OPCE-STR.vp Acta Bot. Croat. 68 (2), 239–250, 2009 CODEN: ABCRA 25 ISSN 0365–0588 Cytological characteristics of the Sellaphoraceae DAVID G. MANN*, ALAN J. STICKLE Royal Botanic Garden Edinburgh, Inverleith Row, Edinburgh EH3 5LR, Scotland, UK Chloroplast and pyrenoid structure are described for Sellaphora, Fallacia and Rossia (Sellaphoraceae). All have a single chloroplast per cell, which is basically H-shaped, con- sisting of two large girdle-appressed plates connected by a central isthmus, though the plates may also bear longitudinal or transverse lobes; one or two invaginated pyrenoids are always present. Fallacia chloroplasts are more variable than those of Sellaphora but always differ fundamentally from the valve-appressed chloroplasts of the unrelated but superficially similar genus Lyrella. Outside Sellaphoraceae, invaginated pyrenoids are uncommon except in the Pinnulariaceae, closely related to the Sellaphoraceae according to molecular data and sharing a similar raphe structure, consistent with the classification of both in the suborder Sellaphorineae. However, invaginated pyrenoids are also found in Diploneis. Key words: diatom, Diploneis, Fallacia, Rossia Sellaphora, Pinnulariaceae, Sellaphora- ceae, chloroplast, pyrenoid Introduction Several changes to the genera of raphid diatoms were made by ROUND et al. (1990) and set in the framework of a new suite of families and orders. A brief description was provided for each genus, but the format did not allow detailed discussion and more detailed accounts of each new or remodelled raphid genus were planned for separate publication. Few of these have been completed, except for Sellaphora (MANN 1989), Lyrella (MANN and STICKLE 1993) and Petroneis (JONES et al. 2005). Meanwhile, molecular biology has pro- vided new tools to probe relationships at all taxonomic levels (e.g. MANN and EVANS 2007), providing important new insights that quite frequently contradict the ROUND et al. (1990) classification at the family level and above. Among the new taxa described by ROUND et al. (1990: 657) was the suborder Sella- phorineae, which comprised two families: Sellaphoraceae (Sellaphora, Fallacia, Rossia and Caponea) and Pinnulariaceae (Pinnularia, Diatomella, Oestrupia and the extinct ge- nus Dimidiata) (ROUND et al. 1990: 128). Small-subunit rDNA (BEHNKE et al. 2004, MEDLIN and KACZMARSKA 2004, SORHANNUS 2007) and rbcL gene trees (EVANS et al. 2008) support the view that Sellaphora, Fallacia, Rossia and Pinnularia, together with some ACTA BOT. CROAT. 68 (2), 2009 239 * Corresponding author: d.mann@rbge.org.uk U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 9. listopad 2009 13:09:45 Color profile: Disabled Composite 150 lpi at 45 degrees small-celled naviculoid species currently referred to Mayamaea and Eolimna (both de- scribed after 1990), comprise a monophyletic group. As noted by ROUND et al. (1990: 657, 'Sellaphorineae'), the raphe in Sellaphoraceae and Pinnulariaceae exhibits similar charac- teristics, with internal central endings deflected towards the primary side (except where hidden by extra silica deposition) and external central endings similarly deflected but also expanded. In this paper, we make a preliminary survey of protoplast structure in the Sellaphorineae, as a first step to determine whether any characteristics are autapomorphic for the Sellaphoraceae. Material and methods Sellaphorineae are generally epipelic. To obtain freshwater epipelon from shallow (< 1 m) sites, sediment and overlying water were collected using a glass tube as described by ROUND (1953); deeper sediments were sampled using an Ekman grab. For marine species, exposed damp sand was sampled at low tide. Sample preparation was as described by MANN and STICKLE (1993) and MANN and CHEPURNOV (2005). Bright field (BF) and differ- ential interference contrast (DIC) light microscopy (LM: planapochromat lenses, nominal numerical aperture 