Jennifer L Ripley
email: jripley@mix.wvu.edu

Christy M Foran
email: cmforan@mail.wvu.edu

Influence of estuarine hypoxia on feeding and sound production by two sympatric pipefish species (Syngnathidae)

Keywords: Hypoxia, Feeding, Sound Production, Syngnathus fuscus, Syngnathus floridae

This research utilizes the acoustic behavior of two sympatric pipefish species to assess the impact of hypoxia on feeding. We collected northern, Syngnathus fuscus, and dusky pipefishes, Syngnathus floridae, from the relatively pristine Chincoteague Bay, Virginia, USA and audiovisually recorded behavior in the laboratory of fish held in normoxic (> 5 mg/L O 2 ) and hypoxic (2 and 1 mg/L O 2 ) conditions. Both species produced high frequency (~ 0.9 -1.4 kHz), short duration (3 -22 msec) clicks. Feeding strikes were significantly correlated with both wet weight of ingested food and click production. Thus, sound production serves as an accurate measure of feeding activity. In hypoxic conditions, reduced food intake corresponded with decreased sound production. Significant declines in both behaviors were evident after 1 day and continued as long as hypoxic conditions were maintained. Interspecific differences in sensitivity were detected. Specifically, S. floridae showed a tendency to perform head snaps at the surface.

Syngnathus fuscus exhibited a breakdown in the coupling of sound production with food intake in 2 mg/L O 2 with clicks produced in other contexts, particularly choking and food expulsion.

Reductions in feeding will ultimately impact growth, health, and eventually reproduction as resources are devoted to survival instead of gamete production and courtship. This work suggests acoustic monitoring of field sites with adverse environmental conditions may reflect changes in feeding behavior in addition to population dispersal.

Introduction

No environmental factor of such ecological importance to coastal and estuarine systems has changed as rapidly as dissolved oxygen (DO; [START_REF] Diaz | Overview of hypoxia around the world[END_REF][START_REF] Goldberg | Emerging problems in the coastal zone for the twenty-first century[END_REF][START_REF] Nixon | Marine eutrophication: a growing international problem[END_REF].

The Chesapeake Bay is the largest and most productive estuary in the United States, yeilding more than 70% of the nation's blue crab catch [START_REF] Wang | Assessment of best management practices for improvement of dissolved oxygen in Chesapeake Bay estuary[END_REF]. Hypoxia, or DO concentrations below 2 mg/L, dominates the summer months with conditions declining to anoxic levels [START_REF] Diaz | Overview of hypoxia around the world[END_REF][START_REF] Diaz | Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF][START_REF] Nixon | Coastal marine eutrophication: a definition, social causes, and future concerns[END_REF][START_REF] Breitburg | Near-shore hypoxia in the Chesapeake Bay: patterns and relationships among physical factors[END_REF][START_REF] Holland | Long-term variation in a mesohaline Chesapeake Bay macrobenthos: spatial and temporal patterns[END_REF][START_REF] Officer | Chesapeake Bay anoxia: origin, development and significance[END_REF]. The catalyst for current troubles dates to the 1930s when the human population in the Bay watershed nearly doubled and inorganic fertilizer use tripled [START_REF] Boesch | Chesapeake Bay eutrophication: scientific understanding, ecosystem restoration, and challenges for agriculture[END_REF][START_REF] Zimmerman | A geochemical record of eutrophication and anoxia in Chesapeake Bay sediments: anthropogenic influence on organic matter composition[END_REF][START_REF] Davidson | Chesapeake waters: four centuries of controversy, concern, and legislation[END_REF][START_REF] Cornwell | A sediment chronology of the eutrophication of the Chesapeake Bay[END_REF][START_REF] Newcombe | Oxygen poor waters in the Chesapeake Bay[END_REF]. Today, DO is a central factor decreasing the capacity of the Chesapeake Bay to support economically important fisheries.

Hypoxia can severely stress, and in some instances eliminate, populations by decreasing growth rate, altering behavior, disrupting endocrine function, and interfering with other physiological activities [START_REF] Wu | Aquatic hypoxia is an endocrine disruptor and impairs fish reproduction[END_REF][START_REF] Taylor | Physiological performance of juvenile southern flounder, Paralichthys lethostigma (Jordan and Gilbert, 1884), in chronic and episodic hypoxia[END_REF]). Most fishes immediately depress feeding activity to conserve energy and reduce oxygen consumption in hypoxic conditions. Food intake decreased significantly in Atlantic cod (Gadus morhua) and rainbow trout (Oncorhynchus mykiss) held at 60% water oxygen saturation [START_REF] Chabot | Reduced growth of Atlantic cod in non-lethal hypoxic conditions[END_REF][START_REF] Neji | Relationships between feeding, hypoxia, digestibility and experimentally induced furnunculosis in rainbow trout[END_REF]. Reduced food consumption results in growth impairment for juvenile turbot (Scopthalmus maximus) and Europen sea bass (Dicentrarchus labrax) in DO less than 3.5 mg/L [START_REF] Pichavant | Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass[END_REF][START_REF] Pichavant | Effects of hypoxia on growth and metabolism of juvenile turbot[END_REF].

Hypoxia also significantly reduced energy allocated to somatic growth and reproduction in carp (Cyprinus carpio; [START_REF] Zhou | Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio) under hypoxia[END_REF]. Thus, depressed feeding activity under hypoxic conditions is potentially harmful to population stability.

Acoustic signals play a key role in intraspecific and interspecific communication. Sound production by the family Syngnathidae (pipefishes and seahorses) involves bony edges of the skull, particularly the supraoccipital-coronet articulation [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF][START_REF] Fish | Sounds of western North Atlantic fishes[END_REF][START_REF] Fish | The production of underwater sound by the Northern seahorse, Hippocampus hudsonius[END_REF][START_REF] Fish | Studies on the production of underwater sound by North Atlantic coastal fishes[END_REF]. Seahorse sounds, described as loud clicks resembling the snapping of a finger against the thumb, have been recorded during introduction to new surroundings, courtship and spawning, and feeding [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF][START_REF] Bergert | Morphology and kinematics of prey capture in the syngnathid fishes Hippocampus erectus and Syngnathus floridae[END_REF][START_REF] Fish | Sounds of western North Atlantic fishes[END_REF][START_REF] Tarasov | Zhivyye zvuki morya (The live sounds of the sea)[END_REF][START_REF] Fish | The character and significance of sound production among fishes of the Western North Atlantic[END_REF][START_REF] Fish | The production of underwater sound by the Northern seahorse, Hippocampus hudsonius[END_REF][START_REF] Gill | The life history of the sea horses (Hippocampids)[END_REF]. Behavioral observations revealed a positive correlation between sound production and feeding strikes in two seahorse species [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF]. Reports on sound production by the closely related pipefishes are considerably rare. Studies have recorded clicking by Syngnathus louisanae [START_REF] Burkenroad | Notes on the sound-producing marine fishes of Louisiana[END_REF] and S. fuscus [START_REF] Fish | Studies on the production of underwater sound by North Atlantic coastal fishes[END_REF], but the context, mechanism, frequency, and duration of pipefish sounds have not been documented. We recorded sounds produced during feeding by northern, Syngnathus fuscus, and dusky pipefishes, Syngnathus floridae, of the Chesapeake Bay region. Our first study explored the relationship between sound production and feeding. Next, under hypoxic conditions, we hypothesized food intake and,temporal frequency of sound production would decrease. Finally, by studying two sympatric species, we examined potential interspecific differences in hypoxia sensitivity. The purpose of this investigation is to propose the utilization of acoustic monitoring as an indicator of population changes in feeding.

Materials and methods

Study animals

We collected pipefish by seining in the seasonally abundant shallow eelgrass beds (Zostera marina) of the Chincoteague Bay, VA, USA from May through September 2004-2005. Details of the site, collection technique, and reproductive condition of the pipefishes including sex ratios, growth rates, population size structure, and spawning synchrony are presented in Ripley and Foran (in press). Brooding S. fuscus and S. floridae males, along with other pipefish species, have been shown to feed less than conspecific gravid females [START_REF] Teixeira | Trophic ecology of two congeneric pipefishes (Syngnathidae) of the lower York River, Virginia[END_REF][START_REF] Steffe | Habitat selection and diet in two species of pipefish from seagrass: sex differences[END_REF][START_REF] Svensson | Reproductive costs in two sex role reversed pipefish species (Syngnathidae)[END_REF]. To eliminate this potential source of variation, we used only non-brooding males in this study. Dissolved oxygen levels, water temperature, and salinity were recorded with a YSI 85 handheld meter (YSI Incorporated, Yellow Springs, OH, USA) and are presented in Ripley and Foran (in press). Pipefish were housed for a maximum of three days at the field station in aerated tanks with daily water changes before being transported to the laboratory in Kordon® breathing bags (Novalek Inc., Hayward, CA, USA) chilled with ice.

Baseline feeding behavior

Acclimation to laboratory conditions and diet spanned at least four weeks during which pipefish were housed in aerated, filtered 38 liter tanks at 24 ± 1 °C on a 14:10 photoperiod (6:00 on; 20:00 off). The purpose of the lengthy acclimation period was to minimize influences on respiratory physiology and/or behavior stemming from adverse field conditions, travel stress, and laboratory housing. Photoperiod mimicked the average seasonal trend for the collection location, while temperature was maintained on the low end of the seasonal range to ward off disease and parasite infections. We used artificial seawater (Instant Ocean) in the laboratory and decreased salinity from Bay averages of 30 to 20-25 parts per thousand to prevent parasite transfer between animals (Ripley & Foran, in press). Fish were separated by species and sex and maintained at a density of less than ten per tank. Pipefish were fed frozen mysid shrimp and Artemia to satiation three times daily.

