Diversity and temporal pattern of Pseudo-nitzschia species (Bacillariophyceae) through the molecular lens

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Harmful Algae 42 (2015) 15–24

Diversity and temporal pattern of Pseudo-nitzschia species(Bacillariophyceae) through the molecular lens

Maria Valeria Ruggiero *, Diana Sarno, Lucia Barra 1, Wiebe H.C.F. Kooistra,Marina Montresor, Adriana Zingone

Department of Ecology and Evolution of Plankton, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Napoli, Italy

A R T I C L E I N F O

Article history:

Received 22 July 2014

Received in revised form 1 December 2014

Accepted 1 December 2014

Keywords:

Diatoms

Diversity

Environmental DNA

LTER-MC

Pseudo-nitzschia

Temporal patterns

A B S T R A C T

In diatoms, as in other organisms, many genetically distinct and reproductively isolated species may

show identical or highly similar morphological features. Such groups of species are defined as cryptic and

pseudo-cryptic species, respectively. The difficulty of discriminating them with optical means impairs

the study of their temporal patterns and geographic ranges. This is also the case for Pseudo-nitzschia, a

worldwide distributed planktonic diatom genus which includes several toxigenic species. Using a

Pseudo-nitzschia-specific pair of large sub-units ribosomal DNA (LSU rDNA) primers, we generated clone

libraries from 19 samples collected at the Long Term Ecological Station MareChiara (LTER-MC) in the

Gulf of Naples (GoN) from 2009 to 2010 and compared sequence records with light microscopy (LM)

counts from the same samples. Our aim was to elucidate the diversity and the seasonal patterns of taxa

within Pseudo-nitzschia. Most of the Pseudo-nitzschia species already known from the GoN were

identified within the 1643 obtained sequences. In addition, two species known from elsewhere and three

un-described ribotypes were detected. Several cryptic species showed distinct temporal patterns of

occurrence, with most species confined to restricted periods and only a few present year-round.

Microscopic and molecular results generally concurred for species recognizable using LM, while clone

libraries tended to overestimate the relative abundance of some of the species. Due to its high resolution

and detection power, the DNA-barcoding approach used in our study is an optimal tool to trace the

distribution of cryptic and toxigenic Pseudo-nitzschia species and the diversity of this key diatom genus

in the natural environment.

� 2014 Elsevier B.V. All rights reserved.

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jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/ha l

1. Introduction

Diatoms are among the best known marine protists. Their silicainvestment, the frustule, permits a variety of shapes and ornamen-tations, which has facilitated species identification based onmorphology since the introduction of the first microscopes (Agardh,1830–1832; Ehernberg, 1838). Morphology-based studies ondiatom diversity and distribution patterns have flourished overthe last century along with studies on their ecology and evolution

Abbreviations: ASP, amnesic shellfish poisoning; DA, domoic acid; EM, electron

microscopy; GoN, Gulf of Naples; ITS, internal transcribed spacer; LM, light

microscopy; LSU, large sub-unit; LTER-MC, Long Term Ecological Research Station

MareChiara; rDNA, ribosomal DNA.

* Corresponding author. Tel.: +39 081 5833 2121; fax: +39 0817641355.

E-mail address: [email protected] (M.V. Ruggiero).1 Present address: Institute of Biosciences and Bioresources (CNR-IBBR), Via

Universita 133, 80055 Portici, Italy.

http://dx.doi.org/10.1016/j.hal.2014.12.001

1568-9883/� 2014 Elsevier B.V. All rights reserved.

(reviewed in Mann, 1999; Mann and Chepurnov, 2004). On the otherhand, molecular phylogenetic studies have mainly focused on theoverall topology of the diatom trees and rarely have they includedmultiple strains within the same morphologically defined species.Recent studies integrating molecular phylogenetics, morphological,ultrastructural and biological information have uncovered numer-ous cases of genetically distinct and at times reproductively isolatedgroups of strains that could not be distinguished easily or at all withmicroscopy (e.g. Sarno et al., 2005; Amato et al., 2007; Nanjappaet al., 2013). Such groups of specimens indeed deserve the status ofspecies and are designated as cryptic or pseudocryptic species incase of null or subtle morphological differences, respectively. At thesame time, evidence is accumulating that such cryptic species canexhibit remarkable physiological and ecological differences (e.g.Vanelslander et al., 2009; Degerlund et al., 2012; Huseby et al., 2012;Mann and Vanormelingen, 2013). Molecular approaches constitutethe most straightforward way to identify these taxa and elucidatetheir distribution over space and time. In addition, molecular

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M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–2416

approaches permit the investigation of the actual genetic diversityamong and within species, tracing phylogenetic relationships andshedding light on evolutionary patterns and speciation mechanisms.

The planktonic pennate diatom Pseudo-nitzschia (Heterokonta,Bacillariophyceae) is a cosmopolitan genus commonly found inneritic and oceanic waters. It represents a typical case in which thenumber of genetically distinct lineages is markedly higher than thenumber of taxa recognizable in LM. The genus includes at present43 species of which 39 have been genetically characterized. At least12 of these produce domoic acid (DA), a neurotoxin responsible foramnesic shellfish poisoning (ASP) mainly in mammals and birds(Lelong et al., 2012; Trainer et al., 2012). In LM, Pseudo-nitzschia

species can only be differentiated based on cell shape and size andon the shape of frustule ends in cell chains. In EM, a wealth ofultrastructural details of the frustule have permitted a far finerresolution of diversity, generally concurring with differencesdetected with nucleotide markers such as the nuclear encodedrDNA internal transcribed spacer region (ITS1 and ITS2 and thehyper-variable domains D1–D3 of the LSU; e.g. Hasle andSyvertsen, 1997; Lundholm et al., 2003, 2006; Amato et al.,2007). In addition, an ever-increasing number of cryptic andpseudo-cryptic species have been found within most of themorphologically delineated species recognizable in LM (Amatoet al., 2007; Quijano-Scheggia et al., 2009; Lim et al., 2012, 2013;Lundholm et al., 2012; Trainer et al., 2012; Orive et al., 2013; Tenget al., 2014), prompting the use of these molecular markers as DNAbarcode (Evans et al., 2007; Moniz and Kaczmarska, 2010;Hamsher et al., 2011). Besides its value in ecological andbiogeographic studies, correct identification of Pseudo-nitzschia

species has relevant implications for monitoring and managementpurposes, considering that DA-producing species in the genus maybe morphologically similar or identical to non-toxigenic ones.

