Ecological Considerations of Diatom Cell Motility. I. Characterization of Motility and Adhesion in Four Diatom SPECIES1

J . Phycol. 32, 928-939 (1996)

ECOLOGICAL CONSIDERATIONS OF DIATOM CELL MOTILITY. I. CHARACTERIZATION OF MOTILITY AND ADHESION IN FOUR DIATOM SPECIES'

S t a n l e y A. Cohn2 and Roy E. Weitzell, Jr. Department of Biological Sciences, DePaul Unlversity, 1036 West Belden Avenue,Chicago, Illinois 60614

ABSTRACT

T o better d e t e r m i w the rcologicnl role of viotilttj i)i pennate diatoms, u'e qua ntita tiilelj chn ra cteri:ed sfiieral viotilitj and adhesiotz properties o f f o u r species of motile p n i i a t e clintoins (Craticula ~ p . , Pinnularia sp., Nitzsch- ia sp., and Stauroneis sp . ) isolated f r o m the same f r e sh - uluter p o n d . Csiizg computer-n ~sis tr t l i'ideo microscopj, U'P

i n easu red speed, size / shape , f u )i ction a 1 ndh esio n, p a t h curzature, aiid light sensitii'itj f o r these species, each of u'kich shouNs a distinctii~e set of inotile brhailiors. T h e ail- rrage speeds o f Stauroneis, Pinnularia, Xitzschia, a n d Craticula cells a re 4.6, 3.3, 1 0 . 4 , aiid 1 0 . 0 pm. s - ' , respectizlelj. Craticula a ) i d Nitzschia cells inoi'e i u a relatizielj straight pa th (<4 degrees rotntio,i per 1 0 0 p )n rnouement), Stauroneis exhibits ) n i u o r rotntioii (about 7 degrees p e r 1 0 0 p)ii ino:'ei)irjit), a r i d Pinnularia rotates considerablj duriug inoilemeiit (about 22 degrees p e r 1 0 0 pin inoiled). Fu)ictional adhi2sioTi (as ineosiired b j the release rate of attachecl cells Jro)n thr uiiderside of au i n - zlerted coilerslip) shoilis a hnlJ tinip f o r cell ,-Please of ap - p r o s i m t e l j 50 iniiz for Craticula, 1 9 2 jnin f o r Pinnu- laria, am1 > I daj f o r Nitzschia nizcl Stauroneis. Di- rection reuersal a t light I d a rk b o u d n rips, il~hicli appea rs to be the m a i n contributor to iliutoiii phototnsis, is most wsporzsiz~e f o r C ra t icu la, Pin n u lar ia , N i i (1 IS i t zsc h ia at wazlelengths around 500 n i n . Craticula n ~ c l Nitzschia cells are the most seiisitiibe to the photophobic respowe, with ozfer 6 0 % of these cells respoiidiiig to n 30-1.u l i gh t / dark boutidarj at 500 nvi , u l i e i w a Pinnularia cells are o n l j moderatelj responsizie (it this irrcidia I I C P , slioxing a tnaxiwial rcsponsr of nppro.yivinte/! 30Q of cells a t 450 ? i i n . Stauroneis cells, i n cojitrnst, hod a innsiinal pho- tosmsitizle reJponse a t 700 in, sirggestitig that this cell t jpe ina j use ri differeut resf3o)ise uiechn,iisin t h a n the other three cell tjpes. ZH ndditiou, Craticula a n d Pinnularia shoul (I w t v io iwt ie i i t out oft l ie light spot d i e t i illuininated a t 650 wn, whereas Stauroneis shoas a )let ~ ~ i o ~ l e ~ n e i ~ t out of the light spot il4ieiz illuiniiinted nt 4 5 0 nm. Such qnmntitati i~e chu racteri-atiow ofspecies-sprclfic responses to eu i i ro pi 1ne)i ta 1 stim u li should giire us a fir i n f o ii iz da tie,?

f o r f u t u r e studies ana1j:i)ig the behni'ior of iiiterspecies dintoin coinpetitio)) f o r limited light or nutr ieut resources.

Kej i d e x u~rcls: Bacillariophjcetre; Craticula; ecologj; motilit!; Xitzschia: ~hoto))iui 'ei i ie)i t; phototaxis; Pinnu- laria: Stauroneis

I Recelled 8 Decembcr 1993. Accepted 29 Ju l j 1996 Author for reprint request\.

Diatoms are among the most abundant and ubiq- uitous members of the plant-like protists and are responsible for a substantial portion of the earth's primary production. As such, diatoms are vital to the maintenance of a large number of marine and freshwater ecosystems (Darley 1982, Caduto 1990). T h e significance of diatoms in the food chain is further reinforced by recent poisonings in both humans (Per1 et al. 1990) and pelicans (Fritz et al. 1992), which were ultimately the result of diatom toxicity. Of additional ecological importance, diatom secre- tions and adhesion are thought to play a role in the stabilization of some coastal sediments (Patterson 1989) and in rendering new substrates competent for further algal colonization (Stevenson and Peter- son 1989).

However, despite their ecological importance, Iit- tle work has been done on the differences in physiology and behavior among different diatom species. For example, although approximately 100,000 species of diatoms have been described, mainly on the basis of their unique morphological characteristics, the natural variability of species morphology due to their ecological conditions suggest that the number of true species may be as low as 15,000 (Schmid 1995). This level of uncertainty is but one example illustrating how little information is available on actual physiological differences between individual species. Yet, knowledge of the individual behaviors of species, and the effect of environmental conditions on their behavior, is crucial to discerning the viability and ecological success of different diatom species and how they can exploit different ecological niches.

T h e spatial distributions of benthic diatoms within their local habitat and how these distributions are generated and maintained are particularly intrigu- ing for several reasons. For example, the first cells colonizing a newly available area have easy access to light. and nutrients. As the community develops, however, the initial colonizers become covered with additional growth of other cells and material. Many benthic cells such as diatoms can avoid the lowered level of light and suspended nutrients by raising themselves upward within the algal mat by one of several processes: stalk formation (e.g. secretion of enough mucilage differentially at one end to push itself upward), epiphytic behavior (e.g. diatom adhesion to other algae that are growing upward or moving through the mat), or active motility of in-

928

929 CHARACTERIZATION OF DIATOM MOTILITY

dividual cells. On the other hand, some cells show little or no change in relative position as the algal community develops, preferring to take advantage of potentially higher organic nutrients (from waste material and breakdown products) deeper within the community.

