Planktonic diatoms vary in diameter by over three orders of magnitude, from Ethmodiscus cells that can be over 2 mm in diameter (Round et al. 1990) down to species with cells that have diameters of 2 μm (e.g. Vaulot et al. 2008). Size is important in planktonic environments for a variety of reasons, including trade-offs in terms of nutrient uptake, light interception, growth rate, grazing, and suspension (e.g. Geider et al. 1986, Vaulot et al. 2008, Winder et al. 2009, Litchman et al. 2009, Barton et al. 2013, Reavie and Barbiero 2013, Clark et al. 2013, Narkov et al. 2014, Jewson et al. 2015). However, for most diatoms, an additional factor is that cell diameters in a population usually decrease with time. This was first observed by Macdonald (1869) and Pfitzer (1869) and was thought to be an inevitable consequence of their having a rigid silica cell wall structure. However, we now know that this decline can be avoided (see Chepurnov et al. 2004), but that it is retained in most species as an elegant solution to timing the length of their life cycles (Lewis 1983, 1984, Jewson 1992a, b, D’Alelio et al. 2010, Mann 2011, Jewson and Granin 2015). What we do not know is whether the cycle of size decline and subsequent size restoration still operates in a similar way in species at the lower size limit, when cell volume approaches the theoretical minimum for eukaryote cells of just under one micron in diameter (e.g. Raven 1998).

Minidiscus comicus Takano, order Thalassiosirales, is one of the smallest marine diatom species. It is widespread along the Pacific and Atlantic coasts, as well as in the Adriatic and Mediterranean Seas (Takano 1981, Lange 1985, Hasle and Syvertsen 1997, Tomas 1997, Bérard-Therriault et al. 1999, Shevchenko and Orlova 2002, Kaczmarska et al. 2009, Percopo et al. 2011, Lee et al. 2012). Miniduscus comicus belongs to a small marine genus of seven species (Aké-Castillo et al. 2001, Kang et al. 2003, Quiroga and Chrétiennot-Dinet 2004, Kaczmarska et al. 2009). The first report of M. comicus was in large aggregated flocks in Tokyo Harbour (Takano 1981), but since then it has usually been found as solitary cells or in short chains of 2 to 3 cells (Kaczmarska et al. 2009). Kaczmarska et al. (2009) reported cells of M. comicus with diameters of 2.1 to 3.2 μm and Percopo et al. (2011) found cells of 2.7 to 4.3 μm, but Quiroga and Chrétiennot-Dinet (2004) found broader diameter ranges of 1.9 to 7.0 μm and Takano (1981) 2.0 to 7.0 μm. In this study, we used samples of M. comicus collected from the western Mediterranean (see Estrada et al. 2014), to investigate how a change from discoid to spherical cell shape in small cells can help offset loss of volume when diameter decreases as part of the life cycle.

Detailed site and sampling information are reported in Estrada et al. (2014), so only a brief summary is given here. The samples were collected during three cruises of the FAMOSO project carried out in mid-March, late April-May and September 2009, on board the R.V. Sarmiento de Gamboa in the central zone of the NW Mediterranean. The region was delimited approximately by coordinates 41°30’ to 42°N and 4° to 5°E. Water samples were obtained with 12-L Niskin bottles mounted on a rosette. The samples in this study were collected from 20 m depth on 21 March 2009, from 25 m on 22 March 2009, from 80 m on 11 May 2009, and from 80 m on 19 September 2009. For each sample, either 150 or 250 mL of water was filtered onto 0.8- or 2-μm polycarbonate Nucleopore filters. These were stored dry in Petri dishes, which were kept in the dark and under partial vacuum. The filters were attached to stubs using a colloidal silver gel and then sputter coated (Polaron SC-500) with a gold-palladium layer less than 10 nm thick. The scanning electron microscope (SEM) was a Hitachi S-3500N operated at 5 Kv. Cells were orientated and photographed in both girdle and mantle views to enable measurements of cell shape. All estimates of cell volume were based on non-dividing cells. In the largest cells, with diameters above 5 μm, their shape approximated to a cylinder, with the girdle band region having a height of 0.8 μm. In cells with smaller diameters (below 4 μm), the valves of non-dividing cells abutted (see below), so no allowance was needed for the girdle region. Most small cells below 3 μm approximated to the shape of a sphere and their cell volume was then estimated using the standard formula. However, for cells with valves that were domed but not completely hemispherical, their volume was calculated using the formula for a ‘spherical cap’ of a sphere, V=0.33h²(3r-h), where h is the height of the domed valve and r is the radius of the sphere that would be formed if the curvature of the valve was extended to form a full sphere. This could be estimated from the SEM photographs, because the curvature of the doming was consistent in all but a few cells, enabling simple geometry to reconstruct the appropriate circle size for each individual valve in a cell.