The genetic diversity of Pseudo-nitzschia in the Gulf of Naples(GoN, Tyrrhenian Sea, Mediterranean Sea), has been studiedextensively by means of culturing techniques, combined with LMand electron microscopy observations and mating compatibilityexperiments. These studies have resulted in the description of newspecies and have provided insight in the genetic composition ofblooms (Orsini et al., 2004; Amato et al., 2007; Amato andMontresor, 2008). DNA barcoding approaches based on clonelibraries, which by-pass underestimation due to cultivation biasesand species rarity, was first tested on a limited number of samplesfrom the GoN to assess Pseudo-nitzschia genetic diversity

Table 1Pseudo-nitzschia diversity per sample. Sample = denomination of the LTER-MC sample; d

diversity; Ds�1 = inverse Simpson index; Chao1 = Chao index; C = coverage; cells L�1 = ligh

Season Sample name Date N

Summer 2009 MC878 08 September 2009 112

Autumn 2009 MC882 06 October 2009 94

MC883 13 October 2009 60

MC886 04 November 2009 86

MC890 02 December 2009 81

Winter 2010 MC895 12 January 2010 89

MC899 09 February 2010 96

MC903 09 March 2010 86

MC904 16 March 2010 74

Spring 2010 MC908 13 April 2010 92

MC909 20 April 2010 78

MC913 18 May 2010 75

MC917 15 June 2010 82

Summer 2010 MC921 13 July 2010 101

MC924 03 August 2010 98

MC930 14 September 2010 100

Autumn 2010 MC934 12 October 2010 95

MC938 11 November 2010 71

MC941 14 December 2010 73

Total dataseta 1643

a Nh, Hd, Ds�1, Chao1 and coverage calculated over the whole dataset.

(McDonald et al., 2007), while microarrays provided contrastingresults, highlighting the challenges of appropriate probe designand hybridization conditions (Barra et al., 2013). At the Long TermEcological Research Station MareChiara (LTER-MC, GoN), Pseudo-

nitzschia species are present basically year round, with concentra-tions up to 106 cells L�1 (Ribera d’Alcala et al., 2004). Differentspecies or species-complexes, as recognized by LM, bloom atdifferent times of the year, while some species show multiplepeaks over the year (Zingone et al., 2003, 2006; Cerino et al., 2005).

In the present study we investigated the genetic diversity ofPseudo-nitzschia in environmental clone libraries from 19 planktonsamples collected over 16 months at the LTER-MC station usinggenus-specific LSU rDNA primers (McDonald et al., 2007) andcompared the molecular identification results with those obtainedby LM cell counts on the same dates. The aims of this DNA-barcoding study were: (i) to study the diversity of Pseudo-nitzschia,possibly detecting taxa unknown to date in the GoN or anywhere;(ii) to unveil patterns of intraspecific diversity in the species in thegenus; (iii) to depict the seasonal patterns for the different crypticspecies in the genus.

2. Materials and methods

2.1. Study site and sample collection

Water samples were collected at the LTER-MC station(40848.50 N, 148150 E; bottom at �75 m) in the GoN (Fig. S1).The station is located 2 nautical miles off the coast of the city ofNaples, at the boundary between the eutrophic coastal zone andthe oligotrophic Tyrrhenian Sea waters (Ribera d’Alcala et al.,2004). The samples were collected at 0.5 m depth with a 12 LNiskin bottle mounted on an automatic Carousel sampler on19 sampling dates from September 2009 to December 2010(Table 1). Temperature and salinity values in surface waters variedbetween 13.39 8C (16 March 2010) and 28.92 8C (20 July 2010), andbetween 36.58 (20 July 2010) and 38.20 (28 September 2010),respectively. Average values of nutrient concentrations in theupper layer (0.5–5 m depth) were 0.09 � 0.07 mM for phosphates,2.93 � 1.91 mM for silicates and 2.88 � 2.72 mM for inorganicnitrogen (NH4 + NO3 + NO2); unpublished results, courtesy of MECAservice).

Supplementary Fig. 1 related to this article can be found, in theonline version, at doi:10.1016/j.hal.2014.12.001.

ate = sampling date; N = number of sequences; Nh = number of ribotypes; Hd = gene

t microscopy cell counts. Seasons are defined according to the Gregorian calendar.

