CRYPTIC AND PSEUDO-CRYPTIC DIVERSITY IN DIATOMS-WITH DESCRIPTIONS OF PSEUDO-NITZSCHIA HASLEANA SP....

CRYPTIC AND PSEUDO-CRYPTIC DIVERSITY IN DIATOMS—WITH DESCRIPTIONSOF PSEUDO-NITZSCHIA HASLEANA SP. NOV. AND P. FRYXELLIANA SP. NOV.1

Nina Lundholm2

The Natural History Museum of Denmark, University of Copenhagen, Sølvgade 83S, 1307 Copenhagen K, Denmark

Stephen S. Bates

Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick E1C 9B6, Canada

Keri A. Baugh, Brian D. Bill

Marine Biotoxin Program, Environmental Conservation Division, Northwest Fisheries Science Center, NOAA Fisheries, 2725

Montlake Boulevard East, Seattle, Washington 98112, USA

Laurie B. Connell

School of Marine Sciences, University of Maine, Orono, Maine 04469, USA

Claude L�eger

Fisheries and Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick E1C 9B6, Canada

and Vera L. Trainer

Marine Biotoxin Program, Environmental Conservation Division, Northwest Fisheries Science Center, NOAA Fisheries, 2725,

Montlake Boulevard East, Seattle, Washington 98112, USA

A high degree of pseudo-cryptic diversity wasreported in the well-studied diatom genus Pseudo-nitzschia. Studies off the coast of Washington Staterevealed the presence of hitherto undescribeddiversity of Pseudo-nitzschia. Forty-one clonal strains,representing six different taxa of the P. pseudodelica-tissima complex, were studied morphologically usingLM and EM, and genetically using genes from threedifferent cellular compartments: the nucleus(D1–D3 of the LSU of rDNA and internal tran-scribed spacers [ITSs] of rDNA), the mitochondria(cytochrome c oxidase 1), and the plastids (LSU ofRUBISCO). Strains in culture at the same time wereused in mating studies to study reproductive isola-tion of species, and selected strains were examinedfor the production of the neurotoxin domoic acid(DA). Two new species, P. hasleana sp. nov. andP. fryxelliana sp. nov., are described based on mor-phological and molecular data. In all phylogeneticanalyses, P. hasleana appeared as sister taxa to aclade comprising P. calliantha and P. mannii,whereas the position of P. fryxelliana was moreuncertain. In the phylogenies of ITS, P. fryxellianaappeared to be most closely related to P. cf. turgidu-la. Morphologically, P. hasleana differed from mostother species of the complex because of a lower

density of fibulae, whereas P. fryxelliana had fewersectors in the poroids and a higher poroid densitythan most of the other species. P. hasleana did notproduce detectable levels of DA; P. fryxelliana wasunfortunately not tested. In P. cuspidata, productionof DA in offspring cultures varied from higher thanthe parent cultures to undetectable.

Key index words: cox1; cryptic; diatom; domoicacid; ITS; Pseudo-nitzschia; rbcL

Abbreviations: CBCs, compensatory base changes;cox1, cytochrome c oxidase 1; DA, domoic acid;HCBCs, hemi-CBCs; ITS, internal transcriberspacer; MP, maximum parsimony; NJ, neighbor-joining method; RBA, receptor-binding assay;rbcL, LSU of RUBISCO; SNP, single nucleotidepolymorphism

Identification and delineation of diatoms isbecoming an even more demanding task with therecognition that morphology may mask greatgenetic diversity and that cryptic and pseudo-crypticspecies are a more commonly encountered phenom-enon than hitherto considered. Cryptic species aregenetically different, but morphologically identicalspecies, whereas pseudo-cryptic species, apart fromthe genetic diversity, show minor morphologicaldifferences that are only detected by detailed

1Received 28 March 2011. Accepted 31 August 2011.2Author for correspondence: e-mail [email protected].

J. Phycol. 48, 436–454 (2012)� 2012 Phycological Society of AmericaDOI: 10.1111/j.1529-8817.2012.01132.x

436

examination (Mann and Evans 2007). By combiningmorphological and molecular data, cryptic andpseudo-cryptic species have been recognized inmany different groups of algae (e.g., Lundholmet al. 2003, Saez et al. 2003, Sarno et al. 2005,Boenigk et al. 2006, Medlin and Zingone 2007). Inan evolutionary context, cryptic and pseudo-crypticspecies may represent species that (i) have notevolved evident morphological differences becausethere is no selective pressures on morphology (Bick-ford et al. 2007), (ii) are under selection that pro-motes morphological stability (Bickford et al. 2007),or (iii) represent examples of convergent morpho-logical evolution (Potter et al. 1997). Cryptic orpseudo-cryptic species are important to identify, notonly from a taxonomic point of view, but alsoecologically, as they may differ with respect to physi-ological traits (e.g., growth rate, nutrient uptake,toxin production; see, e.g., Gallagher 1982, Woodand Leatham 1992, Quijano-Scheggia et al. 2009,Thessen et al. 2009). The importance of identifyingcryptic species is understood in terrestrial biology,where cryptic species complexes have been discov-ered—for example, in parasitic wasps that are usedfor biological control, in human and crop plantpathogens, in malaria mosquitoes that have differ-ent host preferences, and in poisonous snakeswhere the importance of cryptic species is relevantfor snakebite treatment and the production of anti-venom serum (see references in Bickford et al. 2007).

Diatoms are ecologically widespread and are con-sidered among the most species-rich group of algae(Mann 1999). Cryptic and pseudo-cryptic diatomspecies are now found in genera like SellaphoraMereschkowsky (Mann et al. 2004), SkeletonemaGreville (Gallagher 1982, Sarno et al. 2005, Zingoneet al. 2005), Cyclotella (Kutzing) Brebisson (Beszteriet al. 2007), and Pseudo-nitzschia H. Peragallo 1899(Lundholm et al. 2003, 2006, Orsini et al. 2004,Amato et al. 2007, Quijano-Scheggia et al. 2009).These genera represent the best-studied among dia-toms, indicating that cryptic ⁄ pseudo-cryptic speciesare widespread and that more cryptic species areexpected to be discovered in the future.

The genus Pseudo-nitzschia has attracted attentionbecause at least 12 of its 30+ species can producedomoic acid (DA) (Trainer et al. 2008, 2012, Lund-holm 2011). A number of pseudo-cryptic specieshave been described in Pseudo-nitzschia: P. arenysen-sis, P. caciantha, P. calliantha, P. decipiens, P. dolorosa,P. mannii, and three varieties of P. pungens (Lund-holm et al. 2003, 2006, Orsini et al. 2004, Amatoet al. 2007, Amato and Montresor 2008, Casteleynet al. 2008, Churro et al. 2009, Quijano-Scheggiaet al. 2009). The genus Pseudo-nitzschia harbors evenmore diversity than presently described, as morpho-logical variation that does not agree with anypresent species description has been detected (e.g.,Lundholm et al. 2003, Kaczmarska et al. 2005,Sahraoui et al. 2009). Similarly, genetic diversity

revealed by sequencing strains (Stehr et al. 2002) orby applying genus-specific primers to environmentalsamples (McDonald et al. 2007) shows that theintrageneric and intraspecific diversity is greaterthan presently recorded.

The observation of field samples off the coast ofWashington State revealed the presence of hithertoundescribed morphotypes of Pseudo-nitzschia similarto P. calliantha. This called for a thorough study of thediversity around P. calliantha. Hence, clonal strainsisolated off the Washington coast were studied mor-phologically and genetically, and were used in matingstudies as well as examined for the production of DA.

MATERIALS AND METHODS

Sampling and culturing. The strains (Table 1) were estab-lished from field samples using single-cell isolation techniquesand maintained, depending on the isolate, at 15�C or 20�C at16:8 or 10:14 h light:dark (L:D) photoperiods and at a photonflux density of 20–100 lmol photons Æ m)2 Æ s)1 provided bycool-white fluorescent lamps. Growth media were f ⁄ 2 (Guillardand Ryther 1962), amended with 107 lM Si and 10)8 M Se, orL1 (Guillard and Hargraves 1993), containing a 3x highersilicate concentration.

Molecular methods. Cells were harvested by centrifugation,frozen at )20�C, and afterward the DNA was extractedfollowing Lundholm et al. (2003). Four different regions wereamplified: The ITS1-5.8S-ITS2 region of the rDNA was ampli-fied using primers 1380F,ITS4 and ITS055R and sequencedusing ITS1,1400F (White et al. 1990, Lundholm et al. 2003)and ITS4. The D1–D3 region of the LSU rDNA was amplifiedusing the primers D1R-F, D3B-R for PCR plus D2C forsequencing (Lundholm et al. 2002). The LSU of RUBISCO(rbcL) was amplified using the primers DPrbcL1 and DPrbcL7(Daugbjerg and Andersen 1997) plus rbcL 1F and rbcL 1R(Amato et al. 2007) for sequencing. Parts of the mitochondrialcytochrome c oxidase 1 (cox1) were amplified using either theprimers LCO1490 and HCO2198 (Folmer et al. 1994) or GazF2and KEdtmR (Evans et al. 2007) or COXF (Iwatani et al. 2005)and DM1R (Kaczmarska et al. 2008) for PCR and sequencing.

