Morphology and Phylogeny of the Marine Bipolar Centric Diatom Pseudoleyanella Lunata (Cymatosiraceae) with Special Reference to the Diatotepum

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 N 1†, Tomoko Y 2 and Shigeki M 1*

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, slightly 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 gradually lost their dorsiventral nature, and eventually became almost circular. Although P. lunata was rectangular in girdle view in small cells, large vegetative cells, particularly those generated soon aer auxosporulation, 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 girdle 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 Campylosira cymbelliformis CCC-1 HQ912623/HQ912487 Cerataulina pelagica ECT3845 HQ912669/HQ912533 cf. Minutocellus sp. CCMP1701 AY485520/N/A Chaetoceros muellerii CCMP1316 HQ912558/HQ912422 Cymatosira belgica ECT3892 N/A/KC309563 Cymatosira belgica 25VI12-1 N/A/KJ577891 Cymatosira lorenziana ECT3874 KC309490/KC309562 Extubocellulus cribriger CCAP1026/2 N/A/FJ002150 Extubocellulus cribriger CCMP391 HQ912571/FJ002116, HQ912435 Extubocellulus sp.1 CCAP1018/1 N/A/KC309570 Extubocellulus spinifer CCMP393 AY485504/FJ002117 Hemiaulus sinensis CCH-2 HQ912624/HQ912488 Leyanella arenaria CCMP471 HQ912570/FJ002146, HQ912434 Leyanella arenaria R23 N/A/HQ413688 Minutocellus polymorphus CCAP 1049/1 FR865497/N/A Minutocellus polymorphus CCMP3303 KF925333/N/A Minutocellus polymorphus CCMP497 AY485478, HQ912568/FJ002145, HQ912432 Minutocellus polymorphus ECT3920 KC309498/KC309572 Minutocellus sp. CCMP1701 N/A/FJ002118 Papiliocellulus elegans clone 1127 JF790987/N/A Papiliocellulus simplex CS431 HQ912630/HQ912494 Pierrecomperia catenuloides R10 HQ413684/HQ413686 Pierrecomperia catenuloides R2 HQ413685/HQ413687 Plagiogrammopsis sp. TN-2014 N/A/KJ577906 Plagiogrammopsis vanheurckii ECT3856 KJ577870/KJ577907 Plagiogrammopsis vanheurckii ECT3885 KC309504/KC309578 Pseudoleyanella lunata NG0001 LC164820/LC164794 Terpsinoë musica NHOP43 HQ912682/HQ912546 uncultured Minutocellus clone 1103 JF790976/N/A uncultured Minutocellus clone 7922 JF791106/N/A Urosolenia eriensis Y98-8 HQ912577/HQ912441

(Toyobo, Osaka, Japan) using MiniCycler (Bio-Rad, aligned with publicly available sequences retrieved from Hercules, CA, USA) with an initial denaturation step at GenBank (Table 2). For the SSU rDNA, sequences were 95°C for 3.5 min, followed by 35 cycles of 95°C for 30 s, rst aligned with MAFFT version 7 (Katoh & Daron 55°C for 30 s, and 68°C for 1.5 min. e primers used for 2013) and rened by referring to a secondary struc- PCR amplications and sequencing are listed in Table 1. ture model of the 18S rRNA molecule (Van de Peer et e PCR products were puried by the Wizard SV Gel al. 2000). Finally, ambiguously aligned positions were and PCR Clean-Up System (Promega, Madison, WI, excluded using BioEdit 7.0.2 (Hall 1999), resulting in a USA) and directly sequenced using BigDye Terminator data set of 1681 positions. Alignment of the rbcL dataset v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster was conducted manually and resulted in 1473 positions. City, CA, USA) using the following PCR protocol: ini- e datasets consisted of 30 and 31 operational taxo- tial denaturation step at 95°C for 1 min, followed by 25 nomic units (OTUs) for SSU rDNA and rbcL, respective- cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. ly, including multiple outgroups (Table 2) selected based e products were analyzed with an ABI 3130 DNA Se- on the multigene phylogeny by eriot et al. (2010). quencer (Applied Biosystems, Foster City, CA, USA). Maximum likelihood trees were calculated with RAxML e SSU rDNA and rbcL sequences obtained were 7.0.3 (Stamatakis et al. 2005). e model used was GTR- 4 Noriaki Nakamura, Tomoko Yuasa and Shigeki Mayama