Nh Hd Ds�1 Chao1 C Cells L�1

12 0.20 1.26 34.5 0.89 4.41 � 104

16 0.71 3.47 34.0 0.83 2.60 � 105

6 0.33 1.49 6.0 0.90 3.59 � 105

19 0.86 7.18 26.2 0.78 9.88 � 103

13 0.79 4.81 15.5 0.84 2.96 � 104

15 0.74 3.81 20.3 0.83 9.88 � 103

20 0.84 6.30 35.0 0.79 5.01 � 104

15 0.72 3.56 18.3 0.83 1.12 � 105

15 0.85 6.67 25.5 0.80 4.22 � 105

20 0.67 3.00 39.5 0.78 8.05 � 105

10 0.56 2.28 15.0 0.87 1.22 � 105

9 0.58 2.39 24.0 0.88 0.00

16 0.77 4.35 71.0 0.80 6.65 � 104

12 0.34 1.51 40.0 0.88 1.52 � 106

14 0.33 1.50 18.7 0.86 5.14 � 106

14 0.72 3.51 17.8 0.86 1.58 � 105

13 0.61 2.54 15.5 0.86 1.91 � 105

17 0.87 7.58 26.3 0.76 0.00

17 0.84 6.21 22.6 0.77 0.00

84 0.86 7.36 84.0 0.95

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M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–24 17

For genetic analyses, 2–5 L of seawater was filtered on cellulose-ester filters (47 mm diameter, 1.2 mm pore size, EMD Millipore,USA). Filters were cut immediately into two halves, frozen in liquidnitrogen and stored at �80 8C until further processing.

2.2. Light microscopy cell counts

Water samples for phytoplankton cell enumeration werecollected at the same depth and fixed with 0.8% neutralizedformaldehyde. One to 50 mL of fixed sample was allowed tosediment in an Utermohl chamber. Phytoplankton cells wereidentified and enumerated using an inverted light microscope (LM,Zeiss Axiovert, Carl Zeiss, Oberkochen, Germany) at 400�magnification. Counts were performed generally on two transeptsand the volume of seawater inspected ranged from 0.02 to 1.52 mL.Only few of the species present in our samples could be identifiedat the species level in LM. This was the case of Pseudo-nitzschia

multistriata (Orsini et al., 2002) and Pseudo-nitzschia galaxiae, forwhich small and medium-large morphotypes were distinguished(Cerino et al., 2005). Pseudo-nitzschia fraudulenta cannot bedistinguished from Pseudo-nitzschia subfraudulenta (Traineret al., 2012). Cells with a transapical diameter �3 mm wereassigned to the Pseudo-nitzschia pseudodelicatissima- or P. delica-

tissima-complex (hereafter indicated as P. cf. pseudodelicatissima

and P. cf. delicatissima) based on the presence of pointed orrounded/truncated ends when seen in girdle view, respectively.

2.3. DNA extraction and PCR amplification

Total genomic DNA was extracted from half filters using CTABextraction buffer as described in McDonald et al. (2007). ThePseudo-nitzschia LSU primers D1-186F and D1-548R (McDonaldet al., 2007) were used to amplify a 348 bp fragment within thenuclear-encoded LSU rDNA from the total genomic DNA extractedfrom the environmental samples. In detail, total reaction volume of50 ml contained approximately 40 ng environmental DNA, 200 mMdeoxynucleoside triphosphates, 1 mM of each primer and 2.5 U Taqpolymerase in 1� enzyme buffer with MgCl2 added (RocheDiagnostics GmbH, Mannheim, Germany). The PCR conditionswere 94 8C for 4 min followed by 35 cycles of 94 8C for 1 min,primer annealing at 62 8C for 35 s and elongation at 72 8C for 1 min20 s, with a final elongation step at 72 8C for 5 min.

In three cases in which no visible PCR product was obtained(samples MC913, MC938 and MC941), a nested PCR approach wasused: total genomic DNA was used as template for amplification ofthe D1–D3 LSU fragment using universal primers D3Ca and DIR(Lenaers et al., 1989; Scholin et al., 1994); the purified PCR-productwas then used as a template for the amplification of the 348 bpfragment (as in McDonald et al., 2007).

The amplified LSU-fragment is able to distinguish all Pseudo-

nitzschia species described to date, with two exceptions: Pseudo-

nitzschia micropora and Pseudo-nitzschia dolorosa, which areidentical for this fragment, and Pseudo-nitzschia cuspidata andPseudo-nitzschia pseudodelicatissima, which are identical for thewhole D1–D3 LSU rDNA marker (ca 800 bp), commonly used forspecies discrimination. While P. cuspidata has been previouslyidentified in the GoN based on other markers (Amato et al., 2007),P. micropora has never been reported in the Mediterranean Sea. Theprimers selected also amplify LSU sequences of the genusFragilariopsis, which is recovered as a clade inside Pseudo-nitzschia

(Lundholm et al., 2002a).

2.4. Clone libraries

PCR fragments were purified using a QIAquick PCR purificationkit (Qiagen Ltd., Venlo, The Netherlands) and cloned using TA

Cloning1 kit (InvitrogenTM Life Technologies, Carlsbad, California).Approximately 100 clones were manually picked for each sample.Plasmids were purified using the Millipore Montage PlasmidMiniprep Kit (Millipore Corporate, 290 Concord Road, Billerica,MA 0182, USA) and a robotic station, Beckman Coulter’s Biomek1 FXLaboratory Automation Workstation, equipped with ORCA1 roboticarm (Beckman Coulter, Fullerton, CA). Single strand sequences wereobtained with the BigDye Terminator Cycle Sequencing technology(Applied Biosystems, Foster City, CA), using a standard T7 primer,and purified in automation using the Millipore Montage SEQ96Sequencing Reaction Cleanup Kit (Millipore Corporate) and theBiomek1 FX. Products were run on an Automated CapillaryElectrophoresis Sequencer 3730 DNAAnalyzer (Applied Biosys-tems). These sequence data have been submitted to the GenBankdatabase (accession numbers in Table S1).

Supplementary Table 1 related to this article can be found, inthe online version, at doi:10.1016/j.hal.2014.12.001.