For the baseline feeding observations, pipefish were moved to acoustically buffered 38 liter tanks maintained as described above. Acoustic buffering was achieved by standing tanks on a 4 cm rubber platform. Tanks held a single, large piece of non-living coral in the center to buffer pH, thereby circumventing any pH changes from the build-up of carbon dioxide [START_REF] Sagasti | Effects of periodic hypoxia on mortality, feeding and predation in an estuarine epifaunal community[END_REF][START_REF] Burnett | The challenges of living in hypoxic and hypercapnic aquatic environments[END_REF]. Artificial eelgrass fashioned from nylon rope filled the right side of the tank while the left contained the back filter but was otherwise bare. Pairs of conspecific, same sex fish were moved one at a time with 5 minutes separating the introduction of each. We recorded acoustic and visual behavior during this introduction event beginning 1 minute before the first fish was added to 10 minutes after the second addition for a total taping of 16 minutes. Food was not introduced during this time. Synchronous audiovisual data was recorded by an omnidirectional hydrophone (Aquarian Audio Products, Anacortes, WA, USA; model AQ-6; frequency range 20Hz-100kHz) connected to a Sony Hi8 video camera recorder (model no. CCD-TRV43) and a Hewlett Packard Pavilion zv5000 laptop computer. The video camera stood on a tripod approximately 1.5 m in front of the tank. We digitized acoustic data by the connection to the laptop with CoolEdit 2000 (Syntrillium Software Corporation, Phoenix, AZ, USA) and Raven 1.2 sound analysis software (Cornell Laboratory of Ornithology, Ithaca, NY, USA). Sounds were digitized at a 16-bit resolution and a sampling rate of 44.1 kHz. All pumps, filters, and heaters were turned off during recording sessions.

Single sex, conspecific pairs were acclimated to the acoustic tanks for three to five days during which food was always introduced on the right, barren side of the tank to allow unobstructed viewing. Dissolved oxygen levels were documented prior to audiovisual recording.

We videotaped and acoustically digitized the feeding event for 15 minutes starting with the introduction of food. Food was added to the right section of the tank in 0.33 mL increments every 5 minutes with a graduated, disposable pipette to ensure constant movement of available food. To quantify food consumption, we immediately anaesthsized both fish with 3aminobenzoic acid ethyl ester (MS222; Sigma Aldrich) in saltwater. Standard length (tip of the snout to the caudal peduncle) and weight were recorded. [START_REF] Ryer | Feeding chronology, daily ration, and the effects of temperature upon gastric evacuation in the pipefish, Syngnathus fuscus[END_REF] showed the time required for S. fuscus to completely evacuate a meal was 14.1 hours at 23°C with time decreasing at higher temperatures. The recording session was always the morning episode.

Thus, the last feeding event was at least 16 hours earlier, indicating the entire content of the gut was consumed during the recording. Syngnathus fuscus and S. floridae possess relatively undifferentiated gastrointestinal tracts [START_REF] Teixeira | Trophic ecology of two congeneric pipefishes (Syngnathidae) of the lower York River, Virginia[END_REF][START_REF] Ryer | Feeding ecology of the Northern pipefish, Syngnathus fuscus, in a seagrass community of the lower Chesapeake Bay[END_REF].

Therefore, the contents of the whole gastrointestinal tract were extracted and wet weight recorded. We repeated these procedures on 10 males and females of each species for a total of 40 individuals examined.

Behavior and sound analysis

Videotapes were reviewed noting sound production, behavioral accompaniment, context, position in the water column and tank, individual identity, and counter time. The frequency of behavioral pairing with sound production was calculated for each individual and averaged for the species. Non-parametric Spearman correlations compared sound production with food intake, or the number of Artemia consumed, and wet weight of gut content to investigate the accuracy of sound production as a measure of feeding behavior for S. fuscus and S. floridae.

Five sounds produced by each pipefish were randomly selected for acoustic analysis.

Only sounds produced when the fish was positioned in the water column were analyzed.

Background noise and artifacts characteristic of small tanks complicate acoustic recordings [START_REF] Okumura | Analyses of small tank acoustics: empirical and theoretical approaches[END_REF][START_REF] Yager | Underwater acoustic communication in the African pipid frog Xenopus borealis[END_REF][START_REF] Parvulescu | The acoustics of small tanks[END_REF]. Reflections and reverberations off the tank walls may interfere with parameter measurements when fish are resting on the bottom or against the side. This subset of analyzed sounds also ensured consistent sample sizes between exposure days during the hypoxia studies when sound production decreased. We measured two parameters: sound duration and peak frequency. Sound duration quantifies the temporal length of the sound and is calculated from the waveform. Peak frequency is the frequency at peak amplitude read from the spectrogram. Peak frequency is less affected by tank characteristics than other frequency assessments, making it the preferred measure in this study employing tanks of rather small size (de [START_REF] De Amorim | Sound production in the blue-green damselfish, Chromis viridis (Cuvier, 1830) (Pomacentridae)[END_REF]. We used Raven 1.2 to analyze sounds (FFT = 4095, Hamming).

Feeding and acoustic behavior in hypoxic waters

Syngnathus fuscus and S. floridae pipefish were moved to acoustic tanks and videotaped as described in the baseline study. After a three to five day acclimation in normoxic conditions, DO levels were recorded and a 15-minute morning feeding session videotaped. This recording was designated day 0. Dissolved oxygen levels were then lowered over a four hour period by bubbling nitrogen gas. Similar abrupt oxygen changes take place in the Chesapeake Bay [START_REF] Sagasti | Effects of periodic hypoxia on mortality, feeding and predation in an estuarine epifaunal community[END_REF][START_REF] Kuo | Hypoxia and salinity in Virginia estuaries[END_REF]. Two hypoxic levels were tested: 2 and 1 mg/L O 2 . Hypoxia, the level at which many animals suffocate, is defined as DO concentrations below 2 mg/L [START_REF] Diaz | Overview of hypoxia around the world[END_REF][START_REF] Diaz | Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF][START_REF] Nixon | Coastal marine eutrophication: a definition, social causes, and future concerns[END_REF]. We chose 1 mg/L O 2 as a second treatment because this level is lethal to Bay grasses [START_REF] Holmer | Photosynthetic and growth response of eelgrass to low oxygen and high sulfide concentrations during hypoxic events[END_REF][START_REF] Smith | Effects of anaerobiosis on root metabolism of Zostera marina (eelgrass): implications for survival in reducing sediments[END_REF]. Dissolved oxygen levels were maintained within ± 0.2 mg/L of the target concentration. The YSI 85 meter was calibrated every morning and experimental DO levels checked five times daily. As the typical duration of hypoxic episodes in the Chesapeake Bay lasts 5 days [START_REF] Sagasti | Effects of periodic hypoxia on mortality, feeding and predation in an estuarine epifaunal community[END_REF][START_REF] Kuo | Data report: hypoxia in the York River[END_REF], the study spanned the same period. We videotaped morning feeding sessions on days 1, 3, and 5. On the even days, 50% water changes were performed to maintain water quality and prevent the buildup of metabolic byproducts as the filters did not run during exposures. We established reservoirs of water of the appropriate DO level to be utilized during water changes to avoid large fluctuations in oxygen concentrations. Water quality was tested on odd days and measures kept within an optimum range (ammonia < 0.50 parts per million (ppm), nitrate < 10 ppm, nitrite < 0.50 ppm, pH = 8.2 -8.6). Following the feeding event on day 5, we anaesthsized fish and recorded standard length and weight. Ten males and females of both species were tested at each hypoxia level for a total of 80 experimental pipefish.

Videotapes were reviewed noting the same measurements listed for the baseline study.

The frequency of behavioral pairing with sound production was calculated for each individual and averaged for the species at both DO concentrations. Interspecific changes in food intake and sound production with hypoxia exposure were investigated for the two DO levels. Differences in sound production between oxygen concentrations were examined for each species. We randomly selected 5 sounds produced on day 0 and 3-5 sounds from each hypoxia recording for sound duration and peak frequency analysis. Differences in sound duration and frequency on day 0 versus days 1, 3 and 5 were investigated for individual pipefish. Individuals that did not produce sound on one or more days or only produced sound when resting on the bottom of the tank were excluded from this investigation of potential changes in sound structure due to the lack of data. All statistical analyses were performed with JMP 5.1.

Results

Dissolved oxygen concentrations in the Chincoteague Bay, VA, USA measured 4.6-9.3 mg/L during May-September 2004-2005 collections (Ripley & Foran, in press). We assumed the site did not experience extended periods of low DO and experimental fish had not been previously exposed to chronic hypoxia.

This study recorded 53.85 hours of feeding activity, 19.05 hours of introduction to new surroundings, and 1.72 hours of courtship and spawning by S. fuscus and S. floridae with observation time divided equally between the two species.

Feeding behavior

Food intake by S. fuscus and S. floridae was accompanied by a head snap in which head and snout elevation was followed by a rapid return to the resting position. Behavioral observations determined each feeding strike resulted in the ingestion of one Artemia. Tallying feeding strikes therefore estimates food consumption. In both species, food intake was always accompanied by sound production and a head snap (Table 1). However, pipefish produced sound without ingesting food (Table 1). Each time a sound was produced, a head snap was also performed. Yet S. fuscus and S. floridae performed head snaps without sound accompaniment (Table 1).

Sound production was positively correlated with feeding strikes in S. fuscus (r = 0.85, p < 0.0001; Fig. 1(a)) and S. floridae (r = 0.97, p < 0.0001; Fig. 1(a)). The number of sounds produced also showed a significant positive correlation with the wet weight of food in the gut of S. fuscus (r = 0.60, p < 0.01; Fig. 1(b)) and S. floridae (r = 0.51, p < 0.001; Fig. 1(b)).

Sex differences in feeding behavior were not detected for S. floridae. Female S. fuscus ingested more food (2-way ANOVA, F = 12.62, p < 0.0001; post-hoc Tukey HSD, Q = 2.69, = 0.05 (post-hoc test identical for subsequent comparisons)), contained a greater wet weight of food in the gut (F = 6.76, p = 0.001), performed more head snaps (F = 21.09, p < 0.0001), and produced more sounds (F = 22.96, p < 0.0001) than conspecific males. Syngnathus fuscus females were also heavier (F = 18.63, p < 0.0001) and longer (F = 17.91, p < 0.0001). This size dimorphism is characteristic of the species (Ripley & Foran, in press). Weight was positively correlated with food intake (r = 0.68, p = 0.001) and wet weight of ingested food (r = 0.72, p < 0.001). The sex differences in S. fuscus behavior can be attributed to larger females eating more food. Since size distinctions explain the behavioral differences, only interspecific distinctions were examined in the remaining analyses.