Sequences similar to P. fryxelliana and P. hasleana foundusing the BLAST algorithm (Altschul et al. 1997) in GenBankwere included in the alignments (see Table S1 in the supple-mentary material) with some of the presently publishedtaxonomical units of Pseudo-nitzschia.

The ITS rDNA sequences were aligned using Clustal W(Thompson et al. 1994) and adjusted using information onsecondary structure (see below). The alignment included 927positions of which 620 were unambiguous and analyzed. Aninitial alignment (210 taxa) was analyzed using neighborjoining (NJ) in PAUP* version 4.0b.8 (Swofford 2003) andreduced to the final alignment (53 taxa). Separate analyses ofITS2 were also performed. The ITS analyses were unrooted.

The LSU rDNA sequences were aligned using Clustal W andmanually corrected using information on secondary structure(Ben Ali et al. 1999, Wuyts et al. 2004). Of the 829 alignednucleotide positions (including gaps), 783 were consideredunambiguous and analyzed. An initial alignment (120 taxa)was, after phylogenetic analyses using NJ in PAUP*, reduced tothe final alignment of 53 taxa with eight outgroup taxa (seeTable S1).

The rbcL sequences were aligned using Clustal W. Thealignment included 22 taxa and 1,484 nucleotide positions,with Nitzschia frustulum as outgroup taxon. The cox1 alignment,aligned using Clustal W, included 10 taxa and 648 nucleotidepositions with three outgroup taxa.

CRYPTIC DIVERSITY IN PSEUDO-NITZSCHIA 437

A concatenate dataset using sequences of strains for whichLSU, ITS, and rbcL were sequenced was gathered for 10 speciesthat were morphologically or phylogenetically similar toP. hasleana and P. fryxelliana (see Table S1). Apart fromanalyses on the concatenate dataset, the parts LSU, ITS,ITS2, and rbcL were also analyzed separately.

Bayesian analyses were performed using MrBayes 3.1.2(Ronquist and Huelsenbeck 2003), using four chains run for1,200,000 generations (rbcL and cox1) or 2,000,000 genera-tions (LSU, ITS, and concatenate). The temperature was setto 0.2, except the LSU dataset (0.1). Sample frequency was100, and the number of burn-in generations was 3,000, exceptfor the ITS and the LSU dataset (5,000). All distance,parsimony, and likelihood analyses were performed usingPAUP* version 4.0b.8 (Swofford 2003). Maximum-parsimony(MP) analyses were done with 1,000 heuristic searches (rbcL,cox1, ITS, and concatenate) or 100 (LSU). All distanceanalyses used NJ (1,000 replicates) with the GTR (generaltime reversible) model. The optimal model for ML analyseswas found with a 99% level of significance using Modeltest

version 3.7 (Posada and Crandall 1998); the proposed modelsused for the ML analyses of all dataset included 1,000 (cox1)or 100 (rbcL, concatenate, ITS, LSU) random additionreplicates. In all NJ analyses and the MP and ML analyses ofcox1, rbcL, and concatenate datasets, we made 1,000 boot-strap replicates; 100 were used for MP and ML analyses of ITSand LSU datasets.

The secondary structure of ITS was predicted using themfold server at http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi (Zuker 2003). Helices were rec-ognized by comparing the ITS regions of several Pseudo-nitzschia species. The helices were named according to Maiand Coleman (1997) and Amato et al. (2007). The ITS2 ofP. calliantha, P. fryxelliana, P. hasleana, and P. mannii wascompared to identify compensatory base changes (CBCs)(changes of base pairs at both sides of a helix, which thusconserve the pairing) and hemi-CBCs (HCBCs) (changes ofbase pairs at one side of a helix).

Morphological studies. The cultures were studied using LM(Olympus BX60 microscope with an Olympus DP10 camera

Table 1. Strain designation, isolation location and date, and accession numbers for cultures of Pseudo-nitzschia establishedor acquired for the present study.

Species Strain designation

Origin of strain

Location Date Provided by

P. calliantha CL-187 Miramichi Bay (New Brunswick), Canada Sept 30, 2002 C. Leger & S. BatesCL-188 Miramichi Bay (New Brunswick), Canada Sept 30, 2002 C. Leger & S. BatesNCP-1 North Carolina, USA June 22, 2005 S. MortonNCP-2 North Carolina, USA June 22, 2005 S. MortonNCP-4 North Carolina, USA June 22, 2005 S. MortonNCP-5 North Carolina, USA June 22, 2005 S. MortonNL1 The Sound, Denmark Sept 28, 2005 N. LundholmNL2 The Sound, Denmark Sept 28, 2005 N. LundholmNL4 The Sound, Denmark Sept 28, 2005 N. LundholmNL5 The Sound, Denmark Sept 28, 2005 N. LundholmNL7 The Sound, Denmark Sept 28, 2005 N. LundholmNL10 The Sound, Denmark Sept 28, 2005 N. LundholmICL-1 Bizerte Lagoon, Tunisia Oct 3, 2005 I. SahraouiPN-13 Chesapeake Bay (Maryland), USA Oct 26, 2005 A. Thessen

P. cuspidata NWFSC 189 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerNWFSC 190 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerNWFSC 191 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerNWFSC 193 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerNWFSC 194 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerCLN-36 Cross between NWFSC 190 and 191 Feb 11, 2005 C. Leger & S. BatesCLN-37 Cross between NWFSC 190 and 191 Feb 11, 2005 C. Leger & S. BatesCLN-38 Cross between NWFSC 190 and 191 Feb 11, 2005 C. Leger & S. BatesCLN-39 Cross between NWFSC 190 and 191 Mar 4, 2005 C. Leger & S. BatesCLN-41 Cross between NWFSC 190 and 191 Mar 4, 2005 C. Leger & S. BatesCLN-43 Cross between NWFSC 190 and 191 Mar 4, 2005 C. Leger & S. BatesCLN-44 Cross between NWFSC 190 and 191 Mar 4, 2005 C. Leger & S. BatesCLN-45 Cross between NWFSC 190 and 191 Mar 4, 2005 C. Leger & S. Bates

P. fryxelliana NWFSC 239 Teawhit Head, WA, USA June 20, 2006 B. Bill & V. TrainerNWFSC 241 Teawhit Head, WA, USA June 20, 2006 B. Bill & V. TrainerNWFSC 242 Teawhit Head, WA, USA June 20, 2006 B. Bill & V. Trainer

P. hasleana NWFSC 095 Sequim Bay, WA, USA Aug 8, 2002 B. Bill & V. TrainerNWFSC 184 Twin Harbors, WA, USA May 18, 2004 B. Bill & V. TrainerNWFSC 185 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerNWFSC 186 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerNWFSC 187 Coastal WA, NE Pacific Ocean Sept. 20, 2004 B. Bill & V. TrainerNWFSC 252 Teawhit Head, WA, USA July 6, 2006 B. Bill & V. TrainerNWFSC 253 Teawhit Head, WA, USA July 6, 2006 B. Bill & V. TrainerOFP41014-2 Ofunato Bay, Iwate, Japan Oct 14, 2004 Y. Kotaki

P. lineola NWFSC 188 Coastal WA, NE Pacific Ocean Sept 20, 2004 B. Bill & V. TrainerP. cf. turgidula NWFSC 251 Ocean Station Papa, NE Pacific Ocean May 25, 2006 B. Bill & V. Trainer

NWFSC 255 Ocean Station Papa, NE Pacific Ocean June 6, 2006 B. Bill & V. Trainer

WA, Washington State, USA.Strain designations in bold indicate that the strain was sequenced for the present study.

438 NINA LUNDHOLM ET AL.

and Olympus DP-soft version 3.0, Olympus, Hamburg,Germany). Samples were cleaned following Lundholm et al.(2002). For TEM, drops of cleaned material were placed onFormvar-coated copper grids, dried, and studied in JEOL-100SX and JEM-1010 electron microscopes (Jeol, Tokyo,Japan). Morphometric measurements were performed onTEM images. The valve width and the density of interstriae,fibulae, poroids, and band striae (Table 2) were measured atthe middle of the valve. Morphometrics and structure of theband striae refer to the valvocopula unless otherwise stated.

