J. Phycol. 42, 1002–1006 (2006) r 2006 by the Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00260.x

NOTE

AUXOSPORE FORMATION BY THE SILICA-SINKING, OCEANIC FRAGILARIOPSIS KERGUELENSIS (BACILLARIOPHYCEAE)1

Philipp Assmy2, Joachim Henjes, Victor Smetacek Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany and Marina Montresor Stazione Zoologica ‘‘A. Dohrn’’, Villa Comunale, 80121 Napoli, Italy

Size restoration by the auxospore that develops central role played by the sexual phase in the diatom from the is a crucial stage in diatom life life history dictated by its peculiar morphology. cycles. However, information on sexual events in Diatom cells are enclosed in two siliceous thecae and pelagic diatom species is very limited. We report for during mitotic division each daughter cell retains one the first time auxospore formation by the pennate maternal theca and synthesizes a new one internally. It diatom Fragilariopsis kerguelensis (O’Hara) Hustedt follows that the two daughter cells differ slightly in size, during an iron-induced bloom in the Southern which causes a progressive reduction of the average Ocean (EIFEX, European Iron Fertilization EXper- cell size in a growing population (MacDonald 1869, iment). Auxospores of F. kerguelensis resembled Pfitzer 1869). This progressive size reduction can be those described for Pseudo-nitzschia species. The curtailed by the onset of the sexual cycle and the pro- auxospore was characterized by an outer coating, duction of the auxospore. Within the auxospore, which the perizonium; two caps, one at each distal end; is not surrounded by rigid siliceous thecae, a large- and four chloroplasts, one at each end and two in sized initial cell is formed. Thus, in , the sexual the central part. Different stages of auxospore elon- phase combines two crucial events of the species life gation were recorded, with a length of 24–91 lm, but cycle: the occurrence of meiosis, and thus genetic re- only the largest auxospores contained the initial combination, and the restoration of large-sized cohorts cell, whose apical axis ranged between 76 and of cells in the population. 90 lm. Gametangial cell walls were often attached We report for the first time on the finding of sexual to the auxospores and ranged from 10 to 31 lmin stages of the pennate diatom Fragilariopsis kerguelensis length. Auxospore abundances were consistently (O’Meara) Hustedt, which is one of the dominant higher in the fertilized patch, where an increase in diatom species of the ice-free Antarctic Circumpolar the F. kerguelensis population was observed, as com- Current (ACC) (Hart 1934, Smetacek et al. 2004). pared with surrounding waters. It forms curved, ribbon-like chains that can be m Key index words: auxospore; diatoms; Fragilario- over 300 m long and is easily differentiated from con- psis kerguelensis; life history; ; generic species occurring in the Southern Ocean by Southern Ocean virtue of its heavily silicified with a low density of well-marked striae (Hasle 1965, Hasle and Syvertsen 1997). The thick frustules are remarkably strong and have likely evolved as mechanical protection against Diatoms are major contributors to marine primary crustacean . They hence ensure long-term production and play a key role in the ocean carbon and persistence in the surface layer (Hamm et al. 2003). silica cycles (Smetacek 1999). Although the intricate life F. kerguelensis is a major contributor to the diatom ooze histories of several diatoms have been described forming the Antarctic opal belt (Zielinski and Gersonde (Drebes 1977, Edlund and Stoermer 1997, Chepur- 1997), the largest depot of biogenic silica in the world nov et al. 2004), field observations of sexual stages in ocean (Tre´guer et al. 1995). Thus, its ecological prop- marine planktonic species are extremely scant (Mann erties make F. kerguelensis by far the most important 1988, Jewson 1992a, b, Waite and Harrison 1992, diatom species in the global silicon cycle and an indica- Crawford 1995). This is surprising, considering the tor species of a silica-sinking regime in an otherwise iron-limited ecosystem (Smetacek et al. 2004). 1Received 14 December 2005. Accepted 28 June 2006 Seawater samples were collected during the in situ 2Author for correspondence: e-mail [email protected]. iron fertilization experiment European Iron Fertiliza-

