The Responses of Amphibolis Griffithii to Reduced Light Availability Kathryn

The responses of Amphibolis griffithii to reduced light availability

A report on the outcomes of the SRFME Collaborative Research Project: Ecophysiology of benthic primary producers

Final Report to SRFME / WAMSI and Geraldton Port Authority

Kathryn McMahon and Paul Lavery Centre for Marine Ecosystems Research Edith Cowan University

Report No. 2008-01

This report has been prepared to summarise the findings and management implications of the SRFME Collaborative Research Project: Ecophysiology of benthic primary producers. No portion of this material may be reproduced or communicated without the permission of ECU, unless the reproduction or communication is authorised by law.  ECU 2008.

The responses of Amphibolis griffithii to reduced light availability Final Report on the SRFME Collaborative Research Project: Ecophysiology of benthic primary producers. Kathryn McMahon and Paul Lavery Centre for Marine Ecosystems Research Edith Cowan University 100 Joondalup Dr, Joondalup, WA Australia

Cite as: McMahon, K. and Lavery, P.S. (2007). The responses of Amphibolis griffithii to reduced light availability. Final Report on the Strategic Research Fund for the Marine Environment (SRFME) Collaborative Research Project: Ecophysiology of benthic primary producers. 148 p. Centre for Marine Ecosystems Research, Edith Cowan University, Joondalup, Western Australia.

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ii. Table of Contents i. Preface...... ii. Table of Contents ...... v iii. List of Figures...... vii iv. List of Tables ...... xi v. Acknowledgements ...... xiii 1. Executive Summary...... 1 2. Rationale and Background ...... 9 3. Research Framework ...... 14 4. Report Format ...... 15 5. Methodology ...... 17 5.1 Experimental design...... 17 5.2 Site location ...... 19 5.3 Environmental parameters measured...... 20 5.4 Biological parameters measured ...... 20 5.5 Data quality ...... 21 5.6 Statistical analysis ...... 22 6. Environmental Responses ...... 25 6.1 Light (PPFD) ...... 25 6.2 Water temperature...... 25 7. Seagrass Response to PPFD Reduction Treatments...... 27 7.1 Biomass...... 27 7.2 Density ...... 32 7.3 Morphology ...... 36 7.4 Growth ...... 42 7.5 Sexual reproduction ...... 45 7.6 Physiology ...... 46 7.7 Impact – response pathway ...... 52 8. Canopy Response to 3-months PPFD Reduction Post-summer ...... 57 8.1 Biomass and density ...... 58 8.2 Morphology ...... 59 8.3 Growth ...... 60 8.4 Physiology ...... 61 9. Recovery From 3-month PPFD Reduction Treatments ...... 69 9.1 Biomass...... 69 9.2 Density ...... 73

9.3 Morphology ...... 74 9.4 Growth ...... 78 9.5 Sexual reproduction ...... 80 9.6 Physiology ...... 81 9.7 Recovery - response pathway of A. griffithii after 3-months of PPFD reduction ...... 88 10. Recovery From 6-month PPFD Reduction Treatments ...... 91 10.1 Biomass and density ...... 91 11. Recovery From 9-month PPFD Reduction Treatments ...... 97 11.1 Biomass and density ...... 97 12. Sub-lethal Indicators...... 102 12.1 Sub-lethal indicators of PPFD reduction ...... 102 12.2 Sub-lethal indicators of recovery from PPFD reduction ...... 104 12.3 Summary statistics of possible sub-lethal indicators ...... 106 12.4 Potential indicators of sub-lethal reduction in light availability ...... 109 13. Management Application of the Research Results...... 115 13.1 Environmental modeling ...... 116 13.2 Impact prediction ...... 125 13.3 Impact management...... 130 14. References...... 138 15. Publications and Presentations From This Research...... 142 16. Graphic Appendix...... 143

iii. List of Figures

Figure 1. Effects of different intensities and durations of light reduction on the leaf biomass, leaf density and density of leaf clusters of Amphibolis griffithii...... 3