2.5. Data analyses

Sequence chromatograms were inspected in Bioedit (Hall,1999). ClustalW (Thompson et al., 1994) implemented in Bioeditwas used to align sequences. Putative chimeras were identified asgenerating different hits for different portions of the sequence inthe Blast analysis, further checked by eye and finally eliminatedfrom the dataset. Singletons over the whole dataset wereconsidered as Taq errors and eliminated from the dataset.

2.5.1. Sample diversity

MOTHUR (Schloss et al., 2009) was used to calculate samplediversity and richness. The inverse Simpson diversity index (Ds�1)takes into account the number of species present and their relativeabundance (Simpson, 1949). The non-parametric estimator Chao1(Chao, 1984) was employed to assess ribotype richness (95% CI),correcting for abundance of rare ribotypes, i.e. counted once ortwice (Hughes et al., 2001). Coverage of each clone library and oftotal samples was calculated as C = 1 � (Nh/N), where Nh is thenumber of observed ribotypes and N is the total number ofsequences in that sample (Romari and Vaulot, 2004). Genediversity (Hd, Nei, 1987) was calculated by means of the softwareDNAsp (Librado and Rozas, 2009); this parameter takes intoaccount both the number and the frequency of each ribotype.Sampling dates were assigned to seasons according to theGregorian calendar.

2.5.2. Species diversity

Taxonomic assignation was performed by blasting each ribotypeagainst the GenBank database (NCBI BLAST version 2.2.9, Altschulet al., 1997) and attributing the ribotypes to the species whosereference sequence in GenBank was showing the highest matchingscore. (Table S1). Within Pseudo-nitzschia galaxiae, ribotypes wereassigned to ribogroups corresponding to the LSU clades identified inMcDonald et al. (2007). In order to assess within-species diversityand temporal patterns of occurrence, all ribotypes assigned tospecies were gathered from all sampling dates. Gene diversity Hd

and pairwise nucleotide diversity (p, Nei, 1987) were calculated bymeans of the software DNAsp.

3. Results

3.1. Diversity

Of the19 clone libraries obtained, 16 were from samples in whichdifferent abundance values of Pseudo-nitzschia species were recordedby LM counts and 3 from samples in which no Pseudo-nitzschia weredetected by LM counts. (Fig. 1; Table 1). Of the 1885 sequences

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Fig. 1. Cell counts from LTER-MC dataset over the whole sampling period (67 dates) for total phytoplankton, diatoms and Pseudo-nitzschia spp. Dots indicate the dates for

which a clone library was obtained.

M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–2418

obtained, 242 (12.9%) consisted of unreliable reads, chimeras orsequences containing single base changes. The coverage value foreach clone library was generally high; the maximum value wasobtained over the complete dataset, indicating that the geneticdiversity of Pseudo-nitzschia in the GoN was sampled exhaustively inthis study. Gene diversity was correlated (R2 = 0.779) with theInverse Simpson Index. The Chao1 index ranged between 6 and71. Diversity indices for the samples generated through a nested PCRdid not differ markedly from the average of all other samples.

Blast results of the ribotypes revealed 15 taxa, among which12 were assigned to taxonomically described species. Theribotypes with the highest number of occurrences were generallyidentical to a reference sequence of a described species (Table S1).

In the P. delicatissima species-complex, three known cryptic orpseudo-cryptic species were retrieved, namely Pseudo-nitzschia

arenysensis, P. delicatissima and Pseudo-nitzschia dolorosa. Inaddition, 259 sequences (ribotypes #14–20, Table S1) wereattributed to a taxon herein called P. delicatissima IV, alreadyknown from the GoN (McDonald et al., 2007, as ‘‘P. delicatissima

new genotype’’, Lamari et al., 2013, as P. cf. delicatissima). Anothergroup of sequences formed a clade close to the reference sequenceof P. delicatissima in a phylogeny inferred from the D1–D3 domainof the LSU (Fig. S2). These sequences included one ribotype (#12)identical to a sequence of a Namibian strain (SZN-B341) attributedto P. delicatissima and a sequence from an Australian strain CHBnamed Pseudo-nitzschia arenysensis in Ajani et al. (2013), as well astwo ribotypes (#7 and #11) close to those Namibian/Australiansequences (Table S1 and Fig. S1). These sequences are attributed toa new taxon henceforth referred to as P. delicatissima V. The wholeP. delicatissima species-complex accounted for 29% of the totalnumber of sequences, of which about half was close or identical tothe sequence of P. delicatissima IV, which showed the highest geneand nucleotide diversity (Table 2).

Supplementary Fig. 2 related to this article can be found, in theonline version, at doi:10.1016/j.hal.2014.12.001.

Within the Pseudo-nitzschia pseudodelicatissima species-com-plex, environmental sequences identical or close to referencesequences of four cryptic species were retrieved, namely those ofPseudo-nitzschia calliantha, Pseudo-nitzschia mannii, Pseudo-nitzschia

hasleana and P. pseudodelicatissima/cuspidata. These sequencesaccounted for 10% of the total, about half of which belonging to P.

calliantha. Each species except P. hasleana was represented by tworibotypes. Within both P. mannii and P. calliantha two differentribotypes were found; in both cases, the two ribotypes matcheddifferent GenBank accessions attributed to each species. Among thespecies in this complex, P. calliantha showed the lowest gene andnucleotide diversity (Table 2).

Sequences belonging to Pseudo-nitzschia galaxiae were the mostabundant (49% of the total) and showed the highest geneticdiversity (Table 2), with 45 distinct ribotypes forming fourribogroups (following McDonald et al., 2007): ribogroup I (closestreference sequence: P. galaxiae SM1); ribogroup II (P. galaxiae

SM26), ribogroup III (P. galaxiae SM10), and ribogroup IV (P.

galaxiae 220604_F04). Ribogroup II included the vast majority ofthe ribotypes (36), while ribogroup IV harbored the highestribotype, gene and nucleotide diversity (Table 2).