The bilaterally symmetric, or pennate diatoms, are particularly interesting specimens for investi- gation, because they often display active regulated movement within the benthic habitat. The manner of movement for diatoms is somewhat unique among the protists because the hardened silica cell wall pre- cludes amoeboid or ciliated driven motility (Jarosch 1962, Harper 1977, Werner 197'7, Roundetal. 1990, Hader and Hoiczyk 1992). Among the several mod- els for explaining the movement of pennate diatoms (e.g. Drum and Hopkins 1966, Gordon and Drum 1970, Edgar and Pickett-Heaps 1984), there is con- sensus that the movement is coupled to mucilage secretion and substratum adhesion along the slit in the cell wall (raphe) characteristic of motile species (Webster et al. 1985, Round and Crawford 1990, Round et al. 1990). Such movements may require underlying actin filaments (Edgar and Zavortink 1983) and be similar to other actin-based cytoplas- mic movements in plants (Preston et al. 1990). While attempts to determine several aspects of diatom motile responses to ecological conditions have been per- formed (e.g. Nultsch 197 1, B. Cooksey and Cooksey 1980, 1988), little is known about the role of cell motility in the ecological success of motile diatoms.

Clearly, the way in which motility contributes to the ecological success of any individual species is going to depend on a number of physiological and ecological conditions (e.g. differences in the type or strength of adherent mucilage, differences in their sensitivity to light intensity or wavelength). Envi- ronmental characteristics of nutrients, shading, and speed of water current have already been shown to affect diatom immigration ability (Stevenson and Pe- terson 1989, Stevenson et al, 1991) in a manner that appears to be also correlated with aspects of raphe morphology.

This paper begins to address the possible roles of cell motility in the ecological success of pennate diatoms by characterizing the motility of four diatom species isolated from the same pond, with respect to several physical and environmental variables. These individual characteristics can then be correlated in future studies to the ability of the diatoms, in mixed populations, to successfully compete with one another at ecologically relevant activities (e.g. light- directed migration or substrate adherence during active water How). In this way, we hope to better understand the different ecological conditions best exploited by different pennate diatom species.

MATERIALS AND METHODS

All cell cultures were isolated from pond samples from the same pond in Boulder, Colorado. Cells were individually isolated

and transferred to a glass spot plate well using a drawn glass micropipet, washed several times with distilled H,O, and then transferred to small polystyrene Petri dishes containing Diatom medium [DM: 300 pM Ca(NO,), 400 r M KH,PO,, 100 r M MgSO,, 0.01%v/vsaturatedNa,SiO8.pH8.5, 1 pMFeSO,,O.l fiM MnCI, 5% v/v soil extract, 1 pg.mL-' niacinamide, 1 pg.mL-l thiamine, 0.1 pg.mL-l biotin, and 1 pg.mL-l B,,], pH. 6.7 (Cohn and Pickett-Heaps 1988, Cohn and Disparti 1994). Cell cultures were incubated at 10"- 12" C on a 10: 14 h LD cycle. DM in the culture plates was changed every 1-2 weeks, and cells were subcultured into new plates every 2-4 weeks. For all assays requiring cells to be tested at distinct time intervals or for prolonged incubations, cells were kept on an electric cooling plate to keep the cells at 8"-12" C between measurements.

Motility assay Cells to be assayed for speed were transferred via micropipet from culture plates to ,1.5-mL wells of a glass spot plate containing fresh DM, washed several times with fresh DM, transferred to a glass slide containing two thin spacing strips of VALAP (vaseline : lanolin : paraffin 1 : 1 : 1 by weight) to prevent crushing of the cells, and covered with a glass coverslip, which was sealed onto the slide with more VALAP.

Motility was measured using a computer-assisted video microscope system consisting of a Zeiss Axioscope equipped with standard DIC optics and a halogen-tungsten light source and fitted with a Dage-MTI 68 video camera. The video signal of the camera was interfaced with a Commodore Amiga 2000 computer through a GenLock device allowing superimposition of the computer screen onto the video monitor. Using a BASIC computer program writ- ten by SAC and Dr. W. Schreuers (Teledyne-Water Pik, Fort Collins, Colorado), the distance or speed of the cells could be measured by tracking the movement of the cells using the mouse- driven computer cursor (as used previously: Cohn et al. 1987, 1989, 1993; Cohn and Disparti 1994). Static measurements of cell dimensions could also be obtained in this way. The length was considered to be the largest distance as measured along the apical axis (tip to tip), the width was the largest distance as measured along the transapical axis at the center of the cell (side to side), and the depth as the largest distance as measured along the pervalvar axis (valve face to valve face). For analysis of path curvature, path tracings from recorded images were made onto acetate sheets that were marked with the cell location every 5-10 s. Surface areas of valve faces were calculated using Image1 soft- ware (Universal Imaging, West Chester, Pennsylvania).

To quantify the differences in path curvature between the four cell types, we measured the relative orientation of the apical axis of a cell over several 5- or 10-s intervals, from a prerecorded videotape, and calculated the rate at which the apical axis rotated from its previous position. The distance moved by the cell in each interval was compared to the rotation of the apical axis to obtain the amount of path curvature in degrees rotated per micrometer of movement.

Adhesion tests. Cultures of cells to be used for the adhesion assay were checked for active cell motility prior to cell isolation in order to ensure that differences in adhesion values were not due to a lack of cell movement in a particular test culture. Many cells from culture were then collected via micropipet and rinsed two times by consecutive placement in spot plate wells containing distilled water, aspirating, and mixing the cells several times in each well to ensure breakup and release of any large amounts of extracellular mucilage attaching groups of cells together. Using another clean micropipet, a number of the washed cells (usually 50-400) were then isolated and transferred onto the center of a 24- X-40- mm glass coverslip (Kimble) submerged in DM and resting on two plastic spacers. After allowing the cells to settle onto the coverslip for 15 min (during which they were again observed for active motility), the coverslip was inverted by hand, with cells and coverslip remaining submerged in DM during the rotation and placed back onto the spacers.

The inverted coverslip was then observed using a Wild MZ8

930 STAKLEY A. COHN A N D ROY E. WEITZELL, JK.

TABLE 1 . Comparison of shape a n d m o ~ i l i t ~ i n f o u r diatom species. All values reported are mean i 1 SE. Speeds were determined f r o m a large number of speed measurements, at both 30 n n d 1000 Is, a l l light frequencies combined, >550 cells measuredfor each species. All other columns are calculated from independent populntiorls of cells nnn1j:ed speczjrallj f o r that property, with > 10 cells of each species measured.