1002 AUXOSPORE FORMATION IN FRAGILARIOPSIS KERGUELENSIS 1003 tion EXperiment (EIFEX) conducted in the Atlantic being immature, having been fixed before expansion Sector of the Southern Ocean (021E, 491S) in late aus- was complete. Initial thecae were present only in tral summer to early austral fall (11 February–19 auxospores that had reached 76–90 mm (aver- March 2004) during cruise ANT XXI-3 of R.V. age 5 84 mm; n 5 18) and we consider this as the size Polarstern (Smetacek 2005). The ‘‘in-patch stations’’ range of mature auxospores. The width of the initial were placed at the sites of the highest photosynthetic thecae varied between 7 and 13 mm. The auxospores efficiency (Fv/Fm) and/or the lowest pCO2 values ob- were slightly inflated in their central part (Fig. 1, c served, hence closest to the center of the iron-fertilized and d). This central bulge was not evident in the initial patch. ‘‘Out-patch stations’’ were located in adjacent, thecae (Fig. 1, d–f), indicating that this feature was not iron-limited waters with low Fv/Fm ratios and equilib- inherited during thecae deposition. The auxospores rium pCO2 concentrations. were delimited by a perizonium consisting of trans- Water samples for the estimation of verse siliceous bands (Fig. 1, c and d) and one cap was cell concentration were obtained from Niskin bottles present at each distal end (Fig. 1d). Auxospores in attached to a conductivity temperature depth (CTD) which the thecae of the initial cell were not yet formed rosette from seven depths between 10 and 150 m at contained four chloroplasts: one at each end and two nine ‘‘in-patch’’ and five ‘‘out-patch’’ stations. Water in the central part of the auxospore (Fig. 1c). In stages samples of 200 mL were preserved with hexamine- in which the deposition of the first theca of the buffered formaldehyde solution at a final concentration initial cell became evident, the four chloroplasts were of 2% and stored at 41 C in the dark for subsequent aligned along the wall (Fig. 1d). The initial thecae were counting in the home laboratory. A volume of 50 mL not formed in contact with the inside of the per- was settled in sedimentation chambers (Hydrobios, izonium (Fig. 1, d and e). In most cases, auxospores Kiel, Germany) for 48 h. Cells and sexual stages were were attached to gametangial thecae. It was thus pos- identified and enumerated using inverted light and sible to estimate the size of the gametangia that ranged epifluorescence microscopes (Axiovert 200 and Axi- from 10 to 31 mm in length (n 5 48). We exclusively overt 135, Zeiss, Oberkochen, Germany) according to observed gametangia bearing only one auxospore the method of Utermo¨hl (1958). Auxospores were pho- and, in a few cases, we observed one auxospore con- tographed and measured with a Zeiss AxioCam MRc5 nected to two gametangial thecae that differed in and the Zeiss AxioVision software 4.1. Auxospore abun- length (Fig. 1b). dances (number of auxospores Á m À 2) were obtained Auxospores of F. kerguelensis were recorded both in- from trapezoidal integration of values from the 100 m side and outside the iron-fertilized patch and through- deep surface mixed layer. out the water column, from the surface to 150 m F. kerguelensis was one of the dominant species dur- depth. Interestingly, one auxospore was even found ing the iron-induced bloom and contributed up to 14% at 1000 m depth. Depth-integrated abundances over of the total diatom biomass (P. Assmy, unpublished the upper 100 m of the water column ranged between data). Cells were mainly joined in chains of variable 1.8 and 11.4 Â 106 auxospores Á m À 2 inside the patch length and were 10–90 mm long (Fig. 1a). In the water and 0–3.5 Â 106 auxospores Á m À 2 outside the patch samples collected during the bloom, we repeatedly re- (Fig. 2). Auxospore abundances were consistently high- corded auxospores of a pennate diatom (Fig. 1, b–e). er inside the patch, in accordance with the iron-induced The identification of these sexual stages as F. kergue- increase of the F. kerguelensis population. Auxospores lensis was unequivocal and based on the fact that au- accounted for 0.03%–0.4% of the total population, with xospores were often still attached to the gametangia the highest relative contribution inside the patch. (Fig. 1b) or that the initial cell was visible inside the F. kerguelensis belongs to the family Bacillariaceae auxospore (Fig. 1e) and after release from the auxo- and is genetically closely related to species of the genus spore (Fig. 1f). It has thus been possible to observe the Pseudo-nitzschia (Lundholm et al. 2002). Its auxospore distinctive morphological features of the vegetative, presents morphological similarities to the auxospores gametangial, and initial cells, which were characterized of Pseudo-nitzschia and those of many other pennate by a low density of striae (4–7 in 10 mm) always clearly diatom species (Fryxell et al. 1991, Davidovich and visible both in girdle view and in cells containing Bates 1998, Kaczmarska et al. 2000, Amato et al. chloroplasts (Fig. 1, a, b, e and f; Hasle 1965, Hasle 2005). These similarities include the presence of a and Syvertsen 1997). Except for a single finding of a transversal perizonium with conspicuous bands and much slimmer auxospore, most probably attributable caps at the extremities of the auxospore. In the auxo- to a Pseudo-nitzschia species, the auxospores of spore of F. kerguelensis, we observed four chloroplasts F. kerguelensis were the only sexual stages of a pennate instead of the two characteristic of the vegetative cells, diatom recorded in our samples. a feature also described for P. multiseries and P. del- Auxospores of F. kerguelensis had an elongate shape. icatissima (Davidovich and Bates 1998, Amato et al. Different sizes of auxospores (24–91 mm; n 5 48) were 2005). In F. kerguelensis, we always recorded only one present in the preserved samples and had widths of auxospore attached to the gametangium. Two auxo- 7–16 mm (average 5 11 mm). The shorter auxospores spores have been observed for each gametangial cou- were slightly wider than the longer ones and never ple in Pseudo-nitzschia delicatissima (Amato et al. 2005) contained initial cells. We therefore interpret them as and P. pseudodelicatissima (Davidovich and Bates 1998), 1004 PHILIPP ASSMY ET AL.