Feeding click characterization

Syngnathus fuscus and S. floridae produce high frequency, short duration clicks with food intake (Table 2; Fig. 2). Body weight was not related to peak frequency in either species (S. fuscus; r = 0.02, p = 0.81; S. floridae; r = 0.05, p = 0.44). Interspecific differences were detected in sound duration (Nested 2-way ANOVA, F =2.51, p = 0.02; post-hoc Student's t, t = 1.97, = 0.05) and peak frequency (Nested 2-way ANOVA, F = 6.67, p < 0.0001; post-hoc Student's t, t = 1.97, = 0.05). Syngnathus fuscus produce longer sounds of higher frequency.

Click production by S. fuscus and S. floridae was largely limited to feeding activity.

Neither S. fuscus nor S. floridae clicked when moved individually or in same sex pairs to a new aquarium. Males aligned parallel and quivered with one another when courting females, but clicks were not produced. Courting pairs also exchanged quivers. Although low frequency sound production most likely accompanied aggressive and courtship quiver behavior, it was not quantified in this study. Both S. fuscus and S. floridae clicked when held out of water during field collections and in the laboratory.

Feeding behavior in 2 mg/L O 2

Syngnathus fuscus and S. floridae exhibited significant decreases in food intake after one day in 2 mg/L O 2 (2-way ANOVA, F = 9.36, p < 0.0001; post-hoc Tukey HSD, Q = 2.60, = 0.05; Fig. 3(a)). Over the five day period of hypoxia, the frequency of feeding strikes did not change (Fig. 3(a)). Sound production by S. fuscus and S. floridae decreased significantly following one day of 2 mg/L O 2 and the number of clicks did not recover or wane during the hypoxia period (2-way ANOVA, F = 6.80, p < 0.0001; post-hoc Tukey HSD, Q = 2.60, = 0.05; Fig. 3(b)). Species differed in the number of sounds produced with S. floridae clicking significantly less often than S. fuscus (post-hoc Student's t, t = 1.98, = 0.05; Fig. 3(b)). In this hypoxia study, pipefishes did not significantly differ in standard length (2-way ANOVA, F= 2.08, p = 0.12; Table 2) or weight (2-way ANOVA, F = 1.38, p = 0.26; Table 2) based on species or sex.

Many clicks produced at 2 mg/L O 2 by S. fuscus and S. floridae coincided with behaviors other than food intake. Every feeding strike was accompanied by sound production in both species. Sound production without food intake was greater for S. fuscus (2-way ANOVA, F = 4.97, p < 0.0001; post-hoc Student's t, t = 1.98, = 0.05; Fig. 4(a-b)). The significant interruption in the correlation of sound and food intake for S. fuscus appeared one day in 2 mg/L hypoxic conditions and showed the greatest disruption at day 5 (post-hoc Tukey HSD, Q = 2.60, = 0.05; Fig. 4(a)).

When food intake did not accompany sound production, one of four other behaviors corresponded with clicks in this study. Behaviors included a series of head snaps with a piece of food moving back and forth in the snout that eventually led to food expulsion, head snaps coupled with food expulsion, and head snaps at the water surface. The fourth behavior was solitary head snaps without any observable context. Head snaps in a series leading to food expulsion were the most performed of these behavioral accompaniments for both species (S.

fuscus; 1-way ANOVA, F = 498.18, p < 0.0001; post-hoc Tukey-Kramer HSD, Q = 2.58, = 0.05; Fig. 4(a); S. floridae; 1-way ANOVA, F = 179.93, p < 0.0001; post-hoc Tukey-Kramer HSD, Q = 2.59, = 0.05; Fig. 4(b)). Interspecific and hypoxia-induced differences were not observed in the performance of food expulsion (F = 0.61, p = 0.75), repetitive head snaps (F = 0.96, p = 0.46), or solo head snaps (F = 0.35, p = 0.93; Fig. 4(a-b)). However, S. floridae coupled clicks with head snaps at the surface more often than S. fuscus during both the normoxic and hypoxic periods (2-way ANOVA, F = 2.77, p = 0.01; post-hoc Student's t, t = 1.98, = 0.05; Fig. 4(a-b)).

Feeding behavior in 1 mg/L O 2

Food intake by S. fuscus and S. floridae declined significantly in 1 mg/L O 2 (2-way ANOVA, F = 25.56, p < 0.0001; Fig. 5(a)). Feeding strikes decreased after one day in 1 mg/L O 2 and did not change over the duration of exposure (post-hoc Tukey HSD, Q = 2.60, = 0.05; Fig. 5(a)). Overall S. fuscus performed more feeding strikes than S. floridae (post-hoc Student's t, t = 1.98, = 0.05; Fig. 5(a)). Sound production by S. fuscus and S. floridae also decreased significantly in 1 mg/L O 2 (2-way ANOVA, F = 11.25, p < 0.0001; Fig. 5(b)). Following one day of 1 mg/L O 2 , sound production declined, remaining consistently low over the exposure (post-hoc Tukey HSD, Q = 2.60, = 0.05; Fig. 5(b)). Syngnathus fuscus produced significantly more clicks than S. floridae (post-hoc Student's t, t = 1.98, = 0.05; Fig. 5(b)). Syngnathus fuscus females in this hypoxia treatment were longer (2-way ANOVA, F = 6.18, p = 0.001; posthoc Tukey HSD, Q = 2.69, = 0.05; Table 2) and heavier (2-way ANOVA, F = 9.12, p = 0.0001; post-hoc Tukey HSD, Q = 2.69, = 0.05; Table 2) than S. fuscus males.

Both species showed a similar breakdown in the correlation of sound production with food intake. All feeding strikes were accompanied by clicks, but after one day of 1 mg/L O 2 , S. fuscus and S. floridae coupled sound production with food intake less often (2-way ANOVA, F = 9.18, p < 0.0001; Fig. 4(c-d)). The pairing of sound production with feeding strikes did not change over the course of hypoxia exposure for either species (post-hoc Tukey HSD, Q = 2.6, = 0.05; Fig. 4(c-d)).

When sound production did not coincide with food intake, repetitive head snaps leading to food expulsion were the most performed behavioral accompaniment to click production for S. fuscus (1-way ANOVA, F = 112.34, p < 0.0001; Fig. 4(c)) and S. floridae (1-way ANOVA, F = 41.65, p < 0.0001; Fig. 4(d)). Syngnathus floridae exhibited a significant decline in the frequency of food expulsion when DO was decreased to 1 mg/L (2-way ANOVA, F = 3.71, p = 0.001; post-hoc Tukey HSD, Q = 3.09, = 0.05; Fig. 4(d)). Although both species clicked at the surface more frequently under 1 mg/L O 2 (F = 3.45, p = 0.002; post-hoc Tukey HSD, Q = 2.61, = 0.05), S. floridae performed this behavior more often than S. fuscus (post-hoc Student's t, t = 1.98, = 0.05; Fig. 4(c-d)). Interspecific and hypoxia-induced differences were not observed in the performance of repetitive (F = 1.14, p = 0.34) or solo head snaps (F = 0.67, p = 0.70; Fig. 4(c-d)).

Characterization of clicks produced in hypoxic conditions

Analysis of sounds produced in 2 mg/L O 2 did not reveal any significant hypoxia-induced changes in sound duration or peak frequency for either species (Tables 2 and3). Duration and peak frequency also did not change in the 1 mg/L O 2 study (Tables 2 and3). Comparisons between clicks produced during feeding strikes versus those accompanying head snaps before food expulsion did not uncover significant differences in sound parameters (Table 3).

Both species responded to low DO levels with a reduction in sound production.

Discussion

Here we detail the results of two studies. The first study assessed the utility of sound production as a measure of feeding activity. The context of sound production and the characters of these sounds (i.e. duration and peak frequency) were documented. In S. fuscus and S. floridae, sound production was not recorded during competition, courtship and spawning, or the introduction to new surroundings. Sound always accompanied food intake with both species performing head snaps with sound production. The sound is a high frequency, short duration click which does not differ with body size, sex, or species. The second experiment recorded changes in sound production and feeding activity when DO levels were hypoxic. Both species displayed significant reductions in feeding behavior and sound production within 24 hours of hypoxic conditions. Syngnathus fuscus produced fewer sounds and feed less than S. floridae, which were observed to click at the water surface in oxygen limited conditions. These results suggest altered feeding behavior should be considered in acoustic monitoring of populations under hypoxic conditions. Sound production serves as an accurate measure of food intake in S. fuscus and S. floridae. Regardless of DO concentration, 100% of feeding strikes were accompanied by sound production. Observations of Syngnathid seahorses noted 93% of Hippocampus zosterae and 100% of H. erectus feeding strikes were accompanied by clicks [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF]. Feeding strikes by H. zosterae that failed to produce detectable clicks showed reduced relative angular movements of skull components [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF]. The prey capture sequence for Hippocampus seahorses involves a nearly synchronous, extremely rapid head elevation and mouth opening with clicks produced within 2 msec of head elevation [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF][START_REF] Bergert | Morphology and kinematics of prey capture in the syngnathid fishes Hippocampus erectus and Syngnathus floridae[END_REF]. Considering the similarity with S. fuscus and S. floridae feeding strikes, restricted movement may explain the performance of head snaps unaccompanied by sound in this study.

We believe S. fuscus and S. floridae feeding clicks are produced by a stridulation mechanism. Identifying traits of stridulation sound include a non-harmonic structure (i.e. the spectrogram does not resolve into a series of horizontal, parallel bars at harmonic intervals) and a wide frequency spread with the maximum energy located in the higher end of the spectrum [START_REF] Tavolga | Sonic characteristics and mechanisms in marine fishes[END_REF][START_REF] Fish | The character and significance of sound production among fishes of the Western North Atlantic[END_REF][START_REF] Fish | Studies on the production of underwater sound by North Atlantic coastal fishes[END_REF]. Spectrograms of clicks produced with any behavioral accompaniment by S. fuscus and S. floridae never revealed harmonics. Pipefish peak frequency measurements were high, matching the ranges of H. zosterae (2650-3430 Hz) and H. erectus (1960-2370 Hz;[START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF]. Swimbladder and pharyngeal teeth-generated sounds provide an indication of male size in courtship and competitive interactions as larger fish produce sounds of lower frequency (Lobel & Mann, 1995;[START_REF] Myrberg | The sounds of bicolor damselfish (Pomacentrus partitus), predicotrs of body size and a spectral basis for individual recognition and assessment[END_REF].