Pseudo-nitzschia multistriata (5% of the total sequences) includedsix different ribotypes, showing moderately high gene and nucleo-tide diversity (Table 2). Pseudo-nitzschia fraudulenta (3% of the totalsequences) showed high gene diversity (0.45) but only moderatenucleotide diversity. Its sister species, Pseudo-nitzschia subfraudu-

lenta, was present with 2 sequences of a single ribotype. Pseudo-

nitzschia linea was detected as 10 sequences of a single ribotype.Nine sequences (ribotypes #22, #23 and #24) were similar to a

sequence from South Africa (Table S1) and clustered in a singleribogroup, herein called Pseudo-nitzschia sp., with high gene andnucleotide diversity (Table 2).

One ribotype (#21) was similar to Neodenticula seminae

(96.24%) and Fragilariopsis curta (95%) and was excluded fromfurther analyses.

3.2. Seasonality

Almost all species, and the 4 ribogroups of Pseudo-nitzschia

galaxiae, were recorded in two or more consecutive seasons. Thespecies found in single seasons were Pseudo-nitzschia subfraudu-

lenta, only found in winter, and Pseudo-nitzschia mannii and thethree ribotypes not attributable to any described species yet

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Table 2Intraspecific diversity within the genus Pseudo-nitzschia; N = number of sequences; Nh = number of ribotypes; S = number of segregating (polymorphic) sites; Hd = gene

diversity; p = pairwise nucleotide diversity. Cell counts per species, species complex or morphotypes are summed over sampling dates. na = not applicable.

N Nh S Hd p Species complex/morphotype Cells L�1

Pseudo-nitzschia delicatissima species-complex

P. arenysensis 150 2 2 0.04 0.0002

P. delicatissima 67 4 2 0.41 0.0012 Pseudo-nitzschia cf.

delicatissima

1.92 � 106

P. dolorosa 8 1 na na na

P. delicatissima IV 251 6 5 0.10 0.0003

P. delicatissima V 44 3 3 0.46 0.0033

Pseudo-nitzschia pseudodelicatissima species-complex

P. pseudodelicatissima/cuspidata 19 2 2 0.20 0.0011

P. calliantha 89 2 1 0.04 0.0001 Pseudo-nitzschia cf.

pseudodelicatissima

5.42 � 105

P. mannii 33 2 1 0.22 0.0006

P. hasleana 29 1 na na na

Pseudo-nitzschia galaxiae

Ribogroup I 176 5 3 0.19 0.0006 P. galaxiae small morphotype 1.28 � 105

Ribogroup II 613 36 32 0.34 0.0016

Ribogroup III 7 1 na na na P. galaxiae medium-large

morphotype

6.59 � 106

Ribogroup IV 8 3 3 0.69 0.0034

P. galaxiae tot 804 45 38 0.58 0.0025

P. fraudulenta 51 4 2 0.45 0.0013 P. fraudulenta/subfraudulenta 5.27 � 103

P. subfraudulenta 2 1 na na na

P. multistriata 75 6 7 0.27 0.0011 1.16 � 105

P. linea 10 1 na na. na na na

Pseudo-nitzschia sp. 9 3 7 0.72 0.0097 na na

Neodenticula/Fragilariopsisa 2 1 na na na na na

Total diversity 1643 84 68 0.86 0.0178

a Ribotype not attributable to the genus Pseudo-nitzschia, not included in following analyses.

M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–24 19

(herein grouped as Pseudo-nitzschia sp., see above), only found inautumn (Figs. 2 and 3).

Pseudo-nitzschia galaxiae was present in all samples and seasons(Figs. 2A and B and 3). Among the different morphotypes known forthe species (Cerino et al., 2005) the small-sized cells were presentin winter-early spring and the medium-large sized in late spring-summer (Fig. 2A). Accordingly, ribogroup I, corresponding to thesmall morphotype (McDonald et al., 2007), reached the highestabundance in winter while ribogroup II (medium and largemorphotypes) dominated in summer and was substituted again byribogroup I in autumn (Fig. 2B). Ribogroups III and IV, alsocorresponding to medium and large morphotypes, were much lessabundant and were observed from autumn to spring and insummer/autumn, respectively.

Three peaks of P. cf. delicatissima were observed by LM (Fig. 2C),in the autumns 2009 and 2010 and in spring 2010. Based on clonelibrary results, P. delicatissima and Pseudo-nitzschia arenysensis

overlapped to a large extent in their occurrences, being the maincontributors to the spring peak of this morphotype (Fig. 2D). P.

arenysensis appeared and decreased later than P. delicatissima.Pseudo-nitzschia dolorosa was present in low abundances in earlyspring, while P. delicatissima IV dominated in the early autumnsamples (Fig. 2D), both in 2009 and 2010. Pseudo-nitzschia

delicatissima V occurred in low numbers in all seasons.Within the Pseudo-nitzschia pseudodelicatissima species-com-

plex (Fig. 2E–F), the spring peak recorded by LM was mainly due toPseudo-nitzschia calliantha with minor contributions of Pseudo-

nitzschia hasleana and P. pseudodelicatissima/cuspidata. P. calliantha

was also abundant in autumn (Fig. 2F); P. hasleana and Pseudo-

nitzschia mannii were detected both in spring and autumn, thelatter species more abundant in 2010 than in 2009. P. pseudode-

licatissima/cuspidata and P. hasleana were present in winter), whenno P. pseudodelicatissima morphotype was detected by LM.

