The relative importance of amphipod and gastropod grazers in Posidonia sinuosa meadows

A_qU~Llc oo~ny ELSEVIER

Aquatic Botany 56 (1997) 183-202

The relative importance of amphipod and gastropod grazers in Posidonia sinuosa meadows Peter Jernakoff *, John Nielsen 1 CSIRO Division of Fisheries, P.O. Box 20, North Beach, W.A. 6020, Australia

Accepted 12 November 1996

Abstract

The relative importance of amphipod and gastropod grazers in controlling periphyton and epiphytes of the seagrass Posidonia sinuosa Cambridge and Kuo, was assessed using exclosure chambers in the field with an amphipod assemblage and the herbivorous gastropod, Thalotia conica Gray. Periphyton were considered to be bacteria, diatoms and algal propagules whereas epiphytes were defined as larger algal forms visible to the naked eye. Subtle grazing effects were detected after 14, 21 28 and 35 days despite chamber artefacts. Seagrass mortality was three times lower in the "no-chamber" controls than inside the chambers. It was 24% lower in the gastropod-inclusion chambers, suggesting that gastropod grazing enhanced seagrass leaf survival. Grazing effects on periphyton were varied. Amphipods had no significant effect on periphyton biomass or chlorophyll a, but they reduced the ratio of dry weight to ash weight by 54% and the taxonomic richness by 12%, suggesting an active selection of taxa during grazing. Gastropods reduced periphyton chlorophyll a levels by 51% after 35 days although they had no clear effects on periphyton biomass. Gastropods also reduced the ratio of chlorophyll a to ash-free dry weight by 99% which indicated a change in the periphyton community composition. Grazing impacts on epiphytes also differed with the different grazers. Gastropods reduced epiphyte biomass by 44% but had no effect on the number of taxa, while amphipods increased the number of taxa by 29% but had no impact on epiphyte biomass. There was no evidence of larger grazing impacts on periphyton or epiphytes when both grazers were present. Gastropods are more efficient but less selective grazers than amphipods. However, because amphipods are highly mobile with rapid production rates their impact on epiphytes may be important, particularly in affecting species composition rather than biomass. However, spatial and

* Corresponding author. CSIRO Division of Marine Research, P.O. Box 20, North Beach, W.A. 6020, Australia. Tel.: 0011 61 9 4228288; Fax: 0011 61 9 4228222; E-mail: [email protected] 1Present address: Kinhill Engineers,47 Burswood Road, VIC. PK, WA 6100, Australia. 0304-3770/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved. PII S0304-3770(96)01112-6

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temporal patchiness in abundance, which was not examined in the present study may also play a role in determining the relative effectiveness of these two types of grazers. Keywords: Grazing;Periphyton;Epiphytes; Posidonia

1. Introduction

Grazers are fundamental in maintaining healthy seagrass meadows (Klumpp et al., 1992; Neckles et al., 1993). Grazing enhances seagrass growth and survival by regulating the abundance and growth of epiphytic algae that compete with the seagrasses for light (Orth and van Montfrans, 1984; van Montfrans et al., 1984). Grazing can also modify periphyton and epiphyte composition (Scott and Russ, 1987; Mazzella and Russo, 1989) and provide a link in the food chain between these primary producers and higher trophic levels (Orth, 1992). There are many grazers of seagrass epiphytes, with a significant portion being epifaunal invertebrates such as amphipods, isopods and gastropods (see van Montfrans et al., 1984; Klumpp et al., 1989; Lanyon et al., 1989; Orth, 1992). The majority of studies have focused on these types of grazers because these are the most common and they have a high productivity. However, despite numerous studies on invertebrate grazers, we know little about the relative importance of amphipods compared with gastropods in controlling epiphytic growth in the field. Both taxa occur within the seagrass community and probably use similar food resources. While laboratory studies may provide some indication of the relative effects of these grazers, they do not expose the animals to the full range of environmental conditions which may affect their production, biomass, and grazing rates. Thus, laboratory studies may be of limited use for finding their relative importance in controlling periphyton and epiphyte communities in the field. The aim of the present paper was to determine the relative grazing impact of amphipod and gastropod herbivores on the production and composition of the periphyton and epiphyte community of Posidonia sinuosa Cambridge and Kuo seagrass meadows. We carried out this study in situ, enclosing seagrass plants in acrylic and mesh chambers to ensure that grazers and epiphytes were exposed to as many natural environmental factors as possible (e.g. natural light, temperature and nutrients).