The absence of this correlation in S. fuscus and S. floridae and other stridulation species can be attributed to the wide frequency range of these sounds [START_REF] Ladich | Comparative analysis of swimbladder (drumming) and pectoral (stridulation) sounds in three families of catfishes[END_REF][START_REF] Tavolga | Sonic characteristics and mechanisms in marine fishes[END_REF][START_REF] Fish | The character and significance of sound production among fishes of the Western North Atlantic[END_REF][START_REF] Fish | Studies on the production of underwater sound by North Atlantic coastal fishes[END_REF]. [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF] surgerically filed the bony supraoccipital-coronet articulation of H. zosterae and H. erectus, eliminating sound production and confirming a stridulation mechanism. Analogous coupling of food intake with clicks, feeding strike head movements, and sound parameters suggest a comparable skull articulation, but differences in pipefish head morphology and posture warrant further investigations to identify the specific structures and mechanism. The functional significance of Syngnathid feeding clicks is not known. Causal observations did not indicate a role of feeding clicks in communicating the presence of food or deterring conspecifics [START_REF] Amorim | Sound production during competitive feeding in the grey gurnard[END_REF][START_REF] Amorim | Growling for food: acoustic emissions during competitive feeding of the streaked gurnard[END_REF].

Seahorse studies have documented clicks during courtship and spawning [START_REF] Colson | Sound production during feeding in Hippocampus seahorses (Syngnathidae)[END_REF][START_REF] Fish | Sounds of western North Atlantic fishes[END_REF][START_REF] Fish | The character and significance of sound production among fishes of the Western North Atlantic[END_REF]. We did not detect sound production during reproduction in S. fuscus and S. floridae. Synganthid species are generally divided among polygamous pipefish and monogamous seahorses with a few pipefish species exhibiting monogamy [START_REF] Carcupino | Functional significance of the male brood pouch in the reproductive strategies of pipefishes and seahorses: A morphological and ultrastructural comparative study on three anatomically different pouches[END_REF][START_REF] Vincent | Pipefishes and seahorses: are they all sex role reversed?[END_REF].

Distinctions in reproductive strategies may translate into differences in click production.

Syngnathid feeding clicks could be similar to the feeding sounds of rainbow trout and may be an unintentional product of the feeding mechanism, playing no communicative role at all [START_REF] Phillips | The feeding sounds of rainbow trout, Salmo gairdneri Richardson[END_REF]. Future studies should investigate the ecological and behavioral implications of sound production in Syngnathidae.

The Chincoteague Bay mirrors the habitat and community structure of the neighboring Chesapeake without the water quality problems [START_REF] Kiddon | Ecological condition of US Mid-Atlantic estuaries, 1997-1998[END_REF][START_REF] Dauer | Long-term trends in the macrobenthos and water quality of the lower Chesapeake Bay (1985-1991)[END_REF]. Dissolved oxygen concentrations rarely measured below state water quality standards of 5 mg/L in our monitoring or Kiddon et al.'s (2003) data from 1997-98, granting the Chincoteague Bay an index of environmental integrity rating of "good" with less than 20% of the area exhibiting impairment (Ripley & Foran, in press). The pipefish in this study, accordingly, were collected from a relatively pristine site and the behaviors observed are assumed to be unaltered by prior exposure to adverse environmental conditions.

Pipefishes are ideal model organisms for studying disturbances to estuarine habitats due to their seasonal abundance and behavioral responses. Syngnathidae is among the most abundant fish families occupying estuarine grass beds worldwide, including the Atlantic-Mediterranean and Indo-Pacific regions [START_REF] Howard | Population dynamics and feeding ecology of pipefish (Syngnathidae) associated with eelgrass beds of Western Port, Victoria[END_REF][START_REF] Pollard | A review of ecological studies on seagrass-fish communities, with particular reference to recent studies in Australia[END_REF]. In the Chesapeake Bay, S. fuscus ranks second and S. floridae sixth in abundance among fishes [START_REF] Orth | Structural components of eelgrass (Zostera marina) meadows in the lower Chesapeake Bay -fishes[END_REF]. The migration of S. fuscus and S. floridae from deeper waters to shallow eelgrass beds overlaps summer hypoxic events (Ripley & Foran, in press;[START_REF] Wang | Assessment of best management practices for improvement of dissolved oxygen in Chesapeake Bay estuary[END_REF][START_REF] Lazzari | Northern pipefish, Syngnathus fuscus, occurrences over the Mid-Atlantic Bight continental shelf: evidence of seasonal migration[END_REF][START_REF] Holland | Long-term variation in a mesohaline Chesapeake Bay macrobenthos: spatial and temporal patterns[END_REF][START_REF] Officer | Chesapeake Bay anoxia: origin, development and significance[END_REF]. As swimming speed and elevated predation risk to brooding males limits movement, pipefish are not able to flee to more oxygenated areas as is common for other fish species [START_REF] Ashley-Ross | Mechanical properties of the dorsal fin muscle of seahorse (Hippocampus) and pipefish (Syngnathus)[END_REF][START_REF] Wannamaker | Effects of hypoxia on movements and behavior of selected estuarine organisms from the southeastern United States[END_REF][START_REF] Roelke | Gender-based differences in habitat selection and activity level in the Northern pipefish (Syngnathus fuscus)[END_REF][START_REF] Breitburg | Episodic hypoxia in Chesapeake Bay: interacting effects of recruitment, behavior, and physical disturbance[END_REF]. Adult S. fuscus and S. floridae survived and, in some cases, continued to feed at 1 mg/L O 2 . This study demonstrated these two pipefish species are tolerant to DO concentrations below levels lethal to most commercially important species of the Bay including the bay grasses which serve as pipefish spawning habitat [START_REF] Miller | Determination of lethal dissolved oxygen levels for selected marine and estuarine fishes, crustaceans, and a bivalve[END_REF][START_REF] Diaz | Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF][START_REF] Davis | Minimal dissolved oxygen requirements of aquatic life with emphasis on Canadian species: a review[END_REF]. Accordingly, acoustic monitoring of pipefish during hypoxic events is a rather conservative estimate of disturbance because the consequences to commercial species are probably more severe.

Behavior is very sensitive to contaminants, demonstrating links between effects at different levels of organization as changes at the biochemical level may be readily translated into population level impacts [START_REF] Shumway | A neglected science: applying behavior to aquatic conservation[END_REF][START_REF] Weis | Altered predator/prey behavior in polluted environments: implications for fish conservation[END_REF][START_REF] Morgan | Effect of fenitrothion on the foraging behavior of juvenile Atlantic salmon[END_REF]. Hypoxia immediately reduced feeding in S. fuscus and S. floridae. For S. fuscus, energy assimilation depends on temperature. Higher water temperatures speed gastric evacuation and increase the number of prey items processed [START_REF] Ryer | Feeding chronology, daily ration, and the effects of temperature upon gastric evacuation in the pipefish, Syngnathus fuscus[END_REF]. With summer hypoxic events correlating to periods of high prey availability, S. fuscus energy assimilation can be greatly limited by decreased food intake following only one day of hypoxia. The pipefish lifetime spans 2 years, while most Chesapeake Bay epifaunal species live only one season (Ripley & Foran, in press;[START_REF] Able | The first year in the life of estuarine fishes in the middle Atlantic Bight[END_REF][START_REF] Campbell | Life history characteristics of the Northern pipefish, Syngnathus fuscus, in Southern New Jersey[END_REF][START_REF] Abbe | Ecological studies in the middle reach Chesapeake Bay: Calvert Cliffs[END_REF]. Short disruptions in feeding could impact a considerable portion of an animal's lifetime. Even brief reductions in food intake may profoundly influence growth, health, or reproduction as resources are devoted to survival instead of gamete production.

Pipefish feeding activity is an important component in the transfer of carbon from planktonic to benthic food webs [START_REF] Ryer | Feeding chronology, daily ration, and the effects of temperature upon gastric evacuation in the pipefish, Syngnathus fuscus[END_REF]. Long snouts characterize S. floridae (1.65-1.85 in head) and shorter snout-to-head lengths distinguish S. fuscus (2-2.5 in head; [START_REF] Murdy | Fishes of the Chesapeake Bay[END_REF][START_REF] Teixeira | Trophic ecology of two congeneric pipefishes (Syngnathidae) of the lower York River, Virginia[END_REF][START_REF] Herald | Studies on the Atlantic American pipefishes with descriptions of new species[END_REF][START_REF] Breder | Field book of marine fishes of the Atlantic coast[END_REF]. Accordingly, S. floridae in the Chesapeake Bay region prefer to prey upon various sized grass shrimp (Palaemonetes pugio), while S. fuscus appear to be more gape-limited, only feeding on small crustaceans [START_REF] Teixeira | Trophic ecology of two congeneric pipefishes (Syngnathidae) of the lower York River, Virginia[END_REF][START_REF] Ryer | Feeding ecology of the Northern pipefish, Syngnathus fuscus, in a seagrass community of the lower Chesapeake Bay[END_REF][START_REF] Ryer | Feeding chronology, daily ration, and the effects of temperature upon gastric evacuation in the pipefish, Syngnathus fuscus[END_REF]. Amphipods, particularly Gammarus mucronatus, comprise the majority of the diet of S. fuscus and in fact, S. fuscus predation is believed to largely control populations of this amphipod [START_REF] Ryer | Feeding ecology of the Northern pipefish, Syngnathus fuscus, in a seagrass community of the lower Chesapeake Bay[END_REF]. Reduced predation associated with hypoxia could impact community dynamics in estuarine food webs.

