Responses of settling invertebrate larvae to bioorganic films: Effects of large-scale variation in films

Journal of Experimental Marine Biology and Ecology,

ELSEVIER

JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY

207 (1996) 59-78

Responses of settling invertebrate larvae to bioorganic films: Effects of large-scale variation in films Michael J. Keough*, Department of Zoology, University

Peter T. Raimondi’

ofMelbourne, Parkville, WC 3052, Australia

Received 8 August 1995; revised 16 April 1996; accepted 3 May 1996

Abstract Results of previous studies have shown that at two sites in southeastern Australia (Williamstown and Momington), some sessile invertebrate species recruit at higher densities onto surfaces that have a microbial film present, and that some species recruit in proportion to the age (O-6 days) of that film. Other sessile species, notably colonial ascidians, do not respond to the presence of these films, and some barnacles may avoid the films. We tested whether larvae can detect or respond to differences in the microbial films that develop in different geographic localities and on longer time scales. To do this, we first confirmed that laboratory-developed microbial films induced higher recruitment of benthic invertebrates at a third site, Queenscliff, in Port Phillip Bay, Victoria, Australia. Approximately two-thirds of the sessile species settling at the time of the experiment were more abundant on the filmed surfaces. We then allowed microbial films to develop at all three sites on experimental substrata that were covered with plankton mesh to exclude larvae. The sites were chosen to represent three different rates of development of microbial films. After 1 week, we removed the plankton meshes and reciprocally transplanted experimental surfaces between all three sites. At the same time, we brought a sample from each site back to the laboratory to estimate the number of recruits passing through plankton meshes. We tested two hypotheses using these data, first that settling larvae at a particular site respond more strongly to locally-developed films than to films originating elsewhere and second, that it is the density of the microbial film, rather than its origin, that determines recruitment. Williamstown appeared to have the fastest-growing, most luxurious microbial films, followed by Momington and Queenscliff. Locally-developed films were not significantly more attractive than foreign films at Mornington or Queenscliff; none of the 1.5 taxa tested showed higher recruitment rates to the local treatment, and highest recruitment occurred on substrata filmed at Williamstown. At Wilhamstown, there were differences in recruitment rates to plates with films of different origin, but in each case, films from Williamstown received the most recruitment. These latter results could not separate our two hypotheses, but those from Momington and Queenscliff suggest that larvae do not recognize and/or respond to films from their local area, but that more heavily-filmed surfaces *Corresponding author ‘Present address: Biology Department, 0022-0981/96/$15.00 Copyright PII SOO22-0981(96)02632-9

0

University

of California,

Santa Cruz, CA 95064, USA.

1996 Elsevier Science B.V. All rights reserved.

60

M.J.

Keoqh,

P.T. Raimondi

I J. Exp. Mar.

Bid.

Ed.

207 (1996)

59-78

may be more attractive to settling larvae. The species showing the strongest responses were polychaetes, encrusting bryozoans, and some solitary ascidians. We also compared the attractiveness of laboratory-developed films varying in age up to 1 month and found that some species (set&id polychaetes and encrusting bryozoans) recruited onto these surfaces at variable rates, while other species, notably ascidians, did not distinguish between the different substrata. Keywords:

Bioorganic

tilm; Microbial film; Settlement; Bryozoans; Ascidians; Polychaetes

1. Introduction Settling marine invertebrate larvae are presented with a wide range of cues as they approach a substratum. These cues may be physical ones, such as light or surface texture, or they may be biologically-derived. Biological cues may be the presence or absence of particular animals and plants, including microbial organisms. The bioorganic films that cover exposed surfaces rapidly have long been thought to be a strong cue for settlement (ZoBell and Allen, 1935 and see recent literature cited by Pawlik, 1992; Rodriguez et al., 1993; Todd and Keough, 1994), although it is becoming clear that responses of individual species to the presence or absence of such films are highly variable (Todd and Keough, 1994). A number of authors have demonstrated highly specific responses by individual invertebrate species to individual microbial organisms (Roberts et al., 1991; Holmstrom et al., 1992), or to the presence or absence of general films (e.g., Todd and Keough, 1994). However, in a natural situation, the nature of the bioorganic film changes rapidly as the microbial organisms undergo their own successional changes (Little, 1984). As clearings are made and recolonized on hard surfaces, we might therefore expect invertebrate larvae to be exposed to a mosaic of microbial cues, and their responses to these cues may have major effects on settlement, and ultimately adult distributions. We have attempted to determine the responses of invertebrates to this variation in cues, with two general aims. First, to assess the extent of larval responses to bioorganic films, we have aimed to examine as many species within an assemblage as possible, effectively taking a random sample of species, rather than focusing on conspicuous species that may be exceptionally abundant or show unrepresentatively strong settlement patterns. Second, we have attempted to vary the films at a number of temporal and spatial scales, and to characterize the responses of individual taxa to these kinds of variation. In earlier work, we have shown the extent of species’ responses, across a number of phyla, to the presence or absence of microbial cues (Todd and Keough, 1994). In that work, we have shown that species’ responses are generally consistent between two locations in southern Australia, and that there is also temporal consistency. Also, we found little small-scale spatial variation in the attractiveness of either natural or laboratory-developed films. In later work (Keough and Raimondi, 199_5), we showed that some species respond to variation in the age of microbial films over the scale of a few days, with some recruitment positively correlated with age of the bioorganic film, a