Mating experiments. Crosses were made between pairs ofstrains isolated from the different locations, when strains wereavailable at the same time (Table 3); methodology according toDavidovich and Bates (1998), with cells growing in f ⁄ 2 mediumat 20�C, 10:14 L:D and �100 lmol photons Æ m)2 Æ s)1. Thecrosses were made during February to May 2005, using 0.3–0.8 mL of 3- to 6-day-old (exponential phase) cultures. Petridishes containing the crosses were examined for evidence ofsexual activity (presence of gametes, auxospores, and largeinitial cells) on days 1–5 and 7. Crosses were carried out fromone to six times each (Table 3), and it was ensured that onlycells within the cell size range for sexualization were used, asevidenced by the successful crosses observed within the samespecies of opposite mating type.

Toxin analyses. DA was analyzed using one or more of threemethods (see Table 4): by receptor-binding assay (RBA) (Baughet al. 2004) (limit of quantification in culture = 0.6 ng Æ mL)1;detection limit in culture = 0.3 ng Æ mL)1); by competitiveenzyme-linked immunosorbent assay (cELISA) (Litaker et al.2008) (detection limit in culture �4 pg Æ mL)1) or by HPLCusing the FMOC fluorescence derivatization technique (HPLC-FMOC) (Pocklington et al. 1990) (detection limit�2 ng Æ mL)1).

For cultures analyzed by the RBA or ELISA, growth exper-iments were initiated by transferring �1 mL of culture (orappropriate volume to yield an initial density of 20–200 cells ÆmL)1) to 100 mL of medium in a 250 mL flask. Once the cellculture reached the midexponential phase, �5 mL (or appro-priate volume to attain an initial cell density of 20–200 cellsÆ mL)1) of culture was transferred to 1.5 L of medium in a 2 Lborosilicate flask. The culture was sampled several times ateach growth stage to obtain total cell counts and particulateDA, quantified using the RBA or ELISA. Samples wereprocessed for DA detection first by filtering 100 mL of culturethrough a Nuclepore HA filter (0.45 lm pore size, Nuclepore,Pleasanton, CA, USA). Filters were minced in 4 mL of distilledwater with a thin metal spatula and sonicated for 2 h in a bathsonicator, to lyse cells.

The RBA followed the method by Van Dolah et al. (1997)and Baugh et al. (2004). All samples were analyzed in triplicateand mean values were used for quantification of unknowns.

For single toxicity measurements of strains, single 20 mLaliquots from stationary-phase (days 22–32; Table 4) culturesgrowing in f ⁄ 2 medium at 20�C, 10:14 L:D and �100 lmolphotons Æ m)2 Æ s)1 were frozen until analyzed. The sampleswere sonicated using a high-intensity ultrasonic processor(Vibra-Cell, Meyrin ⁄ Satigny, Switzerland), followed by filtrationthrough a disposable polycarbonate filter (0.45 lm pore size)to remove the debris. DA in the whole culture (cells plusmedium) was then analyzed using HPLC-FMOC. To verify thatthe HPLC-FMOC peak of positive samples was DA, the sampleswere spiked with 31.4 ng DA Æ mL)1; this gave a retention timeidentical to the original sample and also increased thesuspected DA peak by the value of the added DA.

RESULTS

Forty-one different monoclonal strains morpho-logically similar to P. calliantha ⁄ P. caciantha were

established or acquired; 16 of these were fromcoastal Washington State (Table 1). Several strainswere morphologically and genetically identical toP. calliantha, corresponding to the description inLundholm et al. (2003), although slightly higherdensities of interstriae and band striae were alsoobserved (Table 2). Five strains were morpho-logically identical to P. cuspidata as emended inLundholm et al. (2003) and further described inTrainer et al. (2009). One strain was identified asP. lineola as in Hasle (1965) and will be described indetail below. Finally, several strains were found tobelong to two previously undescribed species; thesewill be described below as P. hasleana sp. nov. andP. fryxelliana sp. nov.

Pseudo-nitzschia hasleana Lundholm sp. nov.(Fig. 1, A–M).

Cellulae symmetricae et lanceolatae, aspectu val-vari cingularique apicibus acutis. Longitudo cellula-rum 37–79 lm, latitudo valvarum 1.5–2.8 lm.Raphe excentrica, in medio nodulo centrali divisa.Fibulae nonnumquam irregulariter dispersae, 13–20per 10 lm, interstriae 31–40 per 10 lm. Fibulae inLM visibiles. Striae una serie pororum rotundorum,5–6 pori per 1 lm. Membrana pororum 2–6 seg-mentis magnitudinis variabilis. Pallium valvae simile,2–3 poris altum. Cingulum copulis tribus apertis.Valvocopula 37–46 striis per 10 lm, unaquaequestria 2 poris lata et 3–6 poris alta. Copula secundastriis 2 poris latis et 3–4 poris altis. Copula tertiastriis 1–2 altis et 1–2 poris latis.

Holotype : Slide of the strain NWFSC 252 depos-ited at the herbarium of the National HistoryMuseum of Copenhagen, Denmark, registered asCAT2464.

Isotype : Fixed material of NWFSC 252 depositedat the herbarium of the National History Museumof Copenhagen, Denmark, registered as CAT2465.

Epitype : Molecular characterization: DNAsequences for ITS1, 5.8S, and ITS2 of nuclear rDNAof strain NWFSC 252 deposited in GenBank usingaccession number JN085962.

Type locality : Isolated from the coastal PacificOcean off Teawhit Head, Washington State, USA,on July 6, 2006.

Etymology : The species is named in honor ofGrethe Rytter Hasle, Norway, because of her contri-butions to diatom taxonomy in general, and Bacil-lariaceae and Pseudo-nitzschia in particular.

Molecular signature : Synapomorphy in helix IV ofITS2 of the nuclear rDNA: 5¢-CTATGATCTAGG-TATTGGATTATCCGA-3¢ (27 bp). This sequenceincludes a CBC as well as a HCBC and a SNP (singlenucleotide polymorphism) that differentiates P. has-leana from P. calliantha and P. mannii. A test for theuniqueness of the diagnostic signature of P. hasleanais, was confirmatory. The sequence was found to


Table 2. Morphometric data obtained from LM and TEM examination of Pseudo-nitzschia calliantha, P. fryxelliana, P. hasleana, and P. lineola.

Species Reference Valve shapeWidth(lm)

Length(lm)

Interstriaein 10 lm

Fibulaein 10 lm

Poroidsin 1 lm

Sectorsin poroids

Band striaein 10 lm

Band striastructure

P. calliantha Present study Linear 1.3–1.81.6 ± 0.2

nd 35–4138.0 ± 2.0

18–2420.3 ± 1.6

4–54.9 ± 0.4

7–108.5 ± 1.0

42–5046.6 ± 2.5

2–3 x var

Lundholm et al. (2003) Linear 1.4–1.8 41–98 34–39 15–22 4–6 7–10 42–48 2–3 x var

Amato et al. (2007) nd 1.7–2.42.1 ± 0.2

nd 32–3936.8 ± 1.7

18–2419.7 ± 1.4

4–64.7 ± 0.4

3–105.6 ± 1.4

nd nd

P. fryxelliana Present study Lanceolate 2.1–2.52.2 ± 0.2

30–5437.6 ± 8.7

34–4036.7 ± 1.4

(17)18–2520.6 ± 1.9

5–6(7)5.7 ± 0.5

(1) 2–32.3 ± 0.6

41–5044.1 ± 2.3

2 · 1–3

P. hasleana Present study Lanceolate 1.5–2.82.0 ± 0.4

37–7953.3 ± 11.2

31–4035.4 ± 2.1

13–2016.3 ± 1.6

5–65.3 ± 0.5

2–63.6 ± 1.2

37–46 (47)42.4 ± 2.3

2 · 3–6

P. lineola Present study Linear-lanceolate 2.0–2.82.4 ± 0.2

71–9583.2 ± 5.9

22–3125.0 ± 2.4

10.5–15.513.2 ± 1.0

3–64.3 ± 0.7

1–2 rowsporoids

22–3425.4 ± 2.8

2–3 · 2–3

Cleve (1897) Linear-lanceolate 2 100–110 24 14 nd nd nd nd

Hasle (1965) Linear-lanceolate 1.8–2.7 56–112 22–28 11–16 3–7 1–2 rowsporoids

nd nd

P. caciantha Lundholm et al. (2003) Lanceolate 2.7–3.5 53–75 28–31 15–19 3.5–5 4–5 33–38 2 x 3–5

Amato et al. (2007) nd 2.2–3.02.6 ± 0.2

nd 33–3734.9 ± 1.5

18–2320.7 ± 1.5

3–54.6 ± 0.1

2–63.1 ± 1.0

nd nd

P. cuspidata Lundholm et al. (2003) Lanceolate 1.4–2.0 30–73 35–44 19–25 4–6 2 47–53 Splitporoids

P. pseudodelicatissima Lundholm et al. (2003) Linear 0.9–1.6 54–87 36–43 20–25 5–6 2 48–55 Splitporoids

Amato et al. (2007) nd 1.5–1.91.8 ± 0.1

nd 34–4541.8 ± 1.9

20–2923.5 ± 1.8

4–75.6 ± 0.6

2 nd nd

P. mannii Amato andMontresor (2008)

Linear 1.7–2.62.1 ± 0.2

nd 30–4035.8 ± 2.1

17–2520.2 ± 2.0

4–64.8 ± 0.3

2–73.5 ± 1.0

47 2 · 4

nd, no data; var, variable.Data are given as minimum and maximum range (above), and mean value ± standard deviation (below).Data in parentheses indicate seldom-recorded measurements. For comparison, data on band striae in P. mannii are taken from fig. 5 in Amato and Montresor (2008).