Our observations revealed choking-like repetitive head snaps and food expulsion prevailed in hypoxic conditions when sound coincided with activities besides food consumption.

Limited energy availability and expenditure may be preventing food from traveling the entire distance of the snout during ingestion. Differences in interspecific snout length did not translate into increased performance of these behaviors by S. floridae [START_REF] Murdy | Fishes of the Chesapeake Bay[END_REF][START_REF] Teixeira | Trophic ecology of two congeneric pipefishes (Syngnathidae) of the lower York River, Virginia[END_REF][START_REF] Herald | Studies on the Atlantic American pipefishes with descriptions of new species[END_REF][START_REF] Breder | Field book of marine fishes of the Atlantic coast[END_REF]. Measurement of angular movements during head snaps would indicate reductions in feeding force. Energy wasted choking on and expelling food may exceed energy acquisition. The danger of having food caught in the snout as well as these energy costs may account for reduced food intake. Decreased locomotion conserves energy and thus, reduced swimming may also be the cause for the decline in feeding as searching for and capturing prey would be limited [START_REF] Wu | Hypoxia: from molecular responses to ecosystem responses[END_REF]. Both species, but more so for S. floridae, gulped air at the surface in hypoxic conditions. Naked gobies effectively use aquatic surface respiration (ASR) to escape low oxygen in the field [START_REF] Breitburg | Episodic hypoxia in Chesapeake Bay: interacting effects of recruitment, behavior, and physical disturbance[END_REF]. By swimming to the surface, fish can irrigate their gills with more highly oxygenated surface water [START_REF] Gee | Reactions of gobioid fishes to hypoxia: buoyancy control and aquatic surface respiration[END_REF][START_REF] Kramer | Dissolved oxygen and fish behavior[END_REF].

Due to inherent and hypoxia-induced swimming constraints, pipefish appear to employ the same behavior. Anatomical specializations associated with surface respiration in pipefishes were not investigated in this study.

Syngnathus fuscus and S. floridae show interspecific differences in hypoxia sensitivity.

Compared to S. floridae, S. fuscus demonstrated more frequent production of clicks accompanying choking, food expulsion, and solitary head snaps and a decreased tendency to perform ASR. Differences in geographic distributions may offer an explanation for these distinctions. A northern range from Halifax to North Carolina characterizes S. fuscus, while S. floridae are common from the Chesapeake Bay to Panama [START_REF] Dawson | Family Syngnathide[END_REF]. These distributions translate into differences in seasonal recruitment to the Chesapeake and Chincoteague Bays.

Syngnathus fuscus abundance peaks an entire month before S. floridae in late July and early August (Ripley & Foran, in press;[START_REF] Mercer | The comparative ecology of two species of pipefish (Syngnathidae) in the York River, Virginia[END_REF]. Hypoxia problems dominate late summer in warm, southern bodies of water with large human populations and abundant agricultural activity [START_REF] Diaz | Overview of hypoxia around the world[END_REF]. Common hypoxia events will correspond to when and where S. floridae are abundant. Hence, prevalence in areas plagued with low DO is consistent with decreased sensitivity and/or adaptation.

The practicality of acoustically monitoring pipefish feeding clicks is confounded by the variety of sympatric sound producing organisms. Snapping shrimp (Crangonidae), mussels (Mytilidae), sea urchins (Echinometridae), and various crabs produce stridulation sounds in estuarine and coastal ecosystems [START_REF] Fish | Biological sources of sustained ambient sea noise[END_REF]. Still, acoustic monitoring is feasible and a valuable tool to employ [START_REF] Shumway | A neglected science: applying behavior to aquatic conservation[END_REF]. Passive acoustic detection of the damselfish, Dascyllus albisella, in the field has allowed reproductive activity to be monitored over long periods and wide spatial scales (Mann & Lobel, 1995). Rather than specifically pinpoint pipefish feeding clicks, an array of hydrophones could record overall noise levels for comparisons between sites. Quieter locales resulting from either a decline in sound production as illustrated in this study or a decrease in density from physical avoidance would suggest adverse environmental conditions. Table 1 Behavioral pairings for Syngnathus fuscus (bold text) and Syngnathus floridae * The mean ± SE frequency (%) of coupling the actions listed down the left column with the actions listed along the top are reported. For example, 68.6% of the sounds produced and 66.0% of the head snaps performed by S. fuscus accompany food intake.

  Syngnathus fuscus and S. floridae in 2 mg/L O 2 produced more clicks than conspecifics in 1 mg/L O 2 (S. fuscus; post-hoc Student's t, t = 1.98, = 0.05; Fig. 6(a); S. floridae; post-hoc Student's t, t = 1.98, = 0.05; Fig. 6(b)).

Figure Captions Fig. 1 .

 Captions1 Figure Captions

Fig. 2 .

 2 Fig. 2. Spectrograms (top) and waveforms (bottom) of clicks produced during food intake by (a)

Fig. 5 .Fig. 6 .

 56 Fig. 5. Effects of five days of 1 mg/L O 2 on (a) food intake and (b) sound production by

  Summary of p-values investigating individual differences in sound parameters over the course of hypoxia exposure and between clicks produced during food intake versus repetitive head snaps leading to food expulsion

	Table 3
		Sound Production S. fuscus p-values	Food Intake*		Head Snap S. floridae	p-values
	Parameter	n		Median	Min	Max	n	Median	Min	Max
	Sound Production Changes with exposure duration 2 mg/L		68.6 ± 3.4 82.8 ± 4.1		100 100
	Duration Food Intake Peak Frequency	14 14	100 100	0.4006 0.3452	0.2112 0.1527	0.9834 0.7147	100 20 100 20	0.4377 0.4186	0.1371 0.2279	0.9947 0.7807
	1 mg/L Head Snap Duration	13	96.2 ± 1.8 93.9 ± 1.4 0.3777	66.0 ± 2.8 77.7 ± 4.2 0.1980	0.5993	12	0.4686	0.2082	0.6931
	Peak Frequency	13		0.3774	0.1708	0.7386	12	0.4288	0.2119	0.9863
	Comparison between feeding strikes and series leading to food expulsion
	2 mg/L
	Duration	11		0.3776	0.1984	0.7352	8	0.5540	0.1434	0.9109
	Peak Frequency	11		0.4797	0.1155	0.9211	8	0.4870	0.1076	0.8703
	1 mg/L
	Duration	8		0.2837	0.1511	0.4868	13	0.3913	0.1646	0.4501
	Peak Frequency	8		0.6512	0.4367	0.9484	13	0.4040	0.1977	0.8954

Acknowledgements

Pipefish collection was approved by the Commonwealth of Virginia Marine Resources

Commission (permits 04-22 and 05-18). All animal care and methods complied with regulations

enforced by the West Virginia University Animal Care and Use Committee (protocol number 03-0501, November 2003). We thank the Marine Science Consortium of Wallops Island, Virginia for the use of their facility and equipment. We also thank E. Anderson, D. Hardee, J. Marshall, A. Martin, W. Sites, T. Stueckle, and R. Torres for assistance in field collections and laboratory maintenance. This research was supported in part by a student research award to JR from the Chesapeake and Potomac Regional Chapter of the Society of Environmental Toxicology and Chemistry, the Oak Ridge Associated Universities, the West Virginia University Research Corporation, and the West Virginia University Department of Biology.


David J Blockley
email: dblockley@eicc.bio.usyd.edu.au

Effect of wharves on intertidal assemblages on seawalls in Sydney Harbour, Australia

Keywords: Algae, Artificial habitats, Coastal structures, Grazing molluscs, Sessile invertebrates Shading, Sydney Harbour, Temperature, Urbanisation

Worldwide, urbanisation has resulted in extensive replacement of natural habitats with manmade habitats. In Sydney Harbour, Australia, approximately half of the natural foreshore has been replaced by seawalls. Many of these have wharves built over part of their length, which could affect intertidal assemblages on seawalls beneath the wharves. This was tested by sampling and comparing assemblages under and not under wharves in Sydney Harbour. Assemblages differed between the two habitats, with greater cover of macro-algae and abundance of grazing molluscs on seawalls without a wharf and, to a lesser extent, greater cover of sessile invertebrates on seawalls under a wharf. There was, however, considerable spatial variability among locations in composition of assemblages and the species dominating differences between the two habitats. The impact of multiple artificial structures in close proximity and the variability among apparently homogeneous artificial habitats must be considered for the management of urbanised estuaries.

Introduction

Urbanisation of coastal waters has resulted in proliferation of a variety of different artificial marine structures (e.g. pontoons, pilings and seawalls), each supporting different assemblages (Connell & Glasby, 1999;Glasby & Connell, 1999). Seawalls are among the most common marine habitat within urbanised estuaries and bays [START_REF] Davis | Artificial armored shorelines: sites for opencoast species in a southern California bay[END_REF][START_REF] Chapman | Paucity of mobile species on constructed seawalls: effects of urbanization on biodiversity[END_REF], but often have other types of artificial structures built in close proximity, such as wharves (Blockley & Chapman, In press ). These structures might influence the assemblage living on the seawall by increasing or decreasing abundances or covers of different species.

The sections of seawalls under wharves are potentially subject to different environmental conditions (e.g. light, temperature, rain and wind) to those in the open. Wharves, jetties and similar structures have been found to reduce the growth of seagrasses [START_REF] Short | Quantifying eelgrass habitat loss in relation to housing development and nitrogen loading in Waquoit Bay[END_REF][START_REF] Burdick | The effects of boat docks on eelgrass beds in coastal waters of Massachusetts[END_REF] and density of salt marsh plants [START_REF] Sanger | Cumulative impacts of dock shading on Spartina alterniflora in South Carolina estuaries[END_REF]. The effect of wharves and jetties on assemblages living on other artificial habitats has also been examined in subtidal habitats (e.g. for pilings), with reductions in the cover and types of algae in shaded areas and an increase in sessile invertebrates (Glasby, 1999a;[START_REF] Glasby | Effects of shading on subtidal epibiotic assemblages[END_REF]. The effect of wharves on intertidal hard substrata has not, however, received much attention. This is an important consideration because wharves are most profuse in areas that are already urbanised and so are most likely to be built in association with other artificial structures, frequently seawalls (Blockley & Chapman, In press ).