Figure 1.2: The relationship between the cumulative hours of deviation from HSAT and leaf biomass of A. griffithii...... 5 Figure 2.1: Locations of proposed or existing dredging and port expansion in Western Australia ...... 12 Figure 2.2: PPFD conditions (Observations below saturating irradiance (55 µmol m-2 s-1(Masini & Manning 1997)) from March – April 2003 and associated seagrass effects...... 13 Figure 5.1: Photographs showing experimental design and sampling methodology...... 18 Figure 5.2: Map showing experimental study site in Jurien Bay, near Boullanger Island...... 19 Figure 7.1: Photographs of representative plots of A. griffithii at the end of each treatment ...... 28 Figure 7.2: Photographs of stems from representative above-ground biomass samples from plots at the end of each PPFD reduction treatment...... 29 Figure 7.3: Biomass (g DW m-2) of A. griffithii and algal epiphytes following PPFD reduction treatment. 31 Figure 7.4: Density (m-2) of A. griffithii following PPFD reduction treatments...... 33 Figure 7.5: Morphology of A. griffithii following PPFD reduction treatments...... 37 Figure 7.6: Additional morphology of A. griffithii following PPFD reduction treatments...... 39 Figure 7.7: Growth of A. griffithii following PPFD reduction treatments...... 43 Figure 7.8: Branching frequency of A. griffithii following Post-summer PPFD reduction treatments...... 44 Figure 7.9: Seedling production in A. griffithii following PPFD reduction treatments ...... 45 Figure 7.10: Carbohydrate content (% DW) of A. griffithii following PPFD reduction treatments...... 47 Figure 7.11: Leaf nutrient content (% DW) of A. griffithii following PPFD reduction treatments...... 48 Figure 7.12: Rhizome nutrient content (% DW) of A. griffithii following PPFD reduction treatments ...... 49 Figure 7.13: Carbon and nitrogen stable isotope ratio (δ13C, δ15N) of A. griffithii following PPFD reduction treatments...... 50 Figure 7.14: Response pathway showing morphological, growth and physiological responses of A. griffithii meadows following PPFD reduction treatments...... 54 Figure 7.15: Response of A. griffithii to increased intensity of stress (PPFD reduction) in the 3-month PPFD reduction treatments...... 55 Figure 8.1: Total seagrass, leaf and algal epiphyte biomass (g DW m-2) and cluster and leaf density (m-2) of A. griffithii at 10 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments...... 58 Figure 8.2: Leaf length, width, leaves per cluster and internode length of A. griffithii at 10 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments ...... 59 Figure 8.3: Cluster growth, leaf extension and productivity of A. griffithii at 10 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments...... 60 Figure 8.4: Leaf sugar and starch content of A. griffithii at 20-30 and 40-50 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments ...... 61 Figure 8.5: Leaf ∂15N, Leaf ∂13C, Leaf N & C at 20-30 and 40-50 cm height categories through the A. griffithii seagrass canopy after 3-months of PPFD reduction treatments...... 62

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Figure 8.6: Photosynthetic measures of A. griffithii leaves in the upper and lower canopy derived from Rapid Light Curves...... 65 Figure 8.7: Photosynthetic measures of A. griffithii leaves derived from Rapid Light Curves...... 67 Figure 8.8: Photosynthetic measures of A. griffithii leaves derived from Rapid Light Curves...... 68 Figure 9.1: Photographs of plots over the recovery period for 3-month treatment both Post-summer and Post-winter...... 70 Figure 9.2: Biomass (g DW m-2) of A. griffithii and algal epiphytes following recovery from 3-months of PPFD reduction treatments...... 71 Figure 9.3: Density (m-2) of A. griffithii following recovery from 3-months of PPFD reduction...... 72 Figure 9.4: Morphology of A. griffithii following recovery from 3-months of PPFD reduction...... 75 Figure 9.5: Additional morphology of A. griffithii following recovery from 3-months of PPFD reduction. 76 Figure 9.6: Growth of A. griffithii following recovery from 3-months of PPFD reduction...... 79 Figure 9.7: Seedling production in A. griffithii following recovery from 3-months of PPFD reduction...... 80 Figure 9.10: Carbohydrate content (% DW) of A. griffithii following recovery from 3-months of PPFD reduction...... 83 Figure 9.11: Leaf nutrient content (% DW) of A. griffithii following recovery from 3-months of PPFD reduction...... 84 Figure 9.12: Rhizome nutrient content (% DW) of A. griffithii following recovery from 3-months of PPFD reduction...... 85 Figure 9.13: Carbon and nitrogen stable isotope ratio (δ13C, δ15N) of A. griffithii following recovery from 3- months of PPFD reduction...... 86 Figure 10.1: Photographs of 6-month plots at the end of the impact treatment and then in August 2007, 23 months (Post-summer) and 17 months (Post-winter) following re-exposure to ambient PPFD...... 92 Figure 10.2: Biomass (g DW m-2) of A. griffithii following recovery from 6-months of PPFD reduction. ..93 Figure 10.3: Density (m-2) of A. griffithii following recovery from 6-months of PPFD reduction...... 94 Figure 10.4: Canopy height – 80th percentile (cm) of A. griffithii following recovery from 6-months of PPFD reduction...... 95 Figure 10.5: Stems with leaves expressed as % of number of stems with leaves at end of PPFD reduction treatments and categorised into stem heights for the Post-summer (PS) and Post-winter (PW) 6-month plots...... 96 Figure 11.1: Photographs of 9-month plots at the end of the impact treatment and then in August 2007, 21 months (Post-summer) and 15 months (Post-winter) following re-exposure to ambient PPFD...... 97 Figure 11.2: Biomass (g DW m-2) of A. griffithii and algal epiphytes following recovery from 9-months of PPFD reduction...... 98 Figure 11.3: Density (m-2) of A. griffithii following recovery from 9-months of PPFD reduction...... 99 Figure 11.4: Canopy height – 80th percentile (cm) of A. griffithii following recovery from 6-months of PPFD reduction...... 100 Figure 11.5: Counts of stem with leaves categorised into stem heights for the Post-summer (PS) and Post- winter (PW) 9-month plots...... 101 Figure 13.1: Effect of reduced light (PPFD as % of the ambient PPFD at the surface of the canopy) on the leaf biomass of Amphibolis griffithii, for shading after summer (Post-summer, commencing March) and shading after winter (Post-winter, commencing September)...... 117