440N

INA

LU

ND

HO

LM

ET

AL

.

Table 3. Matrix of crosses. Number of crosses performed among the different Pseudo-nitzschia strains.

Species Strain

P. calliantha P. cuspidata P. hasleana P. lineola

CL-187

CL-188

NCP-1

NCP-2

NCP-4

NCP-5 NL4 NL5

ICL-1

PN-13

NWFSC189

NWFSC190

NWFSC191

NWFSC192

NWFSC193

NWFSC194

NWFSC184

NWFSC185

NWFSC186

NWFSC187 OFP41014-2

NWFSC188

P. calliantha CL-187 –P. calliantha CL-188 2 –P. calliantha NCP-1 4 1 –P. calliantha NCP-2 3 2 2a –P. calliantha NCP-4 1 1 2a 6 –P. calliantha NCP-5 1 1 2a 6 6 –P. calliantha NL4 1 2 1 2 1 1 –P. calliantha NL5 2 1 2 1 1 1 4 –P. calliantha ICL-1 nt nt 2 2 2 2 nt nt –P. calliantha PN-13 nt nt 2 2 2 2 nt nt 4 –P. cuspidata NWFSC

189nt nt nt nt nt nt nt nt nt nt –

P. cuspidata NWFSC190

nt nt nt nt nt nt nt nt nt nt 1 –


nt nt nt nt nt nt nt nt nt nt nt 4b –


nt nt nt nt nt nt nt nt nt nt nt nt nt –


nt nt nt nt nt nt nt nt nt nt 1 2 4b nt –


nt nt nt nt nt nt nt nt nt nt 1 2 4b nt 4 –

P. hasleana NWFSC184

nt nt nt nt nt nt nt nt nt nt 2 2 2 2 2 2 –


nt nt nt nt nt nt nt nt nt nt 2 nt nt nt nt nt 4 –


nt nt nt nt nt nt nt nt nt nt 2 nt nt nt nt nt 2 4 –


nt nt nt nt nt nt nt nt nt nt 2 nt nt nt nt nt 2 4 4 –

P. hasleana OFP41014-2 1 1 1 1 2 2 2 2 nt nt nt nt nt nt nt nt nt nt nt nt –P. lineola NWFSC

188nt nt nt nt nt nt nt nt nt nt 2 nt nt nt nt nt 2 2 2 2 nt –

aOnly a few gametes produced.bAll sexual stages produced and successful isolates obtained.nt, crosses not made.Bold values = successful sexual reproduction observed.

CR

YP

TIC

DIV

ER

SIT

YIN

PS

EU

DO

-NIT

ZS

CH

IA441

have an E-value of 2e-05 for two P. hasleanasequences in GenBank (GQ228394 and AM183801).The next five hits had E-values of 3.7 and above andwere fungi, higher plants, and bacteria.

Morphology: The cells are lanceolate in both gir-dle and valve view (Fig. 1, A–C). They are 37–79 lmlong and 1.5–2.8 lm wide in valve view (Table 2).The valve ends are pointed in both girdle and valveview (Fig. 1, A–E). The fibulae are not always regu-larly spaced (Fig. 1, D and E) and are seen usingLM. The distance between the two central fibulae islarger than between the other fibulae and containsa central nodule (Fig. 1, A, G, and L). The densitiesof the fibulae and interstriae are 13–20 and 31–40in 10 lm, respectively (Table 2). The striae containone row of more or less rounded poroids (Fig. 1, G,L, and M), 5–6 poroids in 1 lm. Each poroid is splitin 2–6 sectors (Fig. 1, G, K, L, and M) and the num-ber may vary among valves of a clonal culture andeven among the poroids within a single stria (Fig. 1,K and L). Similarly, the size of the sectors is greatlyvariable. The perforation pattern of each sector ishexagonal. The mantle is structured as the valve,being 2–3 poroids high (Fig. 1G). The girdle mostoften comprises three bands. The valvocopula

contains 37–46 band striae in 10 lm. Each bandstria is 2 poroids wide and 3–6 poroids high (Fig. 1,F, J, and M). The next two bands contain striae witha decreasing number of poroids in the height; thesecond band often being 2–4 poroids high (Fig. 1J)and the third band most often being 1–2 poroidshigh and not always 2 poroids wide (Fig. 1H). Theperforation of the girdle bands is also hexagonal(Fig. 1F). An abnormal band pattern was sometimesseen where the band pattern was longitudinallyinterrupted (Fig. 1I). This was mainly observed inolder cultures and accompanied other deformationson the valve.

Taxonomic remarks: The above description isbased on culture material of cultures listed in boldin Table 1.

Pseudo-nitzschia fryxelliana Lundholm sp. nov.(Fig. 2, A–K).

Cellulae aspectu cingulari symmetricae et lanceo-latae. Cellulae aspectu valvari lanceolatae in specimi-nibus magnis, ad rhomboides in speciminibusminoribus. Apices cellularum late rotundati. Longi-tudo cellularum 40–54 lm, latitudo valvarum 2.1–2.5 lm. Raphe excentrica medio nodulo centrali

Table 4. Domoic acid (DA) content of Pseudo-nitzschia species in stationary-phase batch culture as analyzed by two differentmethods: HPLC-FMOC (for DA in cells plus medium) and either cELISAa or receptor binding assay (RBAb) (both for DAin cells only).

Species Strain

HPLC-FMOC cELISAa or RBAb

Strainage (d)

DA(ng Æ mL)1)

Dayharvested

Strainage (d)

DA(ng Æ mL)1)

DA(fg Æ cell)1)

Dayharvested

P. calliantha CL-187 63 ND 28P. calliantha CL-188 63 ND 28P. calliantha NCP-1 156 ND 29P. calliantha NCP-2 156 ND 29P. calliantha NCP-4 156 ND 29P. calliantha NCP-5 156 ND 29P. calliantha NL4 310 ND 29P. calliantha NL5 310 ND 29P. calliantha ICL-1 53 ND 29P. calliantha PN-13 30 ND 29P. cuspidata NWFSC 189 204 ND 32 224 3.1b 26.2 12P. cuspidata NWFSC 190 122, 148, 204 ND 22, 29, 29 344 0.8a 31.2 8P. cuspidata NWFSC 191 148, 204 ND 29, 29 170 5.4b 19.9 19P. cuspidata NWFSC 193 122, 148, 204 ND 22, 29, 29 343 0.1b 2.9 7P. cuspidata NWFSC 194 122, 148, 204 ND 22, 29, 29P. cuspidata CLN-36 60 ND 29P. cuspidata CLN-37 60 ND 29P. cuspidata CLN-38 60 22.8 29P. cuspidata CLN-39 39 19.1 21P. cuspidata CLN-41 39 ND 21P. cuspidata CLN-43 39 ND 21P. cuspidata CLN-44 39 ND 21P. cuspidata CLN-45 39 ND 21P. hasleana NWFSC 184 329 ND 32P. hasleana NWFSC 185 122, 148, 204 ND 22, 29, 29 845 NDa ND 10P. hasleana NWFSC 186 122, 148, 204 ND 22, 29, 29P. hasleana NWFSC 187 122, 148, 204 ND 22, 29, 29 170 <LoQb <LoQ 17P. lineola NWFSC 188 148, 204 ND 29, 29

When two or three strain ages and days harvested are given, samples for toxin analysis were taken on those harvest days. Whenno day harvested is given, toxin samples were taken during stationary phase. < LoQ = toxin detected but not quantifiable.ND = toxin below the limit of detection (see text); blank cell = not tested.


divisa. Fibulae nonnumquam irregulariter dispersae,18–25 per 10 lm, interstriae 34–40 per 10 lm.Fibulae in LM visibiles. Unaquaeque stria serie una

pororum rotundorum ad quadratorum, 5–6 (non-numquam 7) per 1 lm. Membrana pororum in 1–3(raro 0–4) sectores divisa, saepe vix visibiles. Pallium

FIG. 1. Pseudo-nitzschia hasleana sp. nov. (A) Valve width mantle. (B and C) Valve of different length and shape. (D and E) Valve endsof slightly different shape. (F) Details of valvocopula, showing pores. Note part of valve at left side. (G) Stria and poroid structure of valveand mantle. Note central nodule. (H) Details of third girdle band. (I) Abnormal girdle band. (J) Details of valvocopula (right; V) and sec-ond girdle band (left; II). (K) Details of poroid hymnate structure. (L) Detail of valve showing variation in poroids within a single valve.(M) Valve stria structure. TEM micrographs. (A, G, H–J) NWFSC 253. (B and L) NWFSC 252. (C) NWFSC 186. (D) NWFSC 187. (E)NWFSC 184. (F and K) NWFSC 185. (M) OFP41014-2.


valvae superficei valvae simile, 2–3 poris altum.Cingulum copulis 4 apertis. Valvocopula striis 41–50per 10 lm. Unaquaeque stria satis variabilis, poris 2lata et 1–3 alta, ad irregulariter in duas sive plures

partes divisa. Copula secunda aut tertia striis divisis,in numero partium decrescentibus, parte abvalvarisine aut cum paucis perforationibus. Copula quartanon perforata.