Speed Path curvature Length Width Depth Area of valve face Cell type @rn 5-I) rotated pm-l moved) (pm, apical axis) (rm, transapical axis) (rm, pervalvar axis) (pm* x lo3) Len@h/width ratio

Craticula 10.0 f 0.1 0.04 f 0.01 109.9 ? 0.8 24.9 i 0.4 16.0 -t 1.0 1.62 5 0.02 4.4 * 0.1 Pinnularia 5.3 f 0.1 0.22 * 0.02 166.4 i 1.4 31.8 * 0.5 28.8 i- 3.0 3.99 f 0.07 5.3 * 0.1 Stauroneis 4.6 4 0.1 0.07 * 0.03 139.6 i 1.3 34.9 i 0.6 33.5 * 2.9 2.95 i- 0.06 4.0 i 0.1 Nitzschia 10.4 f 0.1 0.038 i 0.004 129.9 ? 3.3 7.3 i- 0.6 8.0 i 0.4 0.94 i 0.04 17.8 5 1.8

dissecting microscope, and the number of cells remaining adhered to the underside of the coverslip were counted at regular time periods (usually every 15-30 min over 3-5 h). Concurrently, a centralarea of the coverslip was usually recorded on a Panasonic AG-6040 time-lapse video recorder to confirm the rate of fall off as well as observe the motile characteristics of the moving cells during release and reattachment.

Light responsc assaj. Cultures of cells to be assayed for their light/dark boundary response were first checked for active motility and then placed in a light-tight box in the incubator for 2 days prior to the assay. Once ready to be tested, the boxes containing the cultures were placed onto the electric cooling plate for the remainder of the assay. For each test, a small sample of cells from the desired culture was quickly removed and mounted on slides in dim room light (<30 Ix, < 1 min for mounting). Slides with mounted cells were placed back in the dark for 15-20 min to reduce any transient light effects from the mounting process. Cells were then placed onto the microscope stage in a darkened room (<1 Ix) and placed into the center of a small 30-IX spot (350-360 Prn diameter) created by closing the field diaphragm down to the appropriate size. T h e irradiating wavelength of light was controlled using 5-nm band\\idth interference filters or (for full spectrum) a silvered neutral densit, filter, from Oriel Corp. (Stratford, Connecticut). As the cells moved tow-ard the light/ dark boundary, their speed was measured. Upon arrival at the light/dark boundary, each cell was classified as to whether or not it reversed direction within one cell length past the light/dark boundary. For each wavelength, at least 10 cells were measured in each of two trials.

As a control to assess the basal rate at which the cells naturally reversed their direction, cells were tested in a similar “mock” assay where no light/dark boundary was present. For this test, cells were placed into the center of the field of view at the same wavelength and intensity as the light/dark tests but with the field diaphragm fully open (i.e. no boundary). A circle was placed over the video image of cells using either an acetate tracing or computer superimposition, corresponding to the same size as the test spots of light. Motile cells crossing the circle tracing were scored as before for whether or not they reversed direction within one cell length past the circle. T h e net photophobic light response was then calculated by subtracting the unstimulated frequency of cell reversal (mock response) from the measured frequency of cell reversals at the spot boundary (spot response).

Light levels (in lux) were determined using an INS DX-100 Lx Meter. The calibration of Ix readings on our meter with actual light quanta was carried out using a Li-Cor LI-189 photometer and a SA-190 quantum sensor. All comparative statistics were carried out using ANOX’A factorial analysis, using StatView soft- ware (Abacus Concepts, Berkeley, California) and Scheffe’s post- hoc analysis.

RESULTS

Siw nnd \ h a p . T o correlate any differences in motile behavior rvith size or shape, the length, width, depth, and valve face surface area of each cell type were measured (Table l) , and the resulting length-

to-width (L/W) ratio was calculated. Each of the four species in this study had a distinctive size and shape. From longest to shortest, respectively, the cell types were Piiznularia (about 170 pm long), Stau- roneis, Sitzschia, and Craticula (about 110 pm long). In terms of width, from widest to narrowest, the cell types were Stauroneis (about 35 pm wide), Pinnularia, Craticula, and ,\$‘tzschia (about 7 pm wide), respectively. While the length and width distribution for each of the cell types was relatively narrow, the distribution of depths within each of the cell types showed a somewhat wider distribution. Stauroneis, Pintzularia, Craticula, and Nitzschia, showed the same order, respectively, from the thickest to the least thick (Table 1).

The valve shape of Craticula and Stauroneis was most similar, with both showing a L/W ratio of about 4-4.5. The L/W of Pinnularia was somewhat higher, at about 5. The long and slender Nitzschia cells showed the greatest L/Mi at about 18. Pinnularia, Sitzschia, and Stauroneis all displayed a depth that was insignificantly different from their width. How- ever, Craticula cells were significantly wider than they were deep (P < O.OOl), so that the cross-sectional profiles of Pinnu la ria, Nitzschia, and Stauroneis were approximately square, whereas the profile for Craticulu was essentially a flattened rectangle.

Using the average values of the width and depth, the cross-sectional areas (i.e. the cell profile looking down the apical axis) were approximately 1 170,900, 400, and 60 pm2 for Stauroneis, Pinnulnria, Craticula, and Sitzschia, respectively. The estimated cell vol- umes (in pmS) were approximately 1 15,000 for Pin- izularia, 100,000 for Stauroneis, 25,000 for Craticula, and 7500 for Sitzschia.

S P P P ~ a n d path cunlature. The average speed of moving cells was determined for each of the four cell types (i.e. measurements did not include any stationary cells, as opposed to the distribution of total cell populations, including stationary cells, as done in Cohn and Disparti [ 19941). The fastest cells were Croticula and ATitzschia, with Pinnularia and Stnurotwis being the slowest (Fig. 1, Table 1). All four cell types showed a relatively normal unimodal distribution of speeds. Sitzschia and Craticula cells showed the widest distribution in speeds, ranging from about 2 pm.s-’ to occasional cells moving as fast as 18-20 pm.s-’; the distribution of Craticula speeds showed a somewhat smaller standard devia-

CHARACTERIZATION OF DIATOM MOTILITY 93 1

tion. The other two cell types showed a somewhat narrower range of speeds, with the greatest number ofPinnularia cells moving at around 4-6 pm.s-' and a range of 1 to 10-12 pm.s-', whereas the largest group of Stauroneis cells moved at 2-5 pm.s-' with a range from 1 to 12 pm.s-'.

Measurement of the path curvature showed that Craticula and iVitzschia cells had the least deviation from a straight path, with both types rotating less than 5" over the distance of one cell length. Stau- roneis showed a slight curvature, with a curvature of just less than 10" over one cell length. In contrast, Pinnularia showed a large amount of rotation, such that movement of only one cell length would result in over 35" of rotation.

