Gametogenesis and Auxospore Development in Actinocyclus (Bacillariophyta)

Gametogenesis and Auxospore Development in Actinocyclus (Bacillariophyta) Masahiko Idei1*, Keigo Osada2, Shinya Sato3, Kensuke Toyoda4, Tamotsu Nagumo5, David G. Mann3 1 Department of Biology, Bunkyo University, Saitama, Japan, 2 Department of Biology, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan, 3 Royal Botanic Garden Edinburgh, Edinburgh, United Kingdom, 4 Department of Biology, Keio University, Kanagawa, Japan, 5 Department of Biology, The Nippon Dental University, Tokyo, Japan Abstract cGametogenesis and auxospore development have been studied in detail in surprisingly few centric diatoms. We studied the development of sperm, eggs and auxospores in Actinocyclus sp., a radially symmetrical freshwater diatom collected from Japan, using LM and electron microscopy of living cultures and thin sections. Actinocyclus represents a deep branch of the ‘radial centric’ diatoms and should therefore contribute useful insights into the evolution of sexual reproduction in diatoms. Spermatogenesis was examined by LM and SEM and involved the formation of two spermatogonia (sperm mother-cells) in each spermatogonangium through an equal mitotic division. The spermatogonia produced a reduced ‘lid’ valve, resembling a large flat scale with irregular radial thickenings. Sperm formation was merogenous, producing four sperm per spermatogonium, which were released by dehiscence of the ‘lid’ valve. The sperm were spindle-shaped with numerous surface globules and, as usual for diatoms, the single anterior flagellum bore mastigonemes. One egg cell was produced per oogonium. Immature eggs produced a thin layer of circular silica scales before fertilization, while the eggs were still contained within the oogonium. Sperm were attracted in large numbers to each egg and were apparently able to contact the egg surface via a gap formed between the long hypotheca and shorter epitheca of the oogonium and a small underlying hole in the scale-case. Auxospores expanded isodiametrically and many new scales were added to its envelope during expansion. Finally, new slightly-domed initial valves were produced at right angles to the oogonium axis, after a strong contraction of the cell away from the auxospore wall. At different stages, Golgi bodies were associated with chloroplasts or mitochondria, contrasting with the constancy of Golgi–ER–mitochondrion (G-ER-M) units in some other centric diatoms, which has been suggested to have phylogenetic significance. Electron-dense bodies in the vacuole of Actinocyclus are probably acidocalcisomes containing polyphosphate. Citation: Idei M, Osada K, Sato S, Toyoda K, Nagumo T, et al. (2012) Gametogenesis and Auxospore Development in Actinocyclus (Bacillariophyta). PLoS ONE 7(8): e41890. doi:10.1371/journal.pone.0041890 Editor: Ross Frederick Waller, University of Melbourne, Australia Received March 14, 2012; Accepted June 29, 2012; Published August 1, 2012 Copyright: ß 2012 Idei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: reports are the series of five ‘Entwicklungsgeschichtliche Untersuchungen’ by von Stosch and colleagues on species of Melosira, Odontella, Stephanopyxis and Chaetoceros ([10], [11], [12], [13], [14]; see also [15]) and there is also a detailed early account of Cyclotella reproduction [16]. More recent papers deal with Bacteriastrum [17], Attheya [18], Melosira [19], [20], Skeletonema [21], Coscinodiscus [22], and Thalassiosira [23]. The centric diatoms that have been studied in detail represent several of the major lineages detected by molecular systematic studies (e.g. [24]). However, there are also lineages – such as Hydrosera and Terpsinoë, the Toxarium clade (Toxarium, Climacosphenia and Ardissonea), Lampriscus, Leptocylindrus, and the subject of the present paper, Actinocyclus – for which little or nothing is known of the sexual phase. It is ironic that Hasle et al. [25] excluded the Cymatosiraceae from the pennate diatoms largely because of their oogamy (which was clearly well-known to von Stosch, judging by his comments in [25] and [26]) and yet the details of this process and the resulting auxospores have never been published. Thus, much basic observation and description remains to be done before it will be possible to determine the evolution of the life cycle and sexuality in centric diatoms. Introduction For many years the nature of sexual reproduction in centric diatoms was not understood. It was known that cell size was restored via auxospores, as in pennate diatoms, but it was thought that the auxospores were produced asexually from small vegetative cells. It was also known that the cells of some centric diatom species sometimes divided up to produce small naked cells, which were referred to as ‘microspores’ (e.g. [1], [2]), but these were not considered to be involved in auxosporulation. Following the discovery of meiosis during the formation of microspores in Coscinodiscus by Hofker [3] and observations of apparent attraction between small released flagellate cells and undivided cells of Chaetoceros by Went [4], Geitler [5] suggested that centric diatoms might be oogamous. However, it was not until 1950 that von Stosch [6] finally demonstrated oogamy, in Melosira. Since 1950, there have been many reports of spermatogenesis or auxospores in centric diatoms, leaving little doubt that oogamy is the only method of allogamous sexual reproduction in the group. However, most of these reports are of individual stages (e.g. in [7] or [8], [9]) and there are remarkably few centric diatoms in which the whole of the sexual process has been documented. The most complete PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e41890 Gametogenesis and Auxosporulation in Actinocyclus using Leica Reichert Ultracut S, and examined using a JEOL JEM-1200EX. For voucher rbcL sequences, DNA was extracted from two Actinocyclus sp. clones (A5 and D3) from Lake Akan and one (A6) from Lake Ogawara, using the method described in [34]. The rbcL sequences obtained were c. 1,480 bp long, representing most of the gene, and have been deposited in GenBank as accessions AB723750 (A-5), AB725385 (D-3) and AB725386 (A-6). Apart from one possible nucleotide difference between one of the Ogawara sequences and the other two, the sequences were identical. One of us (M.I.) discovered an undescribed freshwater Actinocyclus associated with sandy sediments in Lake Ogawara, Japan. This was isolated into clonal culture and its sexual reproduction studied by light microscopy (LM) of living and DAPI-stained cells, SEM observations of critical point dried material, and TEM of thin sec (...truncated)