The ecology of sporulation by the macroalga Ulva lactuca L. (chlorophyceae)

Aquatic Botany, 32 (1988) 155-166 Elsevier Science Publishers B.V., Amsterdam-- Printed in The Netherlands

155

THE E C O L O G Y OF S P O R U L A T I O N BY T H E M A C R O A L G A ULVA L A C T U C A L. ( C H L O R O P H Y C E A E )

RICHARD A. NIESENBAUM* Department of Marine Sciences, University of Connecticut, Avery Point, Groton, CT 06340 (U.S.A.) (Accepted for publication 10 May 1988)

ABSTRACT

Niesenbaum, R.A., 1988. The ecologyof sporulation by the macroalga Ulva lactuca L. (Chlorophyceae). Aquat. Bot., 32: 155-166. A fieldpopulation of Ulva lactuca L. in Mumford Cove in Groton, CT, U.S.A., was monitored from May through October 1985. During warmermonths, a significantportion of the macroalga's biomass is allocated to the formation of zoospores and gametes. Laboratory experiments show that U. lactuca could significantlyincrease the chlorophylla concentration of the water column through the releaseof zoosporesand gametes.These findingssuggestthat the traditional paradigm that marine macrophytescontribute mostly to detritus-based foodchainsshould be extended to include the macroalgalcontribution of zoospores and gametesto the phytoplankton.

INTRODUCTION Estuarine productivity is thought to be dominated by either phytoplankton or macrophytes, depending upon environmental parameters such as depth and nutrient concentrations. Where p h y t o p l a n k t o n predominates, most of the productivity is either passed along to grazing zooplankton, which in t u r n are consumed by organisms at higher trophic levels (Odum, 1980), or is taken up by benthic filter-feeders in coastalwaters (Cloern, 1984). In contrast, macroalgae are thought to contribute mostly to detritus-based food chains with the organic detritus serving as the primary link between autotrophic and heterotrophic parts of macroalgae-dominated ecosystems (Mann, 1972, 1973; Odum, 1980). Although grazers are associated with macroalgae, their food requirements normally a m o u n t to less t h a n 10% of macroalgal productivity (Mann, 1972). Macroalgal productivity may make a direct contribution to the water column through the release of zoospores and gametes (collectively called swarmers). *Present address:BiologyDepartment, Universityof Pennsylvania,Philadelphia,PA 19104-6018, U.S.A.

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© 1988 Elsevier Science Publishers B.V.

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This mechanism has not been widely considered by ecologists, even though certain biological features of macroalgal swarmers suggest they have a great ecological potential as a food source for filter-feeders. Swarmers are similar in size and form to many species of phytoplankton. They lack the heavier cell wall of the parent plant, possessing only a thin plasmalemma (Chapman, 1979). If a large amount of macroalgal productivity is allocated to the formation of swarmers, then the trophic role of the macroalgae should be extended to include their contribution of swarmers to planktonic foodchains. Littler and Littler (1980) have postulated that opportunistic species of marine algae produce great numbers of swarmers, and an abundance and diversity of seaweed spores have been measured in coastal waters (Amsler and Searles, 1980) and in the intertidal zone (Hruby and Norton, 1979; Hoffmann and Ugarte, 1985). The objective of this study was to measure the amount of reproductive tissue in the green macroalga Ulva lactuca L. (Chlorophyceae) over one season, and to estimate its potential contribution to the phytoplankton in the surrounding water column. Ulva lactuca possesses several characteristics that could make it a significant contributor to planktonic food chains. It is ubiquitous in temperate regions, and inhabits both intertidal and subtidal zones (Taylor, 1957 ). It survives a wide range of salinities (2-35%o), and lives on a variety of substrata. The Ulotrichales in the Chlorophyceae of which Ulva is a member, form zoospores and gametes directly by transformation of vegetative cells to sporangia or gametangia (Lerstein and Voth, 1960). Thus, reproduction is not restricted to limited areas of the thallus as in the laminarians. Littler and Littler (1980) classify Ulva as an opportunistic species which suggests that it could have a large output of swarmers over extended reproductive seasons. Smith (1947) has shown that Ulva species have long reproductive seasons, and that sporulation may be related to periodic increases of exposure to light and desiccation associated with spring and neap tides. MATERIALS AND METHODS

Standing crop measurements The amount of reproductive tissue occurring in the field over one season was estimated in Mumford Cove, a shallow eutrophic estuary in Groton, CT, U.S.A., from May through October 1985. Ulva lactuca was collected at 4 permanent stations along 2 non-parallel, subtidal transects. Stations were not evenly spaced, but were located along each transect at the point where the depth corresponded to 1.0, 1.1, 1.2 and 1.3 m below mean low water. At each station, all of the algae within a 0.5-m 2 quadrat were collected. On each successive sampling date, every 7-10 days, the quadrat was rotated 90 ° about each station on the transects, to minimize the impact of one sampling date on another.

