Pattern and Process in a Threatened Seagrass Community: Dynamics and Habitat Use by Sessile Epifaunal Invertebrates in Posidonia Australis

University of Wollongong Research Online

University of Wollongong Thesis Collection 2017+ University of Wollongong Thesis Collections

2017

Pattern and process in a threatened seagrass community: dynamics and habitat use by sessile epifaunal invertebrates in Posidonia australis

Marie-Claire A. Demers University of Wollongong

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Recommended Citation Demers, Marie-Claire A., Pattern and process in a threatened seagrass community: dynamics and habitat use by sessile epifaunal invertebrates in Posidonia australis, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2017. https://ro.uow.edu.au/theses1/43

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PATTERN AND PROCESS IN A THREATENED SEAGRASS COMMUNITY: DYNAMICS AND HABITAT USE BY SESSILE EPIFAUNAL INVERTEBRATES IN POSIDONIA AUSTRALIS

A thesis submitted in fulfilment of the requirements for the award of the degree DOCTOR OF PHILOSOPHY from University of Wollongong

by Marie-Claire A. Demers B Env Sc (Hons Class 1)

Faculty of Science, Medicine and Health Department of Biological Sciences

2017

CERTIFICATION

I, Marie-Claire Demers, declare that this thesis, submitted in fulfilment of the requirements for the award of Doctor of Philosophy, in the Department of Biological Sciences, Faculty of Science, Medicine and Health, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution.

Marie-Claire A. Demers 08 February 2017

TABLE OF CONTENTS

CERTIFICATION ...... i TABLE OF CONTENTS ...... ii LIST OF TABLES ...... vi LIST OF FIGURES ...... ix ABSTRACT ...... xv ACKNOWLEDGEMENTS ...... xviii CHAPTER 1 : General introduction ...... 1 1.1 Ecological role of sponge and ascidians ...... 2 1.2 Habitat bias ...... 3 1.3 Seagrass importance and decline...... 4 1.4 Seagrass associated species and ecosystem function ...... 5 1.4.1 Seagrass associated species ...... 5 1.4.2 Seagrass ecosystem functioning ...... 6 1.5 Patterns and drivers of sessile epifaunal invertebrate distribution in seagrass meadows ...... 8 1.5.1 Factors driving sponge distribution ...... 8 1.5.2 Abiotic factors ...... 9 1.5.3 Biotic factors ...... 11 1.6 Species of interest...... 13 1.7 Aims and structure of this thesis ...... 14 CHAPTER 2: Under the radar: Small-scale distributional patterns of sessile epifaunal invertebrates in the seagrass Posidonia australis...... 16 2.1 Abstract ...... 17 2.2 Introduction ...... 18 2.3 Materials and methods ...... 20 2.3.1 Study area, study species and sampling methodology ...... 20 2.3.2 Statistical analyses ...... 24 2.4 Results ...... 25 2.4.1 Overall patterns of diversity and abundance ...... 25 2.4.2 Patterns of abundance; individual taxa ...... 34 2.4.3 Substrata on which epifauna attached ...... 35 2.5 Discussion ...... 39 2.5.1 Patterns of distribution ...... 39 2.5.2 Substratum used for attachment and the impact of humans ...... 41

2.6 Conclusions ...... 42 CHAPTER 3 : Are pilot studies worth the trouble? A case study using recalcitrant organisms ...... 43 3.1 Abstract ...... 44 3.2 Introduction ...... 45 3.3 Materials and methods ...... 46 3.3.1 Pilot study ...... 46 3.3.2 Main study ...... 47 3.3.3 Sampling method ...... 47 3.3.4 Statistical analyses ...... 47 3.4 Results ...... 49 3.4.1 Measures of diversity and abundance ...... 49 3.4.2 Assemblage composition ...... 49 3.4.3 Variability among spatial scales ...... 53 3.5 Discussion ...... 56 3.5.1 Effectiveness of the pilot study ...... 56 3.5.2 Limitations of the Pilot study ...... 57 3.6 Conclusions ...... 58 3.7 Appendix ...... 59 CHAPTER 4 : Seagrass attributes and seascape patterns as drivers of sessile invertebrate distribution in a coastal biogenic environment ...... 62 4.1 Abstract ...... 63 4.2 Introduction ...... 64 4.3 Materials and methods ...... 67 4.3.1 Study area ...... 67 4.3.2 Sampling method ...... 67 4.3.3 Statistical analyses ...... 69 4.4 Results ...... 69 4.5 Discussion ...... 74 4.5.1 Seagrass density and frond length ...... 74 4.5.2 Hydrodynamics and dispersal ...... 74 4.5.3 Depth ...... 74 4.5.4 Seascape effects ...... 74 4.5.5 Role of predators ...... 74 4.5.6 Anthropogenic effects ...... 74

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4.6 Conclusions and recommendations ...... 78 CHAPTER 5 : Picky eaters or prickly entrées? Effect of predation, palatability and availability of substrata on the sponge community of Posidonia australis seagrass meadows ...... 79 5.1 Abstract ...... 80 5.2 Introduction ...... 81 5.3 Materials and methods ...... 85 5.3.1 Study area ...... 85 5.3.2 Predator exclusion experiment ...... 85 5.3.3 Palatability experiment ...... 88 5.3.4 Statistical analyses ...... 91 5.4 Results ...... 91 5.4.1 Predator exclusion cages ...... 91 5.4.2 Palatability test ...... 91 5.5 Discussion ...... 98 5.5.1 Predation and substratum availability ...... 98 5.5.2 Palatability ...... 99 5.5.3 Cage accessibility ...... 100 5.6 Conclusions and recommendations ...... 101 5.7 Appendix ...... 102 5.8 Supplementary material...... 102 CHAPTER 6 : Discussion and conclusions ...... 103 6.1 Immediate challenges ...... 104 6.2 Overall trends ...... 107 6.3 Limitations ...... 107 6.4 Conservation challenges ...... 108 6.5 Future challenges...... 109 6.6 Conclusions ...... 112 REFERENCES...... 115 APPENDIX 1: A guide to sponge and ascidian identification ...... 131 A1.1 Sponge identification ...... 133 A1.2 Ascidian identification...... 163 A1.3 Bryozoan identification ...... 171 APPENDIX 2: A comparison of the impact of ‘seagrass-friendly’ boat mooring systems on Posidonia australis...... 172

A2.1 Abstract...... 173 A2.2 Introduction ...... 174 A2.3 Materials and methods ...... 177 A2.3.1 Study area ...... 177 A2.3.2 Seagrass species ...... 179 A2.3.3 Sampling method ...... 179 A2.3.4 Statistical analyses ...... 180 A2.4 Results ...... 181 A2.4.1 Conventional ‘swing’ moorings ...... 181 A2.4.2 Seagrass-friendly ‘screw’ moorings ...... 183 A2.4.3 Seagrass-friendly ‘cyclone’ moorings ...... 187 A2.4.4 Size of mooring impact ...... 187 A2.5 Discussion...... 189 A2.6 Conclusions and recommendations ...... 190 A2.7 Supplementary data ...... 191 A2.8 References ...... 192 APPENDIX 3: Filtering the unknown: sampling sessile epifaunal invertebrates in seagrass meadows...... 195 A3.1 Introduction ...... 196 A3.2 Outcomes ...... 202 A3.3 References ...... 204

LIST OF TABLES

Table 2.1: Number of transects where (A) sponge and (B) ascidian individual species were present out of six transects for each of the two sites at each of the six locations. Species were organized in descending order based on their percentage of occurrence pooled across sites (n=72)...... 29 Table 2.2: Analyses of variance comparing variation among locations and sites for (A) overall sponge abundance, volume and diversity, (B) abundance of five main sponge taxa, (C) volume of five main sponge taxa, (D) ascidian abundance and diversity. NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001...... 31 Table 2.3: Summary of the two factor nested PERMANOVA for sponge abundance, sponge volume and ascidian abundance of each species among sites and locations in the seagrass meadows of Jervis Bay. Factors were locations with six levels (random) and sites nested in locations with two levels (random). Transects at each sites served as replicates. Analysis was based on Bray-Curtis dissimilarities with permutation of the raw data. NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001...... 33 Table 3.1: Summary of the three factor nested PERMANOVA for (A) the sponge and ascidian assemblage (untransformed and presence/absence transformed), (B) the diversity of sponge and ascidian species and (C) the abundance of sponge and ascidian species. I used a Bray-Curtis distance measure for the multivariate analyses and Euclidean distance resemblance matrix for the univariate analyses. Factors were ‘study’ with two levels (fixed) the pilot and the main study, ‘location’ was nested in ‘study’ with two levels for the pilot study and six levels for the main study (random) and ‘site’ was nested in ‘location’ with two levels (random). Transects at each sites served as replicates. Post-hoc pooling was used to increase the power of the main tests for P>0.25 (Underwood, 1997). NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001...... 52 Table 3.2: Analyses of variance comparing variation at a hierarchy of spatial scales for (A) sponge diversity, (B) ascidian diversity, (C) sponge abundance, (D) ascidian abundance. The factors were: ‘Location’ a random orthogonal factor with up to six levels (km apart), ‘Site’ a random orthogonal factor with two levels nested within ‘Location’ (100s m apart) and ‘Transect’ or residuals (10s m apart) (n=6). NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001...... 55 Table 3.3: Summary of the nested ANOVAs and magnitude of effects comparing diversity and abundance of sponges and ascidians for (A) the pilot study and (B) the main

study, as well as the abundance of dominant taxa for the pilot study. Magnitude of effects (Ϭ2) is represented as a percentage (ω2), calculated using procedures detailed in Graham and Edwards (2001). The factors were: ‘Location’ a random and orthogonal factor with up to six levels (km apart), ‘Site’ a random orthogonal factor with two levels nested within ‘Location’ (100s m apart) and ‘Transect’ or residuals (10s m apart) (n=6). The symbol * in the MS column indicate where pool-the-minimum logarithm was applied...... 59 Table 4.1: Predictive variables used in the statistical models in addition to their individual units, scale, transformation (trans.), a brief description, sample size (n), minimum value (min), maximum value (max), mean and standard deviation (SD)...... 71 Table 4.2: Summary of the model transformation, distribution and outcomes for sponge abundance (N), sponge volume, ascidian diversity, Haliclona spp. and Tedania (Tedania) sp. abundance (N)...... 72 Table 5.1: Analyses of variance comparing variation for (A) the total number of fish bites taken of explants, (B) total number of shrimps that interacted with explants and (C) total maximum number of shrimps that interacted with explants in a single frame at one time (MaxN). The factor was ‘Treatment’ a random orthogonal factor with three levels (unmanipulated plot, cage control and predator exclusion cage) (n=4). NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001...... 97 Table 5.2: Materials used for the construction of the predator exclusion cages ...... 102 Table 6.1: Area (ha) of endangered P. australis populations in NSW, Australia. This includes exclusive P. australis meadows as well as a combination of P. australis and other seagrass species. Note that these values were acquired from maps that were based on air photos and field surveys dated between 2000 and 2009 (NSW Department of Primary Industries, 2010)………………………….……………………...... …108 Table A1.1: Summary of Sponge ID code, binomial name, expert that examined and confirmed the identification, photo, presence of spicule and specimen/voucher availability. Species were arranged by taxonomic order according to the new organisation proposed by Morrow and Cárdenas (2015)...... 132 Table A2.1: Analyses of variance comparing ‘seagrass-friendly’ ‘screw’ and conventional ‘swing’ mooring systems at Callala Bay. Tr: Treatment, Si: Site, Di: Distance. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001...... 181

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Table A2.2: Analyses of variance comparing ‘seagrass-friendly’ ‘screw’ mooring systems at Bindijine Beach. Tr: Treatment, Si: Site, Di: Distance. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001...... 184 Table A2.3: Analyses of variance comparing ‘seagrass-friendly’ ‘cyclone’ mooring systems at Callala Bay. Tr: Treatment, Si: Site, Di: Distance. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001...... 187 Table A3.1: The techniques, equations and associated references used to determine the appropriate transect length...... 198 Table A3.2: The outcome of the binomial sampling theory (Dennison & Hay, 1967) for each of the species present in order of increasing sample size (n). The sample size (n) is the number of replicates and the appropriate unit size (m2) is the size of the sampling unit to allow greater probability of detection (0.05). The equation and details of calculation can be found in Table A3.1...... 200 Table A3.3: Replication at each spatial scale derived from cost-benefit analyses for sampling sponge and ascidian diversity and abundance using the pilot study. The variance estimates were obtained from ANOVAs of untransformed data. Values were rounded to the nearest whole unit of sampling (except for variances). I used a standard design of six locations, two sites and six transects when calculating the expected variance and relative efficiency. For instance, when comparing amongst locations I assumed two sites and six transects...... 201

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LIST OF FIGURES

Figure 1.1: Compilation of peered-reviewed studies (1971-2012) expressed as a A) percentage of studies that investigated sponge distribution patterns in different habitats and substrata and, B) percentage of studies that investigated exclusively the spatial distribution of sponges within seagrass ecosystems and two specific seagrass species. This review of 111 papers was sourced from ‘web of science’. The search string included: ‘sponge’, ‘Porifera’, ‘process’ for A and ‘seagrass’, ‘spatial’, ‘distribution’ were added to obtain B. In order to be selected, the paper had to clearly specify the habitat from which sponges were sampled. Papers that studied more than one habitat were divided into percentages of each habitat sampled for a total of 100%. Spatial distribution within seagrass (non-Posidonia), Spatial distribution within Posidonia australis, Spatial distribution within Posidonia oceanica, Other seagrass studies...... 7 Figure 2.1: Jervis Bay, showing each of the six locations I sampled: 1) Booderee Waters, 2) Plantation Point, 3) Callala Bay, 4) Hare Bay, 5) Long Beach and 6) Bindijine Beach. The prevalent clockwise gyre, important in nutrient circulation in the embayment, is indicated by the grey arrow (Wang & Wang, 2003)...... 21 Figure 2.2: (A) Sponges and hydroids occupying a Eunicid polychaete worm tube located outside of the P. australis meadow. Note the other worm tubes in the background (Scale is 10cm). (B) Multiple sponge species inhabiting a single Eunicid polychaete worm tube (Scale is 3cm). (C) Callyspongia (Toxochalina) sp. 1 attached to the sheath and frond of P. australis (Scale is 5cm). (D) Encrusting Phoriospongia sp. 2. growing on shells (Scale is 1cm). (E) Botrylloides leachii growing on the sheath and frond of P. australis (Scale is 5cm). (F) Bivalve clump (Trichomya hirsuta) bearing multiple sponge species (Scale is 4cm)...... 23 Figure 2.3: Comparison of sponge and ascidian species richness, abundance and volume at the two sites sampled for each of the six locations within Jervis Bay. (A) mean (±SE) estimates of sponge diversity expressed as number of species present per site. (B) mean (±SE) estimates of sponge abundance expressed as number of individuals per site. (C) mean (±SE) estimates of sponge volume expressed as cm3 per site. (D) mean (±SE) estimates of ascidian diversity expressed as number of species present per site. (E) mean (±SE) estimates of ascidian abundance expressed as number of individuals per site. Each bar represents a mean of six transects each of 25m2 (n=6)...... 27

Curtis Similarity measures. Callala Bay site 2 was removed from (C).

Bindijine Beach, Callala Bay, Hare Bay, Booderee Waters,

Plantation Point, Long Beach...... 28 Figure 2.5: Comparison of abundance and volume for the five main sponge taxa at the two sites sampled for each of the six locations within Jervis Bay. Mean (±SE) estimates of abundance expressed as number of individuals per site for (A) Phoriospongia sp. 1 (B) Haliclona spp. (C) Callyspongia (Toxochalina) sp. 1 (D) Tedania (Tedania) sp. (E) Halichondria sp. Mean (±SE) estimates of volume expressed as cm3 per site for (F) Phoriospongia sp. 1 (G) Haliclona spp. (H) Callyspongia (Toxochalina) sp. 1 (I) Tedania (Tedania) sp. (J) Halichondria sp. Each bar represents a mean of six transects each of 25m2 (n=6)...... 37 Figure 2.6: Comparison of substratum of attachment for sponges, five dominant sponge taxa and ascidians at the two sites sampled for each of the six locations within Jervis Bay. Mean (±SE) estimates of number of substrata occupied by (A) sponges (B) ascidians (C) Phoriospongia sp. 1 (D) Haliclona spp. (E) Callyspongia (Toxochalina) sp. 1 (F) Tedania (Tedania) sp. (G) Halichondria sp. Each bar represents a total of six transects of 25m2 (n=6)...... 38 Figure 3.1: A comparison of the mean (±s.e.) (A) species richness and (B) abundance of sessile epifaunal invertebrates pooled across transects from all sites and locations between the pilot (n=24) and the main study (n=72)...... 50 Figure 3.2: Non-metric multidimensional scaling (nMDS) plots showing dissimilarities among the pilot and the main study for the abundance of sponge and ascidian

species without transformation. Pilot Study; Main Study...... 51 Figure 3.3: Magnitude of effects, expressed as percentage of variation (ω2), calculated from the diversity and abundance of sponges and ascidians for (A) the pilot study and (B) the main study. Location (km apart) random and orthogonal with up to six levels, Site within Location (100s m apart) random orthogonal with two levels and Transect (10s m apart) expressed as residuals (n=6) (See Appendix Table 2.3 for additional details)...... 54 Figure 4.1: Scatterplot of (A) Sponge abundance and leaf length of P. australis, (B) Sponge volume (sqrt transformed) and the quantity shoots of P. australis, (C) Ascidian

species richness and the area of reef within a 1500m radius (log transformed), (D) Haliclona spp. abundance and depth, (E) Tedania (Tedania) sp. and the number of reefs neighbouring the edge of the meadow...... 73 Figure 5.1: Map showing the distribution of predator exclusion cages, cage controls and unmanipulated plots between Murray’s Boat Ramp and Hole-in-the-wall in Booderee National Park, Jervis Bay, NSW, Australia...... 84 Figure 5.2: (A) predator exclusion cage, (B) cage control and (C) diver installing star picket to secure the cage to the substratum...... 87 Figure 5.3: Photographs of (A) sponge palatability underwater video (SPUV) unit with mounted GoPro camera and affixed sponge explants, and (B) isolation containers holding sponge explants...... 90 Figure 5.4: Fish species that consumed sponges; (A) Chinanaman leatherjacket (Nelusetta ayraudi), (B) Little weed whiting (Neoodax balteatus), (C) Six spine Leatherjacket (Meuschenia freycineti) and (D) yellow sabretooth blenny (Plagiotremus tapeinosoma). The unidentified leatherjacket species was barely visible due to poor light and was not included in this figure. I recorded for a period of 84 min using sponge palatability underwater videos (SPUVs) in predator exclusion cages, cage controls and unmanipulated plots (n=4)...... 93 Figure 5.5: Organisms seen on the surface of sponges: (A) small and, (B) large adult shrimp of the same species (Palaemon intermedius), and (C) unidentified worm...... 94 Figure 5.6. Mean (±SE) number of bites by fish taken from explants within a 84 min period, including all three sponge species combined, for the three treatments investigated (n=4)...... 95 Figure 5.7: Mean (±SE) number of shrimps (Palaemon intermedius) that interacted with explants within a 84 min period, including all three sponge species combined, for the three treatments investigated (n=4). small and, large adult shrimps of the same species (Palaemon intermedius)...... 95 Figure 5.8: Mean (±SE) maximum number of shrimps (Palaemon intermedius) that interacted with explants in a single frame at one time (MaxN) within a 84 min period, including all three sponge species combined, for the three treatments investigated (n=4). I did not differentiate between small and large shrimps in this instance...... 96 Figure 6.1: Unknown ascidian species observed in the P. australis seagrass meadows of Maianbar, Port Hacking, NSW. Photograph of (A) an adult specimen covering the entire seagrass frond and (B) close-up view showing individual siphons...... 114

Figure A1.1.1: Chelonaplysilla sp...... 151 Figure A1.1.2: Euryspongia sp...... 146 Figure A1.1.3: Thorectidae family sp.1 ...... 156 Figure A1.1.4: Thorectidae family sp.2 ...... 136 Figure A1.1.5: Thorectidae cf Hyrtios sp.3 ...... 161 Figure A1.1.6: Callyspongia (Toxochalina) sp.1 ...... 148 Figure A1.1.7: Callyspongia (Callyspongia) sp.2 ...... 159 Figure A1.1.8: Chalinula sp ...... 149 Figure A1.1.9: Chalinidae Haliclona spp...... 134 Figure A1.1.10: Stelletta sp...... 157 Figure A1.1.11: Chondropsidae family sp.4...... 147 Figure A1.1.12: Chondropsis sp...... 142 Figure A1.1.13: Phoriospongia sp.1 ...... 152 Figure A1.1.14: Phoriospongia sp.2 ...... 139 Figure A1.1.15: Coelosphaeridae, Lissodendoryx (Lissodendoryx) sp ...... 133 Figure A1.1.16: Lissodendoryx (Waldoschmittia) sp...... 153 Figure A1.1.17: Tedaniidae Tedania (Tedania) sp...... 160 Figure A1.1.18: Tethya cf bergquistae...... 144 Figure A1.1.19: Halichondria sp ...... 140 Figure A1.1.20: Unidentified Demospongia sp.1 ...... 137 Figure A1.1.21: Unidentified Demospongia sp.2 ...... 154 Figure A1.2.1: Botrylloides leachii ...... 167 Figure A1.2.2: Cnemidocarpa pedata and associate Halisarca laxus sponge...... 168 Figure A1.2.3: Didemnum sp...... 163 Figure A1.2.4: Herdmania grandis ...... 165 Figure A1.2.5: Pyura praeputialis ...... 164 Figure A1.2.6: Pyura spinifera with associate Halisarca laxus sponge ...... 169 Figure A1.3.1: Unidentified Bryozoan ...... 171 Figure A2.1: Schematic representation of a typical (A) ‘screw’ mooring system, (B) ‘swing’ mooring system and (C) ‘cyclone’ mooring system...... 176 Figure A2.2: (A) Aerial photograph of the mooring area at Callala Bay showing distinctive circular areas denuded of seagrass, (B) underwater photograph of the ring of bare seagrass observed around ‘screw’ moorings at Bindijine Beach...... 178

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Figure A2.3: Comparison of seagrass density and cover for ‘screw’ and ‘swing’ moorings with reference areas at Callala Bay. (A) mean (+SE) estimates of Posidonia density. These estimates were expressed as percentages of the maximum density recorded in the reference area for each site. Each bar represents a total of four quadrats; two quadrats of 0.25 m2 for each of the two transect lines (n = 4). (B) mean (+SE) percent cover of Posidonia. Each bar represents 20 points per distance for each of the two transect lines (n = 2). (C) mean (+SE) percent cover of Halophila ovalis and Zostera spp. Each bar represents 20 points per distance for each of two transect lines (n = 2). Distances (0-2, 3-5 and 6-8 m) are from the centre of each mooring. Reference; ‘Screw’ mooring; ‘Swing’ mooring...... 182 Figure A2.4: Comparison of seagrass density and cover for ‘screw’ moorings with reference areas at Bindijine Beach. (A) mean (+SE) estimates of Posidonia density. These estimates were expressed as percentages of the maximum density recorded in the reference area for each site. Each bar represents a total of four quadrats; two quadrats of 0.25 m2 for each of the two transect lines (n = 4). (B) mean (+SE) percent cover of Posidonia. Each bar represents 20 points per distance for each of the two transect lines (n = 2). Distances (0-2, 3-5 and 6-8 m) are from the centre of each mooring. Reference; ‘Screw’ mooring...... 185 Figure A2.5: Comparison of seagrass density and cover for ‘cyclone’ moorings with reference areas at Callala Bay. (A) mean (+SE) estimates of Posidonia density. These estimates were expressed as percentages of the maximum density recorded in the reference area for each site. Each bar represents a total of four quadrats; two quadrats of 0.25 m2 for each of the two transect lines (n=4). (B) mean (+SE) percent cover of Posidonia. Each bar represents 20 points per distance for each of two transect lines (n = 2). (C) mean (+SE) percentage cover of Halophila ovalis and Zostera spp. Each bar represents 20 points per distance for each of two transect lines (n = 2). Distances (0-2, 3-5 and 6-8 m) are from the centre of each mooring. Reference; ‘Cyclone’ mooring...... 186 Figure A2.6: Relationship between mooring chain length and the mean (+SE) radius of areas scoured of seagrass for ‘swing’ and ‘cyclone’ mooring systems at Callala Bay (n = 2). ‘Screw’ mooring; ‘Swing’ mooring; ‘Cyclone’ mooring...... 188 Figure A3.1: Variation for increasing transect lengths (m) for sampling sessile epifaunal invertebrate in the seagrass meadows of Jervis Bay using (A) Coefficient of variation (Kingsford & Battershill, 1998), (B) Replication with a precision of 15%

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(Andrew & Mapstone, 1987), (C) Sample size (Snedecor & Cochran, 1980) with a

95% confidence, (D) Wiegert’s method with C0=140 min (Krebs, 1999). See Table A3.1 for equations...... 199

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ABSTRACT

This is the first detailed study to uncover the patterns of distribution and abundance of the sessile epifaunal invertebrate community inhabiting seagrass meadows on the south-eastern coast of Australia. It is also one of the few studies worldwide to investigate patterns and processes of sessile epifaunal invertebrate distribution in seagrass meadows. The target seagrass species, Posidonia australis, is threatened and historically has shown rapid decline in the Sydney region. The extensive seagrass meadows selected for this study exhibited negligible anthropogenic disturbances so as to construct a reliable baseline. Sessile epifaunal invertebrates inhabiting seagrass ecosystems, particularly sponges and ascidians, have been poorly studied due to their taxonomic complexity. They are considered difficult to identify because of their great morphological plasticity and a general lack of taxonomic expertise. This study constitutes a thorough and statistically robust approach to investigating patterns and processes in a poorly described community. This study addresses an important knowledge gap and widens our understanding of a community associated with a threatened seagrass ecosystem facing tremendous anthropogenic pressure. I used a step by step framework, from preliminary data to manipulative experiments, as to uncover baseline information on patterns of distribution and community dynamics. I encountered 20 sponge and eight ascidian species within Jervis Bay, which has the most extensive and near pristine P. australis seagrass meadows of the south eastern Australian coast. The adequacy of my sampling procedures of sessile invertebrate diversity and abundance, was established for this specific assemblage through preliminary sampling and cost effective way of sampling. I established the sampling guidelines, the ideal sampling units and their dimensions, as well as the most effective experimental design. This included replication at the appropriate scales of variability whilst ensuring the efficient allocation of resources. I used a hierarchy of spatially nested scales to determine the patterns of distribution of sponges and ascidians inhabiting P. australis. This assemblage was sporadically distributed in the seagrass meadows with high variability at multiple spatial scales. A few sponge and ascidian species dominated the assemblage and were widespread across the largest spatial scale sampled. The remaining species were mostly rare and sparsely distributed. Sponges attached to a variety of substrata but most notably shells, P. australis and polychaete tubes. No obligate seagrass species were recorded although three species predominantly used P. australis as a substratum. Due to the high variability seen in the distribution of this assemblage, I assessed the robustness of my sampling design by comparing the outcome of my study with that of my initial pilot study. Overall, the pilot study was a valuable tool in the establishment of an effective sampling design. Despite intrinsic limitations and high levels of variation, the pilot study reflected the distribution of the patchy assemblage seen in the main study.

Once the patterns of distribution of this assemblage were established, the factors driving these patterns of distribution were investigated as ecological processes driving community dynamics are paramount to our understanding of species interactions. Small-scale biogenic characteristics of the seagrass and large-scale seascape patterns of the meadow were found to influence sessile epifaunal distribution, although a marked disparity occurred between sponges and ascidians. Sponge distribution relied on seagrass characteristics, including the length and density of seagrass shoots. In contrast, ascidian species richness was influenced by the area of nearby reefal habitats, not seagrass characteristics. Distributional patterns of sessile epifaunal invertebrates in seagrass meadows were not influenced by the abundance of a common fish species, noted as a consumer of invertebrates. This provided an initial investigation of the factors driving this threatened community and should be perceived as an important stepping stone in designing future manipulative experiments. The processes responsible for the patterns of distribution observed were then uncovered using manipulative experiments. The biotic factors driving sponge distribution were tested since predation was observed in situ and is a known driver of sponge distribution in reefal habitats. I investigated the array of predator species present within the seagrass meadows, the palatability of seagrass-associated sponge species and their level of deterrence to predators, as well as the availability of hard substrata, such as shells, within the seagrass meadow. A predator exclusion experiment showed no detectable effect of fish predation or substratum availability on the seagrass sponge community. Predator exclusion cages successfully excluded large fish predators but were breached by organisms small enough to traverse the mesh, such as fish and shrimps. Diurnal palatability assays showed negligible predation for the three sponge species investigated (Callyspongia (Toxochalina) sp., Haliclona spp. and Halichondria sp.). Shrimps (Palaemon intermedius) were observed interacting with sponge explants although further research is needed to ascertain whether shrimps were feeding on sponge tissue or on their epifaunal community. Future studies should aim to uncover the effect of sponge recruitment and larvae dispersal on sponge distribution, as they are thought to influence the patterns seen in the predation exclusion cages. Nocturnal predators should be investigated further. In conclusion, this study uncovered a previously poorly known assemblage from one the most threatened ecosystems worldwide. This is the first study in the world to 1) establish comprehensive sampling procedures in this study systfem, 2) identify the species present, 3) determine patterns of distribution, 4) examine the abiotic and biotic factors driving such distribution, and 5) use manipulative experiments to understand the key processes influencing this community, specifically for the sessile epifaunal invertebrate assemblage of seagrass ecosystems. This extensive assemblage was found to rely heavily on seagrass and is therefore likely prone to

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disturbances impacting its host habitat. Examining the response of sessile epifaunal invertebrates to the degradation of their seagrass habitat remains a key challenge for the future.

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ACKNOWLEDGEMENTS

Firstly I would like to thank my supervisors; Nathan Knott and Andy Davis. Without you, I would not have started on this crazy ride. Nathan encouraged me to pursue a PhD in the first place, making it sound exciting, rewarding and everything a PhD isn’t. Andy kindly reminded me, on the day, that it was the cut-off date to apply for a PhD scholarship and to ‘whip-up’ a project proposal within a few hours. But most importantly, you kept me going throughout the years. You picked me up when I was down and you believed in me when I doubted myself. Thank you Andy for your patience and constant positive feedback. Thank you Nathan for pushing me to think critically and for your useful, but endless, phone conversations. I would like to thank numerous volunteers that spent countless hours diving and shivering by my side or helping out with field work. Your assistance was crucial to the success of my project and no amount of tiramisu can come close to repaying you. Thank you Axton Aguiar, Lucia Aguilar, Nathan Bass, Allison Broad, Aimee Bullock, Elizabeth Carron, Daniel Coleman, Guy Demers, Corrine DeMestre, Jaqueline Fenwick, Bridget Harvey, Anne Martha Hellgren, Lara Hindmarsh, Rhiannon Kiggins, Russell McWilliam, Kai Paijmans, Naomi Paquette, Matthew Rees, Helene Roy, Kozue Saito, Mari Spildrejorde, Margaret Whalen and Caitlin Woods for assistance in the field. Above all, I would like to acknowledge my mom Helene Roy, self-proclaimed Field Safety Officer, who donated time, efforts and tasty food. Thank you for your support. Also, Corrine DeMestre and I shared the hardest field day of my life, and bonded over the electroshock therapy. I wish you and your lovely family all the best. I want to thank colleagues and professionals that provided valuable assistance. I acknowledge; Dr. Stephen Keable for lodging sponge vouchers at the Australian Museum Research Institute, Zhi Huang from GeoScience Australia for providing the SST and ChlA data, Chris Owers for providing assistance with spatial data analysis, Lisa Goudie and Dr. Jane Fromont who unlocked the secret identity of these elusive sponge species, Andréanne Demers for her skills in graphic design, Marijka Batterham for assistance with statistical analysis, Carol Cordonis for assistance with sponge digestion, Corrine DeMestre for her boating skills, Geoffrey Hurt and Steve Cooper for assistance with field equipment, Shane Ahyong for last minute shrimp identification, Beecroft Weapons Range and Booderee National Park rangers for their patience and understanding, especially Martin Fortesque, and all my colleagues at University of Wollongong who generously donated their time and precious advice, especially Ben Gooden and Allison Broad for their moral support. Also, Matthew Rees for his help with statistics in R and for his continuous motivation through the ups and downs of our PhDs.

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I would like to thank the research team and technicians from CEAB-CSIS, Catalonia, Spain for your support and kind welcome. Although I have not included our data in my thesis, it has been a fulfilling experience to visit your beautiful seagrass meadows. Thank you Dr. Manuel Maldonado, Cèlia Sitjà Poch, Maria López-Acosta, Marta García-Puig, Ferran Crespo Cano, David Barba and Manel Bolivar Basart. I wish you the best of luck in your future endeavours. I would like to thank University of Wollongong and the Department of Primary Industries for funding my research. Additional funding was generously provided by Global Challenges and ACA Scientific, which allowed me to travel to Spain and acquire supplementary data. Thank you. Also, I would like to acknowledge animal ethics approval (permit AE 12/07) and Booderee National Park permission to perform research (permit F95/269 & OUT11/2524). This research has been conducted with the support of the Australian Government Research Training Program Scholarship. Finally, I would like to thank my better half, Axton, my family (Guy, Helene and Andréanne) and friends (Hala, Hayley, Caity, Clara and Michelle) that have supported and motivated me to finally cross the finish line.

‘A PhD is like a calcareous sponge, it is hard, boring and unpalatable but when you look closely you can see the beauty in its intricate spicule design that can only be achieved with a tremendous amount of dedication.’

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Chapter 2

CHAPTER 1

General Introduction

Chapter 1

1.1 ECOLOGICAL ROLE OF SPONGE AND ASCIDIANS Sponges (Phylum Porifera) belong to a diverse phylum of an estimated 15,000 species (Rützler et al., 2007) and consist of a ‘sedentary filter-feeder metazoan which utilise a single layer of flagellated cells (choanocytes) to pump a unidirectional water current through its body’ (Bergquist, 1978). A recent study suggests that sponges may have been the first metazoan animal to inhabit the Earth as they appeared 640 million years ago (Gold et al., 2016). They are the most primitive animal on Earth and are thought to be the ancestors of all complex animals (Nichols et al., 2009). Sponges lack a complex nervous, digestive, or circulatory system, although this does not prevent them from having crucial ecological roles. For instance, they are believed to have helped aerate early oceans by means of their filtering capability (Zhang et al., 2015). Some sponges are known to filter a quantity of water 100 000 times their volume daily (Schrope, 2009). Some sponges cultivate photosynthetic symbionts to complement their nutritional needs (Wilkinson, & Vacelet, 1979). Sponges are easily affected by their environment and have specific abiotic requirements (Wilkinson & Vacelet, 1979). There are a variety of interactions between sponges and other organisms in marine ecosystems with sponges acting as competitors, consumers, predators, symbionts, the host of symbionts, an important food source and provide key habitat for an array of epibiota (Wulff, 2006a). Ascidians (Phylum Chordata, Subphylum Tunicata, Class Ascidiacea), also known as sea squirts, are colonial or solitary benthic suspension feeders, and exhibit a wide variety of forms, colours and structures (Kott, 1989). They feed by filtering water through a branchial basket enveloped in a mucus net which traps particulate matter (Millar, 1971). Water is pumped through the inhalant siphon and is expelled through the exhalent siphon (MacGinitie, 1939). Like sponges, some ascidians are highly dependent on endosymbionts for their energy needs (Sybesma et al., 1981). Their abundance is determined by the nature of the substratum, amount of suspended matter and light (Millar, 1971). Most ascidians thrive in eutrophic environments and favour hard substratum such as artificial surfaces, rocks, reefs, shells, seagrass and algae (Dybern, 1963; Shenkar & Swalla, 2011). Ascidians also compete inter and intra specifically for food and space (Dalby, 1995; Yakovis et al., 2008), they serve as an important food source and host a multitude of organisms (Millar, 1971). Sessile epifaunal invertebrates, including sponges and ascidians, are an important structural and functional component of reefal habitats (Diaz & Rützler, 2001). They play an influential role in a multitude of ecological processes such as primary production, nitrification, nutrient cycling, food chains, habitat provision, competition for space and predation (Diaz & Rützler, 2001; Rützler,

Chapter 1

2004). Importantly, they impact nutrient fluxes and particulate organic matter availability via their incomparable filtering capacities (Diaz & Rützler, 2001).