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Figure 13.2: Effect of number of hours per day above saturating irradiance (HSAT) on the leaf biomass of Amphibolis griffithii, for Post-summer (commencing March) and Post-winter (commencing September) periods...... 118 Figure 13.3: The relationship between the cumulative hours of difference between the hours of saturating irradiance of control plots versus shaded plots with the leaf biomass of A. griffithii expressed as a percentage of the control...... 119 Figure 13.4: Hypothetical scenarios demonstrating loss of function in an ecosystem with different extent and duration of impact and recovery...... 121 Figure 13.5: Timescales of loss of ecological function (expressed as the loss of leaf biomass relative to the control) over the duration of the Impact and Recovery phase...... 122 Figure 13.6: A comparison of the loss of function estimate in the experimental Amphibolis griffithii seagrass meadows after 6 months of light reduction PW (post-winter) and PS (post-summer) with the loss of function estimate for Posidonia sinuosa based on Collier (2006)...... 126 Figure 13.7: A comparison of the loss of function estimate in the experimental Amphibolis griffithii seagrass meadows after 9 months of light reduction with the loss of function estimate at a number of sites exposed to a turbid plume from dredging during the Geraldton Port Enhancement Project. 127 Figure 16.1: Algal epiphyte biomass (g DW m-2) on A. griffithii following PPFD reduction treatments ...143 Figure 16.2: Faunal epiphyte biomass (g DW m-2) on A. griffithii following PPFD reduction treatments. 144 Figure 16.3: Leaf cluster morphology of A. griffithii following PPFD reduction treatments...... 145 Figure 16.4: Canopy height (cm) of A. griffithii following PPFD reduction treatments ...... 146 Figure 16.5: Leaf cluster morphology of A. griffithii following recovery from 3-months of PPFD reduction treatments ...... 147 Figure 16.6: Canopy height (cm) of A. griffithii following recovery from 3-months of PPFD reduction...148

iv. List of Tables

Table 1.1 Summary of the plant and algal parameters in Amphibolis griffithii habitats which were responsive to shading treatments within 3 months ...... 7 Table 5.1: Timing of sample collection in this experiment...... 19 Table 5.2: Parameters measured in the experiment...... 22 Table 6.1: PPFD summary data for all treatments in the experiment determined from in situ PPFD loggers...... 26 Table 6.2: PPFD attenuation through the seagrass canopy...... 26 Table 6.3: Water temperature expressed as average over the experimental period...... 26 Table 7.1: Results of statistical analysis to determine significant effects of Timing, Duration and Intensity of PPFD reduction treatments on Amphibolis griffithii seagrass meadow biomass and density parameters...... 34 Table 7.2: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass morphology parameters...... 40 Table 7.3: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass growth parameters...... 42 Table 7.4: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass carbohydrate parameters...... 46 Table 7.5: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass nutrient parameters...... 51 Table 9.1: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass meadow biomass and density parameters...... 73 Table 9.2: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass morphology parameters...... 77 Table 9.3: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass growth parameters...... 78 Table 9.4: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass carbohydrate parameters...... 82 Table 9.5: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass nutrient parameters...... 87 Table 9.6: Summary of recovery response-pathway variables following 3 months shading post-summer and post-winter with 3 and 10 months re-exposure to ambient light...... 89 Table 12.1: Response of all parameters measured to different timings, durations and intensity of PPFD reduction...... 103 Table 12.2: Recovery of all parameters from 3-months PPFD reduction Post-summer and Post-winter....105 Table 12.3: Summary statistics of parameters proposed as useful sub-lethal indicators...... 107 Table 13.1 Summary of the plant and algal parameters in Amphibolis griffithii habitats which were responsive to shading treatments within 3 months ...... 133