FIG. 2. Pseudo-nitzschia fryxelliana sp. nov. (A–D) Whole valves. (E) Part of valve showing stria structure and mantle. Note central nodule.(F) Valve end. Note striae lacking poroids. (G and H) Poroid structure. Note missing poroids and sectors. (I) Poroid hymenate structure.(J) Valvocopula striae with details of poroids. (K) Girdle bands. Arrows indicate borders of bands. V, II and III indicate valvocopula, secondand third band, respectively. TEM micrographs. (A, E, F, H–K) NWFSC 242. (B and G) NWFSC 241. (C and D) NWFSC 239.


Holotype: Slide of the strain NWFSC 241 depos-ited at the herbarium of the National HistoryMuseum of Copenhagen, Denmark, registered asCAT2466.

Isotype: Fixed material of NWFSC 241 depositedat the herbarium of the National HistoryMuseum of Copenhagen, Denmark, registered asCAT2467.

Epitype: Molecular characterization: DNAsequences for ITS1, 5.8S, and ITS2 of nuclear rDNAof strain NWFSC 241 deposited in GenBank usingaccession number JN050288.

Type locality: Isolated from the coastal PacificOcean off Teawhit Head, Washington State, USA,on June 20, 2006.

Etymology: The species is named in honor ofGreta A. Fryxell, USA, because of her contributionsto diatom taxonomy and ecology.

Molecular signature: Synapomorphy in helix IV ofITS2 of the nuclear rDNA: 5¢-CTAAGTCTGGCTTGCAGTGGTGAACATTAGTTTATCACCTGCTCG-3¢(45 bp). This sequence includes CBCs and HCBCs,differentiating it from the morphologically mostsimilar species P. mannii, P. calliantha, and P. hasleana.

FIG. 3. Pseudo-nitzschia lineola, TEM micrographs. (A) Whole valve. (B–D) Valve ends. Note differences in striae and poroids. (E) Partof valve with a shift in stria structure. (F) Valve end and stria structure variation. (G) Central part of valve with central nodule. (H) Poroidstructure, mantle, and valvocopula. (I) Second and third girdle band. (J) Valvocopula. Note stria structure. (K) Valve and valvocopula.Note band stria structure. (A–K) NWFSC 188.


A test for the uniqueness of the diagnostic signatureof P. fryxelliana is, was confirmatory. In GenBank,the next closest hits had E-values of 2.9 and were allmammals.

Morphology: The cells are lanceolate in girdleview, and in valve view they are lanceolate in longerspecimens, tending towards being more rhomboidin smaller specimens (Fig. 2, A–D). The valve endsare broadly pointed (Fig. 2, A–D, and F). They are40–54 lm long and 2.1–2.5 lm wide (Table 2). Thefibulae are not always regularly spaced. The two cen-tral fibulae have a larger interspace and the raphe ishere interrupted by a central nodule (Fig. 2E). Thedensities of fibulae and interstriae are 18–25 and34–40 in 10 lm, respectively (Table 2). The striaecontain one row of rounded to squared poroids thatare more or less confluent, 5–6 (sometimes 7)poroids in 1 lm (Fig. 2, E and I). Each poroidmost often contains 1–3 sectors (Fig. 2, E–I), butsometimes 4 sectors are seen (Fig. 2, E and I). Inaddition, some striae are simply composed of morelightly silicified areas without any perforations(Fig. 2, F and H). The exact number of sectors mayoften be difficult to discern as the striae are lightlysilicified (Fig. 2E). The sections are often of varyingsize (Fig. 2I) and the perforations within them arearranged in a hexagonal pattern (Fig. 2I). The man-tle is often 1–2 poroids high and has compositionsimilar to the valve (Fig. 2E). The girdle comprises3–4 bands (Fig. 2K). The valvocopula contains onelongitudinal row of 41–50 band striae in 10 lm.The structure of the striae on the valvocopula isvariable, even within the same band, varying frombeing 2 poroids wide and 1–3 poroids high to beingirregularly divided into two or more parts (Fig. 2, Jand K). In the second and the third band, the bandstriae are split in a decreasing number of partsand both bands have an abvalvar part that is eitherwithout perforations or contains few transverse slitperforations. The fourth band is not perforated andhas a more silicified line in the middle (Fig. 2K).The bands are of decreasing width in abvalvardirection.

Pseudo-nitzschia lineola (Cleve) (Hasle 1965)Hasle (Fig. 3, A–K).

The cells are lanceolate and more or less sigmoidin girdle view and linear to slightly lanceolate invalve view (Fig. 3A), with a length of 71–95 lm andwidth of 2.0–2.8 lm (Table 2). The ends arepointed in both girdle and valve view (Fig. 3, A–D,and F). The fibulae and interstriae have densities of10.5–15.5 and 22–31 in 10 lm, respectively(Table 2). The distance between the two central fib-ulae is larger and contains a central nodule (Fig. 3,A and G). The overall valve pattern may be differentin one part of the valve compared to other parts ofthe same valve, for example, striae mainly with onerow of poroids in one part of the valve and two rowsin other parts (Fig. 3, E and F). It is not consistent

FIG. 4. Phylogenetic tree of neighbor joining analyses of ITSrDNA, with bootstrap values of neighbor-joining, Bayesian, maximum-parsimony, and maximum-likelihood analyses indicated in that order.The tree is unrooted. Bold branches indicate a bootstrap support on100% in all types of analyses. e indicates bootstrap support on >90%in all analyses, * indicates bootstrap support on >75% in all analyses,d indicates bootstrap support on >75% in at least two of the analyses.

FIG. 5. Phylogenetic tree from Bayesian concatenated analysisof D1-D3 of LSU ITS1, 5.8S, and ITS2 and rbcL, with bootstrapsupport from neighbor-joining (NJ), Bayesian, maximum-parsi-mony (MP), and maximum-likelihood (ML) analyses indicated inthat order. Support from separate analyses of ITS2, ITS, rbcL,and LSU analyses are shown as additional branches. Branches areshown only if bootstrap support is above 80% in three of theanalyses: NJ, MP, ML, and Bayesian analyses of the respectivedataset. The tree is unrooted.


which parts of the valves have a certain pattern.Sometimes, what seems to be a single poroid is splitinto two parts (Fig. 3, B, E, and G). There is hencea transition in stria pattern from one single poroid,to one row of split poroids and further to two rowsof poroids. The transition from mainly one tomainly two rows of poroids is sometimes sharp(Fig. 3, D and E). The size of the poroids is also var-iable (Fig. 3, B, E, and F). The density of the por-oids varies between 3–6 poroids in 1 lm. Themantle is structured as the valve and is 1–2 poroidshigh. The girdle appears to be composed of threegirdle bands. The valvocopula has 22–34 striae in10 lm, with each stria being 3 (seldom 2 or 4) por-oids wide and 2–3 poroids high (Fig. 3, H, J, andK). The next two bands have band striae of decreas-ing width and height (Fig. 3I), with the latter bandsometimes having the band striae reduced to asingle poroid (Fig. 3I).

Mating studies: Not all strains were available atthe same time, so crosses could only be made with aselection of the strains (Table 3). Of the P. callian-tha strains, most crosses did not result in sexualreproduction, as evidenced by the absence ofgametes, auxospores or initial cells. An exceptionwas between strain NCP-1 and strains NCP-2, NCP-4,and NCP-5, in which a few gametes, but no furthersexual stages, were found. None of the crosses wassuccessful between the four P. hasleana strains fromcoastal Washington State, nor between the latterand P. hasleana strain OFP41014-2 from Japan.Other unsuccessful crosses were expected, becausethey were between strains of two different Pseudo-nitzschia species (i.e., between P. calliantha strainsand P. hasleana strain OFP41014-2, and between anytested strains of P. cuspidata and those of P. hasleanaor P. lineola).