2. Methods 2.1. Study site and seagrass

The study site was in the Marmion Lagoon off Perth, Western Australia (31°14'S, 115°42'E), in a 4 m deep Posidonia sinuosa meadow. The seagrass, P. sinuosa, has strap-like leaves 4-11 mm wide which live for between 84 and 168 days (D. Walker, personal communication, 1994). One to two leaves grow in a shoot covered by a sheath and separate just above the substratum. Leaf density at the study site was approximately 1177 _ 57 (SE) per square metre.

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185

2.2. Exclosure chambers Grazers were excluded using acrylic chambers. The chambers consisted of a clear acrylic top which snapped into a clear acrylic cylinder. The cylinder was 42 cm long with an internal diameter of 14cm. It had nine 5 0 m m diameter holes covered by 400 p,m stainless steel mesh that allowed water flow but restricted the entry of grazers. This size of mesh was used because in pilot studies (P. Jernakoff and J. Nielsen, unpublished data, 1994) a smaller mesh (142 Ixm) significantly restricted the flow of water through the chambers. There was a large slot from the centre to the edge of the clear acrylic base through which ten seagrass leaves from attached plants could be inserted. A sliding gate filled the slot and foam at the inner edge of the gate sealed the leaves from the outside. The base had two large 400 p~m mesh panels that allowed water to flow and fine particulate matter that entered through the mesh panels in the wall of the cylinder to fall out. Although the base and top fitted snugly into the chambers, they were also secured by elastic bands attached to lugs on the sides of the cylinder. Each chamber housed ten intact P. sinuosa leaves. The chambers were attached to randomly placed star pickets within the meadow.

2.3. Experimental design Treatments consisted the following chamber treatments: " n o grazers"; "amphipods only"; "gastropods only"; and "both types of grazers". Additional no-chamber control treatments consisted of groups of ten P. sinuosa leaves which had also been standardised for periphyton, epiphytes and grazers at the start of the experiment. Before the start of the study, divers removed all visible periphyton and epiphytes from the seagrass leaves to standardise the amount of epiphytic material across all treatments. The seagrasses were then left for three days to allow amphipod grazers to recolonise; any gastropods were removed from the seagrass leaves and the chambers were installed. The most numerous amphipods found in the seagrass meadows and chambers were species of Tethygeneia, Ampithoe, and Hyale. The amphipod exclusion chambers were gassed with CO 2 which kills adult and juvenile amphipods, although it appears to have no effect on amphipod eggs (P. Jernakoff and J. Nielsen, unpublished data). All other chambers were gassed with air to act as a control for any physical disturbance from the bubbling gas. A single adult gastropod (Thalotia conica Gray) was added to each "gastropod" chamber to mimic approximately the natural density of large gastropods within the seagrass meadow. Thalotia was chosen as it is a typical gastropod within these meadows and its diet and grazing is similar to that of other gastropods within local seagrass meadows (Nielsen and Lethbridge, 1989). Chambers were cleaned every two days to remove periphyton growing on the outer surface of the chambers and their mesh panels thus maximising the flow of water through the mesh panels. Chambers were gassed with either CO 2 or air, depending upon treatment, every seven days to ensure that any amphipods hatching from eggs were killed. Four replicates of each of the five treatments were sampled at 14, 21, 28 and 35 days after deployment of the chambers. Thus, there were a total of 64 chambers deployed at the start of the experiment in addition to the 16 control sites that had also

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been cleared of periphyton, epiphytes and grazers. Four replicates of other " n o chamber" controls were also sampled four days before deploying the chambers (i.e. just before cleaning of seagrass leaves) and then also immediately before chamber deployment to estimate initial periphyton, epiphyte and fauna abundance and verify standardisation techniques respectively. 2.4. Sample processing

