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ABSTRACT: There is an extensive literature on chrysophyte stomatocysts from diverse habitats and their use as
paleolimnetic indicators of past environmental conditions in lakes. The majority of these cysts are unidentified, and more
laboratory studies are needed to link stomatocyst morphotypes to vegetative stages. A laboratory procedure that resulted
in stomatocyst production in the chrysophyte alga Ochromonas pinguis (Chrysophyceae) was tested to determine if the
protocol would also stimulate the production of stomatocysts in six other chrysophytes. Chrysolepidomonas
dendrolepidota, Chrysosaccus cf. sphaericus, Dermatochrysis sp., Synura cf. americana, Synura cf. macropora and a
second species of Ochromonas all produced stomatocysts within two to three weeks. Morphology of the stomatocyst was
described using scanning electron microscopy (SEM). This was the first report of stomatocysts from Chrysosaccus and the
first SEM description of O. pinguis cysts. These autecological studies provide a greater linkage between sediment
stomatocysts and their biological origin, strengthening the interpretive value of these microfossils and broadening our
understanding of chrysophyte biogeography.
KEY WORDS: Chrysolepidomonas, Chrysophytes, Chrysosaccus, Cyst, Dermatochrysis, Ochromonas, Statospore,
Stomatocysts, Synura
INTRODUCTION 1981; Smol 1988; Kamenik & Schmidt 2005; Pla & Anderson
2005). Nevertheless, only about 5% of chrysophyte stoma-
As part of their life history, many algae produce resting cysts tocysts have been linked to their vegetative counterparts;
during unfavorable environmental conditions. Although cyst that is, they are unidentified. These unidentified stomatocyst
development is also involved in the sexual cycle of some morphotypes are grouped together and categorized simply as
algae (Sandgren & Flanagin1986), the production of resting chrysophyte stomatocysts and assigned numbers as de-
cysts as a result of environmental stress, including predation scribed by the guidelines of the International Statospore
(Rengefors et al. 1998), increases the likelihood of vegetative Working Group (Cronberg & Sandgren 1986). Although
cell survival and, in addition, provides a seed bank for calibration sets (Charles & Smol 1994) have been developed
population recruitment when conditions improve. Within the for use in models correlating stomatocyst morphotypes to
Chrysophyceae and Synuophyceae, two chrysophyte classes specific physical and chemical lake conditions (Zeeb & Small
separated based on biochemical and ultrastructural differ- 1995; Duff et al. 1997; Wilkinson et al. 1999; Pla et al. 2003),
ences (Andersen 1987), this resting stage appears particularly a clearer picture of chrysophyte distribution patterns would
important, as most if not all genera produce silica cysts. The emerge if vegetative stages and cysts were linked.
term stomatocyst is a more specific term for chrysophyte In 2008, Ochromonas pinguis Conrad was isolated from an
cysts, but the terms cyst and statospore are also used oligotrophic lake in northeastern Pennsylvania and was
interchangeably. maintained under constant illumination in a temperature-
Stomatocysts are typically spherical to ovoid shape, their controlled incubator. When this alga was switched to a 16:8
outer surface may be smooth or ornamented, and there is a day/night light cycle as part of a feeding study, stomatocyst
single pore that is sometimes surrounded by a collar. Viable formation was observed. Because there is no prescribed
stomatocysts have an organic plug that fills the pore. The method for inducing stomatocysts, the intent of this study
significance of silica cysts is exemplified by the high was to evaluate the day/night laboratory procedure for
concentrations of cysts observed in whole lake water producing stomatocysts in other species. Chrysolepidomonas,
(Firsova et al. 2008) and in sediments (Rybak 1986; Chrysosaccus, Dermatochrysis, two Synura taxa and a second
Kamenik et al. 2001; Betts-Piper et al. 2004). Because the species of Ochromonas were subjected to the same laboratory
biogeography of many chrysophyte species is influenced by regiment as O. pinguis. Stomatocysts were produced in all
limnological variables (Sandgren 1988; Siver 1995; Škaloud cultures. This article describes the specific laboratory
et al. 2013), cyst deposition and accumulation in lake technique and scanning electron microscopy (SEM) of the
sediments leave a historical record of chrysophyte assem- stomatocysts.
blages that can be used as paleolimnological indicators of
past chemical and physical conditions (Adam & Mahood
MATERIAL AND METHODS
* Corresponding author (dah13@psu.edu).