Cell adhesion. Differences in cell adhesion were determined using an inverted coverslip assay. By measuring the number of cells remaining adhered to the inverted coverslip over time, we determined the rate at which cells fell from the coverslip when subjected to a relatively constant force (i.e. the force of their own weight under gravity).

Craticula and Pinnularia cells appeared to fall off at a rate approximating an exponential decrease. This was confirmed using a plot of the logarithm of the cells stuck versus time, standardized by plotting the data as ln[N/No] versus time (where No is the initial number of cells adhering onto the coverslip after inversion, and N is the number of cells remaining adhered at each measured timepoint). Such plots appeared linear for all four cell types (Fig. 2), and for Craticula and Pinnularia (with slopes < -0.001 min-l) the plots had regression values of r2 2 0.9; the slopes of the plots were determined by standard linear regression to obtain the exponential rate constant k (= - slope). The time for one-half of the cells to fall off (t1/2) was determined by calculating ln(2)/k. Although the plots for Nitzschia and Stauroneis also appeared linear, the slopes were so close to zero that the correlation values r2 were all <0.8 (in four of the trials the r2 < 0.4).

Craticula cells released most rapidly from the coverslip, with an average tIl2 of under 60 min (Table 2). Pinnularia cells showed the next most rapid loss, with an average tl/2 of about 3 h. Both Stauroneis and Nitzschia cells showed almost no loss from the coverslip under the conditions of our assay, so that the slope of the ln(N/No) vs. time plot was virtually zero (Fig. 2, Table 2). Due to the variability of the slope about zero, and the sensitivity of the resulting calculation for the half-time of cell loss (tl/2 = In 2/-slope), it was impossible to arrive at an accurate value for the t,,2 for Stauroneis and Nitzschia. But, based on the most rapid cell loss of the four trials for each of these two types, their half-time was greater than 24 h.

Motile behavior of inverted cells. All four cell types were filmed under time-lapse videomicroscopy to determine their behavior when moving on the underside of a coverslip (i.e. when they were adhering on their upper valve surface and thus free to fall).

120 1 I00 -

5 x o - g 60 - u 40 -

20 - 0 -

0 2 4 6 X 10 12 14 16 18 20

Craticula Velocities

0 2 4 6 8 1 0 12 14 16 18 20

Pinnularia Velocities

0 2 4 6 X 1 0 12 14 I6 18 20

Stauroneis Velocities

70 60

0 L 0 2 4 1) X 10 12 14 16 18 20

Nitzschia Velocities FIG. 1 . Speed distribution of diatoms. These histograms show

the distribution of speeds (prn.s-l) for Cratzcula, Pinizularia, Stau- roneis, and Nitzschia. At least 300 cells were measured for each species, and each measurement represents the average speed determined for a motile diatom over a 2-5-s interval. T h e average speeds for each of the cells type is as indicated in Table 1 .

932 STANLEY .4. C O H N A N D R O Y E. WEITZELL, J R

J

-.? - - 6 -

- 8 -

- 1 -

2 1

- 2 .-+ - 1 . 2 1

+ -0- + -A-

Nitzwhia Pinnuldri'i Stauronei\ Craticulii

h - 1 4 4 1 I ' I ' I . I ' ' I ' 4 ' '

0 50 100 150 Z(x) 250 300 350 400

Time (min) FIG. 2 . Comparison of cell adhesion. Loss of adhered cells

from the underside of an inverted coverslip over time for Cra- ticula (A), PiniiuIaria (0), Stauroneis (A), and .Yitmhia (0). Data points represent the natural logarithm of the fraction of adhering cells remaining [ln(iX/No)] versus time after inversion of the coverslip. The data for Craticula and Pinnularia show a linear correlation with rz > 0.9. The data for Stauroizeis and .Yitzsrhia show no significant correlation. T h e data in the first two columns of Table 2 were determined from the average of four such trials for each cell type.

Under such conditions, we observed several distinctive types of motile behavior.

Moving Craticuia and Slnuroaeis cells often displayed a partial loss of contact, followed by recovery (Fig. 3 ) . Under such situations, a moving cell would briefly stop and then lose contact with most of the valve face so that only one tip of the cell wall would remain attached to the coverslip. That is, one end of the cell would stay attached, and the rest of the valve would fall away from the coverslip, such that the apical axis would be pointing straight down. In

virtually all of the cases where Stauroneis showed this loss of contact (and in most of the cases of Craticula), the cell would be able to recover by pulling itself back up, such that the valve face would regain contact with the coverslip, and the cell would start to move.

Interestingly, when this type of contact loss and recovery occurred, the tip that remained attached to the coverslip was virtually always at the trailing end of the cell prior to the loss of contact and at the leading end of the cell upon reattachment and motile recovery (Fig. 3 , Table 2). In the case of Crati- cula, the valve face that reattached upon recovery )\.as usually (but not always) the same one that had initially lost contact, whereas the valve face that reattached was more variable with Stauroneis, with many Stciiiroizeis cells rotating around the attached tip during the period of single end attachment.

The ability to recover was different between Stau- roneis and Craticula. While Stauroneis cells virtually always recovered from the loss of valve face contact, the recovery of Craticula was less frequent, account- ing for the loss of cells over time. In fact, almost all of the Craticula cells observed to fall off of the coverslip did so when they were attached to the coverslip by only one tip. For those Stauroneis and Cra- t icula cells that did recover, the rate of recovery also differed, with Craticula cells being able to recover more than five times faster on average than Stau- roueis (Table 2).

Piii)iularia cells never showed this type of drop and recovery. Pimular ia cells occasionally lost valve face contact in a similar manner (attached by only one end), but they were never observed to recover from this position. Instead, such single end-attached Pinnula ria dropped off from the coverslip, either directly or by lowering themselves away from

TABLE 2. Characterization of adhesion and rnotile behai'ior upon inwrsion. Values reported are mean i 1 SE. Adhesion values were determined f rom at ieust f our trial3 o f a t [east 100 iiiitrall> adhrrrd rrlls (.Yo) earh triut. The rate constant (k) was determined by calculating the slope o f the ln(N/ LVo) 11s. time plots; k = -slope, and awraged for four ti-iols. The hal f t ime f o r cell loss was determined by averaging the values f o r the individual hay - time calculated for earh trial, d e t e r n i r d b> calculnting lii(2)/ k. Rerowrj t ime is determined f r o m the recorded video images and defined as the time betuseen thefirst detectable loss of :w lc , r fn r r roiitart and thr tiinr a t u,hichfi t l l r ' a lw face contact appeared restored. Orientation o f ends relative to cell direction ujere determined z ~ i s u a l l ~ nf thP tiiiie of i'alr,r face dissociatioti or at time of re11 rerorlery. T h e classzjication of "ambiguous" is used when accurate drterniiriations could riot he mudr due to rapid osrrllafians a t the t ime of loss or recoi'erj or, for valve face determination, due to cells rotating around rapid11 while attached a t O I I C e n d .

