Two New Species in the Chaetoceros Socialis Complex (Bacillariophyta): C

TWO NEW SPECIES IN THE CHAETOCEROS SOCIALIS COMPLEX (BACILLARIOPHYTA): C. SPOROTRUNCATUS AND C. DICHATOENSIS, AND CHARACTERIZATION OF ITS RELATIVES, C. RADICANS AND C. CINCTUS1

Chetan C. Gaonkar, Wiebe H. C. F. Kooistra Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy Carina B. Lange Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy Department of Oceanography, Centers COPAS Sur-Austral and FONDAP-IDEAL, University of Concepcion, Concepcion, Chile Marina Montresor, and Diana Sarno2 Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy

The diatom genus Chaetoceros is one of the most Abbreviations: BI, Bayesian inference; BPP, Baye- abundant and diverse phytoplankton in marine and sian posterior probability; GTR, general time rever- brackish waters worldwide. Within this genus, sible; LTER-MC, Long Term Ecological Research Chaetoceros socialis has been cited as one of the most station MareChiara; ML, maximum likelihood; TBR, common species. However, recent studies from tree-bisection reconnection different geographic areas have shown the presence of pseudo-cryptic diversity within the C. socialis complex. Members of this complex are characterized by curved chains (primary colonies) The diatom genus Chaetoceros is one of the most aggregating into globular clusters, where one of the abundant marine phytoplankton in coastal and four setae of each cell curves toward the center of oceanic waters worldwide (Malviya et al. 2016). The the cluster and the other three orient outwards. genus is highly diverse with about 200 species New light and electron microscopy observations as reported (Guiry and Guiry 2016). One of the most well as molecular data on marine planktonic common species is Chaetoceros socialis Lauder (e.g., diatoms from the coastal waters off Chile revealed Leblanc et al. 2012, Harrison et al. 2015), a suppos- the presence of two new species, Chaetoceros edly ubiquitous and cosmopolitan species, docu- sporotruncatus sp. nov. and C. dichatoensis. sp. nov. mented from polar to tropical waters (Hasle and belonging to the C. socialis complex. The two Syvertsen 1997). This species is characterized by new species are similar to other members of the colonies with a secondary spherical structure formed complex (i.e., C. socialis and C. gelidus)in by intermingled curved chains. Cells possess three the primary and secondary structure of the colony, curved, short setae and one long, straight seta; the the orientation pattern of the setae, and the valve chain curvature is due to the convergence of the ultrastructure. The only morphological characters long setae toward the center of the spherical col- that can be used to differentiate the species of this ony. complex are aspects related to resting spore Recent studies on strains collected in different morphology. The two newly described species are geographic areas have shown the presence of geneti- closely related to each other and form a sister clade cally distinct entities within the morphologically sim- to C. gelidus in molecular phylogenies. We also ilar species C. socialis. Strains isolated from the provide a phylogenetic status along with the North Atlantic/Arctic (northern strains) and the morphological characterization of C. radicans and Tyrrhenian Sea (southern strains) showed clear dif- C. cintus, which are genetically related to the ferences in spore morphology, physiological traits C. socialis complex. (growth rate, photosynthetic yield, metabolomics proﬁles) and genetic proﬁle (28S rRNA; Degerlund Key index words: Chaetoceros; Chaetoceros dichatoensis et al. 2012, Huseby et al. 2012). In their detailed sp. nov.; Chaetoceros sporotruncatus sp. nov.; diatoms; morphological and molecular analysis of the C. so- phylogeny; spores; taxonomy cialis species complex, Chamnansinp et al. (2013) summarized the intricate taxonomic history of this species. The authors amended the description of

1 C. socialis based on epitype material collected from Received 21 December 2016. Accepted 28 May 2017. 2Author for correspondence: e-mail [email protected]. warm waters off Japan, whereas the species recorded Editorial Responsibility: R. Wetherbee (Associate Editor) in colder waters was described as Chaetoceros gelidus.

1 2 CHETAN C. GAONKAR ET AL.

A few morphological characters differentiate the water to remove all traces of acid (modified from Round two species: C. gelidus has narrower apertures et al. 1990). Acid-cleaned material was mounted on Formvar- between the cells and the setae emerge very close to coated grids and observed with a LEO 912AB (LEO, Oberko- chen, Germany) transmission electron microscope (TEM) or the valve margin whereas they emerge from inside mounted on stubs, sputter-coated with gold-palladium and the valve in C. socialis. Finally, the spores of C. so- observed with a JEOL JSM-6500F (JEOL-USA Inc., Peabody, cialis are spiny as compared to the smooth spores of MA, USA) scanning electron microscope (SEM). Samples C. gelidus (Chamnansinp et al. 2013, Balzano et al. containing spores were prepared for SEM examination using 2017). the same protocol. We investigated the ultrastructure of strains with a Sequence generation and alignment. DNA extraction of the gross morphology resembling that of C. socialis isolated strains was performed using a modified cetyltrimethyl ammonium bromide method (Doyle and Doyle 1987, Cul- within the framework of assessing the diversity of lings 1992). Two marker regions from the nuclear encoded Chaetoceros species at the Long Term Ecological ribosomal subunit (rDNA) were used. The D1–D3 region of Research station MareChiara (LTER-MC) in the the nuclear-encoded large subunit ribosomal DNA (partial Gulf of Naples (Tyrrhenian Sea, Mediterranean 28S rDNA) was PCR-amplified in 25 lL-volumes containing Sea), along the French Atlantic coast and the south- 10–250 ng DNA, 1 mM dNTPs, 0.5 lm of the D1R forward central Chilean coast. Results of phylogenetic analy- primer (Scholin et al. 1994; Table S1 in the Supporting Infor- mation), 0.5 lm of the D3Ca reverse primer (Auwera and de ses of the 18S and 28S rDNA regions confirmed that Wachter 1998; Table S1), 19 Roche diagnostics PCR reaction C. socialis was present in the Gulf of Naples and was buffer (Roche Diagnostics GmbH, Mannheim, Germany), recorded also in the temperate Atlantic waters, and 1 unit Taq DNA Polymerase (Roche Diagnostics GmbH). while sequences of strains isolated from Chilean The thermocycler (C1000 Touch; Bio-Rad, Foster City, CA, waters clustered in two distinct clades new to USA) was preheated at 98°C prior to the PCR cycling. PCR conditions included an initial denaturation step at 94°C for science. The structure of the resting spores was the ° only distinctive morphological character of the two 3 min, 35 cycles of denaturation at 94 C for 35 s, annealing at 54°C for 35 s, and extension at 72°C for 2 min, followed new species within the C. socialis complex, by a final extension at 72°C for 15 min. The nuclear encoded C. sporotruncatus sp. nov. and C. dichatoensis sp. nov. small subunit (SSU) ribosomal DNA (18S rDNA) was ampli- A sister clade to that of the C. socialis species com- fied usually with primer pair SSU-F (Hamsher et al. 2011) plex included sequences of strains identified as and SSU-R (Ki et al. 2007). The PCR mix was prepared as C. radicans and C. cinctus. We illustrate the ultra- described for the 28S rDNA PCR mix; only the primers being structure of vegetative cells and spores of these spe- different. PCR conditions included an initial denaturation step at 94°C for 3 min, 40 cycles of denaturation at 94°C for cies, thus providing a sound morphological 35 s, annealing at 52°C for 35 s and extension at 72°C for description of the strains from which sequences 3 min, followed by a final extension step at 72°C for 10 min. have been obtained. PCR products were purified using low melting agarose TAE (Tris-acetate-EDTA) buffer gel electrophoresis, excision MATERIALS AND METHODS of the target band under low UV light, and subsequent purifi- cation using DNA Isolation Spin Kit Agarose (PanReac Appli- Strain isolation and morphological analyses. Strains were col- chem GmbH, Darmstadt, Germany) following manufacturer’s lected along the coast of Chile, at the LTER-MC in the Gulf instructions. The obtained PCR products were sequenced of Naples (Italy, Mediterranean Sea) and in Roscoff (France, using the BigDye Terminator Cycle Sequencing technology Atlantic coast; Table 1). Clonal cultures were established by (Applied Biosystems, Foster City, CA, USA), purified using a isolating short chains or single cells from plankton net sam- ‘Biomek FX’ (Beckman Coulter, Fullerton, CA, USA) robotic ples. Cultures were grown in f/2 culture medium (Guillard station, and analyzed on an Automated Capillary Elec- 1975) prepared using oligotrophic natural seawater amended trophoresis Sequencer ‘3730 DNA Analyzer’ (Applied Biosys- with Guillard’s (f/2) Marine Water Enrichment Solution tems). Forward and reverse sequences were combined into (Sigma-Aldrich, St. Louis, MO, USA). To stimulate the forma- contigs and aligned using BioEdit v7.0.0 (Hall 1999). Any site tion of resting spores, cultures were inoculated in f/2 med- showing an ambiguity in the forward and reverse sequence ium to which the nitrogen stock was not added. At times, was recorded as such, if the surrounding sites read without spores were also recorded in old cultures. Cultures were any difficulties. maintained at 20°C, 12:12 h L:D photoperiod, and at an irra- Phylogenetic analyses. Sequences assigned as C. socialis and diance of 50 lmol photons m 2 s 1 provided by cool C. gelidus in GenBank were retrieved and included in the white fluorescent tubes. For the examination of vegetative alignment. The final alignment comprised sequences of C. so- cell morphology, subsamples of cultures in exponential cialis, C. gelidus, C. sporotruncatus sp. nov., and C. dichatoensis growth phase were observed alive using light microscopy, and sp. nov., along with sequences of C. radicans, C. cinctus, and fixed with neutralized formaldehyde at a final concentration C. costatus. Sequences of C. tenuissimus, C. neogracilis clade I, of 1.6% for electron microscopy observations. and Chaetoceros sp. strain CCMP1315 (identified as C. calci- Light microscopy observations were done using a Zeiss trans in the NCMA culture collection) were used as outgroup Axiophot microscope (Carl Zeiss, Oberkochen, Germany) based on previous phylogenetic information (Kooistra et al. equipped with Nomarski differential interference contrast, 2010, Degerlund et al. 2012, Chamnansinp et al. 2013). For phase contrast, and bright field optics. Micrographs were C. socialis, only a few representative sequences from different taken using a Zeiss Axiocam HRc digital camera. For electron geographic locations were included in the analysis, as most of microscopy observations, selected fixed samples (Table 1) these sequences were identical to the ones generated in the were treated with nitric and sulfuric acids (1:1:4, sample: 70% present study (Table 1). – HNO3: 95% 98% H2SO4), boiled for some seconds to Maximum likelihood (ML) and Bayesian inference (BI) remove organic matter and washed 5–7 times with distilled were used to infer phylogenies as well as bootstrap values or TWO NEW CHAETOCEROS SPECIES 3