Three peaks of Pseudo-nitzschia multistriata, in December, Juneand September, were detected in both cell counts and clone

libraries (Fig. 2G–H). Pseudo-nitzschia fraudulenta/subfraudulenta

was observed by LM on a single date in winter 2010, while in clonelibraries it was also detected in low abundance in December 2009,January, March and June 2010 (Fig. 2I–J). P. subfraudulenta was onlypresent in December 2010, while P. linea was detected from Marchto April (Fig. 2K). The three ribotypes of Pseudo-nitzschia sp. wereall detected in autumn.

4. Discussion

4.1. Pseudo-nitzschia diversity at LTER-MC

Our dataset of 1643 partial LSU sequences collected over16 months covered the specific and intraspecific diversity of thegenus Pseudo-nitzschia in the GoN with unprecedented resolution.As a complement to a series of studies on Pseudo-nitzschia diversityin the GoN genotyping several hundred strains (Orsini et al., 2004;Amato et al., 2007; Cerino et al., 2005; Tesson et al., 2014), clonelibraries on 6 dates (175 sequences, McDonald et al., 2007) and amicroarray study over one year sampling (Barra et al., 2013), thepresent study expands the known generic diversity in the GoN,adding new species and ribotypes never before found there. Theseinclude P. linea, described from the East Atlantic coasts (Gulf ofMexico and Narragansett Bay, Lundholm et al., 2002b) andreported from the Western Mediterranean (Catalan coasts,Quijano-Scheggia et al., 2010), which was probably overlookedin previous observations of the GoN plankton because of its smallsize (ca 10 mm length), and solitary and epiphytic habit. Indeedfollowing its detection in the clone libraries P. linea was alsoobserved in net samples as epiphyte on Chaetoceros sp. Pseudo-

nitzschia hasleana, described from coastal waters of WashingtonState (Eastern Pacific Ocean, Lundholm et al., 2012) and detectedalong Greek and East Mediterranean coasts (Moschandreou et al.,2012), was also found for the first time in the GoN. Other newlyfound ribotypes, probably belonging to un-described species,

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Fig. 2. Abundance of Pseudo-nitzschia species and sequence counts from clone libraries (CL). A: P. galaxiae (* = right axis), LM; B: P. galaxiae, CL; C: P. delicatissima-complex, LM;

D: P. delicatissima-complex, CL; E: P. pseudodelicatissima- complex, LM; F: P. pseudodelicatissima- complex, CL; G: P. multistriata, LM; H: P. multistriata, CL; I: P. fraudulenta/

subfraudulenta, LM; J: P. fraudulenta and P. subfraudulenta, CL; K: P. linea and Pseudo-nitzschia sp., CL.

M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–2420

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Fig. 3. Seasonal signal for Pseudo-nitzschia species and ribogroups in the Gulf of Naples as detected by clone libraries. Bars indicate the proportional abundance of the

sequences for each species/ribogroup in the different seasons (as defined in Table 1).

M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–24 21

include P. delicatissima V, similar to Namibian and Australianstrains, and Pseudo-nitzschia sp., close to a strain from South-African waters. These species may simply have been overlooked bythe LM-monitoring because of their apparent rarity. Alternatively,the new findings of genotypes previously detected in regionsremote from the GoN (Namibian, South African and Australianwaters) might indicate a recent introduction into the Mediterra-nean Sea through anthropic-driven (ballast waters) routes as wellas natural routes such as migratory birds, aerosols, hydrodynamicprocesses etc. The hypothesis of a man-mediated introduction hasbeen proposed for Pseudo-nitzschia multistriata (Zenetos et al.,2010) a species now fairly common, but only observed since1996 in the GoN and initially only known from Japanese waters.

Some species previously found in the GoN did not appear in thisstudy.

Pseudo-nitzschia subpacifica, identified by LM and EM, wascommon at station LTER-MC in the spring period from thebeginning of the long-term series (1984) until 1990, but it hasnever been seen again since. Two strains of Pseudo-nitzschia

caciantha were isolated in a previous study (Amato et al., 2007) butthis species was not detected in the previous clone library-basedstudy (McDonald et al., 2007), nor in any other isolation andobservation efforts in the GoN. Finally, this study did not find agenotype close to Pseudo-nitzschia inflatula (strain B26), onlyencountered once and misidentified as Pseudo-nitzschia pseudo-

delicatissima in Orsini et al. (2002) based on the resemblance inporoid ultrastructure with taxa in the P. pseudodelicatissima

complex. It is difficult to assess whether P. caciantha and P. cf.inflatula are still present but extremely rare in the GoN, or if theyrepresent a case of local extinction, like Pseudo-nitzschia sub-

pacifica.Four other Pseudo-nitzschia species known from other localities

in the Mediterranean Sea (Pseudo-nitzschia pungens, Pseudo-

nitzschia brasiliana, Pseudo-nitzschia multiseries and Pseudo-

nitzschia australis), have never been recorded in the phytoplanktoncounts at our LTER in the GoN and were absent in our clonelibraries as well. At least one of them, P. pungens is quite frequentand abundant in the Adriatic Sea (Penna et al., 2013) and in Greekwaters (Moschandreou et al., 2012) and has been found recentlyalso in the nearby Gulf of Gaeta (unpublished data). The other threespecies have been recorded only rarely and basically at single sitesin the Mediterranean.