1.32) were carried out using a Reichert Polyvar 1 photomicroscope and Kodak Technical Pan film, or a Polyvar 2 fitted with a Polaroid DMC2 digital camera. Film negatives were digitized using a Nikon Super Coolscan 5000. Contrast and brightness of digital and digitized images were manipulated in Adobe Photoshop CS2 (http://www. adobe.com/) by global application of the Levels and Curves tools; limited burn-in of the chloroplasts and valve detail in figures 14–28; and use of the Unsharp Mask filter at 60%, 4.5 pixels radius on final-size images at 300 dpi. The observations were made between Oc- tober 1984 and August 2008, initially by A.J.S. (Figs. 29–43) but after 1986 by D.G.M. (Figs. 1–20, 44–52). Results The interphase plastid structure was determined for selected species of Sellaphora, Fallacia and Rossia within the Sellaphoraceae. In order to check whether the characteris- tics found may occur outside the Sellaphoraceae, we also studied Pinnularia and Diploneis species, to confirm and extend literature records. Identifications follow MANN et al. (2008) for Sellaphora, HUSTEDT (1964) for Fallacia (as Navicula spp.), and HENDEY (1964) for Pinnularia. Sellaphora bacillum (Ehrenb.) D.G. Mann (Figs. 1–13): Two optical sections of S. bacillum cells are provided, in girdle and valve views (Figs. 1–7, 8–13, respectively), to fa- cilitate understanding of chloroplast structure in Sellaphoraceae. Each cell contains a sin- gle saddle-like, H-shaped chloroplast (Figs. 8, 9), which lies with its central isthmus against the epivalve. The single large pyrenoid is tetrahedral (here and in most other larger Sellaphora species, it is usually close to being a regular tetrahedron) and lies eccentrically within the chloroplast with one vertex projecting through the central isthmus into the oppo- site chloroplast lobe. The outer edges of the pyrenoid are entire (Figs. 1, 8), but the edge facing the nucleus is penetrated by a branching system of cavities and tubes (Figs. 2–6, 9–11). The nucleus, which lies as a broad cytoplasmic bridge separating the two apical vac- 240 ACTA BOT. CROAT. 68 (2), 2009 MANN D. G., STICKLE A. J. U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:30 Color profile: Disabled Composite 150 lpi at 45 degrees uoles is displaced towards the corner of the cell opposite the pyrenoid (Figs. 7, 12). Two prominent spherical volutin granules are present, one in each vacuole (Figs. 5, 6, 13), usu- ally associated with one of the raphe slits. ACTA BOT. CROAT. 68 (2), 2009 241 CYTOLOGY OF SELLAPHORACEAE Figs. 1–13. Sellaphora bacillum, living cells (Ashford-in-the-Water, Derbyshire, England). 1–7 – Successive foci of a cell in girdle view (epivalve at the top). The pyrenoid is entire and trian- gular in surface view (1) but penetrated by branching tubular channels (2–6) extending from the nucleus (6, 7); the cell also contains two prominent volutin granules (4–6, arrow). 8–13 – Successive foci of a cell in valve view, from near the epivalve (8) to near the hypovalve (13). Again, the pyrenoid is entire and triangular in surface view (8) and cytoplasmic invagi- nations become visible in deeper foci (10, arrow). The nucleus (with single spherical nucleo- lus, arrow 12) lies on the opposite side of the cell from the pyrenoid. Scale bar = 10 mm. U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:34 Color profile: Disabled Composite 150 lpi at 45 degrees Sellaphora pupula (Kütz.) Mereschk. (Fig. 49): Cell structure in S. pupula and its al- lies (see MANN et al. 2008 for a taxonomic review) is like that of S. bacillum (compare fig- ures 8 and 49). The chloroplast is again saddle-like. The observation of this species led MERESCHKOWSKY (1902) to propose the genus. He unaccountably overlooked the promi- nent, tetrahedral and invaginated pyrenoid. The nucleus is eccentric. Sellaphora pupula is the generitype. Rossia (Figs. 14–17): Rossia valves bear lyre-shaped or