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Before drying, each sample was examined under a compound microscope (200 × ) for the presence of swarmers. Tissue composed of swarmer-containing cells were severed from vegetative tissue using a razor blade. Reproductive and vegetative components of each quadrat were washed with fresh water, dried at 60°C for 12 h, and weighed. Standing crop was reported on a dry weight per m e basis. Total biomass was computed as the sum of the vegetative and reproductive components. Laboratory experiments to determine swarmer output Two laboratory experiments were conducted to determine the rate of swarmer production by parent thalli, the first in July when reproductive thalli could be obtained from the field, and the second in winter, when reproductive thalli could be obtained only through laboratory induction. The number of swarmers was estimated as the concentration of chlorophyll a because it was not possible to count them accurately. For the first experiment U. lactuca was collected from the study site on 7 July 1985. The thalli were washed with filtered seawater, patted dry and placed into experimental containers. Each container received 30-g wet weight of either reproductive or vegetative portions of thalli and 2 1 of filtered seawater. Vegetative and reproductive treatments were each replicated 3 times. Three containers, each with 2 1 of filtered seawater but no algae, served as controls. The 9 containers were placed randomly on a shelf of a controlled environmental chamber, where they were lighted from above with a 15/9 h light/dark cycle and exposed to a day/night temperature regime of 23/18°C. At 12, 36, 60, 84 and 156 h all of the water in each container was poured through a 100-~m mesh which retained the thallus, and filtered through Millipore AA filters to retain the swarmers. The thalli were returned to their respective containers, and 2 1 of fresh, filtered seawater were added to each. Chlorophyll a was extracted from the Millipore AA filters using 90% acetone as described in Parsons et al. (1984), and measured using a Turner fluorometer, model 110, with a F4T4-BL lamp, a Corning CS.5-60 filter for excitation light, and a Corning CS.2-64 filter for emitted light. The fluorometer was calibrated using a known concentration of chlorophyll a as determined by spectrophotometry, and all measured concentrations of chlorophyll a were corrected for the presence of phaeopigments (Parsons et al., 1984). To convert the chlorophyll a measurements from mg of chlorophyll a per gram wet weight of Ulva to a dry-weight basis, a wet weight to dry weight conversion factor for U. lactuca was needed. This factor was determined by weighing 20 samples of algae (ranging in wet weight from 5 to 50 g) after excess water was removed by blotting, and again after drying them at 60°C for 12 h. For the second experiment, the algae were collected on 15 January 1986 from a deeper portion of the cove, the only area where plants were still present. None

158 were reproductive. The algae were placed in trays with filtered seawater, and for 24 h maintained at light and temperature regimes similar to those in which the algae were collected, approximately 2 °C and 9 h of light per day. After 24 h, temperature was then increased 2 °C day-1 to 22 ° C. The cultures were held at 3500 lux, at a pH between 8.0 and 8.5, and at nutrient concentrations of 0.6 ttmol m1-1 NaS03, 250 #mol m1-1 PO4, 15 #mol m l - ' Fe-EDTA and 2.5/tmol ml-1 TRIS buffer. These conditions are considered optimal for reproduction (Nordby, 1977 ). The medium was changed 3 times a week, and the cells of the algae were inspected daily, to determine if they had become reproductive. When no reproduction had occurred after 30 days, reproduction was stimulated by temperature shocking the plants with 2 ° C seawater. Plants were removed from their containers which were at 22 °C and washed thoroughly in filtered seawater at 2 ° C, and returned to clean culture chambers with fresh medium at 22°C. In this experiment two reproductive treatments, and one control were used. The design was restricted to this because 18 h after the 2 ° C wash, all of the algae had become reproductive so no vegetative algae were available. The amount of U. lactuca in each container was 12.5 g wet weight per container. Water in the containers was not periodically replaced as in the first experiment, allowing chlorophyll a to accumulate over the entire experiment. Non-destructive sampling of chlorophyll a was accomplished by the in vivo fluorometric method (Lorenzen, 1966). Measurements were made every 15 min over a 13-h period with 10-ml aliquots of seawater from each treatment. After measurement, the aliquot was returned to its respective container. RESULTS Standing-crop measurements The pattern of abundance of Ulva biomass (Fig. 1 ) was distinctly seasonal. Plants first appeared at the end of May. From the end of May through July mean biomass remained at 250 g dry weight (DW) m -2. In late July, there was a sudden increase in the total biomass, to as high as 1000 g DW m -2 in some quadrats. This increase was followed by a sharp decline which was coincident with high August temperatures as measured by Curtis ( 1986, Fig. 4). After the August decline, the standing crop recovered, but remained less than in June and July. There was no biomass at this site by mid-October. The coefficient of variation between samples on each date ranged from 28 to 91%, and was greatest on the date of the late July increase. Differences in total biomass were highly significant among sampling dates, and total biomass did not vary between transects or among subtidal depths on individual sampling dates (Table 1). Reproductive tissue was first evident in early June, and increased through