GAMMA. 10000 bootstrap replicates were performed to tion of medium to small cells. Cells formed ribbon-chain assess the robustness of the nodes. colonies (Figs 1, 2, 4). One chloroplast was appressed to the ventral side of frustule (Fig. 3). In girdle view, large Results and Discussions cells were slightly bent, one valve being convex and the other concave (Fig. 1). Following cell size reduction, Living cell and chloroplast however, cells became rectangular with a slight elevation Strain NG0002 exhibited homothallic auxosporulation at each end (Fig. 2, arrowheads). During chloroplast in our culture condition and recovered large cell sizes. division, a cleavage furrow extended from the center of erefore, observations of large cells were made based one margin of the chloroplast to the middle of the cell on NG0002, whereas NG0001 was used for the observa- (Fig. 5a), and nally split the chloroplast in half. As the

Figs 1–5. Pseudoleyanella lunata, live cells. LM. Fig. 1. Girdle view of larger cells forming a chain-like colony aer auxosporulation in culture. Figs 2, 3. A chain of smaller cells (Fig. 2) and a single cell in valve view (Fig. 3). Arrows indicate cleavage furrows of chloroplast and arrow heads indicate slight elevation. Fig. 4. Chain of the shortest cells. Fig. 5. Diagram of chloroplast and cell division. a, b: chloroplast division through formation and development of a cleavage furrow. c: completion of chloroplast division and continued rotation of the two daughter chloroplasts. d, e: chloroplasts become parallel to one another. f: cytokinesis. Scale bars＝10 µm.

Figs 6–23. Pseudoleyanella lunata. Figs 6–15. Variation in valve size. Figs 6–10. LM. Figs 11–23. SEM. Figs 11, 13–15. Valve exterior. Fig. 12. Valve interior. Figs 16, 17. Dierent oblique views in the same valve. View from dorsal (Fig. 16) and ventral sides (Fig. 17). Figs 18, 19. Detailed view of both valve ends in the same specimen, showing an ocellulus at each pole (arrow). Fig. 20. Areolae occluded by rotae (with spinulae: arrows) and marginal ridge with decussate latticework. Fig. 21. Marginal ridge and valve mantle in girdle view. Fig. 22. Partly crushed sibling valves interlocked with marginal ridge, girdle view. Fig. 23. Sibling valves of the smallest cell interlocked with marginal ridges. EB: epithecal bands. EV: epivalve. m: marginal ridge. SV1: sibling valve 1. SV2: sibling valve 2. Scale bars＝10 µm (Figs 6–10); 5 µm (Figs 11–15); Scale bar＝2 µm (Figs 16, 17, 21, 22); 1 µm (Figs 18–20, 23). Morphology and phylogeny of Pseudoleyanella lunata 5 6 Noriaki Nakamura, Tomoko Yuasa and Shigeki Mayama

