Protist, Vol. 165, 81–92, January 2014 http://www.elsevier.de/protis Published online date 18 December 2013 ORIGINAL PAPER Polykrikos tanit sp. nov., a New Mixotrophic Unarmoured Pseudocolonial Dinoflagellate from the NW Mediterranean Sea 1 Albert René˜ , Jordi Camp, and Esther Garcés Institut de Ciències del Mar (CSIC) Pg. Marítim de la Barceloneta, 37-49 08003 Barcelona (Spain) Submitted July 1, 2013; Accepted December 4, 2013 Monitoring Editor: Mona Hoppenrath Pigmented pseudocolonies initially identified as Polykrikos hartmannii Zimmermann were detected at several locations of the Catalan coast (NW Mediterranean Sea) in April–June of 2012 and April–May of 2013. To further explore the several remarkable morphological discrepancies between these organisms and P. hartmannii, we carried out a detailed morphological study and used single-cell PCR to obtain par- tial LSU and SSU rDNA sequences. The resulting phylogenies showed that our isolates occupy a basal position within the Polykrikos clade, close to P. hartmannii, but do not correspond to any described polykrikoid species. P. barnegatensis Martin is controversially considered to be synonymous with P. hartmannii. The organisms studied in this work were similar to P. barnegatensis but showed signifi- cant morphological differences with its original description such as the torsion of the pseudocolony, more pronounced overhanging of the cingula, stepped fusion border of the zooids, and number and shape of nuclei. Consequently, we propose that the isolates constitute a new species, which we named Polykrikos tanit sp. nov. The observed characters, pigmented, same number of zooids and nuclei, sulci not fused, and its phylogeny suggest that the species is an early evolutionary Polykrikos species. © 2013 Elsevier GmbH. All rights reserved. Key words: Acrobase; mixotrophy; Pheopolykrikos; phylogeny; SEM; single-cell PCR. Introduction and autotrophic as well as heterotrophic species are known. The polykrikoid organisms are included within the Polykrikos species were primarily characterized Gymnodiniales sensu stricto clade (Hoppenrath on the basis of their even number of zooids, with and Leander 2007a,b; Kim et al. 2008) and are half the number of nuclei. Every zooid has its own grouped within two genera: Polykrikos, erected by cingulum and a pair of flagella. The sulci of both Bütschli (1873), and Pheopolykrikos, erected by zooids are fused. In addition to the type species of Chatton (1933) and later emended by Matsuoka the genus, P. schwartzii Bütschli, members include and Fukuyo (1986). Both genera comprise unar- P. kofoidii Chatton, P. lebourae Herdman emend. moured multinucleated pseudocolonial organisms Hoppenrath et Leander, P. herdmanae Hoppenrath et Leander, and P. hartmannii Zimmermann. The 1 Corresponding author; fax +34 93 230 9555 validity of P. grassei Lecal is highly dubious and P. e-mail [email protected] (A. René).˜ auricularia Bergh is considered to be synonymous © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.protis.2013.12.001 82 A. René˜ et al. with P. schwartzii. P. barnegatensis was erected wide with a length:width ratio of 1.4–2 (n=20). by Martin (1929) following observations of a single Pseudocolonies were ovate and nearly circular in specimen from Barnegat Bay (USA). This two-zooid cross-section. The sides of the pseudocolonies pseudocolonial photosynthetic organism contains were convex, with a constriction at the junction of only a single, large nucleus and lacks nemato- the two zooids (Figs 1A, B; 2A). The border of this cysts. It was synonymized with P. hartmannii by junction was stepped, with the left side being higher Chatton (1952), although he described two nuclei. than the right one (Figs 1A, B; 2A, B). The epi- However, this nomenclature was not adopted by cone of the anterior zooid was round (Fig. 1A) to Hulburt (1957) because of the difference in the conical (Figs 1F; 2A, B). The apex was blunted number of nuclei. Nevertheless, the synonymy of and protuberant (Fig. 2A, B). The hypocone of the P. barnegatensis with P. hartmannii is accepted posterior zooid was round, slightly bilobated, and (Gómez 2012; Guiry and Guiry 2013), albeit with the antapex was flattened. Pseudocolonies showed uncertainty (Hoppenrath and Leander 2007a). torsion of the cell body to the left (Fig. 2A). The The genus Pheopolykrikos comprises Ph. cingula were displaced about two to three times beauchampii Chatton as the type species, char- their width, with overhanging ends (Figs 1A, B; acterized by having four zooids and four nuclei. 