Successful crosses were observed between P. cuspi-data strains NWFSC 190, 191, 193, and 194(Table 3), which produced abundant gametes, aux-ospores, and large new cells. The latter were iso-lated and became 10 successfully growing F1generation cultures, eight of which are shown inTable 1. The morphology of these F1 cultures wasnot examined. Later, crosses were made betweenP. cuspidata strains NWFSC 189 and NWFSC 190,192, 193, 194 and F1 progeny CLN-36, 37, 38, 41,and 44. Gametes and then auxospores were pro-duced in crosses between NWFSC 189 versus CLN-37 (progeny of NWFSC 190 vs. 191). Crossesbetween NWFSC 189 versus CLN-44 (progeny ofNWFSC 191 vs. 194) gave large new cells (F2 gener-ation), although isolates of these did not survive.

Toxin analyses: The single samples collected dur-ing the stationary phase and analyzed using HPLC-FMOC showed that for the strains of P. calliantha(CL-187, CL-188, NCP-1, NCP-2, NCP-4, NCP-5, NL4,NL5, ICL-1, PN13), P. hasleana (NWFSC 184, 185,186, 187), P. cuspidata (NWFSC 189, 190, 192, 193,

194), and P. lineola (NWFSC 188), DA was below thelimit of detection (Table 4). The cELISA ⁄ RBA analy-ses on the batch culture experiments showed low lev-els of DA produced by four strains of P. cuspidata(NWFSC 189, 190, 191, 193) from midexponentialphase (not shown). For the P. hasleana strains, DAwas below the detection limit, in agreement with theHPLC-FMOC results. Pseudo-nitzschia cuspidata strainsCLN-38 and CLN-39 (which are progeny of strainsNWFSC 190 and NWFSC 191; see below) produced22.8 and 19.8 ng DA Æ mL)1, respectively, on days 29and 21 of the stationary phase in the single-sampleanalyses (Table 4). Strains CLN-36, 37, 41, 43, 44,and 45 (also progeny of strains NWSC 191 andNWFSC 194), however, produced no detectable DA(Table 4). Unfortunately, no strains of P. fryxellianawere available for the DA analyses.

Phylogenetic inference: The phylogenetic analysesusing LSU, ITS, and rbcL showed in all analysesP. calliantha and P. mannii clustering as highly sup-ported sister taxa, with P. hasleana as the closestrelated species in a common highly supported clade(Figs. 4 and 5). Similarly, a clade comprising P. ca-ciantha and P. cf. subpacifica was highly supported inall analyses. In the ITS analyses, the taxa of thesetwo latter clades (comprising the taxa P. calliantha,P. mannii, P. hasleana, P. caciantha, and P. cf. subpaci-fica) clustered together, supported by bootstrap val-ues of 82, 98, 63, and 61% (NJ, B, MP, ML). In theconcatenate analyses, there was high support forP. caciantha clustering with P. hasleana, P. mannii,and P. calliantha (Fig. 5), also in the analyses of theindividual genes. In analyses of the rbcL dataset,there was low-moderate support for this branchingand no support in the analyses of the LSU dataset.In all analyses, the P. fryxelliana strains appeared asa well-supported end clade that was distantly relatedto P. calliantha, P. mannii, P. hasleana, and P. cacian-tha. The position of P. fryxelliana was more uncer-tain; it varied among the analyses and was notsupported by bootstrap values above 50%, except inthe ITS analyses, where it clustered in a well-sup-ported clade with P. cf. turgidula (Fig. 4) The P. cu-spidata strains grouped in a highly supported cladecomprising strains of P. pseudodelicatissima and P. cu-spidata. The cox1 analyses comprised few taxa, butshowed support for P. hasleana and P. fryxelliana asseparate entities (not illustrated).

The strains of P. hasleana were all identical inrbcL, LSU, and ITS. The ITS sequence of strainIEO-PS50V, designated as P. calliantha in GenBank(AM183801) (Penna et al. 2007), was identical withthe P. hasleana sequences, except for a SNP, a transi-tion in ITS1 (T fi C). The sequence (GQ228394)of strain Ner-D8, designated Pseudo-nitzschia sp.(Orive et al. 2010), was 100% identical with P. hasle-ana. Both clustered in all analyses of ITS with theother P. hasleana strains, supported by at least 90%bootstrap (Fig. 4). They should hence be reassignedto P. hasleana. In cox1, the two sequenced strains of


P. hasleana (NWFSC 186 and NWFSC 252) differedin five transitions: two (GMA) and three (CMT).

The strains of P. fryxelliana were identical in ITS,rbcL, LSU, and cox1. The LSU sequence of strainNWFSC 047, designated as P. pseudodelicatissima(AF440774) (Stehr et al. 2002), was identical to theP. fryxelliana strains and should hence be referred toP. fryxelliana.

The strains of P. calliantha varied in six positionsin ITS1: four of the positions had the same transi-tion (CMT), one position comprised either of threedifferent bases (C, T, G) and one position com-prised one of three other (G, A, C). All the strainswere identical in 5.8S and ITS2. In LSU, the

variation among the strains included one transition(GMA) and three indels of one basepair each.

Calculating distances among the strains in the ITS,dataset revealed distances among P. calliantha strainsbeing larger (0–0.008) than distances among eitherP. hasleana: (0.002) or P. mannii strains (0–0.003).The distances were smaller than those among P. deli-catissima or P. arenysensis strains, being 0–0.022 and0–0.045, respectively. Distances between P. hasleanaand P. mannii (0.074–0.079) were slightly larger thanthose between P. calliantha and P. hasleana (0.062–0.070) and between P. calliantha and P. mannii(0.045–0.052). The distance between P. fryxellianaand P. cf. turgidula was even larger (0.093). For

Wid

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FIG. 6. Morphometric data on (A) valve width, (B) densities of interstriae, (C) fibulae, (D) poroids, (E) number of sectors in the valveporoids, and (F) band striae of species in the pseudodelicatissima complex: P. lineola, P. calliantha, P. hasleana, P. fryxelliana, P. mannii, P. ca-ciantha, and the morphologically similar P. pseudodelicatissima ⁄ P. cuspidata. The data on P. lineola, P. calliantha, P. hasleana, and P. fryxellianaare from the present study; data on P. caciantha and P. pseudodelicatissima, from Amato et al. (2007); and data on P. mannii, from Amatoand Montresor (2008). The density of band striae of P. mannii was measured from fig. 5 in Amato and Montresor (2008).


comparison, the distances between P. delicatissima,and P. micropora (0.038–0.043), P. delicatissima andP. arenysensis (0.062–0.078) and P. arenysensis andP. micropora (0.056–0.069), were similar or smaller.

Secondary structure of ITS2. Folding of the ITS2revealed the typical four-helix structure (I–IV) withthe additional helix IIa, similar to previous results(Amato et al. 2007, Casteleyn et al. 2008, Lundholmand Hasle 2008). Comparisons of the secondarystructure showed the presence of one CBC andseven HCBCs between P. hasleana and each of theother two phylogenetically closely related species(i.e., P. calliantha and P. mannii). The CBC was situ-ated in helix IV (A-U in P. hasleana M C-G in P. cal-liantha and P. mannii), and the HCBCs were foundas: two in helix I, three in helix II, one in helix III,and one in helix IV. The single CBC and six of theHCBCs (three G-UMG-C and three U-AMU-G)were the same when comparing P. hasleana withP. calliantha and P. mannii. The last HCBC differedfor P. hasleana versus P. calliantha (A-UM C-U) andP. hasleana versus P. mannii (A-UM A-C).

Comparison between P. calliantha and P. manniirevealed no CBCs but four HCBCs (two in helix Iand two in helix III), similar to the results of Amatoand Montresor (2008). The species P. fryxelliana,which was more distantly related to the other threespecies according to the phylogenetic studies,showed four CBCs (one in helix III and three inhelix IV) and a varying number of HCBCs (9, 10, or11, spread in all four helices), when compared toP. calliantha, P. mannii, and P. hasleana, respectively.

DISCUSSION

We have delineated six distinct genetic units in asampling of 41 clonal strains, revealing increaseddiversity in the P. pseudodelicatissima complex. Twonew pseudo-cryptic species, P. hasleana and P. fryxelli-ana, are described using morphological, molecular,reproductive, and physiological characters. Theneed for further study is evoked because P. cuspidatastrains show considerable variation in toxicityamong offspring strains and between parents andoffspring. This is the first demonstration of sexualreproduction in P. cuspidata.

Comparison of P. hasleana and P. fryxelliana withsimilar species. The P. pseudodelicatissima complex(P. caciantha, P. calliantha, P. cuspidata, P. pseudodelica-tissima, and P. mannii) is here defined as those speciesthat are more or less lanceolate to linear in valve view,comparatively narrow (�1.5–2.5 lm) and possess acentral nodule and uniseriate striae (Figs. 1, 2, 3, and6A). Pseudo-nitzschia hasleana differs from P. fryxellianaby having a lower density of fibulae (13–20 vs. 18–25)and a tendency for more sectors in the poroids(Figs. 1, 2, and 6, C and E; Table 2). The poroids ofP. hasleana appear uniform and regularly silicified,whereas in the striae of P. fryxelliana, some of the por-oids and the poroid sectors appear to be ‘‘missing,’’

giving the striae a less uniform appearance. Further-more, the valve shape of P. fryxelliana is more lanceo-late than P. hasleana (Figs. 1, A–C vs. 2, A–D) and theband striae are higher (more poroids in transvalvarorientation) in P. hasleana than in P. fryxelliana (Figs.1J vs. 2J).