1.2 HABITAT BIAS Although our knowledge is limited in regards to the sessile epifaunal invertebrates inhabiting seagrass ecosystems, communities from other habitat have been well-studied. Sessile epifaunal invertebrates occur in many habitats such as rocky reefs (Roberts & Davis, 1996), mangrove roots (Farnsworth & Ellison, 1996), kelp forests (Wright et al., 1997), urchin dominated barrens (Wright et al., 1997), caves (Bibiloni et al., 1989), artificial structures (Knott et al., 2004), sand (Barnes, 1999), mollusc shells (Keough, 1984) and gorgonians (Reiswig, 1973; McLean & Yoshioka, 2007). The sessile epifaunal invertebrate community inhabiting coral reefs is perhaps the most extensively studied. Sponges associated with coral reefs are considered abundant and highly diverse, sustaining a large sponge-associated infaunal community (Diaz & Rützler, 2001). Effectively, sponge diversity often exceeds that of corals and algae on coral reefs (Wulff, 2008). The sessile epifaunal invertebrate community of seagrass meadows has been found to be relatively low compared to other habitats such as reefs (Kuenen & Debrot, 1995; Barnes, 1999). The spatial distribution of sessile epifaunal invertebrates in Posidonia meadows has rarely been investigated (but see, Lemmens et al., 1996; Barnes et al., 2006, 2013). As shown by Barnes et al. (2006, 2013), sponges and ascidians inhabiting coastal saline lakes in NSW, Australia, were patchily distributed at a range of spatial scales (Barnes et al., 2006, 2013). These studies however were not strictly inclusive of seagrass meadows and included other habitat such as bare sediment, macroalgae and shells. A patchy distribution was also documented in sponges and ascidians inhabiting other habitats such as reefs (Roberts & Davis, 1996; Ferdeghini et al., 2000; Hooper & Kennedy, 2002) and mangrove roots (Farnsworth & Ellison, 1996; Guerra-Castro et al., 2011). Variation in the abundance of sessile epifaunal invertebrates occurres at multiple scales ranging from 10s of metres to 100s of kilometres (Barnes et al., 2006, 2013). This variation was also reported for sessile epifaunal invertebrates settling on artificial surfaces (Glasby, 1998) and natural rock walls (Davis et al., 2003). It has been suggested that the sporadic distribution of sessile epifaunal invertebrates in seagrass meadows may be associated with the nature and availability of substrata (Millar, 1971; Keough & Downes, 1982; Keough, 1984; Knott et al., 2004; Barnes et al., 2006; Wulff, 2008). It may be informative to examine the patterns and subsequently the processes driving sessile epifaunal invertebrates within seagrass ecosystems. Overall, there is an extensive gap in the literature on the composition, distribution and dynamics of the sessile epifaunal invertebrate assemblage of seagrass meadows. This knowledge is particularly relevant to the threatened seagrass species P. australis owing its alarming decline in the

Chapter 1

NSW region. Although sponges and ascidians have often evaded biologists due to their taxonomic difficulties, they are now in need of immediate scrutiny.

1.3 SEAGRASS IMPORTANCE AND DECLINE Extinction rates are accelerating globally and have reached 1000 times that of pre-human levels (Pimm et al., 2014). This biodiversity decline is the result of intensifying pressures from overexploitation, habitat loss and climate change, as well as inadequate efforts to protect biodiversity (Butchart et al., 2010; Pimm et al., 2014). Although the exact number of species at risk of extinction is unknown (Pimm et al., 2014), studies suggest that millions of species are expected to go extinct over the next century (Dirzo & Raven, 2003; Pereira et al., 2010). We face an urgent need to protect threatened habitats worldwide (Dirzo & Raven, 2003). Species-rich habitats are especially important as their communities are too, at risk of extinction. These communities may comprise unknown species that could disappear before being discovered or described scientifically (Dirzo & Raven, 2003). Seagrass meadows are disappearing at an alarming rate and are now amongst the most threatened ecosystems worldwide (Waycott et al., 2009). Seagrass meadows are considered a pivotal marine habitat and provide valuable ecosystem services, estimated at US$3.8 trillion annually (Costanza et al., 1997). Seagrass meadows make significant contributions to coastal productivity worldwide and rank amongst the highest recorded for marine and terrestrial ecosystems (Hillman et al., 1989). Their intrinsic value stems from the dis-proportionately abundant and diverse communities they support, relative to other marine habitats (Hillman et al., 1989). Seagrass meadows provide nourishment and protection to an array of organisms through complex species interactions. Seagrasses are particularly important in sustaining juvenile species due to their function as nursery grounds (Hemminga & Duarte, 2000). Many organisms rely on seagrass habitat for survival including commercially valuable species (Heck et al., 2003; Gillanders, 2006), as well as rare and threatened species (Short et al., 2011). It is likely that damage to seagrass will diminish stocks of important commercial and recreational fisheries, molluscs and crustaceans (Bell & Pollard, 1989; Butler & Jernakoff, 1999; McArthur & Boland, 2006). As ecosystem engineers, seagrasses indirectly control the availability of resources by physically changing, maintaining and creating habitats for other organisms (Costanza et al., 1997; Bos et al., 2007). Seagrasses modify their environment in four different ways; 1) through filtration of suspended sediments and nutrients, 2) stabilization of sediments, 3) reduction of wave action, and 4) the diurnal cycle of oxygenation followed by carbonation of the surrounding waters (McRoy & Helfferich, 1980; McComb et al., 1981; Fonseca, 1989; Costanza et al., 1997; Bos et al., 2007). These processes result in high water clarity, promoting an ideal environment for marine tourism and

Chapter 1 associated benefits for the community (Badalamenti et al., 2000; Samonte-Tan et al., 2007; Cullen- Unsworth et al., 2014). Most importantly, this ecosystem is an intensive carbon sink with a carbon storage capacity 35 times more efficient than rainforest (McLeod et al., 2011) and the ability to bind carbon for millennia (Macreadie et al., 2012). The destruction of seagrass meadows leads to the release of this trapped blue carbon into the atmosphere, therefore exacerbating the effect of climate change (Fourqurean et al., 2012). Despite the well-established importance of seagrass ecosystems, they have been declining globally at an accelerating rate over recent decades (Short & Wyllie-Echeverria, 1996; Waycott et al., 2009). Approximately 30% of the world’s seagrass species are in decline with 14% of seagrass species having a high risk of extinction and three species listed as endangered (Short et al., 2011). Australian seagrasses are no exception, seagrass meadows have suffered extensive declines in several states in the past (Shepherd et al., 1989; Walker & McComb, 1992). In NSW, seagrasses are protected under the NSW Fisheries Management Act 1994 as a way to minimize local seagrass decline (NSW Department of Primary Industries, 2010). Seagrass loss has been attributed to a broad spectrum of anthropogenic and natural disturbances (Duarte, 2002; Ruiz & Romero, 2003), including herbivory (e.g. Klumpp et al., 1989), inter and intra species competition (Shepherd et al., 1989), restriction of solar radiation (e.g. Fyfe & Davis, 2007), eutrophication (Orth et al., 2006; Burkholder et al., 2007), mechanical damage (e.g. Demers et al., 2013) and climate change (Short & Neckles, 1999).

1.4 SEAGRASS ASSOCIATED SPECIES AND ECOSYSTEM FUNCTION

1.4.1 Seagrass associated species Seagrass ecosystems are a key habitat for an array of organisms including infaunal species1, sessile and motile epifaunal species2 and epibenthic species3 (Howard et al., 1989). Seagrass residents may be permanent or temporary, while other species may visit seagrass meadows at regular or occasional intervals (Hemminga & Duarte, 2000). A considerable amount of research has focused on epibenthic species within seagrass, such as fish, decapods, cephalopods, especially those of commercial importance (Boström et al., 2006; Gillanders, 2006). In contrast, sessile epifaunal invertebrates have been poorly documented in seagrass ecosystems despite being known permanent inhabitants (Barnes et al., 2006). The sponge and ascidian community of seagrass meadows is essentially unknown and is often deliberately excluded from studies due to the multitude of challenges they present. They are

1 fauna living in the sediment 2 fauna living on the surface of the stems and leaves of seagrasses 3 free-moving organisms 5

Chapter 1 considered to be taxonomically difficult to identify because of their great morphological plasticity and the presence of numerous undescribed species worldwide. In addition, sponges display complex 3-dimensional growth forms that are difficult to define and quantify (Bergquist, 1978; Wulff, 2001). The disparity in the morphological features of sponges make species description and identification arduous as researchers must contend with a range of morphs of different colours, textures and structures (Bergquist, 1978; Wulff, 2001). Ascidians identification is also difficult as specimens often require dissection for positive identification and the prevalence of cryptic species presents an additional challenge (Davis et al., 1999; Shenkar & Swalla, 2011). The sessile epifaunal invertebrate community of seagrass ecosystems is currently poorly documented. A review of papers investigating sponge processes (n=111) revealed that sponge research primarily focuses on reefal habitats and constituted 80% of the papers investigated (Figure 1.1A). In contrast, papers examining sponge processes in seagrass ecosystems represented just 9% of the literature reviewed (Figure 1.1A). The spatial distribution of sponges in seagrass ecosystems contributed to more than half (5%) of this 9% (Figure 1.1B). A mere two papers focused on the sponge spatial distribution within the seagrass genus Posidonia. This was for Posidonia oceanica in the Mediterranean, and the Australian Posidonia australis (Hook f.) (Figure 1.1B). Importantly, the sessile epifaunal invertebrate assemblage inhabiting P. australis meadows has not been vigorously investigated despite the rapid decline of this species in Australian waters (Meehan & West, 2002; Waycott et al., 2009).

1.4.2 Seagrass ecosystem functioning Foundation species are the basis of entire ecosystems. They mediate ecosystem functioning and influence community composition through complex interactions with community members (Bracken et al., 2007). Species interactions between a foundation species and the community it supports may be context dependant (Chamberlain et al., 2014) and generally involve positive relationships (Bracken et al., 2007). Species interactions between seagrasses, which are important foundation species, and sponge inhabitants have been poorly documented (but see Archer et al., 2015). It is crucial to establish the nature of the relationship between sessile epifaunal invertebrate community and its seagrass host so as to uncover the role sponges play in seagrass ecosystem structure and function. This will provide a more complete understanding of how sessile epifaunal invertebrates influence seagrass ecosystem and vice versa. Positive relationships have been recorded between coral reefs and the sponge community it hosts (e.g. Wulff, 2012). Through this association, sponges acquire suitable substrata in exchange for increased coral survival. Other foundation species such as mangrove and algae species use their

Chapter 1

Chapter 1 sponge community as beneficial sources of bioavailable nutrients, therefore promoting plant growth (Ellison et al., 1996; Davy et al., 2002). The interactions between sponges and seagrass could also involve similar benefits (adequate hard substratum for attachment and increased nutrient availability) as seen in these other habitats. Deleterious interactions between sessile epifaunal invertebrates and their host habitat have also been documented. Sponges often chemically or mechanically compete with corals for space (Wulff, 2012). For instance, the sponge family Clionidae is known to excavate corals and mollusc shells (Rützler, 1975). Sponge bioerosion of corals not only has direct negative impact on the host species but also weakens the reef framework, leaving this habitat highly susceptible to disturbances, such as storm damage (Macdonald & Perry, 2003). Bioerosion of shells by sponges may also affect substratum availability in seagrass meadows. Species interactions within seagrass ecosystems maintain species diversity and define seagrass condition. Studies have recorded positive interactions between seagrass and sessile epifaunal invertebrates in the past. For instance, a symbiotic relationship was established between seagrass and a bivalve as well as its gill bacteria (van der Heide et al., 2012). A study by Archer et al., (2015) established a commensal interaction between a subtropical seagrass Thalassia testudinum and its sponge inhabitants. Sponges were impartial to seagrass as the negative impact of shading cancelled the positive effect of nutrient transfer. Alternatively, sponges benefited from a stable substratum and enhanced food availability as particulate matter was concentrated at the base of the seagrass (Archer et al., 2015). This context-dependent interaction has the potential to change to a parasitic interaction if eutrophication reaches antagonistic levels (Archer et al., 2015). Such beneficial relationships may also take place in the sessile epifaunal invertebrate assemblage of P. australis meadows.

1.5 PATTERNS AND DRIVERS OF SESSILE EPIFAUNAL INVERTEBRATE DISTRIBUTION IN SEAGRASS MEADOWS

1.5.1 Factors driving sponge distribution Ecological processes driving community dynamics are paramount to our understanding of species interactions and the role these species play in alleviating anthropogenic pressure on our coastal ecosystems. Rapidly declining ecosystems such as seagrass meadows are likely to experience changes in ecosystem structure and function due to the cascading effect of human disturbances. Food chain dynamics, a subset of ecological theory, propose that interacting factors influence ecosystem functioning (Fretwell, 1987). The role of sessile epifaunal invertebrates in this seagrass trophic network is unclear and could have important ramifications for this threatened

Chapter 1 ecosystem. A myriad of factors may be driving the sessile epifaunal invertebrate community of seagrass meadows. These drivers of spatial distribution are often interrelated and vary depending on the spatial scale being investigated (Hooper & Kennedy, 2002).

1.5.2 Abiotic Factors The abiotic factors influencing the distribution of sessile epifaunal invertebrates in seagrass meadows have rarely been investigated (but see Corriero et al., 1989; Lemmens et al., 1996). Physio-chemical parameters are likely drivers of seagrass faunal distribution patterns as they have been known to alter seagrass-associated species composition, other than sessile epifaunal invertebrates. For instance, the distribution of soft-bottom polychaetes in seagrass meadows was found to be mainly influenced by salinity and depth (Quintas et al., 2013). In addition to these two environmental factors, the species composition and richness of epibenthic macrofauna (crustaceans, molluscs and fishes) inhabiting a seagrass meadow also varied with seagrass species and water temperature (Young & Wadley, 1979). Other known factors of benthic species distribution include the nature of the substratum, organic content and disturbances (i.e. Quintas et al., 2013). The abiotic factors driving the distribution of sessile epifaunal invertebrates inhabiting reefal habitats have been well documented, unlike seagrass meadows. It is unknown whether these factors may also play a role in the distribution of sponges and ascidians in seagrass ecosystems. Physio-chemical parameters were also found to influence the distribution of sessile epifaunal invertebrates in reefal habitats (Wilkinson & Evans, 1989; Roberts & Davis, 1996; Duckworth et al., 1997; Duckworth et al., 2004), especially gradients in water quality (Wilkinson & Cheshire, 1989; Roberts et al., 2006). Also, the growth and survival of sponges was found to rely heavily on water flow and associated food availability (Wilkinson & Vacelet, 1979; Duckworth et al., 2004), which is to be expected due to their filter-feeding nature. These parameters are therefore likely to influence the distribution of sessile epifaunal invertebrates in seagrass meadows. The environmental parameters associated with a specific sponge habitat can drive population dynamics. For instance, sponges inhabiting illuminated habitats such as shallow reefs are affected by competition for light (Rützler, 2004). In contrast, sponges from deep, light-deprived environments such as caves, crevices and overhangs, are disinclined to compete for solar radiation. Sponges inhabiting these deep stable habitat structures have different morphology to shallow reef sponges as their environment offers protection against surge and periodic storm disturbances (Roberts & Davis, 1996; Rützler, 2004). Effectively, a higher risk of toppling was associated with arborescent growth forms and a low proportion of spongin in sponge skeletal composition (Wulff, 1995a). The environmental variables characteristic of seagrass meadows are also likely to impact the distribution of sessile epifaunal invertebrate in this habitat.

Chapter 1

Nutrient availability is likely an important driver for the distribution of sessile epifaunal invertebrates within seagrass meadows. As mentioned previously, seagrass meadows have the ability to trap suspended sediments and nutrients (Burkholder et al., 2007; Bos et al., 2007). Most sponge species are filter-feeders with an outstanding ability to capture particulate materials from the water column (Bergquist, 1978). They are likely to benefit from the increased sedimentation associated with seagrass meadows. Increased nutrient availability is known to favour sponge growth and survival rates (Morris & Keough, 2003; Duckworth et al., 2004), at least to a threshold (Roberts et al., 1998). A study by Corriero et al. (1989) showed that the hydrodynamic regime and nutrient load of a seagrass meadow played a deterministic role in the spatio-temporal distribution of two sponge species. The inner meadow showed weak currents and high sedimentation rate resulting from the high density of seagrass shoots (Corriero et al., 1989). Also, nutrient availability has been linked to the spatial distribution of large suspension feeders such as sponges in the P. australis meadows of Cockburn Sound, Western Australia (Lemmens et al., 1996). The availability of this important food source was tightly associated with the current baffling ability of seagrass meadows (Lemmens et al., 1996). The nature and availability of suitable substrata are important community components for the distribution of sessile epifaunal invertebrates (Sarà & Vacelet, 1973; Schubauer et al., 1990; Adjeroud, 1997; Davis et al., 2003; Fromont et al., 2006). A study by Pansini and Musso (1991) reported that the orientation and size of the available substratum affect sponge settlement and growth in muddy and detritic environments. The orientation and nature of the substratum was also found to influence the distribution of ascidians (Knott et al., 2004). Fromont et al. (2006) established that the configuration of the substratum, such as platform versus dissected reef also have an effect on the composition of sponge assemblages. As an environmental engineer, seagrass might also provide suitable substratum for sponge settlement and persistence in an otherwise species-poor landscape. The biogenic characteristics of seagrass habitats are expected to influence the spatial distribution of sponges and ascidians. This assemblage relies heavily on its host habitat when found on biogenic habitats such as mangrove roots, kelp forests and gorgonians (e.g. Reiswig, 1973; Wulff, 2003). Multiple studies have established the importance of habitat structure in shaping seagrass invertebrate communities. Biogenic seagrass factors such as shoot density, meadow size and configuration were previously found to influence the density, diversity and survival of epifauna (e.g. Irlandi et al., 1995; Hovel & Lipcius, 2002). The hydrodynamic forces associated with meadows of different sizes and orientations were established as important drivers of larvae dispersal for a wide range of epifaunal species in seagrass (Tanner, 2003). The structural complexity

Chapter 1 provided by epiphytic algae on seagrass leaves (Gartner et al., 2013), the seagrass species present (Nakaoka et al., 2001) and the level of habitat fragmentation of the meadow (Healey & Hovel, 2004) also play an important role in determining the distribution of some invertebrates. Anthropogenic disturbances alter these abiotic spatial drivers and often indirectly impact the patterns of distribution of sessile epifaunal invertebrates (Rützler et al., 2004). For instance, changes in sponge species composition, a reduction in species richness and sponge cover were associated with the increased siltation, reduced light availability and salinity that stemmed from the installation of a sewage outfall (Roberts et al., 1998). Varying levels of human disturbance may contribute to the distributional patterns of the sessile epifaunal invertebrate assemblage, as species associated with seagrass meadows are most likely prone to disturbances impacting their host habitat. Eutrophication is the most prominent source of seagrass degradation and is likely to affect seagrass- associated communities (Burkholder et al., 2007). Direct physical disturbances such as anchoring and mooring are highly damaging to seagrass (Milazzo et al., 2004; Demers et al., 2013) and are therefore prone to impact the seagrass invertebrate community.

1.5.3 Biotic Factors Biotic factors have been shown to influence the spatial heterogeneity in sponge assemblages (i.e. Wulff, 2006a). It is likely that these biotic factors also play a key role in driving sessile epifaunal invertebrate patterns of distribution within seagrass environments. Little is known of the biotic factors affecting sessile epifaunal distribution in seagrass meadows (but see Wulff, 2008; Archer et al., 2015) as most studies concentrate on reefal habitats. Predation can modulate sponge community structure (Wulff, 1997b; Pawlik, 2013). Benthic organisms are unable to evade predation and therefore attempt to tolerate or reduce predation (Lubchenco & Gaines, 1981). Predation deterrence and avoidance have initiated several prey adaptations, including: 1) favouring an opportunistic life cycle, 2) selecting habitats with low predation pressure, 3) producing defences, and 4) regenerating rapidly after partial mortality (Connell, 1975; Brosnan, 1992; Hoppe, 1998; Wulff, 2006a; Wulff, 2010). These adaptations likely incur costs and are often associated with a reduction in growth rate. An array of mechanical and chemical defences are used by sponges such as collagen fibres, spicules and noxious chemical compounds (Sarà & Vacelet, 1973, Thacker et al., 1998; Ferguson & Davis, 2008; Pawlik et al., 2013). Sponge predators include molluscs, echinoderms, crustaceans, fishes and turtles. Spongivorous species can be categorised into three main groups based on their diet. ‘Smorgasbords’ are restricted in their sponge intake to small fragments of multiple species. ‘Specialists’ consume a few target species exclusively. ‘Opportunistics’ rarely ingest sponges and prefer poorly defended species (Wulff, 2006a). The presence and identity of spongivorous species within seagrass

Chapter 1 ecosystems, as well as the adaptations seagrass-associated sponge species have developed to deter predation are poorly understood. Sponge palatability can be an important determinant of sponge ecosystem dynamics and processes (Wulff, 2005; Pawlik et al., 2013). Population and community dynamics, of an Antarctic sponge assemblage, were driven by predation and mediated by chemical defences (Dayton, 1979; McClintock et al., 2005). Structurally or chemically defended sponges have specific physical characteristics associated with low predation pressure such as exposed, colourful and branching species. More palatable species, however, have a different appearance and are generally more cryptic (Wulff, 1997b; Wulff, 2006a; Pawlik et al., 2013). It is unknown whether seagrass- associated sponges display specific physical characteristics. Competition among sponge species is also an important driver of sponge dynamics. Sponge competition stems from limited resources, most frequently from restricted substratum availability (Jackson & Buss, 1975; Branch, 1984). Competition for space often involves elimination by overgrowth, in which case an individual of disproportionate size or growth rate engulfs its neighbour (Wulff, 2006a). Most sponges use chemical defences against a variety of other taxa such as corals, bryozoans and tubeworms (Keough, 1984; Rützler & Muzik, 1993; Aerts & Van Soest, 1997). Rapid regeneration is an important strategy employed by sponges to obtain competitive superiority in space-limited systems. This ability often allows a sponge species to successfully obtain or retain a substratum, although it is often energetically expensive to do so (Wulff, 2010). Ascidians are also efficient competitors on coral reefs (Bak et al., 1981) and among bivalve aggregations (Bullard et al., 2007; Daigle & Herbinger, 2009). The colonial ascidian Didemnum vexillum, for instance, is a successful invasive species that dominates and negatively impacts coastal habitats worldwide (Janiak et al., 2013). It is unknown whether sessile epifaunal invertebrates inhabiting seagrass meadows use seagrass blades as a substratum of attachment, whether this assemblage competes for space or other resources and the resulting species dynamics. The energetic trade-offs between resistance to competition and likelihood of predation influence community structure. Transplant experiments have shown that although sponges may be well adapted to a suite of predators and competitors from their native environment, this may not be the case once transplanted into a foreign environment (e.g. Wulff, 2005). For instance, reef predators preferred consuming mangrove species over their typical reef sponge assemblage. Unlike reef species, these mangrove species were defence-less against predation (Wulff, 2005). However, once transplanted in a mangrove ecosystem, reefal sponges competed poorly for space due to their slow growth rate. Fast growing mangrove sponge species easily outcompeted reef species and monopolized mangrove root space (Wulff, 2005). Our knowledge of the biotic factors driving the

Chapter 1 sessile epifaunal invertebrate assemblage of seagrass meadows is highly limited and requires further investigation.

1.6 SPECIES OF INTEREST Posidonia australis Hook.f. is distributed from Shark Bay, Western Australia, following the southern coast of Australia to Lake Macquarie, New South Wales, and along the northern coast of Tasmania (Edgar, 2000). In New South Wales, this species is present in approximately 20 estuaries located southwards from Wallis Lake extending to Twofold Bay (NSW Department of Primary Industries, 2010). Posidonia australis generally forms vast, dense and continuous communities in clear saline water 0.5m to 7m deep (Cambridge, 1975). This species favours locations with reduced wave exposure and the absence of hyposaline conditions, such as sheltered bays, lagoons and estuaries (Cambridge, 1975; West et al., 1989; den Hartog & Kuo, 2006). These specific habitat requirements renders Posidonia australis particularly prone to anthropogenic disturbances. In addition, Posidonia australis does not generally recover once disturbed (Clarke & Kirkman, 1989) or recovers at a rate of just 21cm per year assuming no further disturbances (Meehan & West, 2000). This stems from its slow rhizome expansion, growth and colonizing rates (Kirkman, 1985; Kirkman & Kuo, 1990; Gobert et al., 2006). This species has a long life span and a very low turnover rate (West & Larkum, 1979; Hemminga & Duarte, 2000). Although Posidonia australis produces large numbers of seeds this dispersal method is rarely effective. Posidonia australis mainly disperse through vegetative propagation via horizontal rhizome extension (McComb et al., 1981; Gobert et al., 2006). In this case, a horizontal subterranean stem slowly spreads and progressively produces a new system of shoots and roots. Posidonia australis, is listed as ‘threatened’ under the amended (1997) NSW Fisheries Management Act, while populations of Port Hacking, Botany Bay, Port Jackson (Sydney Harbour), Pittwater, Brisbane Waters, Lake Macquarie, Wallis Lake, Port Stephens, Hawkesbury River and the lee of Broughton Island have been recently listed as ‘endangered populations’ (Fisheries Management Amendment (Threatened Species Conservation) Order (No 1) 2010 of the NSW Fisheries Management Act). Human derived climate change has been listed as a key threatening process for these specific populations. Direct conservation actions have been introduced by the Marine Parks Authority NSW such as the installation and monitoring of seagrass-friendly ‘screw’ moorings (Marine Parks Authority NSW, 2012). These permanent boat mooring systems appear to have virtually no effect on seagrass (Demers et al., 2013). Boat exclusion zones and restricted anchoring zones have also been established to minimise seagrass damage (Marine Parks Authority NSW, 2012).

Chapter 1

1.7 AIMS AND STRUCTURE OF THIS THESIS Sessile epifaunal invertebrates inhabiting seagrass ecosystems, particularly sponges and ascidians, have been poorly studied due to their taxonomic complexity. The sessile epifaunal invertebrate assemblage inhabiting Posidonia australis, a threatened seagrass species, is currently unknown, even though it is likely prone to suffer from the same disturbances as its host habitat. The importance of this assemblage has been underestimated and further investigation is evidently needed. The aims of this study were formulated based on the limited information available on the spatial distribution of seagrass-associated sessile epifaunal invertebrates, and studies that have investigated the sponge and ascidian assemblage of other habitats.

The aims of this study are to; 1) identify the sponge and ascidian species within seagrass Posidonia australis, 2) determine the distributional patterns of this assemblage at a hierarchy of spatial scales, 3) examine how abiotic and biotic factors influence the distribution of this assemblage.

The first two aims are addressed in Chapter 2, where I identified the sponge and ascidian species within seagrass Posidonia australis within Jervis Bay, NSW, Australia. The distributional patterns of these invertebrates were determined at a hierarchy of spatial scales. Based on previous studies, this community was expected to be characterised by an anomalous and patchy distribution at a range of spatial scales. The availability and suitability of substrata was also determined since this variable is likely to influence the settlement and survival of sessile epifaunal invertebrates in seagrass meadows. I established whether sponge and ascidian species occur consistently on certain substrata within seagrass habitats, and identified seagrass obligate species. This chapter is now published in the Journal of the Marine Biological Association of the United Kingdom (Demers et al., 2016). Chapter 3 was an investigation of the effectiveness of a pilot study in developing a robust sampling design for a spatially variable assemblage; sessile epifaunal invertebrates in seagrass meadows. I determined whether the pilot study effectively reflected the spatial distribution of the assemblage seen in the main study. I predicted that the pilot study may be limited in reflecting sessile epifaunal invertebrate distribution due to the patchy distribution and presence of rare taxa in this assemblage. I contrasted the pilot and the main studies for: 1) the diversity and abundance of sessile invertebrates, 2) the composition of the assemblage, 3) the variability apparent across multiple spatial scales, and 4) significant outcomes. This chapter has been submitted to the Journal of Experimental Marine Biology and Ecology and is currently in review.

Chapter 1

The third aim was addressed in chapter 4, where correlations between an array of factors and the spatial distribution of sessile epifaunal invertebrates inhabiting seagrass meadows were uncovered. I investigated seagrass attributes (depth, shoot count, leaf length and epiphyte cover), seascape of the meadow (meadow area, perimeter, amount of reefs neighbouring the edge of the meadow, distance to nearest reef, perimeter shared between meadow and reef, area of seagrass and reef habitat at various radii scales (100m, 500m, 1000m, 1500m) surrounding each site), and abundance of a common seagrass-associated fish species. I determined how these abiotic and biotic factors influence the highly variable distribution of sponges and ascidians in seagrass meadows. As chapter 4 was a correlative chapter, I adopted an experimental approach for chapter 5. The aim of this chapter was to determine whether the sessile epifaunal invertebrate community of the seagrass meadow was driven by predation, palatability and the availability of suitable hard substratum on which to establish. This was determined using 1) a predator exclusion experiment and 2) palatability assays. Using a predator exclusion experiment, I determined the impact of removing predators from the system and the impact of providing differential levels of substratum in absence of predators. Using palatability assays, I determined the level of palatability of seagrass sponges, identify sponge predators, and test whether predators could access the predator exclusion cages used for the exclusion experiment. Finally, I conclude with a general discussion in chapter 6, where the immediate, conservation and future challenges of the sessile epifaunal invertebrate inhabiting seagrass ecosystems were recognised. The conservation challenges of this assemblage were tightly linked to its host habitat, as seagrass is one of the most threatened ecosystem worldwide. Future challenges were identified and included to 1) determine the temporal distribution of sessile epifaunal invertebrates in seagrass meadows, 2) uncover additional processes driving this assemblage via manipulative experiments and 3) identify the sessile epifaunal invertebrate assemblage inhabiting disturbed meadows.

Chapter 2

CHAPTER 2

Under the radar: Small-scale distributional patterns of sessile epifaunal invertebrates in the seagrass Posidonia australis.

Final version: Demers, M-C. A., Knott, N. A. and Davis A. R. (2016). Under the radar: Small-scale distributional patterns of sessile epifaunal invertebrates in the seagrass Posidonia australis. Journal of the Marine Biological Association of the United Kingdom, 96, 363-377.

I am the lead author of this chapter. Data collection and the first draft of this chapter were completed without the assistance of my supervisors. My supervisors contributed to 1) the conception of the project and development of the experiment, 2) data analysis and 3) manuscript editing. Chapter 2

2.1 ABSTRACT Despite the current global decline in seagrass, sessile epifaunal invertebrates inhabiting seagrass ecosystems, particularly sponges and ascidians, have been poorly studied due to their taxonomic complexity. Understanding patterns of distribution of sessile epifaunal communities in seagrass meadows is an important precursor to determining the processes driving their distribution and species interactions. This study 1) identified the sponge and ascidian assemblage associated with Posidonia australis meadows and 2) determined distributional patterns of these invertebrates at a hierarchy of spatial scales in Jervis Bay, Australia. I used a fully nested design with transects distributed in the seagrass (10s m apart), two sites (100s m apart), and six locations (km apart). Within these transects, I recorded the abundance, volume, diversity and substratum used for attachment by sponges and ascidians. I encountered 20 sponge species and eight ascidian species; they were sporadically distributed in the seagrass meadows with high variability among the transects, sites and locations. A few sponge and ascidian species dominated the assemblage and were widespread across the largest spatial scale sampled. The remaining species were mostly rare and sparsely distributed. Sponges attached to a variety of substrata but most notably shells, P. australis and polychaete tubes. No obligate seagrass species were recorded although three species predominantly used P. australis as a substratum. These sponge species relying heavily on seagrass for their attachment are likely prone to disturbances impacting their host habitat. Examining the response of sessile epifauna to the degradation of their seagrass habitat remains a key challenge for the future.

Keywords: Sessile epifaunal invertebrates, Posidonia australis, seagrass; sponges, ascidians, spatial distribution, substratum used for attachment.

Chapter 2

2.2 INTRODUCTION Seagrass meadows are economically and ecologically valuable as dis-proportionately high levels of biodiversity rely on seagrass habitat for survival relative to other marine habitats (Hemminga & Duarte, 2000; Beck et al., 2001; Short et al., 2011). Despite the importance of seagrass meadows, they have been disappearing at an alarming rate globally in recent years. One- third of the world’s seagrass species are in decline and a third of all seagrass species are considered at high risk of extinction (Waycott et al., 2009; Short et al., 2011). Disturbingly, seagrass-associated species inhabit one of the most threatened ecosystems worldwide. Epifaunal abundance and diversity have been found to decline as a result of habitat loss and fragmentation associated with relatively large seagrass disturbance (Reed & Hovel, 2006). Seagrass ecosystems are a key habitat for an array of organisms which rely on seagrass for food and their habitat requirements (Howard et al., 1989). A considerable amount of research has focused on epibenthic species within seagrass, especially those of commercial importance (Boström et al., 2006; Gillanders, 2006). In contrast, sessile invertebrates have been poorly documented in seagrass ecosystems (Barnes et al., 2006). The assemblage of sponges and ascidians associated with seagrass is essentially unknown and often deliberately excluded from studies due to the multitude of challenges they present. They are considered to be taxonomically difficult because of their great morphological plasticity and the presence of numerous undescribed species. In addition, sponges display complex 3-dimensional growth forms that are difficult to define and quantify (Bergquist, 1978; Wulff, 2001). Ascidians identification is also difficult as specimens often require dissection for positive identification and the prevalence of cryptic species presents an additional challenge (Davis et al., 1999; Shenkar & Swalla, 2011). The spatial distribution of sponges and ascidians in seagrass ecosystems has rarely been investigated (but see, Lemmens et al., 1996; Barnes et al., 2006) even though this assemblage has been the subject of several manipulative experimental studies (e.g. Corriero et al., 1989; Kuenen & Debrot, 1995; Diaz, 2005; Wulff, 2008). Descriptive studies of ecological pattern provide the starting point from which ecological explanations and theories arise. They ensure the validity of the patterns observed, and establish the scale and scope of processes operating in a specific system (Andrew & Mapstone, 1987; Underwood et al., 2000). In addition, the scales at which spatial patterns are operating must be determined so as to broaden our understanding of processes involved in shaping species interactions (Underwood & Chapman, 1996). A review of papers investigating sponge processes (n=111) revealed that sponge research primarily focuses on reefal habitats and constituted 80% of the papers investigated (Figure 1.1A). In

Chapter 2 contrast, papers examining sponge processes in seagrass ecosystems represented just 9% of the literature reviewed (Figure 1.1A). The spatial distribution of sponges in seagrass ecosystems contributed to more than half (5%) of this 9% (Figure 1.1B). A mere two papers focused on the sponge spatial distribution within the seagrass genus Posidonia. This was for Posidonia oceanica in the Mediterranean, and the Australian Posidonia australis (Hook f.) (Figure 1.1B). Importantly, the sessile epifaunal invertebrate assemblage inhabiting P. australis meadows has not been vigorously investigated despite the rapid decline of this species in Australian waters (Meehan & West, 2002; Waycott et al., 2009). The aims of this research were to 1) identify the sponge and ascidian species inhabiting P. australis meadows and 2) provide estimates of the spatial distribution patterns of this assemblage in the most extensive P. australis meadow in NSW 3) establish if sponge and ascidian species occur consistently on certain substrata within seagrass habitats. This is the first study to comprehensively quantify the abundance, diversity and substratum used for the attachment of sessile epifaunal invertebrates inhabiting P. australis. I elected to ignore other sessile taxa (e.g. bryozoans) as they were rarely encountered in my surveys.