Size is a key factor in planktonic life and many studies have looked at its functional implications (e.g., Geider et al. 1986, Vaulot et al. 2008, Winder et al. 2009, Litchman et al. 2009, Barton et al. 2013, Reavie and Barbiero 2013, Clark et al. 2013, Jewson et al. 2015). Among diatoms there is the added complication of having to cope with the cycle of size decline and size restoration used to time the length of their life cycle over periods longer than one year (Lewis 1983, 1984, Jewson 1992a, b, Jewson and Granin 2015). The interest in M. comicus was to see whether this cyclic clocking mechanism could still operate in very small cells, where the rigid cell wall structure might be expected to constrain the volume necessary for cell organelles, such as nuclei, mitochondria and chloroplasts, and for gamete production. The solution used by M. comicus, which avoids interfering with diameter decline, is to become spherical or even oblong to maintain cell volume as diameter declines. Essentially, it is a change in cell length, which is an adaptation used by some other centric diatoms, such as Aulacoseira baicalensis, to make room for storage products during dormancy (e.g. Jewson et al. 2010, Jewson and Granin 2015).

A longer sequence of sampling would be needed to accurately determine the length of the life cycle of M. comicus, but from the rate of size decline observed during 2009, it is probable that some cells could reach the 3.1 μm threshold after size restoration in two years. For example, in September 2009 there were predominately two age classes (Fig. 5C). The size-restored cohort in September had a modal diameter of around 5 μm, while the second peak at 3 μm was probably mainly made up of cells that had regenerated their size in the previous year (2008). The diameter of these cells would then have declined further to be below the threshold for inducing size restoration by March 2010, and thus be ready to begin completing the cycle of size restoration by May, with cell diameter distributions similar to those found, respectively, for March 2009 (Fig. 5A) and May 2009 (Fig. 5B). This is just the ‘average’ situation, because the population would contain cells from other age classes, as the rate of size decline depends very much on conditions for growth and division experienced by individual cells, with some declining at slower rates and some at higher rates, which ensures an overlapping of age classes (see Jewson 1992a). The likely length of the life cycle of M. comicus is therefore probably two or three years. The timing of size restoration in M. comicus is also interesting because it occurred in summer when larger diatoms were scarce and it was likely to have been a nutrient-poor period, especially for silica, which suggests that M. comicus can survive and reproduce at times when other, larger diatoms have ceased growth. Although it seems likely, we do not know yet whether size restoration in M. comicus is linked to sexual reproduction.

In addition to the large changes in cell shape, another striking feature of the M. comicus cell wall was the relatively large size of its exterior processes (Figs 1-4). There were two types. One was the exterior fluted tube-structures of the fultoportulae. Kaczmarska et al. 2009 showed that these structures produce chitin filaments. Similar chitin filaments are found in other small Thalassiosira (Martin-Cereceda and Cox 2011) and Cyclotella (Jewson et al. 2015), and they have been linked to reducing sinking rate (e.g. Walsby and Xypolyta 1977, Walsby and Reynolds 1980). However, in M. comicus it is unlikely that this is their sole purpose, because in such small cells, sinking is minimal. An alternative possibility is that the chitin filaments could interfere with grazing by flagellates and ciliates, because M. comicus lives in a nanno-habitat along with other small species, such as Parmales. Other possible functions might come from an observation by Kaczmarska et al. (2009) that cells of a close relative, Minidiscus chilensis, which were collected from surface sediments, had projecting chitin filaments. It is possible that the filaments, which can be over 30 μm long, could help in the resuspension of cells back into the water column by bridging across the boundary layer at the sediment surface. One property of the external tubes is that they restrict and support the angle at which the chitin filaments are deployed. However, the reason for this and their function is yet to be determined.

One consequence of the doming and production of the large external fultoportulae tubes during division is that they create a problem for keeping cells together. They require a relatively large distance of up to 5 μm, which is the same as or bigger than the cell diameter. In this study, the cingula protecting the lengthening cell during division offered little rigid support. Therefore, holding the two daughter valves together is probably the role of the other prominent structure, the central rimoportula. One reason for this adaptation may be that M. comicus grows after the main spring diatom bloom, when there is a lack of sufficient silica to build rigid cingula, so an alternative solution is required to keep daughter cells together during division. However, in nutrient sufficient conditions, such as in culture, M. comicus does appear to form cingula with a rigid structure (Percopo et al. 2011).

In conclusion, the main result from this study is that M. comicus demonstrates the versatility of diatoms and shows how their apparently rigid cell wall is not restrictive, with changes in shape possible over their life cycle. In the case of M. comicus, this is done by changing from discoid to spherical shape to maintain cell volume during diameter decline, thereby making it possible to avoid interference with the life cycle clocking mechanism in a very small species.