DOI: 10.2216/14-001.1 Ochromonas pinguis strain Giles 1 was isolated from Lake
Ó 2014 International Phycological Society Giles, an oligotrophic lake located in northeastern Pennsyl-
426
Holen: Inducing chrysophyte stomatocyst formation 427
vania, and Ochromonas sp. strain Sem 1 was isolated from stomatocysts. The stomatocyst’s surface was heavily orna-
Sweeny Pond, a small brown-water reservoir (0.052 km2) mented with thick lunate ridges of varying lengths and heights.
also located in northeastern Pennsylvania (Williamson et al. These projections ranged from short verrucae to long, curvy
1997; Holen 2010). Chrysolepidomonas dendrolepidota Peters ridges. Some of the ridges were bifurcated and others so
et Andersen strain CCMP 293, Dermatochrysis sp. strain prominently elevated as to appear more plate-like in appear-
A12,609, Chrysosaccus cf. sphaericus Bourrelly strain CCMP ance. The ridges were uniformly distributed over the surface of
1162 Synura cf. americana Kynčlová & Šlakoud strain the stomatocyst and are slightly thicker at their base. Assuming
A12,391 and Synura cf. macrospora Šlakoud & Kynčlová a random distribution over its surface, the mean number of
strain A12,220 were kindly provided by Dr. Robert these projections on the stomatocyst was estimated to be 57 6
Andersen. All cultures except Synura cf. americana, which 6.1 (n ¼ 12).
died, are available from the author upon request. Ochromonas sp. was an undescribed species. The vegeta-
Prior to the experiment, nonaxenic cultures were main- tive cell was small (4–6 lm diameter) and pleomorphic; the
tained on DY-V medium (Andersen et al. 2005) at 258C on a typical cell shape was spherical to oval with a wide anterior
16:8 day/night light cycle at 100 lmol m2 s1. To induce flagellar depression that gave it a heart-shaped appearance.
stomatocyst formation, a 1-ml sample in stationary phase of The anterior side, over which the longer flagellum arcs, was
growth was added to 50 ml of sterile-filtered DYV medium more rounded and slightly elevated. The long and short
in 125-ml glass, sterile Erlenmeyer flasks. The foil-topped flagella were approximately 8 lm and 2 lm in length,
flasks were placed in a temperature-controlled incubator set respectively. The chloroplast was ribbon-like and positioned
at 218C on a 16:8 day/night cycle and illuminated from one off center from the more rounded anterior side of the cell to
side by a bank of cool-white fluorescent bulbs at 100 lmol the posterior end of the cell; the plastid was sometimes
m2 s1. The cultures were manually shaken daily. Cyst twisted. There was a noticeable stigma. The shape of the
production was monitored using visual observation every stomatocyst was spherical to oval (5.6–9.6 lm long, 3.8–8.3
two to three days by scanning a wet mount at 3600 and lm wide) with a distinct collar (n ¼ 10; Fig. 3). The mature
31000 using an Olympus BX51 microscope fitted with 360 cyst was smooth (devoid of ornamentation) and had a simple
UPlanFLW and 3100 UPlanApo objective lenses (Olympus conical collar that arose abruptly from the cyst body. The
Corp., Tokyo, Japan). If no stomatocysts were observed in conical collar had a basal diameter of 2.3–2.9 lm with an
wet mount preparations, then a 5-ml subsample was pelleted apex diameter of 1.3–1.5 lm and a collar height of 0.8–1.2
by centrifugation at 1800 3 g. The supernatant was discarded lm (n ¼ 10). A plug was never observed. The collar had a
and the pellet vortexed in 30% H2O2 and immersed in a 508C rounded apex and a regular pore with a pore diameter of
water bath for 24 hours following the procedure of Battarbee 0.2–0.4 lm (n ¼ 10). Collar morphology was variable; some
cysts exhibited a true collar that entirely encircled the pore,
(1986). Samples were sedimented by centrifugation, the pellet
but other cysts possessed a collar that was interrupted and
was resuspended in distilled water, and a small aliquot was
left a small break or groove that was continuous from its
removed to scan microscopically for stomatocysts at 3600 to
apex to the base (Fig. 4).