Data collected during each sampling period included the biomass (dry weight (DW) and ash weight (AW)), chlorophyll a and species composition of periphyton; the biomass and species composition of epiphytes; and the surface area, biomass and ratio of dead to live sections of P. sinuosa leaves. The contents of each chamber were sieved through a 500 ~m sieve immediately after harvesting to separate them from flora. The gassing was found to significantly reduce the density of amphipods in amphipod exclosure treatments (mean = 14.7, SE = 2.7) compared with enclosure treatments (mean = 38.9, SE = 4.2) (ANOVA: d f = 1,61, P < 0.001). No large gastropods were found in gastropod exclusion treatments although very small molluscs (shell lengths less than 2 mm) were found in all treatments. Reliable density estimates of these small molluscs were not possible because their small shell width allowed an unknown number to pass through the 500 Ixm sieve. Floral samples were placed in plastic bags and stored in an ice-chilled cooler. At the laboratory, the seagrass leaves from each chamber were placed in a shallow tray filled with chilled seawater and divided into two subsamples, each consisting of five leaves. One subsample was used for periphyton chlorophyll a determinations and the other for ash-free dry weight (AFDW) biomass estimates. All sorting and separation was done in a darkened room to minimise any changes in photosynthetic pigments that would affect chlorophyll a analyses. The seagrass leaves in each subsample were scraped using a razor blade to remove all attached flora. The scraped material was washed through a 500 p~m sieve to separate larger epiphyte fragments from the microscopic periphyton. Fleshy epiphytes on the sieve were separated by hand from the periphyton, which consisted of the remaining material on the sieve (calcareous encrusting algae) and the smaller periphyton which had passed through the sieve. Both components of the periphyton were combined and then collected by filtering onto a pre-dried and pre-weighed GFC filter-paper. Samples for chlorophyll a analysis were stored at - 2 0 ° C until processed using the trichromatic method (Strickland and Parsons, 1972). The species composition of periphyton and epiphyte communities from the remaining samples was recorded. The periphyton samples were then washed with 250 ml of distilled water to remove any crystallised salt which could bias AFDW measurements. The AFDW biomasses of epiphytes from the two subsamples were combined to provide a single measure of total epiphyte biomass within the chambers and the no-chamber control groups. All periphyton and epiphyte measurements were standardised to a unit weight of seagrass biomass (grams DW) before analyses; these measurements are directly related to seagrass surface area (seagrass surface area (cm 2) = 40.50 + 377.15 X seagrass biomass (grams DW); R 2 = 1.000, n = 87). Estimates of seagrass surface area, biomass and leaf mortality were carried out separately for each subsample but these data were later pooled.

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2.5. Data analyses Data were analysed by three factor analyses of variance (ANOVA) with asymmetrical controls (Table 1). Assumptions of the ANOVA (homogeneity of variances and normality of error terms) were tested respectively by Bartlett's test and by correlating residuals with N-scores of the residuals. Where necessary, data were transformed to satisfy the ANOVA assumptions. If the assumptions were still violated after transforming, the offending data cells were identified and omitted from the analyses, which were then rerun and checked to ensure that ANOVA assumptions were met.

3. Results 3.1. Periphyton 3.1.1. Biomass Unfortunately the data from several samples (14 out of 24, and 1 out of 24 samples from the 21 and 28 day sampling periods, respectively) were lost. There was no significant difference in biomass AFDW between the no-chamber control and chamber treatments, and the only significant effects were between times, and an interaction between time and amphipod grazing. However, no clear trends were apparent from the analyses (Table 1; Fig. 1). 3.1.2. The ratio of dry weight to ash weight This ratio indicates if there has been a change in the relative amount of inorganic material compared to the total (organic and inorganic) material in the periphyton. A change in this ratio could be due, for example, to a relative increase in encrusting coralline algae compared to diatoms. The periphyton DW:AW ratio was analysed (log X + 0.005 transform) after excluding the 21 day sampling period to satisfy the ANOVA assumptions. The analysis indicated significant differences between the no-chamber controls and the chamber treatments; between amphipods and no amphipods; and between the time-amphipods interactions and the time-amphipods-gastropods interactions. Tukey's tests indicated that the DW:AW ratio was significantly lower in the control treatments than in the enclosed treatments (Tukey's Family error rate P = 0.05, individual error rate P = 0.05). Tukey's analysis of the three-way interaction was difficult to interpret and the analysis was therefore carried out on the time-amphipods interaction. While the ratio of DW:AW was not significantly different between amphipod grazer treatments by 14 days, it was significantly lower where amphipods were present by 28 (and also 35) days (Tukey's Family error rate P = 0.05, individual error rate P = 0.001). Overall, the ratio of DW:AW in chambers where amphipods were enclosed was lower by 54% compared to chambers where amphipods were excluded. 3.1.3. Chlorophyll a There were significantly higher amounts of chlorophyll a in no-chamber controls compared with chamber treatments (Table 1; Fig. 2). There was no significant difference

P. Jernakoff, J. Nielsen~Aquatic Botany 56 (1997) 183-202

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in the levels of chlorophyll a b e t w e e n c h a m b e r treatments apart from a significant interaction b e t w e e n a m p h i p o d and gastropod grazers (Table 1). T u k e y ' s multiple range test indicated that, in chambers where gastropods were enclosed, there was 51% less chlorophyll a than in chambers where they were excluded ( F a m i l y error rate P = 0.05, individual error rate P = 0.01).