This study was done in Sydney Harbour, Australia, where most wharves are built over parts of the extensive network of seawalls. Sections of seawalls that were either shaded or unshaded by wharves were predicted to have different assemblages and covers or abundances of individual taxa would differ between shaded or unshaded seawalls. Specifically, it was predicted that; (1) the cover of algae would be greater on unshaded seawalls where there is more light (e.g. [START_REF] Clark | Effects of shade from multiple kelp canopies on an understory algal assemblages[END_REF], (2) grazing invertebrates would be more abundant on unshaded seawalls because this is where their food source was predicted to be greater and (3) sessile invertebrates would have greater cover on shaded seawalls because they would not have to compete for space with algae, nor be dislodged by mobile invertebrates and might be benefited by the presence of a wharf (e.g. reduction of thermal stress and desiccation). It was also predicted that these patterns would be spatially and temporal consistent throughout Sydney Harbour.

Methods

This study was done at seven locations in Sydney Harbour: Athol Bay, Cremorne Point, Rushcutters Bay, Hermit Point, Parriwi Head, Little Manly Point and Quarantine Station (Figure 1).

All locations had seawalls with adjacent sections either with or without wharves. At each location, one section of seawall, approximately 5 m wide, under a wharf and one not under a wharf were sampled. Despite considerable variability in the design of wharves in this study, each 5 m section under a wharf was completely in shade and at least 1 m from the edge of the wharf. For each section, ten replicate 20 x 25 cm quadrats were sampled at the mid-(0.9 -1.1 m above chart datum) and low-tidal (0.6 -0.8 m above chart datum) heights. Percentage cover of all algae and sessile invertebrates were measured and all mobile invertebrates counted. Where possible, without destructive sampling, organisms were identified to species. Where complexes were formed, so that individual species could not be distinguished (e.g. some of the small foliose algae and sponges), these were assigned to broad groups (e.g. mixture of sponges referred to as "orange sponge").

Sampling was repeated four times; in March, June, October, 2003 andFebruary, 2004. Even though the same sections of seawall were sampled each time, these times are far enough apart to provide independent data [START_REF] Underwood | Spatial analyses of intertidal assemblages on sheltered rocky shores[END_REF][START_REF] Bulleri | Intertidal assemblages on seawalls and vertical rocky shores in Sydney Harbour (Australia)[END_REF].

Replicates were sampled randomly across the wall each time, so quadrats were not fixed. At the low-tidal height, only six of the locations were sampled because the lower shore of Parriwi Head was buried by a sand bank after the first sampling.

Environmental conditions on seawalls under or not under wharves were measured, with incident light on the wall and the surface temperature of the substratum the two variables recorded. These variables were considered to most likely differ between seawalls under or not under a wharf and are known to be important in structuring assemblages. The amount of incident light has a strong influence on the growth and survival of algae (Glasby, 1999a;[START_REF] Goldberg | Settlement and post-settlement processes limit the abundance of the geniculate coralline alga Calliarthron on subtidal walls[END_REF][START_REF] Clark | Effects of shade from multiple kelp canopies on an understory algal assemblages[END_REF] and recruitment of invertebrates [START_REF] Pomerat | The influence of surface angle and light on the attachment of barnacles and other sedentary organisms[END_REF][START_REF] Saunders | Interactive effects of shade and surface orientation on the recruitment of spirorbid polychaetes[END_REF].

Incident light was measured (in Lux) using a light meter placed at 10 random areas on seawalls under or not under wharves at each of the 7 locations. Sampling was repeated 5 times each month from April, 2003until March, 2004. Surface temperature of the substratum has been closely linked to the body temperature of many sessile invertebrates [START_REF] Wethey | Biogeography, competition, and microclimate: the barnacle Chthamalus fragilis in New England[END_REF] and is an important indirect measure of thermal stress.

The surface temperature of the seawall was also recorded for each habitat and location using an infra-red electronic thermometer. This measures the temperature of the surface of the substratum directly and is unaffected by air temperature or whether or not the device is in the sun. As for incident light, 10 random areas were sampled in each habitat for each of the 7 locations, with sampling repeated 5 times each month for a year.

All measurements of light and temperature throughout the sampling period were collected at approximately the same time of day and during low tide. It was not possible to collect incident light data during high tide and it would not have made sense to collect thermal data when organisms were immersed. During high tide, the surface temperature would have been controlled by water temperature and would not have differed between shaded and unshaded seawalls. For each month, there were 50 replicate measures of surface temperature and incident light collected for each habitat and location. These were averaged to give a mean monthly value. Collection of continuous data was not feasible with the resources available over such a large number of sites. There are also many restrictions on what can be attached to seawalls, being publically or privately owned structures.

The assemblages were compared using PERMANOVA [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. Matrices of dissimilarity among samples were calculated using the Bray-Curtis dissimilarity coefficient [START_REF] Bray | An ordination of the upland forest communities of Southern Wisconsin[END_REF]. The method of permutation used for all analyses was the permutation of residuals under a full model because this gives the best power and accurate Type 1 error while being reasonably quick [START_REF] Anderson | An empirical comparison of permutation methods for tests of partial regression coefficients in a linear model[END_REF]. Each of the 4 times of sampling was treated as an independent test of the hypothesis that difference between assemblages on seawalls shaded or unshaded by wharves was general to all locations, while in separate analyses each of the 7 locations were used as independent tests of the hypothesis that patterns would be temporally consistent. The data were visualised with nMDS ordinations [START_REF] Clarke | Non-parametric multivariate analysis of changes in community structure[END_REF]. Analyses of variance tested hypotheses that covers or abundances of taxa differed between the two habitats (fixed, orthogonal), among locations (random, orthogonal) and through time (random, orthogonal) for algae, mobile invertebrates and sessile invertebrates. Generally, it was found that heterogeneous variances for percentage covers could not be stabilised by transformation and so analyses were done on untransformed data with a more conservative significance level (see tables). ANOVA is, however, robust to heterogeneity of variance and non-normal data and so is an appropriate analytical technique [START_REF] Underwood | Experiments in Ecology. Their logical design and interpretation using analysis of variance[END_REF].

Results

Comparisons among assemblages

In general, at each time, the assemblages of the two habitats plotted apart on the nMDS graph for each location (Figure 2). Despite some pattern of grouping by habitat, particularly at times 1 and 4, there was a great deal of overlap between habitats shown in the plots among locations (i.e. some points representing assemblages on shaded seawalls plotted close to the cluster of points representing those on unshaded seawalls from different locations (Figure 2)).

There was a significant interaction of Location and Habitat for the mid-and low-tidal heights for the analyses of each time (Table 1a). Therefore, the hypothesis that the patterns of difference would be general to all locations was rejected, although a posteriori pairwise comparisons showed that these habitats were always different for each location. This result is supported by the Bray-Curtis dissimilarities, which show that the dissimilarity between habitats is much greater than the average dissimilarity within each habitat for both tidal heights (Table 2). The dissimilarity among locations for each habitat is also large, supporting the lack of generality of the pattern among locations (hence the variability in Figure 2). Furthermore, the variability among locations for seawalls under wharves was greater than for seawalls not under a wharf at the midtidal height, but similar at the low tidal height (Table 2).

The analyses of each location separately across all times gave interactions between time and habitat for both heights in each location (Table 1b) showing that patterns of difference changed through time. Nevertheless, the plot for each location shows strong separation between habitats across all times indicating differences between the habitats remained large (Figure 3). The temporal changes, illustrated in the nMDS plots, did not show a consistent pattern among locations or between habitats.

Comparisons of individual taxa

Despite no general pattern of difference between habitats for whole assemblages, it was evident that there were consistent patterns for individual taxa among locations at the mid-(Table 3, Figure 4) and low-tidal height (Table 4, Figure 5). The encrusting alga, Hildenbrandia rubra was the dominant alga at the mid-tidal height (Figure 4a), while the turf, Corallina officinalis or foliose green algae, formed by complex patches of Enteromorpha intestinalis, Ulva lactuca, Cladophora sp., Chaetomorpha sp. and Bryopsis sp., dominated the low-tidal height (Figure 5a). Hildenbrandia rubra showed Time x Location and Location x Habitat interactions (Table 3a) while the low-shore algae had significant Time x Location x Habitat interactions (Table 4a). Despite this, the general patterns did not vary much among times, as shown by Hildenbrandia rubra (Figure 4a). For this reason and for the sake of brevity, only one time is shown to illustrate patterns for the other two alga (Figure 5a). There were large differences in cover among locations and the difference between habitats was only significant at some times and locations, but there was a strong pattern of greater cover on seawalls not under wharves (Figure 4a & 5a). Binomial tests indicated that, despite the lack of significant differences at some locations or times, the general pattern was significant (Figure 4a & 5a).

The oyster Saccostrea glomerata was the dominant sessile invertebrate at most locations at the mid-tidal height. The mussel Mytilus galloprovincialis, the tubeworm Galeolaria caespitosa, the barnacles Tesseropora rosea and Tetraclitella purpurascens and an encrusting orange sponge also had relatively large covers at many locations (Figure 4b). The pattern was similar at the low-tidal height, although S. glomerata was not as dominant and the barnacle Austrobalanus imperator was also present (Figure 5b). As for the algae, there were significant Time x Location x Habitat interactions for most analyses of cover of sessile invertebrates (Table 3b &4b). Exceptions were M. galloprovincialis at the mid-tidal height and A. imperator and T. rosea at the low-tidal height, which showed higher-order interactions. Despite this, the patterns were fairly consistent through time, shown for S. glomerata and so only one time is presented for the remaining taxa to illustrate the pattern (Figure 4b). There was, however, considerable difference in the covers of the various species among locations. Covers of S. glomerata were most consistent among locations mid-tidally and were significantly greater on seawalls that were not under a wharf (binomial tests, Figure 4b). This is opposite to the pattern predicted. Similarly, mid-tidally T. rosea had a greater cover on seawalls not under wharves at many locations, although the general pattern was not significant (Figure 4b). The other sessile invertebrates showed the predicted pattern of greater covers on seawalls under wharves at many, but not all locations, however the difference was still significant (Figure 4b).