TABLE 1. Strains and sequences of Chaetoceros species included in this study. Species name, strain code, collection site, collection date, and morphological data (LM, SEM, TEM); GenBank accession number of 18S and 28S rDNA sequences.

GenBank Species name Strain code Collection site Collection date Morphology images 18S 28S Chaetoceros socialis YL1* China 15 March 2009 Chamnansinp NA # KF219701 et al. 2013 No_1 Thailand 31 October 2008 Chamnansinp NA # KF219700 et al. 2013 KMMCC B734 South Korea 6 July 2004 NA # JQ217339 # JQ217339 RR NA NA NA # AY485446 NA newLC4 Gulf of Naples (Italy) 16 April 2013 NA # KY852274 As KY852295 newEA3 Gulf of Naples (Italy) 16 April 2013 NA # KY852277 As KY852295 newCB4 Gulf of Naples (Italy) 16 April 2013 NA # KY852276 # KY852295 newEC4 Gulf of Naples (Italy) 16 April 2013 NA # KY852275 # KY852296 Na2C4 Gulf of Naples (Italy) 26 November 2013 LM, SEM NA # KY852293 Na4C4 Gulf of Naples (Italy) 26 November 2013 LM, SEM NA As KY852295 Na12C2 Gulf of Naples (Italy) 19 March 2014 LM NA As KY852295 Na33B1 Gulf of Naples (Italy) 14 July 2015 LM NA As KY852295 Na33C3 Gulf of Naples (Italy) 14 July 2015 LM NA As KY852295 Ro4A1 Roscoff (France) 11 August 2014 LM NA As KY852294 Ro4A2 Roscoff (France) 11 August 2014 LM NA # KY852294 C. gelidus CNCIII51_13 Central Arctic NA NA # HM581777 NA WC-13-MC1138 Bering Sea, Paciﬁc 23 March 2010 NA # KC771205 NA Ocean RCC1992 Beaufort Sea June 2009 NA # JF794042 NA D8* Skovshoved Harbour, June 2009 Chamnansinp NA # KF219703 Denmark et al. 2013 CPH22 Denmark 16 December 2010 Chamnansinp NA # KF219714 et al. 2013 AMB-66 Finnmark coast NA NA NA # HE573573 (Norway) RCC1990 Beaufort Sea July 2009 NA NA # JQ995407 E65PG4 Beaufort Sea June 2009 NA NA # JQ995393 C. sporotruncatus Ch2A4* Las Cruces (Chile) 16 October 2013 LM, SEM # KY852270 # KY852297 Ch9C4 Concepcion (Chile) 29 October 2013 LM, SEM, TEM NA # KY852298 CCMP172 San Juan Island (USA) NA Chamnansinp NA # EF423466 et al. 2013 UNBF-P38C1 New Brunswick 22 June 2010 NA NA # KC986102 (Canada) C. dichatoensis Ch4A4* Las Cruces (Chile) 16 October 2013 LM, SEM, TEM # KY852271 # KY852300 Ch1B3 Las Cruces (Chile) 16 October 2013 LM, SEM # KY852272 # KY852299 Ch9B4 Concepcion (Chile) 29 October 2013 LM, SEM # KY852273 # KY852301 C. radicans Ch1B4 Las Cruces (Chile) 16 October 2013 LM, SEM, TEM # KY852260 # KY852288 Ch2A2 Las Cruces (Chile) 16 October 2013 LM # KY852259 # KY852289 Ch3B1 Las Cruces (Chile) 16 October 2013 LM # KY852261 # KY852290 Ch10A3 Las Cruces (Chile) 29 October 2013 LM, SEM # KY852263 # KY852291 Ch11A4 Las Cruces (Chile) 1 November 2013 LM # KY852262 # KY852292 CCMP197 Fladen Ground, 1 January 1977 NA # AB430592 # AB430626 North Sea C. cinctus Ch3A1 Las Cruces (Chile) 16 October 2013 LM # KY852265 # KY852281 Ch3C4 Las Cruces (Chile) 16 October 2013 LM # KY852266 # KY852282 Ch6A2 Las Cruces (Chile) 16 October 2013 LM # KY852264 # KY852283 Ch10B1 Las Cruces (Chile) 29 October 2013 LM # KY852267 # KY852284 Ch10B3 Las Cruces (Chile) 29 October 2013 LM, SEM, TEM # KY852268 # KY852285 Ch10B4 Las Cruces (Chile) 29 October 2013 LM NA # KY852286 Ch11C3 Las Cruces (Chile) 1 November 2013 LM # KY852269 # KY852287 C. costatus Na32B1 Gulf of Naples (Italy) 22 May 2015 Kooistra # KY852258 # KY852280 et al. 2010 C. tenuissimus newCA3 Gulf of Naples (Italy) 16 April 2013 Kooistra # KY852257 # KY852279 et al. 2010 C. neogracilis RCC2275 Beaufort Sea July 2009 NA # JN934684 # JQ995449 Chaetoceros sp. CCMP1315 NA 1960 NA # KY852256 # KY852278 An asterisk marks the type strain of the different species. “#” indicates sequences used for phylogenetic analysis. NA = Not available.