3.1.4. Relationship between periphyton chlorophyll a values and biomass There was a small but significant correlation b e t w e e n periphyton chlorophyll a and b i o m a s s (correlation coefficient = 0.207; n = 88; P < 0.001).

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P. Jernakoff, J. Nielsen~Aquatic Botany 56 (1997) 183-202

Table 2 List of periphyton taxa found in the study and their presence within chamber treatments pooled over sampling times and replicates Species name

Rhopalodia gibberia Mastogloia sp. A Mastogloia binotata Grammatophora oceanica Actinoptychus splendens Diploneis chersonensis Cocconeis scutellum Paralia sulcata Nitzschia spp. Amphora ventricosa Trachyneis aspera Striatella interrupta Grammatophora serpentina Mastogloia brauni Gomphonema valentinica Stephanodiscus sp. A Entomoneis tenuistriata Pseudonitzschia australis Nitzschia closterium Stepahnopyxis sp. A Amphora bigibba Licmophora ehrenbergii Licmophora paradoxa Amphora australiensis Cocconeis spp. Achnanthes exigua Amphora veneta Rhoicosphenia sp. A Navicula tripunctata Campyloneis grevilleii Navicula sp. A Diatom sp. A Pleurosigma barbadense Nitzschia panduriformis Mastogloia smithii Coscinodiscus sp. A Cocconeis disculus Mastogloia mauritiana Opephora schwartzii Opephora martyi Diploneis suborbicularis Synedra fasciculata Rhaphoneis amphiceros Striatella unipunctata Amphora mexicana

Taxon

Presence of the taxon

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Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom

0 days b

No chamber control

No grazers

Gastropods only

Amphipods only

Both grazers

P. Jernakoff, J. Nielsen/Aquatic Botany 56 (1997) 183-202

191

Table 2 (continued) Species name

Taxon group

Presence of the taxon 4 0 No No Gastro- Amphi- Both days ~ days b chamber grazers pods pods grazers control only only -

Campylodiscus sp. A Surierella fastuosa Achnanthes haukiana Cocconeis sp. A Ausliscus sculptus Cymbella nat,iculiformis Thalassionema nitzschoides Hyalodiscus sp. A Nitzchia incurva Campylodiscus sp. B Plagiogramma appendiculatum Nitzschia hummii Pleurosigma formosum Mastogloia exigua Mastogloia psuedoexigua Diploneis vacillans Diploneis ot,alis Oscillatoria s p p . Lyngbya spp. Cladophora spp. Heterocystic algae Coccoid B-G algae Polykrikos sp. A Ceratium sp. A Fosliella sp. A

Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Diatom Cyanobacteria Cyanobacteria Green algae Cyanobacteria Cyanobacteria Dinoflagellate Dinoflagellate Coralline red algae

Unidentified algal propagules Algal epiphyte fragments a Four days before the chambers were deployed (and prior to standardising floral levels on seagrass leaves). b Immediately before deploying chambers. • indicates the presence of a species/taxon within a treatment. Diatoms identified from John (1983) and Round (1981). Algae identified from Huisman and Walker (1990), Millar (1990) and Price and Scott (1992).

The analysis o f the ratio o f c h l o r o p h y l l a to b i o m a s s (data transform = l o g ( x + 0.01); 21 day sampling time r e m o v e d f r o m the analysis to satisfy A N O V A assumptions) indicated significant differences b e t w e e n controls and e n c l o s e d treatments (Table 1): the ratio for c h a m b e r treatments was significantly higher ( T u k e y ' s F a m i l y error rate P = 0.05, individual error rate P = 0.05). In addition, there was a significant effect o w i n g to t i m e and to gastropods. The ratio o f c h l o r o p h y l l a to b i o m a s s was significantly greater after 35 days c o m p a r e d with 14 days ( T u k e y ' s F a m i l y error rate P = 0.05, individual error rate P = 0.0196). This ratio was significantly less in treatments w h e r e

P. Jernakoff, J. Nielsen~Aquatic Botany 56 (1997) 183-202

192 44

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gastropods were present than where they were absent (0.001 compared to 0.097), irrespective of the presence or absence of amphipods (Tukey's Family error rate P = 0.05, individual error rate P = 0.05).

