Diatom 32: 1–10. December 2016 Morphology and phylogeny of Pseudoleyanella lunata 1 DOI: 10.11464/diatom.32.1 Morphology and phylogeny of the marine bipolar centric diatom Pseudoleyanella lunata (Cymatosiraceae) with special reference to the diatotepum Noriaki N1†, Tomoko Y2 and Shigeki M1* 1 Department of Biology, Tokyo Gakugei University, 4–1–1 Nukuikita-machi, Koganei, Tokyo 184–8501, Japan 2 Department of Earth Science, Tokyo Gakugei University, 4–1–1 Nukuikita-machi, Koganei, Tokyo 184–8501, Japan * Corresponding Author. E-mail: [email protected] †Present address: Fukui Prefectural University, 1–1 Gakuen-cho, Obama, Fukui 917–0003, Japan Abstract Pseudoleyanella lunata Takano is a marine diatom with dorsiventral valves belonging to the family Cy- matosiraceae. We studied the range of variation in valve morphology throughout the life cycle. We also observed the chloroplast division of this species. In large cells, the valves were narrowly lanceolate, slight- ly capitate at the apices, and asymmetrical with respect to the apical plane, i.e. with almost straight and convex margins on the ventral and dorsal sides, respectively. During cell size reduction, the valves gradu- ally lost their dorsiventral nature, and eventually became almost circular. Although P. lunata was rectan- gular in girdle view in small cells, large vegetative cells, particularly those generated soon aer auxosporu- lation, were slightly bent, as in Leyanella Hasle et al. However, in contrast to Leyanella, P. lunata lacked both pili and tubular processes at all stages of its life cycle. In phylogenetic analyses (SSU rDNA and rbcL), P. lunata was sister to Leyanella. We also observed the whole structure of the diatotepum which was a sheet-like structure underlying each theca. Under the transmission electron microscope, dot-like marks with high electron density were observed: their position and pattern corresponded to valve poroids. In addition, the diatotepum bore electron-dense lines corresponding to the internal sutures between the gir- dle bands. Key index words: Cymatosiraceae, diatotepum, heterovalvy, Leyanella arenaria, Pseudoleyanella lunata Introduction colonies that grow attached to sand grains in a similar manner to the genus Leyanella, which is a member of e Cymatosiraceae is a family of bipolar marine Cymatosiroideae and also monotypic with L. arenaria centric diatoms, originally established by Hasle et al. Hasle et al. (1983). Pseudoleyanella and Leyanella also (1983) to include Cymatosira Grunow (the type genus) share frustule characteristics, most notably marginal and Campylosira Grunow ex Van Heurck along with ridges with an elaborate lattice of decussate ribs lying seven newly established genera, Arcocellulus Hasle et along the junction between valve face and mantle. How- al., Brockmanniella Hasle et al., Extubocellulus Hasle et ever, apart from its isovalvate frustules, Pseudoleyanella al., Leyanella, Minutocellus Hasle et al., Papiliocellulus can also be distinguished from Leyanella by its dorsiven- Hasle et al. and Plagiogrammopsis Hasle et al. Since 1983, tral valve and absence of pili and tubular processes. further genera have been added to the family as follows: ere has been no record of P. lunata since its original Pseudoleyanella Takano (1985), Lennoxia omsen & description by Takano (1985), probably because of its Buck (omsen et al. 1993), Hyalinella Witkowski et al. small cell size and rather simple morphology, so that (2000), Pierrecomperia Sabbe et al. (2010), Cymatosirella the species may have been overlooked under light mi- Dąbek et al. (2013) and Syvertsenia Witkowski & Gomes croscopy (LM). Recently we collected sand grains that (Gomes et al. 2013). e Cymatosiraceae is subdivided bore P. lunata cells, allowing us to establish monoclonal into two subfamilies, Cymatosiroideae for heterovalvate cultures. In the present study we report the frustule genera and Extubocelluloideae for isovalvate ones. morphology and the cellular structures of P. lunata, as e genus Pseudoleyanella is a member of Extubocel- well as the phylogenetic position of the strain among luloideae and currently consists of a single species, P. lu - the Cymatosiraceae using SSU rDNA and rbcL. We have nata Takano (1985). Pseudoleyanella forms ribbon-chain given special attention to the structural characteristics of diatotepum, which is an organic layer underlying the Received 16 March 2016 siliceous structure of diatoms (von Stosch 1981) and is Accepted 30 May 2016 found in P. lunata and also in other cymatosiracean gen- 2 Noriaki Nakamura, Tomoko Yuasa and Shigeki Mayama era, including Arcocellulus, Minutocellus and Papiliocellu- OPC40 osmium plasma coater (Filgen, Nagoya, Japan). lus (Hasle et al. 1983, Kociolek et al. 1990, Gardner et al. SEM