Chapter 2

2.3 MATERIALS AND METHODS

2.3.1 Study area, study species and sampling methodology Jervis Bay is an open embayment of approximately 12,400 ha situated on the south eastern Australian coast. The prevalent seagrass species in Jervis Bay is P. australis which forms extensive meadows totalling 5.7 km2 in depths of 2-10 m (West, 1990). These meadows constitute the largest continuous areas of this seagrass species along Australia’s south eastern coast (Meehan & West, 2000), and are considered some of the most pristine seagrass meadows on this coastline (West et al., 1989, Kirkman et al., 1995). When compared with other seagrass species, Posidonia meadows have a long life span and a low turnover rate (West & Larkum, 1979; Hemminga & Duarte, 2000). Posidonia australis is particularly prone to anthropogenic disturbances due to its specific habitat requirements (West et al., 1989; den Hartog & Kuo 2006) and its low likelihood of recovery following disturbance (Clarke & Kirkman, 1989; Gobert et al., 2006). It is currently listed as a threatened ecosystem in NSW with endangered populations in the Sydney region (Fisheries Management Amendment (Threatened Species Conservation) Order (No 1) 2010 of the NSW Fisheries Management Act). Seagrass meadows were sampled at six locations in Jervis Bay Marine Park (Figure 2.1). These were Bindijine Beach (35°03’12S 150°46’41E), Booderee Waters (35°07’44S 150°44’59E), Callala Bay (35°00’09S 150°43’39E), Hare Bay (35°00’31S 150°45’59E), Long Beach (35°01’55S 150°47’03E) and Plantation Point (35°04’16S 150°41’36E) (Figure 2.1). Seagrasses in most locations in Jervis Bay have not been extensively affected by anthropogenic disturbances except for Callala Bay and Long Beach. Callala Bay has suffered substantial damage from boat moorings in recent decades (Demers et al., 2013). While, the northern end of Long Beach is a popular anchoring area where seagrass is sparse and absent in places (authors’ personal observations). Sampling locations differ in their exposure to wind, wave, and current regimes, nutrient input and water temperature. By sampling areas with varying environmental conditions, I sought to capture the entire spectrum of sponge diversity within the seagrass meadows of Jervis Bay. In situ sampling by SCUBA divers was used to determine the abundance, diversity and volume of sponges and ascidians as well as the substratum to which they were attached within P. australis meadows. I sampled between November 2011 and February 2012 at the six locations (km apart) which were divided into two sites (100s m apart) where I haphazardly sampled six transects (10s m apart) (n=6). My data provide a snapshot of sessile invertebrate diversity and abundance, as I did not sample locations more than once.

Chapter 2

Figure 2.1: Jervis Bay, showing each of the six locations I sampled: 1) Booderee Waters, 2) Plantation Point, 3) Callala Bay, 4) Hare Bay, 5) Long Beach and 6) Bindijine Beach. The prevalent clockwise gyre, important in nutrient circulation in the embayment, is indicated by the grey arrow (Wang & Wang, 2003).

Chapter 2

Abundance was measured by recording the number of sessile invertebrates encountered within 25m2 transects (25m x 1m) haphazardly placed in the meadow. A sponge individual was defined as a singular physiologically independent entity (Wulff, 2001). Individuals smaller than 1cm3 were treated as recruits and were excluded from this study. Sponges and ascidians with at least 50 % of their body located within the transect were included. Individuals located under substrata or buried in sand were sampled by turning the substratum over and fanning the sand to exposed as much of the animal as possible. I also quantified the number of species present within each transect to the lowest taxonomic level possible, and later confirmed this by examining spicule mounts. Volume (cm3) was estimated by measuring the maximum height, width and thickness of each individual. These measures were taken at 90o angle from each other to cover all three planes of view (x, y, z vectors). In order to provide a precise estimate of the size of unevenly-shaped individuals, ‘branches’ or ‘extensions’ were measured separately. Two taxa were combined for the purpose of this study, Haliclona spp. comprised two species Haliclona (Gellius) and Haliclona (Haliclona) which were indistinguishable in the field. The substratum on which each individual occurred was also recorded. Substrata were grouped into categories; these were 1) shells, 2) components of P. australis and 3) polychaete tubes (Figure 2.2). Shells encompassed various calcium carbonate structures mainly bivalve clumps including the mussel Trichomya hirsuta and the flat oyster Ostrea angasi. The P. australis components comprised the entire plant including the leaf blade, sheath and rhizomes. These three seagrass components were grouped because sponge individuals generally overgrew all of these components (authors’ personal observations). Polychaete tubes are flexible branched structures constructed and permanently inhabited by Eunicid polychaete worms. These tube-dwelling worms were previously found to be distributed in clusters in the soft sediment habitats of Jervis Bay (Anderson et al., 2009). Sponges occurred on the outer surface of these tubes along with other organisms such as algae, crinoids and molluscs (author’s personal observations). Minor substrata included boulders, objects (manmade objects such as a concrete block and natural objects such as branches), other invertebrates (ascidians and sponges) and algae, while in a few cases sponges were unattached (free living). These highly infrequent substratum categories were not included in my analysis.

Chapter 2

Figure 2.2: (A) Sponges and hydroids occupying a Eunicid polychaete worm tube located outside of the P. australis meadow. Note the other worm tubes in the background (Scale is 10cm). (B) Multiple sponge species inhabiting a single Eunicid polychaete worm tube (Scale is 3cm). (C) Callyspongia (Toxochalina) sp. 1 attached to the sheath and frond of P. australis (Scale is 5cm). (D) Encrusting Phoriospongia sp. 2. growing on shells (Scale is 1cm). (E) Botrylloides leachii growing on the sheath and frond of P. australis (Scale is 5cm). (F) Bivalve clump (Trichomya hirsuta) bearing multiple sponge species (Scale is 4cm).

Chapter 2

2.3.2 Statistical analyses A fully-nested analysis of variance (ANOVA) was used to assess the levels of variation for each of the variables sampled (GMAV5, University of Sydney). Two factors were analysed: Location was a random orthogonal factor with six levels (Bindijine Beach, Booderee Waters, Callala Bay, Hare Bay, Long Beach, Plantation Point); Site was a random factor with two levels and was nested within Location. Six transects were sampled at each site and constituted replicates. Variances were tested for homogeneity using Cochran’s C-test and normality of the data were assessed visually as recommended by Quinn and Keough (2002). The data were transformed using double square root (x0.25) to minimise the effect of skewed distributions (Quinn & Keough, 2002). In the case of heterogeneous data, the analyses were still performed since ANOVA is robust to heterogeneity with balanced experimental designs and large numbers of replicates (Underwood, 1997). I used Student-Newman-Keuls (SNK) tests for a-posteriori comparisons where factors were significant (α ≤ 0.05). The use of a balanced design and adequately transformed data allowed for moderate non-normality in my data resulting from patchily distributed taxa (McGuinness, 2002). Multivariate techniques were used to examine patterns in the composition of sessile epifaunal invertebrates at different locations in Jervis Bay (PRIMER 6, Plymouth Marine Laboratory). Differences in the abundance and volume of each species among sites were tested using nested multivariate PERMANOVAs (Anderson, 2001) on Bray-Curtis dissimilarities calculated from untransformed data with the same statistical design as the ANOVA. So as to avoid undefined resemblance matrix values, transects lacking sponges or ascidians were removed (Callala Bay site 2 Transect 2, 3, 4, 5 and 6, Booderee Waters site 1 Transect 6 and site 2 Transect 6). Thus, seven transects out of 72 were removed. Non-metric multidimensional scaling (nMDS) ordinations were used to graphically illustrate the patterns of distribution. The data were pooled for each site to provide a centroid based on Bray Curtis Similarity measures. The most widespread species were determined using the number of transects per site, from a total of six transects (Table 3.1). The percentage of ‘rare’ species was calculated as per Hooper and Kennedy (2002). They defined ‘rare’ species as those occurring at two sites or less.

Chapter 2

2.4 RESULTS

2.4.1 Overall patterns of diversity and abundance I encountered 20 sponge and eight ascidian species in the P. australis meadows of Jervis Bay. A total of 1110 sponge individuals and 119 ascidian individuals were recorded. The most widespread sponge species recorded were Tedania (Tedania) sp., Phoriospongia sp. 1 and Haliclona spp. (Table 3.1). Sponge species were patchy in their distribution with the most common species occurring at 58% of the transects sampled (Table 3.1). Approximately half the sponge species (47%) were found at five or more locations of the six locations sampled (Table 3.1). Species that occurred at two sites or less were deemed ‘rare’ and represented 57% of ascidians and 24% of sponges sampled. The most widespread species were also the most abundant and the largest. The five most abundant taxa were Phoriospongia sp. 1 (27% of individuals encountered), Haliclona spp. (21%), Callyspongia (Toxochalina) sp. 1 (15%), Tedania (Tedania) sp. (14%) and Halichondria sp. (5%). These species also provided the largest contribution to the total sponge volume sampled, Tedania (Tedania) sp. (32%), Phoriospongia sp. 1 (20%), Haliclona spp. (17%), Halichondria sp. (10%) and Callyspongia (Toxochalina) sp. 1 (6%). Sponge abundance was averaged over six transects (150 m2) at each site and varied by an order of magnitude across sites; it was greatest at Hare Bay site 2 with a mean of 34 ±7 individuals (x̄ ±SE) and lowest at Callala Bay site 2 (0.33 ± 0.21 individual). Patterns of abundance were similar among sites in Jervis Bay, except for Callala Bay which had a significantly greater abundance at site 1 than that at site 2 (Figure 2.3A, Table 2.2A, SNK: Callala Bay site 1 > site 2). The multivariate analysis detected significant differences in sponge abundance among sites and locations (Figure 2.4, Table 2.3). The nMDS plots (stress 0.09) clearly showed similarity in the assemblages among sites within most locations and that Callala Bay site 2 differed from the other sites in Jervis Bay (Figure 2.4A). Sponge volume varied by two orders of magnitude among sites; it was greatest at Callala Bay site 1 (110.63 ± 19.90 cm3) and Bindijine Beach site 1 (106.57 ± 34.20 cm3). Sponge volume was lowest at Callala Bay site 2 (4 ± 2 cm3). There was little difference in sponge volume across sites, with the exception of Callala Bay which, again, showed a significantly greater volume at site 1 (Figure 2.3B, Table 2.2A, SNK: Callala Bay site 1 > site 2). PERMANOVA confirmed that significant differences existed among sites and locations (Table 2.3). The nMDS (stress 0.06) supported this pattern as there was consistent separation between assemblages among locations and clustering at the site level. It also clearly showed that Callala Bay site 2 was an outlier (Figure 2.4B). 25

Chapter 2

Sponge species richness across sites was greatest at Bindijine Beach site 1 (6.5 ±0.33 species) followed by Long Beach site 2 (6.17 ±0.17 species). The lowest mean sponge diversity was measured at Callala Bay site 2 (0.33 species). Sponge diversity also significantly differed between Callala Bay site 1 and site 2 (Figure 2.3C, Table 2.2A, SNK: Callala Bay site 1 > site 2). Ascidians inhabiting the seagrass meadows of Jervis Bay were also patchy in their distribution. The three most widespread species were Herdmania grandis, Pyura praeputialis and Didemnum sp. (Table 2.1). Ascidians were patchily distributed given that they occurred at 1% to 36% of the transects sampled (Table 2.1). In addition, half the species sampled occurred within a single transect (57%). Ascidian abundance was highest at Callala Bay site 1 (4 ± 3.6 individuals) and lowest at Callala Bay site 2 (Figure 2.3D). Ascidian abundance differed significantly between sites at Bindijine Beach, Callala Bay, Plantation Point and Long Beach (Figure 2.3C, Table 2.2D, SNK: Bindijine Beach and Callala Bay site 1 > site 2; Plantation Point and Long Beach site 1 < site 2). The multivariate analysis and nMDS plot (stress 0.12) showed significant differences in ascidian abundance among sites and locations (Figure 2.4C, Table 2.3). Callala Bay site 2 was removed from the nMDS to display the remaining sites more clearly. Ascidian diversity across sites was highest at Long Beach site 2 (1.83 ± 0.37 species). A significant difference in species richness was recorded between sites at Long Beach (Figure 2.3D, Table 2.2D, SNK: Long Beach site 1 < site 2).

Chapter 2

Figure 2.3: Comparison of sponge and ascidian species richness, abundance and volume at the two sites sampled for each of the six locations within Jervis Bay. (A) mean (±SE) estimates of sponge diversity expressed as number of species present per site. (B) mean (±SE) estimates of sponge abundance expressed as number of individuals per site. (C) mean (±SE) estimates of sponge volume expressed as cm3 per site. (D) mean (±SE) estimates of ascidian diversity expressed as number of species present per site. (E) mean (±SE) estimates of ascidian abundance expressed as number of individuals per site. Each bar represents a mean of six transects each of 25m2 (n=6).

Chapter 2

Figure 2.4: Non-metric multidimensional scaling (nMDS) plots for centroids of (A) sponge abundance (B) sponge volume (C) ascidian abundance for each species among sites. The data were pooled to provide a centroid for each site based on Bray Curtis Similarity measures. Callala Bay site 2 was removed from (C). Bindijine Beach, Callala Bay, Hare Bay, Booderee Waters, Plantation Point, Long Beach.

Chapter 2

56 Phoriospongia sp. 1 ● ● • ●

a 46 Haliclona spp. ● ● ● ● ●

• ● • ● ● ● Halichondria sp. 31 ● • ● ● ● ● • ● Chelonaplysilla sp. Callyspongia (Toxochalina) sp. 1 ● • ● • ● 26

Thorectidae family sp. 1 • ● • ● 22

• • ● • • 15 Lissodendoryx (Lissodendoryx) sp. • • • ● • • 11 Unidentified Demospongiae sp.1 • • • ● ● 10 Euryspongia sp. • • • • • • 8 Chondropsis sp. ● ● 8 Thorectidae family sp. 2 ● • ● 7 Callyspongia (Callyspongia) sp. 2 ● • • 7 Stelletta sp. ● 3 Unidentified Demospongiae sp. 2 • 1 Lissodendoryx (Waldoschmittia) sp. • 1 Phoriospongia sp. 2 Chalinula sp. * Tethya cf bergquistae * Thorectidae cf Hyrtios sp. 3 * a this is a complex of species including Haliclona (Gellius) and Haliclona (Haliclona).

Chapter 2

B Locations Bindijine Beach Callala Bay Hare Bay Booderee Waters Plantation Point Long Beach Transect Sites Site 1 Site 2 Site 1 Site 2 Site 1 Site 2 Site 1 Site 2 Site 1 Site 2 Site 1 Site 2 (%)

● ● ● ● • ● Herdmania grandis 36

● • • • • • Pyura praeputialis 21

• ● • • Didemnum sp. 14 • Styela plicata 7 • Cnemidocarpa pedata 1 • Pyura spinifera 1 • Distaplia c.f. stylifera 1

Botrylloides leachii *

Chapter 2

Table 2.2: Analyses of variance comparing variation among locations and sites for (A) overall sponge abundance, volume and diversity, (B) abundance of five main sponge taxa, (C) volume of five main sponge taxa, (D) ascidian abundance and diversity. NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

A Sponge Abundance Sponge Volume Sponge Diversity Source d.f. MS F p MS F p MS F p Location 5 2.180 1.47 NS 1.341 0.29 NS 0.671 1.06 NS Site (Loc) 6 1.486 6.38 *** 4.684 7.44 *** 0.634 6.50 *** Residual 60 0.233 0.630 0.098

Cochran’s test 0.268 (P < 0.05) 0.266 (P < 0.05) 0.289 (P < 0.05) Transformation X0.25 X0.25 X0.25

B Phoriospongia sp. 1 Haliclona spp. Halichondria sp. Tedania (Tedania) sp. 1 Callyspongia (Toxochalina) sp. 1 Source d.f. MS F p MS F p MS F p MS F p MS F p Location 5 26.40 2.39 NS 6.576 2.40 NS 0.231 0.55 NS 2.768 3.73 NS 12.421 0.97 NS Site (Loc) 6 11.58 4.14 *** 2.744 1.36 NS 0.416 2.04 NS 0.742 0.96 NS 12.839 12.0 *** Residual 60 1.064 2.012 0.204 0.774 1.074

Cochran’s test 0.688 (P < 0.01) 0.321 (P < 0.01) 0.448 (P < 0.01) 0.580 (P < 0.01) 0.818 (P < 0.01) 0.25 Transformation X0.25 X0.25 X0.25 X0.25 X

Chapter 2

C Phoriospongia sp. 1 Haliclona spp. Halichondria sp. Tedania (Tedania) sp. 1 Callyspongia (Toxochalina) sp. 1 Source d.f. MS F p MS F p MS F p MS F p MS F p Location 5 7.100 2.39 NS 4.620 1.05 NS 3.740 0.70 NS 10.352 6.31 * 6.741 2.36 NS Site (Loc) 6 2.971 4.14 ** 4.380 3.90 ** 5.376 2.95 * 1.640 0.80 NS 2.854 3.44 ** Residual 60 0.717 1.124 1.823 2.048 0.829

Cochran’s test 0.509 (P < 0.01) 0.204 (NS) 0.214 (NS) 0.309 (P < 0.05) 0.745 (P < 0.01) 0.25 Transformation X0.25 X0.25 X0.25 X0.25 X

D Ascidian Abundance Ascidian Diversity Source d.f. MS F p MS F p Location 5 0.554 0.47 NS 0.506 0.60 NS Site (Loc) 6 1.179 3.74 ** 0.842 3.72 ** Residual 60 0.315 0.226

Cochran’s test 0.201 (NS) 0.168 (NS) Transformation X0.25 X0.25

Chapter 2

Table 2.3: Summary of the two factor nested PERMANOVA for sponge abundance, sponge volume and ascidian abundance of each species among sites and locations in the seagrass meadows of Jervis Bay. Factors were locations with six levels (random) and sites nested in locations with two levels (random). Transects at each sites served as replicates. Analysis was based on Bray-Curtis dissimilarities with permutation of the raw data. NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

Sponge Abundance Sponge Volume Ascidian Abundance Source d.f. MS Pseudo F P (perm) d.f. MS Pseudo F P (perm) d.f. MS Pseudo F P (perm) Location 5 13434 1.97 ** 5 9549.7 1.59 * 5 8732.4 4.06 * Site (Loc) 6 6850.9 3.22 *** 6 6057.5 1.86 *** 5 2150.2 0.98 NS Residual 54 2125.4 54 3257.8 30 2186.9

Transformation none none none

Chapter 2

2.4.2 Patterns of abundance; individual taxa Phoriospongia sp. 1 (AM Z.7219) was most abundant at Hare Bay site 1 (24.17 ± 4.84 individuals) and largest at Callala Bay site 1 (240.65 ± 46.24 cm3), when averaged across six transects (150 m2) at each site. There was a marked disparity between abundance and volume among sites for this species (Figure 2.5A, Table 2.2BC). A significant difference in abundance between sites was recorded at Hare Bay, Bindijine Beach and Callala Bay (Figure 2.5A, Table 2.2B, SNK: Hare Bay site 1 > site 2; Bindijine Beach site 1 < site 2; Callala Bay site 1 > site 2), and in volume at Callala Bay (Figure 2.5F, Table 2.2C, SNK: Callala Bay site 1 > site 2). Haliclona spp. (AM Z.7218) were most abundant at Plantation Point site 1 (9.33 ± 3.57 individuals) and largest at Hare Bay site 2 (192.38 ± 71.89 cm3). Hare Bay site 1 had no Haliclona spp. present resulting in a significant difference between sites at this location (Figure 2.5G, Table 2.2C, SNK: Hare Bay site 1 < site 2). Due to the high variability in abundance for these taxa, there was no significant difference among sites or locations (Figure 2.5B, Table 2.2B). Halichondria sp. (AM Z.7217) was abundant at Long Beach site 2 (2.67 ± 1.48 individuals) and Bindijine Beach site 1 (2.5 ± 1.71 individuals) but abundance did not vary significantly among sites or locations (Figure 2.5C, Table 2.2B). The volume of Halichondria sp. individuals was largest at Plantation Point site 2 (245.5 ± 244.5 cm3) and differed significantly across sites at Bindijine Beach and Callala Bay (Figure 2.5H, Table 2.2C, SNK: Bindijine Beach and Callala Bay site 1 > site 2). Tedania (Tedania) sp. (AM Z.7220) was most abundant at Bindijine Beach site 1 (7 ± 1.32 individuals) and Long Beach site 1 (5.12 ± 3.79 individuals). There was no significant difference in the abundance of this species among sites or locations (Figure 2.5D, Table 2.2B). Individuals of Tedania (Tedania) sp. were largest at the Booderee Waters site 1 (207.46 ± 77.46 cm3). Bindijine Beach, Booderee waters and Long Beach showed significantly greater volume of this species than that at Callala Bay (Figure 2.5I, Table 2.2C, SNK: BB, BW and LB > CB). Callyspongia (Toxochalina) sp. 1 (AM Z.7216) was most abundant at Hare Bay site 2 (20.5 ± 5.30 individuals) and was significantly more abundant there than at site 1 (Figure 2.5E, Table 2.2B, SNK: Hare Bay site 1 < site 2). Callyspongia (Toxochalina) sp. 1 individuals were largest at Bindijine Beach site 1 (633.33 ± 233.33 cm3) and were significantly larger than that of site 2 (Figure 2.5J, Table 2.2C, SNK: Bindijine Beach site 1 > site 2).

Chapter 2

2.4.3 Substrata on which epifauna attached The substratum used for attachment pooled across all locations I sampled revealed that 23% of sponges occurred on shells, 60% on components of P. australis and 10% on polychaete tubes. The substratum of attachment varied across sites and locations (Figure 2.6A) and differed amongst species. The ‘other’ category included ascidians (2%), other sponges (<1%), algae (1%), boulders (<1%), anthropogenic structures (<1%), unknown (1%) and free living (2%). Some taxa were consistent in their use of substrata for attachment, whilst other species showed considerable variability. Of the five dominant sponge taxa, three were predominantly encountered on components of P. australis; Phoriospongia sp. 1 (93%), Callyspongia (Toxochalina) sp. 1 (88%) and Haliclona spp. (87%). In contrast, Tedania (Tedania) sp. and Halichondria sp. showed marked variation in their substrata of attachment among sites and locations. Their occurrence on components of P. australis was 27% and 38% respectively (Figure 2.6). Tedania (Tedania) sp. occurred equally on shells (36%), seagrass components (27%) and polychaete tubes (29%) (Figure 2.6E). This species used shells solely at Plantation Point, seagrass components at Callala Bay and polychaete tubes at Long Beach Site 1 (Figure 2.6). A similar trend occurred with Halichondria sp., which primarily occurred on shells (44%) and seagrass components (38%) depending on its location (Figure 2.6). It is worth noting that the five dominant sponge taxa were found on shells, seagrass components and polychaete tubes at least once in Jervis Bay, therefore, no sponge species was established as an obligate occupant of P. australis. Ascidians predominantly occurred on shells (56%) and seagrass components (41%) but none were observed on polychaete tubes (Figure 2.6G). Negligible occurrences were recorded on other ascidians (2%) and anthropogenic structures (2%). Ascidians were found exclusively on shells at Bindijine Beach, Booderee Waters site 2 and Long Beach site 2 (Figure 2.6G).

Chapter 2

Figure 2.5: Comparison of abundance and volume for the five main sponge taxa at the two sites sampled for each of the six locations within Jervis Bay. Mean (±SE) estimates of abundance expressed as number of individuals per site for (A) Phoriospongia sp. 1 (B) Haliclona spp. (C) Callyspongia (Toxochalina) sp. 1 (D) Tedania (Tedania) sp. (E) Halichondria sp. Mean (±SE) estimates of volume expressed as cm3 per site for (F) Phoriospongia sp. 1 (G) Haliclona spp. (H) Callyspongia (Toxochalina) sp. 1 (I) Tedania (Tedania) sp. (J) Halichondria sp. Each bar represents a mean of six transects each of 25m2 (n=6).

Chapter 2

Figure 2.6: Comparison of substratum of attachment for sponges, five dominant sponge taxa and ascidians at the two sites sampled for each of the six locations within Jervis Bay. Mean (±SE) estimates of number of substrata occupied by (A) sponges (B) ascidians (C) Phoriospongia sp. 1 (D) Haliclona spp. (E) Callyspongia (Toxochalina) sp. 1 (F) Tedania (Tedania) sp. (G) Halichondria sp. Each bar represents a total of six transects of 25m2 (n=6). components of P. australis, Polychaete tubes, Shells.

Chapter 2

2.5 DISCUSSION

2.5.1 Patterns of distribution The P. australis meadows of Jervis Bay had a highly speciose assemblage of sessile epifaunal invertebrates. I encountered 20 sponge species and eight ascidian species within a sampling area totalling 1800m2. I felt it informative to compare these values with diversity in other habitats. These seagrass meadows were more speciose than vegetated and unvegetated sediments of saline coastal lagoons in NSW, Australia (Barnes et al., 2013). Barnes and co-workers (2013) reported 18 sponge species and six ascidian species in a sampling area tenfold larger than my study. Other studies have recorded slightly lower species richness in seagrass habitats, but have sampled much smaller areas. For instance, Lemmens et al. (1996) recorded 11 species of macro-suspension feeders (sponges, ascidians and bivalves) in a Western Australian P. australis meadow. Seagrass meadows have a relatively high sessile epifaunal invertebrate diversity in comparison to the kelp forests of NSW; Wright et al. (1997) recorded 10 algae-associated sponge species. Despite their relatively high species richness, the seagrass ecosystems of Jervis Bay were surpassed by the temperate reefs of NSW, which comprised more than double the sponge and ascidian diversity I observed (Roberts & Davis, 1996; Newton et al., 2007). I found taxonomic similarities among the sessile epifaunal invertebrate assemblage of Jervis Bay and that of estuarine lakes in NSW particularly for ascidians. Both assemblages comprised Botrylloides leachii, Herdmania grandis, Pyura praeputialis, Styela plicata as well as two sponge taxa, Halichondria sp. and Haliclona sp. (Barnes et al., 2013). Although, it is unknown whether the sponge species I have observed correspond to the five species of Halichondria and two species of Haliclona recorded by Barnes et al. (2013). The assemblage was dominated by several patchily distributed but locally widespread species and included sparsely distributed uncommon species. This pattern is consistent to that seen on natural and artificial reefs (Wilkinson & Evans, 1989; Roberts & Davis, 1996; Knott et al., 2004), as well as within mangrove (Farnsworth & Ellison, 1996) and kelp habitats (Wright et al., 1997). The dominant sponge species (Phoriospongia sp. 1, Haliclona spp., Callyspongia (Toxochalina) sp. 1, Tedania (Tedania) sp. and Halichondria sp.) were widespread over the largest spatial scale sampled, with three of the five most common sponge taxa found at every location. Although widespread, species were patchily distributed with the most common species occurring at 58% of the transects sampled. Ascidians were not as widespread, with only one ascidian species (Herdmania grandis) found at all locations and two species (Pyura praeputialis and Didemnum sp.) occurred at five or more locations. 39

Chapter 2

‘Rare’ species (those species occurring at less than two sites) constituted 24% of sponges and 57% of ascidians from the entire community sampled. Hooper and Kennedy (2002) recorded up to 60% of ‘rare’ sponge species on the reefs of the Sunshine Coast, eastern Australia. In my seagrass study, uncommon species were sparsely distributed amongst sites and locations; most species occurred at less than half the transects per site. A markedly low abundance of sponges and ascidians was recorded at Callala Bay Site 2 with only two sponge individuals (Phoriospongia sp. 1) found within 150 m2 of habitat; the reason for this discrepancy was not clear. High variation in sponge and ascidian distribution at the smallest spatial scale I sampled was clearly apparent. Heterogeneity in sessile epifaunal invertebrate distribution at small spatial scales has also been recorded in coastal lakes (Barnes et al., 2006), on mangrove roots (Guerra-Castro et al., 2011), in reefal habitats (Roberts & Davis, 1996; Hooper et al., 2002; Hooper & Kennedy, 2002; Knott et al., 2004; Newton et al., 2007) and even on bare substratum in deep Antarctic waters (Barthel & Gutt, 1992). Similarity in sessile assemblages between sites was observed in spite of high levels of heterogeneity. Marked clustering at the site level was evident from the nMDS plots. This concurred with previous work (Corriero et al., 1989; Barthel & Gutt, 1992) and most likely reflects limited dispersal by these taxa, among other explanations, although it has not been formally tested. The patchy nature of sponge distribution has been linked to their low probability of long-distance dispersal, asexual propagation and lack of connectivity among populations (Hooper et al., 2002; Hooper & Kennedy, 2002). Like sponges, some ascidians are limited in their dispersal abilities and are likely to have highly restricted gene flow (Davis & Butler, 1989). These patterns of dispersal may also see rare sponge species form clusters in some habitats (Fromont et al., 2006). The three metrics I used in assessing sponges (their abundance, volume and level of spread) produced contrasting patterns of spatial distribution. This has also been documented by other studies (Wulff, 2001; Bannister et al., 2010). Despite marked disparity in these metrics among and within taxa, two sponge taxa were relatively abundant, voluminous and widespread; Phoriospongia sp. 1 and Haliclona spp. It is noteworthy that sponge species displayed a range of functional growth forms including encrusting, simple massive, cryptic massive, erect laminar and erect branching (concept by Schonberg & Fromont, 2014). This range of growth forms, commonly seen in other habitats, ensures that volume is complex and time-consuming to quantify compared to the number of sponge individuals present. However, a combination of abundance and volume measurements may provide more meaningful estimates of the distributional patterns of sponges and ascidians (Wulff, 2001; Bannister et al., 2010; Wulff, 2012).

Chapter 2

Sites within Jervis Bay showed marked disparity in their sessile epifaunal invertebrate distribution. The highest values for the overall sponge abundance (Hare Bay site 2), sponge volume (Callala Bay site 1), sponge diversity (Bindijine Beach site 1), ascidian abundance (Callala Bay site 1) and ascidian diversity (Long Beach site 2) almost all occurred at different sites. The site with the highest sponge abundance had relatively low sponge volume and vice versa. Bindijine Beach site 1 was the only site that had relatively high abundance, volume and diversity of sponges and ascidians in Jervis Bay. The five dominant sponge taxa also showed no concordance in their abundance and volume among sites. Most sponges are filter feeders and capture particulate materials from the water column (Bergquist, 1978). Increased nutrient availability and water flow favour sponge growth and survival rates (Morris & Keough, 2003; Duckworth et al., 2004). Nutrient availability is a likely key driver of sponge and ascidian distribution. The locations with greatest sponge and ascidian abundance, diversity and volume were mainly located on the east side of Jervis Bay (Hare Bay, Long Beach and Bindijine Beach) which coincides with a high nutrient load. In the Bay, oceanic water enters at Booderee and circulates clockwise (Figure 2.1) around the Bay, accumulating nutrients and sediments (Wang & Wang, 2003). Plankton levels have been linked to the spatial distribution of large suspension feeders such as sponges, ascidians and bivalves in the P. australis meadows of Cockburn Sound, Western Australia (Lemmens et al., 1996). A correlation was established between the density of sponges and ascidians, and the phytoplankton levels in this coastal embayment. The effect of the clockwise gyre carrying nutrients and sediments in Jervis Bay should be further investigated.

2.5.2 Substratum used for attachment and the impact of humans Substrata used for attachment by sessile epifaunal invertebrates in P. australis meadows comprised mainly P. australis, shells and polychaete tubes, in order of importance. Attachment to components of P. australis was prevalent for three sponge taxa Phoriospongia sp. 1, Callyspongia (Toxochalina) sp. 1 and Haliclona spp., although none were seagrass obligate species. Seagrass components were commonly used as a substratum, with sponges occurring on all P. australis components; rhizome, sheath and blade. Ascidians occurred equally on shells and P. australis components but were not observed on polychaete tubes. My findings contrast with those of Corriero et al. (1989). They compared two Tethya sponge species in the Posidonia oceanica meadows of a Mediterranean lagoon; both were found on seagrass rhizomes but were not reported on other plant components.

Chapter 2

Anthropogenic disturbances to seagrass ecosystems are expected to have an impact on the distribution of sessile epifaunal invertebrate species, especially those relying heavily on seagrass for their attachment. Disturbances most likely to produce negative effects on seagrass-associated fauna include habitat fragmentation (Healey & Hovel, 2004), siltation stemming from meadow patchiness (e.g. Shepherd et al., 1989; Short & Wyllie-Echeverria, 1996) and other sources of siltation (Wilcox & Murphy, 1985; Reed & Hovel, 2006). Direct human-induced siltation was found to alter sponge species composition and to reduce species richness as well as sponge cover (Roberts et al., 1998). Despite relatively low levels of human disturbance within Jervis Bay, seagrass meadows have already suffered from negative boating practices, a known contributor to habitat fragmentation (Demers et al., 2013).

2.6 CONCLUSIONS Sessile epifaunal invertebrates inhabiting the seagrass meadows of Jervis Bay were patchy in their distribution. A few of the taxa in the assemblage were widespread over large spatial scales (km apart). In contrast, the majority of species were rare and sparsely distributed. Variation in sessile epifaunal invertebrate distribution was greatest at small spatial scales (100s m apart). This should be considered in the design of experiments and impact studies as sampling efforts should concentrate on small spatial scale with sufficient sampling intensity to detect rare species. Sponges are rarely recognised as a major constituent of seagrass ecosystems (Wulff, 2001). My study demonstrated otherwise. Although no obligate seagrass species was recorded, three sponge species relied heavily on seagrass for attachment and a total of 20 sponge species and eight ascidian species were encountered in the meadows of Jervis Bay. To our knowledge, no other studies have investigated the importance of P. australis as a substratum for the attachment of these invertebrates. Further studies need to adopt an experimental approach to disentangle the key processes and factors responsible for the patterns observed. Examining the response of sessile epifauna to the degradation of their seagrass habitat remains a key challenge.

Chapter 2

CHAPTER 3

Are pilot studies worth the trouble? A case study using recalcitrant organisms

Final version: Demers, M-C. A., Knott, N. A. and Davis A. R. (in review). Are pilot studies worth the trouble? A case study using recalcitrant organisms. Journal of Experimental Marine Biology and Ecology.

Chapter 3

3.1 ABSTRACT Pilot studies are used to refine sampling protocol by providing an indication (or sample) of the variation which can then be used to determine the appropriate level of replication, often across a range of spatial scales to ensure the efficient allocation of resources. The utility of pilot studies may, however, be questionable for organisms with patchy distribution. Sponges and ascidians are renowned for being highly variable spatially and, here, I did a pilot study to determine at which spatial scales to concentrate my sampling effort. I then compared the mean and variability estimates generated from the pilot study with the main study to determine how well the initial pilot study informed the later study. The data obtained by the pilot study were compared to that of the main study for 1) measures of diversity and abundance, 2) assemblage clustering 3) variability at a hierarchy of spatial scales using magnitude of effects, and 4) significant outcomes using nested analyses of variance. The pilot study was effective in reflecting robust measures of sessile epifaunal invertebrate diversity and abundance. More than half the sponge species encountered in the main study were recorded in the pilot study, indicating that it provided a meaningful estimate of sponge diversity. However, the pilot study was limited in effectively recording ascidian abundance and diversity due to their extraordinarily patchy distribution. Community structure was very similar between the pilot and the main studies for the sessile epifaunal invertebrates. The pilot study successfully detected the scale at which variation prevailed (10s m apart) but failed to acknowledge the spatial complexity seen in the main study (site within location), possibly due to low replication at the level of location (i.e. two in the pilot and six in the main study). Overall, the pilot study was a valuable tool in the establishment of an effective sampling design. Despite intrinsic limitations and high levels of variation, the pilot study reflected the distribution of the patchy assemblage seen in the main study.

Keywords: pilot study, method, variability, experimental design, spatial scale.