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Table 3 ANOVA summary for epiphytes and seagrass Source of variation

Degrees of freedom

Between controls Controls versus rest T Amphipods Gastropods T×Amphipods TXGastropods Amphipods×Gastropods T × Amphipods × Gastropods Residual Total

5 1 3 1 1 3 3 1 3 65 86

Epiphyte biomass

Epiphyte taxonomic Seagrass leaf richness mortality

F

P

F

P

F

P

1.1812 69.4610 24.4161 1.4507 60.7307 7.1089 3.9248 1.2581 3.8755

NS .... .... NS .... ***

9.6382 120.2137 0.5099 19.7458 0.1168 0.0415 0.5901 0.0183 0.7761

.... .... NS .... NS NS NS NS NS

0.3374 119.3903 12.1865 0.0329 16.5184 2.9104 2.1176 0.0016 0.6313

NS .... .... NS ....

NS

NS NS NS

One degree of freedom from the residual and total has been removed because data for one replicate was lost during the collection of samples in the field. F is the F ratio; P is the probability value of the F ratio; T is time. N S i s P > 0 . 0 5 , * P < 0 . 0 5 , ** P < 0 . 0 1 , * * * P<0.005,**** P<0.001.

3.1.5. Number of taxa Of the 70 taxa found, 62 were diatoms, four were cyanobacteria, two were dinoflagellates, one was a green alga and one was a coralline red algal germling (Table 2). Analysis of the taxonomic richness (number of taxa per replicate) indicated a significantly lower richness of taxa in chambers where amphipods were present (29 + 1) compared to in those where they were absent (33 + 1). Although there was a significant difference between sampling times (ANOVA: Table 1), Tukey's multiple range test was

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Table 4 List of epiphyte taxa found in the study and their presence within chamber treatments pooled over sampling times and replicates Species name

Order

Presence of the taxon -4 0 No No Gastro- Amphi- Both days a days b chamber grazers pods pods grazers control only only

Giraudia sp. A Champia cf. viridis Colpomenia spp. Ceramium cf. codii Coeloclonium umbellulum Sphacelaria rigidula Craspedocarpus cf. tenuifolius Griffithsia ovalis Metagoniolithon stelliferum Laurencia filiformis Champia zostericola Hypnea spp. Heterosiphonia spp. Dasya spp. Anotrichium sp. A Polysiphonia spp. Laurencia forsteri Cladophora spp. Liagora sp. A Enteromorpha spp. Antithamnion armatum Asparagopsis armata Ceramium sp. A Semnocarpa minuta Botryocladia sp. A Giffordia irregularis Cryptomenia sp. A Chrysymenia ornata Haliptilon roseum Lenormandia spectabilis Herposiphonia sp. A Herposiphonia sp.B Hymenema sp. A Acrosorium spp. Grateloupia sp. A Lenormandia sp. A Microcoleus spp. Protokeutzingia australasica Dictyota radicans Platysiphonia miniata Ceramium shepherdii

Dictyosiphonales Rhodymeniales Scytosiphonales Ceramiales Ceramiales Sphacelariales Gigartinales Ceramiales Corallinales Ceramiales Rhodymeniales Gigartinales Ceramiales Ceramiales Ceramiales Ceramiales Ceramiales Cladophorales Nemaliales Ulvales Ceramiales Bonnemaisoniales Ceramiales Rhodymeniales Rhodymeniales Ectocarpales Gigartinales Rhodymeniales Corallinales Rhodymeniales Ceramiales Ceramiales Ceramiales Ceramiales Gigartinales Ceramiales Cyanobacteria Ceramiales Dictyotales Ceramiales Ceramiales

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Table 4 (continued) Species name

Order

Presence of the taxon 4 0 No No Gastro- Amphi- Both days a days b chamber grazers pods pods grazers control only only -

Oscillatoria spp. Antithamnion verticale Laurencia c r u c i a t a Antithamnion sp. A Lyngbya spp. Polycerea sp. A Chondria succulenta Helminthora sp. A Ceramium pusillum Chaetomorpha sp. A Spyridia sp. A Ceramium sp. C Centroceras sp. A Ectocarpoid A Chondria sp.B Ceramium australe Platysiphonia intermedia Wrangelia australis Ulva sp. A Antithamnion sp.B Ceramium monocanthum Lophothalia L,erticillata Brogniartella australis Lenormandia marginata

Cyanobacteria Corallinales Ceramiales Ceramiales Cyanobacteria Chordariales Ceramiales Nemaliales Ceramiales Cladophorales Ceramiales Ceramiales Ceramiales Ectocarpales Ceramiales Ceramiales Ceramiales Ceramiales Ulvales Ceramiales Ceramiales Ceramiales Ceramiales Ceramiales