observation was undertaken using a Hitachi S-4500 1995, McConville et al. 1999). microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 15 kV. For the observation of cellular structure Material and Methods under transmission electron microscopy (TEM), cells were xed with 1% glutaraldehyde in cacodylate buer Two samples were collected from Banzu tidal at (0.2 M sodium cacodylate, pH. 7.4) for 60 min at room (35.43876°N, 139.91248°E), Tokyo Bay, Chiba Prefec- temperature (RT), followed by post-x treatment with ture, Japan. A single chain colony of P. lunata was iso- 0.2% OsO4 for 1 min at 5°C. Aer washing three times lated from the surface of a sand grain by Pasteur pipette with the same buer, cells were embedded in Low Vis- in order to establish two cultured strains, NG0001 from cosity Resin (Agar Scientic, Stansted Essex, UK) aer a sample collected on May 28, 2011, and NG0002 from dehydration with an alcohol series, and polymerized for a sample collected on Jun 21, 2013. Both strains were 10 h at 70°C. Sections were cut using an ultramicrotome maintained in f/2 medium with a salinity of 33 (Guillard with a diamond knife and stained with uranyl acetate 1975) under conditions of 18°C, L : D＝12 : 12 h. Since the and Reynolds’s lead citrate (Reynolds 1963). e sec- strains underwent size reduction as a result of vegetative tions were dried on a copper grid with formvar support cell division, cells were xed by 2% formaldehyde at lm and observed using a JEOL 100CX-II (JEOL, Tokyo, several time points to preserve the cells of dierent size Japan) at an accelerating voltage of 80 kV. ranges, as follows: for strain NG0001, August 10, 2011 To remove protoplast and mucilaginous materials to (voucher ID: M-1152), December 23, 2011 (M-1165) isolate diatotepum from the cells, aliquots of cultured and August 30, 2012 (M-1224), and for NG0002, Octo- strain were treated for 15 min at RT with an equal ber 20, 2014 (M-1666). All the voucher specimens are amount of Pipe Unish (Johnson, Yokohama, Japan), kept in the diatom collection at Tokyo Gakugei Univer- which is a domestic drain cleaner containing detergent sity, Japan. and sodium hypochlorite, followed by rinsing in distilled Live cells were observed directly in plastic culture con- water. Some treated specimens were then dried onto a tainers using a Zeiss Axioskop LM (Zeiss, Oberkochen, cover slip for SEM. e rest were further treated with Germany) equipped with a ×63 water immersion lens. 4.6% hydrouoric acid (HF) for 7 min at RT, washed Images were captured using an Olympus DP71 digital with distilled water, and then stained with toluidine blue camera (Olympus, Tokyo, Japan). In order to prepare (pH 7) (Wako Pure Chemical Industries, Osaka, Japan) cleaned frustules, cells were heated in a water bath with for LM. HF-treated specimens were also dried on a grid concentrated sulfuric acid for 30 min, followed by addi- for TEM. Terminology for frustule morphology follows tion of potassium dichromate and boiling for 1 h; they Ross et al. (1979) and Hasle et al. (1983), and for the gir- were then rinsed with distilled water. Cleaned specimens dle, von Stosch (1975). were mounted in Mount Media (Wako Chemical, Osaka, For PCR amplication of molecular markers, cultured Japan) to make a permanent slide and observed under a cells were transferred to 0.2 ml Eppendorf tubes and cen- Nikon SKE microscope (Nikon, Tokyo, Japan) equipped trifuged at 1000×g for 2 min and the pellet was rinsed with an Olympus E-620 digital camera. For scanning twice in distilled water. e pellet was used as a template electron microscopy (SEM), cleaned valves were dried for the amplication of SSU rDNA and rbcL. PCR am- on a cover slip and coated with osmium using an Filgen plications were performed with KOD FX polymerase Table 1. List of primers. Primer name Sequence Reference SSU rDNA Aa 5′-ACCTGGTTGATCCTGCCAGT-3′ Medlin et al. (1988) 570Fb 5′-CGCGGTAATTCCAGCTCC-3′ Hendriks et al. (1991) 1180Fb 5′-AATTTGACTCAACACGGG-3′ Hendriks et al. (1991) 570Rb 5′-ATTACCGCGGCTGCTGGC-3′ Hendriks et al. (1991) 1130Rb 5′-CCGTCAATTTCTTTAAGTTT-3′ Hendriks et al. (1991) Ba 5′-CCTTCTGCAGGTTCACCTAC-3′ Medlin et al. (1988) rbcL NDrbcL2a 5′-AAAAGTGACCGTTATGAATC-3′ Daugbjerg & Andersen (1997) NDrbcL8moda 5′-GACCAATTGTACCACCACCAAAT-3′ Based on NDrbcL8 (Daugbjerg & Andersen 1997) a Primers used for amplication and sequencing. b Primers used for only sequencing. Morphology and phylogeny of Pseudoleyanella lunata 3 Table 2. List of sequences used in phylogenetic analyses. Taxon Locality GenBank Accession (SSU rDNA/rbcL) Arcocellulus cornucervis RCC2270 JN934677/N/A Arcocellulus mammifer CCMP132 EF192989, HQ912569/FJ002152, HQ912433 Brockmanniella brockmannii CCMP151 HQ912565/HQ912429 Brockmanniella brockmannii HK040 KC284711/N/A