Adhesion characteristicr Single-end attachment characteristics

Half-time for Orientation of end Orientation of end Rate constant cell loss maintainin attachment

for cell loss upon imersion (at vafve face Recovery time Valve face reassociating upon maintaining attachment Cell type (k. min-') (th) dissociation) (s) recovery (at recovery)

C m ticula 0.025 * 0.014 50 I 17 min (n = 14) 47 ? 8 13 trailing end, 93% 0 leading end, OR 1 ambiguous, 7%

Piiinulnria 0.0039 IO .0009 192 i 29 min N A" N A Stouroneis 0.00014 i 0.00007 > 1 day (n = 17) 279 * 77

14 trailing end, 82% 1 leading end, 6% 2 ambiguous, 12%

,Vitzsrhiu 0.0004 i 0.0002 > 1 day NA N A

(n = 11) 8 same side, 73%

2 opposite side, 18% 1 ambiguous, 11 %

N A (n = 17)

8 same side, 47% 4 opposite side, 24%

5 ambiguous, 29% N A

~

( n = 11) 0 trailing end, 0%

10 leading end, 91% 1 ambiguous, 9%

NA (n = 17)

0 trailing end, 0% 14 leading end, 82% 3 ambiguous, 18%

N A

a N.4 = nor applicable.

CHARACTERIZATION OF DIATOM MOTILITY 933

FIG. 3. Release and recovery of valve face attachment in Craticula. Still pictures from a time-lapse video sequence in which a moving Craticula cell stops, loses valve face attachment (remaining adhered by only one tip of the cell), regains attachment, and begins to move again. The cell is moving on the underside of a coverslip as in the adhesion assay described in text. Time notations in the lower right corner of each frame represent the time elapsed since frame A. A) Craticula cell is moving forward in the direction of the arrow. B) Cell halts and begins to lose association of the valve face, maintaining attachment by the tip of one end (marked by arrowheads in panels B, C, F, and G ; the same end is attached in panels B-G [the dust particle at the bottom of the frames can be used as a point of reference]). C) The leading end of the cell has visibly lost attachment, falling away from the coverslip. D, E) The leading tip continues to fall down, such that by frame E the cell is attached to the coverslip only by the tip of its initially trailing end. F) The cell begins to recover, pulling its valve face back toward the coverslip. G) The cell regains valve face contact. This cell has flipped over in the process so that the original trailing tip of the cell is now on the lower right side. H, I) The cell begins to move forward (in direction of arrow), leading with the tip that was attached (which was trailing prior to the cell’s drop and release). Upon recovery, Craticula cells virtually always move in the direction of the original trailing end, whether or not the cell flips over (see Table 2). Scale bar = 50 pm.

the coverslip as if they remained attached by a slowly lengthening tether. In addition, many Pinnularia dropped off the coverslip directly from a position where the entire valve face previously seemed to have contact.

The Nitzschia cells also rarely displayed a drop and

recovery behavior, although they often quickly rotated around a single attached tip. Nitzschia were observed to be “hanging” by a single attached end only very rarely and in those cases quickly recovered. In addition, Nitzschia cells had the tendency to aggregate together with other Nitzschia cells much

934 STANLEY A . COHN AND ROY E. WEITZELL, JR.

7 0 1

-20 J L

Cl'lt Pinn Staum Yiiz

Cell Type

FIG. 4. Wavelength-dependent sensitivity of the four cell types to direction reversal at light/dark boundaries. Cells were exposed to a 30-Ix field of light consisting of full-spectrum 100'12' tungsten light (clear bars) or of 400, 450, 500, 550, 600, 650, or 700 nm light generated by interference filters (shaded bars per legend). Moving cells were in the center of either a 350-360-pm-wide diameter spot of light (spot test) or a full field ( > 2 mm diameter) of light with no boundar) (mock test). Cells were then observed to determine the percentage ofcells that changed direction within one cell length of the light/dark boundary (for the spot illumination), moving back into the light, or the percentage reversing direction within one cell length of an artificial boundary drawn on an acetate film or the video screen representing the same spot size (for the mock test). This graph represents the net sensitivity of each of the cell types (Crat = Cmtrcu[a, Pinn = Pirinulnria, Stauro = Stnuronerj, S i t z = . Y i h c / m ) to light/dark boundaries at each of the frequencies(i.e. percentageofcells changingdirection at the actual houndar) minus the percentage of cells changing direction at the mock boundary).

more than the other three cell types self-aggregated. In all of our trials, the only significant loss of L\itzschia from an inverted coverslip occurred when a small group of 10-20 cells aggregated, becoming attached to the coverslip by onlj- one end of one cell in the aggregate; these cells were unable to reassociate with the coverslip and eventually fell off.

Light sensitizfitj. Because previous experiments had suggested to us that the primary mechanism of pho- todependent movement i n diatoms was a wavelength-sensitive photophobic response (Cohn 1993), all four species were tested for their ability to re- sporid to a Iight/dark boundary using a direction reversal assay. Under this test: dark-adapted cells were placed individually into the center of a small spot of light (approximately 350-360 pm diameter) at a specific wavelength. A11 experiments lvere carried out at 30-1x light levels (calibrated \\.ith a quantum light meter to be equivalent to approximately 1 pmol photons.s-1.m-2, which corresponds to ir- radiation energies of 0.2-0.3 \V.m-2). Cells were then allowed to move to the light/dark boundary and were observed to determine \\.hether or not they exhibited a step-down photophobic response (i.e. reversed direction at the boundary, moving back into the light) \vithin one cell length. T o determine the basal rate of direction reversal at each wavelength of light, cells were exposed to an open field

of light containing no light/dark boundary, and the percentage of cells changing direction at a mock boundary drawn only on the video monitor was determined. T h e functional response was then considered to be the number of cells reversing at a real light/dark boundary minus the cells reversing at the mock boundary (overall response shown in Fig. 4, and broken down by real and mock spots in Fig. 4, and broken down by real and mock spots in Fig. 5).