31000. Once stomatocysts were observed in a culture, a 10-
Dermatochrysis sp. had a spherical stomatocyst with a
ml subsample was removed and treated with H2O2 as
smooth surface during its early stage of development, but the
described above for SEM preparation.
cyst surface was uniformly scabrous (approximately 0.1 lm
For SEM, the samples were collected on a 0.22-lm-pore-
in height) on mature cysts (Figs 5, 6). The average cyst
size 13-mm-diameter Millipore polycarbonate filter (Merck-
diameter was 6.6 lm and ranged from 5.9 lm to 7.2 lm (n ¼
Millipore Corp., Billerica, Massachusetts USA). The filter
5). There was no collar development. The cyst contained a
was transferred to 2.5% glutaraldehyde in 0.1 M sodium
regular conical pore with an inner and outer diameter of 0.4–
cacodylate buffer for 1 h at room temperature. The filter was 0.5 lm and 0.8–1.0 lm, respectively (n ¼ 5).
washed three times for 5 min each using 0.1 M sodium The cyst of C. dendrolepidota was spherical to slightly
cacodylate buffer; the filter was then dehydrated in a graded oblate and had a smooth surface. The cyst diameter was 6.1–
ethanol series (50%, 70%, 85%, 90%, 95%, 33 100%) for 5 6.8 lm. There was a regular pore that appeared flush with
minutes each. Samples were critical point dried, mounted on the cyst body (n ¼ 3; Fig. 7). The pore appeared unusually
studs and sputter coated with 10 nm Au/PD. Samples were small in comparison to the cyst body and ranged from 0.4
examined using a JEOL JSM 5400 scanning electron lm to 0.5 lm in diameter (n ¼ 3). The slight anterior
microscope (JEOL Ltd, Tokyo, Japan) operated at 20 kV. depression in the oblate-shaped cysts gave the impression of
a narrow lip surrounding the pore opening (Fig. 8).
Synura cf. americana produced a stomatocyst that was
RESULTS usually spherical with a diameter of 8.4–8.9 lm (n ¼ 3), but
oval cysts have been observed (Fig. 9). The cyst surface was
The mature stomatocyst of O. pinguis was spherical to slightly unornamented and had a shallow concave pore with an inner
oval with a length and width of 17.4–19.2 lm and 15.7–16.9 and outer diameter 0.5 lm and 1.5–2.0 lm, respectively. The
lm, respectively (n ¼ 21; Figs 1, 2). The wide secondary collar identity of the strain was determined using SEM observa-
was cylindrical and complex with a mean height of 2.48 6 0.28 tions of silica scales (Figs S1, S2; Table S1). Unfortunately,
lm (n¼10), and a thick, concave annulus separates the primary the culture died, and further study was not possible.
and secondary collars. The mean collar width is 8.5 6 0.36 lm Synura cf. macropora had spherical stomatocysts that were
(n ¼ 15) and is reflexed at its base. The 1-lm pore appeared to 8.8–9.8 lm diameter (n ¼ 9). Cysts had a smooth surface and
be regular and was often observed with a plug in mature a shallow concave pore (Fig. 10). The depressed pore had an
428 Phycologia, Vol. 53 (5)
Fig. 1. Stomatocyst of Ochromonas pinguis strain Giles 1. Note regular pore (arrow). Scale bar ¼ 10 lm.