2A, B). A large peduncle was always present in Although the validity of the genus has been dis- each zooid, emerging from the upper intercingu- cussed by several authors (Dodge 1982; Loeblich lar area (the area where both ends of the cingulum III 1980; Sournia 1986), Pheopolykrikos species meet) (Fig. 2A, B, C). Peduncles were present in all are defined as having the same number of nuclei specimens observed by SEM (n=30), but we never as zooids and forming phototrophic pseudo- observed them under LM. The sulci ran obliquely colonies that are able to dissociate (Matsuoka and from right to left (Fig. 2B) and had a sigmoid outline Fukuyo 1986). P. hartmannii was transferred into resulting from the overhanging cingula (Figs 1A, Pheopolykrikos because it agreed with all previ- B; 2A). The sulcus of the posterior zooid reached ously cited characteristics (Matsuoka and Fukuyo the antapex, where it widened. The sulcal anterior 1986). However, phylogenetic analyses of LSU end of the anterior zooid penetrated the epicone, in rDNA (Hoppenrath and Leander 2007a; Kim et al. contact with the acrobase, which formed a closed 2008) and SSU rDNA (Hoppenrath and Leander anti-clockwise loop around the apex that re-joined 2007a,b) sequences subsequently showed that P. the sulcus, although at a lower position than its hartmannii clusters with Polykrikos species inde- proximal end (Fig. 2A, B, D). The sulci of both pendently of Ph. beauchampii, thereby confirming zooids were not fused and ran independently. Pseu- the validity of the genus Pheopolykrikos. Moreover, docolonies had two nuclei, one for each zooid, those results together with ultrastructural studies located in the hypocone of the anterior zooid and led to the re-classification of Pheopolykrikos the epicone of the posterior zooid (Fig. 1C, E). hartmannii as Polykrikos hartmannii (Hoppenrath The nuclei were ovate to horizontally lenticular and et al. 2010), although this nomenclature has not nearly touched. Some pseudocolonies contained been adopted by all authors (Tang et al. 2013) only a single nucleus, located centrally (Fig. 1D). Recently, in samplings carried out at several loca- Pseudocolonies had a yellow-greenish colouration, tions along the Catalan coast (NW Mediterranean apparently with numerous small ovate chloroplasts Sea), we detected pigmented pseudocolonies (Fig. 1F, G). Neither nematocysts nor taeniocysts comprising two zooids. Detailed morphological were observed. Large ingestion bodies were com- observations and partial LSU and SSU rDNA monly seen in the posterior zooids (Fig. 1H), in sequencing were used to determine whether these some cases displacing the nuclei. Cyst formation organisms were P. hartmannii or P. barnegatensis was never observed. or constituted a new species. In this study, we show The pseudocolonies differed in width among that our isolates indeed belong to a new species, specimens. Some were wider, with a lower which we have named Polykrikos tanit sp. nov. length:width ratio (≤ 1.5), while others were nar- rower, with a higher length:width ratio (> 1.6). In the wider pseudocolonies (Figs 1H; 2A) the cin- Results gula were more deeply overhanging and torsion was greater than in the narrower pseudocolonies Polykrikos tanit sp. nov. (Figs 1A; 2B). Both morphologies were observed in Morphology natural samples, but healthy specimens and those Unarmoured pseudocolonies consisted of two possessing visible ingestion bodies tended to be zooids. They were 46–76 ␮m long and 26–50 ␮m wider, whereas isolated specimens maintained in Polykrikos tanit sp. nov. 83 Figure 1. Light microscopy images of P. tanit sp. nov. A) and B) Ventral view of pseudocolonies. Note the sig- moid outline of the sulci and the stepped junction of the two zooids (arrowheads). Arrows indicate the acrobase. C) Left lateral view of a pseudocolony showing two nuclei (n) and an ingestion body (i). Epifluorescence images of P. tanit sp. nov. D) Pseudocolony with one nucleus stained with Sybr Green. E) Pseudocolony with two nuclei stained with Sybr Green. F) and G) Images showing the shape and distribution of the autofluorescent plastids. Light microscopy images of P. tanit sp. nov. H) Right lateral view of a pseudocolony showing a large ingestion body (i). I) Ventral view of a single zooid. Scale bars = 10 ␮m. 84 A. René˜ et al. Figure 2. Scanning electron microscopy images of P. tanit sp. nov. A) and B) Ventral view of pseudocolonies, showing the acrobase (black arrows), the stepped junction of the two zooids (white arrowheads), and the presence of peduncles in both zooids (white arrows). Black arrowheads point to the longitudinal flagella. C) Detail of the intercingular area of the posterior zooid of a pseudocolony. The peduncle (arrow) is inserted in the proximal end of the cingulum. Arrowheads point to both longitudinal flagella. D) Apical view of the apex, showing the detail of the acrobase. Scale bars = A) and B) 10 ␮m; C) 5 ␮m; D) 2.5 ␮m. well plates for a few weeks and exhibiting a loss of Phylogeny pigmentation tended to be narrower. Single zooids The sequences obtained for the partial LSU rDNA were only observed once, when an isolated pseu- region were ∼690 bp (KF806602 was ∼585 bp) and ␮ ∼ docolony dissociated. These were 29–32 m long 1340 bp for the partial SSU rDNA region (Table 1). ␮ and 26–27 m wide (Fig. 1I), almost round but All the sequences of each region were identi- with a blunted apex. The cingulum was median, cal, except KF806602, which differed in 1 position displaced three times its width, with overhanging with respect to the other LSU rDNA sequences ends.