FIG.1. Fragilariopsis kerguelensis: light micrographs (phase contrast) of vegetative cells and auxospores. (a) Chain of vegetative cells. (b) Auxospore still connected to the gametangium. (c) Auxospore in which four chloroplasts are visible; the white arrows point to the perizonial bands. (d) Auxospore containing an early stage of the initial cell; the white arrows point to the perizonial bands; the white arrowheads point to the caps at the extremities of the auxospore. (e) Auxospore containing a complete initial cell. (f) An initial cell. Scale bars, 10 mm. whereas formation of only one auxospore was M. Montresor, unpublished data). Moreover, the find- reported for P.multiseries and some strains of P.pseudo- ing of paired gametangial thecae of different size at- delicatissima, due to the weak attachment between the tached to one auxospore suggests that sexual gametangial frustules and the consequent successful reproduction occurs, most probably a ‘‘type II’’ au- pairing of only one couple of gametes (Davidovich and xosporulation (sensu Geitler 1973), in which two game- Bates 1998). The finding of one auxospore attached to tangia with one functional gamete each are produced a single theca in F.kerguelensis might suggest automixis, and gave rise to a single auxospore per gametangial as reported for Nitzschia frustulum var. perpusilla pair. (Rabenh.) Grunow (Geitler 1970). However, we are The length of gametangia recorded in our samples inclined to rule out this hypothesis because we never ranged between 10 and 31 mm. We hence assume that observed the formation of larger cells or auxospores in vegetative cells of F. kerguelensis can become sexually several clonal cultures of F. kerguelensis (P. Assmy and inducible when they reach about 35% of the initial cell AUXOSPORE FORMATION IN FRAGILARIOPSIS KERGUELENSIS 1005

12 In-patch Auxospores were encountered both inside and 2

− Out-patch outside the fertilized patch, suggesting that sex in 10 F. kerguelensis was not the direct result of the iron- 8 induced bloom. Nevertheless, the fact that more auxospores were found inside the patch indicates 6 that higher cell numbers resulted in more sexual 4 stages. If the life cycle of F. kerguelensis proves to be auxospores . m