posterior probabilities for the various clades. ML-trees were mode (default), TBR branch swapping and a GTRGAMMA inferred with RAxML as implemented in raxmlGUI v.1.5beta base substitution model. Bootstrap values were obtained with (Silvestro and Michalak 2012) using heuristic searches (ten- 1,000 bootstrap replicates and one random-addition-of- random-additions-of-sequences-runs), rapid hill climbing sequences-run per bootstrap replicate (thorough bootstrap 4 CHETAN C. GAONKAR ET AL. analysis). Bayesian trees were constructed using MrBayes 3.2.2 Description of two new species in the C. socialis on XSEDE (Ronquist and Huelsenbeck 2003) with GTR + ґ + complex. Five strains of C. socialis isolated from the PINVAR parameters being estimated during the run, using Gulf of Naples and two strains from Roscoff the default-value of four Markov chains and a ‘temperature’ parameter of 0.2. The Monte Carlo Markov Chain length was (Table 1) were examined in LM and EM. Because set at 1 million generations, with a posterior probability of the gross morphology and ultrastructure of the veg- bipartitions sampled every 100 generations and diagnosed etative cells and spores of these strains match the every 1,000 generations. The initial 25% of the sampled trees description of C. socialis (Chamnansinp et al. 2013) were discarded as burn-in because thereafter the BPP was sta- they will not be described further here. bilized. BI-consensus trees were generated from the sampled Chaetoceros sporotruncatus Gaonkar, Kooistra et trees. The number of differences between sequences was cal- – – culated using MEGA v6.06 (Tamura et al. 2013). Lange sp. nov. (Figs. 3, A L and 4, A K; Table 2) Diagnosis. Each cell has three short curved setae and one long, straight seta. Distal ends of long setae converge to a common point forming curved colo- RESULTS nies. Spherical secondary colonies are formed by 18S phylogeny. For the inference of the ML tree, a the interconnection of bundles of long setae from GTR model of base substitutions was estimated dur- multiple chains. Cells are quadrangular in girdle ing analysis, and the same was done for the BI tree. view. Cell apical axis is 7.2–15 lm long, pervalvar Model parameters and tree scores are presented in axis is 7.2–27.8 lm long, and length of aperture in Appendix S1 in the Supporting Information. The pervalvar axis is 2.2–6.7 lm. Contains one chloro- ML and BI trees exhibited the same topology, and plast per cell. Apertures between adjacent cells are therefore, only the ML tree is shown (Fig. 1). The large, hexagonal, and slightly constricted at the cen- sequence of Chaetoceros costatus branched off first. ter. Valves are elliptical to circular, with a central The four species within the C. socialis complex hyaline annulus. Costae radiate from the annulus grouped together in a clade in which C. sporotrunca- and partly converge toward the insertion point of tus and C. dichatoensis were nearest sisters, C. gelidus the setae. Terminal valves have a central rimopor- their next nearest sister, and C. socialis the sister of tula. Setae emerge from the inside of the valve face all three. Chaetoceros cinctus was resolved as sister to with a basal part and cross at the chain edge. The C. radicans, with the clade comprising the C. socialis three short setae and the long seta are ornamented complex as their nearest sister, and C. costatus as with spirally arranged spines and poroids, and with next nearest sister. All clades obtained high support, large, elongate, solitary pores. Spines are absent on except the one containing C. gelidus, C. sporotrunca- the proximal-intermediate part of the long seta. Gir- tus, and C. dichatoensis. dle bands are ornamented with a siliceous ridge Partial 28S phylogeny. For the inference of the ML running along the whole length of the band and tree, a GTR model of base substitutions was esti- with transverse costae alternating with less silicified mated during analysis, and the same was done for areas perforated by minute perforations. Spores are the BI tree. Model parameters and tree scores have biconvex and valves have the shape of a truncated been presented in Appendix S1. As with the 18S- cone. The primary valve is ornamented on its cen- trees, the ML and BI trees inferred from the 28S tral portion with variable number of raised lenticu- sequences exhibited the same topologies, and there- lar-shaped structures. fore, only the ML tree is shown (Fig. 2). The topol- Holotype. A permanent slide of strain Ch2A4, iso- ogy of this tree was the same as that of the 18S tree, lated from Las Cruces, Chile, deposited at the except that C. cinctus and C. radicans did not group Museum of Stazione Zoologica Anton Dohrn, into a clade. Naples, Italy, as no. SZN-Ch2A4. Sequence diversity within clades. Within the C. so- Isotype. SEM stubs, TEM grids and fixed material cialis clade, a partial 28S rDNA sequence of strain of strain Ch2A4, isolated from Las Cruces, Chile, No_1 (Thailand) differed at five positions, and the deposited at the Museum of Stazione Zoologica sequence of the type strain YL1 (South China Sea) Anton Dohrn, Naples, Italy. at one position from the Neapolitan sequences. Molecular characterization. The species is defined by Within C. radicans, the partial 28S rDNA of strain the combined nucleotide sequences of strain CCMP197 (Fladen Ground, North Sea) differed at Ch2A4: D1-D3 of 28S rDNA (GenBank no. four positions from the Chilean sequences over the KY852297) and full length 18S rDNA (GenBank no. 586 shared positions, and their full length 18S KY852270). rDNA sequences differed at 10 positions. No Type locality. Las Cruces, Chile (33°29046″ S and intraspecific sequence differences were observed 71°37039″ W). among the partial 28S and 18S sequences of strains Ethymology. The species name sporotruncatus refers belonging to C. cinctus, C. gelidus, C. sporotroncatus, to the shape of the spore valves, which look like and C. dichatoensis. Notably, the partial 28S rDNA of truncated cones. the C. sporotroncatus strains differed at seven posi- Morphology. Cells have three short, curved setae tions from those of C. dichatoensis and their 18S and one longer, straight seta; occasionally, cells with rDNA sequences differed at 10 positions. four short curved setae were observed. The long TWO NEW CHAETOCEROS SPECIES 5 setae converge to a common point to form a curved long and length of aperture in pervalvar axis is chain (Fig. 3, A and B). The spherical secondary 2.2–6.7 lm. Each cell has a single large chloroplast colonies are formed by interconnecting of several (Fig. 3B). Apertures between adjacent cells have a long setae from multiple chains (Fig. 3A). Single wide hexagonal shape, with a small central constric- cells were also observed in culture. Cells are quad- tion (Fig. 3C). Cells with longer apical axis have rangular in girdle view (Fig. 3B), longer than wide narrower (in pervalvar axis) apertures. Setae emerge or wider than long, depending on the age of the from inside the valve margin; they have a relatively cells, i.e., on the cell size reduction stage. Apical cell long basal part, and cross at the chain edge (Fig. 3, axis is 7.2–15 lm long, pervalvar axis is 7.2–27.8 lm B and C). Setae are circular in cross-section. Short