Four days before the chambers were deployed (and prior to standardising floral levels on seagrass leaves). b Immediately before deploying chambers. • indicates the presence of a species/taxon within a treatment. Taxonomic sources: Christianson et al. (1981), Huisman and Walker (1990), Millar (1990), Borowitzka and Lethbridge (unpublished data, 1989).

unable to distinguish any clear differences ( F a m i l y error rate P = 0.05, individual error rate P = 0.0004; Fig. 3). Cluster analysis o f p e r i p h y t o n groups indicated that although there w e r e several levels o f separation (Fig. 4), the levels o f dissimilarity w e r e not great (less than 0.31) and the associations w e r e difficult to interpret. In general, the periphyton species in the n o - c h a m b e r controls w e r e different f r o m those in c h a m b e r treatments and there were differences b e t w e e n levels o f pre- and post-standardisation o f periphyton, but no clear trends w e r e detected b e t w e e n times or treatments within chambers. W h e n the data were p o o l e d b e t w e e n times, the control groups w e r e separate f r o m each other and separate f r o m the e n c l o s e d treatments. T h e e n c l o s e d treatments w e r e highly similar to each other and, within these groups, the " m i n u s g r a z e r s " and " g a s t r o p o d " treatments fused at 0.11 just as the " a m p h i p o d s " and " a l l g r a z e r s " treatments also fused at 0.11.

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3.1.6. General summary of results Neither type of grazer, nor the chambers (compared with the no-chamber controls) had a significant effect on periphyton biomass over the time of the experiments. However, the ratio of DW:AW was lower (relatively less organic material) in control treatments. Within the chamber treatments, by 28 days, the ratio was lower under amphipod grazing. Chlorophyll a levels were higher in control treatments and lower under gastropod grazing. The ratio of chlorophyll a to biomass was lower in controls than in chambers and lower under gastropod grazing. Within the chambers, this ratio increased with time. The taxonomic richness was lower under amphipod grazing and differences in species were detectable between no-chamber controls and the enclosed treatments, and between pre- and post-standardisation of floral levels at the start of the experiment. 3.2. Epiphytes 3.2.1. Biomass The epiphyte biomass in the no-chamber controls did not vary significantly throughout the study, and was significantly lower than in experimental chamber treatments (Table 3 and Fig. 5). In general, the highest biomass was found in chambers where either gastropods or both grazers were absent. Specific grazing effects were complex and confounded by significant interactions between time, amphipod and gastropod treatments (Table 3). Given this complexity, we examined the two-way interaction between amphipods and gastropods separately for each sampling time, but used Tukey's multiple range test probabilities for the complete three-way interaction (Family error rate P = 0.05, individual error rate P = 0.0007). After 14 days, there was no significant difference in epiphyte biomass between any chamber treatment. However, by 21 days, epiphyte biomass was higher in the absence of both grazers. After 28 days, there was significantly more epiphyte biomass in chambers that had amphipods but no gastropods. By 35 days, however, there was significantly more epiphyte biomass in chambers with no gastropods regardless of whether amphipods were present or not. Overall, gastropods had reduced the biomass of epiphytes by 44%. Tukey's multiple range test was unable to detect any significant effect of amphipod grazing on epiphyte biomass by the end of the experiment. 3.2.2. Number of taxa Epiphyte taxa are listed in Table 4. A total of 65 taxa were recorded, but only 54 could be identified to genus or species. The remaining 11 taxa consisted of epiphytes pooled at the genus level and included common algae such as Hypnea, Polysiphonia, Dasya and Heterosiphonia. Of the identifiable epiphytes, there were eight that occurred in at least 50% of all chambers regardless of the presence of grazers. The majority of these epiphytes were either filamentous red algae or cyanobacteria. The number of epiphyte taxa in the no-chamber control treatments remained relatively constant during the first 21 days of the experiment (Fig. 6) but increased significantly by 28-35 days (Table 3, Tukey's multiple range test Family error rate P = 0.05, individual error rate P = 0.005). There were significantly more taxa in the control samples (26.75 ___1.52) than in the

197

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3.2.3. Clustering The clustering of epiphyte taxa, pooled over replicates, showed a similar trend to that of periphyton. There were differences in epiphyte taxa before and after standardising epiphyte cover on the seagrass leaves and, in general, groups within the no-chamber controls were separate from those of the chamber treatments. There was no clear separation between any grazing treatment at separate times. However, when the data were pooled between times a clearer pattern was obtained (Fig. 7). Overall, there was a high level of similarity between all samples as all groups fused at about 0.39. The control groups were relatively dissimilar from each other and from the chamber treatments. Within the chamber treatments the samples without grazers fused with the gastropod treatments at 0.15, whereas the amphipods treatment fused with the all-grazers treatment at 0.12.