159 1000

"

Total BIomass

800'

600' E a

400'

o~

200"

0

,

200

100

May

June

July

300

Aug

Sept

Oct

1985

Fig. 1. Total biomass estimates as grams dry weight (DW) by date, 1985. Points are the geometric means. Bars indicate standard deviation based on the In (x + 1 ) transformation. TABLE1 Standing-crop variation of analysis of covariance by date, transect and depth Response variables

Source

df

SS (Type 1)

Total biomass

Date Transect Depth Error Date Transect Depth Error Date Transect Depth Error

12 1 1 86 12 1 1 86 12 1 1 86

142.98 1.61 0.10 114.98 199.23 0.06 3.25 131.01 6.50 0.08 0.24 5.28

Reproductive biomass

Percent reproductive

F

Significance

8.91 1.20 0.11

0.005 ns ns

10.86 0.04 2.12

0.005 ns ns

8.82 1.29 3.90

0.005 ns ns

Analysis of covariance comparing the response variables: total biomass, reproductive biomass and percent of total biomass that was reproductive over the sampling dates and between transects. Depth of sampling station was treated as the covariate. Total biomass and reproductive biomass were log transformed, and percent reproductive was arcsine transformed. the e n d of July. T h e a m o u n t o f r e p r o d u c t i v e tissue declined a b r u p t l y in August, b u t i n c r e a s e d a g a i n in t h e middle of S e p t e m b e r (Fig. 2). O n each s a m p l i n g date t h e q u a n t i t y of r e p r o d u c t i v e b i o m a s s was m u c h m o r e variable a m o n g samples t h a n t o t a l biomass; t h e coefficient o f v a r i a t i o n r a n g e d f r o m 48 to 128%. R e p r o d u c t i v e tissue e x p r e s s e d as t h e p e r c e n t o f t o t a l b i o m a s s was variable w i t h i n s a m p l i n g dates b u t t h e m e a n s were fairly c o n s t a n t f r o m J u n e t h r o u g h to t h e e n d o f S e p t e m b e r , w i t h s h a r p increases in m i d - A u g u s t a n d m i d - S e p t e m -

160

i001

Reproductive Biornass

15°1 a

,J,

100

0 100 May

200 June

J~Ly

300 Aug

Sept

Oct

198S

Fig. 2. Reproductivebiomass estimates as grams dry weight (DW) by date, 1985. Points are the geometric means. Bars indicate the standard deviationbased on the in (x + 1) transformation. 100Percent Reproductive Biornoss

6o. ~ . 80-

o m

.~_o go o.

40.

20"

0 100 May

200 June

July

300 Aug

Sept

Oct 1985

Fog. 3. Percentreproductivebiomassestimatesby date, 1985.Points and standarddeviation (bars) based on the arcsine transformation. ber (Fig. 3 ). The quantities of reproductive biomass and percent reproductive tissue did not vary between transects or among subtidal depths (Table 1 ).

Swarmer release experiments Swarmer release by reproductive algae taken from the field in July 1985 was estimated in the laboratory as chlorophyll a accumulated within a test interval. Mean chlorophyll a accumulation in the 3 containers with reproductive algae between 0 and 12 h was 4.94/~g per 30 g wet weight Ulva, nearly three times t h a t in the vegetative and control containers, 1.08 and 1.76/lg, respectively. Chlorophyll a accumulation in reproductive containers was significantly different from t h a t in the vegetative and control containers during the first test interval, but the vegetative containers did not differ from the controls (Table