FIG. 1. 18S rDNA maximum likelihood phylogenetic hypothesis inferred for the species of Chaetoceros socialis complex and the phylogenetically closely related species C. radicans and C. cinctus. Far outgroups have been pruned away from the tree. Bootstrap values (ML) and Bayesian posterior probabilities (BI) are indicated (ML/BI) at their respective internodes. Bootstrap values below 50% and BPP values below 0.95 are marked “-.” Black dots indicate the type strains of the species in the C. socialis complex. 18S sequences are not available for the type strains of C. socialis and C. gelidus. 6 CHETAN C. GAONKAR ET AL. and long setae are ornamented with spirally transverse costae alternating with less siliciﬁed areas arranged poroids and spines, and large, elongate, perforated by minute perforations; a siliceous ridge solitary pores (Fig. 3, D–G). Spines are absent on runs along the whole length of the band (Fig. 3L). the proximal-intermediate part of the long seta Spores are biconvex in shape and have valves (Fig. 3G). Terminal setae have the same ultrastruc- shaped as a truncate cone (Fig. 4, A and B). The ture as the short setae (Fig. 3, D and E). Valves are central portion of the primary valve is ornamented elliptical to circular in outline, ornamented with with raised lenticular-shaped structures of variable branching costae radiating from a central annulus number and size (Fig. 4, A–G). The secondary valve and partly converging toward the two insertion is smooth (Fig. 4H) and presents a single ring of points of the setae (Fig. 3, H and I). The shape of puncta on the advalvar margin of the mantle the annulus is elliptical, at times with an irregular (Fig. 4J). outline; the area within the annulus is hyaline Chaetoceros dichatoensis Gaonkar, Montresor et (Fig. 3, H and I). Terminal valves have a single cen- Sarno sp. nov. (Figs. 5, A–K and 6, A–J; Table 2) tral rimoportula, which has an internal elongated Diagnosis. Each cell has three short curved setae slit-shaped opening (Fig. 3J) and a short external and one long, straight seta. Distal ends of long setae tube (Fig. 3K). The rimoportula is absent on the converge to a common point forming curved colo- intercalary valves. Girdle bands are ornamented with nies. Spherical secondary colonies are formed by

FIG. 2. Partial 28S rDNA maximum likelihood phylogenetic hypothesis inferred for the species of Chaetoceros socialis complex and the phylogenetically closely related species C. radicans and C. cinctus. Far outgroups have been pruned away from the tree. Bootstrap values (ML) and Baye- sian posterior probabilities (BI) are indicated (ML/BI) at their respective internodes. Bootstrap values below 50% and BPP values below 0.95 are marked “-.” Black dots indicate the type strains of the species in the C. socialis complex. TWO NEW CHAETOCEROS SPECIES 7

FIG.3. Chaetoceros sporotruncatus sp. nov., vegetative cells. (A and I–L) strain Ch2A4 and (B–H) strain Ch9C4. (A, B) LM; (C, D, J and K) SEM; (E–I and L) TEM. (A) Spherical colony with multiple chains. (B) Single chain in broad girdle view. (C) Aperture between adjacent cells. (D) Detail of a terminal seta with spirally arranged spines. (E) Detail of the middle portion of a terminal seta with spirally arranged poroids and spines, and solitary pores (arrowed). (F) Detail of the middle portion of a short intercalary seta with spirally arranged poroids and spines. (G) Detail of the proximal portion of a long intercalary seta with spirally arranged poroids and solitary pores marked with an arrow. (H, I) Intercalary valves with radial costae extending from the central annulus and converging at the insertion point of the setae. (J) Internal view of terminal valve with central rimoportula (arrowed). (K) Terminal valve with ﬂattened external tube (arrowed). (L) Detail of a girdle band; the longitudinal ridge is arrowed. Scale bars: (A) = 20 lm, (B) = 5 lm, (C–H, J, K) = 1 lm, (I) = 2 lm, (L) = 0.5 lm. 8 CHETAN C. GAONKAR ET AL.

FIG.4. Chaetoceros sporotruncatus sp. nov., SEM pictures of spores. (B–F) strain Ch2A4 and (A and G–I) strain Ch9C4. (A–B) Spores with the primary valve ornamented on its central part with raised lenticular-shaped structures; the secondary valve is still covered by the vegetative valve in B. (C) Partially formed spores in which the primary valves have been produced. (D–F) Primary valves with different ornamentation. (G) Primary valve in girdle view. (H) Secondary valve. (I) Internal view of a secondary valve with a ring of puncta (arrowed). All scale bars = 1 lm. the interconnection of bundles of long setae from edge. The three short setae and the long seta are multiple chains. Cells quadrangular in girdle view. ornamented with spirally arranged spines and por- Cell apical axis is 3.9–10.0 lm long, pervalvar axis is oids, and with large, elongate, solitary pores. Spines 6.1–22.2 lm long, and size of aperture in pervalvar are absent on the proximal-intermediate part of the axis is 1.1–6.7 lm. Contains one chloroplast per long seta. Girdle bands are ornamented with a silic- cell. Apertures between adjacent cells are large, eous ridge running along the whole length of the hexagonal in shape, and slightly constricted at the band and with transverse costae alternating with less centre. Valves are elliptical to circular, with a cen- siliciﬁed areas perforated by minute perforations. tral hyaline annulus. Costae radiate from the annu- Spores are biconvex, with roughly hemispherical lus and partly converge toward the insertion points valves. Valves are ornamented with spines of vari- of the setae. Terminal valves have a central rimo- able density and size; the basal portion of the spines portula. Setae emerge from the inside of the valve is at times expanded, forming more or less margin with a basal part and cross at the chain extended ridges. TWO NEW CHAETOCEROS SPECIES 9

TABLE 2. Comparison of the morphological characteristics of Chaetoceros socialis (Chamnansinp et al. 2013; number of poroids and spines on the setae are from strains collected in the Gulf of Naples, this study), C. gelidus (Chamnansinp et al. 2013), C. sporotruncatus sp. nov., and C. dichatoensis sp. nov.

C. socialis C. gelidus C. sporotruncatus C. dichatoensis Colony Curved chains joined to form spherical colonies Cell apical 6.8 1.5 (4.3–11.5) 7.1 2.7 (3.6–16.9) 11.2 2.3 (7.2–15.0) 6.4 1.4 (3.9–10.0) axis (lm) n = 20 n = 20 n = 30 n = 57 Cell pervalvar 9.1 1.6 (5.1–12.9) 9.7 1.8 (5.0–14.7) 11.3 4.13 (7.2–27.8) 11.6 2.7 (6.1–22.2) axis (lm) n = 20 n = 20 n = 30 n = 59 Aperture Hexagonal, wide in Hexagonal, narrow in Hexagonal, wide in Hexagonal, wide in pervalvar axis pervalvar axis pervalvar axis pervalvar axis Aperture size in 5.1 1.0 (3.1–6.9) 2.0 0.6 (0.7–3.3) 4.6 1.3 (2.2–6.7) 2.6 0.9 (1.1–6.7) pervalvar n = 20 n = 20 n = 23 n = 52 axis (lm) Intercalary setae: Distant from the Near the valve margin Distant from the Distant from the origin valve margin valve margin valve margin Basal part of the Long Short Long Long setae Short setae: Spirally arranged spines and poroids; large, elongate, ornamentation solitary pores Long seta: As in short setae; spines more sparse and located on the distal part ornamentation Poroids on setae 21 1 NA 20 1 21 1 (#/lm) n = 3 n = 11 n = 16 Spines on setae 3NA33 (#/lm) Spore: shape Biconvex; hemispherical- Biconvex; valve mantle Biconvex; valves as Biconvex; hemispherical- conical valves with crest made of fused truncated cones conical valves projections Spore: Spines; one ring of Smooth; crest on valve Primary valve with raised Spines, often with an ornamentation spines on valve margin margin lenticular-shaped expanded base structures at apex forming ridges between spines Data are given as mean value SD, and as minimum and maximum range in brackets; “n” indicates the number of measured cells. NA = Not available.