All four cell types showed a definite step-down photophobic response when cells were exposed to full-spectrum light (1 OOW tungsten-halogen) at 30 Ix intensity (Fig. 4). Three cell types (Craticula, -\it;schia, and Pi)z)iularin) showed a maximal response at 450-500 nm, whereas Stnuroneis cells seemed most responsive at 700 nm. In addition, C m - ticula and Piiiuularia showed a slight reversal of cells (aw.ay from the light spot) when exposed to light at 650 nm. T h e Crnticula and Sitzschia were the most responsive cells, with about 60-65z of the cells responding at 500 nm and 30-50% responding to full- spectrum light. Pinnularia and Stnuroizeis were less responsive, with only 20-30s of the cells responding to full-spectrum light, and about 30% of the cells responding at their most sensitive wavelength (450 nm for Piu)zulnria, 700 nm for Stauroneis).

When the results of the three similar cell types (Crnt icufn, A\vitzscJiia, and Pirmularia) were combined (Fig. 5A), the trend is even more distinct, showing a maximal response at 500 nm, as well as a slight net movement of cells out of the spot at 650 nm. By analyzing the actual light spot and the mock spot responses separately for each wavelength (Fig. 5B, C), we observed a marked decrease in the basal rate of direction change at 500 nm, relative to the mock spot responses at 400 and 650 nm. Independently, there is also an increase in the total number of cells reversing direction at a real light/dark spot boundary at 500 nm (Fig. 5B). In contrast, Stauroneis cells show a somewhat opposite effect (Fig. 6A, B), with a decreased level of mock spot response at 700 nm and a decreased sensitivity to real light/dark boundaries at 450 nm.

Measurements of cell speed as a function of wavelength (Fig. 7) showed little wavelength-dependent photokinesis (light-dependent changes in cell speed). Crnticuln, Pinizulnriu, and Stauroiwis cells displayed no substantial trend of differences in speed with respect to light wavelength (P > 0.01). T h e Nitzschia cells seemed to be the only species that showed sta- tistically distinct differences in speed (P < O.OOl), with somewhat decreased speeds at 550 and 600 nm and with full spectrum light.

DISCUSSION

T h e results described in this paper show a clear difference in the behavioral responses of the four diatom species with respect to path shape, adhesion, and light response. Such information is useful for both understanding the ecological success of diatom


400nm 450nm 500nm 550nm 600nm 650nm 700nm 17 Whole

500nm 550nm fj00nm 650nm 700nm 0 Whole

A

-10 ’ 1 I

Average Net Response (by Wavelength)

G 70 60 1 . B Stauroneis Spot Response

E) “ 4 so I I F 40 d 3 so v) 30

a 40 % 20 u

2 20

c 0 ?--

m 10 2 30

6 10

$ 0 I

0 d Average Spot Response (by Wavelength)

C 40 1 Stauroneis Mock Response bD ”’ .s 3s

pi 2s

v) 1s % 1 0

5 u

3 30

2 20

ti9 0

0 v)

e

FIG. 6 . The response of Stauroneis cells, by wavelength, to direction changes when exposed to real and mock light/dark boundaries. The net response of Stauroneis cells to changing direction at lightjdark boundaries, shown in Figure 4, was calculated by A) determining the percentage changing direction at an actual light/dark boundary and B) subtracting the percentage of cells changing direction at an artificial mock boundary. Note that although there is a slight increase in direction change for a real boundary at 500 nm (A), there is a significant reduction in the rate of random direction reversal at 700 nm (B).

Average Mock Response (by Wavelength)

species within aquatic communities as well as for the classification of diatoms based on behavioral characteristics.

As with previous studies (e.g. Bertrand 1992, Cohn and Disparti 994)9 we show no ‘Orrelation between the average size of the species and speed. While the two largest species in our study were also the slowest moving cells, the largest cell (Pinnularia) was faster than the next smallest (Stauroneis). Moreover, both

rapidly moving Craticula of previous studies (Cohn

FIG. 5 . Combined response of Craticula, Nztzschia, and Pin- nularia cells to changing direction at light/dark boundaries. Av- erage response for sensitivity to light/dark boundaries for the three blue-green-sensitive species combined (Craticula, Pinnular- ia, Nitzschia). The net response of cells to A) changing direction at light/dark boundaries, as in Figure 4, calculated by determining the percentage B) changing direction at an actual light/dark boundary and C) subtracting the Percentage of cells changing direction in the absence of a boundary (mock test). At 500 nm there is an increase in direction changes at a real boundary (B) and a net reduction in the rate of direction changes with no of these cell types were shorter than the large and boundary (C).

936 STASLEY .4. COHN .4ND ROY E. WEITZELL, JR.

- 1 4 7 1. 450 nm 500 nm I

Crat PiM Staur Nitz

Cell Type

FIG. 7. Effect of light wavelength on cell speed. This figure represents the average speed of cells (Crat = Cral icu la , Pinn = Pi~7nulari0, Staur = Slauroneis, S i t z = .Yitzschia) when illuminated by 30 Ix light at either full spectrum 100W tungsten light or at 450, 500, 550, 600, 650, or 700 nm light (shaded bars as in legend). Only .Yitzschia cells show any significant difference in cell speed as a function of wavelength.

and Disparti 1994). There also appears to be no distinct correlation bet\\.een shape and speed: for example, Croticula and Sto urowis have very similar shapes, yet the former moves twice as fast as the latter.

Our species of Pinnularia also moved similarly to that described by Edgar and Pickett-Heaps (1 984)- in our case, moving in a nearly circular path approximately 250 pm in diameter. Such curved movement suggests that this species of Pititiulnria might be poorly suited to vertical migrations within those algal mats comprised mainl) of linear algal filaments. However, Piiznularicc might prove to be more successful in vertical migrations through loose sediment, where linear paths are not a restriction.

T h e inverted coverslip assay alloLyed us to quantify the functional abilit)- of each species to adhere to a surface while under constant force (in our case, the constant force due to gravity, albeit this force differs for each species due to differences in cell size and cell volume). This contrasts with previous assays that only determined qualitative differences among species (Tanaka 1986) o r that relied on potentially variable external shear forces (K. E. Cooksey 198 1). In our assay, any inconsistencies in the initial dis- turbance due to inverting the coverslip are irrele- vant, because we quantifi- the rate at which the cells fall off, not the fraction of cells remaining adhered during the actual inversion process.

Because the secreted mucilage of many diatoms may act as an external matrix to bind other diatoms, algae, and components of the benthos together (e.g. Round and Cra\\.ford 1990, Round et al. 1990, Hoagland et al. 1993), the cells in our assay were first rinsed thoroughly to remove most external mucilage and ensure that only the adhesion of individual cells was considered. Nonetheless, in all four species, a significant percentage of the cells adhered to the coverslip within our 15 min incubation period. This is in contrast to the 1-3 h period previ-

ously reported for the maximal adhesion of some marine diatoms (K. E. Cooksey 1981), suggesting that some of the adhesion in the latter case may be due to multiple cell contact or cell aggregation.