Chapter 3

3.2 INTRODUCTION Adequate replication provides a precise representation of the taxon being sampled. Determining what is an adequate level of replication (and which spatial scales to sample) is indispensable in sampling spatially patchy organisms and to capture the entire spectrum of organisms or species within an assemblage (Kennelly & Underwood, 1984). Only once a precise and representative method has been designed can biologists investigate distribution patterns and do manipulative experiments (i.e. Kennelly & Underwood, 1984; Underwood et al., 2000). Many studies have used preliminary sampling to determine levels of replication and identify the scales of variability. The scales at which spatial patterns operate must be determined so as to broaden our understanding of processes involved in shaping species interactions (Underwood & Chapman, 1996). Determining spatial variability can be achieved by sampling across a hierarchy of scales, providing estimates of variation (ANOVA) and their fit (Magnitude of effects). Analysis of variance is a widely used and well established statistical model; it is mainly used to estimate the relative importance of individual factors (Graham & Edwards, 2001). Magnitude of effects is a measure of a given factor’s relative contribution to the response variable. It can greatly enhance the description and interpretation of ecological processes, especially when using nested analyses across a number of spatial scales (Graham & Edwards, 2001). Pilot studies are desirable in optimizing sampling designs aiming to achieve appropriate replication and the cost-efficient allocation of resources (e.g. Green, 1979; Andrew & Mapstone, 1987). Design optimization is a compromise between precision and feasibility. It entails the shape and size of the sampling unit, level of replication and scale. It minimises confounding by integrating adequate replication at the appropriate scale, as well as ensuring high levels of randomization and independence among experimental units (Quinn & Keough, 2002). Estimation of the required sample size is dependent upon the level of variability between sampling units of the target population and the effect size required to be detected. In the case of poorly-studied populations, estimates of variation are often obtained through a pilot study and are then used to estimate appropriate sample sizes (Quinn & Keough, 2002). Recalcitrant organisms present difficulties for ecological assessments. Sponges and ascidians fall into this category; they are often difficult to identify and quantify (Davis et al., 1999; Wulff, 2001). This stems from two main taxonomic challenges; 1) morphologically different individuals that are actually members of the same species and 2) individuals of different species that cannot be morphologically distinguished in situ without the help of a specialist (Davis et al., 1999; Wulff, 2001). As a result, this assemblage is often excluded from studies and is generally poorly

Chapter 3 described (Demers et al., 2016). The patterns of distribution of sponges and ascidians also contribute to their recalcitrance. They are often patchily distributed and exhibit high variability (e.g. Keough, 1983; Newton et al., 2007; Wulff, 2012). These characteristics make it difficult to detect potential differences among assemblages (e.g. Roberts & Davis, 1996; Knott et al., 2004; Duckworth et al., 2008). In seagrass meadows, the assemblage of sessile epifaunal invertebrates has been particularly poorly described in the literature (Demers et al., 2016). The lack of interest in these recalcitrant organisms stems from high levels of variation within this system, which includes rare and patchily distributed taxa (Barnes et al., 2006; Demers et al., 2016). This does not undermine the need for pilot studies as a stepping stone to identifying and understanding the processes driving spatial distribution in this habitat however (Underwood et al., 2000; Demers et al., 2016). Indeed, Barnes et al. (2006) used a pilot study incorporating a combination of transects and timed searches to sample the sessile epifaunal invertebrate assemblage within an estuarine environment. However, this work incorporated a variety of habitats and was not in a seagrass dominated system (Barnes et al., 2006). The aim of my research was to assess the effectiveness of a pilot study in developing a robust sampling design for a spatially variable assemblage; sessile epifaunal invertebrates in Posidonia australis Hook F. meadows. I established a methodology for sampling seagrass- associated sessile invertebrates in Jervis Bay, in line with that developed by Barnes et al. (2006). I sought to determine whether the pilot study effectively reflected the spatial distribution of the assemblage seen in my main study. I predicted that the pilot study might be limited in reflecting sessile epifaunal invertebrate distribution due to the patchy distribution and presence of rare taxa in this assemblage. To test this I contrasted the pilot and the main studies for: (i) the diversity and abundance of sessile invertebrates, (ii) the composition of the assemblage, (iii) the variability apparent across multiple spatial scales and, (iv) significant outcomes.

3.3 MATERIALS AND METHODS

3.3.1 Pilot study Our pilot study was focused at two locations: Bindijine Beach and Callala Bay in Jervis Bay, NSW, Australia. These locations were sampled between September and October 2011 and were selected haphazardly amongst the Posidonia australis meadows of Jervis Bay. Each location was divided into two sites (100s m apart), where I sampled six transects (10s m apart). A diver on SCUBA recorded the diversity and abundance of sponges and ascidians along a 25m2 transect (25 m x 1m) haphazardly placed in the meadow.

Chapter 3

3.3.2 Main study Seagrass was sampled at six locations in Jervis Bay Marine Park; Bindijine Beach (35°03’12S 150°46’41E), Booderee Waters (35°07’44S 150°44’59E), Callala Bay (35°00’09S 150°43’39E), Hare Bay (35°00’31S 150°45’59E), Long Beach (35°01’55S 150°47’03E), Plantation Point (35°04’16S 150°41’36E) (Figure 2.1). Sampling locations differ in their exposure to wind, wave and current regimes, nutrient input and water temperature. By sampling areas with varying environmental conditions, I sought to capture the entire spectrum of sponge diversity within the seagrass meadows of Jervis Bay. I sampled between November 2011 and February 2012 at the six locations (km apart) which were divided into two sites (100s m apart) where I haphazardly sampled six transects (10s m apart) (n=6). My data provide a snapshot of sessile invertebrate diversity and abundance, as I did not sample locations more than once.

3.3.3 Sampling method In situ sampling by divers on SCUBA was used to determine the abundance and diversity of sponges and ascidians. Abundance was measured by recording the number of sessile invertebrates encountered within each transects. A sponge individual was defined as a singular physiologically independent entity (Wulff, 2001). Individuals smaller than 1cm3 were treated as recruits and were excluded from this study. Sponges and ascidians with at least 50 % of their body located within the transect were included. Individuals located under substrata or buried in sand were sampled by turning the substratum over and fanning the sand to expose as much of the animal as possible. I also quantified the number of species present within each transect to the lowest taxonomic level possible, and later confirmed this by examining spicule mounts.

3.3.4 Statistical analyses

3.3.4.1 Measures of diversity and abundance As my focus was on contrasting the pilot study and the main study, I calculated the mean species richness and abundance for sponges and ascidians pooled across transects from all sites and locations. Although this approach does not take into account my nested design and is therefore inadvisable (Kromrey & Dickinson, 1996), it provides broad measures of sessile epifaunal invertebrate distribution between studies that cannot be obtained otherwise. Note that this approach is purely complementary to my other analyses.

Chapter 3

3.3.4.2 Assemblage composition Differences in the community composition of sessile epifaunal invertebrates (sponges and ascidians) between studies were tested using nested permutational multivariate analysis of variance (PERMANOVA) (Anderson, 2001) and non-metric multidimensional scaling (nMDS) ordinations with PRIMER 6 (Plymouth Marine Laboratory). Multivariate analyses of sponge and ascidian abundance were done on untransformed and presence/absence transformed data. Unlike untransformed data, the presence/absence transformed data have equal weighting for rare and abundant species (Clarke & Warwick, 1994). Univariate analyses were used to test for differences between the studies and a range of spatial scales for sponge and ascidian diversity and abundance. A second-stage community analysis (2STAGE) was used to determine which transformed data (square root, log and presence/absence) was most appropriate (Olsgard et al., 1997). The square root transformation was closest to the raw data. The data were then square root transformed to homogenize the variance of most taxa given that the assemblage contained a few dominant species and many rare species (Tomasovych & Kidwell, 2009). Post-hoc pooling was used to increase the power of the main tests for P>0.25 (Underwood, 1997) as indicated in the footnote of Table 2.1. I used a Bray-Curtis distance measure to generate similarity matrices for the multivariate analyses and Euclidean distance resemblance matrix for the univariate analyses (Quinn & Keough, 2002). So as to avoid undefined resemblance matrix values, transects lacking sponges or ascidians were removed (Main Study Callala Bay Site 2 Transect 2, 3, 4, 5 and 6, Booderee Waters Site 1 Transect 6 and Site 2 Transect 6). Thus, seven of 96 transects were removed. I used a three factor design: ‘study’ was orthogonal and fixed with two levels (Pilot and Main), ‘location’ was orthogonal, random and nested within ‘study’ with two levels for the pilot study and six levels for the main study, ‘site’ was orthogonal, random and nested within ‘location’ with two levels. Replication was six transects (25m2) per site for the pilot study and the main study.

3.3.4.3 Variability among spatial scales and significant outcomes Two fully-nested analyses of variance (ANOVA) were calculated (GMAV5, University of Sydney). In the first, data were untransformed to allow for calculation of variance components and magnitude of effects. In the second, I assessed the levels of variation for each of the metrics sampled for the pilot and the main study across numerous spatial scales. The contribution of each factor (diversity and abundance of sponges and ascidians) to the response variable was determined via magnitude of effects for the pilot and the main study, in line with Graham and Edwards (2001). For the pilot study, I also calculated the contribution of variation for the abundance of all taxa encountered. The factors tested were: ‘Location’ a random orthogonal

Chapter 3 factor with six levels (km apart), ‘Site’ a random orthogonal factor with two levels nested within ‘Location’ (100s m apart) and ‘Transect’ or residuals (10s m apart) (n=6). I calculated the magnitude of effects expressed as percentages of overall variation using variance components. Negative variance components were present and a remediation procedure called ‘pool-the- minimum-violator’ algorithm was applied (Graham & Edwards, 2001). The levels of variation were tested for each of the metrics sampled for the pilot and the main study. The factors were the same as mentioned previously. Variances were tested for homogeneity using Cochran’s C-test and normality of the data were assessed visually as per Quinn and Keough (2002). The data were transformed using double square root (x0.25) to minimise the effect of skewed data (Quinn & Keough, 2002). In the case of heterogeneous data, the analyses were still performed since ANOVA is robust to heterogeneity with balanced experimental designs and large numbers of replicates (Underwood, 1997).

3.4 RESULTS

3.4.1 Measures of diversity and abundance In the pilot study, I recorded 11 sponge species and two ascidian species. This constituted the majority (65%) of the sponge species encountered in the main study (17 species) despite only covering a third of the sampling area. For the ascidians, however, only a quarter of the species (25%) in the main study (8 species) were detected in the pilot study. The mean (±s.e.) sponge species richness and abundance pooled across transects from all sites and locations showed similarities between the pilot study and the main study (Figure 3.1). The mean (±s.e.) ascidian species richness and abundance across transects from all sites and locations were greater for the main study than that of the pilot study (Figure 3.1).

3.4.2 Assemblage composition The nMDS analysis clearly showed the similarity in the sessile epifaunal invertebrate assemblages between studies (stress 0.19) (Figure 3.2). The pilot study fell well within the bounds of the main study and data from both studies were dispersed over the same scale. I did not detect a significant difference between studies for the entire sessile epifaunal invertebrate assemblage, nor the diversity and abundance of sponges and ascidians (Table 3.1). Variation occurred at the location level for the untransformed multivariate analysis (Table 3.1A) and for the presence/absence transformed multivariate analysis (Table 3.1A). The variation occurred predominantly at the site level for all four of the univariate analyses (Table 3.1B, C).

Chapter 3

Figure 3.1: A comparison of the mean (±s.e.) (A) species richness and (B) abundance of sessile epifaunal invertebrates pooled across transects from all sites and locations between the pilot (n=24) and the main study (n=72).

Chapter 3

Figure 3.2: Non-metric multidimensional scaling (nMDS) plots showing dissimilarities among the pilot and the main study for the abundance of sponge and ascidian species without transformation. Pilot Study; Main Study.

Chapter 3

Table 3.1: Summary of the three factor nested PERMANOVA for (A) the sponge and ascidian assemblage (untransformed and presence/absence transformed), (B) the diversity of sponge and ascidian species and (C) the abundance of sponge and ascidian species. I used a Bray-Curtis distance measure for the multivariate analyses and Euclidean distance resemblance matrix for the univariate analyses. Factors were ‘study’ with two levels (fixed) the pilot and the main study, ‘location’ was nested in ‘study’ with two levels for the pilot study and six levels for the main study (random) and ‘site’ was nested in ‘location’ with two levels (random). Transects at each sites served as replicates. Post-hoc pooling was used to increase the power of the main tests for P>0.25 (Underwood, 1997). NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

A) Multivariate assemblage Source d.f. MS Pseudo F P (perm) MS Pseudo F P (perm) Study 1 10518 0.81 NS 8285 0.71 NS Location(Study) 6 12422 1.91 ** 11090 2.91 ** Site(Loc(Study)) 8 6563 3.03 ** 3853 2.68 ** Residual 80 2167 1436

Transformation none Presence/absence

B) Diversity Sponge Ascidian Source d.f. MS Pseudo F P (perm) MS Pseudo F P (perm) a Study 1 0.07 0.04 NS 1.25 1.83 NS Location(Study) 6 1.94 1.13 NS 0.53 0.67a NS Site(Loc(Study)) 8 1.72 6.32 *** 0.79 2.55a * Residual 80 0.27 0.31

Transformation Square root Square root

C) Abundance Sponge Ascidian Source d.f. MS Pseudo F P (perm) MS Pseudo F P (perm) a Study 1 3.06 0.21 NS 3.15 2.55 NS Location(Study) 6 14.57 2.06 NS 0.58 0.34a NS Site(Loc(Study)) 8 7.09 2.91 ** 1.72 2.46a * Residual 80 2.43 0.70

Transformation Square root Square root a identifies the MS that were pooled and F ratios that changed due to pooling.

Chapter 3

3.4.3 Variability among spatial scales In both studies (pilot and main), the variation attributed to transects (i.e. the smallest spatial scale investigated) was the main contributor towards the total variation (Figure 3.3). It accounted for 100% of the variation in five of the eight variables I investigated. Magnitude of effects calculations confirmed that the second highest contributor was the variation attributed to locations but, generally this was <25% of the total variation. Variation attributable to sites was also apparent in both studies but it was relatively small and was not observed for the same variables (Figure 3.3). The pilot study and the main study were only in agreement for the magnitude of effects for ascidian diversity. The ascidian diversity was driven entirely by variation attributable to transects for both studies (Figure 3.3). In the main study, locations and sites within locations contributed a small proportion to the total variation, but solely for sponge abundance (Figure 3.3B). This pattern was not reflected in the pilot study (Figure 3.3A). For patterns among individual taxa, I observed the most variation at the ‘transect’ level for nine of the 13 sessile epifaunal invertebrate taxa investigated in the pilot study (Appendix Table 3.3A). While, the abundance of the four remaining taxa (3 sponges and one ascidian) showed predominant variation at the largest spatial scale of ‘location’ (Appendix Table 3.3A). It should be noted that the ‘pool-the-minimum-violator’ algorithm was applied to more than half the magnitude of effects calculations for the pilot study (Appendix Table 3.3A), as well as three of four magnitude of effects calculations for the main study (Appendix Table 3.3B). This was due to negative variance components. The pilot study failed to detect significant effects among sites or locations for diversity and abundance of sessile epifaunal invertebrates as confirmed by the analyses of variance (Table 3.2). In contrast, the main study showed consistently significant differences in diversity and abundance of sessile epifaunal invertebrates among sites within locations (Table 3.2).

Chapter 3

Figure 3.3: Magnitude of effects, expressed as percentage of variation (ω2), calculated from the diversity and abundance of sponges and ascidians for (A) the pilot study and (B) the main study. Location (km apart) random and orthogonal with up to six levels, Site within Location (100s m apart) random orthogonal with two levels and Transect (10s m apart) expressed as residuals (n=6) (See Appendix Table 2.3 for additional details).

Chapter 3

Table 3.2: Analyses of variance comparing variation at a hierarchy of spatial scales for (A) sponge diversity, (B) ascidian diversity, (C) sponge abundance, (D) ascidian abundance. The factors were: ‘Location’ a random orthogonal factor with up to six levels (km apart), ‘Site’ a random orthogonal factor with two levels nested within ‘Location’ (100s m apart) and ‘Transect’ or residuals (10s m apart) (n=6). NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

A) Pilot Main Source d.f. MS F p d.f. MS F p Location 1 0.314 3.52 NS 5 0.671 1.06 NS Site (Loc) 2 0.089 3.24 NS 6 0.634 6.50 *** Residual 20 0.028 60 0.098

Cochran’s test 0.4401 (NS) 0.2886 (P < 0.05) Transformation X0.25 X0.25

B) Pilot Main Source d.f. MS F p d.f. MS F p Location 1 0.079 1.90 NS 5 0.506 0.60 NS Site (Loc) 2 0.042 0.14 NS 6 0.842 3.72 ** Residual 20 0.300 60 0.226

Cochran’s test 0.2865 (NS) 0.1683 (NS) Transformation X0.25 X0.25

C) Pilot Main Source d.f. MS F p d.f. MS F p Location 1 0.005 0.02 NS 5 2.180 1.47 NS Site (Loc) 2 0.267 1.35 NS 6 1.486 6.38 *** Residual 20 0.198 60 0.233

Cochran’s test 0.5301 (NS) 0.2679 (P < 0.05) Transformation X0.25 X0.25

D) Pilot Main Source d.f. MS F p d.f. MS F p Location 1 0.146 4.50 NS 5 0.554 0.47 NS Site (Loc) 2 0.033 0.10 NS 6 1.179 3.74 ** Residual 20 0.340 60 0.315

Cochran’s test 0.3237 (NS) 0.2007 (NS) Transformation X0.25 X0.25

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3.5 DISCUSSION

3.5.1 Effectiveness of the pilot study Pilot studies are routinely used to inform the design of studies, but rarely do researchers consider the effectiveness of pilot studies. These small-scale preliminary studies are often conducted to evaluate effect size and develop a sampling design. However, it is often unknown whether they are precise or effective at reflecting robust measures of distribution. I contrasted outcomes of the pilot study with the main study used to sample a spatially variable assemblage of sessile epifaunal invertebrates. The pilot study provided reliable variance estimates, despite a limited sample size and patchily distributed taxa. Accurate variance estimates ensured that what appear to be spatial differences in this assemblage were actual differences rather than the reflection of a poor sampling design (Miller & Ambrose, 2000). Outcomes of the pilot study were consistent with a sufficient sampling effort at a small spatial scale despite the highly variable nature of this assemblage (Miller & Ambrose, 2000) and proved to be a useful tool in designing my larger study with confidence. Importantly, the scales at which variation prevailed (10s m apart) were successfully detected by the pilot study. Variation in the spatial distribution of sessile epifaunal invertebrates was consistently high and occurred predominantly at the transect level. It is worth noting that there were slight discrepancies between the pilot and the main study, probably due to the high variability in this system. For instance, variation at the location and site levels for sponge diversity and ascidian abundance were detected solely by the pilot study. Despite these minor discrepancies, the pilot study effectively reflected the dominant scale of variation. These outcomes reinforce pilot studies as an important step in ensuring precision in developing sampling designs (Andrew & Mapstone, 1987; Benedetti-Cecchi et al., 1996). Barnes et al. (2006) stated the importance of using pilot studies, for patchily distributed taxa, as a way to optimize the precision of their sampling design. The dominance of spatial variation at small spatial scales (the level of transects) underscores the need for numerous replicates at the smallest spatial scale, while replication at the level of site appeared adequate, despite sampling just two sites. Similarly, little variation was associated with the largest spatial scale, location. The addition of more locations in the main study produced little change in our interpretation. Since most of the variation in these assemblages occurs at the smallest spatial scales, this is the scale that would be most profitable for manipulative experimentation. It should also be emphasised that the pilot study produced robust estimates of sponge diversity and abundance. The pilot study recorded a relatively high sponge species richness and more than half the species recorded by the main study, despite covering just a third of the area. Sponge species richness and abundance, averaged across transects, were similar between the pilot

Chapter 3 and the main study. Analogous community structure was revealed between the pilot and the main study in the MDS plot. The large proportion of species overlap between studies suggests that sample units were adequately sized and replicated (Barbiero et al., 2011). Infrequent and patchily distributed species necessitate larger sample areas (Barbiero et al., 2011). A well represented assemblage presumably captures a greater proportion of these rare species.

3.5.2 Limitations of the Pilot study Spatial variability differed between the studies and the significant spatial effects observed in the main study were not acknowledged by the pilot study. This is not surprising given that the main study had a greater level of replication at the location level, and hence much greater statistical power at this level of the analysis. It is important to note that variance estimates were slightly smaller for the pilot study than that of the main study. A limitation of my work is that it focuses on spatial variability at the risk of ignoring temporal variability. These assemblages were clearly patchily distributed in space but we know little of their variation in time. Sponges and ascidians of other habitats can have highly variable temporal distributions (e.g. Wulff, 2006b; Ponti et al., 2011; Wulff, 2016), often associated with seasonal changes (e.g. Rützler, 1987; Bingham & Young, 1995). Ascidians were extraordinarily patchy in their distribution and were particularly recalcitrant. This explains why the pilot study was limited in effectively recording ascidian abundance. For instance, a single transect (<1.5% of the area sampled), yielded 22 individuals, which constituted 18% of the total ascidian abundance encountered in the main study. Also, ascidian abundance at the two locations sampled in the pilot study consisted of 17% and 55% of the total ascidian abundance recorded at the very same locations in the main study. However, much greater variation occurs when comparing studies at the site level. The total ascidian abundance recorded by the pilot study ranged from <10% to 150% of that of the main study. Notably the PERMANOVA did not reflect this level of recalcitrance probably because the square root transformed data supressed the effect of the major shifts in abundance (Quinn & Keough, 2002). As further evidence of the patchy nature of ascidians, their diversity also varied among studies. However, the two locations sampled for the pilot study had the lowest ascidian diversity in the main study with just two ascidian species recorded (Demers et al., 2016). Importantly, no further ascidian taxa were added at these two locations during the main study. This suggests that the pilot study reflected ascidian diversity reliably although it was hindered by the highly patchy nature of their distribution.

Chapter 3

3.6 CONCLUSIONS I conclude that the pilot study provided useful estimates of variation across a range of spatial scales even for such a highly variable and patchy group of organisms. Overall, my modest pilot study reflected the composition and distribution of a patchy assemblage and detected the predominant scale of variation found in the main study. Despite some limitations, the pilot study was deemed a valuable tool in the establishment of an effective sampling design and can seemingly provide a level of design confidence even with such a patchy and variable assemblage. My other key finding was the predominance of variation at the smallest spatial scale I investigated – the level of transects. This argues that ecological processes may be directed at this scale, and it should be the focus of future sampling.

Chapter 3

3.7 APPENDIX

Table 3.3: Summary of the nested ANOVAs and magnitude of effects comparing diversity and abundance of sponges and ascidians for (A) the pilot study and (B) the main study, as well as the abundance of dominant taxa for the pilot study. Magnitude of effects (Ϭ2) is represented as a percentage (ω2), calculated using procedures detailed in Graham and Edwards (2001). The factors were: ‘Location’ a random and orthogonal factor with up to six levels (km apart), ‘Site’ a random orthogonal factor with two levels nested within ‘Location’ (100s m apart) and ‘Transect’ or residuals (10s m apart) (n=6). The symbol * in the MS column indicate where pool-the-minimum logarithm was applied.

Chapter 3

A) Cochran Transfor Variables Factor df 's C test MS F P Ϭ2 ω2 -mation Diversity Location 1 0.3333 26.04 2.22 0.27 1.19 24 none Sponge Site (Loc) 2 NS 11.71 5.26 5.26 1.58 32 Residual 20 2.23 2.23 45 Diversity Location 1 0.3684 0.38 9 0.10 0 0 none Ascidian Site (Loc) 2 NS 0.04* 0.09 0.92 0 0 Residual 20 0.48 0.43 100 Abundance Location 1 0.5527 24* 0.08 0.78 0 0 none Sponge Site (Loc) 2 NS 285.42 1.39 0.27 0 0 Residual 20 205.85 204.86 100 Abundance Location 1 P < 0.01 2.04 49 0.02 2.36 65 none Ascidian Site (Loc) 2 0.04* 0.03 0.97 0 0 Residual 20 1.38 1.25 35 Porifera Location 1 P < 0.05 0.33* 0.7 0.49 0 0 x0.25 Phoriospongia Site (Loc) 2 0.47 2.53 0.10 0 0 sp. 1 Residual 20 0.19 0.22 100 Haliclona spp. Location 1 P < 0.01 228.17* 0.79 0.47 0 0 none Site (Loc) 2 287.42 2.68 0.09 0 0 Residual 20 107.12 128.06 100 Halichondria Location 1 P < 0.01 15.04 21.2 0.04 41.10 97 none sp. Site (Loc) 2 0.708* 0.5 0.61 0 0 Residual 20 1.41 1.34 3 Tedania Location 1 P < 0.01 96 2.71 0.24 181.75 93 none (Tedania) sp. Site (Loc) 2 35.42 3.96 0.04 4.41 2 Residual 20 8.95 8.95 5 Chondropsidae Location 1 P < 0.01 1.5 1 0.42 0 0 none sp. 2 Site (Loc) 2 1.5 2.5 0.11 0.15 20 Residual 20 0.6 0.6 80 Thorectidea Location 1 P < 0.01 16.67 1.23 0.38 9.50 69 none sp. Site (Loc) 2 13.5 5.44 0.01 1.84 13 Residual 20 2.48 2.48 18 Chondropsis Location 1 P < 0.01 0* 0 F=0 0 0 none sp. 3 Site (Loc) 2 1.5 1.76 0.20 0 0 Residual 20 0.85 0.867 100 Euryspongia Location 1 P < 0.01 0.04 1 0.42 0 0 none sp. Site (Loc) 2 0.04 1 0.39 0 0 Residual 20 0.04 0.04 100 Tethya sp. Location 1 P < 0.01 0.04 1 0.42 0 0 none Site (Loc) 2 0.04 1 0.39 0 0 Residual 20 0.04 0.04 100 Stelletta sp. Location 1 P < 0.01 0.17 1 0.42 0 0 none Site (Loc) 2 0.17 1 0.39 0 0 Residual 20 0.17 0.17 100 Callyspongia Location 1 P < 0.01 0.17 1 0.42 0 0 none (Toxochalina) Site (Loc) 2 0.17 1 0.39 0.02 20 sp. 1 Residual 20 0.07 0.07 80 60

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Ascidiacea Location 1 0.5 0.17* 2 0.29 0 0 none Herdmania Site (Loc) 2 NS 0.08 0.24 0.79 0 0 grandis Residual 20 0.35 0.32 100 Pyura Location 1 P < 0.01 1.04 5 0.15 0.95 57 none praeputialis Site (Loc) 2 0.21* 0.27 0.77 0 0 Residual 20 0.778 0.73 43

B) Cochran Transfor Variables Factor df 's C test MS F P Ϭ2 ω2 -mation Diversity Location 5 0.1917 15.20* 0.81 0.58 0 0 none Sponge Site (Loc) 6 NS 18.72 6.18 0.00 0 0 Residual 60 3.03 5.21 100 Diversity Location 5 P < 0.01 1.10* 0.61 0.70 0 0 none Ascidian Site (Loc) 6 1.81 2.60 0.03 0 0 Residual 60 0.69 0.82 100 Abundance Location 5 P < 0.01 836.97 4.11 0.06 52.78 24 none Sponge Site (Loc) 6 203.61 1.3 0.27 7.83 4 Residual 60 156.62 156.62 72 Abundance Location 5 P < 0.01 1.18* 0.08 0.99 0 0 none Ascidian Site (Loc) 6 14.65 1.57 0.17 0 0 Residual 60 9.34 9.22 100

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CHAPTER 4

Seagrass attributes and seascape patterns as drivers of sessile invertebrate distribution in a coastal biogenic environment

Chapter 4

4.1 ABSTRACT

Understanding the patterns of distribution of sessile epifaunal invertebrate communities in seagrass meadows is an important precursor to determining the ecological processes shaping ecosystem function in this disappearing habitat. To uncover patterns in the spatial distribution of sponges and ascidians within Posidonia australis meadows, I investigated small-scale biogenic characteristics, large-scale seascape patterns, and the abundance of a common seagrass-associated fish species. Linear regressions and generalised linear models were used to determine the relationships between 21 predictor variables and several response variables. Response variables included; 1) sponge diversity, abundance and volume, 2) ascidian diversity and abundance, and 3) the abundance of two common sponge species (Haliclona spp. and Tedania (Tedania) sp.). Distributional patterns of sessile epifaunal invertebrates in seagrass meadows were influenced by both small-scale seagrass characteristics and large-scale seascape patterns. They were not affected by the abundance of a common fish species. The scale over which patterns were apparent differed greatly between sponges and ascidians, suggesting that these different phyla respond to differing drivers of pattern. The biogenic characteristics of seagrass such as the density and length of shoots were found to be important determinants of sponge distribution. Hydrodynamic baffling and nutrient availability are thought to play a role in this interaction. The distribution of a common sponge species complex, Haliclona spp. was found to be influenced by depth. These species are suspected phototrophs and their distribution is believed to be dependent on irradiance associated with depth. Seascape patterns influenced the distribution of ascidians inhabiting seagrass meadows. Meadows neighbouring abundant reefal habitats consistently harboured low ascidian species richness. This study provided an initial investigation of the factors driving this threatened community and should be perceived as an important stepping stone in designing future experiments. I conclude by highlighting the importance of manipulative experiments to further explore the underlying processes driving the patterns identified in this study.

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4.2 INTRODUCTION Rapidly declining ecosystems such as seagrass meadows are likely to experience changes in ecosystem structure and function due to the cascading effect of human disturbances. Hence, it is crucial to establish the nature of the relationship between sessile epifaunal invertebrate community and its seagrass host so as to uncover the role sponges play in seagrass ecosystem structure and function. This will provide a more complete understanding of how sessile epifaunal invertebrates influence the seagrass ecosystem and the biodiversity that it hosts. The factors influencing the distribution of sessile epifaunal invertebrates in seagrass meadows have rarely been investigated (but see Wulff, 2008; Archer et al., 2015). This probably stems from the patchy distribution of this community, which is highly variable at a hierarchy of spatial scales from meters to kilometres apart (Barnes et al., 2006; Demers et al., 2016). Determining the spatial distribution of a highly heterogeneous community is a challenging task, even more so to determine the factors are driving such spatially and temporally variable distributions (Demers et al., 2016). Nonetheless, this information is crucial to further our understanding of this threatened ecosystem and the biodiversity that it houses. A myriad of abiotic and biotic factors may be driving the distribution of sponges and ascidians in seagrass meadows. Most drivers of spatial distribution are interrelated and vary at a multitude of spatial scales (Hooper & Kennedy, 2002). An array of abiotic and biotic factors was shown to influence the distribution of sessile epifaunal invertebrates in non-seagrass habitats, such as reefs. These variables are therefore likely to influence the distribution of sessile epifaunal invertebrates in seagrass meadows. Physio-chemical parameters have also been found to influence the distribution of sessile epifaunal invertebrates in reefal habitats (Wilkinson & Evans, 1989; Roberts & Davis, 1996; Duckworth et al., 1997; Duckworth et al., 2004), especially the gradients in water quality such as light, turbidity and siltation (Wilkinson & Cheshire, 1989; Roberts et al., 2006). Also, the growth and survival of sponges was found to rely heavily on water flow and associated food availability (Wilkinson & Vacelet, 1979; Duckworth et al., 2004), which is to be expected due to their filter- feeding nature. Depth was also found to influence patterns of distribution in sessile epifaunal invertebrates inhabiting reefs (Wilkinson & Evans, 1989). Depth is a likely driver of sponge and ascidian distribution in seagrass meadows as it is a known determinant of species composition in this habitat (i.e. Young & Wadley, 1979). These physio-chemical parameters are therefore likely to influence the distribution of sessile epifaunal invertebrates in seagrass meadows. Biotic factors have been shown to influence the spatial heterogeneity in sponge assemblages (i.e. Wulff, 2006a). It is likely that these biotic factors also play a key role in driving sessile

Chapter 4 epifaunal invertebrate patterns of distribution within seagrass environments. Little is known of the biotic factors affecting sessile epifaunal distribution in seagrass meadows (but see Wulff, 2008; Archer et al., 2015) as most studies concentrate on reefal habitats. Important biotic factors that have received attention include; predation (Wulff, 1997b; Wulff, 2006a), physical and chemical defence abilities (Pawlik et al., 1995; Wright et al., 1997; Ferguson & Davis, 2008), inter and intra specific competition (Thacker et al., 1998; Rützler, 2002), mutualism (Wulff, 1997a), symbiotic relationships (Thacker, 2005), dispersal ability of larvae (Uriz et al., 1998), larval supply and recruitment (Zea, 1993). The biogenic characteristics of seagrass habitats are expected to influence the spatial distribution of sponges and ascidians. The nature and availability of suitable substrata are important community components for the distribution of sessile epifaunal invertebrates (Sarà & Vacelet, 1973; Schubauer et al., 1990; Adjeroud, 1997; Davis et al., 2003; Fromont et al., 2006). This assemblage relies heavily on its host habitat when found on biogenic habitats such as mangrove roots, kelp forests and gorgonians (e.g. Reiswig, 1973; Wulff, 2003). Multiple studies have established the importance of habitat structure in shaping motile invertebrate communities in seagrass. Biogenic seagrass factors such as shoot density, meadow size and configuration were previously found to influence the density, diversity and survival of epifauna (e.g. Irlandi et al., 1995; Hovel & Lipcius, 2002). The hydrodynamic forces associated with meadows of different sizes and orientations were established as important drivers of larvae dispersal for a wide range of seagrass epifaunal species (Tanner, 2003). The structural complexity provided by epiphytic algae on seagrass leaves (Gartner et al., 2013), the seagrass species present (Nakaoka et al., 2001) and the level of habitat fragmentation of the meadow (Healey & Hovel, 2004) also play an important role in determining the distribution of some invertebrates. Seascape patterns are also likely to influence sponge and ascidian distribution in seagrass meadows. Seascape patterns can be defined as spatial variation in the physical structure of a habitat (Wedding et al., 2011), and have been well documented for seagrass meadows and reefal habitats (Boström et al., 2011). Seascape patterns consist of the configuration and arrangement of patch habitats (Wedding et al., 2011). Many studies have focused on the heterogeneity of reefal habitats over fine spatial scales (e.g. Wright et al., 1997) and confirmed that complex habitats harboured a greater abundance and diversity of fish (e.g. Gratwicke & Speight, 2005). The level of heterogeneity of the meadow is therefore likely to influence the distribution of sessile epifaunal invertebrates. A recent study by Rees et al. (2014) confirmed that seascape patterns were correlated with the abundance and diversity of sessile epifaunal invertebrates in a temperate rocky reef

Chapter 4 environment. Such correlations have not been investigated for the sessile epifaunal invertebrate assemblage of seagrass ecosystems, although they are likely to be present in this habitat. Sponge palatability can be an important determinant of sponge ecosystem dynamics and processes (Wulff, 2005; Pawlik et al., 2013). For instance, predation by a starfish species was a key driver of population and community dynamics of an Antarctic sponge assemblage (Dayton, 1979; McClintock et al., 2005). Fish predation could be an important driver of sessile epifaunal invertebrate distribution as seen in reefal habitats (Pawlik et al., 1995; Wulff, 1997b, 2006c). It is unknown whether sponge palatability has such an important role on the distribution of sessile epifaunal invertebrates in austral seagrass meadows. The identity of spongivorous species within seagrass ecosystems is also poorly documented. Leatherjackets (Monacanthids) are commonly found in the P. australis meadows (Bell et al., 1978). Leatherjackets are omnivorous and are known to feed on seagrass leaves and its encrusting fauna. Gut content analysis revealed that at least three species of leatherjackets from the Sydney region, Monacanthus chinensis, Meuschenia freycineti and Meuschenia trachylepis, feed on ascidians (Bell et al., 1978). The leatherjacket species, Nelusetta ayraudi, is a resident of the P. australis meadows of Jervis Bay and was observed feeding on a sponge specimen (Demers per. obs.). This species is likely to feed on the sessile epifaunal invertebrate assemblage inhabiting seagrass meadows. The aim of this study was to uncover correlations between an array of abiotic and biotic factors (predictor variables) and the spatial distribution of sponge and ascidian inhabiting seagrass meadows (response variables). The three broad factors investigated were; seagrass attributes, seascape of the meadow and the abundance of a common seagrass-associated fish species. This is the first study to investigate the potential factors driving the highly variable distribution of sponges and ascidians in seagrass meadows, in an attempt to fill a research gap in the ecology of this rapidly declining ecosystem.