Holotype. A permanent slide of the strain Ch4A4 curved chain (Fig. 5, A and B). Multiple curved isolated from Las Cruces, Chile, deposited at the chains are formed by the interconnection of bun- Museum of Stazione Zoologica Anton Dohrn, dles of long setae from multiple chains (Fig. 5A). At Naples, Italy, as no. SZN-Ch4A4. times, single cells were observed in culture. Cells Isotype. SEM stubs, TEM grids, and fixed material are quadrangular in girdle view (Fig. 5B), longer of the same strain Ch4A4 isolated from Las Cruces, than wide or wider than long, depending on the Chile, deposited at the Museum of Stazione Zoolog- age of the cells, i.e., on the cell size reduction stage. ica Anton Dohrn, Naples, Italy. Apical axis is 3.9–10.0 lm long, pervalvar axis is Additional material. Living culture of strain Ch4A4 6.1–22.2 lm long and size of aperture in pervalvar was deposited as CCM UDEC 288B at the Ficolab, axis is 1.1–6.7 lm. Each cell has a single large Grupo de investigacion microalgal, Facultad Cien- chloroplast. Apertures between adjacent cells are cias Naturales y Oceanograficas of the University of wide in pervalvar axis and hexagonal in shape, gen- Concepcion, Chile (www.ficolab.cl). erally with a central constriction (Fig. 5C). Aper- Molecular characterization. The species is defined by tures are narrower in cells with longer apical axis. the combined nucleotide sequences of strain Ch4A4 Setae emerge from the inside of the valve margin D1-D3 28S rDNA (GenBank no. KY852300) and full with a basal part and cross at the chain edge. Setae length 18S rDNA (GenBank no. KY852271). are circular in cross section. All setae are orna- Type locality. Las Cruces, Chile (33°29046″ S and mented with a spiral row of small poroids and 71°37039″ W). spines, and large, elongate solitary pores (Fig. 5, D– Etymology. The species name dichatoensis is dedi- G). Spines are absent on the proximal-intermediate cated to the University of Concepcion Marine Sta- part of the long seta (Fig. 5E). Terminal setae have tion in Dichato, which was destroyed by the tsunami the same ultrastructure of the short setae (Fig. 5, F of February 27, 2010. and G). The valve face is elliptical to circular in out- Morphology. Cells have three short, curved setae line. The valves are ornamented with costae radiat- and one longer and straight seta; occasionally, cells ing from the central annulus and partly converging with four short curved setae were observed. The toward the insertion points of the setae (Fig. 5H). long setae converge to a common point to form a The annulus is elliptical or sometimes irregular in 10 CHETAN C. GAONKAR ET AL.

FIG.5. Chaetoceros dichatoensis sp. nov., vegetative cells. (A–K) strain Ch4A4. (A, B) LM; (C, D, F, I, and J) SEM; (E, G, H, and K) TEM. (A) A spherical colony with multiple chains. (B) A chain of cells in broad girdle view. (C) Aperture between adjacent cells. (D) Detail of the proximal portion of a short intercalary seta; spines are arrowed. (E) Detail of the proximal portion of a long intercalary seta with spirally arranged poroids and scattered large pores (arrowed). (F, G) Detail of terminal setae. (H) Intercalary valves with radial costae extending from the central annulus and converging at the insertion point of the setae. (I) External view of a terminal valve with flattened external tube (arrowed). (J) Internal view of a terminal valve with the central slit-shaped rimoportula (arrowed). (K) Detail of girdle bands; the longitudinal ridge is arrowed. Scale bars: (A) = 20 lm, (B) = 10 lm, (C–H, I, J) = 1 lm, (K) = 1 lm. shape and the area within the annulus is hyaline bands are ornamented with transverse costae alter- (Fig. 5H). Terminal valves have a central rimopor- nating with less silicified areas perforated by minute tula, which has a flattened external tube (Fig. 5I) perforations; a siliceous ridge runs along the whole and an internal slit-shaped opening (Fig. 5J). The length of the band (Fig. 5K). Spores are biconvex rimoportula is absent on intercalary valves. Girdle in shape, with more or less hemispherical valves TWO NEW CHAETOCEROS SPECIES 11

FIG.6. Chaetoceros dichatoensis sp. nov., spores. (A–J) strain Ch4A4. (A) LM and (B–J) SEM. (A) A chain of cells, many of which contain spores. (B–E) Spores; note the variability of spine size, shape and density. (F) Opened spore. (G) View of the secondary valve in which almost exclusively ridges are present. (H) Valve in which both spines and ridges are present. (I, J) Secondary valves with the ring of puncta arrowed. Scale bars: (A) = 10 lm, (B–J) = 1 lm.

(Fig. 6, A–J). Both the primary and the secondary Vegetative cells and spores of Chaetoceros cinctus and valves are covered with spines of variable length and C. radicans. Chaetoceros cinctus Gran (Figs. 7, A–I density (Fig. 6, B–J). The basal portion of the spines and 8, A–F) can be considerably expanded, forming more or less We examined the morphology of seven strains of extended ridges that connect several spines. At C. cinctus isolated from Las Cruces (Chile; Table 1). times, only ridges have been observed on the sec- Chains are straight (Fig. 7A), curved or slightly ondary valve (Fig. 6G). A single ring of puncta is twisted. In girdle view, cells appear rectangular and present on the advalvar margin of the mantle of the apertures are narrow hexagonal (Fig. 7B). The cell secondary valve (Fig. 6, I and J). apical axis is 3.3–16.7 lm long, the pervalvar axis is 12 CHETAN C. GAONKAR ET AL.

FIG.7. Chaetoceros cinctus, vegetative cells. (A–I) Strain Ch10B3. (A) LM; (B, E and F) SEM; (C, D and G–I) TEM. (A) A chain in broad girdle view. (B) Aperture of adjacent cells. (C) Detail of the basal part of a terminal seta. (D) Intercalary seta ornamented with spirally arranged spines and poroids, and larger solitary pores (arrowed). (E) Detail of terminal seta with spirally arranged spines. (F) Terminal valve with central external ﬂattened tube (arrowed). (G) Valves with radially branched costae and the central annulus; the arrow marks the hyaline rim. Note the terminal valve with central rimoportula on the left. (H) Detail of a valve with costae and scattered poroids. (I) Detail of girdle bands; the longitudinal ridge is arrowed. Scale bars: (A) = 10 lm, (B, C, E, F,) = 1 lm, (D, H) = 0.5 lm, (G, I) = 2 lm.