The cover of S. glomerata, M. galloprovincialis, G. caespitosa, and A. imperator did not differ between habitats significantly at the low-tidal height, although the cover of A. imperator was significantly greater on walls under wharves at Athol Bay and Cremorne Point at all times (Table 4b, Figure 5b). T. rosea showed a significant pattern, although as for the mid-tidal height, it was opposite to that predicted (Figure 5b). T. purpurascens and orange sponge, however, showed a significant pattern of greater cover on walls under wharves as predicted (Figure 5b).

Of the mobile invertebrates, only Chiton pelliserpentis and the limpet, Siphonaria denticulata occurred in large enough numbers to be analysed. Analyses showed that each were more abundant on walls not under a wharf, although not significantly at all locations or times (Table 3c, Figure 6). Nevertheless the general pattern was consistent through time (Figure 6). There were many locations with a significantly greater abundance of both species on unshaded seawalls (Table 3c, Figure 6), although, at some times and locations, the opposite was found. Despite this, the predicted pattern of greater abundance of grazing invertebrates was significant (Figure 6).

Measurements of surface temperature and light on seawalls

The mean surface temperature on walls not under wharves was greater than on walls under wharves at each location (Figure 7). Nevertheless, the magnitude of the difference between the two habitats was not consistent among locations (e.g. the difference between the two habitats at Athol Bay was relatively small, while the difference at Hermit Point was much larger). There was also a difference in range of mean temperatures among locations (e.g. the seawall not under a wharf at Rushcutters Bay had a maximum temperature of approximately 28° C, while the same habitat at Cremorne Point was about 23° C).

Despite the mean surface temperature on seawalls not under wharves being greater than that on walls under wharves, the magnitude of the difference in temperature between habitats changed through time. The greatest difference was in the Austral summer months (December through to February), when the greatest mean surface temperatures were recorded.

The change in mean light, measured in Lux, on seawalls shows some similarity to the pattern seen for mean temperature (Figure 8). Incident light was, as would be expected, greater on seawalls not under wharves at all locations and at all times. There was considerable variability among locations in the magnitude of difference between the two habitats, as well as the range of values recorded and a great deal of variability through time. Unlike temperature, there was no easily discernible pattern for the temporal variability of the light. There did not appear to be a consistent pattern among locations or in relation to seasonal change.

Discussion

In the current study, algae were virtually absent from walls under wharves and there was considerable covers of algae, such as Corallina officinalis and foliose green algae (e.g.

Enteromorpha intestinalis), on unshaded seawalls. Shade can be important in structuring assemblages and has been shown to have important effects on the growth and survival of terrestrial plants [START_REF] Weih | The nitrogen economy of mountain birch seedlings: implications for winter survival[END_REF][START_REF] Valladares | Interactions between water stress, sun-shade acclimation, heat tolerance and photoinhibition in the sclerophyll Heteromeles arbutifolia[END_REF][START_REF] Sans | Positive vs. negative interactions in Picris hieracioides L., a mid-successional species of Mediterranean secondary succession[END_REF], marine algae [START_REF] Glasby | Effects of shading on subtidal epibiotic assemblages[END_REF][START_REF] Goldberg | Settlement and post-settlement processes limit the abundance of the geniculate coralline alga Calliarthron on subtidal walls[END_REF][START_REF] Clark | Effects of shade from multiple kelp canopies on an understory algal assemblages[END_REF] seagrasses [START_REF] Burdick | The effects of boat docks on eelgrass beds in coastal waters of Massachusetts[END_REF] and saltmarsh [START_REF] Sanger | Cumulative impacts of dock shading on Spartina alterniflora in South Carolina estuaries[END_REF]. Animals on intertidal shores have also been shown to be affected by shading, with greater recruitment or survival where there is shade [START_REF] Denley | Experiments on factors influencing settlement, survival, and growth of two species of barnacles in New South Wales[END_REF][START_REF] Harper | Variations in abundance and distribution of the chiton Acanthopleura japonica and associated molluscs on a seasonal, tropical, rocky shore[END_REF]. Shading alone may, therefore, explain the absence of algae and the greater cover of most species of sessile invertebrates under wharves in the present study, although this can not be concluded categorically without manipulative experiments.

Unlike many other sessile invertebrates in this study, the barnacle T. rosea had greater cover on unshaded walls. [START_REF] Denley | Experiments on factors influencing settlement, survival, and growth of two species of barnacles in New South Wales[END_REF] found that T. rosea would settle on unshaded and shaded shores, but survived better in the sun. The oyster S. glomerata, one of the dominant space occupiers, also had greater cover on unshaded walls. [START_REF] Bulleri | Intertidal assemblages on seawalls and vertical rocky shores in Sydney Harbour (Australia)[END_REF] found that S. glomerata was able to dominate space on seawalls in Sydney Harbour, although the pattern was variable among locations. Beds of S. glomerata can be quite thick, providing oysters with protection from environmental extremes (e.g. thermal stress) in a similar way to the presence of a wharf. The lesser cover of other sessile invertebrates on unshaded seawalls may also have meant that oysters had less competition for space and so were able to maintain greater covers than on shaded seawalls.

The present study has shown that there is less incident light reaching seawalls with wharves compared to sections without wharves. [START_REF] Glasby | Effects of shading on subtidal epibiotic assemblages[END_REF] found that the degree of shading may be important in structuring assemblages of epibiota on subtidal pier pilings, with similar results as in the present study. The amount of shade can also influence the surface temperature of rocky intertidal areas, with unshaded areas having much greater surface temperatures than adjacent shaded areas during low-tide leading to physiological affects (e.g. greater mortality through desiccation or thermal stress [START_REF] Garrity | Some adaptations of gastropods to physical stress on a tropical rocky shore[END_REF][START_REF] Harper | Variations in abundance and distribution of the chiton Acanthopleura japonica and associated molluscs on a seasonal, tropical, rocky shore[END_REF]). The provision of shade can ameliorate thermal stress and increase recruitment and survival [START_REF] Denley | Experiments on factors influencing settlement, survival, and growth of two species of barnacles in New South Wales[END_REF][START_REF] Williams | The relationship between shade and molluscan grazing in structuring communities on a moderately-exposed tropical rocky shore[END_REF][START_REF] Bertness | Climate-driven interactions among rocky intertidal organisms caught between a rock and a hot place[END_REF]. According to [START_REF] Helmuth | Microhabitats, thermal heterogeneity, and patterns of physiological stress in the rocky intertidal zone[END_REF], however, it is extreme temperature events (extremes of heat and cold) rather than the mean temperature that causes physiological stress. The design of the experiment was unlikely to capture temperature extremes because it unavoidably involved "snap-shot" measurements. The data collected does give a good indication that these extreme high temperatures would occur on unshaded seawalls, where surface temperature was always greater, whereas wharves would buffer seawalls beneath them from these events.

Recruitment of algae and invertebrates to seawalls has been shown to be affected by wharves, which shade the wall directly under them (Blockley & Chapman, In press ). The greater cover of most sessile animals in the lower light conditions under wharves might represent a negative phototactic response at the time of settlement (e.g. [START_REF] Pomerat | The influence of surface angle and light on the attachment of barnacles and other sedentary organisms[END_REF][START_REF] Wisely | Factors influencing the settling of the principal marine fouling organisms in Sydney Harbour[END_REF]. O'Donnell (1984) showed that G. caespitosa recruit in greater numbers to shaded habitats, possibly explaining the patterns in the current study. Algae has also been shown to have greater recruitment to unshaded habitats [START_REF] Goldberg | Settlement and post-settlement processes limit the abundance of the geniculate coralline alga Calliarthron on subtidal walls[END_REF][START_REF] Clark | Effects of shade from multiple kelp canopies on an understory algal assemblages[END_REF]. It is, therefore, possible that the patterns found in the present study are determined at the time of recruitment rather than by post-recruitment processes.

Despite support for patterns of difference between assemblages on seawalls under or not under wharves, there was great variability among locations and to, a lesser extent, among times.

Large-scale spatial (at the scale of km) and temporal differences in intertidal assemblages are a feature of the rocky shores of New South Wales, Australia [START_REF] Dakin | A study of certain aspects of the ecology of the intertidal zone of the New South Wales coast[END_REF][START_REF] Underwood | Spatial analyses of intertidal assemblages on sheltered rocky shores[END_REF][START_REF] Benkendorff | Identifying hotspots of molluscan species richness on rocky intertidal reefs[END_REF]. However, seawalls, unlike rocky shore, are homogeneous structures and so would be predicted to be less variability among locations.

Previous research on seawalls in Sydney Harbour by [START_REF] Bulleri | Intertidal assemblages on seawalls and vertical rocky shores in Sydney Harbour (Australia)[END_REF] has, however, shown that the variability of assemblages on seawalls can be as great as, or greater than that found on rocky shores among locations. Although [START_REF] Bulleri | Intertidal assemblages on seawalls and vertical rocky shores in Sydney Harbour (Australia)[END_REF] found that the taxa responsible for patterns on seawalls varied among locations, general patterns were consistent for each location,. This is similar to the present study, with assemblages consistently differing between the shaded and unshaded seawalls at each location, but species responsible for patterns differing, resulting in the overall variability among locations. In their study, [START_REF] Bulleri | Intertidal assemblages on seawalls and vertical rocky shores in Sydney Harbour (Australia)[END_REF] only examined three locations and did not directly compare among locations. The present study has therefore expanded on this by examining a greater number of locations, comparing among locations and studying a common, yet so far largely ignored, artificial habitat, that is seawalls under wharves.

One explanation for the large-scale spatial variability is that the orientation and position within the harbour of seawalls differed among locations, such that physical conditions on the seawalls could have varied among locations. Seawalls that faced north, for example, would be subject to more direct sunlight than those facing south, resulting in greater surface temperatures. This is evident in the measures of incident light and surface temperature of unshaded seawalls (e.g.