M.J. Keough, P.T. Raimondi I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78

61

few species showing negative correlations, and others showing little response (see also Wieczorek et al., 1995, for an example of changing attractiveness of films). In addition to these small-scale effects, we might expect large scale variation. Microbial succession might occur over time scales of weeks, rather than just days (Pearce and Scheibling, 1991; Becker, 1993) and different locations, with different nutrient and hydrodynamic regimes, might have very different bioorganic films. Settling larvae might be expected to respond to this latter variation in a number of ways. If the likelihood of settlement is proportional to the amount of microbial films, they might recruit most heavily onto surfaces with the heaviest filming. Alternatively, larvae might distinguish between films of different origins, using the films as indicators of habitats in which conspecific adults have grown successfully (e.g., Strathmann et al., 1981). Most of the species involved have very restricted pelagic phases, raising the possibility of local differentiation and/or adaptation (e.g., Burton, 1983; Keough, 1989; Ayre, 1990; Raimondi and Keough, 1990; Ayre et al., 1991; Ayre and Dufty, 1994; Ayre, 1995; Burnett et al., 1995). In this paper, we report the results of testing the effects of large-scale variation, using bioorganic films that developed at three different sites and, separately, over longer time scales.

2. Study sites We did field work at three sites around Port Phillip Bay in Victoria, Australia. Two of those sites, Breakwater Pier, at Williamstown, and Mornington have been used for other experiments (Todd and Keough, 1994; Keough and Raimondi, 1995), and descriptions of the sites and their faunas are provided elsewhere. Both sites are semi-protected habitats, where the sessile animals live attached to wooden pier pilings. Williamstown is a turbid site at the northern end of Port Phillip Bay (Fig. l), and it receives nutrient input from the Yarra River (e.g., EPA, 1995). The fauna is dominated by the solitary ascidian Pyura stolonifera. Mornington faces west, but is protected from southwesterly swells by a rocky headland and a row of closely-spaced pier pilings that serve as a breakwater. The largest animals on the pier pilings are P. stoloniferu and the mussel Mytil~s edulis. Mornington is a marina that caters to commercial and recreational boating, and turbidity and, nutrient levels are moderate (EPA, 1995); water visibility averages 3-4 m. We have already shown that sessile animals at these two sites respond to the presence of microbial films. To make a better test of the effect of the origin of a film, we selected a third site, at which we expected both lower recruitment rates and less dense bioorganic films. As a preliminary step, we tested whether the invertebrates at this third site responded to bioorganic films in a manner similar to those at our other field sites. The third site was Queenscliff, which is at the entrance to Port Phillip Bay (Fig. 1). The pier at Queenscliff is exposed to moderate wave action, has relatively coarse sediments and low nutrient levels (EPA, 1995) and visibility often exceeds 5 m. No single species dominates the sessile fauna, and pier pilings are covered by a mixture of sponges, solitary ascidians (Molgula, Ascidia), colonial ascidians (Podoclavella cylindrica, Arnphicarpa meridiana, Sycozoa cerebriformis, BotrylluslBottylloides, and a suite of

62

M.J.

Keough,

P.T. Raimondi

Fig. I. Port Phillip Bay, showing

I J. Exp. Mar.

locations

Biol. Ecol.

207 (1996)

of study sites, and directions

59-78

of reciprocal

transplants

didemnid species), encrusting bryozoans (three species of Celleporaria, two of Parasperfragilis), arborescent bryozoans mittina, Mucropetraliella ellerii, Membranipora (Bugula dentata, Caberea grandis and Tricellaria porteri), sponges and the clonal anemone Anthothoe albocincta. Water depth is 4-5 m at low tide and the site receives frequent input of clean water from Bass Strait. We believe that this water is much lower in nutrients, and hence a less favourable environment for microbial growth, than at the other two sites.

3. Materials

and methods

3.1. Preliminary

experiment

We tested whether bioorganic films influenced settlement at Queenscliff using a single experiment in February 1991. We used 11 cm X 11 cm black perspex ( = Plexiglas) substrata (panels), 6 mm thick, which had been roughened with coarse sandpaper. These substrata were attached, using stainless steel bolts, in groups of four to perspex mounting plates that were nailed vertically to pier pilings at a depth of 4 m, 1 m above the seafloor. We used two experimental treatments, filmed and unfilmed panels. Unfilmed panels had been scrubbed, soaked in freshwater and then in filtered, autoclaved seawater, while filmed panels had been immersed in a recirculating seawater system at the University of Melbourne for 1 week. Previous work at Williamstown (Keough and Raimondi, 1995) had shown that such films have similar effects to naturally occurring ones.