3.3–16.7 lm long, and the length of the aperture in corresponding basal plate (Fig. 8, B and D). The pervalvar axis is 1.0–3.4 lm. Each cell contains a sin- two adjacent basal plates are held together by the gle large chloroplast (Fig. 7A). Setae emerge inside fusion of their specialized setae (Fig. 8, C–E). These the valve margin with a basal part and cross at the setae have a slightly larger diameter, are more silici- chain edge (Fig. 7B). Setae diverge with variable fied and have a distinct orientation: they join at the angles. Setae are circular in cross section and are chain margin, fuse for a length a couple of times ornamented with spirally arranged spines and por- their diameter, then separate and turn around the oids (Fig. 7, C–E), along with larger solitary pores cell (Fig. 8, C–E). The setae of the basal plate are (Fig. 7, C and D). Valve face is elliptical to circular, ornamented with spirally arranged small spines with a central annulus from which dichotomously (Fig. 8, C and E). The surface of the primary valve branching costae radiate toward the valve margins is covered with numerous spines, often with an (Fig. 7G). The valves are perforated with minute expanded base that connects adjacent spines pores (Fig. 7H). A small hyaline rim is present on (Fig. 8, A and C). The secondary valve of the spore the edge of the valve face (arrowed in Fig. 7G). The is smooth and possesses two notches (Fig. 8, A, B terminal valve has a central rimoportula, with a slit- and F), which fit in the insertion points of the spe- like opening on the inner valvar face (Fig. 7G) and cialized setae of the basal plates (Fig. 8, D and E). a short flattened tube on the outer valvar face Chaetoceros radicans Schutt€ (Figs. 9, A–L and 10, (Fig. 7F). Girdle bands are ornamented with trans- A–C) verse costae alternating with less silicified areas per- We examined the morphology of five strains of forated by minute perforations (Fig. 7I); a siliceous C. radicans isolated from Las Cruces (Chile; ridge runs along the whole length of the band. Table 1). Chains are straight (Fig. 9A), curved or Spores have a plano-convex shape (Fig. 8A) and are slightly twisted. Cells are rectangular in broad girdle organized in pairs held together by two specialized view, separated by a narrow hexagonal aperture; valves, named basal plates (Fig. 8B). Each spore occasionally a broader aperture is observed (Fig. 9I). adheres with its secondary valve to the The cell apical axis is 5.4–12.8 lm long, the TWO NEW CHAETOCEROS SPECIES 13 pervalvar axis is 4.3–16.7 lm long, and the length of and C. sporotruncatus sp. nov., and to provide addi- the aperture in pervalvar axis is 1.1–3.9 lm. Cells tional morphological and molecular information on contain one large chloroplast (Fig. 9A). Intercalary two phylogenetically closely related species, C. cinc- setae emerge inside the valve margin with a basal tus and C. radicans. part and cross at chain edge (Fig. 9, A and D). They Taxonomy and phylogeny of C. socialis complex. The are differentiated into normal (Fig. 9A) and special- phylogenies inferred from the 18S and the partial 28S ized setae (Fig. 9, B and C). Specialized setae are rDNA genes revealed that C. socialis complex is mono- thicker and lie in the valvar plane forming an angle phyletic. Strains included in this species complex of 90° with the apical plane (Fig. 9, B and C). Nor- cluster into four distinct clades, each with high boot- mal setae diverge with variable angles. Both special- strap support. The four clades comprise: C. socialis, ized and normal setae are circular in cross-section C. gelidus and the two new species C. sporotruncatus and are ornamented with spirally arranged rows of and C. dichatoensis. The clade of C. sporotruncatus also small poroids and spines along with large, elongate includes the sequence of strain CCMP172 that has solitary pores (Fig. 9, D and F–H). In the proximal been used in previous studies based on 28S phyloge- part of the specialized setae, the spines extend into nies (Kooistra et al. 2010, Degerlund et al. 2012) capilli of different length, at times branched showing its distinctness from C. socialis and C. gelidus (Fig. 9D). Capilli are absent on the distal part (Chamnansinp et al. 2013). Chamnansinp et al. (Fig. 9D). Terminal setae have similar ultrastructure (2013) studied the morphology of this strain but did as the normal intercalary setae, with a denser pat- not describe it as a new species as only one strain was tern of spines at their base (Fig. 9E). Strains available, which failed to produce resting spores. Rest- stopped producing specialized setae after a few gen- ing spore information became available for the strains erations in culture. The valve face is elliptical to cir- from the Chilean central coast, and therefore, strain cular, with a central hyaline annulus from which CCMP172 is now assigned to C. sporotruncatus sp. nov. dichotomously branching costae radiate toward the The absence of any 28S rDNA sequence differ- valve margins (Fig. 9, J and K). A narrow hyaline ences among the Neapolitan and Roscoff strains of rim was present on the edge of the valve surface C. socialis suggests that they all belong to the same (Fig. 9, J and K). Terminal valves have a central species. The one base pair difference with the 28S rimoportula, with slit-shape internally (Fig. 9K) and rDNA of strain YL1 from the South China Sea desig- with a short flattened tube externally (Fig. 9L). Gir- nated as epitype of C. socialis, could be due to the dle bands are ornamented with transverse costae pronounced geographic distance. In contrast, the alternating with less silicified areas perforated by five base pairs difference between all these strains minute perforations (Fig. 9M); a siliceous ridge runs and strain No_1 from Thailand (Chamnansinp et al. along the whole length of the band. Spores are 2013) suggests that the latter belongs to a different smooth and have a plano-convex shape (Fig. 10, A species. The absence of any 28S rDNA sequence and C). The primary valve is dome-shaped and the variation among the strains within C. gelidus, secondary valve is probably flat (not observed, based C. sporotruncatus and C. dichatoensis indicates intra- on the presence of the flat basal plate and similarity specific genetic homogeneity. However, this needs with C. cinctus) and presents a single ring of puncta to be checked on a higher number of strains from on the advalvar margin of the mantle (Fig. 10C). As different geographic areas, especially for the two in C. cinctus, spores are organized in pairs held new species. together by two specialized valves, named basal The Chilean strains of Chaetoceros dichatoensis and plates (Fig. 10, B and C). Each spore adheres with C. sporotruncatus from Las Cruces were collected on its secondary valve to the corresponding basal plate. the same dates and from the same net samples, i.e., The two adjacent basal plates are held together by their populations occurred in sympatry at the time the fusion of their specialized setae. These setae of collection. Nevertheless, the Sanger sequence have a slightly larger diameter, are more silicified reads of the two groups of strains showed unam- and have a distinct orientation: they join at the biguous differences, suggesting that these sympatric chain margin, fuse for a length a couple of times populations belong to biologically separated enti- their diameter, then separate and turn around the ties. cell (Fig. 10, B and C). The setae of the basal plate The morphological characters of vegetative cells are ornamented with spirally arranged small spines and spores of the species included within the C. so- (Fig. 10, B and C). cialis complex are summarized in Table 2. The similar morphology of the vegetative cells of the four species in the C. socialis complex makes their differ- DISCUSSION entiation and correct identification in light and Results of detailed ultrastructural investigations electron microscopy almost impossible. These spe- coupled with molecular phylogenetic analyses of sev- cies all share the same primary and secondary struc- eral strains isolated from Chilean coastal waters ture of the colony, orientation pattern of the setae, allowed us to describe two new species within the and valve and setae ultrastructure, with only minor Chaetoceros socialis complex, C. dichatoensis sp. nov., differences. In all four species, the longer, inward- 14 CHETAN C. GAONKAR ET AL.

FIG.8. Chaetoceros cinctus, SEM pictures of spores. (A–F) strain Ch10B3. (A) A spore with spiny primary valve (arrow) and smooth secondary valve. The notch on the secondary valve is arrowheaded. (B) Spore detached from its basal plate (arrow). The notch on the secondary valve is arrowed. (C) Primary valve (arrow) of a spore attached to its basal plate. Note the spirally arranged small spines on the setae of the basal plates. (D) Lateral view of the two adjacent basal plates (arrow and arrowhead). (E) Internal view of the basal plate with specialized setae. Arrow indicates the insertion point of the specialized seta where the notch of the spore ﬁts. (F) A spore with notches on the secondary valve (arrowed). Scale bars: (A–F) = 1 lm.

pointing setae exhibit generally a lower density of varies over the size reduction process and the maxi- spines than the short setae, and the spines are mum size is that of the initial cell produced in the mainly conﬁned to the distal part of the long seta. auxospore after sexual reproduction (Montresor This feature was not reported in the amended et al. 2016). Moreover, along cell size reduction the description of C. socialis (Chamnansinp et al. 2013), progressive decrease of the apical axis can be but has been observed in the strains from the Gulf accompanied by an increase in the transapical axis of Naples examined in this study. Minor differences (Assmy et al. 2008). in setae position were only detected between Spore morphology appears as the distinctive char- C. gelidus and the other three species; in the former acter to differentiate among the four species of the species, the setae emerge closer to the margin of C. socialis complex (Table 2). The spores of C. so- the valves with a shorter basal part and thus form cialis are ornamented with spines on both valves narrower apertures between adjacent cells in a chain (Chamnansinp et al. 2013). These spores may (Chamnansinp et al. 2013, Balzano et al. 2017). resemble those of C. dichatoensis, which are also Although cell size ranges provided for the different ornamented with spines of variable length and num- species are different, they should be considered ber. However, spines in C. dichatoensis are frequently only as indicative characters. Cell size in diatoms connected at their base by siliceous ridges of TWO NEW CHAETOCEROS SPECIES 15

FIG.9. Chaetoceros radicans vegetative cells. (A and B) strain Ch11A4, (C) strain Ch10A3 and (D–L) strain Ch1B4. (A, B) LM; (C–GandI) SEM; (H and J–L) TEM. (A) Chain in broad girdle view. (B) Chain in valve view, note the specialized setae with spirally arranged capilli. (C) Intercalary valve with specialized setae; arrow marks branched capilli. (D) Detail of a specialized seta with capilli (arrowed) in the proximal part and spines (arrowheads) in the distal part. Note the spiral arrangement of both capilli and spines. (E) Detail of the proximal portion of a terminal seta; spirally arranged spines are visible and a large pore is arrowed. (F) Detail of the smooth proximal portion of a normal intercalary seta. (G and H) Detail of the middle portion of normal setae with spirally arranged spines and poroids; the arrow marks a large pore. (I) Aperture between adjacent cells. (J) Intercalary valve with radially branching costae and central annulus; the arrow marks the hyaline valve rim. (K) A terminal valve with central, slit-shaped rimoportula; the arrow marks the hyaline valve rim. (L) External view of a terminal valve with central, ﬂattened tube. (M) Detail of girdle bands. Scale bars: (A, B, C) = 10 lm, (D–I, K–L) = 1 lm, (J) = 2 lm. 16 CHETAN C. GAONKAR ET AL.

