara (MC, Ribera d’Alcalà et al., 2004) and compared to sequences from known strains and to the species abundance results from the phytoplankton counts. It was also investigated whether the Pseudo-nitzschia specific primers might be useful for Single Strand Conformation Polymorphism (SSCP) analyses, which would be a more effective method than clone libraries for routinely assessing diversity.