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v. Acknowledgements

This project has benefited from the contributions of numerous people in the development, implementation, conduct of the research and discussions on the findings. Firstly our co- investigators, Ray Masini and Cameron Sim from Department of Environment and Conservation and Russ Babcock from CSIRO, and industry partner, Geraldton Port Authority led by Michael Mulligan. Initial design and construction of the large field experiment at Jurien Bay was piloted by Paul Mackey in an Honours project with invaluable input from Michael Mulligan. Michael Mulligan was integral to the establishment of the main field experiment. Throughout the duration of the experiment key assistance was provided by research assistants Andrew Tennyson, Paul Mackey and Peter Quintana and Masters student Michael Mulligan. The two-year intensive field program required numerous divers working in challenging field conditions. Thank-you to all who assisted: Russ Babcock, Mark Westera, Karen Crawley, Sofie Harrison, Jason How, Craig Koltasz, Alex Watson, Andrew Limbourn, Griffin Grounds, Helen Barwick, Lachlan MacArthur, Rebecca Kennah, John Eyres, Jay Hender, Bronwen McKay, Emily Gates, Matt Gorski, Christine Hanson, Haslett Grounds, Marianne Nyegaard, Erin D’Raine, Travis Hurley, Wesley Manson, Nikki Hortin, Nicholas Lynch, Chris Doropoulos, Kellie Holloway, Michelle Newport, Michael Raykos, Anne Brearley, Adam Gartner, Adrian Abelardo, David Holley, Wesley Alport, Peter Kis, Francois DeLane and Michael de Ridder. To anyone we may have missed, apologies and thanks. Support in Jurien Bay from Kevin Crane, Greg Inglis and Tim Daly, Department of Environment and Conservation was greatly appreciated. The fieldwork could not have been completed without the fantastic assistance of Ian and Sharon Stiles from Jurien Bay Dive. Paul Baines at Jurien Bay Mobile Marine carried out numerous boat repairs and maintenance efficiently. Many hours were spent in the laboratory processing samples with the assistance of numerous people including Helen Barwick, Bronwen McKay, Wesley Alport, Michelle Newport, Niki Hortin and Petra Mrossi. All your inputs are greatly appreciated

Kathryn & Paul

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1. Executive Summary

Background Light availability is a key determinant of coastal benthic primary productivity and reduction in light availability a major cause of loss of benthic primary producer habitat. Among the significant causes of reduced light availability is dredging, through the suspension of sediments in the water column. Negative effects of dredging on benthic communities have occurred in Western Australia, including the large-scale loss or reduction in biomass of seagrass meadows. In this context, the understanding of light and its co-variates as a driver of ecosystem structure is of fundamental importance for the management of the State’s marine environment. Regulatory agencies have identified nine key information gaps limiting the capacity to predict the impacts of light reduction on benthic ecosystems and design management strategies for activities that reduce light availability, such as dredging. The gaps relate to: • Understanding the physiological requirements of primary producers; • Understanding the levels of susceptibility to reduced light availability, the drivers of resilience and how it changes over time; • Knowledge of appropriate indicators of light-limitation, the levels of stress associated with different indicators; and • Timescales and processes of recovery following periods of reduced light availability. While regulatory agencies recognised the information needs for several benthic primary producer habitats, Amphibolis seagrass habitat was identified as a priority, since dredging has previously impacted these ecosystems and the response to light reduction events was not well understood.

The effects of reduced light availability on Amphibolis griffithii To address the information gaps for Amphibolis griffithii, a manipulative experiment was carried out in Jurien Bay to simulate the light reduction impacts. Three factors (time of year, duration and intensity of light reduction) were manipulated to simulate different dredging scenarios and help in understanding how different times of year (start at end of summer or winter), durations (3, 6, 9 months) and intensities would impact A. griffithii seagrass ecosystems. Light was reduced such that moderate treatments received between 13-19% of ambient light and high treatments 5-11% of ambient light. These are severe reductions but comparative to those observed in dredging operations such as the Geraldton Port Enhancement project.

The key findings were: • All experimental treatments negatively impacted A. griffithii seagrass meadows. • The response-pathway to light stress for A. griffithii commences with physiological changes, such as reductions in leaf and rhizome carbohydrates and leaf ∂15N and increases in leaf nitrogen content and a reduction in leaf growth. • With increasing intensity or duration of light reduction there was then morphological changes, such as reductions in leaf biomass, driven by reductions in the numbers of leaves per leaf cluster, the density of leaf clusters and canopy height (Figure 1.1).

• There was recovery of A. griffithii seagrass meadows (return of leaf biomass to control conditions) 10 months after the removal of light reduction, but only in the treatments shaded for three months.