M.J. Keough, P.T. Raimondi I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78

63

The substrata were left in the field for 5 days, collected and transported in insulated containers back to the laboratory. We then counted and identified all recruits under a dissecting microscope. The data were analysed by two factor analysis of variance, after visual inspection of boxplots and residual plots to assess normality and heterogeneity of variance, because the replicates were attached to mounting blocks on different pilings. The two factors were Filming (present/absent at start of the experiment) and Blocks (four blocks, random factor), with two replicates per block. Main effects of filming were tested against the Filming X Blocks interaction. For rarer species, we counted all recruits2, and made a subjective decision about whether individual recruits could be regarded as independent settlement events. We made this decision according to whether they were clumped or distributed haphazardly across replicates. If the distribution appeared clumped (most recruits on a few panels or within small regions of panels), individual larvae could not be regarded as independent settlement events. If recruits were spread evenly, we could assume that they represent multiple settlement. We analysed those species for which we found no more than two recruits per panel. These data were analysed by a simple G-test of whether recruits occurred equally frequently on filmed and unfilmed surfaces. 3.2. Transplant

experiment

We tested whether the origin of a microbial film influences recruitment using a transplant experiment at Williamstown, Queenscliff and Mornington. Our qualitative impression was that bioorganic films developed faster and more densely ‘at Williamstown, followed by Mornington and Queenscliff, although the latter two sites were both very different from Williamstown. Although we did no quantitative analysis of the biofilms, casual inspection by scanning electron microscope showed much denser and more diverse biofilms on panels from Williamstown, followed by Momington, with panels filmed at Queenscliff having sparse biofilms. We used the same experimental substrata described above, suspended vertically at Queenscliff and Mornington. At Williamstown, the panels were attached in groups of 16 to large perspex plates, and suspended horizontally, with the panels facing downwards (see Todd and Keough, 1994 for details). These orientations were forced on us by an inability to suspend horizontal plates stably at Queenscliff and Mornington and a desire to keep the methods at Williamstown and Momington consistent with our earlier work. This difference in method of deployment is not important, because our hypotheses were tested by comparisons, within, rather than between, sites. The basic experimental protocol involved allowing films to develop at all three sites, and then transplanting panels reciprocally between sites after 1 week. The detailed protocol was: We visited all three sites on the same day, and suspended groups of four substrata. All substrata were covered with 230 p.m nylon plankton mesh to prevent most larvae from settling on the surfaces, while allowing microbial organisms to colonize. The mesh size ‘With a 5-7 day exposure period, we believe that our data reflect settlement, but use recruits to reflect the possibility of post-settlement mortality over this period. Todd and Keough (1994) discuss this point.

M.J. Keough, P.T. Raimondi I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78

64

was a compromise between mesh sizes small enough to restrict larvae and those large enough to allow water flow. We have shown that 150 and 230 pm meshes have similar effects (Todd and Keough, 1994). After 4 weeks, we again visited each site. Experimental substrata were all removed from the water and placed immediately into insulated containers of seawater. We then removed all plankton meshes. We had randomly allocated one panel from each group of four as a control for larvae passing through the net. This panel was transported back to the laboratory for counting, and replaced with a new, unfilmed surface. The other three panels were transplants, one to each other site, and home panels, to be returned to the same locality. We tried to equalize the handling of panels, with constraints imposed by the distances between sites, and no panel was out of the water for more than 2 min. Panels were transported between sites by car and ferry. Mornington and Queenscliff home panels were thus generally out of the water for l-2 h in their insulated containers, while Williamstown home substrata travelled to Queenscliff and back. After a further week, we returned and collected all panels, transported them back to the laboratory in insulated containers, and counted and identified all recruits. 3.3.

Longer

term variution

in age

of ,$lms

We tested whether any longer term changes in films affected recruitment rate by allowing tilms to develop in the laboratory for periods up to 4 weeks. There were four treatments, with film ages of 1, 2 and 4 weeks. Panels were kept in a laboratory holding tank in a recirculating aquarium, with the 4 week treatment added first, followed by the 2 week treatment 14 days later, and the 1 week treatment a further 7 days later. The unfilmed treatment was kept in filtered, autoclaved seawater at the same temperature as the filming treatments for 4 weeks. Panels were attached to two mounting plates, as described earlier, with four replicates of each treatment on each plate. The plates and panels were suspended beneath Breakwater Pier, Williamstown, for 1 week, then collected and the recruits counted and identified.

4. Data analysis The species present at each site were different, as were the overall levels of recruitment, so we analysed each site separately. Our null hypothesis was that, for a given site, the three Film treatments did not differ in their effects. We identified two alternative hypotheses that were of interest. First, if local films are preferred by settling larvae, at each site we expected more settlement on films from that site (i.e., at Williamstown), W > Q, M. Based on our qualitative impressions of the films developing at each site, if larvae respond to the amount of film, we expected our means to be ordered W > M > Q, regardless of the site at which the panels were placed. To confirm the general effectiveness of films, we also analysed all four treatments (Films + sterile control). The alternative hypothesis for most species was that films increased settlement, i.e., Lab < W,Q,M. Our control panels from the first week were used to estimate the number of smaller

M.J. Keough, P.T. Raimondi

I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78

65

larvae passing through the plankton nets. We calculated the mean recruitment onto control panels at each site for the first week, and subtracted this value from all panels that were filmed at that site, regardless of where the panel was placed in the second week. This adjustment was necessary for species such as serpulid polychaetes and some solitary ascidians that have small larvae. Species with relatively large larvae, such as barnacles, colonial ascidians, and most bryozoans, required no adjustment, as no recruitment occurred beneath nets. A more detailed description of the species passing through plankton nets can be found in Todd and Keough (1994). Data for each species were analysed by single-factor analysis of variance and separately for each site. As a preliminary step, we inspected box plots of untransformed, log- and square-root-transformed data and plots of variance versus mean. We used either log- or untransformed data, but the choice varied between taxa. Following the analyses, we used planned comparisons to test the two above hypotheses, except at Williamstown, where the two alternative hypotheses were similar in predicting highest recruitment onto Williamstown-derived substrata. Both contrasts were one-tailed tests. The two hypotheses differed in their predictions about the relative recruitment to Mornington and Queenscliff panels at Williamstown, but the biggest difference in microbial films was between Williamstown and the other two sites, with a smaller difference between Mornington and Queenscliff. We decided that the secondary predictions of the two hypotheses may not be sufficiently distinct for us to test them unequivocally. In the long-term films experiment, data were analysed using one-way analysis of variance, followed by fitting an unevenly-spaced linear polynomial to test for linear changes in recruitment with the age of film. All analyses were done using SYSTAT version 5 (SYSTAT, Evanston, IL, USA (now SPSS)).