161

2). Differences after 12 h were not significant (Table 2), though increased concentrations of chlorophyll a between 60 and 84 h in one of the containers is evidence that a second release may have occurred. The wet weight to dry weight ratio was determined to be 7.44 (SD=0.7, n = 20). The maximum input corrected for the controls and converted to a dryweight basis was 0.31/~g chlorophyll a g-1 DW of reproductive thallus. In the second experiment, no significant difference existed between the two reproductive containers, and both were significantly greater than the seawater control (Table 3). Swarmer release measured as chlorophyll a in the reproTABLE2 Results of analysis of variance from the first swarmer-release experiment Time interval Source

df SS

F

Significance Mean

(h) Control Vegetative Reproductive 0-12 12-36 36-60 60-84 84-156

Treatment Error Treatment Error Treatment Error Treatment Error Treatment Error

2 6 2 6 2 6 2 6 2 6

6.39 0.89 0.39 0.07 0.06 0.55 0.43 0.52 0.06 0.09

1.76a

21.49 0.005

1.08a

4.94b

0.33 ns 0.33 ns 2.51 ns 0.33 ns

One-way analysis of variance comparing swarmer input as mg of chlorophyll a l - 1 of seawater in the control, vegetative and reproductive treatments (containers) for each time interval. The Tukey Studentized Range Comparison of means was performed for the significant analysis of variance. Means (in the units of mg l - 1) with the same letter are not significantly different. TABLE3 Results of analysis of variance from the second swarmer-release experiment Source

Treatment Error

df

2 57

SS

1230 1382

F

25.4

Significance

0.005

Mean Control

Reproductive No. 1

Reproductive No. 2

1.2 a

20.6 b

20.2 b

One-way analysis of variance comparing input of chlorophyll a (mg 1-1 ) over 13 h for the seawater control and two reproductive treatments. Tukey's Studentized Range Comparison of means was performed to determine differences between means, means with the same letter are not significantly different. Means are over the 13 h and have the units mg l - 1.

162 30"

~

20-

3

~ 10" i--

o

i 200

100 May

June

July

i 300 Aug

Sept

Oct

1985

Fig. 4. Temperature in Mumford Cove 1985, from Curtis (1986).

40

O•

30

o 25

0 Q.

20

o _o tO

15

0o o

10

• oo GO

•

°o• o

•

• 0

~

0 0 I

D E I [] 2

0

0 3

O0

o~ o

o 4

$

6

0

7

8

9

IQ

11

12

13

Time (hours)

Fig. 5. Swarmer input as chlorophyll a (•g) versus time the two reproductive t r e a t m e n t s (solid and open circles), a n d seawater control (boxes).

163

ductive containers was highly variable during the first 2 h (Fig. 5 ). Between 2 and 11 h it increased linearly from 6 to 38 ttg. After 11 h, chlorophyll a decreased to about 32 ttg. Chlorophyll a measurements in the seawater control were fairly constant between 0.0 and 1.5 pg l-1 throughout the experiment. The mean maximum chlorophyll a accumulation for the two reproductive treatments was 37.6 ttg. That maximum, corrected for controls and on a dryweight basis, amounted to 21.7 pg g- 1 DW Ulva. This value is approximately 10 times the input observed between 0 and 12 h by reproductive Ulva taken from the field in July. DISCUSSION

This study has shown that the magnitude of formation and release of swarmers by the macroalga U. lactuca has potentially great ecological significance at this location. The process of sporulation is important because it can limit the biomass of the macroalga and can potentially supplement the standing crop of phytoplankton in the water column. The swarmer-release experiments were essential in both quantifying and understanding the formation and release of swarmers. In two experiments, reproductive algae significantly increased the chlorophyll a concentration of the surrounding water. Releases were massive and occurred within 12 h after the visibly reproductive cells were isolated and placed in fresh seawater. In the July experiment, swarmers accumulated over a 12-h period, and an apparent generation of new reproductive cells occurred after approximately 3 days. In the February experiment, the conversion of vegetative tissue to a reproductive state was almost immediate after the algae were washed in water at 2 °C. Swarmer accumulation within the 12 h was rapid and linear. The decrease in chlorophyll a after 12 h may have been due to the settlement of swarmers. In order to assess the contribution of Ulva swarmers to the plankton directly, it would be best to sample the plankton and measure the proportion that is swarmers. Attempts to examine water samples taken at the beginning of daytime flood tide for each date that the transects were sampled were unsuccessful because it was impossible to differentiate the swarmers from the other plankton in a quantitative manner. The method of preservation, 10 drops of Lugol's solution per 200 ml of sample, seemed to cause the destruction of the swarmers. Refinement of this approach will be attempted in future studies. The standing-crop data also indicate that sporulation is an important ecological process. They show that there can be a substantial amount of reproductive tissue in the field at certain times. Also, when the allocation of thallus biomass to the formation and release of swarmers is high, thallus biomass declines. That allocation can be a significant proportion of total U. lactuca production. Thus when estimating production in Ulva and other species of macroalgae, swarmer output is an important consideration.