Hermit Point and Quarantine Station, north facing wall, compared to Cremorne Point and Athol

Bay, south facing wall). In locations where seawalls were oriented towards the sun, the difference in temperature between shaded and unshaded seawalls would be expected to be greater than where seawalls were not oriented towards the sun. In the present study, the difference in temperature between shaded and unshaded sections of seawall that faced north, towards the sun, was up to 10 o C, while the difference between habitats for south facing walls was less than 5 o C. This could explain some of this dissimilarity in the composition of assemblages among locations.

Urbanised waterways are heavily impacted by anthropogenic activity, with many sources of disturbance [START_REF] Kullenberg | Contributions of marine and coastal area research and observations towards sustainable development of large cities[END_REF][START_REF] Vallega | Urban waterfront facing integrated coastal management[END_REF][START_REF] Kennish | Environmental threats and environmental future of estuaries[END_REF]. The multiple impacts of artificial structures built on, or in the vicinity of, other artificial structures and the additive effects of their associated impacts has generally been overlooked in studies of disturbance ecology. Although this study did not examine how the assemblages associated with two different types of artificial structures (i.e. seawalls and wharves) differed from those on natural shores, previous studies have shown that assemblages differ between seawalls and natural shores [START_REF] Chapman | Intertidal seawalls: new features of landscape in intertidal environments[END_REF][START_REF] Chapman | Paucity of mobile species on constructed seawalls: effects of urbanization on biodiversity[END_REF][START_REF] Bulleri | Intertidal assemblages on seawalls and vertical rocky shores in Sydney Harbour (Australia)[END_REF]. The present study has shown that the presence of another structure (wharves) can further alter assemblages on seawalls. Whether the difference to assemblages on seawalls caused by the presence of wharves translates to making assemblages more or less different from those on natural shores is an area of further study.

The potential of wharves to provide or alter habitats for marine organisms needs to consider the possible positive effects against negative impacts. The presence of a wharf not only means that the composition of assemblages or relative covers or abundances of taxa differs from adjacent unshaded seawalls, but also results in assemblages that are more variable at small and large spatial scales. The shaded surfaces may act as substitutes for microhabitats that are missing from seawalls, as well as adding to the amount and variety of habitats because of structures, such as pilings, associated with wharves. The negative impacts on surrounding habitats and the fact that the assemblages on these structures do not necessarily represent natural assemblages must, however, be considered. If seawalls with wharves do not support assemblages that are representative of those on natural shores, then they cannot necessarily be said to have a positive impact, regardless of increasing local diversity. This is an important issue if, as [START_REF] Cole | Extending the generality of ecological models to artificial floating habitats[END_REF] suggest, we attempt to apply ecological theories from the study of natural shores to the management of artificial structures.

Table 1. Results from analyses by non-parametric MANOVA on assemblages on sections of seawall under or not under wharves (a) among locations for each time of sampling and (b) through time at each location, at mid-and low-tidal heights. 5000 permutation of residuals were used for all analyses. NS = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Where analyses showed significant interactions, F-ratios are not given for main effects and lower order interactions because these cannot be logically interpreted. Because Cochran' s test was significant (P < 0.01) a conservative significance level was used was used NS = P > 0.01, * = P < 0.001

F Cremorne Point F Rushcutters Bay F Hermit Point F Parriwi Head F Little Manly Pt F Quarantine Station Time = T 3 - - - - - - - Habitat = H 1 - - - - - - - T x H 3 3.

Figure captions

  472because these cannot be logically interpreted. NS = P > 0.05, * = P < 0.05, ** = P < 0.01, *** = P < 0.001 except where

Figure 1 .

 1 Figure 1. Map of field sites within Sydney Harbour, Australia

Figure 3 .

 3 Figure 3. nMDS ordination of centroids of assemblages on seawalls under wharves (circles) or not

Figure 4 .

 4 Figure 4. Mean (+SE) percentage cover of algae and sessile invertebrates on seawalls under

Figure 5 .

 5 Figure 5. Mean (+SE) percentage cover of algae and sessile invertebrates on seawalls under

Figure 6 .

 6 Figure 6. Mean (+SE) abundance of mobile invertebrates on seawalls under (shaded) or not under

Figure 7 .

 7 Figure 7. Mean surface temperature (± S.E.) of seawalls under (circle, solid line) or not under

Figure 8 .

 8 Figure 8. Mean light (Lux) on seawalls under (circle, solid line) or not under (triangle, hashed line)

  Fig. 1 525

Table 2 .

 2 Average Bray-Curtis dissimilarities within and among locations for each habitat and between the two habitats 466 at each time for mid-and low-tidal height. W= Wharf and NW = No-wharf habitats.

			54 *** 11.07 ***	5.14 ***	6.75 ***	4.71 ***	2.64 **	3.53 ***
	Residual	72
	Low-tidal Height
	Source	df	F	F	F	F		F	F
			Athol	Cremorne	Rushcutters	Hermit		Little	Quarantine
			Bay	Point	Bay	Point		Manly Pt	Station
	Time	3
	Habitat	1
	T x H	3 3.18 *** 2.80 *** 7.55 *** 8.39 ***		6.66 ***	5.98 ***
	Residual	72

Table 3 .

 3 Analyses of individual taxa of (a) algae, (b) sessile invertebrates and (c) mobile invertebrates on sections of 469 seawall under or not under wharves at the mid-tidal height. F-ratios are not given for main effects and lower order interactions

	470
	471
	(a)	H. rubra
	Source	df	MS	F
	Time = T	178.1
	Location = L	3767.1
	Habitat = H	10386.8
	T x L	281.8	2.7 *
	T x H	85.4	0.4 NS
	L x H	3333.0 14.1 *
	T x L x H	237.1	2.3 NS
	Residual	104.3
	Transformation	None
	Cochran' s test	**
	(b)	S. glomerata		M. galloprovincialis	G. caespitosa		T. rosea
	Source	df	MS	F	MS	F	MS	F	MS	F
	Time = T	2795.5		42.8		1767.6		35.8
	Location = L	49571.3		1469.9		6301.4		2151.8
	Habitat = H	207587.9		1980.2		13519.5		192.5
	T x L	1481.0		29.2 1.2 NS	646.6		18.9
	T x H	927.3		68.4 1.2 NS	1823.2		224.1
	L x H	13918.3		1329.2 22.6 *	11220.4		82.1
	T x L x H	1683.8	5.9 *	58.9 2.4 NS	730.0 4.14 *	199.9 5.1 *
	Residual	286.0		25.0		176.2		39.0
	Transformation	None			None		None		None
	Cochran' s test	*			**		**		**
		T. purpurascens	Orange sponge
	Source	df	MS	F	MS	F
	Time	441.0		26.0
	Location	13137.8		124.3
	Habitat	40683.8		536.3
	T x L	243.7		18.5
	T x H	639.4		27.2
	L x H	10876.1		113.6
	T x L x H	313.8	3.2 *	18.6 3.2 *
	Residual	97.5		5.9
	Transformation	None			None
	Cochran' s test	**			**
	(c)	C. pelliserpentis	S. denticulata
	Source	df	MS	F	MS	F
	Time		3.5		1.3
	Location		9.1		2.0
	Habitat	11.4		11.8
	T x L		1.3		0.5
	T x H		2.8		0.2
	L x H		7.1		5.9
	T x L x H		1.2	5.4 ***	0.8 2.9 ***
	Residual		0.2		0.3
	Transformation	Ln(X + 1)		Ln(X + 1)
	Cochran' s test	NS			NS
	Where analyses showed significant interactions,

Table 4 .

 4 Analyses of individual taxa of (a) algae and (b) sessile invertebrates on sections of seawall under or not under wharves at the low-tidal height.

	(a)		C. officinalis		Foliose green algae
	Source	df	MS	F	MS	F
	Time = T	3	4468.2		4.2
	Location = L	5	10590.6		13.3
	Habitat = H	1	76329.8		44.7
	T x L	15	1163.2		3.9
	T x H	3	4446.4		7.5
	L x H	5	10518.0		5.5
	T x L x H	15	1164.7 6.5 *	2.7 7.1 *
	Residual	432	179.4		0.4
	Transformation	None		None
	Cochran' s test		**		**
	(b)		S. glomerata		M. galloprovincialis	G. caespitosa	A. imperator
	Source	df	MS	F	MS	F	MS	F	MS	F
	Time = T	3	2129.6		721.5		977.9		56.8
	Location = L	5	10070.7		698.2		3987.7		450.1 10.6 *
	Habitat = H	1	8535.0		286.8		1117.3		763.4
	T x L	15	2270.7		756.4		415.9		P 35.9
	T x H	3	6362.0		799.4		58.1		71.6 1.7 NS
	L x H	5	7786.4		969.5		2347.6		299.7 7.1 *
	T x L x H	15	2610.3 10.1 *	736.1 41.9 *	316.0 2.6 *	P 45.8
	Residual	432	259.3		17.6		122.0		42.6
	Pooled	462							42.5
	Transformation	None		None		None		None
	Cochran' s test		**		**		**		**
			T. rosea		T. purpurascens	Orange sponge
	Source	df	MS	F	MS	F	MS	F
	Time	3	110.4		12.4		45.2
	Location	5	809.5		1252.6		6107.6
	Habitat	1	145.8		3370.8		18710.1
	T x L	15	109.8 4.3 *	285.4		258.2
	T x H	3	18.0 0.7 NS	10.3		107.1
	L x H	5	105.7 4.1 *	1084.2		4437.5
	T x L x H	15	P 19.5		289.2 7.9 *	364.3 3.2 *
	Residual	432	25.9		74.4		113.5
	Pooled	447	25.7
	Transformation	None		None		None
	Cochran' s test		**		**		**
	P indicates pooling (P > 0.25).

Cochran' s test was significant (for C with P < 0.05, * = P < 0.01, ** = P < 0.001; for C with P < 0.01, * = P < 0.001)

Acknowledgments

This study was supported by the Australian Research Councils Special Centres Programme and the University of Sydney Postgraduate Awards. Gee Chapman, Vicky Cole, Paris Goodsell and two anonymous referees offered useful comments on an earlier draft of this manuscript.