cleavage furrow developed, the chloroplast rotated (Fig. one side of the apex, the two ocelluli of a valve being di- 5b) and the separated chloroplasts continued to rotate agonally oset in a clockwise direction (as viewed from until they were parallel to one another (Fig. 5c, d) and the exterior) (Figs 18, 19). Each ocellulus comprised lay appressed to each valve face. Karyokinesis and cyto- 13–17 porelli in large valves, and 7–10 in smaller valves kinesis started aer chloroplast division. It took 150 min (Fig. 25). Along both sides of the valve face, P. lunata from the start of chloroplast division to the completion had marginal ridges composed of crisscross (decussate) of cytokinesis. meshes (ca. 4 holes/µm measured at right angles to the apical axis; Figs 20–23). Each hole of the mesh was itself Valve observation lled with irregular-shaped and occasionally branching Valves were narrowly lunate in large cells with acute bars (Figs 20, 21). e height of the marginal ridges was apices (Figs 6, 11, 12). e lunate outline and the acute almost constant throughout length of the valve, except apices were gradually lost as the cell became smaller, near the valve ends, where the ridges were discontinuous small cells having bilateral symmetry and obtuse apices (Figs 21, 28) and interlocked with the ocelluli of the op- (Figs 7–9, 13, 14). e smallest valves had an almost posite valves when the cells formed ribbon-like colonies circular outline (Figs 10, 15). Valves were 2.9–42.1 µ m (Figs 22, 23). e decussate framework of the marginal long and 1.6–3.7 µm wide. e valve was perforated by ridge was less evident in the smallest cells, although they circular areolae 168–440 nm in diameter, which were still formed chains (Fig. 23, see also Fig. 4). Internally arranged in transapical rows (14–16 in 10 µm) on the the valve face had a smooth surface with no projections greater part of the valve face (Figs 11–14, 16, 17) except or processes (Fig. 24). e rotae occluding the areolae in the smallest valves, where some areolae fused with were composed of 6–12 rod-like struts (Fig. 26). e cin- each other (Fig. 15). e valve center had a small hyaline gulum was composed of 5–8 plain bands (Figs 27, 28). area of indenite shape (Figs 11–14). e areolae were Since no closed band was found in cleaned specimens occluded externally by rotae with spinulae (Fig. 20). An under SEM, we assumed that all bands were open. e ocellulus was observed at or near each apex of the valve; pleura (i.e. the band furthest from the valve in the epi- it was surrounded by a thick rim and placed slightly to theca) was narrow, and ornamented with a row of small

Figs 24–28. Pseudoleyanella lunata. SEM. Fig. 24. Internal view of a whole valve. Fig. 25. Internal views of apices, showing the ocellulus (arrow) of smaller valve. Fig. 26. Detail of areolae occluded by rotae, internal views. Fig. 27. Internal view of a valve with a valvocopula. e arrow indicates opening. Fig. 28. Detailed view of band conguration. 1: rst (most advalvar) band. 2–6: further bands (including the ‘pleura’, band 6 with a row of small granules, arrows). m: valve mantle. Scale bar＝3 µm (Fig. 27), 2 µm (Fig. 24); 1 µm (Fig. 28); 500 nm (Figs 25, 26). Morphology and phylogeny of Pseudoleyanella lunata 7

granules (Fig. 28, arrows). Figs 13, 16, and 17). e correspondences between the The morphological and ecological features of this diatotepum and valve structure were also detected at specimen agreed well with those of Pseudoleyanella lunata the position of the ocellulus. Here, however, rather than (Takano 1985). bearing thickenings, the diatotepum was perforated, each hole corresponding to one of the pores of the ocel- Cell structure and diatotepum lulus (Fig. 33). Lines of high electron density were also In the cell sections, we observed diatotepum, an or- observed, each corresponding to the inner edge of a gir- ganic layer underlining the siliceous thecae (Fig. 29). dle band, i.e. the suture of a band’s pars interior (Fig. 34). e diatotepum seemed to be intact aer the removal of e diatotepum underlay the internal surface of the cell contents (Fig. 30) and HF treatment to dissolve the theca (Figs 29, 30) and stained with toluidine blue, silica structure enabled us to observe the shape of the which mainly stains acidic tissue components including diatotepum, which reected the structure of the theca sulfated polysaccharide (Sridharan & Shankar 2012). In (Figs 31, 32). Stained with toluidine blue, the isolated these respects, the diatotepum we observed agrees with diatotepum possessed intensively stained spots (Fig. 31), the denition of diatotepum proposed by von Stosch which corresponded to the areola pattern on the valve (1981). Besides P. lunata, three other species of cymato- face. e same circular spots were also observed under siracean diatoms are also known to possess diatotepum TEM as high electron density areas (Fig. 32, compare (Hasle et al. 1983). Tesson & Hildebrand (2013) detected

