J. Phycol. 41, 151–176 (2005) r 2005 Phycological Society of America DOI: 10.1111/j.1529-8817.2005.04067.x DIVERSITY IN THE GENUS SKELETONEMA (BACILLARIOPHYCEAE). II. AN ASSESSMENT OF THE TAXONOMY OF S. COSTATUM-LIKE SPECIES WITH THE DESCRIPTION OF FOUR NEW SPECIES1 Diana Sarno,2 Wiebe H. C. F. Kooistra Stazione Zoologica ‘‘Anthon Dohrn,’’ Villa Comunale, 80121 Naples, Italy Linda K. Medlin Alfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, Germany Isabella Percopo and Adriana Zingone Stazione Zoologica ‘‘Anthon Dohrn,’’ Villa Comunale, 80121 Naples, Italy The morphology of strains of Skeletonema Grev- Abbreviations: AIC, Akaike information criterion; ille emend Sarno et Zingone was examined in LM, CCAP, Culture Collection of Algae and Protozoa; TEM, and SEM and compared with sequence data CCMP, The Provasoli-Guillard National Center for from nuclear small subunit rDNA and partial large Cultures of Marine Phytoplankton; FP,fultoportula; subunit rDNA. Eight distinct entities were identi- FPP, fultoportula process; hLRTs, hierarchical like- fied, of which four were known: S. menzelii Guillard, lihood ratio tests; IFPP, intercalary fultoportula Carpenter et Reimann; S. pseudocostatum Medlin process; IRP, intercalary rimoportula; IRPP, inter- emend. Zingone et Sarno; S. subsalsum (Cleve) Bet- calary rimoportula process; LSU, large subunit; hge; and S. tropicum Cleve. The other four species ML, maximum likelihood; MP, maximum parsimo- were new: S. dohrnii Sarno et Kooistra sp. nov., S. ny; RP, rimoportula; RPP, rimoportula process; grethae Zingone et Sarno sp. nov., S. japonicum Zin- SSU, small subunit; SZN, Stazione Zoologica ‘‘A. gone et Sarno sp. nov., and S. marinoi Sarno et Zin- Dohrn’’ of Naples; TFP, terminal fultoportula; gone sp. nov. Skeletonema species fell into four TFPP, terminal fultoportula process; TRP, terminal morphologically distinct groups corresponding to rimoportula; TRPP, terminal rimoportula process four lineages in the small subunit and large subunit trees. Lineage I included S. pseudocostatum, S. trop- icum, S. grethae, and S. japonicum. All have external In 1973 Hasle wrote ‘‘To most marine planktolo- processes of the fultoportulae with narrow tips that gists, the name Skeletonema is synonymous with the connect with those of sibling cells via fork-, knot-, or specific name Skeletonema costatum.’’ This species fea- knuckle- like junctions. Lineage II included only tures in numerous research studies across all biological the solitary species S. menzelii. Lineage III com- disciplines, and of 588 papers published since 1990 prised S. dohrnii and S. marinoi. This latter pair that mention Skeletonema in their title or abstract, 548 have flattened and flared extremities of the process- refer specifically to S. costatum (Greville) Cleve. The es of the fultoportulae, which interdigitate with picture that emerges from this body of literature is of those of contiguous valves without forming knots an easily culturable widely tolerant species that thrives or knuckles. Lineage IVonly contained the brackish in nutrient-rich coastal waters throughout the world. water species S. subsalsum. Some species also differ In addition, results of these different studies are inter- in their distribution and seasonal occurrence. These preted and compared, with the implicit assumption findings challenge the concept of S. costatum as a that all strains and populations are part of a single single cosmopolitan and opportunistic species and species. calls for reinterpretation of the vast body of re- Skeletonema costatum is easily identified by its cylin- search data based on this species. drical cells with a ring of long processes emerging from Key index words: diatoms; diversity; LSU rDNA; the edge of the valve face (originally called costae by morphology; new species; phylogeny; Skeletonema; Greville [1866]). These processes are aligned parallel S. costatum; S. dohrnii; S. grethae; S. japonicum; to the longitudinal axis of the cell and connect with S. marinoi; S. menzelii; S. pseudocostatum; S. subsal- similar processes of adjacent sibling valves, thus form- sum; S. tropicum; SSU rDNA; taxonomy ing chains. The species was described originally as Me- losira costata Greville (Greville 1866) and subsequently transferred to Skeletonema (Cleve 1873) because of the 1Received 5 May 2004. Accepted 1 November 2004. similarity of the ‘‘longitudinal costae’’ (processes) to 2Author for correspondence: e-mail [email protected]. those observed in the type species of that time, the 151 152 DIANA SARNO ET AL. fossil Skeletonema barbadensis Greville. More recently, pette isolation. Additional strains were obtained from the Sims (1994) demonstrated that several fossil Skeleton- Culture Collection of Algae and Protozoa (CCAP, Oban, UK), ema