T h e adhesion differences we observe among species are not clearly due to any one specific shape or motile characteristic. For example, of the two fastest cell types (Craticula and Sitzschia), one showed the most rapid release from the inverted coverslip (Cra- i icula) , whereas the other (,l’itzschia) showed virtually no loss of cells after >4-5 h. T h e adhesion also does not seem to be correlated to the surface area covered by the valve face or to the cell volume, because the adhesion ability of Pinizularia (with the largest valve face area, cell volume, and raphe length) was intermediate in adhesion between the Craticula (the shortest cell, having the next to smallest valve face area) and the Sitzschia and Stauroneis (with Nitzschia having the smallest valve face area).

We also considered the possibility that the cell length-to-cell volume ratio (L/V) might be important for adhesion because longer raphes have more potential substratum contact sites per cell. T h e L/V ratio might therefore be a measure of the number of active contact sites per unit mass. However, we found no relationship between L/V ratio and cell adhesion; Craticula cells also have a much higher ratio of cell length to cell volume than Stauroneis, yet they fall off at a much more rapid rate, whereas S i t x h i a has the highest L/V ratio and essentially does not fall off at all.

I t is still unknown whether the difference in functional adhesion is due to differences in adhesion of the mucilage (i.e., stickiness to the substratum), co- hesion of the mucilage (i.e. the degree to which the mucilage strands break), number of adhesive contact sites, or of sloughing rate (i.e. the rate at which cells release old mucilage strands in order to make new connections). Motile Cmtirula cuspiclata cells are known to build up large amounts of mucilage in culture (Edgar and Pickett-Heaps 1982, 1983), so it may be that their relatively low adhesion could be due to a high sloughing rate. We hope to address this problem in future experiments by measuring the adhesion of cells placed under conditions that reduce their motility (e.g. low temperature, meta- bolic, inhibitors, high external osmolarity).

In any case, the dynamic characteristics of cell motility on an inverted coverslip also seem to tell us something important about the cellular mechanisms underlying diatom motility. In particular, the results seem to indicate that cell attachment sites are lo- calized to discrete areas on the raphe and that some diatoms (e.g. Craticula, Staurotwis, Pitinularia) can often move with only one major mucilage connection site to the substratum. Such conclusions seem substantiated by the movements of Craticula cells on the underside of the coverslip; the cells undergo frequent loss of contact over most of the valve face, only to remain connected by the tip of the trailing


end of the cell. These cells are usually able to pull themselves back up and begin moving, with the tip that was attached now leading the cell.

The drop and recover behavior strongly suggests that the cell is often connected by only a single mucilage connection to the substratum. This single site, in a moving diatom, would eventually be translo- cated to the tip of the raphe, where, on an inverted coverslip, the majority of the valve face is free to fall down and pivot from the connecting end. If the attachment site now reverses direction and begins moving down the raphe back toward the opposite end, the cell is able to pull itself up and begin moving, with the end that was adhered (the former trailing end) now becoming its leading end. Stauroneis cells also showed the same behavior, although the recovery time for Stauroneis to pull itself up, regain contact, and start moving was somewhat longer. Rel- atively distinct mucilage strands or bundles (as in Edgar and Pickett-Heaps 1984) would likely be re- quired for this behavior, because it would be difficult to explain the drop and recovery activity using a capillarity mechanism for motility (e.g. Gordon and Drum 1970, Gordon 1987).

The curved nature of the Pinnularia path also suggests that very few substratum connecting sites are used for this species, because the large amount of turning during movement would shift the raphe considerably off its prior position with only a short forward displacement. If there were many connecting sites along the entire raphe fissure, this would require that nearly all of the contacts would have to continually break and reform as the cell moves forward. Nitzschia, on the other hand, may very well be that with strongly adhering diatoms such as Stau- roneis and the lack of cell release may have a greater number of connecting mucilage strands along the raphe. The relatively rapid release and recovery behavior of Craticula and Stauroneis is in distinct contrast to the behavior of Nitzschia, which shows virtually no release from the substratum despite constant active motion and frequent direction changes. We hope in the near future to determine directly the number of substratum attachment sites in each species by using techniques such as reflectance interference microscopy.

Our light sensitivity assay agrees generally with previous studies (e.g. Nultsch 1971, 1975). In the reports by Nultsch cells collected in light spots of 400-550 nm, whereas in our case Craticula, Pinnu- laria, and Nitzschia all showed greatest sensitivity at light of 450-500 nm. Craticula and Pinnularia cells also showed a net tendency to move out of the light at 650 nm, similar to the movement of cells out of 670 nm light (Nultsch 197 1). Interestingly, Stauro- neis shows a distinctly different response curve from the other three species, with a maximal response into the spot at 700 nm and a net movement out of the light at 450 nm. Considering the fact that Stau- roneis and Craticula seem to be closely related in

many respects (Mann and Stickle 1991), the different light responses may provide a mechanism for each of the cell types to move into separate micron- iches. The spectral sensitivities of all the species suggest that the same wavelengths of light are used for the directional responses (blue-green at 450-500 nm and red at 650-700 nm) but are used in two different ways (blue-green positive and red negative, or vice versa).

As with previous studies (Nultsch 197 l), w e used low light level illuminations to better simulate the conditions a cell would be subjected to in early morn- ing when it is deeper within the sediment and first responding to the light. In our case, our illumination level was approximately 0.5-1 .O pmol phot0ns.s-’. m-* (bright enough to allow us to observe and measure the behavior of individual cells) as compared to approximately 0.1 pmol photon.s-’.m-* in the studies by Nultsch (1 97 1).

Our data essentially indicate that when cells are exposed to a positively stimulating wavelength of light two effects occur: 1) the cells experience a drop in their natural rate of changing direction, and 2) the cells experience an increased ability to sense light/dark boundaries and change direction at the boundaries. Both of these effects have adaptive ben- efits. While a cell frequently changing direction is likely to help it migrate into new areas with potentially increased levels of nutrients or light, it is ad- vantageous to reduce changing direction when in regions of higher effective light in order to spend more time moving into those areas. For Nitzschia, there may also be additional effects due to photokinesis, whereby the cells slow down when they are detecting light of the photoeffective wavelength, in order to further increase their time in the light-rich areas.