• No recovery was observed in the treatments shaded for six and nine months, up to almost two years following the removal of light reduction. The study was terminated before recovery was observed in these treatments, so the timescale of recovery, if it occurs at all, cannot be estimated. • The timing of light reduction also impacted the response of A. griffithii, most obviously when plants were shaded for three months. Plants shaded at the end of winter through to early summer were least affected and had faster recovery than those plants shaded for the same duration but at the end of summer through to early winter. This is a complex interaction potentially driven by water temperature and total daily light. From a management perspective of predicting responses to light stress, the total amount of light received is important to know, not just the percent reduction. • Shading also impacted algal epiphyte biomass, a source of food and habitat for other organisms. The changes in both seagrass morphology and algal biomass caused by shading have important consequences for the habitat- and food-provision roles of A. griffithii meadows. These changes result in significant loss of macroinvertebrate abundance and biomass, with likely adverse consequences for higher trophic levels.

Figure 1. Effects of different intensities and durations of light reduction on the leaf biomass, leaf density and density of leaf clusters of Amphibolis griffithii. Light reductions were imposed at the end of summer (left) or the end of winter (right).

Implications and Conclusions Large-scale dredging projects have been shown to cause intense (acute and chronic) and widespread impacts on benthic light climate due to the light attenuating effects of sediments liberated to the water column by dredging. This study imposed durations and intensities of light reductions commonly associated with large-scale commercial dredging operations. At these durations and intensities, A. griffithii was significantly affected, with loss of biomass, productivity and the associated ecological functions of habitat and food provision. Similar impacts could be expected to occur during large-scale dredging operations in WA. The research outcomes allow the identified research gaps to be addressed to varying degrees, as summarised below.

What are the physiological requirements of A. griffithii and its environmental tolerances to varying levels of light and suspended sediment? A. griffithii responded to all of levels of light reduction imposed in the study. The minimum light requirements of A. griffithii will be higher than the amount of light provided in this study. With respect to tolerance, A. griffithii responded negatively to all levels of shading imposed, but recovered to control (unshaded) levels after 10 months if the light reduction was restricted to three months. Shading for six months or more resulted in severe impacts and no recovery after almost two years when monitoring of recovery ceased. The tolerance can be summarised as a function of the time plants experience intensities of light above those required to saturate photosynthesis (Hsat) (Figure 1.2). Shading can reduce the time above Hsat. While there is a seasonal effect, it is clear that duration of shading is crucial to the effect on A. griffithii. How far a dredging operation causes a deviation from the ambient Hsat will be a function of the intensity and duration of turbidity generated and the ambient light intensities, a function of time of year.

Figure 1.2: The relationship between the cumulative hours of deviation from HSAT and leaf biomass of A. griffithii. Red lines indicate the points from which there was no recovery observed. The data also includes results from another shading experiment on A. griffithii (Mackey et al. 2007).

Based on the findings of the study, if dredging operations were predicted to reduce light to the same level as this experimental study, then restricting the duration to three months or less will induce less severe impacts and is more likely to allow recovery within a 12 month period than longer periods of light reduction, which will induce greater impacts with the prospect of little, if any recovery, and associated loss of ecological function, for at least two years.

What drives the susceptibility / resilience? The response pathway is consistent with a response to reduced light availability driven by reduced carbon fixation leading to a carbon deficit, which, if prolonged, cannot be offset by drawing on stored carbohydrate reserves. This results in the plant transitioning through physiological and then morphological changes that have the effect of reducing carbon fixation requirements and increasing light availability to the remaining tissue. This improved knowledge has underpinned the discussion of potential parameters for use in the monitoring of A. griffithii habitats.

Does the susceptibility / resilience vary with time of year? Time of year clearly affects the susceptibility of A. griffithii to shading and to its subsequent recovery. Plants responded more rapidly to shading imposed at the end summer than at the end of winter, probably due to the lower ambient light levels at this time which compounded the shading effect. The implication for managers is that the absolute PPFD that plants will receive, rather than the anticipated percent reduction of ambient light, is the key determinant of effect size and this will vary with time of year.

Based on the findings of the study, if dredging events were to last three months or less, timing operations to start at the end of winter or during spring are likely to induce a lower impact than operations commencing at the end of summer or into autumn. Importantly, the strongest effects of timing of light reduction occurred at moderate intensities of shading. Once light reductions become severe, the responses are similarly large and negative at both times of year.

How long does it take for Amphibolis griffithii to recover? Plants shaded during spring-early summer showed rapid recovery, approaching or reaching control levels within three months and with almost complete recovery of all parameters by 10 months. This probably reflects the higher ambient light conditions during the shade and recovery period. Plants shaded for six or nine months at either moderate or high intensities showed no recovery during the study. In these treatments leaf biomass fell to below 25% of the control, possibly a threshold for potential recovery.

How does the resilience of Amphibolis griffithii compare with other seagrass communities? Based on its eco-physiological attributes, A. griffithii has been presumed to be relatively resilient to disturbance and, relative to ‘large’ seagrass species, such as Posidonia spp, is expected to show greater susceptibility to disturbance but a greater potential to recover. This study does not support the above hypothesis as impact and recovery effects and timescales are similar to P. sinuosa (Collier 2006). This is further corroborated by monitoring of A. griffithii following widespread losses from a dredging event in Geraldton (CSIRO 2007), which has shown comparable timescales of recovery to those observed in the experimental study.

