J. Phycol. 43, 156–170 (2007) r 2007 by the Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00305.x DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE): III. PHYLOGENETIC POSITION AND MORPHOLOGICAL VARIABILITY OF SKELETONEMA COSTATUM AND SKELETONEMA GREVILLEI, WITH THE DESCRIPTION OF SKELETONEMA ARDENS SP. NOV.1 Diana Sarno2, Wiebe H. C. F. Kooistra, Sergio Balzano Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy Paul E. Hargraves Graduate School of Oceanography, University of Rhode Island, Narragansett, Rhode Island 02882-1197, USA and Adriana Zingone Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy Skeletonema costatum (Grev.) Cleve emend. tional Center for Cultures of Marine Phytoplankton; Zingone et Sarno and S. grevillei Sarno et Zingone CSIRO, Commonwealth Scientific and Industrial were known only from the type material collected Research Organisation; FPP, fultoportula process; from Hong Kong waters more than a century ago. GTR, general time reversible; IFPP, intercalary ful- Both species have now been collected as live mate- toportula process; IRP, intercalary rimoportula; rial, and their morphology and phylogenetic posi- IRPP, intercalary rimoportula process; ML, maxi- tion are investigated in this study. Eight Skeletonema mum likelihood; MP, maximum parsimony; NJ, strains isolated from Florida, USA; Uruguay; and neighbor joining; RELL, resampling of estimated Brazil are attributed to S. costatum, while one ln likelihood; SZN, Stazione Zoologica A. Dohrn of strain from Oman is ascribed to S. grevillei based Naples; TFPP, terminal fultoportula process; TRP, on morphological similarity to the type material of terminal rimoportula; TRPP, terminal rimoportula these species. In addition, a new Skeletonema spe- process cies, S. ardens Sarno et Zingone, is described for a strain from Singapore and two from northern Aus- tralian waters. Skeletonema ardens has terminal ful- toportula processes ending in a tapered, undulate The centric diatom Skeletonema costatum has long protrusion and long intercalary fultoportulae with been considered one of the most conspicuous and 1:1 junctions. The rimoportula of terminal valves is widespread members of the coastal marine phyto- located at the margin of the valve face. No major plankton. Recently, it was discovered that a number morphological variations were observed within S. of distinct species were included under this name grevillei and S. ardens along a salinity gradient, (Sarno et al. 2005). Skeletonema costatum was originally whereas in S. costatum, the processes shortened described by Greville (1866) as Melosira costata, based and the valves came into close contact at low on light microscopy (LM) of material from Hong Kong salinities, as already described for S. subsalsum Bay (Zingone et al. 2005). LM and EM examination of (Cleve) Bethge. Consistent with their morphology, the original material revealed the presence of two Skeletonema costatum and Skeletonema subsalsum Skeletonema species with a markedly distinct frustule also had similar rDNA sequences. Skeletonema gre- ultrastructure. By comparison with the original draw- villei and S. ardens were distinct in the large subunit ings (Greville 1866), one was identified as S. costatum (LSU) phylogeny. Skeletonema ardens exhibited con- sensu stricto. Morphological analyses showed that it has sistent intraspecific genetic differences in both the a unique combination of ultrastructural features, in- LSU and small subunit (SSU) rDNA. cluding flattened and closed tubular processes emer- Key index words: diatoms; LSU rDNA; morphol- ging from the fultoportulae of the intercalary valves ogy; phylogeny; salinity; Skeletonema ardens; Skele- (intercalary fultoportula processes, IFPPs), open term- tonema costatum; Skeletonema grevillei; SSU rDNA inal valve fultoportula processes (terminal fultoportula processes, TFPPs), asymmetrical 1:2 junctions between Abbreviations: CCAP, Culture Collection of Algae sibling valves, and marginal terminal rimoportulae and Protozoa; CCMP, the Provasoli-Guillard Na- (TRPs) (Zingone et al. 2005). The other morph re- quired the establishment of a new species, S. grevillei 1Received 10 April 2006. Accepted 2 November 2006. Sarno et Zingone, differing from S. costatum by having 2Author for correspondence: e-mail [email protected]. open IFPPs that mostly connect to adjacent cells with 156 SKELETONEMA DIVERSITY 157 1:1 junctions. A unique feature of S. grevillei is the Morphological variability of Skeletonema species in relation to presence of silica bridges joining the basal parts of the salinity. The growth of S. costatum (strains SZN B202 and SZN fultoportula processes (FPPs). Like S. costatum, this B211), S. grevillei (strains CCMP794 and CS347), and species has marginal