Hargraves 1979, Kooistra et al. 2010, Degerlund et al. 2012), which is absent in C. dichatoensis. Spores of C. gelidus have smooth valves and the mantle of both valves has a distinctive crest of more or less fused projections (Chamnansinp et al. 2013). Variability in the presence and extension of the crest has been reported in other studies (as C. socialis ‘northern strains’ in Degerlund et al. 2012, Balzano et al. 2017). In C. sporotruncatus, the spores are biconvex with truncated valves and the primary one is ornamented with raised lenticular-shaped structures. Hargraves (1979) reported a high diversity in spore morphology within what was called ‘varieties of C. sociale’ and illustrated four spore morphotypes from samples collected in different geographical areas. Spores covered by spines and with a row of spines at the margin between the valves and the mantle were reported from Narragansett Bay and Chesapeake Bay and attributed to C. sociale var. radians (Schutt)€ Proschkina-Lavrenko (figs. 42–47, in Hargraves 1979), which is now a synonym of C. socialis (Chamnansinp et al. 2013). Indeed, these spores match the description of the spores of C. socialis. A second spore morphotype with smooth valves and surrounded by a perforated collar was recorded in material from Trondheim (Norway) and attributed to C. sociale var. sociale (figs. 48–50, in Hargraves 1979). This spore morphotype corre- sponds to C. gelidus. The spore morphotype collected along the coast of Peru presented ‘raised slots’ on the top of one valve (figs. 55 and 56, in Hargraves 1979). This latter morphotype corre- sponds to C. sporotruncatus. The other morphotype collected in summer in Narragansett Bay, presented a collar and circular or ovoid pores with a thick rim ornamenting the valves; some of the pores pene- trated to the spore interior (figs. 52–54, in Har- graves 1979). This morphotype does not correspond to any spore of the C. socialis species complex known to date. Spores comparable to those of C. sporotruncatus have previously been reported from the Pacific Mexican coast (Plate 51, figs. 4 and 5, in Hernandez-Becerril 1996) and from the Atlantic Ocean in coastal waters of Buenos Aires Province, Argentina (fig. 13H, in Sunesen et al. 2008). Diatom spores attributed to C. socialis (as C. sociale in fig. 5f, Pitcher 1986) were also reported from the southern Benguela upwelling system. The morphology of this spore seems to be intermediate FIG. 10. Chaetoceros radicans, SEM pictures of spores. (A) Strain Ch10A3 and (B, C) strain Ch1B4. (A) Spore (arrow) still partially between C. sporotruncatus and C. dichatoensis; i.e., the embedded in the parental theca (arrowhead). (B) Internal view valve is moderately convex, the lenticular-shaped of the basal plate (arrow) with specialized setae ornamented with structures are not very high and are not restricted short spines. (C) Internal view of the flat secondary valve, with to the central portion of the valve as they appear in the ring of puncta (arrow), inserted on the basal plate (arrow- C. sporotruncatus, and somehow resemble the ridges head). All scale bars = 1 lm. of C. dichatoensis. Taxonomy and phylogeny of Chaetoceros cinctus and variable shape and thickness. Furthermore, spores Chaetoceros radicans. Chaetoceros radicans was of C. socialis generally have a ring of spines on the described from the Atlantic Ocean by Schutt€ margin of the valves (as C. sociale var. radians in (1895), who only included a single drawing TWO NEW CHAETOCEROS SPECIES 17 illustrating two cells with the characteristic setae Due to the absence of unambiguous type material ornamented with capilli. Since then, it has been of C. radicans, an epitype needs to be designated. reported from various geographical areas, and is The Chilean material cannot be considered as epi- currently considered a cosmopolitan species (Hasle type because the type locality of the species is and Syvertsen 1997). The morphology of C. radicans “Atlantic Ocean.” The North Sea strain CCMP197 has been described as extremely variable, especially could fulfill that role, but unfortunately, no mor- regarding the presence and density of the capilli on phological information is available for it. For these the intercalary setae, ranging from numerous and reasons an epitype is not proposed here. thick to absent (Rines and Hargraves 1988), and Gran (1897) described Chaetoceros cinctus from the their presence on terminal setae (Jensen and Atlantic and North Sea. He provided a detailed Moestrup 1998, Berard-Therriault et al. 1999, Shev- description and illustration of both vegetative cells chenko et al. 2006). In field material, stiff chains and spores matching our findings. The species has a with both intercalary and terminal setae orna- cosmopolitan distribution (Hasle and Syvertsen mented by numerous capilli (named “spines” in 1997) and can be easily differentiated from C. radi- Hernandez-Becerril 1996) have been observed. It is cans as it lacks the characteristic setae ornamented however important to remark that capilli are no with capilli. However, when these specialized setae longer formed in culture after a few weeks from are absent, it is almost impossible to discriminate strain isolation (Stockwell and Hargraves 1986, between these two species. In this case, the only dis- Rines and Hargraves 1988, this study). tinctive character that remains is the morphology of General morphology and ultrastructure of the the spores. The spores have the same distinctive Chilean strains examined in the present study organization in pairs held together by specialized matches previous descriptions (e.g., Fryxell and basal plates, but the primary valves are ornamented Medlin 1981, Sunesen et al. 2008, Lee et al. 2014) with small spines in C. cinctus, whereas they are with only minor differences in the ultrastructure of smooth in C. radicans. The high similarity in mor- setae. Field material from the Atlantic Ocean along phology of vegetative cells and the presence of the Argentinian coast exhibits smooth setae on the paired spores with a characteristic orientation of the basal plate holding the pair of spores (fig. 12, C setae in both C. radicans and C. cinctus suggest that and F, in Sunesen et al. 2008), while in the Chilean they are indeed a single species (Rines and Har- strains the setae of this basal plate are ornamented graves 1988, Jensen and Moestrup 1998). However, with numerous short spines. In the Chilean strains, our molecular data confirm that they are two clearly the normal intercalary setae, i.e., the ones without distinct species. capilli, are ornamented with rows of spirally Chaetoceros furcillatus has been reported under var- arranged spines and poroids, and with few large ious different names, including C. furcillatum, C. fur- elongate pores, whereas we do not have this infor- cellatus, and C. furcellatum. The valid name is mation for the intercalary setae with capilli. The Chaetoceros furcillatus, in accordance with the Latin C. radicans strain WK-140306 isolated from the West “furcillatus” and the masculine gender of the genus Sea of Korea lacks pores on intercalary setae with noun (McNeill et al. 2012). It is a species dis- capilli, but this observation could have been due to tributed in northern cold waters and its spores are the incomplete removal of organic material (figs. very similar to those of C. cinctus and C. radicans.In 113–115 and 134, in Lee et al. 2014). These mor- C. furcillatus, spores can be smooth or spiny and are phological differences among strains from different held together by basal plates (figs. 74–79, in Shev- regions suggest either the existence of multiple spe- chenko et al. 2006). Single spores, that could repre- cies within a species complex of C. radicans, or a sent spores formed in the terminal vegetative cell of morphologically plastic species. the chain, have also been observed (figs. 78 and 79, The sequence differences between the Chilean in Shevchenko et al. 2006). Differently from