What are robust indicators of Amphibolis ecosystem health (primary and secondary indicators of sub-lethal stress) for adoption by managers? The study points to several indicators of sub-lethal, light limitation of A. griffithii with potential for application in environmental monitoring and management. These are summarised in Table 1, categorised by the consistency of response across different intensities of shading and at different times of shading. Priority was given to parameters that respond quickly (by three months), at both times of year and to moderate or at least high levels of shading. Fourteen parameters met these general requirements, and of those, only two (leaf extension rate and leaf ∂15N) had responses that were consistent at both times of year and to moderate levels of light reduction. Issues associated with the application of these variables in monitoring programmes are summarised in Table 1.1. The final choice of variables to use in any programme will depend on the aims and practicalities associated with the particular application.

Table 1.1 Summary of the plant and algal parameters in Amphibolis griffithii habitats which were responsive to shading treatments within 3 months, showed high levels of consistency in the response and have the greatest potential for development as indicators of plant and ecosystem condition in relation to light reductions.

Parameter Advantages Uncertainties/comments Leaf tissue ∂15N Consistent responses. Requires comparison against ‘reference’ data. Low variation and large effect size. Processing is time consuming/costly. Leaf extension Consistent response. Requires comparison against ‘reference’ data. rate Varies with height in the canopy. Requires large replication; time consuming. Leaves per cluster Largely consistent and rapid response. Effect size is small. Relatively low variability. Varies with height in the canopy. Leaves per stem Generally consistent & rapid response. Requires comparison against ‘reference’ data The effect size is large and easy to measure. Difficult to measure non-destructively. Leaf Moderate effect size. Requires comparison against ‘reference’ data. biomass/density Reflect changes in several other variables Varies with canopy height. Destructive sampling; time consuming. Rhizome sugars Generally responsive. Requires comparison against paired ‘reference’ site data. Effect size small for moderate shading. Time consuming and costly. Algal epiphyte Fast response to PPFD reduction. Requires comparison against ‘reference’ data. biomass Varies with canopy height and time of year. Destructive sampling - labour intensive.

The study has provided information on the sample parameters for each variable (percentiles, mean, median etc) under control conditions over a complete annual cycle and under treatment conditions. These summary statistics provide the capacity to develop initial alert and action criteria in keeping with the approaches outlined in ANZECC (2000) and the State Environmental (Cockburn Sound) Policy (Govt. of WA 2005).

2. Rationale and Background

Light – a key driver of benthic ecosystems Light is among the most important environmental factors controlling coastal benthic primary productivity. The wavelengths of light used by benthic plants for photosynthesis (photosynthetic photon flux density or PPFD) vary seasonally, and are also affected by water depth and levels of suspended sediment or plankton density [i.e. Total Suspended Solids (TSS)]. As a consequence of this, benthic plant communities and associated fauna will change both qualitatively and quantitatively across gradients of PPFD and TSS. Examples of such gradients are inshore-offshore changes in water properties, or from sheltered to more exposed waters.

Management of human developments that impact light A broad range of coastal and marine developments can lead directly or indirectly to a reduction in light availability at the seabed. Direct effects include dredging for ports and harbours, breakwater construction, land reclamation, increased ship traffic, aquaculture developments, and domestic wastewater and industrial effluent discharges. The availability of PPFD can be reduced indirectly through increased phytoplankton biomass in the water column or excessive algal growth on aquatic plants, an increase in the amount and change in the characteristics of sediment suspended in the water column (turbidity) and elevated deposition of sediment on benthic organisms themselves (smothering). In this context, the understanding of light and its co-variates as a driver of ecosystem structure is of fundamental importance for the management of the marine environment. Meaningful indicators of sub-lethal environmental stress must underpin such management; indicators based on sound understanding of how ecosystems respond to the pressures that threaten their value. This understanding can be improved through research programs that fill gaps in our understanding of how ecosystems respond to variation in light or associated environmental variables. This will improve our ability to understand variations in habitat structure at the ecosystem level and to assess and manage impacts associated with human use of these systems.

Information gaps in management of light reduction events Key benthic habitats along the central west coast of Western Australia for which scientific research is required are primarily seagrass meadows and macroalgal reefs. Both species of the seagrass genera Posidonia and Amphibolis are important ‘meadow’ forming species, providing key habitat in shallow coastal waters. There are particular gaps in the understanding of the effects of light and sediment stress on Amphibolis species (A. antarctica and A. griffithii). It is reasonably understood what effect reduced PPFD has on photosynthetic organisms: a reduction in growth, biomass and potentially death (Dennison & Alberte 1982, Cambridge et al. 1986, Gordon et al. 1994). How different timings, durations, intensity of PPFD reduction interact to affect key benthic habitats is not well known. In the context of allowing a correct balance to be struck between protecting the environment without unnecessarily constraining development,

quantitative assessments of the degree of light reduction that can be sustained without irreversible or long-term damage are urgently required. This type of information is essential to better predict and manage human impacts. The development of key indicators of sub-lethal stress in these habitats and the establishment of management triggers (impact management) cannot be reliably undertaken in the absence of this basic information.