terminal rimoportulae. S. ardens (strain CCMP1685) was monitored at salinities between 0 and 35 psu. Media with different salinities were After the establishment of S. costatum by Greville prepared with f/2 and Erd-Schreiber medium in different (1866), at least four other species were validly de- proportions. To adapt strains to different salinities, minute scribed, namely S. tropicum Cleve, S. subsalsum (Cleve) amounts of cultures were transferred every third day to media Bethge, S. potamos (Weber) Hasle, and S. menzelii with a salinity of 2.5 psu higher or lower than the one in which Guillard, E. J. Carp. et Reimann. Yet, notably, neither they were growing. Exponentially growing cultures of each S. costatum sensu stricto nor S. grevillei were ever ob- strain were observed with LM and EM at their optimal, mini- mum, and maximum salinity tolerance values. served or illustrated again, nor were they among the Light microscopy. Light microscope observations were taxa examined by Medlin et al. (1991). The latter made on exponentially growing cultures or natural samples described S. pseudocostatum as distinct from what they with a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, believed to be S. costatum, based on morphology and Germany) equipped with Nomarski differential interference small subunit (SSU) rDNA sequences. Sarno et al. contrast (DIC), phase contrast, and bright-field optics. Light (2005) examined large subunit (LSU) and SSU rDNA micrographs were taken using a Zeiss Axiocam digital cam- era. To remove organic matter, samples from cultures or field sequences and morphological traits of 35 Skeletonema material were treated with acids (1:1:4, sample:H- strains, recognizing four new species (S. dohrnii Sarno NO3:H2SO4), boiled for a few seconds, and then washed et Kooistra, S. grethae Zingone et Sarno, S. japonicum with distilled water. Permanent slides were prepared by Zingone et Sarno, and S. marinoi Sarno et Zingone), mounting the dry, clean material in Hyrax (Custom Research but neither S. costatum nor S. grevillei were found. and Development, Auburn, CA, USA; Hasle 1978). Zingone et al. (2005) only had access to fixed material Electron microscopy. Acid-cleaned material was mounted on of S. costatum and S. grevillei from a net sample obtained stubs, sputter coated with gold-palladium, and observed using a Philips 505 SEM (Philips Electron optics BV, Eind- from a single site, and the lack of further samples or hoven, the Netherlands), or mounted on Formvar-coated cultured strains constrained the documentation of grids (Agar Scientific, Stanstead, Essex, UK) and observed morphological variability of the two species, while their using a Philips 400 TEM. Fixed samples not subjected to phylogenetic position was inferred from their ultra- cleaning were dehydrated in an ethanol series, critical point structural characteristics. During the course of a study dried substituting 100% ethanol with CO2, sputter coated on the global biodiversity and biogeography of the with gold-palladium, and observed with SEM. The terminology used to describe ultrastructural features of genus Skeletonema, a number of strains closely resem- Skeletonema species follows Anonymous (1975) and Ross et al. bling S. costatum and S. grevillei were collected, allowing (1979), while rimoportulae, fultoportulae, and their processes more detailed morphological analysis of these are named as in Sarno et al. (2005). species (Kooistra et al., submitted). In addition, three DNA extraction, PCR amplification, sequencing, and phyloge- strains showing ultrastructural features that were netic analyses. For molecular analysis, cultures of Skeletonema not previously encountered are described as a new species were harvested during the exponential growth phase by filtration on 0.45 mm pore-size polycarbonate filters. DNA species: S. ardens. The phylogenetic positions of the extraction and purification were performed as in Kooistra three taxa are inferred from their LSU and SSU rDNA et al. (2003). PCR amplification of the nuclear-encoded SSU sequences and discussed alongside the characters that rDNA was carried out as described in Kooistra et al. (2003); are thought to be phylogenetically informative in PCR products were sequenced with forward primers 1F 50- Skeletonema. AACCTGGTTGATCCTGCCAGT-30, 528F 50-CGGTAATT- CCAGCTCC-30, and 1055F 50-GGTGGTGCATGGCCG-30; and reverse primers 1528R 50-TGATCCTTCTGCAGGT- 0 0 0 MATERIALS AND METHODS TCACCTAC-3 , 1055R 5 -CGGCCATGCACCACC-3 ,and 690R 50-AGAATTTCACCTCTG-30. The PCR amplification Isolation of strains and culture conditions. Cultures of the of the nuclear-encoded LSU rDNA was carried out as in strains SZN B202 and SZN B203 were established by micro- Orsini et al. (2002), and products were sequenced using