species, including S. barbadensis, differ markedly The Provasoli-Guillard National Center for Cultures of Ma- from the extant ones. Therefore, she established a new rine Phytoplankton (CCMP, Bigelow, ME, USA), or kindly provided by colleagues mentioned in the acknowledgments. genus, Skeletonemopsis, for these fossils, conserving the All cultures (Table 1) were maintained at the Stazione Zoo- genus Skeletonema for the extant ones and with S. cos- logica ‘‘Anton Dohrn,’’ Naples (SZN). Skeletonema subsalsum tatum as the type species (Ross et al. 1996). was grown in a medium prepared with 14% f/2 (Guillard After the transfer of M. costata to Skeletonema (Cleve 1975), 22% soil extract (1 kg garden soil boiled with 1000 mL 1873), five more species were described in the genus distilled water to give 500 mL extract), and 64% bidistilled based on morphological characters: S. tropicum, S. sub- water, with a final salinity of 5 psu. All other strains were maintained in f/2 medium, adjusted to a salinity of 36 psu. salsum, S. potamos (Weber) Hasle, S. cylindraceum Prosh- Cultures were kept in glass tubes at 20–221 C and a 12:12-h kina-Lavrenko et Makarova, and S. menzelii. A detailed light:dark cycle at 100 mmol photons Á m À 2 Á s À 1 emitted from morphological investigation using EM documented cool white fluorescent tubes. considerable variation in S. costatum and other conge- Preserved material. Several natural samples from the Gulf neric species (Hasle 1973). Gallagher and coworkers of Naples (Tyrrhenian Sea) and the Adriatic Sea were exam- (Gallagher 1980, 1982, Gallagher et al. 1984) demon- ined to establish the morphological features of Skeletonema species. Tyrrhenian samples were mostly collected at station strated high diversity in allozyme patterns among MC (see above). Adriatic samples were collected in the north- strains of S. costatum and suggested that several species ern part, offshore from Venice, in winter and spring 2000– might be hidden within this taxon. Medlin et al. (1991) 2002. A few samples collected in winter 2001 offshore of An- were the first to recognize a new species, S. pseudo- cona, mid-Adriatic Sea, were also examined. costatum Medlin, based on morphological and molecu- LM. Light microscope observations were made on expo- lar information. However, their concept of S. costatum nentially growing cultures or natural samples with an Axiop- does not agree with the type and a new species must be hot microscope (Carl Zeiss, Oberkochen, Germany) equipped with Nomarski differential interference contrast, described for it (see below). phase contrast, and bright-field optics. Light micrographs In many areas, species attributed to S. costatum (sensu were taken using a Zeiss Axiocam digital camera. To remove lato) are among the most important contributors to organic matter, samples from cultures or field material were phytoplankton blooms (Karentz and Smayda 1984, treated with acids (1:1:4, sample:HNO3:H2SO4), boiled for Cloern et al. 1985, Estrada et al. 1985). In the Gulf few seconds, and then washed with distilled water. Perma- of Naples, the species blooms in late spring (Ribera nent slides were prepared by mounting the dry clean mate- rial in Hyrax (Hasle 1978). d’Alcala` et al. 2004), whereas in the nearby Adriatic EM. Acid-cleaned material was mounted on stubs, sputter Sea it is a recurrent winter blooming species (Zoppini coated with gold-palladium, and observed using a Philips 505 et al. 1995, Totti et al. 2002, Bernardi Aubry et al. scanning electron microscope (Philips Electron Optics BV, 2004). Preliminary observations revealed morpholog- Eindhoven, Netherlands) or mounted onto Formvar-coated ical differences between specimens obtained from grids and observed using a Philips 400 transmission electron these two regions, none of which completely matched microscope. Fixed samples not subjected to cleaning were any described species. These results prompted more dehydrated in an ethanol series, critical point dried substi- tuting 100% ethanol with CO2, sputter coated with gold-pal- detailed morphological and molecular analyses of sev- ladium, and observed with SEM. The terminology used to eral Skeletonema strains obtained from various places. describe ultrastructural features of Skeletonema species follows Results of these investigations revealed previously un- Anonymous (1975) and Ross et al. (1979). suspected morphological and genetic diversity within DNA extraction, PCR, sequencing, and phylogenetic analy- the genus. To assess which strains belonged to S. cos- ses. For molecular analysis, cultures of Skeletonema species tatum, a reexamination of the type material of this spe- were harvested during the exponential growth phase by cen- cies was needed. The results of this study are reported trifugation