Of the species in the P. pseudodelicatissima com-plex, P. calliantha, P. mannii, and P. caciantha resem-ble P. hasleana and P. fryxelliana the most. They allhave similar densities of interstriae (31–40) on thevalve face (Fig. 6B, Table 2). The main differencebetween P. hasleana and P. caciantha, P. calliantha,and P. mannii is its lower fibula density (Fig. 6C,Table 2). Similarly, the main difference betweenP. fryxelliana and the same three species is its higherporoid density (Fig. 6D). In addition, P. hasleanaand P. fryxelliana differ from P. calliantha by havinggreater valve width (Fig. 6A) and fewer poroidsectors (Fig. 6E), and from P. caciantha by having alower valve width (Fig. 6A) and a higher band striadensity (Fig. 6F) (Table 2). Finally, P. hasleana has ahigher poroid density than P. caciantha and atendency for a lower density of band striae thanP. mannii and P. caciantha (Fig. 6, D and F).

Compared to P. pseudodelicatissima and P. cuspidata,P. hasleana, and P. fryxelliana have a lower density ofinterstriae (Fig. 6B, Table 2). Furthermore P. hasle-ana can be differentiated from P. pseudodelicatissimaand P. cuspidata because it has a lower fibula density(Fig. 6C); P. fryxelliana can be differentiated fromthe same two species because of a wider valve width(Fig. 6A, Table 2).

Pseudo-nitzschia hasleana and P. fryxelliana alsoresemble P. lineola, P. inflatula, and P. turgiduloides,although the latter occurs exclusively in Antarcticwaters and will not be considered further. The valveof P. inflatula differs because it is inflated in themiddle and at the ends. Pseudo-nitzschia lineola has asimilar valve shape, width, and poroid structure butcan be differentiated from P. hasleana and P. fryxelli-ana because of its tendency to have 1–2 rows of por-oids, whereas the two other species always have onerow. In addition, P. lineola has simple poroidswhereas the other two have more complex poroids(Figs. 1K, 2I, and 3, G and H; Table 2). Pseudo-nitzs-chia lineola also has a lower density of interstriae andfibulae (Fig. 6, B and C; Table 2), as well as poroidsand band striae, than the two other species (Fig. 6,D and F).

Our study provides more morphological detailson P. lineola than previously published (Hasle1965), with data on the structure of the poroidhymen, the mantle, and the different girdle bands.We found a transition in stria pattern from one rowof single poroids, to one row of split poroids andthen to two rows of poroids, even within a singlevalve (Fig. 3, D–F). In that respect, P. lineola differsfrom the other species discussed in this article butis similar to, for example, P. dolorosa (Lundholmet al. 2006). The valvocopula has a striate structure,


but in contrast to P. calliantha, P. hasleana, P. fryxelli-ana, and P. caciantha, the band striae are oftenthree, and seldom two, poroids wide.

Thus, for this group of pseudo-cryptic species,identification is not an easy task and a combinationof several morphological characters is needed. Cer-tain characters can be used initially to reject or con-firm species identity: for P. hasleana, the density offibulae, the valve width, and the number of poroidsectors; for P. fryxelliana, the density of poroids andthe number of poroid sectors; for P. lineola, the den-sities of interstriae, fibulae, poroids, and band striae,as well as the changing number of rows of poroids.

Mating studies. One aspect of the biological spe-cies concept is that a species is ‘‘a group of inter-breeding natural populations’’ (reviewed by Mann1999). In the strictest sense, successful crosses canbe defined as those producing a viable F1 genera-tion (Amato et al. 2007). This criterion was met bythe crosses between three of the five P. cuspidatastrains (Tables 1, 3). Furthermore, crosses betweena parent strain and one of its progeny gave largenew cells (F2 generation), although isolates did notsurvive. Examination of the matrix results shows thatthe unsuccessful crosses were between strains of thesame sex, or between P. cuspidata and the other spe-cies investigated (i.e., P. calliantha, P. hasleana, andP. lineola).

Crosses between the various P. calliantha strainswere unsuccessful, with the exception of the produc-tion of a few gametes in crosses between strainsNCP-1 versus NCP-2, 4, and 5 (Table 3); theseisolates were all from the same date and location.The unsuccessful crosses indicate that the strainswere either of the same sex, that there was someother unknown sexual incompatibility, or that theconditions were not suited for sexual reproductionof the species. This was the case, for example, withP. delicatissima and P. arenysensis, where it was shownthat appropriate temperatures are important formating to occur (Kaczmarska et al. 2008, Quijano-Scheggia et al. 2009).

Molecular comparisons. Descriptions of the twonew species, P. hasleana and P. fryxelliana, werehighly supported by the molecular analyses of genesfrom all three separate cellular compartments(Figs. 4 and 5). Comparison of the secondary struc-ture of ITS2 showed one CBC and seven HCBCsbetween P. hasleana and each of the other two spe-cies, P. calliantha and P. mannii. By comparison, themore phylogenetically closely related P. mannii andP. calliantha differed by only four HCBCs and noCBCs (Amato and Montresor 2008, present study),but even then mating did not result in viable prog-eny (Amato et al. 2007). Hence, in that case eventhe HCBCs indicated species separation.

Coleman (2007 and therein) showed that for awide array of eukaryotes there was agreementbetween the absence of CBCs in conserved pairingregions of ITS2 and the ability of the strains to mate

successfully. Recently, it has been found that thepresence of one CBC is congruent with matingincompatibility and thus sufficient for differentiat-ing species (Muller et al. 2007, Coleman 2009). Thepresence of one CBC between P. hasleana, and bothP. calliantha and P. mannii, thus strongly supportsP. hasleana as a separate species unable to exchangegenetic material by mating with either of the twoother species. This was supported by the lack of sex-ual activity in our crosses between P. hasleanaOFP41014-2 from Japan and P. calliantha strainsfrom eastern Canada, North Carolina, and Denmark(Table 3); P. mannii was not available for testing.Thus, erection of the new species, P. hasleana, issupported by morphological, molecular, and matingdata.

The new P. fryxelliana, which is more distantlyrelated to the other three species according to thephylogenetic studies, shows four CBCs and 9, 10, or11 HCBCs when compared to P. calliantha, P. man-nii, and P. hasleana, respectively. Both morphologi-cal and molecular data support the erection ofP. fryxelliana as a new species.

Apart from the clustering of P. calliantha, P. man-nii, and P. hasleana, the morphological similaritiesamong the species are thus not evidently reflectedin the phylogenetic analyses. Pseudo-nitzschia cacian-tha clusters in a highly supported clade with the dis-similar P. subpacifica, and P. fryxelliana appears moredistantly related to the other species in the com-plex. Similarly, P. lineola also appears as a well sepa-rated taxon, with no close phylogenetic relationshipto any presently described species (Figs. 4 and 5).The morphological characters thus cannot be usedfor a subdivision of the genus above species andnear-species level, a conclusion also reached by oth-ers (Lundholm et al. 2002, Orsini et al. 2002).

Inter- and intraspecific molecular differences. Thestrains of P. hasleana showed no base pair differ-ences among strains with regard to the plastid generbcL and the nuclear LDU rDNA. In ITS, only a sin-gle SNP was found among the nine strains, regard-less of the globally widespread origin of the strains:Washington State (three localities), Japan, andSpain (Pontevedra and Bay of Biscay) (Penna et al.2007, Orive et al. 2010, present study). In the cox1gene, the two sequenced strains of P. hasleana fromtwo localities off Washington State showed fiveSNPs, and thus exhibited a greater diversity thanthe ITS. This makes the use of cox1 as marker forintraspecific differences an interesting choice forfuture studies, as was also demonstrated for P. delica-tissima (Kaczmarska et al. 2008). The P. fryxellianastrains were identical in all genes from three cellu-lar compartments, which might be explained by allthe strains originating off Washington State.

The variation among the globally widespreadP. calliantha strains was larger than in P. hasleanaand P. fryxelliana, as six SNPs were recorded forITS1, none in 5.8S and ITS2, one SNP and three


indels in LSU. However, in spite of a smaller num-ber of strains studied, the P. hasleana strains alsohad a global origin, yet showed no base pair differ-ences. We expect that inclusion of more strains willsupport the greater intraspecific diversity of P. cal-liantha than P. hasleana.

The P. cuspidata strains were all morphologicallyidentical, but P. cuspidata and P. pseudodelicatissimaare taxonomically problematic, as the only morpho-logical difference between the two species is thevalve shape; P. pseudodelicatissima being linear andP. cuspidata lanceolate. After repetitive vegetativedivisions and the resulting reduction in valve length,P. pseudodelicatissima will become lanceolate. Thus,morphologically, the two species must be consid-ered cryptic or pseudo-cryptic, or should be mergedto one single species.