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4.3 MATERIALS AND METHODS

4.3.1 Study area The study was carried out in Jervis Bay, New South Wales (NSW), Australia (Figure 2.1). Jervis Bay is an open embayment of approximately 12,400 ha situated on the south eastern Australian coast. The dominant seagrass species in Jervis Bay is P. australis which forms extensive meadows totalling 5.7 km2 in depths of 2-10 m (West, 1990). These meadows constitute the largest continuous areas of this species along Australia’s south eastern coast (Meehan & West, 2000), and are considered some of the most pristine seagrass meadows on this coastline (West et al., 1989; Kirkman et al., 1995). Seagrass meadows were sampled at six locations in Jervis Bay Marine Park; Bindijine Beach (35°03’12S 150°46’41E), Booderee Waters (35°07’44S 150°44’59E), Callala Bay (35°00’09S 150°43’39E), Hare Bay (35°00’31S 150°45’59E), Long Beach (35°01’55S 150°47’03E) and Plantation Point (35°04’16S 150°41’36E) (Figure 2.1). These locations correspond to the main seagrass meadows in Jervis Bay with Long Beach and Bindijine Beach sharing a single continuous meadow.

4.3.2 Sampling method The spatial distribution of sessile epifaunal invertebrates was sampled between November 2011 and February 2012. The diversity, abundance, volume and substrata of attachment for sponges and ascidians were recorded in P. australis at the six study locations. The patterns of spatial distribution of seagrass-associated sessile epifaunal invertebrates were published in Demers et al. (2016). It is worth noting that sponge volume was not standardised against leaf length given that sponges occurring on seagrass blades were mostly located at the base of the frond and did not cover the entire length of the blade (Appendix 1). Seagrass condition measurements were sampled between March and April 2012. Divers on SCUBA recorded depth, shoot count, epiphyte cover, leaf length and maximum leaf length within 0.25 m2 quadrats (0.5 m x 0.5 m). I sampled 10 quadrats per site at each of the six locations. Depth is a small-scale abiotic variable, not a true biogenic attribute of the meadow, but was included in this category for the purpose of this study. Shoot count referred to the number of P. australis shoots within one quadrat, a shoot consisting of one to several leaf-blades joined at the base. Shoots with at least 50% of their leaf sheath located inside the quadrat were included. Epiphyte cover was estimated in situ and expressed as a percentage cover. A mature leaf was evaluated against a percentage cover estimate chart. Note that destructive methods were avoided due to the conservation status of P. australis. This ecological community is threatened in NSW.

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The seascape data of the meadow was acquired from a seagrass habitat distribution map (NSW Marine Parks Authority, Jervis Bay Marine Park, 2012). ArcGIS (version 10) was used to calculate meadow area, perimeter, amount of reefs neighbouring the edge of the meadow, distance to nearest reef and perimeter shared between meadow and reef. I also calculated the area of seagrass and reef habitat at various radii scales of 100m, 500m, 1000m and 1500m surrounding each site, using focal statistics under the Spatial Analyst Tool (Table 4.1). Abundance of a common fish species, Nelusetta ayraudi, was obtained for the six locations sampled (Kiggins, 2013). Data was collected in 2012 and 2013 (Kiggins, 2013). Kiggins (2013) used Baited Remote Underwater Videos (BRUVs) to record fish assemblage at multiple locations around Jervis Bay. The use of BRUVs is a well-established technique to sample fish assemblages (Murphy & Jenkins, 2010) consisting of an underwater video camera and a bait bag to attract fish. The BRUV unit consisted of a GoPro Hero 2 camera attached to a brick (23 cm x 11 cm x 7 cm), which served as an anchor. The brick was affixed to a steel grid (45 cm x 30 cm), which was used to flatten the seagrass blades allowing for a greater field of view (Kiggins, 2013). Attached to the steel grid and opposite the brick, was a bait bag (18 cm x 8 cm x 6 cm) made of plastic mesh containing a single pilchard (Sardinops sagax) divided into 4 pieces. Pilchards were replaced after each drop. Note that pilchards are excellent baits to attract a variety of fish species due to their high oil content (Wraith et al., 2013). A rope and buoy were attached to the unit and used for relocation following sampling (Kiggins, 2013). Three sites were sampled for each location. Drops were replicated four times at each site (n=4) and were positioned at least 250 m apart to minimize any overlap of bait plumes. Each GoPro camera was set to the highest possible resolution (Resolution/FPS: 1080-30p; Field of View: 170o/127o; Screen Resolution: 1920 x 1080) and all LED lights were deactivated. In this case, abundance was estimated as the maximum abundance of Nelusetta ayraudi in a single frame during a viewing interval of 30 minutes (Kiggins, 2013). Note that an acclimatization period of five minutes, prior to each video recording, allowed snorkelers to evacuate the area. Predictive variables were obtained within a corresponding timeframe. The spatial distribution of sessile epifaunal invertebrates was sampled between November 2011 and February 2012. Seagrass condition measurements were sampled between March and April 2012. The seascape data of the meadow was obtained on July 2007. The abundance of a common fish species, Nelusetta ayraudi, was obtained on September 2012 and January 2013. Please note that the seascape features are thought to be stable overtime. I sought to minimise the timeframe for the sessile epifaunal invertebrate sampling, but maintain that seasonal effects on their distribution and abundance are minimal.

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4.3.3 Statistical analyses The relationship between sessile epifaunal invertebrate distribution patterns and multiple predictor variables (seagrass attributes, seascape patterns and the abundance of a common fish species) were explored using simple linear regressions and generalised linear models in R 3.1.2 (R Development Core Team, 2014). The response variables were; total abundance of sponges and ascidians, diversity of sponges and ascidians, total volume of sponges, and abundance of the two most common sponge species (Haliclona spp. and Tedania (Tedania) sp.). The response variables were pooled across sites to correspond to the scale of the predictor variables. Normality and homoscedascity were visually assessed using histograms and scatterplots. Response variables were transformed when needed so as to stabilise variances and improve normality for linear regressions. If normality could not be achieved, generalised linear models were employed with a quasipoisson distribution or negative binomial distribution. Prior to analyses, a Pearson’s correlation matrix was calculated to assess multicollinearity. As some predictor variables were collinear, I selected the variable that explained the most variation. For instance, for the seagrass habitat within a circular area of varying radii, a single spatial scale was adopted. A total of 21 variables were modelled with our spatial distribution data using linear models for normal distribution, generalised linear models for poisson and negative binomial distributions. To select the most parsimonious model (minimum adequate model), I used a backward step-wise procedure beginning with the most complex model and ending with the simplest model. The model, with the lowest Akaike’s Information Criteria (AIC) corrected for small sample sizes (AICc), was regarded to be the most parsimonious and the minimum adequate model. Model residuals were tested for spatial autocorrelation using Moran’s I. They were visually assessed using spline correlograms (Bjornstad & Falck, 2001) and the Durbin Watson Test.

4.4 RESULTS Variation was detected in the predictor variables. Leaf length and shoot count varied up to twofold across locations (Table 4.1). Depth ranged from 1.82m to 5.0m (Table 4.1). The area of seagrass and reef around the meadow also showed a high level of variation for radii ranging from 100 to 1500m (Table 4.1). Large variation at fine spatial scales was also recorded for sponge and ascidian distribution, these patterns of distribution were thoroughly described in Demers et al. (2016). Sponge abundance and volume were influenced by small-scale biogenic structures such as the condition of the seagrass sampled. Sponge abundance was positively correlated (Pseudo- R2=0.3931, d.f.=10, P<0.05) with the leaf length of P. australis (Table 4.2, Figure 4.1A). Sponge volume was positively correlated (Pseudo-R2=0.4841, d.f.=10, P<0.01) with the shoot count of P.

Chapter 4 australis (Table 4.2, Figure 4.1B). Predictor variables (seagrass attributes, seascape patterns and the abundance of a common fish species) failed to explain a significant amount of variation in sponge diversity. Ascidian diversity was affected by the large-scale seascape patterns such as the area of neighbouring reefs. Ascidian diversity was negatively correlated (Pseudo-R2=0.36, d.f.=10, P<0.05) with the amount of reef within a 1500m radius (Table 4.2, Figure 4.1C). Based on deviance, I calculated that this factor explained 36% of the total variation. Predictor variables (seagrass attributes, seascape patterns and the abundance of a common fish species) failed to explain a significant amount of variation in ascidian abundance. Note that out of the four scales tested (100m, 500m, 1000m and 15000m radii), the 1500m radius had the lowest AIC.

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Table 4.1: Predictive variables used in the statistical models in addition to their individual units, scale, transformation (trans.), a brief description, sample size (n), minimum value (min), maximum value (max), mean and standard deviation (SD). Variables Units Scale Trans. Description n Min Max Mean SD Small-scale biogenic characteristics Depth m Bathymetry of quadrat 10 1.82 5.00 3.07 0.88 Leaf length cm Length of four leaves within quadrat 40 26.75 45.33 32.47 5.72 Max leaf length cm Length of the longest leaf within quadrat 10 37.8 55.1 44.42 5.32 Shoot count # Quantity of shoots within quadrat 10 49.9 101 69.14 13.51 Epiphyte cover % Percentage of epiphyte cover of a single leaf within 10 64 91.5 75.32 8.96 quadrat Large-scale seascape patterns Area of seagrass ha2 log Area covered by seagrass 6 1.11 2.43 1.72 0.42 Perimeter of meadow ha log+1 Perimeter of the meadow 6 0.27 2.23 0.95 0.66 Area/Perimeter Area divided by perimeter 6 46.99 121.3 71.97 27.08 Number of adjacent reefs # Number of reefs within a 200m of the edge of the 6 2 7 4.67 1.77 meadow Perimeter meet reef ha Perimeter of the meadow in direct contact with reef 6 0.035 0.24 0.13 0.07 Distance to reef m log+1 Distance from the meadow’s edge to the nearest reef 6 0 2.85 1.73 0.78 Area of seagrass within a m 1500 log Area of seagrass within a radius scale surrounding 12 5.18 6.40 5.74 0.35 radius of: m 1000 log each site 12 5.07 6.28 5.56 0.35 m 500 sqrt 12 4.77 5.82 5.26 0.30 m 100 12 3479 31417 20973 8408 Area of reef within a m 1500 log Area of reef within a radius scale surrounding each 12 4.75 6.19 5.26 0.44 radius of: m 1000 log site 12 3.58 6.04 5.02 0.71 m 500 sqrt 12 0 602.4 242.6 184.8 m 100 sqrt 12 0 133.4 48.32 49.36 Seagrass associated fauna Max N Nelusetta 2012 # log+1 Maximum abundance of Nelusetta ayraudi in a single 12 0 1.26 0.23 0.37 Max N Nelusetta 2013 # log+1 frame using BRUVs 12 0 1.62 0.70 0.65

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Table 4.2: Summary of the model transformation, distribution and outcomes for sponge abundance (N), sponge volume, ascidian diversity, Haliclona spp. and Tedania (Tedania) sp. abundance (N). Model and terms Trans. Dist. n Estimate F SE t p r2 Null Residual Deviance deviance deviance explained Sponge N - Normal 10 0.39 - leaf length - 6.36 8.12 2.23 2.85 * Sponge volume sqrt Normal 10 0.48 - shoot count - 1.65 11.32 0.49 3.37 ** Ascidian diversity - Quasi 10 0.36 - reef in 1500m r log poisson -1.00 0.41 -2.44 * 22.89 14.72 36% Haliclona spp. N - Quasi 10 0.61 - depth - poisson -1.21 0.33 -3.70 ** 267.90 105.01 61% Tedania sp. N sqrt Normal 10 0.25 - adjacent reefs - 0.58 4.64 0.27 2.15 NS

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Figure 4.1: Scatterplot of (A) Sponge abundance and leaf length of P. australis, (B) Sponge volume (sqrt transformed) and the quantity shoots of P. australis, (C) Ascidian species richness and the area of reef within a 1500m radius (log transformed), (D) Haliclona spp. abundance and depth, (E) Tedania (Tedania) sp. and the number of reefs neighbouring the edge of the meadow.

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Individual sponge species (Haliclona spp. and Tedania (Tedania) sp.), commonly found in this assemblage, were also affected by seagrass habitat structure. The abundance of Haliclona spp. was negatively correlated (Pseudo-R2=0.61, d.f.=10, P=0.004) with depth (Table 4.2, Figure 4.1D). The abundance of Tedania (Tedania) sp. was correlated with the number of reefs neighbouring the edge of the meadow (Table 4.2, Figure 4.1E), although this was not statistically significant (Pseudo- R2=0.249, d.f.=10, P=0.0567). The remaining factors either violated the assumptions of the analyses or were not correlated with sessile epifaunal invertebrate distribution variables. These included the remaining small-scale biogenic structures (leaf length and epiphyte cover), the remaining large-scale seascape patterns (area and perimeter of the seagrass meadow, distance to the nearest reef, perimeter shared between meadow and reef, the amount of seagrass within varying radii between 100 and 1500m) and the abundance of a common omnivorous fish species.

4.5 DISCUSSION A clear dichotomy was apparent in the scale over which patterns differed between sponges and ascidians. Small-scale biogenic characteristics of the seagrass affected the abundance and size of sponges, and the sponge species complex (Haliclona spp.) was negatively correlated with depth. In contrast, ascidians responded to large-scale seascape patterns. Although sponges and ascidians have a similar mode of feeding, habitat and lifestyles, their patterns of distribution differed greatly. This suggests that these different phyla should be considered separately, combining sponges and ascidians under ‘sessile invertebrates’ could undermine the outcome of a study.

4.5.1 Seagrass density and frond length Seagrass density was found to positively affect sponge volume, and the length of seagrass blades was positively associated with sponge abundance. These findings support spatial distribution patterns reported by Corriero et al. (1989). Corriero et al. (1989) confirmed that the distribution of two Tethya species from a seagrass meadow was correlated with seagrass shoot density and water movement. They suggest that this relationship is closely related to varying sedimentation rates and food availability within the meadow (Corriero et al., 1989). Other studies confirmed that leaf characteristics, such as length, can affect the trapping ability of seagrass by influencing water flow and particle resuspension inside the canopy (Hemminga & Duarte, 2000; Koch et al., 2006; Short et al., 2007; Kennedy et al., 2010). Seagrasses have the ability to change hydrodynamic conditions by reducing flow within the canopy. Peterson et al., (2004) showed that the attenuation of water flow inside the canopy varied inversely with vegetation density. This leads to greater particle deposition and organic matter

Chapter 4 availability, in comparison to the surrounding environment (Hemminga & Duarte, 2000; Peterson et al., 2004). Increasing organic matter, the main food source of sponges (Bergquist, 1978; de Goeij et al., 2013), is known to promote sponge growth and survival rates (Duckworth et al., 2004; Morris & Keough, 2003). Although, excessive levels of nutrients can have an adverse effect on sponge survivorship. For instance, the increased nutrient availabity associated with the commissioning a sewage outfall resulted in a decline in sponge abundance and a complete shift in community composition (Roberts et al., 1998). Seagrass meadows with high shoot density are more effective at reducing water flow (Hemminga & Duarte, 2000; Koch et al., 2006). Canopy height has been previously correlated with a decrease in current velocity and enhanced trapping capacity of the meadow (Gacia et al., 1999). The enhanced sediment deposition ability of dense and long seagrass is likely to increase nutrient availability and therefore benefit sponge energy intake. Plant morphology, such as frond length, is successively impacted by the presence of seagrass inhabitants. Using manipulative experiments, a mutualistic interaction was confirmed between seagrass and the bivalve aggregates that it hosts (Peterson and Heck, 2001). The hydrodynamic abilities of the seagrass influenced bivalve growth, and, in return, the presence of this epifaunal invertebrate significantly affected frond length by providing nutrients to the plant. As a result, seagrass productivity was significantly increased (Peterson and Heck, 2001). The presence of sponges and ascidians, also suspension-feeders, is likely to contribute to a similar positive feedback loop, thereby influencing frond length. It is worth noting that frond length may also be influenced by turbidity. Seagrasses inhabiting high turbidity environments were found to grow longer fronds as a means to harvest light (e.g. Vermaat et al., 1996).

4.5.2 Hydrodynamics and dispersal The dispersal of sponge recruits is likely to be restricted in meadows with greater canopy height, due to enhanced hydrodynamic baffling ability. Leaf structure and blade area were important determinants of hydrodynamic forces exerted by seagrass meadows (Backhaus & Verduin, 2008). These weekly swimming larvae and many other sessile epifaunal invertebrates rely heavily on hydrodynamic factors for larval dispersal (Snelgrove, 1994; Maldonado & Young, 1996). A reduction in current velocity, resulting from long seagrass blades, is likely to reduce larval dispersal and promote sponge aggregation (Woodin, 1986; Butman, 1987; Pawlik & Butman, 1993; Maldonado & Young, 1996). Indeed, the attenuation of water flow associated with seagrass morphology, was found to strongly influence larval recruitment of two bivalve species (Eckman, 1987). I suggest that longer seagrass fronds may restrict the dispersal of recruits and, as a result, influence the patchy and gregarious nature of sponge distribution seen in seagrass meadows (Barnes et al., 2006; Barnes et al., 2013; Demers et al., 2016).

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4.5.3 Depth Depth, and its associated indirect factors, played a key role in sponge distribution. In this study, I established that the abundance of a sponge species complex; Haliclona spp. was negatively correlated with depth. This pattern is supported by the Lambert law of absorption, which states that light intensity decreases exponentially with depth (Lambert, 1760). The light attenuation curve is influenced by the temperature and salinity of the medium, in this case seawater (Pegau et al., 1997). The level of irradiance associated with depth is a major factor influencing sponge distribution on reefal habitats (Wilkinson, 1982; Wilkinson & Trott, 1985). Phototrophic sponges fulfil their energy requirements by cultivating cyanobacterial symbionts (Wilkinson, 1983). Photosymbionts provide nutrition to the sponge host granting adequate levels of UV radiation (Wilkinson, 1983; Wilkinson & Trott, 1985). As Haliclona spp. encountered in this study are suspected to be photosynthetically active, and to benefit from increased light penetration associated with shallow water. Many Haliclona species have been found to host photosynthesizing organisms (Steindler et al., 2002; Grant et al., 2006; Erwin & Thacker, 2007; Lemloh et al., 2009). This photosynthetic activity may also be linked to seagrass condition as the survival of this marine plant relies on adequate light availability (e.g. Ralph et al., 2007). Seagrass cover generally decreases with depth (Duarte, 1991) and variation in shoot density dictates the irradiance levels penetrating the canopy (Hogarth, 2007). This will in turn affect the light harvesting capacity of sponges hosting photosymbionts. It is worth noting that turbidity is negatively related to irradiance, with high turbidity and nutrient levels being one of the most devastating human-driven disturbances for seagrass meadows worldwide (Orth et al., 2006).

4.5.4 Seascape effects Seascape patterns were found to influence ascidian dictribution, with meadows neighbouring abundant reefal habitats consistently harboured low ascidian species richness. This pattern was not supported by any other studies and no known studies have investigated this specific distributional pattern. Greater fish abundance was recently correlated with increasing reef connectivity (Rees et al. 2014), especially that of zooplanktivorous fish (Rees, pers. comm.) that are most likely to feed on ascidian larvae. I suggest that the zooplanktivorous predators associated with reefs may have influenced ascidian species richness within the meadow, by controlling larval availability and dispersal. This effect has been observed previously where planktivorous fish were responsible for the localised depletion of the plankton supply of a temperate reef system (Kingsford & MacDiarmid, 1988). It follows then that ascidian larvae may be at higher risk from predators in seagrass neighbouring abundant reefal habitats. It is unknown whether the species encountered in

Chapter 4 this study have palatable larvae or if chemical defences are used to deter fish predation as seen in other species (Lindquist et al. 1992, Lindquist & Hay 1996). The remaining seascape patterns were not correlated with sessile epifaunal invertebrate distribution. The distributional patterns of sponges were only influenced by small-scale seagrass condition, not the surrounding seascape. This was surprising as meadow size was expected to influence species diversity. One of the most thoroughly documented patterns in ecology is the species-area relationship (SAR), which states that larger areas generally contain more species than smaller areas (e.g. McGuinness, 1984). Based on this concept, larger seagrass meadows should host a greater sessile epifaunal invertebrate diversity than smaller meadows. Also, meadow size is known to affect the capacity of seagrass to stabilise sediments and alter flow (Fonseca & Koehl, 2006). Fluctuating sedimentation rates and nutrient availability between the edge and the centre of a seagrass meadow (Fonseca & Fisher, 1986) are expected to affect the distribution of sessile epifaunal invertebrates (Wulff, 2008). However, none of these relationships were apparent in this study. The abundance of a common sponge, Tedania (Tedania) sp., was almost significantly correlated with the number of reefs neighbouring the edge of the meadow (Table 4.2, Figure 4.1E). Although not significant, this pattern suggests that reefs neighbouring seagrass meadows may contribute to specific sponge abundance within seagrass beds. Further sampling is required to confirm this pattern of distribution.

4.5.5 Role of predators The abundance of a common seagrass-associated fish species was not correlated with the distribution of sessile epifaunal invertebrates inhabiting the seagrass meadows of Jervis Bay. This omnivorous fish species was observed feeding on sponge specimens in situ. Based on this observation, I hypothesised that predation pressure may be influencing the distribution of sessile epifaunal invertebrates. However, the large variation in the abundance of Chinaman leatherjackets (Nelusetta ayraudi) seen in Jervis Bay (Fetterplace, pers. comm.) may be responsible for the lack of correlation. The abundance of this schooling fish species varied greatly through time (yearly averages) and among locations (Fetterplace unpublished data). Predation and palatability have been reported as important drivers of sessile epifaunal invertebrate distribution in other habitats (i.e. Wulff 1997b, 2006c). Many other fish species inhabit seagrass ecosystems in addition to a range of other animals such as molluscs, echinoderms, polychaetes and crustaceans. It is unknown whether these constitute potential sessile epifaunal invertebrate predators and whether they play a role in the distribution of this assemblage. I explore predator-prey responses further in an upcoming chapter.

Chapter 4

4.5.6 Anthropogenic effects Anthropogenic disturbances are likely to influence the distribution of sessile epifauna. My findings suggest that factors affecting seagrass condition, such as seagrass shoot length and density, indirectly determine sponge patterns of distribution. The role of human impacts on the global seagrass decline is undeniable (Waycott et al., 2009; Short et al., 2011) and, this study suggests that it represents a key driver of seagrass sponge distribution. Epifaunal abundance and diversity have been found to decline as a result of habitat loss and fragmentation associated with relatively large seagrass disturbance (Lemmens et al., 1996; Reed & Hovel, 2007). Eutrophication has devastating consequences on seagrass meadows worldwide (Orth et al., 2006) and is likely to have an adverse effect on the sessile epifaunal invertebrate community of this habitat (Roberts et al., 1998). Organisms reliant on photosymbionts, such as some sponge species, are particularly vulnerable to elevated turbidity. Further studies are required to elucidate the effect of anthropogenic disturbance on seagrass-associated sessile epifaunal invertebrate community.

4.6 CONCLUSIONS AND RECOMMENDATIONS The scale over which patterns were apparent differed greatly between sponges and ascidians, suggesting that these different phyla should be analysed separately since they have distinct drivers influencing their patterns of distribution. The distributional patterns of sponges were influenced by small-scale biogenic characteristics of seagrass. Seagrass density and frond length was positively correlated with sponge volume and abundance. I suggest that this pattern was driven by the hydrodynamic conditions imposed by the meadow, and resulted from variation in the hydrodynamic baffling ability of the meadow, nutrient availability and sponge larval dispersal within the canopy. These hypotheses need to be tested experimentally. Depth was negatively correlated with the abundance of a suspected phototrophic sponge species complex, which is thought to benefit from increased light penetration associated with shallow water. Large-scale seascape patterns were found to correlate with ascidian distribution in seagrass meadows. Meadows neighbouring abundant reefal habitats consistently harboured low ascidian species richness. This pattern may be influenced by the high abundance of zooplanktivorous fish associated with large reefal areas and the role of these predators on ascidian larvae dispersal. Further studies need to adopt an experimental approach to disentangle the remaining key processes and factors responsible for the patterns observed. This includes potential drivers of distribution such as environmental factors. An experimental assessment of sessile epifaunal invertebrate diversity and abundance is the focus of the next chapter.

Chapter 3

CHAPTER 5

Picky eaters or prickly entrées? Effect of predation, palatability and availability of substrata on the sponge community of Posidonia australis seagrass meadows

Chapter 5

5.1 ABSTRACT Biotic factors driving sponge distribution in seagrass meadows have been poorly studied and could have important ramifications for this threatened ecosystem. Species interactions between this foundation species and its sponge inhabitants may play a key role in modulating sponge community structure in seagrass meadows. Predation pressure has been found to shape sponge distributional patterns in other habitats such as coral reefs and mangroves. Sponge community dynamics within seagrass meadows may be influenced by; the array of predator species present within this habitat, the palatability of seagrass-associated sponge species, and their level of deterrence to predators. The availability of hard substrata, such as shells, within the seagrass meadow may affect the abundance and diversity of the sponge species present. This study aims to determine 1) whether predation pressure and substratum availability influences the sponge community of Posidonia australis seagrass meadows, 2) whether the common sponge species are palatable to fishes. A predator exclusion experiment showed no detectable effect of fish predation or substratum availability on the seagrass sponge or ascidian community. Diurnal palatability assays showed negligible predation for the three sponge species investigated (Callyspongia (Toxochalina) sp., Haliclona spp. and Halichondria sp.). Predator exclusion cages successfully excluded large fish predators but were breached by small organisms. The fish species seen inside the predator exclusion cages did not readily consume sponge explants and are unlikely to be responsible for the lack of recruits observed in the caging experiment. Shrimps (Palaemon intermedius) were observed interacting with sponge explants. I suggest two possibilities for the lack of recruits in the predator exclusion cages; 1) shrimps or other meso predators consumed sessile epifaunal invertebrate larvae or recruits, or 2) larvae have limited dispersal capacities and were unable to access the predator exclusion cages. Further research should aim to ascertain whether shrimps are feeding on sponge tissue or on their epifaunal community. Future work should also aim to uncover the effect of sponge recruitment and larvae dispersal on sponge distribution Nocturnal spongivory within seagrass meadows should also be investigated.

Chapter 5

5.2 INTRODUCTION Food chain dynamics, a fundamental ecological theory, proposes that interacting abiotic and biotic factors influence ecosystem functioning (Fretwell, 1987). Ecological processes are important determinants of community dynamics and are categorised into either top-down and bottom-up. The ecological process of top-down forcing is initiated by predator pressure indirectly altering food web interactions throughout the system (Pace et al., 1999). Alternatively, bottom-up forcing processes occur in upward-flowing food chains where community dynamics are determined by resource availability (Fretwell, 1987). The trophic transfer of production within a system relies primarily on oceanographic factors such as currents, upwelling and nutrients that dictate plankton abundance, nutrient availability and rate of recruitment (Menge, 2000). These, in return, affect consumer- resource interactions such as abundance and growth at different trophic levels. Many studies have shown that a combination of bottom-up and top-down processes dictate ecosystem dynamics (e.g. Menge, 2000; Mozetic et al., 2012; Whalen et al., 2013). Top-down and bottom-up processes can also act simultaneously through complex populations dynamics at different stages in the life cycle of the organism (Otto et al., 2014). Since benthic organisms lack the ability to escape, their survival relies on tolerating or reducing predation (Lubchenco & Gaines, 1981). Predation plays a key role in modulating sponge community structure in ecosystems other than seagrass meadows (Wulff, 1997b; Pawlik et al., 2013). Predation pressure shapes sponge distributional patterns in coral reefs and mangroves (Wulff, 2003; Wulff, 2005; Pawlik et al., 2013). Sponges use an array of mechanical and chemical defences such as collagen fibres, spicules and noxious chemical compounds (Sarà & Vacelet, 1973; Thacker et al., 1998; Ferguson & Davis, 2008; Pawlik et al., 2013). For instance, the community dynamics of an Antarctic sponge assemblage were mainly driven by predation and were mediated by chemical defences (Dayton, 1979; McClintock et al., 2005). Biotic factors driving sponge assemblages in seagrass meadows have been poorly studied and could have important ramifications for this threatened ecosystem. The sponge community structure inhabiting seagrass meadows is controlled by a multitude of abiotic factors, such as depth, proximity to neighbouring reefs and seagrass condition (Chapter 4). Few studies have examined the effect of predation and substratum availability on sponge community dynamics (but see Wulff, 1995b, 2008; Archer et al., 2015). A study by Wulff (2008) has shown that the sponge assemblage of seagrass ecosystems is influenced by predation pressures. Sponge species inhabiting reefs were unable to survive in seagrass meadows because they were highly vulnerable to a large predatory starfish (Wulff, 2008). Seagrass-associated sponge species were unpalatable and successfully deterred consumption by starfish (Wulff, 2008). A study by Archer et al., (2015) established a

Chapter 5 commensal interaction between a subtropical seagrass Thalassia testudinum and its sponge inhabitants. Sponges were impartial to seagrass as the negative impact of shading cancelled the positive effect of nutrient transfer. Alternatively, sponges benefited from a stable substratum and enhanced food availability as particulate matter was concentrated at the base of the seagrass (Archer et al., 2015). This context-dependent interaction has the potential to change to a parasitic interaction if eutrophication reaches antagonistic levels (Archer et al., 2015). The predator species present in a specific ecosystem and their diet are important determinants of sponge-predator interactions (Wulff, 2006a). Sponge predators include molluscs, echinoderms, crustaceans, fish and turtles. Spongivorous fish were mainly recorded on coral reefs and include angelfishes, parrotfish, wrasses, cowfish, filefish and damselfish (Chanas & Pawlik, 1995; Dunlap & Pawlik, 1996; Andréa et al., 2007; Loh & Pawlik, 2009; González-Rivero et al., 2012). Spongivorous species can fall into 3 consumer categories; ‘smorgasbord’ feeders restrict their intake to small fragments of multiple species, ‘specialist’ feeders consume a few species exclusively and ‘opportunistic’ feeders infrequently ingest poorly defended species (Wulff, 2006a). The type of consumer has implications for the diversity and abundance of sponge in a given habitat. The nature of the tissue being consumed is also key in determining the palatability of a species. Sponge predators were found to be more greatly attracted to inner sponge tissue than outer tissue due to their different coloration and lower spicule content (Wulff, 1997b; Schupp et al., 1999; McClintock et al., 2005; Wulff, 2005). Some predator species specialise in feeding on already scared individuals (Peters et al., 2009). The presence and identity of spongivorous species within seagrass ecosystems are generally poorly understood. Sponge palatability is an important determinant of sponge community dynamics since it is a driver of predation pressure (Wulff, 2005; Pawlik et al., 2013). Different sponge species display specific ability to deter and/or adapt to predation (Wulff, 2006a). Predation deterrence and avoidance have led to multiple adaptations such as the production of mechanical and chemical defences and the ability to withstand predation by rapidly regenerating after partial mortality (Wulff, 2010). These adaptations also vary among a single species across a range of habitats. Individuals of the species Crambe crambe showed different levels of investment in defence mechanisms according to the abundance of predators within their habitat (Turon et al., 1998). These adaptations are often associated with trade-offs in sponge growth (Wulff, 2006a). It is unknown whether the seagrass-associated sponge species have developed these adaptations to deter predation. Competition among sponge species has been directly linked to substratum availability and predation. The competitive ability of a sponge species relies on trade-offs between resistance to competition and resistance to predation (Wulff, 2005). Competition among sponge species

Chapter 5 generally operates under the form of elimination by overgrowth (Jackson & Buss 1975; Wulff, 2006a). Regeneration is an important competitive advantage often employed by sponges to successfully obtain or retain substratum in space-limited system (Kay & Keough, 1981; Wulff, 2010). Though, competitive superiority associated with accelerated regeneration is often energetically expensive. For instance, fast growing competitive dominants easily overgrow other species and monopolize space (Wulff, 2005). However, this elevated growth rate was negatively correlated with defences against predators. Predators preferred consuming competitive dominants as they are not as well-defended against predation as inferior competitors. Inferior competitors are slow-growing and consequently compete poorly for space (Wulff, 2005). Substrata availability within the seagrass meadow is likely to influence biotic processes such as competition for space and predation. The community dynamics of sponges inhabiting mangrove roots, for instance, are affected by substratum availability (Wulff, 2009). Juvenile mangrove roots provide available substrata in a space-limited system. They replenish space for the well-established sponge community inhabiting mature mangrove roots (Wulff, 2009). Sponge species often target a specific substratum (Davis et al., 1996). In seagrass meadows for instance, a distinctive set of species formed clusters on shell aggregations (Demers et al., 2016). The availability of hard substrata, such as shells, within the seagrass meadow may affect the abundance and diversity of the sponge species present. This study aimed to determine if the sessile epifaunal invertebrate community of the Posidonia australis seagrass meadows is driven by predation, palatability and the availability of suitable hard substratum on which to establish. To the best of my knowledge, the effect of predation and the palatability of the sponge community inhabiting the threatened P. autralis seagrass meadows has never been investigated. I used a combination of predator exclusion experiment and palatability assays to test multiple hypotheses; A) The predator exclusion experiment aimed to determine the impact of removing predators from the system and the impact of providing differential availability of substrata in the presence and absence of predators. B) The palatability test aimed to determine the level of palatability of seagrass sponges, to identify sponge predators, and to test whether predators could access the predator exclusion cages used for the exclusion experiment. I hypothesised that the exclusion of predators and substrata availability would have a positive impact on sponge and ascidian recruitment and that sponges were palatable to an array of predators. Please note that prominence was given to the sponge assemblage inhabiting seagrass meadows and that ascidians were not investigated.

Chapter 5

Jervis Bay

Figure 5.1: Map showing the distribution of predator exclusion cages, cage controls and unmanipulated plots between Murray’s Boat Ramp and Hole-in-the-wall in Booderee National Park, Jervis Bay, NSW, Australia (n=18).

Chapter 5

5.3 MATERIALS AND METHODS

5.3.1 Study area Experiments were conducted in Jervis Bay Marine Park, NSW, Australia, an open embayment of the south eastern coast. Extensive P. australis meadows, totalling 5.7 km2, (West, 1990) constitute the most pristine and largest continuous areas of seagrass along this coast (West et al., 1989; Kirkman et al., 1995; Meehan & West, 2000). The predator exclusion cages were installed between Murray’s Boat Ramp and Hole-in-the- wall (35°07’44S 150°44’59E) in Booderee National Park on March 2012 (Figure 5.1). Cages were cleaned and sampled fortnightly until March 2014. The palatability test was conducted at the same location in April 2014. Sponge explants were collected from Hare Bay (35°00’31S 150°45’59E) and Callala Bay (35°00’09S 150°43’39E) for the palatability test in March 2014. I selected Booderee National Park as my study location for three main reasons; 1) I previously observed sponge consumption by omnivorous Chinaman leatherjackets (Nelusetta ayraudi) (author’s personal observation), 2) this area is an ‘anchoring exclusion’ zone, which minimised the risk of cage damage, 3) accessibility was facilitated by a nearby boat ramp. Recreational fishing is permitted at this location and constituted the main risk of disturbance to predator exclusion cages. The depth ranged from 4 to 6 m.

5.3.2 Predator exclusion experiment

5.3.2.1 Experimental design The predator exclusion experiment tested two factors; predation and substratum availability. The factor ‘predation’ had three levels; predator exclusion cages, cage controls and unmanipulated plots. Each of these levels was replicated 18 times (Figure 5.1). Predator exclusion cages prevented fish larger than 3 cm in height and width from gaining access to a 0.75 m3 (50 x 100 x 150 cm) area in the seagrass. Cage controls tested the impact of the cage itself on the assemblage such as current baffling and shading whilst allowing for predation. Unmanipulated plots of seagrass were used as controls. Replicates were haphazardly distributed at least 25 m apart and more than 50 m from the reef. A high level of replication was adopted due to the potential risk of cage loss associated with recreational fishing effort at this location. Each level of predation was exposed to three levels of substratum availability; none, light and heavy addition of oyster shell (n=6). Substratum availability varied in oyster shell addition from none to ≈100 shells for low density and ≈200 shells for high density. These values were estimated based on the density of bivalve clumps within a nearby estuarine seagrass meadow in Lake Conjola, NSW (authors’ personal observations). Oyster shells were chosen as substrata for

Chapter 5 this experiment as they were readily available and sponges were previously recorded on this substratum within the seagrass meadows of Jervis Bay (Demers et al., 2016). Cages were cleaned fortnightly to minimise the establishment of fouling organisms and were sampled for the occurrence and growth of recruits. Cages were repaired when damaged. I recorded the abundance, diversity and volume of recruits on shells and seagrass fronds for all treatment combinations. It is worth noting that seagrass measurements could not be collected. This task could not be achieved from outside the exclusion cages. Accessing the inside of the cage, by opening the mesh, would have allowed fish to enter, which would have interfered with the experiment.