Fig. 2. Stomatocyst of Ochromonas pinguis. Note reflex at the base of the secondary collar. Scale bar ¼ 10 lm.
Fig. 3. Stomatocyst of Ochromonas sp. strain Sem 1 with complete collar. Scale bar ¼ 2 lm.
Fig. 4. Stomatocyst of Ochromonas sp. with incomplete collar. Scale bar ¼ 2 lm.
Holen: Inducing chrysophyte stomatocyst formation 429
inner diameter of 0.6–0.8 lm and an outer diameter of 1.8– grow in the absence of light; whereas, this flagellate exhibits
2.0 lm (n ¼ 7). Identity of the strain was based on SEM a higher growth rate in the dark than the light when fed
observations of the silica scales (Figs S3, S4; Table S1); bacteria (Holen 2010). It also resembles O. minuta (I.F.
however, the scales had significantly smaller keel pores. Lewis) Bourrelly except for the length of the small flagellum,
Chrysosaccus cf. sphaericus produced a smooth stomato- which in O. minuta is exceptionally short in comparison to
cyst that was generally spherical with a diameter of 4.5–6.7 the longer flagellum (Pringsheim & Pringsheim 1959). The
lm (n ¼ 8); however, some cysts appeared slightly ovoid in shape and location of the chloroplast of Ochromonas sp. is
shape (Fig. 11). The cyst had a pronounced conical collar, different from that for Ochromonas minuscula, which has a
and, like Ochromonas sp., some cysts were observed with more cup-shaped plastid that is located in the cell anterior
incomplete collars (Fig. 12). There was an abrupt delineation (Starmach 1985; Curtin 1987). Moreover, the stomatocyst of
between the cyst body and the collar. Complete conical O. minuscula has been previously described, and, although
collars had an acute apex with a sloping inner margin that similar in cyst-body morphology, the collar shape, height
surrounded the regular pore. Incomplete collars exhibited a and width are significantly different (Curtin 1987).
more rounded apex that encircled approximately three- Stomatocyst 67 (Duff et al. 1995) is most certainly the
fourths of the pore with each collar margin sloping same as described here for Dermatochrysis. Both stomato-
asymmetrically toward the cyst body. Collar height ranged cysts have scabrae uniformly distributed over their surface
from 0.5 lm to 0.7 lm (n ¼ 4). The collar base and apex and lack a collar. The diameter of Dermatochrysis cysts (5.9–
diameters ranged from 2.0 lm to 2.4 lm and 1.1 lm to 1.4 7.2 lm) is equivalent to stomatocyst 67 with a diameter of
lm, respectively (n ¼ 4), and the pore diameter ranged from 7.9 lm, and pore diameters are similar. Phaeosphaera
0.3 lm to 0.8 lm (n ¼ 3). perforata Whitford (Whitford 1946) was later considered to
be identical to Tetrasporopsis reticulata Meyer (Whitford
1976), and T. reticulata is the basonym for Dermatochrysis
DISCUSSION reticulata (Meyer) Entwisle & Andersen. Whitford (1946)
describes the stomatocyst as smooth. Either the scabrae were
All seven chrysophytes produced stomatocysts, but it is not unobserved by Whitford, or his description was based on an
clear which induction stimuli were responsible for their immature form like Fig. 5; alternatively, my observations on
formation. Removing 5 ml for centrifugation from the Dermatochrysis sp. may indicate variation within the genus.
Ochromonas cultures concentrated the stomatocysts and Cysts were not previously reported from Chrysosaccus,
expedited their detection. Preliminary work showed that the and this study augments our knowledge of the genus by
encystment frequency (percentage of cells producing stoma- describing its stomatocyst formation. Chrysosaccus cf.
tocysts) for O. pinguis was extremely low, and a similar low sphaericus was previously named Chrysosaccus cf. epilithicus
cyst percentage was found here for Ochromonas sp. Curtin Starmach (Andersen 2007).