6 2 heterothallic, as that of the closely related Pseudo-

10 nitzschia species, gametangia of the opposite mating 0 type will have to come in close contact to allow gamete 0 4 8 1216202428323640 Days since first Fe-release conjugation. This apparently poses a major obstacle for the occurrence and frequency of sex in the pelagic 6 À 2 FIG. 2. Auxospore concentration (auxospores Á 10 Á m ;inte- realm, particularly in the case of pennate diatoms that grated over the upper 100 m) at different in- (full symbols) and lack the flagellated sperms typical of centric diatoms. out-stations (open symbols) during the development of the iron- The formation of aggregates (Buck and Chavez 1994) induced bloom. or the accumulation of cells in thin layers in corre- spondence to physical discontinuities (Rines et al. (76–90 mm). Gametogenesis is generally reported to 2002) have been suggested as possible mechanisms to occur when cells reach a smaller size window, within bring cells into close proximity, thus facilitating the 30%–40% of the initial cell size (Drebes 1977) and encounter rates among gametangia. The possible pro- our in situ data recorded for F. kerguelensis agree with duction of pheromone-like compounds has also been this paradigm. However, in Pseudo-nitzschia, the size hypothesized for planktonic diatoms but clear-cut range for gametangial induction can vary over a wider evidence is still lacking (Davidovich and Bates 2002). size window (Amato et al. 2005). On the other hand, the apparent rarity of sexual stages The link between sex and size restoration in of F. kerguelensis in the field might be of advantage diatoms leaves us with an apparent paradox: why is in reducing mortality. During auxospore formation, evidence for sex so rare in the natural environment? F. kerguelensis is most vulnerable to predation pressure In situ evidence for sexual reproduction comes either because it is deprived of its strong silicified amour. from direct observation of sexual stages (auxospores, Sexual stages are a critical stage in the diatom life cycle gametangia) or from estimates of cell size spectra, and their low density might be a strategy for reducing where the detection of larger size classes—initial encounter rates with potential predators, parasites, cells—is indirect but strong evidence for a recent and pathogens. sexual event (Mizuno and Okuda 1985, Mann 1988, This is, to our knowledge, the first report of sexual Jewson 1992b). Massive synchronous sexual events reproduction in the silica-sinking diatom F. kerguelensis. have been reported for epiphytic and epipelic diatoms Our study indicates that sex in this species is a rare and (see Edlund and Stoermer 1997), but, to our knowl- density dependent event and measurements of the sex- edge, Corethron criophilum Castracane (now C. pennatum ual stages encountered provide an estimate of the two (Grunow) Ostenfeld) is the only marine planktonic di- cardinal points in the life cycle of F.kerguelensis. Further atom for which a mass sexual phase has been reported research on the frequency of size reduction in natural during a bloom in the Southern Ocean (Crawford populations of F.kerguelensis, and therefore the need for 1995). In this species, male gametangia comprised as sex, is required for a better understanding of the life much as 4% of the total cells counted, not accounting history patterns involved. Up to now, the only estimates for the oogonia that were not distinguishable from for the timing of sexual reproduction were derived normal vegetative cells and gametangia that from the examination of cell size spectra and the fre- already discharged their contents (Crawford 1995). quency of sexual phase seems to vary from 2 up to Conversely, other species such as Aulacoseira subarctica possibly 40 years (Edlund and Stoermer 1997). Thus, (O. Mu¨ller) Haworth (Jewson 1992b) and Nitzschia one plausible explanation for the scant reports of sexual sigmoidea (Nitzsch) W. Smith (Mann 1988) show asyn- stages in planktonic diatoms is the infrequency of sex chronous sexuality, occurring at very low frequencies and the small chances of coming across it. The time and over more prolonged time intervals. The percent- span of a cohort of cells to move between the two car- age of auxospores in A. subarctica accounted, on aver- dinal points will obviously depend on growth and size age, for less than 0.1% of the whole population and reduction rates. However, selective grazing on e.g. small these values are in the range of those recorded for cells might influence the frequencies of particular size F. kerguelensis in the present study. The extremely low classes within the population and changes in nutrient concentration of sexual stages in F. kerguelensis lowers availability might speed up or slow down growth rates the probability of recording them in the limited vol- and thus size diminution, thus complicating the ume of water sample used for routine phytoplankton predictability of the life cycle span. Furthermore, size counting. 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(Bacillar- making the experiment possible. We would also like to thank iophyta). J. Phycol. 28:856–66. the two anonymous referees for their helpful and constructive Jewson, D. H. 1992b. Size reduction, reproductive strategy and the comments on the manuscript. The project has been carried life strategy of a centric diatom. Philos. Trans. R. Soc. London B. out in the frame of the MarBEF Network of Excellence ‘‘Ma- 336:191–213. rine Biodiversity and Ecosystem Functioning’’ (contract no. Kaczmarska, I., Bates, S. S., Ehrman, J. M. & Le´ger, C. 2000. Fine GOCE-CT-2003-505446) and the Eur-Oceans ‘‘European structure of the gamete, auxospore and initial cell in the penn- ate diatom Pseudo-nitzschia multiseries (Bacillariophyta). Nova Network of Excellence for Ecosystem Analysis’’ (contract no. Hedwigia 71:337–57. 511106), which are funded in the Community’s Sixth Frame- Lundholm, N., Daugbjerg, N. & Moestrup, Ø. 2002. 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