With 16 taxonomically recognized species retrieved so far(considering Pseudo-nitzschia cuspidata separately from Pseudo-

nitzschia pseudodelicatissima) and three additional ones that areonly delineated genetically, the Pseudo-nitzschia diversity in theGoN is among the highest recorded so far at any site. Consideringonly the recent recognitions, mainly based on strain genotypingand microscopy observations, eight species were recovered fromthe Bilbao estuary (Spanish Atlantic coasts, Orive et al., 2013),10 from the Australian coasts (Ajani et al., 2013), eleven from theNorth western Pacific (Stonik et al., 2011), and 12 from Greekwaters (Moschandreou et al., 2012). Twenty-two species werereported in a study based on electron microscopy (Teng et al.,2013) in Malaysian waters, which however comprise a much moreextensive area compared to the LTER in the GoN. However, thehigher diversity observed in the GoN could simply be due to thefact that the genus Pseudo-nitzschia has been investigated moreintensively here than in other areas of the world (e.g. Riberad’Alcala et al., 2004; McDonald et al., 2007; Amato et al., 2007;Orsini et al., 2004).

Highly diverse phytoplankton assemblages are reported for theGoN in the winter season, despite only occasional increases in theautotrophic biomass (Zingone et al., 2010). Our results also show areverse relationship between abundance and genetic diversity.This is not surprising, as blooming ribotypes may saturate thesample, masking the presence of rare ribotypes. In the absence ofdominating species, as in the case of winter and autumn samples,low abundance species have a higher chance to be detected. As amatter of fact, the samples with a few Pseudo-nitzschia cells or noneat all by LM were the most diverse in clone libraries. Pseudo-

nitzschia species do not form resting benthic stages (Montresoret al., 2013), and hence each species could persist in low densitiesin the water column at any time outside their bloom optimum.

At the intraspecific level, the most striking case of diversity isthat of Pseudo-nitzschia galaxiae, which harbors at least five groupsof LSU-ribotypes (McDonald et al., 2007). Cerino et al. (2005) firsthighlighted the high morphological variability within this species. Inaddition to the type material, constituted by lanceolate cells,25–41 mm long (Lundholm and Moestrup, 2002c), named asmedium morphotype, Cerino et al. (2005) recognized two moremorphotypes: one larger (<82 mm) than the type material, withparallel valve sides named large morphotype, and another muchsmaller (down to 10 mm) non-chain forming, named small

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M.V. Ruggiero et al. / Harmful Algae 42 (2015) 15–2422

morphotype. Although the medium and large morphotypes sharethe same LSU, the three morphotypes are distinct in ITS (McDonald,2006) and rbcL (unpublished data). The observed phenotypicdiversity is thus apparently supported by a genetic basis in thisspecies, possibly highlighting a case of incomplete lineage sortingwhich would identify P. galaxiae as a species-complex. Thishypothesis could be tested collecting data on sexual compatibilitybetween isolates belonging to the different ribogroups.

The high genetic diversity in Pseudo-nitzschia galaxiae could bedue to its larger sample size in terms of sequence counts for thisspecies; the larger the number of sequences obtained for a species,the higher the probability to catch less common ribotypes andhence to have a more exhaustive picture of the genetic diversity ofthe species. This explanation, however, does not apply to otherspecies such as Pseudo-nitzschia fraudulenta, which is alsoremarkably diverse despite being the least represented speciesin the clone libraries, and Pseudo-nitzschia hasleana, which is muchless polymorphic than comparably well represented species.Considering that populations in recently colonized areas are oftenless polymorphic than source populations (Allendorf and Lund-quist, 2003), the low diversity of P. hasleana supports the idea of arecent introduction in the GoN. A recent introduction is alsosuggested by the fact that the species was never recorded duringmultiple isolations with subsequent genotyping carried out in thearea since the beginning of the 2000 s.

The considerable differences in nucleotide and gene diversityobserved even among cryptic sister species (e.g. in the P.

delicatissima species-complex) could also be the effect of intra-genomic variations. rDNA is a multi-copy region with ribosomalgenes arranged in tandemly repeated cistrons (18S-5.8S-28Sintermingled by ITS1 and ITS2) and the number of copies canvary highly among species (Weider et al., 2005; Song et al., 2012).The more the copies, the more likely that concerted evolution failsto homogenize the paralogues within individuals (Alvarez andWendel, 2003; Thornhill et al., 2007). The presence of intra-genomic variation at different regions of the rDNA cistron has beendescribed in several phytoplankton species (Behnke et al., 2004;Alverson and Kolnick 2005; Pillet et al., 2012), including Pseudo-

nitzschia species (McDonald et al., 2007; D‘Alelio et al., 2009).

4.2. Seasonal dynamics and bloom composition

The temporal dynamics that generate patterns of speciesabundance and community structure constitute an importantquestion in ecology (Magurran and Henderson, 2010). However,only a few studies have dealt with temporal changes in diversity inunicellular microalgae, mainly based on isolates from single species(Evans et al., 2005; Rynearson et al., 2006; Godhe and Harnstrom,2010), or focused on the total phytoplankton sample (Behnke et al.,2010) or on size-selected planktonic groups (Massana et al., 2004;Romari and Vaulot, 2004; McDonald, 2006; Piwosz and Pernthaler,2010). More recently, some studies based on phylochips have dealtwith temporal patterns of occurrence in several harmful algae(Dittami et al., 2013; Edvardsen et al., 2013; Kegel et al., 2013),including Pseudo-nitzschia (Barra et al., 2013).