Fig. 29. Cross section of Pseudoleyanella lunata cell. TEM. c: chloroplast. D: diatotepum. m: mitochondrion. n: nucleus. Fig. 30. Valves treated with Pipe Unish. Internal view. SEM. Figs 31–34. Diatotepum of Pseudoleyanella lunata. LM (Fig. 31), TEM (Figs 32–34). Fig. 31. Diatotepum stained with toluidine blue. Fig. 32. Whole structure of diatotepum. Fig. 33. Ocellulus-like structure at one of the ends. Fig. 34. Portion corresponding to the girdle bands. e arrows indicate lines corresponding to suture of the pars interior. Scale bar＝10 µm (Fig. 31); 2 µm (Figs 30, 32); 1 µm (Fig. 34); 500 nm (Fig. 33); 100 nm (Fig. 29). 8 Noriaki Nakamura, Tomoko Yuasa and Shigeki Mayama

an organic layer rich in mannan in ve species of diatoms, which they characterized using gas chromatogra- phy–mass spectrometry. ey also observed the structure in detail using atomic force microscopy (AFM). Based on the biochemical and structural properties they reported, we assume that the layer they found is equiva- lent to what we call diatotepum. It should be noted that Navicula pelliculosa (Kützing) Hilse (a nomenclatural synonym of Mayamaea atomus (Kützing) Lange-Ber- talot) has an ‘organic skin’ that covers the internal and external surfaces of the valve and shows circular marks corresponding to the areolar pattern (Reimann et al. 1966), although von Stosch (1981) described this structure as a ‘coating substance’ to distinguish it from the diatotepum. e presence of intensively stained patches in the diatotepum underlying the areolae (Figs 31, 32) indicates higher concentrations or thicker accumulations of polysaccharide in this area than elsewhere. With AFM, Tesson & Hildebrand (2013) showed that the diatotepum-like organic matrices were thicker under each areola in Amphora Kützing, Coscinodiscus Ehrenberg, Stephanopyxis (Ehrenberg) Ehrenberg, Nitzschia Hassall Fig. 35. Maximum likelihood phylogenetic tree of SSU rDNA. and Triceratium Ehrenberg and suggested that organic Bootstrap support values (≥70) are given at the nodes. matrices act as molds for making the areolae during valve morphogenesis. e functions of diatotepum were originally thought to be to keep each silicied part in place within the frustule, to avoid direct attachment of plasmalemma to silica, or to give extra physical protec- tion to cells (von Stosch 1981). e electron dense areas observed under the areolae and internal sutures of the girdle bands (Figs 32, 34) may function to increase the strength of the frustule by reinforcing it, especially in fragile regions. Small pores observed in the diatotepum at the valve apices of Pseudoleyanella resemble the porelli of the ocelluli (Fig. 33) and may facilitate mucilage se- cretion from the cell.

Phylogenetic analysis Between SSU rDNA and rbcL, there are some incon- gruences such as the positions of genera possessing labiate process and marginal spines, i.e. Brockmanniella brockmannii (Hustedt) Hasle et al., Campylosira cymbelliformis (A. Schmidt) Grunow ex Van Heurck, Cyma- tosira lorenziana Grunow, and Plagiogrammopsis vanheurckii (Grunow) Hasle et al.: they displayed ladder-like diversication at the root of the Cymatosiraceae clade in the SSU tree, whereas they formed a clade with Cymato- Fig. 36. Maximum likelihood phylogenetic tree of rbcL. Boot- sira belgica Grunow in the rbcL tree, although its nodal strap support values (≥70) are given at the nodes. support was low (＜70%) (Figs 35, 36). e rest of Cy- matosiraceae members formed a clade with high support was classied within the subfamily Extubocelluloideae in both trees (100/100 bootstrap supports in SSU rDNA/ (Takano 1985); however, both phylogenetic trees placed rbcL trees). With their isovalvate nature (i.e. epi- and hy- P. lunata outside the clade for Extubocellulus and Pier- povalve are morphologically identical), Pseudoleyanella recomperia, rendering the subfamily paraphyletic. In Morphology and phylogeny of Pseudoleyanella lunata 9