linear, lateral areas, in which striae are present but more delicate than elsewhere. The lateral areas also bear small 'pegs', which appear to link to the edge of a very wide conopeum (SIMS and PADDOCK 1979). How- ever, in LM the raphe system does not possess the 'tear-drop' central endings that are char- acteristic of Fallacia (compare figures 14, 21, 25 etc.). Rossia was described by VOIGT (1960) and has rarely been reported. The species illustrated here is very similar to 'Navicula hyalinula' sensu SIMS and PADDOCK (1979) and needs to be transferred to (or described as a new species within) Rossia. There is a single H-shaped chloroplast with an extremely nar- row connection between the two sides (Fig. 15). Each side contains a slightly angular, invaginated pyrenoid (Figs. 15–17). The nucleus is central, lying in a broad cytoplasmic bridge between the two principal vacuoles. Near each pole, there is a ± spherical compart- ment of the vacuole surrounding a single large volutin granule (Fig. 17). Fallacia pygmaea (Kütz.) Stickle et D.G. Mann (Figs. 18–20): There is a single H-shaped chloroplast per cell, whose proximal (Fig. 18) and distal (Fig. 20) margins are plain or slightly undulate. The connection between the two sides of the chloroplast is nar- row, but not as fine as in Rossia. There is a small cushion-like pyrenoid embedded in the centre of each chloroplast lobe, penetrated by a small, little-branched cavity (Fig. 19). The nucleus was slightly eccentric in the specimens seen (Fig. 19). No volutin granules are present. Fallacia pygmaea is the generitype. Fallacia forcipata (Grev.) Stickle et D.G. Mann type I (Figs. 21–28): Fallacia forcipata is a heterogeneous complex and we illustrate four demes here, each with its own chloroplast and frustule morphology (a valve is illustrated for each deme to facilitate future taxonomic revisions). In type 1, the chloroplast has a complex morphology. Essentially, the chloroplast consists of two large, girdle-appressed plates. It is offset with respect to the api- cal plane, with the isthmus displaced to one side (Fig. 24, arrow; contrast Fig. 18) and the distal margins displaced in the opposite direction (Fig. 22, arrow). The chloroplast isthmus bears two longitudinal extensions beneath the raphe system of the epivalve (Figs. 24, 26), and two similar extensions are formed on the opposite side, by elaboration of the chloro- plast margin (Fig. 22). This arrangement persists during size reduction (Figs. 21–24, 25–28). There are two pyrenoids per chloroplast, each flattened, angular and invaginated; they do not extend into the isthmus (Fig. 24). The nucleus is eccentric (Figs. 23, 28). No volutin granules are present. Fallacia forcipata type II (Figs. 29–33): The chloroplast is simpler than in type I and has a simple or slightly undulate distal margin (compare figures 33 and 20); it is symmetri- cally placed with respect to the apical plane. Unlike others studied here, it has a very wide isthmus (Fig. 30), accommodating a transversely elongate invaginated pyrenoid (Fig. 31) that is rectangular in valve view. There are two volutin granules, one in each vacuole. Fallacia forcipata type III (Figs. 34–37): Type III cells (which have strikingly conver- gent striae at the centre: Fig. 34) have a symmetrically placed chloroplast as in type II, but possess two pyrenoids (Fig. 36), as in type I. The pyrenoids are invaginated and have flat 242 ACTA BOT. CROAT. 68 (2), 2009 MANN D. G., STICKLE A. J. U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:34 Color profile: Disabled Composite 150 lpi at 45 degrees tops, rather than the sloping profiles of type I (contrast figures 36 and 23). The chloroplast bears broad lobes alongside the isthmus (Fig. 35) and on the opposite side (Fig. 37); these have no longitudinal extension. The nucleus is eccentric (Fig. 36). ACTA BOT. CROAT. 