5. Results 5.1. Preliminary

experiment

The preliminary experiment at Queenscliff produced results that were similar to those we have obtained from Williamstown and Mornington (Todd and Keough, 1994; Keough and Raimondi, 1995). Only spirorbid polychaetes, Ascidia, Trididemnum, and the pooled category of erect bryozoans were sufficiently common for analysis of variance. For two of the taxa, we found an interaction between blocks and the presence of a microbial film (Table 1). Inspection of means showed that for spirorbids and erect bryozoans, filmed surfaces always received more recruits than unfilmed ones, with the magnitude of the difference varying among blocks (Fig. 2). For the two ascidians, recruitment varied considerably between blocks, with some receiving IO-20 recruits per panel and others < 2. Our inspection of the means suggested that the interaction occurred because there were substantial differences between treatments on blocks with high recruitment, but, unsurprisingly, no differences when there were few or no recruits (Fig. 2). We conclude that both of these species also recruited more to filmed surfaces than to unfilmed ones. Most of the rarer species also occurred disproportionately on filmed surfaces (Table

66

M.J. Keough,

Table 1 Analyses of recruitment

P.T. Raimondi

patterns

I J. Exp. Mur.

in initial Queenscliff

Biol.

Ecol. 207 (1996)

_i9-7R

exoeriment

TAXON

Filming

Blocks

Interaction

MS,,\,,

Spirorbids

0.002

0.050

0.376

26.4

Trididemnum

0.055

0.052

0.048

0.75

Ascidia

0.133

Erect bryozoans

0.005

0.010 0.883

0.025 0.906

4.6 4.9

The table shows, for four taxa, results of two-factor analyses of variance, with the factors filming of surface (df = 1) and blocks (df = 3). Main effects of filming were tested against MS interaction (df = 3), because blocks were treated as a random factor. P values are one-tailed, because we expected filming to have no effect or a positive effect on these taxa. The residual mean-square (df = 8) is shown to allow reconstruction of a full analysis table.

2). The rare species included serpulid polychaetes and pooled encrusting a group of ascidians: Amphicarpa meridiana, Molgula sp., Botryllus Diplosoma sp. Only Diplosoma showed no response to films.

Spirorbids

and and

Erect bryozoans 5-

5om l

l

l

,g 40 2 ; 30

4

4l

l

l

3 l

b 20~

17 CJ

4

bryozoans, schlosseri

2

” 1

IO/

‘,

c

I7

c ”

”

Trididemnum

Ascidia

12t

Blocks

4

Blocks

Fig. 2. Recruitment onto filmed and unfilmed surfaces at Queenscliff. The figure shows the mean (-tSE) of the number of recruits per panel for filmed (filled symbols) and unfilmed (unfilled symbols) surfaces, plotted which is the variance term used to test the effect separately by block. The error bars represent d/MS InILltlLIIOI’~ of filming, and are shown at the lower left of each graph. Block labels have no meaning and are not shown.

M.J.

Table 2 Analyses

Keough,

of uncommon

P.T. Raimondi

I J. Exp. Mar. Biol. Ecol. 207

taxa in the initial Queenscliff

Species

G

23

8

7.57

0.003

12

3

5.78

0.008

Molgula

17

4

8.66

0.002

Trididemnum

17

3

10.82

0.001

Botryllus schlosseri

15

0

4

8

6

1

15.96 1.36 3.96

0.000 0.122 0.023

polychaetes meridiana

Diplosom

Encrusting

bryozoans

67

P

Unfilmed

Serpulid

59-78

experiment

Filmed

Amphicarpa

(1996)

The table shows the number of recruits on filmed and unfilmed surfaces, and log-likelihood ratio tests of the null hypothesis that recruits are distributed equally on the two surface types, against the alternative that more recruits occur on filmed surfaces.

5.2. Transplant

experiment

The overall levels of recruitment were much higher at Williamstown than at Mornington and Queenscliff, while the number of common taxa was highest at Williamstown, followed by Queenscliff, then Mornington (Fig. 3). Polychaetes were the most common group at Williamstown and Queenscliff, while colonial ascidians were the most common at Mornington (Fig. 3). At Williamstown, the rank order of abundance of the major functional groups was polychaetes, solitary ascidians, colonial ascidians, barnacles, encrusting and then arborescent bryozoans. The recruits at Queenscliff at the time of the experiment were primarily polychaetes, arborescent bryozoans and solitary ascidians, although that is not always the case at that site (Todd and Keough, 1994). At

n Polychaeies

n Barnacles H Encrusting Blyozoans 0 Solitary ascidians

0

Mornington

Queenscliff

Fig. 3. Rates of recruitment of major invertebrate groups abundance of major groups, across all treatments at a site.

q Erect bryozoans Colonial Ascidians

Williamstown

at the three sites. Dates shown

are the mean

68

M.J.