both trees a sister relationship of P. lunata and Leyanella py. Diatom Research 9: 53–63. arenaria Hasle et al. was recovered with moderate nodal Gardner, C., Schulz, D., Crawford, R.M. & Wenderoth, K. 1995. supports (86/84). Stoschiella hebetata gen. et sp. nov. A diatom from intertidal sand. Diatom Research 10: 241–250. Gomes, A., Witkowski, A., Dąbek, P., Boski, T., Delminga, M., Sz- Taxonomic implications kornik, K. & Kurzydlowski, K. 2013. Syvertsenia iberica (Cy- Pseudoleyanella lunata and Leyanella arenaria both matosiraceae): A new estuarine diatom genus characterized have marginal ridges with prominent decussate meshes, by the position of its process. Phytotaxa 142: 25–36. but they dier in valve shape and whether the frustule Guillard, R.R.L. 1975. Culture of phytoplankton for feeding is iso- or heterovalvate (Takano 1985). Unlike the het- marine invertebrate. In: Smith, W.L. & Chanley, M.H. (eds) erovalvate Leyanella, both valves of P. lunata showed Culture of marine invertebrate animals. pp. 26–60. Plenum no ne-structural dierences, such as the presence or Press, New York. absence of pilus/tubular process. us, Pseudoleyanella Hall, T.A. 1999. BioEdit: A user-friendly biological sequence cannot be synonymized with Leyanella based on conven- alignment editor and analysis program for Windows 95/98/ tional taxonomy. Generally in diatom taxonomy, lunate NT. Nucleic Acids Symposium Series 41: 95–98. valve shape has been considered one of the important Hasle, G.R., von Stosch, H.A. & Syvertsen, E.E. 1983. Cymatosir- criteria for dening a genus in tandem with other fea- aceae, a new diatom family. Bacillaria 6: 9–163. tures. Pili and process, which are absent in Pseudoleya- Hendriks, L., De Baere, R., Van de Peer, J., Neefs, A. & Goris, A. 1991. e evolutionary position of the Rhodophyte Porphy- nella, are also considered important criteria for separat- ra umbilicalis and the Basidiomycete Leucosporidium sottii ing genera in Cymatosiraceae (Hasle et al. 1983, Takano among other eukaryotes as deduced from complete sequence 1985, Gardner & Crawford 1994). of small ribosomal subunit RNA. Molecular Biology and In the present study, our phylogenetic analyses indi- Evolution 32: 167–177. cate that P. lunata has a closer relationship with L. are- Katoh, K. & Daron M.S. 2013. MAFFT multiple sequence align- naria than with genera of Extubocelluloideae. is newly ment soware version 7: Improvements in performance and found relationship is also supported by the fact that both usability. Molecular Biology and Evolution 30: 772–780. species share a bent outline in girdle view, albeit only Kociolek, J.P., Sicko-Goad, L. & Stoermer, E.F. 1990. Cytoplasmic slightly and just aer auxosporulation in P. lunata (Fig. ne structure of two Encyonema species. In: Kociolek, J.P. 1). Among the genera of Cymatosiraceae, lunate valves (ed.) Proceedings of the 11th International Diatom Sympo- have evolved only in Pseudoleyanella and Campylosira. sium. pp. 235–245. California Academy of Science, Califor- nia. However, P. lunata loses its lunate outline during size McConville, M.J., Wetherbee, R. & Bacic, A. 1999. Subcellular lo- reduction (Figs 6–10). In Minutocellus and Leyanella, cation and composition of the wall and secreted extracellular pili are not formed when cells become small (Hasle et al. sulphated polysaccharides/proteoglycans of the diatom Stau- 1983). e process also varies in number and position in roneis amphioxys Gregory. Protoplasma 206: 188–200. Arcocellulus and Minutocellus (Hasle et al. 1983). ere- Medlin, L., Elwood, H.J., Stickel, S. & Sogin, M.L. 1988. e char- fore, the stability of these morphological features should acterization of enzymatically amplied eukaryotic 16S-like be veried in future for generic classication. rDNA-coding regions. Elsevier Science Gene 71: 491–499. Reimann, B.E.F., Lewin, J.C. & Volcani, B.E. 1966. Studies on the Aknowledgement biochemistry and ne structure of silica shell formation in diatoms. II. e structure of the cell wall of Navicula pellicu- We thank Prof. David G. Mann (Royal Botanic Gar- losa (Breb.) Hilse. Journal of Phycology 2: 74–84. den Edinburgh, Scotland, UK) for language editing of Reynolds, E.S. 1963. e use of lead citrate at high pH as an electron-opaque stain in electron microscopy. e Journal of Cell the nal version of manuscript. Biology 17: 208–212. References Ross, R., Cox, E.J., Karayeva, N.I., Mann, D.G., Paddock, T.B.B., Simonsen, R. & Sims, P.A. 1979. An amended terminology Dąbek, P., Sabbe, K., Witkowski, A., Archibald, C., Kurzydlowski, for the siliceous components of the diatom cell. Nova Hedwi- K. & Zglobicka, I. 2013. Cymatosirella Dąbek, Witkowski & gia, Beihe 64: 513–533. Sabbe gen. nov., a new marine benthic diatom genus (Bacil- Sabbe, K., Vanelslander, B., Ribeiro, L. Witkowski, A., Muylaert, lariophyta) belonging to the family Cymatosiraceae. Phyto- K. & Vyverman, W. 2010. A new genus, Pierrecomperia gen. taxa 121: 42–56. nov, A new species and two new combinations in the marine Daugbjerg, N. & Andersen, R.A. 1997. A molecular phylogeny of diatom family Cymatosiraceae. Life And Environment 60: the heterokont algae based on analyses of chloroplast-encod- 243–256. ed rbcL sequence data. Journal of Phycology 33: 1031–1041. Sridharan, G. & Shankar, A.A. 2012. Toluidine blue: A review of Gardner C. & Crawford, R.M. 1994. A description of Plagiogram- its chemistry and clinical utility. Journal of Oral and Maxillo- mopsis mediaequatus Gardner & Crawford, sp. nov. (Cymato- facial Pathology 16: 251–255. siraceae, Bacillariophyta) using light and electron microsco- Stamatakis, A., Ludwig, T. & Meier, H. 2005. RAxML-III: A fast 10 Noriaki Nakamura, Tomoko Yuasa and Shigeki Mayama