Background on Amphibolis griffithii seagrass

Distribution and habitat The genus Amphibolis in the seagrass family Cymodoceaceae is endemic to temperate western and southern Australian coastlines (Ducker et al. 1977). It is comprised of two species A. antarctica and A. griffithii, the latter of which has a more restricted distribution and is the focus of this research (Ducker et al. 1977, Kuo & den Hartog 2006). A. griffithii can survive in a variety of conditions from sheltered to exposed environments and on sand, reef and clay (Ducker et al. 1977, Carruthers et al. 2007). It can form continuous monospecific meadows as well as mixed species, patchy meadows (Holmes et al. 2007) in subtidal regions generally down to 12 m (Ducker et al. 1977), but depths of 48 m have been recorded (Carruthers et al. 2007). Amphibolis is placed towards the centre of the seagrass functional form model (Walker et al. 1999). It tends to be faster growing than the other main meadow-forming seagrass in the region, Posidonia, and can persist in disturbed areas (Cambridge 1999). But despite this, it is still categorised as a relatively poor coloniser and has shown little recovery following seagrass loss (Kirkman 1985, Clarke & Kirkman 1989). However, recent studies have demonstrated extensive expansion of A. griffithii meadows over decades, implying they have a greater colonising ability than previously thought (Kendrick et al. 1999, Kendrick et al. 2000).

Morphology and growth This clonal plant is composed of underground roots and rhizomes with a vertical, branching stem that holds terminal leaf clusters (Cambridge 1999). There are generally 2- 5 leaves per cluster and 6-20 clusters per vertical stem (Ducker et al. 1977, Cambridge 1999, Carruthers 1999). The maximum size of leaves is 100 x 10 mm (L x W), stem height ranges from 30-100 cm and diameter is 1-1.5 mm, whilst rhizome diameter is 1-2 mm (den Hartog 1970). Stems are long lived, generally 2-3 years (den Hartog 1970, Coupland 1997) whilst leaves are much shorter lived, generally 90 days (Marba & Walker 1999). The plastochrone interval of vertical stems (short shoots or branches of a stem) is 277 days, horizontal rhizome 509 days and leaves 32 days (Marba & Walker 1999). Upright stems are produced every 4-6 horizontal rhizome internodes and branches are produced every 3- 17 vertical stem internodes (Coupland 1997). A. griffithii, in contrast to other large meadow forming seagrass species like Posidonia, has a higher proportion of its biomass in above-ground tissue. The growth of Amphibolis griffithii is influenced by season with higher production rates in summer when light and

temperature are higher (Walker & Cambridge 1995, Carruthers & Walker 1997), longer internode length on vertical stems (Coupland 1997), and higher biomass (Carruthers 1994), when compared to winter. The energy stores present in the rhizome in winter are unlikely to support A. griffithii for more than a day if there was severe light reduction (Carruthers & Walker 1997).

Light requirements The light requirements of A. griffithii also vary with temperature such that the -2 -1 compensating irradiance (Ec) has a maximum at 18°C of ~ 17 µmol m s and a -2 -1 minimum at 13°C of ~ 14 µmol m s . In contrast the saturating irradiance (Ek) reaches the maximum at 23°C (55 µmol m-2 s-1) and minimum at 13°C (25 µmol m-2 s-1). Net photosynthetic rate is also greater at 23°C (Masini & Manning 1997). This is likely to support the faster growth rates in the summer period when light and temperature are at the maximum. A. griffithii has greater maximum photosynthetic rates and photosynthetic efficiency compared to other dominant seagrass species such as Posidonia which may allow it to persist at deeper depths and lower light environments (Masini et al. 1995).

Habitat provision The complex structure and more persistent habitat provides an ideal environment for algal and faunal epiphytes to colonise (Ducker et al. 1977). Consequently, there is a higher biomass and diversity of algae and fauna living on A. griffithii compared to other seagrass species (Borowitzka et al. 1990, Edgar 1990, Jernakoff & Nielsen 1998, Lavery & Vanderklift 2002). This variation is also reflected at higher trophic levels such that there is a unique composition of fish species and larger fish in A. griffithii compared to P. sinuosa and P. coriacea seagrass meadows (Hyndes et al. 2003). Greater predation rates have also been observed in A. griffithii meadows compared to Posidonia meadows (Vanderklift et al. 2007).