Keough,

P.T. Rnimondi

I .I. Exp. Mar.

Biol. Ecol. 207 (1996) 59-78

Momington, colonial and solitary ascidians dominated recruitment, with smaller contributions from arborescent bryozoans and polychaetes. Within polychaetes, serpulids were most common at Williamstown, in contrast to the preponderance of spirorbids at Queenscliff (Fig. 3). In planned comparisons of filmed versus unfilmed surfaces, we found significant effects of filming at Queenscliff for serpulid polychaetes, erect bryozoans and total recruits, with no significant response for spirorbids, Botryllus schlosseri, Molgula, Trididemnum, Ciorza intestinalis, and Ascidia (Table 3). In each significant comparison, recruitment was higher onto filmed surfaces (Figs. 4 and 5). For the corresponding analysis at Mornington, spirorbid and serpulid polychaetes and Ciona intestinalis were significantly more common on filmed surfaces than unfilmed ones (Table 3), with Trididemrzunz and Ascidia not being influenced strongly. At Williamstown, we found significant effects for spirorbids and serpulids, Botryllus schlosseri, Ascidia, erect bryozoans other than Bugula stolonifera, encrusting bryozoans, and total recruitment (Table 4). Again, most species settled more commonly onto filmed surfaces (Figs. 4-7). Species failing to respond to filming were Elminius modestus and Balanus variegatus, Trididemnum, other didemnid ascidians, Ciona intestinalis and Diplosomu, and an unidentified slime sponge (Table 4), although Ciona intestinalis was close to significant. At Mornington and Queenscliff, we were able to test the pattern of abundance of 15

Table 3 Analyses

of transplant

Taxon

experiment

by site

Overall analysisP

Film > No Film

Local > Foreign

W>Q.M

0.500

0.098

0.049

0.005

N/A 0.481 0.384 0.340 0.393 N/A 0.200 0.472 0.375

0.425 0.192 0.361 0.1 16 0.393 0.006 0.330 0.167 0.239

N/A N/A 0.152 N/A 0.443 0.293

0.001 0.000 0.385 0.002 0.131 0.267

QU&WCliff

Spirorbid polychaetes Serpulid polychaetes Bot~llus

schloswri

Molgula

0.940

0.319

0.176

0.100

Trididemnum

0.907

0.339

Ciona

0.016

0.108

Ascidiu

0.653

0.160

Erect bryozoana Total recruitment

0.155

0.026

0.107

0.0

intestina1i.s

IO

Mornington

Spirorbid polychaetes Serpulid polychaetes Trididemnum Ciona

intestinalis

Ascidict

Total recruitment

0.001 0.000 0.766 0.003 0.665 0.626

0.009 0.002 0.469 0.021 0.490 0.255

The table shows results from Queenscliff (Q) and Momington (M), using one-way analyses of variance. The first P value at each site (with df = 3,12) is for a comparison of all four treatments. Subsequent columns show the results of three planned comparisons to test (a) effects of filming; (b) HI: Settlement onto Local films > Foreign films; (c) that settlement onto Williamstown (W) films was higher than on the other two. Tests (b) and (c) are one-tailed and are shown as N/A for cases in which the pattern of means was in the opposite direction to the predicted one.

M.J. Keough,

P.T. Raimondi

At Mornington

0

LMQW

I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78

At Queenscliff

At Williamstown

LMQW

LMQW

69

Origin of substratum Fig. 4. Recruitment of serpulid polychaetes, spirorbid polychaetes and total panels with films of different origin. For each combination of species and (*SE) of the number of recruits per panel. Origins of plates are denoted substrata, and M, Q and W denote plates filmed at Mornington, Queenscliff,

At Mornington

At Queenscliff

recruits (all species pooled) onto site, the graphs show the mean by L for unfilmed (laboratory) and Williamstown, respectively.

At Williamstown

Trididemnum

0

LMQW

LMQW

LMQW

Origin of substratum

Fig. 5. Recruitment of three ascidian species onto panels with films of different origin. For each combination of species and site, the graphs show the mean (*SE) of the number of recruits per panel. Origins of plates are denoted by L for unfilmed (laboratory) substrata, and M, Q and W denote plates filmed at Momington, Queenscliff, and Williamstown, respectively.

70

M.J.

Table 4 Analyses

of transplant

Keough,

P.T. Raimondi

experiment

I J. Exp.

Mar.

Biol.

Ecol.

207

(1996)

59-78

by site

Taxon

Overall analysis P

Effects of filming

W>Q.M

Spirorbid polychaetes Serpulid polychaetes

0.019 0.019

0.002 0.004

N/A 0.048

Elminius

modestus

0.499

0.097

0.379

Bulanus

variegatus

0.672

0.241

0.336

0.064

0.026

0.520

0.308

Bottyllus

schlosseri

0.698

0.303

0.131 N/A N/A

Cinnu inte.stinalis

0.293

0.060

0.408

Ascidia

0.142

0.034

0.049

Diplosoma

0.093

0.404

0.283

N/A

N/A 0.191

Trididemnum Didemnid

Bugulu

ascidians

stolonifera

Erect bryozoans (excluding Encrusting bryozoans Slime sponge Total recruitment

B. stoloniferu)

0.120

0.03

0.026 0.323 0.000

0.004 0.395 0.000

I

0.077

0.04 1 0.434 0.00 1

The table shows results from Williamstown (W), using one-way analyses of variance. The first P value at each site (with df = 3,12) is for a comparison of all four treatments. Subsequent columns show the results of planned comparisons to test (a) effects of filming; (b) whether settlement onto Williamstown films was higher than on the other two (M,Q). The latter test is one-tailed and is shown as N/A for cases in which the pattern of means was in the opposite direction to the predicted one.