68 (2), 2009 243 CYTOLOGY OF SELLAPHORACEAE Figs. 14–28. Sellaphorineae, living interphase cells in valve view. 14–17 – Rossia valve and succes- sive foci (Portobello Beach, Edinburgh); note the very narrow connection between the two sides of the chloroplast (15), the two invaginated pyrenoids (e.g. 16, arrow), and the two po- lar volutin granules (e.g. 17, arrow), each in a special vacuolar compartment. 18–20 – Fallacia pygmaea (from a freshwater pond at Ashford-in-the-Water, Derbyshire, England), with a simple H-shaped chloroplast and one small, cushion-like, invaginated pyrenoid (e.g. 19, arrow) on each side. 21–28 – Fallacia cf. forcipata, type I: a large (21–24) and a small (25–28) cell from Portobello Beach, Edinburgh. The longitudinal lobes of the complex chloroplast are connected beneath the epivalve (24, arrow) but not beneath the hypovalve (22, arrow). Both sides of the chloroplast contain a flat, angular pyrenoid (e.g. 27, arrow), but the nucleus is nevertheless eccentric (28, arrow). Scale bars = 10 mm (use the bar in 25 for all except 18–20). U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:36 Color profile: Disabled Composite 150 lpi at 45 degrees 244 ACTA BOT. CROAT. 68 (2), 2009 MANN D. G., STICKLE A. J. Figs. 29–43. Fallacia, living cells in valve view. 29–33 – Fallacia forcipata, type II (Cruden bay, near Aberdeen, Scotland), with a relatively simple chloroplast and ± central isthmus (30), a single central rectangular pyrenoid and prominent volutin granules (31), and eccentric nu- cleus (32). 34–37 – Fallacia forcipata, type III (Cruden bay, near Aberdeen, Scotland), with convergent central striae (34), central plastid isthmus (35), transverse chloroplast lobes (35, 37) and one flat pyrenoid on each side (e.g. 36, arrow). 38–41 – Fallacia forcipata, type IV (Portobello Beach, Edinburgh), with narrow valves (38), longitudinal chloroplast lobes (39, 41), eccentric isthmus (39), and a single high tetrahedral pyrenoid (40, arrow). 42, 43 – Postmitotic Fallacia forcipata cells with valve-appressed chloroplasts (Portobello Beach, Edinburgh). Scale bars = 10 mm (use the bar in 43 for all except 42). U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:39 Color profile: Disabled Composite 150 lpi at 45 degrees Fallacia forcipata type IV (Figs. 38–41): The principal cytological difference between this and type I is that type IV has only one pyrenoid, which is tetrahedral and lies to one side of the cell (Fig. 40) as in Sellaphora. Longitudinal lobes are present beneath the raphe sys- tem (Figs. 39, 41). Fallacia cf. subhamulata (Grun.) D.G. Mann (Figs. 44–48): The single H-shaped chloroplast is similar to that of Sellaphora, except that the pyrenoid is more rounded (Figs. ACTA BOT. CROAT. 68 (2), 2009 245 CYTOLOGY OF SELLAPHORACEAE Figs. 44–48. Fallacia cf. subhamulata, living cell in valve view (Threipmuir Reservoir, near Edin- burgh), showing the simple H-shaped chloroplast (45, 48), single eccentric, invaginated, cushion-like pyrenoid (45–47: arrow in 45), and eccentric nucleus (47, with nucleolus at ar- row). 49 – Sellaphora pupula sensu stricto (Malham Tarn, N England) with prominent tetra- hedral pyrenoid and simple H-shaped chloroplast. 50 – Pinnularia cf. cruciformis in girdle view (Portobello Beach, Edinburgh): detail of pyrenoid, penetrated by numerous tubular channels. 51, 52 – Marine Diploneis species (Portobello Beach, Edinburgh) in which the elongate (51) or cushion-like (52) pyrenoids are apparently invaginated (arrows). Scale bars = 10 mm. U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:40 Color profile: Disabled Composite 150 lpi at 45 degrees 45–47) with simpler invaginations. The chloroplast was asymmetrically placed with re- spect to the apical plane in the two specimens photographed (Fig. 48). Pinnularia cf. cruciformis (Donkin) Cleve (Fig. 50): Cells almost always lay in girdle view and possessed two girdle-appressed chloroplasts, each with a single panduriform pyrenoid at its centre (Fig. 50), penetrated by > 100 simple tubular channels. Diploneis spp. (Figs. 51, 52): Two marine Diploneis species apparently had invagi- nated pyrenoids. The invaginations appeared as simple or branched fine striations. Cell cycle changes: Although the chloroplast morphologies described above were con- stant in tens to hundreds of interphase cells examined for each species, major changes in shape and position occur during the cell-cycle. These have been described in detail for Sellaphora by MANN (summarized 1989) and involve premitotic translational movement onto the girdle and postmitotic rotation during the development of the new chloroplast isth- muses. Similarly, in Fallacia, the chloroplast moves across the cell before cell division to lie beneath the valves with simultaneous simplification of shape (Figs. 42, 43). Discussion Some of the observations reported here were used as the basis for the description of Fallacia by ROUND et al. (1990: 554), as follows: »Plastid basically H-shaped, consisting of 2 girdle-appressed plates connected by a narrow isthmus lying against the epivalve; there may also be narrow lobes parallel to the raphe, extending out from the isthmus, and from one of the lateral plates. One or two invaginated pyrenoids present.« Fallacia chloroplasts have also been described briefly by KUYLENSTIERNA (1989–1990), COX (1996) and SABBE et al. (1999), whose descriptions are consistent with our data, and by KARSTEN (1899: 58, 59), who claimed that F. pygmaea and F. reichardtii (Grun.) Witkowski, Lange-Bertalot & Metzeltin had two chloroplasts. We believe he was mistaken, because we collected several populations of the F. pygmaea species complex from fresh and brackish waters, all of which had a single H-shaped chloroplast, as does the morphologically similar F. hudsonis (COX 1996). Cells kept in rough culture, as Karsten's were, often accumulate large amounts of refractile reserve material in the vacuoles, which can obscure fine detail. Fallacia, Rossia and Sellaphora therefore share the following cytological characteris- tics: (1) one chloroplast per cell, (2) chloroplast basically H-shaped, consisting of two large girdle-appressed plates connected by a central isthmus, and (3) pyrenoids invaginated. All Sellaphora species studied so far have a simple chloroplast containing a single offset pyrenoid, which is clearly tetrahedral in the larger-celled species (> 10 mm long). Fallacia is more variable than Sellaphora in chloroplast shape and position (exactly centred with re- spect to the apical plane or slightly displaced); pyrenoid number (one or two) and shape (regular tetrahedron to flattened rectangular to rounded); and pyrenoid position (usually in the lateral plates but sometimes central). Some Fallacia species resemble Sellaphora in having a simple chloroplast and a single eccentric pyrenoid (compare figures 44–48 with 49), but the agreement is not exact (e.g. F. cf. subhamulata has rounded, not strictly polyhe- dral pyrenoids). The single Rossia species examined is unusual among Sellaphoraceae for its extremely narrow chloroplast isthmus, the exactly central nucleus, and the special com- partment in each vacuole, containing a volutin granule. Whether this arrangement is typical of other Rossia species is unknown (the type of Rossia is R. elliptica M. Voigt, which is ap- parently unreported since its discovery). 246 ACTA BOT. CROAT. 