5.3.2.2 Cage construction Cages were 1.50 m2 constructed of a PVC tube frame covered by a net of 25 x 25 mm mesh size and 1 mm thick (Figure 5.2A). Cage controls were exact replicates of predator exclusion cages except for two sides uncovered by mesh (Figure 5.2B). Cages were secured, using cable ties, to four star pickets permanently installed in the seagrass (Figure 5.2C). Many criteria were considered in the construction of these cages; low construction cost, durable for the entire span of the experiment, light weight and foldable to allow for transport by car and boat. The base of the cage had to be in contact with the substratum to eliminate fish intrusion. Star pickets, with the cage attached, were firmly embedded in the substratum leaving no space at the base of the cage (Figure 5.2C). The use of cable ties to secure the cage to the star pickets allowed the cage to be lifted for sampling by divers as cable ties could be easily cut and replaced. The height of the cage could not exceed the seagrass canopy as to avoid navigational hazards, 50 cm high was deemed adequate. I trialled cage prototypes using different mesh sizes and materials (online supplementary mesh trial video). Bait was used to attract fish and to test the resistance of the cages to predation using defrosted pilchards. The aviary polypropylene net 1 mm thick and with a mesh size of 25 x 25 mm was successful in excluding fish and was resistant to fish bites whilst minimising current baffling. A detailed list of the materials used to construct the cages is given in the appendix.

Chapter 5

Figure 5.2: (A) predator exclusion cage, (B) cage control and (C) diver installing star picket to secure the cage to the substratum.

Chapter 5

5.3.3 Palatability experiment

5.3.3.1 Feasibility study A pilot study was used to determine the feasibility of the palatability experiment. The palatability experiment involved collecting sponge individuals from multiple locations in Jervis Bay, cutting the sponge individual into comparable size explants, protecting the sponge against predation while the exposed inner tissue regrows an external layer of cells, and transporting the explants to the location where the predator exclusion experiment took place. This feasibility study aimed to establish if sponges could survive the cutting process and regrow their outer tissue. The reasoning behind the ‘tissue regrowth’ period is that predators were found to be attracted to inner sponge tissue due to their different coloration and lower spicule content. Explants were given time to regrow their outer surface as per other studies (Wulff, 1997b; Schupp et al., 1999; McClintock et al., 2005; Wulff, 2005). The pilot study was carried out in Hare Bay and tested the feasibility for sponges to survive and regrow their exposed inner tissue in situ following transplant. The two sponge species tested (Callyspongia (Toxochalina) sp. and Tedania (Tedania) sp.) survived the initial cutting process and completely replenished their external tissue within two weeks (n=4).

5.3.3.2 Sampling method A palatability experiment was used to 1) record the palatability of common seagrass sponges, and 2) determine if predators were accessing predator exclusion cages (Figure 5.3A). Sponge palatability was tested inside five predator exclusion cages, five cage controls and five unmanipulated plots at a depth of 3-5m. Sponge palatability underwater video (SPUV) units consisted of a Go Pro Hero 3 camera mounted on one end of a brick pointing to a plastic board where sponge explants were attached (Figure 5.3A). A metal mesh, secured to the underside of the brick, flattened the seagrass and minimised obstruction by seagrass fronds. A rope bearing a highly visible float was tied to the brick to facilitate the recovery of the SPUVs. A weight affixed to the rope kept the rope clear of the field of view. SPUVs were assembled using cable ties. Explants were secured to a white corrugated plastic board (corflute) using cable ties. The explants remained submerged in seawater until the SPUVs were ready to be lowered. Lights on the cameras were deactivated to eliminate undesirable stimuli (Rees et al., 2015). I recorded at maximal definition (1080p) for 84 min. Prior to submersion, the camera angle was adjusted so that the field of view encompassed all explants. This was done using the live streaming video feature of the GoPro app on a mobile phone.

Chapter 5

Explants were collected from Hare Bay and Callala Bay from the five most common sponge species (Callyspongia (Toxochalina) sp., Tedania (Tedania) sp., Haliclona spp., Phoriospongia sp. and Halichondria sp.). Sponges were cut to standard sizes of 3 cm x 2 cm x 1 cm (6 cm3) and stored in isolation containers for a period of 12 days at Callala Bay (Figure 5.3B). This allowed the surface tissue to reconstitute before deployment. Note that small holes were made in the ~200 ml isolation containers to allow for water exchange. The Phoriospongia sp. and Tedania (Tedania) sp. explants did not survive the transplant procedures and could not be used in this study. The explants from the remaining three species were transported to Booderee National Park. Isolation containers holding explants were placed in a cool box filled with seawater. The water was kept oxygenated using a portable aquarium pump. Explants were kept submerged until ready to attach to the SPUV units. The abundance and diversity of sponge predators were recorded, as well as the number of bites by fish within a 84 min period for all three sponge species combined. In the case of shrimps (Palaemon intermedius), the mean number of shrimps that interacted with explants was recorded within a 84 min period for all three sponge species combined. It is important to note that shrimps were tracked over time to avoid counting an individual more than once. However, shrimps that exited and re-entered the field of view may have been counted multiple times. In order to minimize recounts, I calculated the maximum number of shrimps that interacted with explants in a single frame at one time (MaxN) within a 84 min period for all three sponge species combined. In this instance, I did not differentiate between small and large shrimp individuals. I also observed a high density of crabs (Nectocarcinus integrifrons) at this location, although quantification was unsuccessful due to their ability to effectively conceal themselves in the sand. It is worth noting that I disregarded one video for each treatment so that four out of five videos were used for my analysis. This was due to either low water visibility or condensation inside the camera housing for one video in each treatment.

Chapter 5

Figure 5.3: Photographs of (A) sponge palatability underwater video (SPUV) unit with mounted GoPro camera and affixed sponge explants, and (B) isolation containers holding sponge explants.

Chapter 5

5.3.4 Statistical analyses I planned to use a two factor ANOVA to contrast the outcome of the predator exclusion experiment with ‘Predation level’ as a fixed orthogonal factor with three levels (predator exclusion cages, cage controls and unmanipulated plots) and ‘availability of substrata’ as a fixed orthogonal factor with three levels (none, light and heavy shell addition). Each treatment was replicated six times (n=6). For the palatability experiment, an analysis of variance (ANOVA) was used to assess the levels of variation for each of the variables sampled (GMAV5, University of Sydney). The factor ‘Predation level’ was random orthogonal with three levels (predator exclusion cages, cage controls and unmanipulated plots). Four SPUV units were analysed for each treatment and constituted replicates (n=4). Variances were tested for homogeneity using Cochran’s C-test and normality of the data were assessed visually as recommended by Quinn and Keough (2002). In the case of heterogeneous data, the analyses were still performed since ANOVA is robust to heterogeneity with balanced experimental designs and large numbers of replicates (Underwood, 1997).

5.4 RESULTS

5.4.1 Predator exclusion cages Sponge recruits were not observed on shells or seagrass fronds over the course of the experiment (two years) despite careful examination of the substratum within each of the treatments. Invertebrate recruits were observed settling on the PVC frames of the cages, after a 24 month period. Species identification was arduous. Most recruits were hardly visible as they settled on the underside of the PVC frame under a layer of mesh material, and amongst algae, barnacles and other molluscs. Recruits were small in size and in their early stage of development. I successfully identified three ascidian species including Herdmania grandis, Pyura praeputialis and Styela plicata. Also, a total of 25% of cages were lost over two years of sampling (six predator exclusion cages and three cage controls).

5.4.2 Palatability test I assessed the palatability of three sponge species; Callyspongia (Toxochalina) sp., Haliclona spp. and Halichondria sp. These sponge species survived the explant cutting process and the outer tissue regrowth period of 12 days. All three sponge species were bitten during the palatability test although none was readily consumed. The spicule content was high for all three sponge species and included mainly oxeas of different sizes.

Chapter 5

Fish did not repeatedly bite explants and showed no interest in the sponge after the initial bite. Sponges were bitten by five fish species; Chinanaman leatherjacket (Nelusetta ayraudi), Little weed whiting (Neoodax balteatus), Six spine Leatherjacket (Meuschenia freycineti), Yellow sabretooth blenny (Plagiotremus tapeinosoma) and an unidentified leatherjacket species (Figure 5.4). In 33% of cases, Chinanaman leatherjackets (Nelusetta ayraudi) were observed regurgitating the sponge explant after ingestion. The only bite recorded by a Six spine Leatherjacket (Meuschenia freycineti) was regurgitated. These two leatherjackets species are opportunistic feeders (NSW DPI, 2015) and anecdotal evidence suggests that they often bite unpalatable objects such as fishing hooks (Holland, 2010). They were also observed biting the equipment such as the plastic rope, brick, camera, white corrugated plastic board, pencils and cable ties. Palatability was tested within three treatments (predator exclusion cages, cage controls and unmanipulated plots) to determine if the lack of recruits observed in the predator exclusion experiment may be due to predators accessing cages. The mean number of explants bitten by fish was consistently low for all three treatments (Figure 5.6). There was no significant difference among treatments in the mean number of bites by fish within a 84 min period, for all three sponge species combined (Table 5.1A). Small sized fish predators such as the Little weed whiting and the Yellow sabretooth blenny were observed biting explants in the predator exclusion cages due to mesh size restrictions. These two fish species were potentially able to access sponge recruits through the predator exclusion cages. Shrimps (Palaemon intermedius) were seen interacting with sponge explants although it is unclear whether they were feeding on sponge tissues or epiphytic organisms (Figure 5.5A, B). I tested whether the lack of recruits observed in the predator exclusion experiment could be related to shrimp predation inside cages. The mean number of shrimps that interacted with explants within a 84 min period for all three sponge species combined did not differ significantly among treatments (Figure 5.7, Table 5.1). Large shrimps were only encountered inside predator exclusion cages (Figure 5.7). The maximum number of shrimps that interacted with explants in a single frame at one time (MaxN) within a 84 min period, for all three sponge species combined, also showed no significant difference among treatment (Figure 5.8, Table 5.1). It is worth noting that shrimps were seen fighting among each other for explants and fleeing from larger shrimps, which may be indicative of feeding behaviour in decapods (Stocker & Huber, 2001). Other organisms were sighted on or near sponge explants. An unidentified worm was observed on Halichondria sp. (Figure 5.5C). Although they did not appear in the SPUVs, I noted a high density of crabs (Nectocarcinus integrifrons) concealed in the seagrass.

Chapter 5

Figure 5.4: Fish species that consumed sponges; (A) Chinanaman leatherjacket (Nelusetta ayraudi), (B) Little weed whiting (Neoodax balteatus), (C) Six spine Leatherjacket (Meuschenia freycineti) and (D) yellow sabretooth blenny (Plagiotremus tapeinosoma). The unidentified leatherjacket species was barely visible due to poor light and was not included in this figure. I recorded for a period of 84 min using sponge palatability underwater videos (SPUVs) in predator exclusion cages, cage controls and unmanipulated plots (n=4).

Chapter 5

Figure 5.5: Organisms seen on the surface of sponges: (A) small and, (B) large adult shrimp of the same species (Palaemon intermedius), and (C) unidentified worm.

Chapter 5

Figure 5.6. Mean (±SE) number of bites by fish taken from explants within a 84 min period, including all three sponge species combined, for the three treatments investigated (n=4).

Figure 5.7: Mean (±SE) number of shrimps (Palaemon intermedius) that interacted with explants within a 84 min period, including all three sponge species combined, for the three treatments investigated (n=4). small and, large adult shrimps of the same species (Palaemon intermedius).

Chapter 5

Figure 5.8: Mean (±SE) maximum number of shrimps (Palaemon intermedius) that interacted with explants in a single frame at one time (MaxN) within a 84 min period, including all three sponge species combined, for the three treatments investigated (n=4). I did not differentiate between small and large shrimps in this instance.

Chapter 5

Table 5.1: Analyses of variance comparing variation for (A) the total number of fish bites taken of explants, (B) total number of shrimps that interacted with explants and (C) total maximum number of shrimps that interacted with explants in a single frame at one time (MaxN). The factor was ‘Treatment’ a random orthogonal factor with three levels (unmanipulated plot, cage control and predator exclusion cage) (n=4). NS P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001.

A) Source d .f. MS F p Treatment 2 1.33 0.63 0.5545 (NS) Residual 9 2.11

Cochran’s test 0.8947 (P<0.01) Transformation none

B) Source d .f. MS F p Treatment 2 123.1 3.39 0.0799 (NS) Residual 9 36.3

Cochran’s test 0.5287 (NS) Transformation none

C) Source d .f. MS F p Treatment 2 28.6 3.99 0.0575 (NS) Residual 9 7.17

Cochran’s test 0.8798 (P<0.05) Transformation none

Chapter 5

5.5 DISCUSSION

5.5.1 Predation and substratum availability No sessile epifaunal invertebrate recruits were recorded by the predation exclusion experiment in Posidonia australis meadows, over a period of two years. A greater recruitment and growth rate of sponges was expected in areas of high substratum availability (Wulff, 2005) and low predation pressure (Wulff, 1997b; Pawlik et al., 2013) as has been seen in other systems. The lack of predation by starfish and/or large fish within seagrass differs from other studies (Wulff, 2008; Barnes, 2009). The complete lack of recruits over a two year period in spite my efforts to minimise competition for space and predation may stem from either limited sponge recruitment at this location or the inadequate exclusion of predators. A lack of sponge recruitment possibly influenced the consistent lack of sponge recruits in all three treatments (predator exclusion cages, cage controls and unmanipulated plots). Sponge larvae mainly disperse locally (Davis et al., 1996) as sessile epifaunal invertebrate larvae dispersion is often restricted by the hydrodynamic regime of the region (Havenhand & Svane, 1991; Maldonado, 2006). I speculate that water circulation patterns inside Jervis Bay are responsible for the limited dispersal of sponge larvae at this location. A prevalent gyre draws oceanic water from the entrance of the Bay and subsequently circulates clockwise accumulating nutrients and sponge recruits (Wang & Wang, 2003). I sampled at close proximity of the Bay’s entrance where the concentration of sponge recruits is likely to be lower than that of other locations in Jervis Bay (Demers et al., 2016). Processes driving sponge recruitment are complex and rely on a range of factors. Patterns of sponge recruitment vary at a multitude of spatial and temporal scales (Keough, 1983). Temporal variations in sponge larvae are challenging to investigate due to their small size and difficulties associated with their collection (e.g. Davis et al., 1991). Successful colonisation is more likely with increasing proximity to the colony and with increasing time since the colony was established (Zea, 1993; Uriz et al., 1998). Variation in recruitment may also stem from low larval survivorship, dispersal ability and patchiness within the plankton (Keough, 1983; Davis et al., 1996; Maldonado, 2006; Ettinger-Epstein et al., 2008). Environmental factors also dictate patterns of recruitment and include light regime, water flow, temperature and substratum availability (Maldonado & Young, 1996; Knott et al., 2004; Maldonado, 2006). Although seasonality was often found to alter recruitment success, the extensive two year sampling period refute that a recruitment episode was overlooked (Bergquist & Sinclair, 1973). Future work should focus on larval capture-and-release experiments using larval traps, as to determine the abundance and diversity of larvae in the meadow. Capturing, releasing and then tracking these larvae over time could provide additional information on the important determinants of spatial and temporal processes influencing their

Chapter 5 recruitment. Similar manipulative experiments were achieved previously for ascidian larvae on reefs (e.g. Davis et al., 1991). Inadequate exclusion of predators may also have contributed to the absence of sponge recruits at my sample location. This experiment exclusively targeted predation pressure by large fish predators. However, other predators may have been able to evade exclusion efforts. For instance, meso predators such as small fish, worms, crabs and shrimps had access to all three treatments and could be responsible for some level of predation pressure on sponge recruits. Specialised feeders have been recorded to inhabit and consume sponge hosts including polychaete worms (Pawlik, 1983; Tsuriumi & Reiswig, 1997), molluscs (Becerro et al., 2006) and shrimps (Rios & Duffy, 1999). Since no recruits were recorded in this experiment, palatability tests were pursued to determine the palatability of common sponge species within the seagrass meadows of Jervis Bay, the identity of sponge predators and whether these predators were accessing predator exclusion cages. The appearance of recruits in highly complex areas with multiple layers of mesh argues for predators accessing exclusion cages.

5.5.2 Palatability Sponges were not palatable and daytime predation pressure is unlikely to influence this community. A total of ±17 hours of footage revealed that sponges were not readily consumed by fish or other organisms. Sponge explants were bitten by five fish species; Chinanaman leatherjacket (Nelusetta ayraudi), Little weed whiting (Neoodax balteatus), Six spine Leatherjacket (Meuschenia freycineti), yellow sabretooth blenny (Plagiotremus tapeinosoma) and an unidentified leatherjacket species. However, the complete lack of interest following the bite and the regurgitation of the sponge tissue suggest that sponges are not an intrinsic part of their diet. Some fish species were found to respond to a novel potential food item by biting on first exposure regardless of their usual diet (Wulff, 2017). It is important to note that nocturnal predators have not been investigated in this study. Inorganic siliceous spicules may have inhibited sponge consumption by fish since all three sponge species (Callyspongia (Toxochalina) sp., Haliclona spp. and Halichondria sp.) tested showed a high spicule content. Spicules were found to be efficient structural deterrent against consumption by reef fish (Burns & Ilan, 2003), crustaceans (Hill et al., 2005) and echinoderms (Ferguson & Davis, 2008). However, in some instances, the presence of spicules did not impede sponge palatability (Chanas & Pawlik, 1995; Waddell & Pawlik, 2000). Palatability assays would be beneficial in investigating the effectiveness of spicule deterrence in seagrass-associated sponge species.

Chapter 5

Shrimps (Palaemon intermedius) may play an important role in the successful settlement and growth of sponges. Shrimps were observed interacting with sponges, although direct consumption of sponge tissue could not be detected due to their small size. Shrimps from multiple habitats including seagrass meadows (Duris et al., 2011), were reported to inhabit sponges by using canals as shelters and sponge tissue as a food source (Rios & Duffy, 1999). These obligatory sponge feeders developed complex morphological adaptations to cut spicules (Duris et al., 2011). Further tests are required to determine whether shrimps were consuming sponge tissue or epiphytic organisms associated with the sponges. Epiphytic organisms covering sponge explants may serve as food source for shrimps. They may be the reason why shrimps were seen interacting with sponges during the palatability tests. Shrimps inhabiting seagrass meadows have a varied diet consisting of crustaceans, bacteria, algae and plant detritus such as seagrass leaves (Schwamborn & Criales, 2000; Abed-Navandi & Dworschak, 2005). These detritivores possibly consume the epiphytic layer of sponges in a similar way to that of seagrass leaves and macroalgae (Quinones-Rivera & Fleeger, 2005; Ebrahim et al., 2014). Sponge nutrient intake and the resulting growth rate may be positively affected by shrimps and their ability to regulate epiphytic fouling. Shading of sponge tissue associated with high epiphytic algae cover was recorded in ecosystems with elevated nutrient levels (Easson et al., 2014). Acute algae cover was also found to hinder sponge growth rate (González-Rivero et al., 2012). The role that shrimps play in sponge community dynamics is unknown and further research is required to establish the nature of such ecological interactions.

5.5.3 Cage accessibility Predator exclusion cages were breached by small fish, shrimps and potentially other organisms. This may explain the lack of recruits observed in my caging experiment. Palatability assays revealed no significant difference among treatments (predator exclusion cages, cage controls and unmanipulated plots) in the number of explants ingested by fish, the number of interactions between shrimps and explants, and the abundance of shrimp estimated from a single frame at one time. This suggests that although large predators were successfully excluded, smaller organisms had access to predation exclusion cages. The occurrence of large shrimps solely inside predator exclusion cages corroborates the effective exclusion of large fish predators inside these cages. Palatability assays suggested that the fish species encountered inside predator exclusion cages (yellow sabretooth blenny and unidentified leatherjacket species) did not readily consume sponge explants. These species are unlikely to be responsible for the lack of recruits observed in the caging experiment. However, a suite of nocturnal seagrass foragers may have been visiting cages and feasting on sponge recruits. Many seagrass inhabitants have nocturnal foraging habits including

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Chapter 5 fish (e.g. Linke et al., 2001), molluscs (e.g. Rueda et al., 2008), crustaceans (e.g. Jernakoff, 1987) and echinoderms (e.g. Ogden et al., 1973; Eklöf, 2008). Further research should target the nocturnal diet of starfish and brittle stars in seagrass meadows as they are known sponge consumers with diel feeding habits (Henkel & Pawlik, 2005; Wulff, 2006a), although echinoderms were not encountered frequently.

5.6 CONCLUSIONS AND RECOMMENDATIONS Overall, predation and substratum availability were not established as important drivers of sponge distribution in seagrass meadows. Multiple factors are thought to be accountable for the absence of sponge recruits over the course of this study. This includes abiotic and biotic factors influencing sponge recruitment success, including low propagule pressure, as well as cage access by small predators. Sponges were not palatable, at least not during the daytime. Shrimps were seen interacting with sponges. I hypothesised that the consumption of sponge epiphyte by shrimps could have an effect on sponge growth and distribution. Also, I suggested that predator exclusion cages may have been inaccessible to colonising larvae due to their known limited dispersal range. Further tests are required to establish this ecological interaction between shrimps and sponges as well as larval dispersal capacities. In addition, manipulative experiments could explore sponge palatability by nocturnal seagrass foragers, although this would require artificial lights.

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5.7 APPENDIX Table 5.2: Materials used for the construction of the predator exclusion cages Mesh anti bird net pro choice (4m x 150m) 25 x 25 Conduit electrical pipe PVC grey 20mm x 4m 10020 Conduit elbow 20mm 2770B Conduit reducer PVC 25-20mm packet of 2 2726B Glue Ring grip PVC Blue jointing Cement 125 ml Cable ties 100mm Qty 500 Star Pickets with holes Cattle Tags (55)

5.8 SUPPLEMENTARY MATERIAL online supplementary mesh trial video https://youtu.be/y-6sjRx2rIw

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CHAPTER 6

Discussion and conclusions

Chapter 6

6.1 IMMEDIATE CHALLENGES I encountered 20 sponge species and eight ascidian species within Jervis Bay, which has the most extensive and pristine P. australis seagrass meadows of the south eastern Australian coast. A total seagrass area of 1800m2 was sampled. Most ascidians were identified to the level of species (88%) as this assemblage corresponded to that of nearby reefal habitats. Sponges were mainly identified to the genus level (75%), as well as family (10%) and class (10%). More tests are required to confirm the identity of the remaining 5%. The sponge assemblage associated with seagrass meadows was challenging to identify in situ as it contained species harbouring similar physical characteristics, such as colour, texture and form, as well as visibly dissimilar morphs that belong to the same species. Spicule mounts were used as a taxonomic aid. Sponge identification was hindered, in some species, by the complete lack of spicules or the incorporation of foreign sand and spicules into their skeletal structures. I sought the expertise of two sponge taxonomic specialists (Lisa Goudie and Dr. Jane Fromont) to complete the identification process. Once identification was completed, the distribution of sessile epifaunal invertebrates in seagrass meadows was investigated. These observational data are the primary basis of any experimental studies. Understanding the patterns of distribution is crucial in developing adequate manipulative experimentation with the purpose of determining ecological processes driving this ecosystem (Underwood et al., 2000). The first step in uncovering the distributional patterns of seagrass-associated sessile epifaunal invertebrates in the meadows of Jervis Bay was to establish adequate sampling procedures. It was crucial to achieve replication at the appropriate scales of variability whilst ensuring the efficient allocation of resources. The optimal sampling design used in this study covered a hierarchy of spatially nested scales. This design was similar to that used to sample sessile epifaunal invertebrates in coastal saline lakes in NSW, Australia (Barnes et al., 2006, 2013) and ranged from meters to kilometres. Another key finding of this study was that the seagrass had a highly speciose assemblage of sponges and ascidians which were sporadically distributed in the seagrass meadows with high variability among spatial scales. This pattern of distribution was expected as it has been previously recorded for the sessile epifaunal invertebrate assemblage of coastal saline lakes (Barnes et al., 2006, 2013). A few sponge and ascidian species dominated the assemblage and were widespread across the largest spatial scale sampled. The remaining species were mostly rare and sparsely distributed. Substrata used for attachment comprised mainly P. australis, shells and polychaete tubes. Sponges have rarely been recognised as a major constituent of seagrass ecosystems (Wulff, 2001); my study demonstrated otherwise. Although no obligate seagrass species was recorded, three

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Chapter 6 sponge species relied heavily on seagrass for attachment and are likely prone to disturbances impacting their host habitat. The high variability seen in the distribution of this assemblage provided an opportunity to further examine of the robustness of my sampling design. The outcome of the main study was compared with that of the initial pilot study. Robust measures of sessile epifaunal invertebrate diversity and abundance were reflected effectively by the pilot study, which also detected the appropriate scale of variation. Despite some limitations, it was deemed a valuable tool in the establishment of an effective sampling design. This assessment also revealed that variation in the sessile epifaunal invertebrates of P. australis was highest at the smallest spatial scales (10s m) investigated. Davis et al. (2003) showed the same outcome for reefal sponges in southern NSW, as have Underwood and Chapman (1996) for molluscs. Replication at the transect level was considered more important in detecting changes in this assemblage, than that of sites or locations. The key factors responsible for the patterns of distribution observed were then investigated. It was crucial to establish the nature of the relationship between the sessile epifaunal invertebrate community and its seagrass host to uncover their role in seagrass ecosystem structure and function. This study provided a primary investigation of the factors driving this threatened community and acted as an important stepping stone in designing future manipulative experiments. Seagrass meadows exhibited substantial variation in seagrass attributes and seascape patterns over a range of spatial scales. The distribution of sessile epifaunal invertebrates in seagrass ecosystems was correlated with both small-scale seagrass characteristics and large-scale seascape patterns. They were not affected by the abundance of a common seagrass-associated omnivorous fish species. The scale over which patterns were apparent differed greatly between sponges and ascidians, suggesting that contrasting drivers may be responsible for patterns in these different phyla. The distributional patterns of sponges were influenced by small-scale biogenic characteristics of seagrass. Seagrass density and frond length was positively correlated with sponge volume and abundance. This pattern was likely driven by the hydrodynamic baffling ability of the meadow, nutrient availability and sponge larval dispersal within the canopy. Depth was negatively correlated with the abundance of a suspected phototrophic sponge species complex, Haliclona spp. I suggest that this sponge benefits from increased light penetration associated with shallow water. Large-scale seascape patterns were found to be important determinants of ascidian distribution in seagrass meadows. Meadows neighbouring abundant reefal habitats consistently harboured low ascidian species richness. This pattern may be influenced by the high abundance of zooplanktivorous fish associated with large reefal areas and the role of these predators on ascidian larval survival. Seagrass condition was found to influence sponge distribution, which raised important concerns in regards to human impact on

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Chapter 6 seagrass condition and the potential deleterious repercussions on its sponge community. Further studies need to adopt an experimental approach to disentangle the remaining key processes and factors responsible for these patterns. Biotic factors were investigated using manipulative experiments to uncover ecological processes shaping seagrass ecosystems and to establish the role of sessile epifaunal invertebrates in the structure and function of their host foundation species. Predation pressure, substratum availability and sponge palatability were investigated. I found no evidence that the sponge community of Posidonia australis meadows was driven by fish predation or competition for space. Predator exclusion cages successfully excluded large fish predators but were breached by small organisms. Diurnal fish species seen inside the predator exclusion cages did not readily consume sponge explants and are unlikely to be responsible for the lack of recruits observed in the caging experiment. Shrimps (Palaemon intermedius) were observed interacting with sponge explants. I suggest two possibilities for the lack of recruits in the predator exclusion cages; 1) sessile epifaunal invertebrate larvae or recruits were consumed by meso predators, or 2) predator exclusion cages were inaccessible to larvae due to their limited dispersal capacities. Further research should aim to ascertain whether shrimps are feeding on sponge tissue or on their epifaunal community. Future studies should also aim to uncover the effect of sponge recruitment and larvae dispersal on sponge distribution. Nocturnal palatability tests could reveal predators that were not detected by this study. Larval dispersal is thought to be an important determinant of sponge and ascidian distribution. A two year investigation (chapter five) showed very limited larval recruitment, despite high substratum availability (bottom-up) and the exclusion of large predators (top-down). Given the lack of evidence for predation on sponge explants, I suggest that a restricted larval dispersal range is responsible for this pattern of distribution. In general, sponges have a low probability of long- distance dispersal in comparison to species with planktotrophic, long-lived larvae (Hooper et al., 2002; Hooper & Kennedy, 2002). A lack of connectivity is often seen among sponge populations (Hooper et al., 2002; Hooper & Kennedy, 2002) and can result in distinct sponge aggregations (Fromont et al., 2006). Like sponges, some ascidians are limited in their dispersal abilities and are likely to have highly restricted gene flow (Davis & Butler, 1989). It is worth mentioning that the dispersal ability of sponges is highly variable among species, depending on their reproductive output, the swimming behaviour of their larvae and larval lifespan (Uriz et al., 1998). For instance, a sponge species was found to have a genetically diverse population and was able to colonise over at least tens of kilometres to reach a specific substratum (Davis et al., 1996). The patterns of larval dispersal and recruitment for sponges and ascidians inhabiting seagrass meadows require further

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Chapter 6 investigation, given that these may be significant drivers of spatial distribution. This is a key knowledge gap for the future and has implications for the recovery of this assemblage.

6.2 OVERALL TRENDS This study clearly showed that the sessile epifaunal invertebrate assemblage of seagrass meadows differed greatly from other habitats and is distinct in its species composition and patterns of distribution. This community has formerly been sampled inadequately; relative to other habitats such as reefs, combined with assemblages from other habitats (e.g. seagrass and mangrove sponge species), and, in cases, purposely ignored due to its complex nature. Sponges and ascidians were not mentioned in the draft of the federal government document describing the community, threats and conservation actions for the P. australis meadows of the Manning-Hawkesbury ecoregion (Threatened Species Scientific Committee, 2015). Subsequently, in consultation with the NSW Fisheries Scientific Committee and correspondence with the Federal Department of the Environment, sponges and ascidians were then added to the list of epifauna inhabiting P. australis meadows. This is the first study to establish comprehensive sampling procedures (Chapter 3, Appendix 3), determine patterns of distribution (Chapter 2), and examine the abiotic and biotic factors driving such distribution (Chapter 4, 5) specifically for the sessile epifaunal invertebrate assemblage of seagrass ecosystems (Appendix 1).

6.3 LIMITATIONS Limitations of my study include the sampling of a single seagrass species, P. australis. This species was selected due to its rapid decline and the resulting ‘endangered’ populations in NSW. Jervis Bay was targeted as my sampling location because this embayment contains the largest and most pristine meadows on the south eastern coast of Australia (Kirkman et al., 1995; Meehan & West, 2000). I avoided sampling other locations as preliminary observations confirmed that seagrass meadows were often small, patchy, damaged and therefore inadequate for a baseline study of this assemblage. These disturbed meadows are nonetheless crucial for future work. This study was also limited by the lack of long term monitoring; my focus was largely on spatial variation. This would have provided estimates of temporal scales of variation and would have been useful in determining patterns of sponge recruitment, settlement and growth rate over time. Limitations also originated from the sporadic nature of patterns of distribution. Indeed, most sessile epifaunal invertebrate species were rare and their abundance could not be used to determine correlations with abiotic and biotic factors. Only the abundance of the two most common sponge taxa (Haliclona spp. and Tedania (Tedania) sp.) were analysed.

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6.4 CONSERVATION CHALLENGES The world faces significant environmental and economic impacts from human driven overexploitation, habitat loss and climate change (Butchart et al., 2010; Pimm et al., 2014). As David Suzuki described brilliantly many years ago; ecology and economy are segregated entities in our current society (Suzuki & McConnell, 1997). Ecological challenges rely partly on the valuable expertise of scientists but ultimately hinges on economic factors such as availability of funding, governmental budgets and general state of the economy. Engaged citizens and organisations also have the power to influence governmental decisions, and channel funding towards specific projects or species. However, this approach has often resulted in charismatic species being given priority over less appealing species or ecosystems. Public interest in coastal marine ecosystems is particularly challenging to stimulate as they lack the visibility and accessibility of terrestrial habitats, which is fuelled by an ‘out of sight and out of mind’ mentality. On the other hand, ecosystems such as coral reefs have gained significant recognition over the years through heightened public awareness. Unlike coral reefs, seagrass meadows are still struggling to achieve recognition by the general public, despite their precarious conservation status and that of their inhabitants. Perhaps, advertising the high monetary value of seagrass ecosystems associated with fisheries, carbon sequestration and tourism (Gillanders, 2006; Fourqurean et al., 2012; Cullen-Unsworth et al., 2014) may revitalise the public’s opinion of seagrass and benefit their survival. The second best scenario entails uncovering additional information on seagrass meadows and their associated community. This would further our understanding of seagrass ecological interactions and associated impacts by anthropogenic disturbances. Incorporating ecology into economics is key to facilitating successful conservation efforts. This remains the greatest challenge of our time, especially for marine biologists and ecologists. When it comes to conservation, seagrass ecosystems deserve undoubtedly higher priority than they currently receive. Seagrass meadows are one of the most threatened ecosystems worldwide, with rapid rates of decline in recent years (Waycott et al., 2009). They are crucial to a large number of rare and threatened species, including dwarf seahorses, green sea turtles and dugongs (Short et al., 2011). Human disturbances are the main drivers of global seagrass decline, especially dredging, mooring scars and runoff of pollutants (Reed & Hovel, 2007; Demers et al., 2016). These destructive methods have caused irreversible damage in the past, such as the disappearance of seagrass-associated species (Waycott et al., 2009). Considering that some seagrass species have a slow and unlikely recovery following anthropogenic disturbances (Clarke &

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Kirkman, 1989), conservation and effective management must be given priority over rehabilitation efforts. The effective and sustainable management of threatened ecosystems requires a multi- facetted and comprehensive knowledge of their ecological functions and services. Although aspects of seagrass meadows have been extensively investigated in the past, substantial gaps in the literature remain, especially in regards to the composition, distribution and dynamics of its sessile epifaunal invertebrate assemblage. This is particularly relevant to P. australis, a threatened seagrass species in rapid decline over its range. Although sponges and ascidians are often overlooked due to their taxonomic difficulties, this study found that they play a crucial part in seagrass ecosystem functioning. There has been a worldwide lack of interest towards seagrass-associated sponge assemblages, including areas where sponges have been studied for decades. For instance, despite considerable expertise in Mediterranean reefal sponge taxonomy and ecology, the sponge species inhabiting the Posidonia oceanica meadows of this region remained uninvestigated. A recent internship in Spain aimed at uncovering the identity of this sponge community (Demers unpublished data). Sponge biology is a challenging field mainly due to the arduous process of taxonomic identification and classification (Bergquist, 1978; Wulff, 2001) although significant advances have been made in recent years (Van Soest et al., 2015). Sponge identification is under constant revision, often involves heated debates and requires many years of expertise. There are currently 8,637 valid sponge species with a large number of undescribed and unknown species (Van Soest, 2015). The number of species that are new to science was estimated at 60 % on reefs below 30 m depth, in South East Australia (Roberts & Davis, pers. comm.), and up to 90% in coastal embayments of NSW (Barnes, 2009). However, the challenges associated with sponges should not undermine their ecological importance (Bell et al., 2015). Consideration should be given to sessile epifaunal invertebrates by managers and policy makers. Sponges and ascidians should be included in faunal surveys of marine ecosystems and should be kept separate from other taxa. Efforts should be devoted to correct species identification via the assistance of taxonomic experts. Surveys would benefit from an overview of the assemblage in its entirety. Diversity, abundance and volume should be measured in order to acquire a truthful pattern of distribution. Attention should be paid that the sampling area is sufficiently large given the high occurrence of rare species within this assemblage.