taxa at different levels of taxonomic resolution. No taxon showed significantly higher recruitment to local than to foreign films (Table 3) and no taxon was even close to significant. When we tested the hypothesis that recruitment was higher to films that developed at Williamstown than to films originating at other sites, we found significantly higher recruitment for eight taxa from the 30 comparisons (Tables 3 and 4). The comparison from Williamstown is difficult to interpret, because we expected local films at this site also to be richer. However, we found four cases at Mornington and Queenscliff in which panels filmed at Williamstown had the highest recruitment (Table 3). At two of the sites, serpulid polychaetes recruited in highest numbers to films from Williamstown (Fig. 4), although the difference between Williamstown and the other films was not significant at Queenscliff (Table 3). Recruitment onto panels filmed at Mornington or Queenscliff was intermediate between unfilmed and Williamstown-filmed surfaces at Williamstown, and close to the values for unfilmed surfaces at Mornington. Spirorbid polychaetes showed no particular pattern at Williamstown or Queenscliff, but recruited predominantly onto Williamstown-derived films at Mornington (Fig. 4), where films from Mornington and Queenscliff had effects only slightly larger than those of unfilmed surfaces. The solitary ascidian Ciona intestinalis recruited to all three sites, but at Mornington and Queenscliff, we found many more individuals on panels bearing Williamstown films than onto any other surfaces (Fig. 5). At Williamstown, we found no significant difference between the three films. Another solitary ascidian, Ascidia sp., showed no patterns at Mornington or Queenscliff, but higher recruitment onto Williamstown-

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0 LMQW

Origin of substratum Fig. 6. Recruitment of species occurring only at Williamstown onto panels with films of different origin. For each combination of species, the graphs show the mean (+SE) of the number of recruits per panel. The origin of each substratum is shown along the base: L = unfilmed, from laboratory; M, Q and W denote plates filmed at Momington, Queenscliff, and Williamstown, respectively. (a) Didemnid ascidians; (b) Unidentified sponge sp.; (c) Elminius modestus; (d) Balanus variegatus; (e) Bugula stolonijera; (f) Encrusting bryozoans.

derived surfaces at Williamstown (Fig. 5). Again, films developing at the other sites had effects intermediate between Williamstown and unfilmed panels. The third ascidian to occur everywhere, Trididemnum, showed no significant differences between films of different origins. A substantial number of species recruited only at Williamstown. Of these, two barnacles, didemnid ascidians, Diplosoma, Bottyllus schlosseri, Bugula stolonijera, other erect bryozoans, and the slime sponge, showed no conspicuous pattern among treatments, or varied in some way other than predicted by our hypothesis (Figs. 7 and 8). Encrusting bryozoans recruited most abundantly onto Williamstown-derived substrata (Fig. 6), and panels filmed at Mornington and Queenscliff were intermediate between Williamstown-derived and unfilmed panels. Although we did not analyse the data, didemnid ascidians appeared to be less common on Williamstown-filmed panels than on other treatments (Fig. 6). At Queenscliff, there were no significant differences in the abundance of Molgula sp., erect Bryozoa, Botryllus schlosseri (Fig. 7), or Amphicarpa meridiana. Total recruitment showed no strong patterns at Mornington or Queenscliff, but at Williamstown, it was highest onto Williamstown-filmed panels, followed by those filmed at Queenscliff, Mornington, and then unfilmed surfaces (Fig. 4).

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a

LMQW

LMQW

Origin of substratum Fig. 7. Recruitment of species occurring only at either Williamstown or Queenscliff onto panels with films of different origin. For each combination of species, the graphs show the mean (5SE) of the number of recruits per panel. The origin of each substratum is shown along the base: L = unfilmed, from laboratory; M, Q and W respectively. (a) Erect bryozoans denote plates filmed at Momington, Queenscliff, and Williamstown, (Queenscliff); (b) Erect bryozoans (Williamstown); (c) Mol&a sp. (Queenscliff); (d) Diplosoma sp. (Williamstown); (e) Botryllus schlosseri (Queenscliff); (f) Botryllus schlnsseri (Williamstown).

40

d

I

Age of Film WW Fig. 8. Recruitment of species on films of different ages at Williamstown. For each taxon, the mean (5SE) number of recruits per panel is shown. (a) Elminius mode.stu.s; (b) C~ptouufa pallasiana; (c) Spirorbid polychaetes; (d) Serpulid polychaetes.

M.J. Keough, P.T. Raimondi I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78 Table 5 Analyses

of the effects of longer term variation

13

in films

Taxon

Overall test

Linear trend

Spirorbid polychaetes Serpulid polychaetes Elminius modestus Didemnid ascidians Cryptosula pallasiana All ascidians (Logged) All bryozoans (Logged) Total recruits (logged) Corophium spp. (Logged)

0.241 0.022 0.100 0.883 0.052 0.894 0.000 0.075 0.017

0.102 0.003 0.132 0.758 0.010 0.898 0.000 0.012 0.012

The table shows the results of single-factor analyses of variance to compare the four treatments (df = 3,28), together with the probabilities associated with a linear trend in recruitment as a function of film age, calculated by fitting an unevenly spaced linear polynomial as a planned comparison (df = 1,28).