Our light sensitivity data can therefore be sum- marized as follows: 1) diatoms appear to be photosensitive (in terms of motility) at two areas of the spectrum (450-500 and 650-700 nm); 2) d‘ iatoms can be classified into at least two groups in terms of their motile response to light (blue-green positive or red positive); and 3) the light response seems to be a combination of a decreased rate of basal (i.e. unstimulated) direction change and an increase in sensitivity to light/dark boundaries.

The wavelengths of maximal photosensitivities for Pinnularia, Craticula, and Nitzschia are close to the absorption peaks for most diatom chlorophylls and photopigments (which are around 420-470 and 650- 700 nm, as indicated by the absorption spectra of acetone-extracted pigments). This suggests that absorption of light by chlorophyll or one of the ac- cessory pigments may be responsible for stimulating photosensitive movements in these cells. However, while the chlorophylls have an absorption peak at around 650-700 nm, it is unlikely that the blue- green and red sensitivities use the same molecule (for the same species) because in each of the species

938 STANLEY A. COHN AND ROY E. WEITZELL, JR.

red illumination and blue-green illumination result in distinctly different responses.

In summary, we find that beyond the physical characteristics, each of the different species we tested can be grouped into one of several different cat- egories based on the measured motile characteristics, namely, speed (slow or fast), adhesion (strong, intermediate, or weak), positive light sensitivity (blue- green-sensitive or red-sensitive), or path curvature (straight or curved). Specifically, the four species can be categorized as follows: Crnticula: fast, weakly adhering, blue-green-sensitive, and straight path; Pin- uularia: slow, moderately adhering, blue-green-sensitive, and curved path; Stil ur0,zeis: slow, strongly adhering, red-sensitive, and straight path: and ,\‘itzschia : fast, strongly adhering, blue-green-sensitive, and straight path. Thus , physical attributes of the valve aside, all of the species can be uniquely determined based on their motile behaviors.

We are now attempting to go further and address the role that each of these motile characteristics plays in actual competitive survival, by placing the cells in competition with each other in several different assays: 1) in an artificial stream setting (flume) where we can see the relative abilities of cells to release and adhere to substrates: 2) in a vertical migration assay where we can assess the relative abilities of the four species to migrate upward through a porous matrix; and 3) in a light-spot cell aggregation assay where we can determine the relative rates of the four species (individually and in mixed populations) to migrate into a defined area of light illumination. Such assays, in combination with the motile characteristics w e have presented, should start to give us a glimpse of the relative importance of some of the motile characteristics in diatom ecological competition and success.

The authors thank Marianne 3lcCollum for obtaining some diatom samples, Dr. N. Tuchman for insights and contributions to the experiments, and Drs. T . Spurck and J . D. Pickett-Heaps for the use of their Image1 system a5 well as valuable comments and discussions. This work was funded in part by S S F grant IBN- 9407279 and the DePaul University Research Council.

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J. Phycol. 32, 939-948 (1996)

COMPETITION FOR PHOSPHORUS AMONG PLANKTONIC DESMID SPECIES IN CONTINUOUS-FLOW CULTURE'

Ell? Spi jkerman2 and Peter F. M. Coesel Department of Aquatic Ecology, University of Amsterdam, Kruislaan 320, NL-1098 SM Amsterdam, The Netherlands

ABSTRACT

W h e n grown under stringent P limitation, a s n i t y f o r P uptake and growth in Staurastrum pingue Teil. and Staurastrum chaetoceras (Schr.) G. M. Smith (both originating f r o m eutrophic lakes) were of the same magnitude, whereas these parameters for Cosmarium abbreviatum Rac. uar. planctonicum W. @ G. S. West (isolated f r o m a meso-oligotrophic lake) were sigrzijicantlj higher in value. O n the other hand, a t allgrowth rates tested, maximum P uptake rates were lower i n C. abbreviatum than i n the two Staurastrum species. The outcome of competition between either Staurastrum species and C. abbreviatum i n mixed chemostats under different levels of continuous P limitation was i n agreement with what could be predicted from the species-specific a f l n i t j parameters: Staurastrum was outcompeted a t dilution rates lower than 0.012 h-', calculated to correspond with external inorganic P con- centrations lower than 0.02 $W P, but won out at higher dilution rates. W h e n P was added i n two pulses of 2.5 pmol. L-I a week instead of continuouslj, S. chaetoceras outcompeted C . abbreviatum at a slow rate. W h e n P was supplied as a daily pulse o f 0.7 pmol.L-', a stable co- exzstence of S. chaetoceras and C. abbreviatum was established, Staurastrum predominating the culture in cell numbers a t ca. 85%. The results shou! that P uptake and growth characteristics of the three species can predict the outcome of competition under various P-limited conditions. Specijic growth kinetic parameters as f o u n d in this s tudj may also explain distribution patterns o f the species observed i n the field. Key index words: Chlorophyta; competition; continuous culture; Cosmarium abbreviatum; desmids; ecophysi- ology; phosphorus limitation; phjtoplankton; pulsed culture; Staurastrum chaetoceras; Staurastrum pingue

* Received 9 February 1996. Accepted 23 August 1996. Author for reprint requests.

Natural selection is thought to favor those algae whose nutrient uptake and growth characteristics are best suited to environmental conditions, so that species originating from habitats differing in trophic state will exhibit different affinities for growth and nutrient uptake kinetics (Hecky and Kilham 1974, Tilman 1977, P. Kilham and Tilman 1979). Con- sequently, nutrient uptake and growth kinetics may help to explain the distribution of algal species in the field.

Since 1980s a lot of experimental documentation has been presented concerning the importance of nutrient uptake and growth kinetic parameters in determining the outcome of phytoplankton competition (e.g. Holm and Armstrong 1981, Tilman 198 1, S. S. Kilham 1984, Healey and Hendzel 1988, Grover 1989a, b, 1991, Olsen et al. 1989, Nicklisch et al. 1991). Yet, what was stressed by Tilman et al. (1982), that is, the need for laboratory studies de- signed specifically to test the validity of conclusions derived from correlation analyses in the field, still holds. Remarkably enough, none of the experimental studies focused on ecophysiological differences among species whose distribution in the field may be linked to the trophic state of the habitat. Most of the preceding experimental studies include species isolated from the same lake. In addition, experiments were often carried out with species be- longing to different algal groups (e.g. cyanobacteria, diatoms, green algae) so that physiological differences observed may be inherent to the groups in question rather than specific to the species level.

Coesel and Wardenaar (1 990) determined growth rates of 12 planktonic desmid species in relation to temperature and irradiance. They found significantly lower maximum growth rates in species characteristic of oligotrophic lakes as compared to those that peak in eutrophic ones. This potential disad-

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