6.5 FUTURE CHALLENGES Future considerations for the research and management of seagrass ecosystems that would address the caveats mentioned above include the acquisition of data on the identification, sampling

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Chapter 6 and long-term monitoring of the sponge and ascidian assemblage inhabiting other seagrass species in multiple embayments along the NSW coast. This would provide a more comprehensive study of this assemblage. The assemblage from different embayments and seagrass species are likely to differ from that of Jervis Bay and new species are likely to be encountered. Temporal variation in this assemblage is currently unknown and would provide important insight into the dynamic nature of this assemblage. The monitoring of sponge individuals over time was attempted in this study, although this was unsuccessful. Relocating individuals was arduous due to the length and density of the seagrass meadow, even though markers were used to facilitate this task. Future work should target the seagrass meadows of Western Australia since they are the largest and most diverse in the world. They are home to 27 seagrass species covering an estimated 20 000 km2 (Department of Fisheries, 2011). The dominant seagrass species, Posidonia sinuosa, is host to a range of unidentified sponge species, mainly (70%) the genus Tethya (Lemmens et al., 1996). It would be beneficial to identify the sessile epifaunal invertebrates inhabiting seagrass meadows from around the world. The patterns of distribution and factors impacting the distribution of the sessile epifaunal invertebrates inhabiting foreign seagrass ecosystems are likely to differ from that found in this study. Importantly, sponges and ascidians that were encountered in the Posidonia meadows of Catalonia, Spain, differed from that encountered in Jervis Bay. The sponge species that occurred in the Catalan Posidonia oceanica meadows lacked spicules and were more abundant than their austral counterparts (Demers unpublished data). Also, the seagrass meadows of the Caribbean were found to host a spongivorous starfish, which played a crucial role in sponge distribution in this system (Wulff, 2008). This predator was not encountered in the seagrass meadows of Jervis Bay, emphasising that ecological systems can differ greatly from one place to another. Further research is needed to uncover the sessile epifaunal invertebrate assemblage of seagrass meadows on an international basis. Future research should also investigate the effect of damaged meadows, prompted by anthropogenic disturbances, on the spatial distribution of seagrass-associated sessile epifaunal invertebrate community. It would be beneficial to compare the seagrass assemblage of Jervis Bay to that of other embayments in NSW, especially that of the ‘endangered’ P. australis populations (Table 6.1). Preliminary sampling revealed that, within the heavily fragmented seagrass of the Sydney region, sponges were infrequent and were not found on P. australis (author’s personal observation). Ascidians were abundant and included Botrylloides leachii, Herdmania grandis, Pyura praeputialis, Styela plicata, Phallusa obesa and an unknown colonial species (author’s personal observation). This unknown ascidian was covering large area of seagrass fronds and its impact on seagrass ecosystem function is currently unknown (Figure 6.1). Sessile epifaunal

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Chapter 6 invertebrates were found to negatively affect seagrass while under anthropogenic stress (Archer et al., 2015). This is likely to be the case within the highly disturbed meadows of NSW. Manipulative experiments such as sponge transplants from pristine to disturbed meadows would provide crucial information on the effect of human disturbances on seagrass-associated fauna. The protection of disturbed seagrass meadows has been a popular trend worldwide in an attempt to facilitate global seagrass recovery. For instance, anchoring has been prohibited in some highly damaged meadows of the Sydney region, prompting the need for seagrass-friendly moorings. A study investigating the impact of boat moorings on seagrass meadows was published during the course of this PhD (Demers et al., 2013; Appendix 2). This study showed that the installation of seagrass-friendly moorings in areas previously damaged by conventional moorings allowed for seagrass recovery. However, it is unknown whether the sessile epifaunal invertebrate community can also recolonise these damaged areas once disturbance ceases. Further research should monitor these newly established patches of seagrass for presence of sponges and ascidians to determine if this assemblage is a useful indicator of habitat recovery following anthropogenic disturbances. This would further our understanding of the timeline required to regain ecosystem function and services in rehabilitated seagrass habitats.

Table 6.1: Area (ha) of endangered P. australis populations in NSW, Australia. This includes exclusive P. australis meadows as well as a combination of P. australis and other seagrass species. Note that these values were acquired from maps that were based on air photos and field surveys dated between 2000 and 2009 (NSW Department of Primary Industries, 2010).

Embayment Area of endangered P. australis (ha) Lake Macquarie 300 Pittwater 580 Brisbane Waters 700 Sydney harbour 100 Botany Bay 960 Port Hacking 340

Future research should use manipulative experiments to investigate the factors responsible for the distribution of sponges and ascidians inhabiting seagrass meadows. Factors investigated in this study included small-scale seagrass attributes and large-scale seascape patterns of the meadow. Manipulative experiments could be used to validate these patterns of distribution. For instance, small-scale biogenic characteristics could be tested by relocating individual sponges to seagrass meadows of varying canopy heights and densities, whilst monitoring their growth rate overtime.

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Artificial meadows could also be used to achieve specific seagrass characteristics (Virnstein & Curran, 1986). Testing the effect of low seagrass density on the distribution of sessile epifaunal invertebrates would reveal if this assemblage can survive in the low seagrass cover associated with disturbed meadows. In addition, sponge species suspected to host photosymbionts could be identified using an underwater fluorometer (dive-PAM). Their ideal level of irradiance could be determined by transplanting individual sponges to artificial meadows located at varying depths. It was predicted that over 65% of temperate reef sponge species possess photosymbionts (Roberts et al., 1999). Describing these species-specific patterns would broaden our understanding of this ecosystem given that our knowledge of seagrass-associated species is limited. Large-scale seascape patterns of the meadow could also be tested in a similar manner. Artificial seagrass meadows located in areas with extensive neighbouring reefs versus areas with minimal neighbouring reefs could be used to monitor sponge and ascidian settlement rate, followed by growth rate overtime. Perhaps, using molecular techniques to better understand connectivity among sponge populations (Davis et al., 1996) would provide important insight in the dynamics of this assemblage. In situ manipulative experiments should be favoured over laboratory-based experiments due to the high sponge mortality rate often associated with sponge captivity. It is crucial to establish the drivers of distribution of this assemblage and at which spatial scale they occur. Indeed, conservation efforts should concentrate on highly speciose seagrass meadows in order to retain the greatest amount of biodiversity. Additional research should also focus on investigating sponge predation and palatability. A laboratory experiment would be ideal to determine the nature of the interactions between sponges and potential predators. The palatability of seagrass-associated sponges may also rely on spicule content. The level of deterrence of each species could be determined using laboratory feeding assays. Investigating the cleaning ability of shrimps and its effect on sponge growth would provide important information on the sponge-shrimp-fish-seagrass relationships. Understanding the biotic factors driving the distribution of this assemblage would clarify its role in the seagrass trophic network, which could have important ramifications for this threatened ecosystem.

6.6 CONCLUSIONS In conclusion, this is the first study in the world to 1) establish comprehensive sampling procedures, 2) identify the species present, 3) determine patterns of distribution, 4) examine the abiotic and biotic factors driving such distribution, and 5) use manipulative experiments to understand the key processes influencing this community, specifically for the sessile epifaunal invertebrate assemblage of seagrass ecosystems. I successfully studied one of the phyla that is most arduous to identify worldwide and, despite the limited amount of previous research on this topic, I

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Chapter 6 succeeded in establishing their distribution patterns and the processes responsible for such patterns. I showed that sponges and ascidians differed greatly in their patterns of distribution with distinct drivers and scales over which patterns were apparent, suggesting that these phyla should be considered separately in any analysis. This study uncovered the previously unknown sessile epifaunal invertebrate assemblage inhabiting the most threatened ecosystem worldwide. This extensive assemblage was found to rely heavily on seagrass and is therefore likely prone to disturbances impacting its host habitat. Examining the response of sessile epifaunal invertebrates to the degradation of their seagrass habitat remains a key challenge for the future.

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Figure 6.1: Unknown ascidian species observed in the P. australis seagrass meadows of Maianbar, Port Hacking, NSW. Photograph of (A) an adult specimen covering the entire seagrass frond and (B) close-up view showing common exhalant siphons.

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APPENDIX 1

A guide to sponge and ascidian identification

Appendix 1

Table A1.1: Summary of Sponge ID code, binomial name, expert that examined and confirmed the identification, photo, presence of spicule and specimen/voucher availability. Species were arranged by taxonomic order according to the new organisation proposed by Morrow and Cárdenas (2015). Australian Code Binomial Examined by Photo Spicule Specimen Museum Voucher # DPu Chelonaplysilla sp. Lisa Goudie Available Not present Available BlueS Euryspongia sp. Lisa Goudie Available Not present Available Thorectidae family Lisa Goudie B/WS Available Not present Available sp.1 (Jane Fromont) Thorectidae family HPro Lisa Goudie Available Not present Available sp.2 Thorectidae cf Hyrtios RWV Lisa Goudie Available Not present Not available sp.3 Callyspongia FibPink Lisa Goudie Available Present Available (Callyspongia) sp.2 Callyspongia Lisa Goudie LS Available Present Available AM Z.7216 (Toxochalina) sp.1 (Jane Fromont) WRH Chalinula sp. Lisa Goudie Available Present Available YS Haliclona spp. (2) Jane Fromont Available Present Available AM Z.7218 GRH Stelletta sp. Lisa Goudie Available Present Available Chondropsidae family PS Lisa Goudie Available Not present Available sp.4 WS Chondropsis sp. Lisa Goudie Available Present Available BYWS Phoriospongia sp.1 Jane Fromont Available Present Available AM Z.7219 NBE Phoriospongia sp.2 Lisa Goudie Available Present Available Lissodendoryx Lisa Goudie GBlue Available Present Not available (Lissodendoryx) sp. (Jane Fromont) Lissodendoryx BLOS Lisa Goudie Available Present Available (Waldoschmittia) sp. OS Tedania (Tedania) sp. Jane Fromont Available Present Available AM Z.7220 PinkS Tethya cf bergquistae Lisa Goudie Available Unknown Not available SS Halichondria sp. Lisa Goudie Available Present Not available AM Z.7217 Unidentified B/BS Lisa Goudie available Present Available Demospongia sp.1 Unidentified B/navB Lisa Goudie available Not present Not available Demospongia sp.2 Unidentified Not Y/B SD Lisa Goudie Present Available Demospongia sp.3 available

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A1.1 SPONGE IDENTIFICATION Scientific name: Chelonaplysilla sp.

Code: DP

Morphology Category: Encrusting

Spicule Morphology: Nitric acid and Bleach (NaOH) revealed no spicules nor sponging fibres. Although 1 specimen showed mucus-like features (long white hair which seem to be coming from the shell the sponge was encrusted on)

Physical Description: • Irregular shape forming mostly on polychaete worms. • Surface uneven with arms/projections giving it a spiky appearance and no visible openings • Elastic/“spongy” consistency

Size: variable

Colour: Dark Purple to black

Habitat: Polychaete worms

Figure A1.1.1: Chelonaplysilla sp.

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Appendix 1

Scientific name: Euryspongia sp.

Code: BlueS

Morphology Category: Solid/massive

Spicule Morphology: Sponge tissue digested in salt water releasing a strong smell and leaving a branching skeleton and a blue slime. Digestion by Nitric acid and bleach (NaOH) showed no spicules and the skeleton entirely dissolved each time. The skeleton is likely to be collagen.

Physical Description: • Irregular shape with multiple postules • Surface uneven and soft with numerous randomly positioned large openings • Elastic/“spongy” consistency

Size: 10s cms

Colour: Bluish purple with distinctive white spots all over the individual

Habitat: coralline algae

Figure A1.1.2: Euryspongia sp.

134

Appendix 1

Scientific name: Thorectidae family sp.1

Code: B/WS

Morphology Category: Solid/massive and encrusting

Spicule Morphology: This is a fibre sponge (Dictyoceratida) so has foreign material and spicules but none of its own. Nitric acid revealed no spicules. Bleach (NaOH) revealed multiple spicules which do not belong to this sponge.

Physical Description: • Irregular shape (elongated, encrusting) • Surface smooth and soft (velvet appearance) with few small openings • Elastic/“spongy” consistency • Glossy outside • Reticulate fibre skeleton with primary fibres cored, secondary fibres uncored. Fibres laminated.

Size: less than 10 cm

Colour: Black with white inner tissue

Habitat: Seagrass sheath, coralline algae

135

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A B

Figure A1.1.3: Thorectidae family sp.1

136

Appendix 1

Scientific name: Thorectidae family sp.2

Code: High Pro

Morphology Categorie: Solid/Massive vertically elongated

Spicule Morphology: Nitric acid revealed no spicules.

Physical Description: • Multiple arms branching vertically • Surface rough and uneven • hard consistency • Characteristics similar to PS

Size: 5-20 cms high

Colour: Brown or black

Habitat: Coralline algae, located near reefs (It has been observed in great quantity on the reef).

Figure A1.1.4: Thorectidae family sp.2

137

Appendix 1

Scientific name: Thorectidae cf Hyrtios sp.3

Code: RWV

Morphology Category: Solid/massive

Spicule Morphology: Nitric acid and Bleach (NaOH) revealed no spicules nor sponging fibre. Sand abundant

Physical Description: • Irregular shape forming at the sheath and extending along the leaves with arms/projections reaching other leaves. • Surface uneven with vein-like texture and small openings • Elastic/“spongy” consistency

Size: 10s cms

Colour: Red with white veins

Habitat: Seagrass sheath and leaf

138

Appendix 1

Figure A1.1.5: Thorectidae cf Hyrtios sp.3

139

Appendix 1

Scientific name: Callyspongia (Callyspongia) sp.2

Code: FibPink

Morphology Categorie: Solid/Massive

Spicule Morphology: • ‘Curved’ Oxea (megasclere diactional, monoaxon, diactine) of 83 μm.

Physical Description: • Visible fibre in the sponge tissue and elongated shape • Surface even and smooth • Elastic/“spongy” consistency

Size: 10 cms

Colour: Pink, beige or orange

Habitat: free-living or seagrass sheath

Figure A1.1.6: Callyspongia (Callyspongia) sp.2

140

Appendix 1

Scientific name: Callyspongia (Toxochalina) sp.1 (Potentially 3 species of Callyspongidae, Callyspongia sp. See individual description below)

Code: LS

Morphology Category: Solid/massive

Physical Description: • Irregular shape forming at the sheath and extending along the leaves with arms/projections reaching other leaves. • Surface smooth and soft with a few small openings • Elastic/“spongy” consistency • Internal pattern: web-like, little holes, honeycombs

Colour: Lavender (purple), white, pink or black

Habitat: Seagrass sheath or leaves, coralline algae and tube worm

Species 1. Physical Description: Black in colour, reticulate fibre skeleton centrally cored with oxeas, primary & secondary fibres are multispicular, no tertiary network.

Spicule Morphology: • Oxea (diactonal) of 130 x 3 μm. Small & thin oxeas. Abundant

Species 2. Physical Description: White colour, primary fibres cored by 2-4 oxeas, secondary fibres unispicular.

Spicule Morphology: • ‘Curved’ Oxea (megasclere diactonal) of short oxeas 100 x 4 μm and thinner forms. Ends slightly rounded.

Species 3. Physical Description: Purple in colour, reticulate fibre skeleton, primary fibres multispicular, secondary fibres unispicular.

Spicule Morphology: • Oxeas 40 x 2 μm.

141

Appendix 1

Figure A1.1.7: Callyspongia (Toxochalina) sp.1

142

Appendix 1

Scientific name: Chalinula sp.

Code: WRH

Morphology Categorie: Solid/Massive

Spicule Morphology: • Oxea (diactional, monoaxon, diactine) of 83 μm. Moderately abundant.

Physical Description: • Irregular shape forming at the sheath with large openings. • Surface uneven, rough and hard (hard to cut)

Size: 10 cms

Colour: White

Habitat: Seagrass sheath & coralline algae

143

Appendix 1

Figure A1.1.8: Chalinula sp.

144

Appendix 1

Scientific name: Chalinidae Haliclona (Gellius) sp.1. Chalinidae ?Haliclona (Haliclona) sp.2.

Code: YSP

Morphology Category: Solid/massive and encrusting

Spicule Morphology: • Oxea (diactonal) of 140 x 4 μm. • Sigmas C & comma 30 μm, rhaphides in trichodragmata, 2 sizes 125/30 μm

Physical Description: • Irregular shape (mostly round, elongated or encrusting) • Surface smooth and soft (velvet appearance) with few/numerous small openings • Elastic/“spongy” consistency • Internal pattern: web-like, little holes (porous), honeycombs • Regular reticulate skeleton of oxeas with thin fibre sheath

Size: variable (few cms to 10s cms)

Colour: Yellow, light yellow

Habitat: tube worms, seagrass sheath, ascidians

Chalinidae ?Haliclona (Haliclona) Irregular plumose to plumoreticulate tracts, faint fibre at nodes of reticulation, oxeas long, think 320x5 μm, also short forms 170x3 μm. Unusual because of apparent 2 size categories of oxeas. Could not be determined if shorter category are localised at the surface, hence tentative genus designation.

145

Appendix 1

Figure A1.1.9: Chalinidae Haliclona spp.

146

Appendix 1

Scientific name: Stelletta sp.

Code: GRH

Morphology Category: Solid/massive

Spicule Morphology: • Oxea (diactional, monoaxon, diactine) of 667 μm. Abundant • Style (diactional, monoaxon, diactine) of 500 μm. Less abundant • Triaene (tetractinal, tetraxon) of 800 μm. Rare ± 5

Physical Description: • Irregular shape • Surface rough (sand paper appearance) with a few small openings • Hard consistency

Size: variable (few cms to 10s cms)

Colour: Gold with pinkish shading

Habitat: coralline algae

Figure A1.1.10: Stelletta sp.

147

Appendix 1

Scientific name: Chondropsidae family sp.4

Code: PS

Morphology Category: Sheath-like and encrusting

Spicule Morphology: Have not been sampled/rare species

Physical Description: • Irregular shape -mostly wave-like thick sheaths in rows or encrusting • Surface smooth (sand paper appearance) with numerous small/large openings • Elastic/“spongy” consistency

Size: variable (few cms to 10s cms)

Colour: Purple to light brown

Habitat: on coralline algae

Figure A1.1.11: Chondropsidae family sp.4

148

Appendix 1

Scientific name: Chondropsis sp.

Code: WS

Morphology Category: Encrusting

Spicule Morphology: • Strongyle (diactional, monoaxon, diactine) of 140 μm. • Bleach (NaOH) showed the presence of Triod of 83 μm.

Physical Description: • encrusting only • Sometimes have siphons protruding • Sometimes is almost completely covered by sand/substratum • Surface smooth or uneven and soft with small openings • Elastic/“spongy” consistency • Thin with fragile appearance

Size: few cms

Colour: White

Habitat: on coralline algae mostly

Figure A1.1.12: Chondropsis sp.

149

Appendix 1

Scientific name: Chondropsidae Phoriospongia sp.1

Code: BYWS

Morphology Category: Small, Solid/massive

Spicule Morphology: • Tylostyle (monactonal) of 205-230 x 1μm. very thin, often with blackened axial canal.

Physical Description: • Irregular shape – form irregular mass at the base of 1 or a few shoots (growing over and joining sheaths together) with arms/projections extending to leaf base • Surface smooth or uneven and soft with few/numerous randomly positioned large openings (oscules). • elastic/“spongy” consistency • Sparse fibres loosely cored with tylostyles • Spicule brushes at surface in 3-4 spicule fans

Size: variable (few cms to 10s cms)

Colour: • Yellow, light yellow to brown, light brown, sometimes both (brown fading to yellow) • Distinctive white spots all over the individual

Habitat: mostly on seagrass sheath

150

Appendix 1

Two colour morphs

B C

Figure A1.1.13: Phoriospongia sp.1

151

Appendix 1

Scientific name: Phoriospongia sp.2

Code: NBE

Morphology Category: Encrusting

Spicule Morphology: • Unknown Tylostyle with flat end (diactional, monoaxon, diactine) of 167 μm.

Physical Description: • Encrusting over shells • Surface smooth and soft with no openings • Elastic/“spongy” consistency • Internal pattern: web-like, little holes, honeycombs

Size: few cms

Colour: Navy Blue

Habitat: On hard substratum such as shell/tube worms

Figure A1.1.14: Phoriospongia sp.2

152

Appendix 1

Scientific name: Coelosphaeridae, Lissodendoryx (Lissodendoryx) sp.

Code: GBlue

Morphology Categorie: Encrusting

Spicule Morphology: • Tylote (diactonal) 175-215 x 5 μm. Most abundant • Style (diactonal, monoaxon, monoactine) of 155 x 5 μm. Abundant

Microscleres: • Isochelae, arcuate 2 sizes 23/15 μm. Abundant. • Sigmas 2 sizes 30/15 μm. Less abundant

Size: few cms

Colour: Greyish Blue

Habitat: coralline algae

Figure A1.1.15: Coelosphaeridae, Lissodendoryx (Lissodendoryx) sp.

153

Appendix 1

Scientific name: Lissodendoryx (Waldoschmittia) sp. Code: BLOS Morphology Categorie: Solid/Massive Spicule Morphology: • Slightly ‘curved’ Tylote (diactional, monoaxon, diactine) of 200 μm. Most abundant • Sigma 20 μm. Rare

Physical Description: • Surface smooth with a brain-like pattern • Rounded protrusions

Size: variable

Colour: Light orange or yellow. Different colour than OS.

Habitat: on mussel clumps

Figure A1.1.16: Lissodendoryx (Waldoschmittia) sp.

154

Appendix 1

Scientific name: Tedaniidae Tedania (Tedania) sp.

Code :OS

Morphology Category: Solid/massive and encrusting

Spicule Morphology: • Tylote (diactonal) of 165 x 3 μm. Very finely microspined, but this should be checked with SEM. • Onychaete of 2 sizes 95/55 μm.

Physical Description: • Irregular shape (mostly round, elongated or encrusting) • Surface smooth and soft (velvet appearance) with few/numerous small/large openings • Elastic/“spongy” consistency • Internal pattern: web-like, little holes (porous), honeycombs • Plumoreticulate skeleton with fibre development centrally cored

Size: variable (few cms to 10s cms)

Colour: Orange

Habitat: drifting, seagrass sheath, on coralline algae, other sponges (e.g. YSP)

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Figure A1.1.17: Tedaniidae Tedania (Tedania) sp.

156

Appendix 1

Scientific name: Tethya cf bergquistae

Code: PinkS

Morphology Category: Solid/massive

Spicule Morphology: Have not been sampled/rare species

Physical Description: • round shape • Surface uneven (small circular lumps) and soft with few small openings • Elastic/“spongy” consistency • Internal pattern: little holes

Size: few cms

Colour: Bright pink

Habitat: coralline algae

Figure A1.1.18: Tethya cf bergquistae

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Appendix 1

Scientific name: Halichondria sp.

Code: SS

Morphology Category: Solid/massive and encrusting

Spicule Morphology: • ‘Curved’ Oxea (megasclere diactional, monoaxon, diactine) of 125 μm.

Physical Description: • Irregular shape (elongated, encrusting) • Surface smooth and soft (velvet appearance) with large openings • Elastic/“spongy” consistency • Thin with fragile appearance

Size: variable (few cms to 10s cms)

Colour: Sandy/beige colour

Habitat: Seagrass sheath, coralline algae

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Figure A1.1.19: Halichondria sp.

159

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Scientific name: Unidentified Demospongia sp.1

Code: B/B

Morphology Category: Solid/Massive

Spicule Morphology: • Oxea (diactional, monoaxon, diactine) of 100 μm. • Style (diactional, monoaxon, diactine) of 250 μm. Less abundant.

Physical Description: • The branching type has an irregular shape forming at the sheath and extending along the leaves with arms/projections reaching other leaves. • The encrusting type has an irregular shape forming on polychaete worms and shells usually in combination with other sponges • Surface uneven • Very elastic/“spongy” consistency (hard to cut)

Size: 10 cms

Colour: Black

Habitat: Seagrass sheath and coralline algae, tube worms and shells

Figure A1.1.20: Unidentified Demospongia sp.1

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Scientific name: Unidentified Demospongia sp.2

Code: B/Navy Blue

Morphology Category: encrusting

Spicule Morphology: Nitric acid and Bleach (NaOH) revealed no spicules.

Physical Description: • Encrusting • Surface uneven with few small openings • Elastic/“spongy” consistency

Size: few cms

Colour: Dark blue or black with navy blue inner tissue

Habitat: coralline algae mostly

Figure A1.1.21: Unidentified Demospongia sp.2

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Scientific name: Unidentified Demospongia sp.3

Code: Y/B SD

Spicule Morphology: • Slightly ‘curved’ Oxea (megasclere diactional, monoaxon, diactine) of 143 μm.

Habitat: free-living

REFERENCE

Morrow, C. and Cárdenas, P. (2015). Proposal for a revised classification of the Demospongiae (Porifera). Frontiers in Zoology. 12(7), 1-27.

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A1.2 ASCIDIAN IDENTIFICATION Scientific name: Botrylloides leachii

Physical Description: • Rows of siphons visible

• Elastic/“spongy” consistency

Size: 5 cms high

Colour: Can be any colour from white to purple to orange to blue.

Figure A1.2.1: Botrylloides leachii

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Scientific name: Cnemidocarpa pedata and associate Halisarca laxus sponge

Code: Purple Asci

Physical Description: • Solitary ascidian found with sponge

• The purple colour comes from the sponge

Size: 20s cms

Colour: Pinkish purple with yellow shading and yellow tips, Darker purple openings

Figure A1.2.2: Cnemidocarpa pedata and associate Halisarca laxus sponge

164

Appendix 1

Scientific name: Didemnum sp.

Code: ALY

Physical Description: • Located along the sheath and leaf

• Narrow band with small openings/lumps

• Elastic/“spongy” consistency

Size: Few cms long

Colour: Cream colour or orange

Figure A1.2.3: Didemnum sp.

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Appendix 1

Scientific name: Distaplia c.f. stylifera

Code: RPP

Physical Description: • Jelly-like texture

• Fragile looking

• Rounded shape

Size: small

Colour: Bright red/orange with lighter tips

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Appendix 1

Scientific name: Herdmania grandis

Code: ART

Physical Description: • Round body covered with epiphyte • Siphons are wrinkly and have a red outer ridge

Size: 10 cms

Colour: White body with red tip siphons

A B

Figure A1.2.4: Herdmania grandis

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Scientific name: Pyura praeputialis

Code: AWT or Cunje

Physical Description: • Body enclosed within hard external shell covered with epiphyte positioned mostly below the substratum (sand)

• Siphons have deep wrinkles

• Usually found buried in the substratum with only the siphon still visible

Size: few cm to10s cms (smaller than ART)

Colour: White A

Figure A1.2.5: Pyura praeputialis

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Appendix 1

Scientific name: Pyura spinifera with associate Halisarca laxus sponge.

Code: Purple Stalk

Physical Description: • Stalked ascidian with an uneven body with protrusions

• found next to Cnemidocarpa pedata

Size: 30s cms

Colour: Pinkish purple with yellow shading and yellow tips, Darker purple openings

Figure A1.2.6: Pyura spinifera with associate Halisarca laxus sponge

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Appendix 1

Scientific name: Styela plicata

Code: O/B asci

Physical Description: • Covered by a thick epiphyte layer, mostly coralline algae –very hard to distinguish.

• flattened shape

Size: small in comparison with other ascidians

Colour: 4 orange bands at the tips of each siphon.

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A1.3 BRYOZOAN IDENTIFICATION Scientific name: Unidentified Bryozoan

Code: Bryozoan

Physical Description: • Thin strip located on shells

Size: Few cms long and mm thin

Colour: Pinkish orange

Figure A1.3.1: Unidentified Bryozoan

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Chapter 3

APPENDIX 2

A comparison of the impact of ‘seagrass-friendly’ boat mooring systems on Posidonia australis.

Final version: Demers M-C.A., Davis A.R. and Knott N.A. (2013) A comparison of the impact of ‘seagrass- friendly’ boat mooring systems on Posidonia australis. Marine Environmental Research, 83, 54-62.

Appendix 2

A2.1 ABSTRACT

Permanent boat moorings have contributed to the decline of seagrasses worldwide, prompting the development of ‘seagrass-friendly’ moorings. I contrasted seagrass cover and density (predominantly Posidonia australis) in the vicinity of three mooring types and nearby reference areas lacking moorings in Jervis Bay, Australia. I examined two types of ‘seagrass-friendly’ mooring and a conventional ‘swing’ mooring. ‘Swing’ moorings produced significant seagrass scour, denuding patches of ∼9 m radius. Seagrass-friendly ‘cyclone’ moorings produced extensive denuded patches (average radius of ∼18 m). Seagrass-friendly ‘screw’ moorings, conversely, had similar seagrass cover to nearby reference areas. My findings reinforce previous work highlighting the negative effects of ‘swing’ and ‘cyclone’ moorings. In contrast, the previously unstudied ‘screw’ moorings were highly effective. I conclude that regular maintenance of moorings and the monitoring of surrounding seagrass are required to ensure that ‘seagrass-friendly’ moorings are operating effectively. This is important, as following damage Posidonia will take many decades to recover.

Keywords: boat mooring system, disturbance, environmental impact, mechanical damage, Posidonia australis, seagrass, ‘seagrass-friendly’ moorings.

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A2.2 INTRODUCTION Seagrass meadows are considered a pivotal marine habitat and provide important ecosystem services (Costanza et al., 1997). Seagrass meadows make significant contributions to coastal productivity worldwide (Orth et al., 1984) and their primary production ranks amongst the highest recorded for marine ecosystems (Hillman et al., 1989). They also support excessively high levels of biodiversity (Hemminga & Duarte, 2000) providing habitat, food and shelter to a diverse array of organisms (Beck et al., 2001). Seagrasses are particularly important in sustaining commercial and recreational fisheries (Heck et al., 2003; Gillanders, 2006) and it is likely that damage to seagrass will diminish stocks of important commercial and recreational fish, molluscs and crustaceans (Bell & Pollard, 1989; Butler & Jernakoff, 1999; McArthur & Boland, 2006). Seagrass meadows also support a large number of rare and threatened species, many of which relying on seagrass habitat for survival (Short et al., 2011). Despite the importance of seagrass, it has been in significant decline worldwide and this appears to have accelerated over recent decades (Short & Wyllie-Echeverria, 1996; Waycott et al., 2009). One-third of the world’s seagrass species are in decline with 10 species having a high risk of extinction (Short et al., 2011). Between 1985 and 1995, approximately 1,200,000 ha of seagrass meadow have been lost globally (Short and Wyllie-Echeverria, 2000). In Australia, seagrass meadows have suffered extensive declines in several states in the past (Shepherd et al., 1989; Walker & McComb, 1992). Seagrass loss has been attributed to a broad spectrum of anthropogenic and natural disturbances (Duarte, 2002) with direct mechanical damage one of the key anthropogenic disturbances (Short & Wyllie-Echeverria, 1996; Duarte, 2002; Ceccherelli et al., 2007; Waycott et al., 2009). Permanent boat moorings are one of the main causes of mechanical disturbance to seagrass, almost always producing scoured areas within seagrass meadows (Walker et al., 1989; Hastings et al., 1995; Montefalcone et al., 2008). These moorings are often highly concentrated across many of the world’s embayments, especially on densely inhabited coastlines, and so represent a large-scale press disturbance (Bender et al., 1984). These impacts will likely increase with the need for more vessel moorings worldwide (Duarte et al., 2008). This threat is heightened as both seagrasses and vessel anchorages favour locations with reduced water movement and wave- action, hence the siting of mooring locations is often in places where seagrass occurs. The state of New South Wales has recognised these declines and has sought to counteract seagrass loss with legislative protection under the NSW Fisheries Management Act 1994. This legislation states that seagrass may not be cut, removed, damaged or destroyed except under the authority of a permit system which is strictly managed (NSW Department of Primary Industries, 2010). This kind of

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Appendix 2 legislative protection has resulted in the need to develop ‘seagrass-friendly’ moorings and still provide mooring opportunities for the burgeoning recreational boating community. Several ‘seagrass-friendly’ mooring systems have been developed worldwide. Nevertheless, to date, I am aware of just two published assessments of the effectiveness of purported ‘seagrass- friendly’ moorings, both of these on ‘cyclone’ moorings (Walker et al., 1989; Hastings et al., 1995). This is surprising as the ability of the mooring systems not to harm seagrass is central to their intended use and without an appropriate assessment it would seem that the claim of ‘seagrass- friendly’ moorings remains unsubstantiated. To provide moorings for the growing numbers of vessels in Jervis Bay (Australia), the Jervis Bay Marine Park in conjunction with the Southern Rivers Catchment Management Authority trialled the use of ‘seagrass-friendly’ ‘screw’ moorings. Several private ‘screw’ moorings were also installed. All of these moorings were installed between January 2008 and July 2009 (Frances Clements, Jervis Bay Marine Park Permitting Officer, pers. comm.). Also present within the marine park were a series of ‘cyclone’ moorings that are also considered ‘seagrass-friendly’ (Walker et al., 1989); installed by private mooring owners. This study presents a quantitative assessment of the effectiveness of the newly designed and never previously assessed ‘seagrass- friendly’ ‘screw’ moorings and older ‘seagrass-friendly’ ‘cyclone’ moorings. Specifically, I compared patterns of seagrass density and cover (Posidonia australis, Halophila ovalis and Zostera spp.) surrounding these ‘seagrass-friendly’ designs (Figure A2.1) relative to conventional ‘swing’ moorings and reference areas lacking moorings. I tested the general prediction that both kinds of ‘seagrass-friendly’ moorings (‘screw’ and ‘cyclone’) would have greater densities and coverage of seagrass in their vicinity than the conventional ‘swing’ moorings. Reference areas were used in order to quantify the effectiveness of the moorings relative to the background conditions of the seagrass which can differ due to environmental conditions and past anthropogenic disturbance. This represents the first published study to examine the effect of ‘screw’ moorings on seagrass condition.

175

Appendix 2

Figure A2.1: Schematic representation of a typical (A) ‘screw’ mooring system, (B) ‘swing’ mooring system and (C) ‘cyclone’ mooring system.

176

Appendix 2

A2.3 MATERIALS AND METHODS

A2.3.1 Study area Seagrass was sampled adjacent to three types of moorings and reference areas in Jervis Bay Marine Park. Jervis Bay is an open embayment of approximately 12,400 ha situated on the southeastern Australian coast. P. australis is the prevalent seagrass species in Jervis Bay with extensive meadows totalling 5.7 km2 in depths of 2-10 m (West, 1990). These meadows constitute the largest continuous areas of this species along Australia’s south eastern coast (Meehan & West, 2000), and are considered some of the most pristine seagrass meadows on this coastline (West et al., 1989; Kirkman et al., 1995). Callala Bay (35°00’S 150°43’E) and Bindijine Beach (35°03’S 150°46’E) (See Figure 1 in Fyfe & Davis, 2007) possess permanent boat moorings and were selected as study areas. At the time of the study, Callala Bay had approximately five ‘screw’ moorings, five ‘cyclone’ moorings and 60 ‘swing’ moorings (Frances Clements, Jervis Bay Marine Park pers. comm). Moorings have been present in Callala Bay for over 30 years and almost all of them are located in P. australis meadow, over a depth range of 3 to 6 m. Previous research has shown that the seagrass at Callala Bay has suffered substantial damage from boat moorings (Figure A2.2A, Crawford, 2003). The level of damage done to the seagrass at Callala Bay presented a complex background over which to objectively assess the effects of permanent mooring systems. In contrast, Bindijine Beach had three ‘screw’ moorings installed in June 2009 (Frances Clements, Jervis Bay Marine Park, pers. comm.). This location has virtually no history of boat mooring (Crawford, 2003) and the seagrass meadow was relatively pristine.

177

Appendix 2

Figure A2.2: (A) Aerial photograph of the mooring area at Callala Bay showing distinctive circular areas denuded of seagrass, (B) underwater photograph of the ring of bare seagrass observed around ‘screw’ moorings at Bindijine Beach.