5.3. Variation in age of films Only six individual taxa recruited in sufficient numbers for analysis: spirorbid and serpulid polychaetes, Elminius modestus, didemnid ascidians, the encrusting bryozoan Cryptosula pallasiana and corophiid amphipods, tubes of which covered substantial parts of the panels. We also analysed the composite groups of ascidians and bryozoans, and total recruitment. Spirorbids, E. modestus, and the ascidians showed no response to the films, whether we tested for overall differences between films of different ages or explicitly for a linear trend in recruitment through time (Table 5, Figs. 8 and 9). Serpulids, C. pallasiana and corophiids all showed strong positive responses to the older films (Table 5, Figs. 8 and 9). Bryozoans also showed a strong pattern, even though Cryptosula contributed about half of total bryozoan abundance. Total recruitment also increased steadily with age of film (Table 5, Fig. 9).

10

0

0

1

2

4

Age of Film (wks) Fig. 9. Recruitment of species on films of different ages at Williamstown. For each taxon, the mean (+SE) number of recruits per panel is shown. (a) All bryozoans; (b) Total recruits; (c) Didemnid ascidians; (d) Percentage cover by tubes of corophiid amphipods.

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6. Discussion

The results from the preliminary experiment are consistent with the earlier findings of Todd and Keough (1994) that larval responses to the presence/absence of bioorganic films vary considerably among species. The experiment with films developed at three different sites provided no evidence for larval recognition of biofilms that developed locally. Approximately one third of species did recruit differentially to biofilms from the three sites, but panels filmed at Williamstown were consistently the most attractive. Such a result is equivocal for panels at Williamstown, because two different hypotheses predicted higher recruitment onto local panels. At Mornington and Queenscliff, however, higher recruitment onto panels from Williamstown allows us to reject the hypothesis about local films. It seems likely, therefore, that the responses we observed were the result of denser biofilms providing stronger cues. It is also possible that the settlement cue is a single component of the biofilm, which varies in abundance (e.g., Holmstrom and Kjelleberg, 1994; Roberts et al., 1991). There were varying responses to the array of substrata with biofilms ranging from 0 to 4 weeks old, with serpulids and encrusting bryozoans responding strongly, and didemnid ascidians showing no strong pattern. The experiment was hampered by having relatively low settlement during the week of the experiment, so data were only available for a few, largely composite, taxa. The original aim of this work was to examine all possible species in an assemblage, rather than to describe strong responses of individual species that show conspicuous patterns. By doing this, we are able to report negative and positive results, and to estimate the proportion of taxa that respond to a given stimulus. We have now described the responses of a range of organisms to 7 day old bioorganic films that developed in the field (Todd and Keough, 1994) and shown that films developing in the laboratory have similar stimulatory effects (Keough and Raimondi, 1995). In addition to simple contrasts between substrata with and without films, larvae of some species are capable of responding to the more subtle variations in biofilms that are associated with short-term successional or developmental changes in the films (Keough and Raimondi, 1995). In all of these experiments, the most conspicuous result has been that individual species differ greatly in their responses. With the data in this present study, we can now examine the responses of these assemblages to temporal changes in biofilms on two different temporal scales, and to biofilms from localities that are widely separated in space. In all of these experiments, we included unfilmed substrata as controls, so we have presence/absence comparisons from a range of times and places. The substrata used were uniform, the experimental conditions were generally consistent, and recruitment was generally measured over 6-7 days, so the data from various experiments are comparable. The most extensive data set is from Williamstown, which has been used in all experiments, and we have summarized those results on Table 6. The limitation of the data set is that some species settle only for short periods, so we have no information about their responses to some cues. One point that we have made in the past and that seems supported by the results in Table 6 is that there is evidence for intraspecific variability in the settlement behaviour of marine invertebrate larvae (Raimondi and Keough, 1990).

M.J. Keough, P.T. Raimondi Table 6 Summary

of responses

Settlers

of invertebrate

species to the presence

Variation presence absence

Serpulid polychaetes Spirorbid polychaetes Elminius modestus Balanus variegatus Bug&a neritina Bugula dentata Bug&a stolonijera Tricellaria occidentalis Encrusting bryozoans Trididemnum Botryllus schlosseri Didemnum Diplosoma Pyura stolonijera Ascidia Ciona intestinalis Sponges Electroma Total recruitment

I J. Exp. Mar. Biol. Ecol. 207 (1996) 59-78

in microbial

Long time (O-4 weeks)

Large spatial (10s of km)

J 0 0

J

0 0 0 0 0 J 0 J

0 0 0 0

J

J 0

films

cues

Short time (O-6 days)

J J X

of microbial

15

J

J

0 0 0 0

0 0

J 0 0 0 0 0

0 J 0 J

0 J

J

Data were compiled from comparisons involving filmed and unfilmed treatments reported by Todd and Keough (1994) and Keough and Raimondi (1995), as well as the present paper. Symbols describe the responses observed: 0 = no effect; J = strong positive; - = weak positive; X = negative effect. a The result for erect bryozoa at Mornington is disproportionately B. dentata, but the result should be interpreted with caution.