The clone library approach used in the present work alloweddisclosing the actual composition of the blooms of cryptic andpseudo-cryptic species as well as the seasonal patterns of theindividual species. As expected, clone libraries showed a far higherdetection power than that achievable by means of LM observa-tions, recording the presence of any Pseudo-nitzschia speciesobserved by LM but also of species not observed in correspondingsamples. However, the relative proportion of the species present inenvironmental samples often diverged from LM observations. Forexample, in comparison with LM cell counts, Pseudo-nitzschia

galaxiae was often over-represented in the clone libraries, while

Pseudo-nitzschia pseudodelicatissima-complex was under-repre-sented. This mismatch can be due to several factors, includingthe exponential nature of the PCR technique (Gonzalez et al., 2012),the variability of the extraction efficiency and the differentnumbers of rDNA copies in the genomes of the different species.Indeed, the rDNA copy number in Pseudo-nitzschia can vary notonly among species but even within a single species sampled atdifferent times, suggesting a relationship with physiologicalactivity and/or adaptive strategies of the strains (Penna et al.,2013).

Interestingly the seasonal patterns revealed by the clone libraryapproach basically match results obtained in previous studies(Orsini et al., 2004; Cerino et al., 2005; Zingone et al., 2006; D‘Alelioet al., 2009) and confirm that most species, including the crypticones, tend to occur in specific periods of the year. Formorphologically distinct species, i.e. Pseudo-nitzschia fraudulenta

and Pseudo-nitzschia multistriata, clone library results matchedthose obtained by LM. Regarding cryptic species, this studyconfirms the high diversity of the P. delicatissima complex in spring(Orsini et al., 2002; McDonald et al., 2007), mainly formed byPseudo-nitzschia arenysensis and P. delicatissima, accompanied bymuch less abundant Pseudo-nitzschia dolorosa and anotherribotype new for the area (P. delicatissima V). In contrast, it wasmainly P. delicatissima IV to form the late summer-early autumnbloom in both 2009 and 2010, matching previous records of thistaxon in late summer in 2004 (McDonald et al., 2007, as ‘‘P.

delicatissima new genotype’’). These results highlight the regularseasonality pattern of this cryptic species and support thehypothesis that P. delicatissima IV can indeed be the species foundto undergo a massive sexual reproduction in September 2006(Sarno et al., 2010). In the Pseudo-nitzschia pseudodelicatissima

complex, Pseudo-nitzschia calliantha, which produces domoic acid(Trainer et al., 2012), appeared to be the main contributor to theautumn and spring peaks, while the other species, including P.

pseudodelicatissima sensu stricto, increased in the clone libraries inlate autumn-winter, when the corresponding morphotypes werevirtually absent from cell counts. Neither morphotypes norsequences of this group were detected in late spring-summer in2010, which indeed is the period of low abundance of P. cf.pseudodelicatissima in the LTER-MC time series. However in a fewyears (1996, 2000 and 2006–2008) there were increases of P. cf.pseudodelicatissima in late May–June, with the highest density(3 � 106 cells L�1) for the whole time series in June 2007 (D.S. andA.Z., unpublished results). This irregular behavior reminds us thatseveral years of observations are needed to fully cover the seasonalpattern of a species.

The peculiar seasonality of the different morphotypes ofPseudo-nitzschia galaxiae described by Cerino et al. (2005) wasalso confirmed by our clone library results which show that theribogroup I, corresponding to the small morphotype, wasresponsible for the early spring increase, while the ribogroup II,encompassing the medium and large morphotypes, was found inlate spring and in summer. This last ribogroup was found toproduce domoic acid in the GoN (Cerino et al., 2005).

The present study shows that distinct but closely related crypticdiatom species may be present in different seasons, over a broadrange of environmental conditions in terms of light, turbulence,temperature and nutrients (Ribera d’Alcala et al., 2004). Seasonaloccurrence of species appears not to be constrained by nutrientlevels because their regularity strongly contrasts with the chemicalvariability in coastal waters (Ribera d’Alcala et al., 2004 and otherunpublished results). This is particularly true for species thatcontribute little to the total phytoplankton abundance and evenless to total phytoplankton biomass. Other factors, such astemperature and photoperiod, which are linked to astronomicalcycles, may be related to species occurrence over the seasons,

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possibly through a direct effect, but also as triggers of endogenousclocks (Anderson and Kaefer, 1987). Advection processes could, intheory, explain the alternation of different blooming speciesduring the year. Yet, the persistence of the blooms over periodsexceeding the time-scales of the complex surface hydrodynamicsof the GoN (Uttieri et al., 2011) suggests that such short-termhydrographic events do not affect species succession.

The alternation of closely related species along seasons raisesquestions about the mechanisms of the speciation process at sea. Infact, populations of closely related lineages can display differentecophysiological characteristics (Rynearson et al., 2006; Degerlundet al., 2012; Huseby et al., 2012), which may reflect adaptations toparticular ecological niches. A reduction of the gene flow due todifferent heritable reproductive times (isolation-by-time, Hendryand Day, 2005) could be an important speciation driver and couldbe advocated to explain the remarkable number of cryptic, closelyrelated lineages in unicellular microalgae.

5. Concluding remarks

Based on our results Pseudo-nitzschia confirms to be among themost diverse diatom genus in the GoN plankton. The molecularlens used in this study highlighted differences in the seasonal andrelative abundance patterns among cryptic sister species and insome cases among ribotypes belonging to the same species.Although we did not investigate their driving factors, eitherenvironmental or endogenous, these patterns point at differencesin the ecological/seasonal niches of cryptic species, stimulatingstudies on their ecophysiological characteristics as well as on theirmode of speciation. In addition, our approach can represent auseful tool for management purposes, revealing the presence ofpotentially toxic species and indicating the most probable periodsfor their occurrence in the plankton.

Acknowledgments

The authors thank the EU-FP7 project MIDTAL for funding andthe Flagship project RITMARE – The Italian Research for the Sea –Program 2011–2013 for partial support; the Molecular BiologyService (SBM-SZN) for DNA sequencing; the Management andEcology of Temperate and Polar Coastal Areas (MECA-SZN) servicefor sampling and providing nutrient data.[SS]

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