3.2.4. General summary of results Overall, the level of epiphyte biomass was less in control treatments than in the chambers. By 21 days there was less biomass under gastropod grazing and under amphipod grazing. The taxonomic richness was greater in control treatments than within chambers and it was greater in amphipod enclosure treatments. There were differences in the species composition between controls and chamber treatments and between preand post-standardisation of epiphyte flora.

3.3. Seagrass The percentage area of dead seagrass leaf was significantly greater within the chambers than for the controls (Table 3; Fig. 8). Although there was a significant

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P. Jernakoff, J. Nielsen/Aquatic Botany 56 (1997) 183-202

199

4. Discussion Amphipod grazers and Thalotia conica had impacts on periphyton and epiphytes, despite some artefacts caused by the presence of the chambers. These artefacts included higher levels of chlorophyll a, lower epiphyte biomass and taxonomic richness, and a higher mortality of seagrass leaves than the no-chamber controls. However, controls for amphipod and gastropod grazing within the chambers permitted the assessment of grazing impacts and the results can be extrapolated to the no-chamber field situations. The impact of grazing will depend on the time of year, the species and the density of grazers and epiphytes (e.g. Jernakoff et al., 1996). In the present study, amphipod grazers consisted of an assemblage with the most numerous species being Tethygeneia, Ampithoe and Hyale. Although these were found in all treatments during the study it is possible that, at different times of the year (and possibly different locations), the relative abundance and composition of the species may vary (Jernakoff et al., 1996) and hence produce results different from those in the present study. Similarly, although the gastropod, Thalotia, was chosen because its grazing and diet was similar to that of other gastropods in local seagrasses (Nielsen and Lethbridge, 1989), grazing impacts by other species may be different. Although we discuss our findings using the generic terms of "amphipods" and "gastropods" it should be recognised that, strictly, these terms refer to the species within the present study. Although periphyton under amphipod grazing showed no significant change in the levels of biomass or chlorophyll a, more subtle effects were apparent. The DW:AW ratio was significantly lower under amphipod grazing, suggesting that there was relatively less organic matter in the periphyton where amphipods were present. This may suggest that amphipods select more palatable food-types, such as diatoms, cyanobacteria, and small turfing algae in contrast to encrusting coralline algae. This suggestion is supported by the findings of Nicotri (1980), Robertson and Lucas (1983), and Agnew and Moore (1986) who report that amphipods prefer diatoms in preference to encrusting algae. Analyses of the taxonomic richness and levels of similarity of periphyton groups between chamber treatments indicated that the periphyton community was different under amphipod grazing from when amphipods were absent. Although this difference was mainly due to the absence of additional diatom species under grazing (Table 2), the one encrusting coralline alga found in the chambers, Fosliella sp. A., was present only under the grazed treatments. This may indicate either an amphipod dislike for Fosliella sp. A, or a resistance of the coralline alga to amphipod grazing, as found by Nicotri (1980) and others. The impact of amphipods on the epiphyte community was also variable. Amphipods significantly reduced the biomass of epiphytes between 21 and 28 days, although no significant grazing effect was detected before or after these times. The number of epiphyte species was greater under amphipod grazing which suggests amphipods may have been selectively feeding on particular species that were competitively dominant. In contrast to amphipods, gastropods significantly reduced the levels of chlorophyll a in periphyton, although they had no significant effect on biomass levels. However, there was proportionally more chlorophyll a relative to the biomass of periphyton where