68 (2), 2009 MANN D. G., STICKLE A. J. U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:41 Color profile: Disabled Composite 150 lpi at 45 degrees Fallacia and Lyrella were formerly classified together in Navicula sect. Lyratae be- cause of the presence in both of a lyre-shaped area of plain or only faintly striated silica. The separation of these genera in ROUND et al. (1990), which is supported by rbcL data (JONES et al. 2005, EVANS et al. 2008), was based on the presence or absence of a conopeum, the kind of pore occlusion, nuclear behaviour during successive cell divisions, and a »com- pletely different plastid structure and arrangement«. The single Fallacia chloroplast occu- pies the whole of the girdle (except small areas close to the poles) and, as shown here, con- sists of two large girdle-appressed plates, connected by an isthmus lying against one valve (apparently always the epivalve, as in Sellaphora). In Lyrella, on the other hand, the chloroplast or chloroplasts lie almost wholly against the valves, leaving the girdle free (MANN and STICKLE 1993, 1997), as in the related genus Petroneis (JONES et al. 2005), and the pyrenoids are never invaginated. The Pinnulariaceae, closely related to the Sellaphoraceae according to molecular data (see Introduction), are not as uniform in cell structure. Some species possess two large gir- dle-appressed chloroplasts per cell, including Pinnularia cf. cruciformis (illustrated here) and other marine species (e.g. MERESCHKOWSKY 1901, Pl. 1, Figs. 14, 21, 22; MANN 1996, Fig. 8), and some freshwater species (e.g. HEINZERLING 1908, Pl. 1, Figs. 1, 12; TSCHERMAK- WOESS 1953, Figs. 1, 7; COX 1996, Fig. 22; SCHMID 2003; POULÍ^KOVÁ et al. 2007, POULÍ^KOVÁ and MANN 2008). Other Pinnulariaceae have H-shaped chloroplasts with an isthmus against one valve (e.g. HEINZERLING 1908, Pl. 1, Fig. 16; TSCHERMAK-WOESS 1953, Figs. 2g, 5, 6; COX 1996, Fig. 26), as in Sellaphoraceae, and the diplastidic Caloneis am- phisbaena exhibits a transient phase during the cell cycle in which there is a single valve-appressed, H-shaped chloroplast (THALER 1972, Fig. 7e). Many Pinnulariaceae, both diplastidic and monoplastidic, possess invaginated pyre- noids (TSCHERMAK-WOESS 1953, THALER 1972, MANN 1996, Fig. 8, SCHMID 2001: 10, POULÍ^KOVÁ et al. 2007, POULÍ^KOVÁ and MANN 2008). The invaginations of the C. am- phisbaena pyrenoid were studied in thin sections by WALKER et al. (1979), EDGAR (1980) and SCHMID (2001). All shown by EDGAR (1980) contained extensions of the cytoplasm, but SCHMID (2001: 10) states that some are extensions only of the periplastidial compartment, i.e. they are lined by the two inner plastid membranes but not the two outer 'chloroplast endoplasmic reticulum' membranes (unfortunately, membranes are not visible in SCHMID’s figures 44 and 45). No other invaginated pyrenoids have been examined ultrastructurally. SCHMID (2001) suggested that this type of pyrenoid represents »significant surface enlarge- ment for localized activity in bi-directional transport phenomena between host and chloro- plast (endosymbiont)«, presumably largely in relation to the pyrenoid’s function as a key site for carboxylation (e.g. ROBERTS et al. 2007). Outside the Sellaphorineae, invaginated pyrenoids are very rare. We have not detected them in any other marine or freshwater genera except Diploneis, two species of which we illustrate here. S.J.M. DROOP also recorded invaginated pyrenoids in other Diploneis spe- cies (archived photographs, Royal Botanic Garden Edinburgh) and TSCHERMAK-WOESS (1953) illustrated them in species identified as Diploneis domblittensis var. subconstricta A. Cleve (in which the invaginations were identical to those shown in our figure 51) and D. oculata (Bréb.) Cleve; however, the drawing of the D. oculata valve given by TSCHERMAK- WOESS (1953, Fig. 4d) suggests misidentification of Fallacia pygmaea or F. hudsonis. So far, no molecular data have been published for Diploneis species, many of which are diffi- ACTA BOT. CROAT. 68 (2), 2009 247 CYTOLOGY OF SELLAPHORACEAE U:\ACTA BOTANICA\Acta-Botan 2-09\Mann.vp 5. listopad 2009 15:23:41 Color profile: Disabled Composite 150 lpi at 45 degrees cult to establish in culture (our unpublished observations), and there are few other pointers to a close relationship between the Sellaphorineae and Diploneis, apart from the pyrenoid structure. 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