(1987) observed that, on average, 0.006% of the population My description of C. dendrolepidota stomatocysts concurs
of Ochromonas minuscula Conrad encysted regardless of with the description reported by Peters & Andersen (1993).
culture conditions, although the percentage increased The stomatocysts for the two Synura species in this study
slightly during its stationary phase of growth. were indistinguishable from each other; however, they were
Doflein (1923) was the first to describe the stomatocyst of considerably smaller than those described by Sandgren &
Ochromonas pinguis, which at that date he named O. crenata Flanagin (1986; 8–9 lm vs 13–16 lm). Minor stomatocyst
Klebs. Because of the limits of light microscopy, the cyst size variation is natural; for example, differences in the
surface was described as being ornamented with spines. The nutritional state of the vegetative cell populations and/or
present study provides the first SEM images of the O. pinguis concurrent cell size at the time of stomatocyst formation may
stomatocyst, and the apparent spines observed in light explain size variation, but this area is not extensively studied.
microscopy are shown to be lunate ridges that are slightly Similar intraspecific size variations in stomatocysts have also
thicker at the base. The stomatocysts of O. pinguis resemble been reported by Cronberg (1988) and Findenig et al. (2010);
cyst 133 (Duff et al. 1995), but O. pinguis cysts are larger. however, in this case, stomatocyst difference could also
The O. pinguis cysts are also nearly identical to the cyst reflect differences between the two S. petersenii-like cryptic
produced by Ochromonas sphaerocystis Matvienko (Ander- species (Škaloud et al. 2012).
sen 1982), but there are differences in collar height (’ 0.6 lm Not all stomatocysts are easily identifiable because many
in O. sphaerocystis vs 2.5 lm in O. pinguis). Because cyst 133 chrysophytes produce simple unornamented cysts that lack a
resembles cysts of both O. pinguis and O. sphaerocystis, cyst collar and are morphologically indistinguishable. For
133 may be produced by Ochromonas or an Ochromonas-like example, the stomatocysts of Synura (Figs 9, 10) are
organism. morphologically similar, and they also resemble the imma-
Morphologically, the small Ochromonas sp. flagellate ture cysts of Dermatochrysis (Fig. 5) and Chrysosphaerella
resembles Ochromonas minima Throndson. However, Flöder longspina Lauterborn (Sandgren 1989). Moreover, the
et al. (2006) reported that O. minima was not to be able to collared and unornamented cysts of Ochromonas sp. and
Fig. 5. Immature stomatocyst of Dermatochrysis sp. strain A12,609 with unornamented surface. Scale bar ¼ 1 lm.
Fig. 6. Developmentally mature stomatocyst of Dermatochrysis sp. with scabrae. Scale bar ¼ 1 lm.
430 Phycologia, Vol. 53 (5)
Fig. 7. Stomatocyst of Chrysolepidomonas dendrolepidota strain CCMP 293. Scale bar ¼ 2 lm.
Fig. 8. Stomatocyst of Chrysolepidomonas dendrolepidota. Note the anterior depression encircling the pore giving the impression of a small
conical collar or lip. Scale bar ¼ 2lm.
Fig. 9. Stomatocyst of Synura cf. americana strain A12,391. Scale bar ¼ 2 lm.
Fig. 10. Stomatocyst of Synura cf. macropora. strain A12,220. Scale bar ¼ 2 lm.
Fig. 11. Stomatocyst of Chrysosaccus cf. sphaericus strain CCMP 1162 with complete collar. Scale bar ¼ 2 lm.
Fig. 12. Stomatocyst of Chrysosaccus cf. sphaericus with incomplete collar. Scale bar ¼ 2 lm.
Holen: Inducing chrysophyte stomatocyst formation 431
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