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gastropods were present. This relative increase in chlorophyll a was not detected in the cluster analyses, suggesting that it may either be an artefact rather than indicating a subtle community change, or that gastropods reduced the biomass of periphyton, although it was not significantly detectable when considered separately from chlorophyll a levels. Gastropods significantly reduced epiphyte biomass by 21 days and again at 35 days. The removal by grazing of epiphyte taxa appeared to be generalised, because grazers had no significant effect on the taxonomic richness of epiphytes, nor were the treatments clearly separated during the cluster analyses. When considering the grazers together, there was broad dietary overlap. This lack of resource partitioning between the two types of grazers is also apparent in other studies (see Hootsmans and Vermaat, 1985; Howard and Short, 1986; Parker and Chapman, 1994). Despite the lack of partitioning in the present study, the impacts of the two types of grazers did vary. It is likely that differences in food selectivity or functional morphology of their mouthparts are responsible for this variation. In terms of the functional morphology of their mouthparts, amphipods are able to exploit a wide variety of food types, shapes and sizes. They appear to be selective feeders which preferentially select softer food-types. Gastropods, appear to be more generalised browsers, with their mouth-part morphology also having a large impact on food selection. Their grazing would be expected to be less selective than that of amphipods, as shown in the present and other studies (for example Klumpp et al., 1992). However, there are also many studies that report feeding preference by gastropod grazers (see review by Jernakoff et al., 1996). Although, the rhipidoglossan radula is broom-like in its action (Steneck and Watling, 1982), Thalotia conica has been seen eating encrusting red algae, such as Fosliella spp., from seagrass leaves (Nielsen and Lethbridge, 1989). Thus, it is likely that the generalised diet of gastropods like T. conica, which includes encrusting algal forms, and the active avoidance of tough food-types by amphipods, as reported in other studies, is responsible for the increased seagrass survival under gastropod grazing. The results of this study were dependent upon its duration (e.g. effects by gastropods on epiphytes only became apparent by 21 days). The question of how long a study should continue before the results can be assumed to approximate natural processes and patterns is a vexing one considering the natural variability in the field. Given the negative influence of the chambers on mortality of Posidonia sinuosa it is evident that the optimum duration of the present experiment is a balance between allowing significant growth of epiphytes without substantial mortality of seagrasses. This problem could be overcome by using artificial seagrass leaves, colonised by epiphytes in the field, and placed in the chambers so that the impact of grazers on periphyton and epiphytes could be studied over longer time-periods. Of course, the ability of artificial substrata to accurately reflect the normal periphyton and epiphyte communities found on seagrass leaves would be a major consideration in this approach. Although this study did not investigate spatial and seasonal variability, these factors may be important (Edgar, 1990; Williams and Ruckelshaus, 1993). Neckles et al. (1993) reported large differences in the relative abundance of amphipods and gastropods during longer-term experiments on grazing. In addition, the growth of epiphytes is a function of nutrients, light and temperature levels that affect plant growth rates. Spatial variation in

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the abundance of grazers may also be important in influencing the relative impact of amphipod and gastropod grazers. Edgar (1990) found that the abundance of amphipod grazers in seagrass varied by two orders of magnitude depending upon the time of year, and this variability can also occur on small spatial scales. P. Jernakoff and J. Nielsen (unpublished data) found that the relative AFDW biomass ratios of amphipods and gastropods varied by two orders of magnitude between seagrass meadows separated by 100 m. The relative impact of amphipod and gastropod grazers on their food sources may thus vary depending upon time and location of the particular study. It was surprising that the grazing impacts on periphyton and epiphytes were not more pronounced in the present study, given the high grazing rates of these types of grazers (Jemakoff et al., 1996). Laboratory studies of these grazers (P. Nielsen and J. Jernakoff, unpublished data) indicate that both amphipods and gastropods have the potential for significant impacts. The differences may be due to what is potentially possible, under laboratory conditions, and what actually occurs under field situations. One possibility is that the natural density of amphipods at the present study site and time was below the threshold level for significant impacts on the flora to occur. In support of this hypothesis, Stoner (1980) found that amphipods had little effect on macroalgae below threshold densities, but when their densities increased, owing to a decrease in the numbers of predators, amphipods had a significant impact. Based on the findings of this study, it appears that the gastropod grazers like Thalotia conica, are more effective in reducing periphyton and epiphyte biomass than the amphipod grazers. They also significantly reduced the mortality of Posidonia sinuosa leaves within the chambers. This may be due to their grazing activities which may help to keep the leaf surface clear of fine sediment (P. Jernakoff and J. Nielsen, unpublished data) that settles within the calmer water within the chamber. Amphipods at the densities within the chambers may, however, have more impact in structuring the type of epiphyte community that develops. In the present study, gastropods have a greater impact than amphipods on periphyton and epiphytes growing on seagrass, but spatial and temporal variation in grazer abundance, may also play a role in determining their relative effectiveness in grazing periphyton and epiphytes on seagrass.

Acknowledgements S. Wooldridge, R. Horbury, S. Braine, provided field assistance. Chambers were manufactured by P. Jolly and I. Cook. Comments by H. Kirkman, D. Walker and G. Edgar improved this manuscript.

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