164

An evaluation of standing crop data from previous studies of various species of Ulva shows that formation of reproductive biomass occurs seasonally (Smith, 1947) and with a semi-lunar periodicity (Smith, 1947; Subbaramaiah, 1970; Rhyne, 1973 ). This study has shown seasonal differences in reproduction, but the field data suggest that reproduction is not limited to two monthly events. The seasonality of both growth and reproduction in U. lactuca could be attributed to temperature, light and the shallowness of this study site. The algae may disappear in shallow portions of the cove between October and May because these areas are more susceptible to the effects of increased winds, increased currents, decreased temperatures and ice formation associated with autumn and winter months. Ulva lactuca is present year round in deeper portions of the cove. The seasonality of reproduction, and changes in the abundance of total biomass and reproductive biomass during the reproductive season could have been a function of temperature. The sharp decline of total biomass in early August, and its low rate of recovery through August and September were probably due to temperature effects on growth and reproduction. The optimal temperature range for growth in U. lactuca is 15-19°C; growth decreases at temperatures above this range, and is completely inhibited as temperatures rise above 25°C (Steffensen, 1976; Fortes and Liining, 1980). The optimal temperature for reproduction in species of Ulva is 21°C (Nordby and Hoxmark, 1972; Nordby, 1977). When temperatures reached seasonal highs, allocation of biomass to the formation and release of swarmers was greatest, while the rate of vegetative replacement diminished as temperatures first became suboptimal and then inhibitory for growth. This could explain the increases in percent reproductive tissue during August and September. Absence of the semi-lunar periodicity of reproduction in this study may be attributable to characteristics of this particular site, such as a relatively small tidal range (mean--.76 meters), and the growth form of Ulva at this site. Studies demonstrating reproduction coincident with spring tides were made on plants that had experienced tidal rhythms. Ulva sampled in this study grew as large, unattached thalli suspended in subtidal waters. Rhyne and Hommersand (1970) reported that the semi-lunar pattern of reproduction in Ulva currata (Kfitz.) De Toni becomes dampened in the laboratory in the absence of tidal conditions. Perhaps unattached thalli in the field, which are buoyant and rise and fall with the tide, have undergone a similar dampening process. The lack of covariance between that station depth and biomass of reproductive Ulva (Table 1 ) suggests that levels of illumination and desiccation are not important factors in the stimulation of reproduction of unattached Ulva in subtidal areas. Sampling thalli along a larger depth gradient would be needed to judge whether depth affects growth and reproduction.

165 ACKNOWLEDGMENTS I e x p r e s s m y sincere a p p r e c i a t i o n a n d g r a t i t u d e to B a r b a r a W e l s h for h e r v a l u a b l e g u i d a n c e a n d a s s i s t a n c e t h r o u g h o u t t h e study. I also t h a n k P . H . Rich, F.R. T r a i n o r , C. Yarisch, M. Curtis, J. K i m a n d B. W a l s h for i n t e l l e c t u a l s t i m u l a t i o n a n d c o n s t r u c t i v e criticism as this r e s e a r c h was b e i n g conducted. I t h a n k B. C a s p e r , P. P e t r a i t i s a n d t w o a n o n y m o u s r e v i e w e r s for t h e i r c o m m e n t s on t h i s m a n u s c r i p t . B o a t s , e q u i p m e n t a n d a d d i t i o n a l s u p p o r t were p r o v i d e d b y P r o j e c t O c e a n o l o g y , G r o t o n , C T . T h i s r e s e a r c h was a i d e d b y a g r a n t - i n - a i d of r e s e a r c h f r o m S i g m a Xi, T h e Scientific R e s e a r c h Society.

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166 Rhyne, C., 1973. Field and experimental studies of the systematics and ecology of Ulva curvata and Ulva rotunda. University of North Carolina, Sea Grant Publication, 125 pp. Rhyne, C., and Hommersand, M. 1970. Laboratory studies concerning growth and reproduction in the seaweed, Ulva curvata. In: Studies of Marine Estuarine Ecosystems Developing with Treated Sewage Wastes. Ann. Rep. 1969-1970, Institute of Marine Sciences, University of North Carolina,pp. 30-55. Smith, G., 1947. On the reproduction of some Pacific coast species of Ulva. Am. J. Bot., 34: 8087. Steffensen, D., 1976. The effect of nutrient enrichment and temperature on the growth in culture of Ulva lactuca L. Aquat. Bot., 2: 337-351. Subbaramaiah, K., 1970. Growth and reproduction of Ulva fasciata in nature and culture. Bot. Mar., 13: 25-27. Taylor, R., 1957. Marine Algae of the Northeast Coast of North America, University of Michigan, MI, 520 pp.