program for maximum likelihood-based inference of large sp. nov. (Diatomophyceae) from South America, California, phylogenetic trees. Bioinformatics 21: 456–463. West Greenland and Denmark. Phycologia 32: 278–283. Takano, H. 1985. A new diatom from sandats in Mikawa Bay, Van de Peer, Y., De Rijk, P., Wuyts, J., Winkelmans, T. & De Wachter, Japan. Bulletin of Tokai Regional Fisheries Research Labora- R. 2000. e European small subunit ribosomal RNA data- tory 115: 29–37. base. Nucleic Acids Research 28: 175–176. eriot, E.C., Ashworth, M., Ruck, E., Nakov, T. & Jansen, R.K. von Stosch, H.A. 1975. An amended terminology of the diatom 2010. A preliminary multigene phylogeny of the diatoms girdle. Nova Hedwigia, Beihe 53: 1–35. (Bacillariophyta): Challenges for future research. Plant Ecolo- von Stosch, H.A. 1981. Structural and histochemical observations gy and Evolution 143: 278–296. on the organic layers of the diatom cell wall. In: Ross, R. (ed.) Tesson, B. & Hildebrand, M. 2013. Characterization and localiza- 6th Diatom-Symposium. pp. 231–252. Otto Koeltz, Koenig- tion of insoluble organic matrices associated with diatom cell stein. walls: insight into their roles during cell wall formation. PLoS Witkowski, A., Lange-Bertalot, H. & Metzeltin, D. 2000. Diatom ONE 8: e61675. ora of marine coasts I. Iconographia Diatomologica 7: omsen, H.A., Buck, K.R., Marino, D., Sarno, D., Hansen, L.E., 1–925. Østergaard, J.B. & Krupp, J. 1993. Lennoxia faveolata gen. et