Serpulid polychaetes responded to variation in microbial cues on every scale that we have used, as well as showing consistent responses through time to presence/absence experiments and consistent responses at Williamstown and Mornington. The other major polychaete group, the spirorbids, generally showed responses to the presence of a film and settled in different numbers in response to short-term temporal variation in films. At a larger spatial scale, however, they showed inconsistent results. This could in part be explained by there being the dominant species at Queenscliff being different from the abundant species at Williamstown and Mornington. However, we also found a strong response at Mornington, but none at Williamstown to the large spatial scale, so we would need to postulate different serpulids at all three sites. We obtained data on a range of arborescent bryozoans, and some encrusting species. We recorded four species of Bugula, which showed a variety of responses. Two other arborescent species, Tricellaria porteri and Caberea grandis, also responded to the presence of films, although we have little information about the responses of these species to larger-scale variation in films. Encrusting species were generally in low numbers, and while we could sometimes test the patterns of individual species (Cryptosula pallasiana, Microporella sp., etc.), we generally had to pool species into a single category. This aggregate category showed a strong positive response in all

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experiments. With no particular species dominant, this result suggests that most species showed settlement patterns similar to the aggregate pattern. The two species of barnacles at Williamstown and Mornington, Elminius modestus and Balanus variegatus, showed weak responses. In some experiments they seemed to avoid films, showing no response in others. We recorded settlement of a wide range of ascidians. Some could be identified easily, but others, notably many of the didemnid ascidians, were impossible to identify when they were only a few days old. In general, we recorded inconsistent results for solitary ascidians, regardless of the species. They tended to show no response with occasional positive ones (e.g., Ciona intestinalis). Colonial ascidians generally showed no responses, except for an occasional positive response to the presence of a film by Bottyllus schlosseri. Sponges were difficult to identify, although we were generally dealing with only two species at Williamstown. As a group, they responded to crude cues or to short-term temporal variation in cues, but not to the larger scale spatial variation. The net result of all of these patterns was that the total number of recruits increased with the age or abundance of the biofilms. The most obvious correlation with this range of responses is the competitive ability of the species concerned. Subtidal sessile assemblages have been studied extensively at a number of locations in southeastern Australia (review by Keough and Butler, 1995) including Port Phillip Bay (Russ, 1980, 1982). The main encrusting species can be grouped according to their competitive ability (Kay and Keough, 1981; Russ, 1982; Keough, 1984a), with serpulids and spirorbids the worst competitors, followed by encrusting bryozoans and with sponges, and especially colonial ascidians the dominant competitors. In our study it is the colonial ascidians that have shown the least response to microbial films, and it may be that they are capable of colonizing and growing successfully in a wide range of clearings. On the other hand, the weak competitors, the polychaetes and encrusting bryozoans, responded to most changes in the biofilms, suggesting that they make considerable use of these films at the time of settlement. It appears likely that the composition of the biotilm is related to the age and/or size of a cleared area on a hard substratum, and may provide a cue to settlement locations that are relatively free of competition. Of the remaining taxa, arborescent bryozoans occupy a range of roles. For example, Bugula dentuta is a perennial species (Keough, unpublished observations) that is not overgrown completely by colonial ascidians, and which may provide spatial refuges for ascidian recruitment (Russ, 1980). Bugula neritinu is an annual species that lives on a range of substrata, and B. stolonijera is a small species that appears to be overgrown easily. These species can, therefore, not be classified easily, and they show inconsistent results to the microbial cues. The other abundant group, the solitary ascidians, are large, mound-shaped animals that appear not to be overgrown much at these sites. Some species, particularly Pyuru stolonifera, provide secondary space, and their tests are heavily colonized by many of the species discussed here (Klemke, 1993; Dalby, 1995). If they are not outcompeted, they may not gain much by using microbial cues, but there is no clear explanation for their weak responses. The remaining common group of animals, barnacles, do not occupy large amounts of space in these assemblages, despite their settling in large numbers at some times. They appear to be overgrown often, and probably colonize bare space quite early, disappearing as space fills up. In one case,

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Elminius

modestus larvae settled preferentially onto unfilmed surfaces, a response that would enable them to exploit very recently cleared areas of substratum. These experiments all focus on the responses of sessile animals to biofilms, but as an area is exposed, it is colonized simultaneously by microbes and invertebrate larvae. An incoming larva, then, will encounter newly established residents, plus any nearby adults, as it explores a surface. Some established animals may repel (e.g., Young and Chia, 1981) or attract (e.g., Jensen and Morse, 1984; Keough, 1984b; Raimondi, 1988, 1991), settling larvae. Todd and Keough (1994) tested for the effects of newly-settled individuals of the same species on settlement of a range of taxa, and found few significant effects. They could not examine interspecific responses and were only able to make ad hoc use of animals that had settled onto surfaces. In assembling a picture of the cues that influence settlement under field conditions, it will be important to examine the effects of established residents of different species and ages. In some cases, cues provided by residents might enhance the effects of microbial cues, while in other cases, we might find that the two sets of cues act in opposite directions. In this latter case, it will be a challenge to determine the relative strengths of the two sets of cues as they act under field conditions.

Acknowledgments This work was supported by grants from the Australian Research Council. We appreciate the permission of the Port of Melbourne Authority to work at Breakwater Pier. Jo Klemke provided assistance with field work for some of the experiments.

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