In phylogenetic analyses, strains designated asP. pseudodelicatissima and P. cuspidata cluster in onehighly supported clade. Studies on the ITS2 second-ary structure of strains from the clade have revealedthat CBCs and HCBCs exist among the strains inthe group, but any subdivision of the clade is preli-minary and not well supported by bootstrap values(Lundholm et al. 2003, Amato and Montresor 2008,Moschandreou et al. 2010). The type locality ofP. cuspidata is Las Palmas, Canary Islands, whereasthat of P. pseudodelicatissima is the Denmark Strait(Hasle 1965). The strain sequenced and studiedthat is closest to any of these localities is strain Ten-erife8 (from Tenerife, Canary Islands), which wasdesignated as P. cuspidata (Lundholm et al. 2003).We thus suggest, for the time being, if sequencedata are present, then similarity with strain Tene-rife8 should be used as reference for P. cuspidata.Future studies that include phylogenies on moregenes, secondary structure analyses, mating studiescomprising several strains, and inclusion of a strainof P. pseudodelicatissima from the type locality, areneeded to finally settle the taxonomic problem.

Toxin studies. Of the 13 available strains of P. cu-spidata, six produced detectable levels of DA(Table 4). Because the limits of detection for cEL-ISA and RBA were lower than for the HPLC-FMOCanalysis, four of the strains that were negativefor DA by the latter technique were positive withcELISA or RBA. Nevertheless, two of the samplesanalyzed by RBA gave results (3.1 and 5.4 ngDA Æ mL)1) that should have been detected by theHPLC-FMOC analysis. However, those cultureswere grown in different laboratories and underslightly different conditions, so this result is notunexpected.

Interestingly, when P. cuspidata strains NWFSC190 and 191, which were only slightly toxic (0.1–5.4 ng DA Æ mL)1), were crossed they produced via-ble offspring (CLN-38 and 39) that were substan-tially more toxic (28.8 and 19.1 ng DA Æ mL)1,respectively) than their parents. None of the othersix offspring, however, produced detectable DA.

This is yet another example of the interclonal toxinvariability seen in Pseudo-nitzschia species (e.g.,Kudela et al. 2004). It would be important to knowif this variability extends to other physiologicalparameters (e.g., nutrient uptake) (Thessen et al.2009) and growth.

The toxin quota for P. cuspidata strain NWFSC191 (calculated as 19.0 fg DA Æ cell)1, also reportedin Trainer et al. 2009) is several orders of magni-tude less than the cellular quotas estimated forP. cuspidata in the field (up to 35 pg DA Æ cell)1;Trainer et al. 2009), supporting the common obser-vation that cells in culture produce less toxin thanproduced by wild populations.

Explaining previous observations. The descriptionof P. hasleana and P. fryxelliana may explain someof the variation that has previously been reported.Cells that were morphologically different from pre-viously described species were recorded in fieldsamples and whale feces collected from the Gulf ofMaine and the Bay of Fundy, western North Atlan-tic (Leandro et al. 2010). The cells most probablybelong to P. hasleana, although the width is slightlynarrower than for P. hasleana. Similarly, a strain(Ner-D8), referred to Pseudo-nitzschia sp. from theBay of Biscay, Spain (Orive et al. 2010), and astrain referred to P. calliantha (IEO-PS50V) fromPontevedra (Atlantic coast of Spain) (Penna et al.2007), are both identified as P. hasleana in thephylogenetic analyses (Fig. 4). Morphologically,the former strain agrees with the description ofP. hasleana, confirming the molecular data. Wesuggest that the names of both sequences arecorrected in GenBank.

The identity of the Pseudo-nitzschia species thatcomprised the 1988 toxic bloom in the Bay of Fun-dy, Canada (Martin et al. 1990) has caused confu-sion and must be reconsidered. In a later study inthe area, Kaczmarska et al. (2005) recorded valveswith three different types of poroids, two of whichwere different from any described species. Thediversity of the P. pseudodelicatissima complex thusseems even more complicated. This agrees withStehr et al. (2002), McDonald et al. (2007) andHubbard et al. (2008), who all reported a highdegree of diversity within this cluster of species.

Biogeographic distribution. Pseudo-nitzschia hasleanahas been recorded from Japan, Washington State(present study), and from Atlantic Spanish waters(Penna et al. 2007, Orive et al. 2010). It thus seemsto be globally widespread, although presently onlyfrom temperate waters. Pseudo-nitzschia fryxelliana hasonly been recorded from two localities in coastalwaters of Washington State: Kalaloch beach (Stehret al. 2002) and Teawhit Head (present study). It istherefore premature to speculate on its distribution.

Hasle (1965) listed P. lineola as occurring in theAtlantic Ocean from the Faeroe Channel in thenorth to 45� S and as south as 45� S in the IndianOcean. The synonymous Nitzschia barkleyi was


described from the Atlantic part of the SouthernOcean, from 51�29¢ S to 66�32¢ S (Hustedt 1952).More recently, P. lineola has been recorded fromwidely distributed localities: Australia (Hallegraeff1994), Pacific coasts of Mexico (Hernandez-Becerril1998); the Drake Passage in the southern AtlanticOcean (Ferrario et al. 2004), the Weddell Sea, Ant-arctica (Almandoz et al. 2008); and the NE Pacificoff Washington State (present study). It thereforeseems to be a cosmopolitan species distributed inboth coastal and oceanic areas.

Many of the pseudo-cryptic Pseudo-nitzschia speciesappear to occur sympatrically. Our study in Wash-ington State coastal waters with several P. pseudodeli-catissima complex species confirms this. Along withthe morphological and molecular data, the sympat-ric occurrence provides indirect evidence that thetaxa are distinctly derived entities that do not andcannot exchange genetic material (see Bickfordet al. 2007).

CONCLUSIONS

Our understanding of the diversity of phytoplank-ton may never be complete, but by selecting a few‘‘model genera,’’ one may study processes that willhelp us understand the general mechanisms of speci-ation. Because of the ecological and economicimportance and the health risks posed by the pro-duction of DA, the genus Pseudo-nitzschia has becomesuch a model genus. This gives further reason toexplore the diversity of the genus. The descriptionof cryptic and pseudo-cryptic species, making it diffi-cult to identify Pseudo-nitzschia species, might be seenas an obstacle for those working on its monitoring,ecology, and physiology. On the other hand, it mightprovide a way to understand complex patterns ofphysiology (including toxin production), biogeogra-phy, and species succession in field studies. Theobstacle posed by cryptic and pseudo-cryptic speciesfor species identification will be removed in thefuture with further developments of PCR tools likemicroarrays or simpler methods like ARISA (Auto-mated Ribosomal Intergenic Spacer Analysis) analy-ses (McDonald et al. 2007, Hubbard et al. 2008,Medlin and Kooistra 2010). However, application ofsuch methods relies on a thorough understanding ofthe underlying taxonomy.

We thank Jørgen Kristiansen for translating the diagnoses toLatin, Monica Moniz for providing unpublished primers,Steve Morton and Yuichi Kotaki for providing cultures, andAnette Hørdum Løth and Amber Bratcher for laboratoryassistance. Jessica Hendrickson assisted with the culturesNWFSC 190 and 193, and Bich-Thuy Eberhart assisted withthe cELISA. We acknowledge the Ecology and Oceanographyin the Pacific Northwest (ECOHAB PNW) program duringwhich many of the Pseudo-nitzschia isolates were collected.ECOHAB is funded jointly by the National Science Founda-tion and the National Oceanic and Atmospheric Administra-tion. This is ECOHAB publication 659 and ECOHAB

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Supplementary Material

The following supplementary material is avail-able for this article:

Table S1. List of stains used in the phyloge-netic analyses of LSU; ITS1, 5.8S, and ITS2; rbcLand cox1, showing species identity, strain desig-nation, origin country and accession numbers.Accession numbers and strain designations inbold indicate that the strain was sequenced forthe present study. * Indicates type strain. £ Indi-cates strains used for concatenate dataset. 1Desig-nated as P. pseudodelicatissima in GenBank.2Designated as P. calliantha (Penna et al. 2007).3Designated as Pseudo-nitzschia sp. (Orive et al.2010). Washington refers to Washington State,USA. nk, data not known.

This material is available as part of the onlinearticle.

Please note: Wiley-Blackwell are not responsi-ble for the content or functionality of any supple-mentary materials supplied by the authors. Anyqueries (other than missing material) should bedirected to the corresponding author for thearticle.


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CRYPTIC AND PSEUDO-CRYPTIC DIVERSITY IN DIATOMS-WITH DESCRIPTIONS OF PSEUDO-NITZSCHIA HASLEANA SP....

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