178

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A2.3.2 Seagrass species The seagrass species present in the meadows of Jervis Bay include P. australis, Halophila ovalis and Zostera spp. P. australis Hook.f. is a slow growing horizontal spreader with slow rhizome expansion and colonisation rates (Gobert et al., 2006). For this reason, Posidonia does not generally recover once disturbed (Clarke & Kirkman, 1989) or recovers very slowly (Meehan & West, 2000). Halophila ovalis (R.Br.) Hook. and the taxonomically difficult to separate Zostera spp. are pioneer species of smaller size and rapid growth rates (Birch & Birch, 1984; Marbá & Duarte, 1998). They have the ability to recolonise disturbed areas and may initiate a sequence of succession (Clarke & Kirkman, 1989; Duarte et al., 2006; Montefalcone et al., 2010). This implies that following a disturbance generated by moorings, if the direct scouring impact is removed, the area is more likely to be re-colonised by pioneer species than P. australis. I predicted greater densities and cover of these pioneer species in areas that had suffered previous scour.

A2.3.3 Sampling method I sampled seagrass density and cover surrounding each mooring type and in reference areas. This included a reference area, a ‘screw’ and a ‘swing’ mooring at two sites at Callala Bay; a reference area and a ‘cyclone’ mooring at three sites at Callala Bay; and a reference area and a ‘screw’ mooring at two sites at Bindijine Beach. Reference areas for each site were located at least 10 m from existing moorings over similar depths and conditions to the adjacent mooring(s). This reduced the spatial variation in seagrass density and cover with which we had to contend. At Callala Bay, the ‘swing’ and ‘screw’ moorings (3-5 m) were shallower than the ‘cyclone’ moorings (5-6 m). Hence, I tested the effectiveness of the two ‘seagrass-friendly’ mooring types separately and sampled separate sets of reference areas. At Bindijine Beach the ‘screw’ moorings were at a depth of 3 m. Seagrass density was quantified within 0.25 m2 quadrats (0.5 m x 0.5 m) at three distances (0-2 m, 3-5 m and 6-8 m) along two transect lines from the centre of each mooring or reference area. I sampled three distances to assess whether impacts diminish from the centre of each mooring. Two quadrats were placed haphazardly within 1 m of the transect line at each of the three distances. Hence, at each mooring, I examined 12 quadrats, four at each distance. Within each quadrat the number of P. australis shoots, consisting of one to several leaf-blades joined at the base, were counted by divers on SCUBA. Shoots with at least 50 % of their leaf sheath located inside the quadrat were included. I sampled from the centre of each mooring; for the ‘cyclone’ mooring system this was identified as the mooring’s central ring, which connects all three ground chains to a riser chain (Figure A2.1).

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Appendix 2

Seagrass cover was also estimated using point counts along the same transect lines. I recorded the nature of the substratum (seagrass or sediment) at 10 cm intervals for 8 m along each transect. The occurrence of any component of seagrass, such as root, rhizome, stem, leaf sheath and leaf-blade under each point on the tape was recorded as seagrass cover. I distinguished among all seagrass species, counting them separately. Additional information I recorded included the GPS coordinates of each mooring and reference area, the height of the base of each ‘screw’ mooring, length of the chain for ‘swing’ and ‘cyclone’ moorings, as well as the radius of the annuli of scoured seagrass surrounding each mooring. I characterised the extent of scoured seagrass as the area of substratum with <5 % seagrass cover.

A2.3.4 Statistical analyses Analysis of variance (ANOVA) was used to test the stated hypotheses regarding the density and cover of seagrass species for each of the mooring comparisons. Three factors were analysed: treatment (mooring type) was a fixed orthogonal factor with three levels (reference, ‘screw’, ‘swing’) at Callala Bay, two levels (reference, ‘cyclone’) at Callala Bay and two levels (reference, ‘screw’) at Bindijine Beach; Site was a random orthogonal factor with three levels for the ‘cyclone’ moorings and two levels for the other comparisons; while distance was a fixed orthogonal factor with three levels (0-2 m, 3-5 m and 6-8 m). The seagrass density values were expressed as percentages of the maximum seagrass density recorded at the reference area for each site (n = 4). A different maximum seagrass density value was used for each analysis, consisting of ‘screw’ and ‘swing’ moorings at Callala Bay, ‘cyclone’ moorings at Callala Bay and ‘screw’ moorings at Bindijine Beach. I standardized the density estimates in this way to account for differences in seagrass density across reference areas at different depths, and mooring histories. I estimated the mean seagrass cover for each distance (0-2 m, 3-5 m and 6-8 m) and analysed these using the same design as the density measurements (n = 2). Variances were tested for homogeneity using Cochran’s C-test and normality of the data were assessed visually as per Quinn and Keough (2002). In the case of heterogeneous data, the analyses were still performed since ANOVA is robust to heterogeneity with balanced experimental designs and large numbers of replicates (Underwood, 1997). I used Student-Newman-Keuls (SNK) tests for a-posteriori comparisons. An estimate of the scoured area (blowout size) was made for each mooring. The mean (n = 2) radius of scoured seagrass at each mooring was plotted against the length of ground chain. I did

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Appendix 2 not include the lighter riser chain of ‘cyclone’ moorings in these estimates as it was generally not in contact with the substratum.

A2.4 RESULTS

A2.4.1 Conventional ‘swing’ moorings I observed ‘swing’ moorings with large circular areas of scour at Callala Bay (Figure A2.2A, Table A2.1, Appendix SNK: Reference > Swing). These moorings consistently lacked seagrass around them (Figure A2.3). These impacts lessened with distance from the mooring anchor at site 1; at 6-9 m I observed a seagrass density and cover similar to the reference area (Figure A2.3A, B, Appendix site1, 6-8m, SNK: Reference = Swing). The presence of Posidonia at this distance at this site produced a significant higher order interaction (Tr x Si x Di).

Table A2.1: Analyses of variance comparing ‘seagrass-friendly’ ‘screw’ and conventional ‘swing’ mooring systems at Callala Bay. Tr: Treatment, Si: Site, Di: Distance. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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A2.4.2 Seagrass-friendly ‘screw’ moorings Patterns of seagrass density and cover surrounding the ‘screw’ moorings at Callala Bay and Bindijine Beach were similar to that at the reference areas at these locations (Figure A2.3, Figure A2.4). This was particularly clear at site 2 at Callala and both sites at Bindijine Beach. At site 2, where Posidonia was most abundant at Callala Bay, there was little difference between the seagrass density and cover of the reference area and the area surrounding the ‘screw’ mooring (Appendix site2, SNK: Reference = Screw > Swing). In contrast, site 1 had a seagrass cover and density that were generally lower surrounding the ‘screw’ mooring than in the reference area (Appendix site1, 0-2m and 6-8m, SNK: Reference > Screw). At Bindijine Beach, a location with virtually no history of mooring, the Posidonia density and cover around the ‘screw’ moorings was very similar to that at the reference areas (Table A2.2, Figure A2.4A, B). ‘Screw’ moorings were not without impacts, however. I observed a small circular scar around most of the ‘screw’ moorings at Bindijine Beach (Figure A2.2B) although I did not detect it in my data set (Figure A2.4). This scoured area, approximately 10 cm in width, corresponded to the coupling of the mooring to the float line. Contact between the coupling and the substratum had created this small-scale disturbance. Such small-scale effects were not observed at Callala Bay where the screw moorings sat higher above the substratum and did not make contact with it. I also noted colonisation of pioneering seagrass Halophila ovalis and Zostera spp. observed around many of the ‘screw’ moorings as well as in some of the reference areas (Figure A2.3C, Figure A2.5C). These species were not observed surrounding moorings at Bindijine Beach, as the meadow was composed entirely of P. australis.

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Appendix 2

184

Appendix 2

A) 100

0 0- 2 3- 5 6- 8 0- 2 3- 5 6- 8 Site 1 Site 2 B) 100

0 0- 2 3- 5 6- 8 0- 2 3- 5 6- 8 Site 1 Site 2 Figure A2.4: Comparison of seagrass density and cover for ‘screw’ moorings with reference areas at Bindijine Beach. (A) mean (+SE) estimates of Posidonia density. These estimates were expressed as percentages of the maximum density recorded in the reference area for each site. Each bar represents a total of four quadrats; two quadrats of 0.25 m2 for each of the two transect lines (n = 4). (B) mean (+SE) percent cover of Posidonia. Each bar represents 20 points per distance for each of the two transect lines (n = 2). Distances (0-2, 3-5 and 6-8 m) are from the centre of each mooring. Reference; ‘Screw’ mooring.

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Appendix 2

Figure A2.5: Comparison of seagrass density and cover for ‘cyclone’ moorings with reference areas at Callala Bay. (A) mean (+SE) estimates of Posidonia density. These estimates were expressed as percentages of the maximum density recorded in the reference area for each site. Each bar represents a total of four quadrats; two quadrats of 0.25 m2 for each of the two transect lines (n=4). (B) mean (+SE) percent cover of Posidonia. Each bar represents 20 points per distance for each of two transect lines (n = 2). (C) mean (+SE) percentage cover of Halophila ovalis and Zostera spp. Each bar represents 20 points per distance for each of two transect lines (n = 2). Distances (0-2, 3-5 and 6-8 m) are from the centre of each mooring. Reference; ‘Cyclone’ mooring.

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Appendix 2

A2.4.3 Seagrass-friendly ‘cyclone’ moorings I observed large areas cleared of seagrass at all of the ‘cyclone’ moorings I sampled. These cleared areas were in the form a Y-shape, closely matching the layout of the mooring itself and generally extended to 18 m from the centre of each mooring (Figure A2.6). As transects were laid in random directions they did not always align with the areas of maximal scour and hence Posidonia was observed in some transects particularly with increasing distance from the centre of the ‘cyclone’ moorings (Figure A2.5A, B). The presence of Posidonia produced a significant Treatment by Site interaction (Table A2.3). Nevertheless, seagrass cover and density was consistently lower around ‘cyclone’ moorings than nearby reference areas (Figure A2.5A, B, Table A2.3, Appendix SNK: Reference > Cyclone).

A2.4.4 Size of mooring impact I observed a strong correlation between the length of the ground chain and the extent of the damage to seagrass as measured by the mean radius of the blowout around the moorings (Figure A2.6, r2 = 0. 73, P = 0.01). The mean radius of the blowout area of conventional ‘swing’ moorings was ~9 m, while for ‘cyclone’ moorings it was ~18 m (Figure A2.6).

Table A2.3: Analyses of variance comparing ‘seagrass-friendly’ ‘cyclone’ mooring systems at Callala Bay. Tr: Treatment, Si: Site, Di: Distance. NS, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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Appendix 2

Figure A2.6: Relationship between mooring chain length and the mean (+SE) radius of areas scoured of seagrass for ‘swing’ and ‘cyclone’ mooring systems at Callala Bay (n = 2). ‘Screw’ mooring; ‘Swing’ mooring; ‘Cyclone’ mooring.

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A2.5 DISCUSSION Dramatic impacts on seagrass were apparent around conventional ‘swing’ moorings with virtually no seagrass found within ~9 m of these moorings. Conversely, seagrass surrounding the ‘seagrass-friendly’ ‘screw’ moorings was similar to that of reference areas. In contrast, ‘seagrass- friendly’ ‘cyclone’ moorings had far greater impact on seagrass than that associated with the ‘swing’ moorings; scouring areas of almost double the size. ‘Screw’ moorings are indeed genuinely ‘seagrass-friendly’ at least over the first 12 months of their operation as we content that this is a crucial period in assessing the efficacy of mooring systems. In addition, Posidonia is highly sensitive to disturbances and responds quickly to physical damage (Ceccherelli et al., 2007). Numerous studies report that cleared areas surrounding ‘swing’ moorings are generated by the continuous dragging of the mooring chains (Walker et al., 1989; Lenihan et al., 1990; Hastings et al., 1995; Creed & Amado Filho, 1999; Francour et al., 1999; Marbà, et al., 2002; Crawford, 2003; Milazzo et al., 2004; Montefalcone et al., 2008). At Callala Bay, I estimate that the patch size of the scoured seagrass was 254 m2 per mooring (Figure A2.2A) and there are ≈60 swing moorings at this location. This estimate of damage per mooring is similar to that reported by Walker et al. (1989) in Western Australia. Surprisingly, there are no quantitative reports of impacts of moorings from other parts of the world, although impacts have been alluded to, especially in the Mediterranean (e.g. Milazzo et al., 2004). Given the large number of moorings and their continued installation worldwide, their overall impacts are alarming. There is an immediate and explicit need to address this issue (Hastings et al., 1995; Smith et al., 1997). The newly designed ‘screw’ moorings were highly successful in conserving seagrass, with seagrass density and cover generally indistinguishable from that observed in reference areas. The patchy nature of the seagrass at Callala Bay, stemming from previous mooring damage (Crawford, 2003), complicated the interpretation of present mooring impacts. It should be noted that small- scale damage was observed around ‘screw’ moorings at Bindijine Beach. This ring of damage (Figure A2.2B) was not detected by my analysis due to its relatively small size; especially when compared to the large areas of scour around conventional ‘swing’ moorings. This small-scale effect highlights the need for regular inspection of these moorings to assess their performance and ensure that they do not malfunction and threaten seagrass. Of major importance were the signs of seagrass recovery around some of the ‘screw’ moorings. Halophila ovalis and Zostera spp., which are considered pioneer species, colonised previously damaged areas either before or since the installation of the ‘screw’ moorings. Either way this indicates that ‘screw’ moorings allow recovery to take place, as observed in Shoal Bay and Pittwater (Gladstone, 2011a, b). This is important as the continued presence of the ‘swing’

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Appendix 2 moorings represent a press disturbance (sensu Bender et al., 1984) where the impact is on-going and recovery is halted by the frequent movement of the anchor chain. The removal of the ‘swing’ moorings and their replacement with effectively operating ‘seagrass-friendly’ moorings will enable seagrass recovery and lead to the restoration of original ecosystem function and services. In the case of Posidonia, recovery will be a very slow process and may take many decades (Meehan & West, 2000). Although considered ‘seagrass-friendly’, I observed large areas devoid of seagrass at all of the ‘cyclone’ moorings I sampled. Scoured areas were in the form a Y-shape, matching the layout of the mooring for distances of 18 m from the centre of the mooring (Figure A2.6). Although seagrass was apparent in some quadrats closer to the mooring anchors than this 18 m value (Figure A2.3B), it should be remembered that these values were estimated from random transects which were not aligned with the areas of scoured seagrass. Hastings et al. (1995) also reported that ‘cyclone’ moorings generated greater seagrass loss than ‘swing’ moorings at Rottnest Island, Western Australia; often generating ‘tri-circle’ sand areas. That is, three 10 m diameter circular blowouts in a triangle formation. These blowouts were assumed to be caused by the dragging of the ground chains and were found to coalesce with neighbouring blowouts in shallow water, producing a greater impact than ‘swing’ moorings (Hastings et al., 1995). The three ground chains of ‘cyclone’ moorings are designed to be under tension and concealed within the substratum (Jeyco, 2010; Adrian Nute, Jervis Bay Commercial Marine, pers. comm.), this was not the case at any of the ‘cyclone’ moorings at Callala Bay. An earlier assessment of the impact of permanent boat mooring systems at Rottnest Island revealed that ‘cyclone’ moorings were less damaging than ‘swing’ moorings (Walker et al., 1989) – only producing a relatively small area of damage (3 m2). Possible explanations for the discrepancy between the results of Walker et al. (1989), my findings and those of Hastings et al. (1995) include time since the installation of the moorings or the level of maintenance they receive. I contend that these differences are due to reduced effectiveness of these moorings over time as tension in the mooring chains dissipates; reinforcing the need for on-going maintenance.

A2.6 CONCLUSIONS AND RECOMMENDATIONS ‘Screw’ moorings were found to work effectively, while ‘cyclone’ moorings did greater damage to seagrass than conventional ‘swing’ moorings. I consider that a lack of regular inspection and maintenance has contributed to this damage. I therefore recommend that all ‘seagrass-friendly’ moorings receive annual maintenance checks and servicing to ensure their effectiveness. Such maintenance checks are especially important considering that failure of a mooring may do irreparable damage to Posidonia beds and contravene laws protecting seagrass (i.e. NSW Fisheries

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Appendix 2

Management Act 1994). Greater accountability of those responsible for those moorings may reduce the likelihood of future damage from faulty mooring systems. Habitat fragmentation has the potential to dramatically impact ecosystem integrity (Wilcox & Murphy, 1985) and indeed, there have been demonstrated impacts of fragmentation in seagrass meadows (Hovel, 2003). Such impacts remain to be examined over the spatial scale of mooring damage that I have observed in temperate seagrass meadows. Although I observed small-scale scour with ‘screw’ moorings, I encourage their use and further improvements in their design and maintenance. In addition, I argue for assessments like ours on ‘seagrass-friendly’ moorings in other parts of the world and other types of systems (e.g. with different vessel sizes, different types of substrata, etc). ‘Seagrass-friendly’ systems are necessary in order to sustainably accommodate the global increase in demand for moorings in coastal embayments. As well, their installation may allow seagrass recovery in areas previously damaged by conventional moorings and the return of the ecosystem function and services in these habitats.

A2.7 SUPPLEMENTARY DATA Supplementary data related to this article can be found at: http://dx.doi.org/10.1016/j.marenvres.2012.10.010

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Gladstone, B. (2011a). Monitoring of Seagrass Friendly Moorings in Shoal Bay. University of Technology Sydney, Newcastle innovation, 29pp. Gladstone, W. (2011b). Effectiveness of Seagrass Friendly Moorings (Pittwater). Newcastle Innovation, 40pp. Gobert, S., Cambridge, M. L., Velimirov, B., Pergent, G., Lepoint, G., Bouquegneau, J-M., Dauby, P., Pergent-Martini, C. and Walker, D. I. (2006). Biology of Posidonia. In Seagrasses: Biology, Ecology and Conservation (eds. A. W. D. Larkum, R. J. Orth, and C. M Duarte). Springer, The Netherlands, 387-408. Hastings, K., Hesp, P. and Kendrick, G. A. (1995). Seagrass loss associated with boat moorings at Rottnest Island, Western Australia. Ocean & Coastal Management, 26(3), 225-246. Heck, K. L., Hays, G. and Orth, R. J. (2003). Critical evaluation of the nursery role hypothesis for seagrass meadows. Marine Ecology Progress Series, 253, 123-136. Hemminga, M. and Duarte, C. M. (2000). Seagrass Ecology. Cambridge University Press, Cambridge, UK, 298pp. Hillman, K., Walker, D. I., Larkum, A. W. D. and McComb, A. J. (1989). Productivity and nutrient limitation. In Biology of Seagrasses – A treatise on the biology of seagrass with special reference to the Australia Region (eds. A.W.D. Larkum, A. J. McComb and S. A. Shepherd). Elsevier, New York, 635-685. Jeyco (2010). accessed 06/07/10. http://www.jeyco.com.au/ Hovel, K. A. (2003). Habitat fragmentation in marine landscapes: relative effects of habitat cover and configuration on juvenile crab survival in California and North Carolina seagrass beds. Biological Conservation, 110, 401–412. Kirkman, H., Fitzpatrick, J., Hutchings, P. A. (1995). Seagrasses. In Jervis Bay – A place of cultural, scientific and educational value (G. Cho, A. Georges and R. Stoutjesdijk). An Australian Nature Conservation Agency Publication, Canberra, 137-142. Lenihan, H. S., Oliver, J. S. and Stephenson, M. A. (1990). Changes in hard bottom communities related to boat mooring and tributyltin in San Diego Bay: a natural experiment. Marine Ecology Progress Series, 60, 147–160. Marbá, N. and Duarte, C. M. (1998). Rhizome elongation and seagrass clonal growth. Marine Ecology Progress Series, 174, 269-280. Marbà, N., Duarte, C. M., Holmer, M., Martinez, R., Basterretxea, G., Orfila, A., Jordi, A. and Tintore, J. (2002). Effectiveness of protection of seagrass (Posidonia oceanica) populations in Cabrera National Park (Spain). Environmental Conservation, 29(4), 509-518. McArthur, L. C. and Boland, J. W. (2006). The economic contribution of seagrass to secondary production in South Australia. Ecological Modelling, 196, 163-172. Meehan, A. J. and West, J. R. (2000). Recovery times for a damaged Posidonia australis bed in south eastern Australia. Aquatic Botany, 67, 161-167. Milazzo, M., Badalamenti, F., Ceccherelli, G. and Chemello, R. (2004). Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, western Mediterranean): effect of anchor types in different anchoring stages. Journal of Experimental Marine Biology and Ecology, 299(1), 51-62. Montefalcone, M., Chiantore, M., Lanzone, A., Morri, C., Albertelli, G. and Bianchi, C. N. (2008). BACI design reveals the decline of the seagrass Posidonia oceanica induced by anchoring. Marine Pollution Bulletin, 56(9), 1637-1645. Montefalcone, M., Parravicini, V., Vacchi, M., Albertelli, G., Ferrari, M., Morri, C. and Bianchi, C. N. (2010). Human influence on seagrass habitat fragmentation in NW Mediterranean Sea. Estuarine, Coastal and Shelf Science, 86, 292-298. NSW Department of Primary Industries (2010). accessed 20/04/10. http://www.dpi.nsw.gov.au/

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Orth, R. J., Heck, K. L. and van Montfrans, J. (1984). Faunal communities in seagrass beds: a review of the influence of plant structure and prey characteristics on predatory prey relationships. Estuaries, 7(4A), 339-350. Quinn, G. P. and Keough, M. J. (2002). Experimental design and data analysis for biologists. Cambridge University Press, Cambridge, 245-249. Shepherd, S. A., McComb, A. J., Bulthuis, D. A., Neverauskas, V., Steffensen, D. A. and West, R. (1989). Decline of seagrasses. In Biology of Seagrasses – A treatise on the biology of seagrass with special reference to the Australia Region (eds. A. W. D. Larkum, A. J. McComb and S. A. Shepherd). Elsevier, New York, 536-564. Short, F. T. and Wyllie-Echeverria, S. (1996). Natural and human-induced disturbance of seagrasses. Environmental Conservation, 23(1), 17-27. Short, F. T. and Wyllie-Echeverria, S. (2000). Global seagrass declines and effect of climate change. In Seas at the Millennium: An Environmental Evaluation (ed. C. R. C. Sheppard). Pergamon, Elsevier, Amsterdam, 3, 10-11. Short, F. T., Polidoro, B., Livingstone, S. R., Carpenter, K. E., Bandeira, S., Bujang, J. S., Calumpong, H. P., Carruthers, T. J. B., Coles, R. G., Dennison, W. C., Erftemeijer, P. L. A., Fortes, M. D., Freeman, A. S., Jagtap, T. G., Kamal, A. H. M., Kendrick, G. A., Kenworthy, W. J., La Nafie, Y. A., Nasution, I. M., Orth, R. J., Prathep, A., Sanciangco, J. C., van Tussenbroek, B., Vergara, S. G., Waycott, M. and Zieman, J. C. (2011). Extinction risk assessment of the world’s seagrass species. Biological Conservation, 144(7), 1961-1971. Smith, A. K., Holliday, J. E. and Pollard, D. A. (1997). Management of seagrass habitats in NSW estuaries. Wetlands (Australia), 16(2), 48-55. Underwood, A. J. (1997). Experiments in Ecology: Their Logical Design and Interpretation Using Analysis of Variance. University Press, Cambridge, 504pp. Walker, D. I., Lukatelich, R. J., Bastyan, G. and McComb, A. J. (1989). Effect of boat moorings on seagrass beds near Perth, Western Australia. Aquatic Botany, 36(1), 69-77. Walker, D. I. and McComb, A. J. (1992). Seagrass degradation in Australian coastal waters. Marine Pollution Bulletin, 25(5-8), 191-195. Waycott, M., Duarte, C. M., Carruthers, T. J. B., Orth, R. J., Dennison, W. C., Olyarnik, S., Calladine, A., Fourqurean, J. W., Heck, K. L., Hughes, A. R., Kendrick, G. A., Kenworthy, W. J., Short, F. T. and Williams, S. L. (2009). Accelerating loss of seagrasses across the globe threatens coastal ecosystems. Proceedings of the National Academy of Sciences of the United States of America, 106(30), 12377-12381. West, R. J. (1990). Depth-related structural and morphological variations in an Australian Posidonia seagrass bed. Aquatic Botany, 36(2), 153-166. West, R. J., Larkum, A. W. D. and King, R. J. (1989). Regional studies – Seagrasses of south eastern Australia. In Biology of Seagrasses – A treatise on the biology of seagrass with special reference to the Australia Region (eds. A. W. D. Larkum, A. J. McComb and S. A. Shepherd). Elsevier, New York, 230-260. Wilcox, B. and Murphy, D. (1985). Conservation strategy: the effects of fragmentation on extinction. American Naturalist, 125, 879-887.

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APPENDIX 3

Filtering the unknown: sampling sessile epifaunal invertebrates in seagrass meadows.

Appendix 3

A3.1 INTRODUCTION In order to sample the sessile epifaunal invertebrate community of Posidonia australis (Hook f.) seagrass meadows I had to establish 1) definitions and sampling guidelines, 2) the ideal sampling units and their dimensions, and 3) the most effective experimental design through a cost- benefit analysis. A single study was found to consider the appropriate sampling design of sessile epifaunal invertebrates inhabiting seagrass meadows (Barnes et al., 2006). They used a combination of transects and timed searches to sample this assemblage within an estuarine environment, composed primarily of seagrass. Spatial variation was examined at four different scales; two Lake (100s km apart), six locations (km apart), four sites (100s m apart) and 20 transects (10 m by 2 m) within sites (10s of metres apart). Based on this study, I used a fully nested pilot study to sample sessile epifaunal invertebrates within six transects (1 m x 25 m) haphazardly distributed in the seagrass (10s m apart), two sites (100s m apart), and two locations (km apart). Transects were employed over quadrats due to their greater effectiveness in detecting patchily distributed species (Miller & Ambrose, 2000). The ideal transect length was determined using the diversity and abundance of sessile epifaunal invertebrates at increasing transect lengths (5, 10, 15, 20 and 25 m). Multiple equations were used to determine the most adequate transect length (Table A3.1). Note that I estimated that transects longer than 25m would not be feasible as they moved with the surge. It was ascertained that a transect width of 1 m was adequate, in accordance with previous seagrass sampling (Demers et al., 2013). The advantages of this width were: 1) time- efficiency by allowing the diver to swim in a straight line, 2) reduction of sediment resuspension as a result of directional sampling, 3) feasible despite low visibility. The experiment design was determined using multiple cost-benefit analyses. I used a fully nested analyses of variance (ANOVA) of untransformed data using GMAV5 (University of Sydney) to calculate variance estimates (Sokal & Rohlf, 1981; Winer et al., 1991; Underwood, 1997). Two factors were analysed: ‘location’ was a random orthogonal factor with two levels (Bindijine Beach and Callala Bay); ‘site’ was a random orthogonal factor with two levels and was nested within Location. The level of replication of six represents the amount of transects sampled at each site (n=6). In addition to the variance estimates I calculated the proportion of the total variation in percent for each of the levels in the analysis. A cost-benefit analysis use preliminary estimates of variance to determine the ideal number of replicate transects per site, the ideal number of replicate sites per locations and the optimal number of replicate locations (Winer et al., 1991; Underwood, 1997). It reveals the optimal design for individual taxa and sample abundances, which might markedly differ. Time was my limiting cost with a limitation of six days of sampling per embayment. I allocated 11 hours (660 min) of

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Appendix 3 sampling time per day which included seven hours (420 min) of travelling and preparation time prior and subsequent to sampling. This included the time it takes to set up on arrival (launch vessel, assemble dive gear, transfer equipment on board) and vice versa on departure. A 90 min period was budgeted for each site assuming a travel time of 60 min per site (30 min each way) and 30 min of traveling between transects. The average time to sample a single transect was 14 min based on preliminary sampling. Based on this budget, I determined the optimal number of replicate locations, sites and transects, using procedures stated in Sokal and Rohlf (1981). In addition, the precision of some designs were calculated using their expected variances and relative efficiencies (Sokal & Rohlf, 1981).

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Table A3.1: The techniques, equations and associated references used to determine the appropriate transect length. Techniques and Reference Equations Coefficient of Variation (CV) 푆퐸 푆퐸 = standard error CV = (1) A low value indicates a high precision (Kingsford 푥̅ x̅ = mean & Battershill, 1998). Replication (n) 휎 2 휎 = standard deviation n = ( ) (2) The lowest replication show greater accuracy for 푝 ∗ 푥̅ 푥̅ = mean abundance that desired precision (Andrew & Mapstone, 1987). 푝 = precision of 15% Sample Size (n) 4 휎2 휎2 = standard deviation squared n = (3) 2 The sample size required to maintain a high level of 퐿2 퐿 = the limit squared calculated by multiplying 95% confidence by accuracy (Snedecor & Cochran, 1980). the mean x̅ Wiegert's Method C = Co + Cx (4) C = Cost of one transect The optimal unit size is the transect length with the (R Cost) ∗ (R Variance) (5) Co = Fixed costs (cost per location of 50 min + cost per site of 90 lowest product of the ‘Relative cost’ and ‘Relative C σ2 min = 140 min) ( ) ∗ ( ) (6) variance’ (Krebs, 1999). Cmin σ2min Cx = Cost of sampling one transect of a size x Cmin = Minimum time taken to sample a transect σ2min = Minimum standard deviation squared 푛 Binomial Sampling Theory Unit size = ( ) (7) n = sample size The size of a sampling unit from a benthic 푚 m = mean density of each species community to allow greater probability of detection (n obtained from a graphical p = proportion of the assemblage species occupy (Dennison & Hay, 1967 from Green, 1979). estimation format using p and Y) Y = proportion of time species will be randomly encountered (0.05)

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Figure A3.1: Variation for increasing transect lengths (m) for sampling sessile epifaunal invertebrate in the seagrass meadows of Jervis Bay using (A) Coefficient of variation (Kingsford & Battershill, 1998), (B) Replication with a precision of 15% (Andrew & Mapstone, 1987), (C) Sample size (Snedecor & Cochran, 1980) with a 95% confidence,

(D) Wiegert’s method with C0=140 min (Krebs, 1999). See Table A3.1 for equations.

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Table A3.2: The outcome of the binomial sampling theory (Dennison & Hay, 1967) for each of the species present in order of increasing sample size (n). The sample size (n) is the number of replicates and the appropriate unit size (m2) is the size of the sampling unit to allow greater probability of detection (0.05). The equation and details of calculation can be found in Table A3.1.

Species Sample Size (n) Appropriate Unit Size (m2) Porifera Phoriospongia sp. 1 6 4 Haliclona spp. 10 9 Tedania (Tedania) sp. 30 62 Halichondria sp. 70 400 Thorectidea sp. 70 420 Chondropsis sp. 3 160 1600 Chondropsidae sp. 2 300 6000 Stelletta sp. 800 4800 Callyspongia (Toxochalina) sp. 1 800 4800 Euryspongia sp. 1500 180000 Tethya sp. 1500 180000 Ascidiacea Herdmania grandis 4 60 Pyura praeputialis 6 103

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Table A3.3: Replication at each spatial scale derived from cost-benefit analyses for sampling sponge and ascidian diversity and abundance using the pilot study. The variance estimates were obtained from ANOVAs of untransformed data. Values were rounded to the nearest whole unit of sampling (except for variances). I used a standard design of six locations, two sites and six transects when calculating the expected variance and relative efficiency. For instance, when comparing amongst locations I assumed two sites and six transects.

Diversity Abundance A) Variance estimates All taxa Sponges Ascidians All taxa Sponges Ascidians Location 1.63 1.19 0.03 -22.26 -21.78 0.17 Site (L) 1.52 1.58 -0.07 8.88 13.26 -0.22 Transect (S(L)) 3.95 2.23 0.48 225.93 205.85 1.38 B) Proportion of total variation (%) Location 30 31 6 0 0 0 Site (L) 28 42 0 4 6 0 Transect (S(L)) 72 58 94 96 88 1 C) Number of replicates Location 5 5 8 6 6 8 Site (L) 2 2 1 1 1 1 Transect (S(L)) 4 3 1 13 10 1 D) Expected variance Location: 3 0.22 0.2 0.01 4.65 4.76 0.06 6 0.18 0.16 0.01 3.88 3.96 0.05 8 0.14 0.12 0 2.91 2.97 0.04 Site: 1 2.18 1.95 0.08 46.53 47.57 0.23 2 1.09 0.98 0.04 23.27 23.78 0.11 Transect: 5 1.42 1.16 0.08 42.09 40.94 0.23 6 1.09 0.98 0.04 23.27 23.78 0.11 9 1.07 1 0.03 17.49 18.80 0.08 12 0.99 0.95 0.02 14.22 15.76 0.06 E) Relative Efficiency % Location: 5 to 6 20 20 20 20 20 20 6 to 8 33 33 33 33 33 33 Site: 1 to 2 100 100 100 100 100 100 Transect: 3 to 6 30 19 100 81 72 100 6 to 9 2 -3 50 33 26 50

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A3.2 OUTCOMES The experimental guidelines and sample units established by the pilot study were deemed adequate granting three improvements; 1) clear definition of a sponge individual, 2) measurement of sponge volume as estimates of sponge distribution, and 3) exclusion of recruits from sponge total abundance estimates. The sample units, transects of 1 m x 25 m, were considered ideal as they were practical and had relatively low variation. A sampling design of six locations, two sites and six transects was elected based on the cost-benefit analyses and spatial constraints associated with small meadow size at certain locations. The methodology developed by this pilot study allows for adequate sampling of sessile epifaunal invertebrates inhabiting P. australis meadows and provides a standardised sampling alternative for future research. The experimental guidelines established by the pilot study were adequate in sampling sessile epifaunal invertebrates in the seagrass meadows of Jervis Bay. Sponge individuals were defined as singular physiologically independent entity (Wulff, 2001). Guidelines to measure sponge individuals were developed and included variables such as abundance, diversity, volume and substratum of attachment. Although volume was more complex and time-consuming than the number of sponge individuals present, a combination of abundance and volume measurements was selected as the most meaningful estimate of sponge distribution, in accordance with other studies (Wulff, 2001; Bannister et al., 2010; Wulff, 2012). Recruits were excluded as they disproportionately increased abundance estimates. Recruitment patterns in sponge and ascidian are highly variable spatially and temporally (Keough, 1983; Bingham & Young, 1995; Maldonado & Young, 1996; Shenkar et al., 2008) therefore having the potential to erroneously skew adult population distribution if included in the study. Sampling units of 1m x 25m were deemed ideal for sampling the distribution of sessile epifaunal invertebrates. Transect dimensions greater than 25m in length and 1 m in width were impractical in situ. The lowest variation across different transect lengths was at 25m when measuring the diversity and abundance of sponges and ascidians in seagrass meadows. The binomial sampling theory showed unrealistic sample sizes for rare species. This was expected since sparsely distributed species are less frequently encountered and would never occur at a 95% occurrence probability. A sampling design of six locations, two sites and six transects was selected based on a compromise between the cost-benefit analyses and spatial constraints associated with small meadow size at Plantation Point. Negative variance estimates were abundant, which was likely engendered by the highly variable distribution of sessile epifaunal invertebrates at the transect level. Further, the pilot study was restricted by a low replication level, with only two locations sampled.

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Restrictions came into play when selecting the optimal number of transects per site that could not be incorporated in the cost-benefit analyses; 1) the maximum amount of transects that could be contained within the smallest seagrass meadow whilst still respecting the minimum distance among transects, 2) the maximal amount of time a diver could safely stay underwater. I selected six transects per site based on those criteria, which provided a relatively low variance of 23 for the abundance of all taxa sampled (Table A3.3D). The size of the smallest seagrass meadow dictated the maximum amount of transect per site. It was estimated that six transects per site were the maximal amount feasible considering the small size of the meadow at Plantation Point. Although a single site per location was established as optimal by the cost-benefit analysis, I selected two sites per location to allow for replication. The use of one site reduced precision by 50% in comparison to two sites (Table A3.3D). Again, the size of the meadow at Plantation Point only allowed for a maximum of two sites per location. The ideal number of locations per embayment varied from five to eight replicates (Table A3.3C). There was a 20% increase in relative efficiency between five and six locations, and a 33% increase between six and eight locations (Table A3.3E). Based on preliminary sampling, a single location per day yielded increased efficiency. I therefore selected six locations as the optimal level of replication to achieve a single location per day for a final design of six locations, two sites and six transects.

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