C. radi- and North Sea strains of C. radicans are comparable cans and C. cinctus, setae of basal plates do not turn to the differences observed between the two Chilean around the cells but remain away from the cell and species in the C. socialis complex (i.e., C. dichatoensis perpendicular to the transapical plane. Unfortu- and C. sporotruncatus), suggesting that these C. radi- nately, molecular information on C. furcillatus is not cans strains represent different species within a spe- available to verify its phylogenetic relationships with cies complex. Such complexes have been often C. cinctus and C. radicans. reported in the genus Chaetoceros: C. contortus Spore morphology. Spore morphology can be a use- (Chamnansinp et al. 2015), C. curvisetus (Kooistra ful character for the identification of Chaetoceros spe- et al. 2010), C. decipiens-lorenzianus complex (Li cies (Ishii et al. 2011) and can help in differentiating et al. 2017), C. neogracilis (Balzano et al. 2017), and closely related species that have very similar vegetative C. socialis (Degerlund et al. 2012, Chamnansinp cells. This is the case of C. radicans and C. cinctus as et al. 2013, this study). However, the genetically dif- well as of the species included in the C. socialis com- ferent C. radicans strains could also represent geo- plex. However, the various degrees of morphological graphically isolated, genetically distinct populations convergence reported among Chaetoceros species has within a single species. to be considered, e.g., the spores of C. gelidus 18 CHETAN C. GAONKAR ET AL. somewhat resembling those of C. contortus, C. protu- Assmy, P., Hernandez-Becerril, D. U. & Montresor, M. 2008. berans, or C. curvisetus (Kooistra et al. 2010). The Morphological variability and life cycle traits of the type species of the diatom genus Chaetoceros, C. dichaeta. J. Phycol. apparent similarity between spores produced by dif- 44:152–63. ferent taxa can result in identification problems, Auwera, G. V. D. & de Wachter, R. 1998. Structure of the large especially when these spores are recorded in sedi- subunit rDNA from a diatom, and comparison between small and large subunit ribosomal RNA for studying Stramenopile ment traps or in sediment cores, without any – remains of the vegetative cells connected to these evolution. J. Euk. Microbiol. 45:521 7. Balzano, S., Percopo, I., Siano, R., Gourvil, P., Chanoine, M., resting stages. For this reason, in most cases, it is Marie, D., Vaulot, D. & Sarno, D. 2017. Morphological and important to consider spore morphology together genetic diversity of Beaufort Sea diatoms with high contribu- with the morphological characters of the vegetative tions from the Chaetoceros neogracilis species complex. J. Phy- – cells. Moreover, it is important to consider the high col. 53:161 87. Berard-Therriault, L., Poulin, M. & Bosse, L. 1999. Guide d’Identifi- level of variability observed in Chaetoceros spores, cation du Phytoplankton Marin de l’Estuarie et du Golfe du Saint- especially concerning the shape and extension of Laurent Incluant Egalement Certains Protozoaires. Canadian Spe- crests, ribs, number of spines or processes (Ishii cial Publication of Fisheries and Aquatic Sciences No. 128. et al. 2011). These differences can be observed even NRC Research Press, 387 pp. within a single strain (see C. dichatoensis and Chamnansinp, A., Li, Y., Lundholm, N. & Moestrup, Ø. 2013. Global diversity of two widespread, colony-forming diatoms C. sporotruncatus, this study) and may be related with of the marine plankton, Chaetoceros socialis (syn. C. radians) different levels of silicification of the spores, or with and Chaetoceros gelidus sp. nov. J. Phycol. 49:1128–41. the different size of the vegetative cell. The latter, Chamnansinp, A., Moestrup, Ø. & Lundholm, N. 2015. Diversity for example, can affect the number of branching of the marine diatom Chaetoceros (Bacillariophyceae) in Thai waters – revisiting Chaetoceros compressus and Chaetoceros contor- processes in spores of C. diadema (Kooistra et al. tus. Phycologia 54:161–75. 2010, Ishii et al. 2011). Cullings, K. W. 1992. Design and testing of a plant-specific PCR The distinct spore morphologies associated with primer for ecological and evolutionary studies. Mol. Ecol. the four clades within the C. socialis complex and 1:233–40. between C. cinctus and C. radicans allows identifica- Degerlund, M., Huseby, S., Zingone, A., Sarno, D. & Landfald, B. 2012. Functional diversity in cryptic species of Chaetoceros tion in routine surveys using LM, as long as the field socialis Lauder (Bacillariophyceae). J. Plankton Res. 34:416–31. material exhibits those spores. The distinct spore Doyle, J. J. & Doyle, J. L. 1987. A rapid DNA isolation procedure morphotypes associated with these species and the for small quantities of fresh leaf tissue. Bull. Phytochem. – reports of similar morphotypes in geographically 19:11 5. Fryxell, G. A. & Medlin, L. K. 1981. Chain forming diatoms, evi- distant locations suggest that each of them is widely dence of parallel evolution in Chaetoceros. Cryptogamie Algol. distributed. The finding of spore morphologies 2:3–29. other than those observed in the four species of the Gran, H. H. 1897. Botanik. Protophyta: Diatomaceae, Silicoflagellata og Cilioflagellata. Den Norske Nordhavs-Expedition 1876-1878. C. socialis complex (see above discussion on summer – morphotype in Narragansett Bay), suggests the exis- Botanik, Christiana, Grøndahl ns, pp. 1 36. Guillard, R. R. L. 1975. Culture of phytoplankton for feeding tence of additional species in the complex. On the marine invertebrates. In Smith, W. L. & Chanley, M. H. other hand, the usefulness of spores for species [Eds.] Culture of Marine Invertebrate Animals. Plenum Press, detection may be severely limited if spore morphol- New York, pp. 29–60. ogy is plastic as a result of environmental factors or Guiry, M. D. & Guiry, G. M. 2016. AlgaeBase. World-wide elec- tronic publication. National University of Ireland, Galway. exhibits geographic variation. These possibilities Hall, T. A. 1999. 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Biological Laboratories) Grant Agreement 227799, staff of Harrison, P., Zingone, A., Mickelson, M., Lehtinen, S., Ramaiah, ECIM, Las Cruces, Chile, P. von Dassow, PUC, Santiago de N., Kraberg, A., Sun, J., McQuatters-Gollop, A. & Jakobsen, Chile, and CBL, COPAS, Concepcion, Chile for hosting him. H. 2015. Cell volumes of marine phytoplankton from glob- CBL acknowledges support by ASSEMBLE and CNRS/LIA- ally distributed coastal data sets. Estuar. Coast. Shelf Sci. – MORFUN Mission Nr. 7569 and Convenio de Desempeno~ 162:130 42. UCO1202, University of Concepcion. The authors wish to Hasle, G. R. & Syvertsen, E. E. 1997. Marine diatoms. In Tomas, C. R. [Ed.] Identifying Marine Phytoplankton. Academic Press, thank Alessandro Manfredonia, Ferdinando Tramontano and – Carmen Minucci (SZN) for media preparation, culture San Diego, California, pp. 5 385. Hernandez-Becerril, D. U. 1996. A morphological study of Chaeto- maintenance and assistance in DNA extraction respectively. ceros species (Bacillariophyta) from the plankton of the Paci- We acknowledge the support provided by the Electron Micro- fic Ocean of Mexico. Bull. Nat. Hist. Mus. Lond. 26:1–73. scopy Service and the Molecular Biology service of SZN, the Huseby, S., Degerlund, M., Zingone, A. & Hansen, E. 2012. Meta- COPAS Oceanographic Time Series off Concepcion, and bolic fingerprinting reveals differences between northern the crew of the L/C Kay-Kay II. This study was supported by and southern strains of the cryptic diatom Chaetoceros socialis. the project FIRB Biodiversitalia (RBAP10A2T4) funded by Eur. J. Phycol. 47:480–9. the Italian Ministry of Education, University and Research Ishii, K. I., Iwataki, M., Matsuoka, K. & Imai, I. 2011. Proposal of (MIUR). identification criteria for resting spores of Chaetoceros species TWO NEW CHAETOCEROS SPECIES 19

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