Western Australian context – dredging management The current economic boom and export of raw materials has led to, or will lead to, massive expansion of port infrastructure, almost all of which requires dredging of the seabed (Figure 2.1). Dredging can result in turbid plumes of fine suspended material, which simultaneously reduces light availability at the seabed and increases sedimentation. Both of these effects are known to be deleterious for seagrass ecosystems, and managing these potential impacts is a current priority in assessment of planned developments.

Figure 2.1: Locations of proposed or existing dredging and port expansion in Western Australia. Locations sourced from Department of Environment and Conservation, Western Australia.

A dredging event in Geraldton, on the central mid-west coast of Western Australia, during 2002-03 provided timely demonstration of the need for a sound understanding of the effects of light reduction on Amphibolis ecosystems. As part of a port expansion project, dredging with a cutter suction dredge began in October 2002 and continued until November 2003 (Mulligan 2005). After 5 months (March 2003), the turbid plume from the dredging was significantly larger than predicted, extending ~ 70 km up the coastline and at times 1-2 km out to sea (Mulligan 2005). PPFD reduction associated with the plume saw many sites regularly below the saturating irradiance required for Amphibolis griffithii growth (Figure 2.2) resulting in significant impact on seagrasses (Mulligan 2005) (Site 1, D68, D90, Figure 2.2). A number of sites north of Geraldton experienced an estimated 72-100% loss of seagrass (CSIRO 2007) (Figure 2.2). Three years post- dredging 1 of 6 sites had recovered and exceeded pre-dredging cover, and all other sites were on a trajectory of recovery, though at different rates of recovery. Sites in deeper water (11-15 m) and closer to the dredging activity have the slowest recovery (Figure 2.2). The environmental impact from this dredging programme highlighted the need for coastal development proponents and regulators to be able to better predict the impacts of these PPFD reduction events. It was also noted that sub-lethal indicators of stress in seagrasses that could be used as a monitoring tool would be useful in future dredging programmes.

Figure 2.2: PPFD conditions (Observations below saturating irradiance (55 µmol m-2 s-1(Masini & Manning 1997)) from March – April 2003 and associated seagrass effects. Instantaneous PPFD data (using a 4π sensor) was supplied by Geraldton Port Authority and converted to downward dwelling PPFD (analogous to using a 2π sensor) from an equation 2π PPFD = 4π PPFD x 0.6073 (Russ Babcock pers. comm.). Changes in seagrass cover were determined from CSIRO (2007).

3. Research Framework

This research had three phases. Phase 1, funded by SRFME and Geraldton Port Authority, was the pilot study examining the impact of a single, high intensity PPFD reduction (>88% reduction relative to ambient) over a 3.5-month duration, with 1.5- month recovery at the end of summer. The outcomes from this experiment are reported in the Final SRFME report (Lavery & McMahon 2006) and by Mackey et al (2007) and Mackey (2004). The second phase, also funded by SRFME and Geraldton Port Authority, with additional funding from Department of Environment and Conservation, Jurien Bay, was a larger experiment examining the interactive effects of three factors, intensity, duration and timing of PPFD reduction on A. griffithii and the recovery from these impacts. Phase 2 of the study parallelled a study by CSIRO into the recovery of seagrass from the actual dredging event in Geraldton (CSIRO 2007). This report presents the findings of Phase 2. Phase 3 of the study is still underway, investigating the trophic consequences of disturbance to seagrass systems. This part of the project has been funded by SRFME and Department of Environment and Conservation, Jurien Bay.

4. Report Format

This final report has been written as a requirement for the funding bodies, Strategic Research Fund for the Marine Environment (SRFME, now Western Australian Marine Science Institution: WAMSI) and Geraldton Port Authority (GPA). However, considering the various dredging proposals planned for the temperate waters of Western Australia (Figure 2.1) and the benefit this research will provide to those in government and industry working in environmental impact, assessment and management, the report has been complied as a complete reference to all data collected and to provide wider access to the information before publication of the planned journal articles. The main body of this report is separated into 16 sections as follows: 1. Executive summary 9. Recovery from 3-months PPFD reduction 2. Rationale and background to the study 10. Recovery from 6-months PPFD reduction 3. Research framework 11. Recovery from 9-months PPFD reduction 4. Report format 12. Sub-lethal indicators 5. Methodology 13. Application of research findings 6. Environmental parameter 14. References 7. Seagrass response to PPFD reduction 15. List of theses and presentations Appendix 8. Canopy response to PPFD reduction 16. Graphic Appendix

The rationale and background to the study, research framework and report format are presented in section 2-4. The methodology (5) is presented briefly. In the results and discussion section (6-12) all data collected from the experiment are presented graphically and in most cases with the associated statistical analyses. The key responses are highlighted and discussed, though not all results are discussed in detail. A summary of the key research findings and their applications are presented in section 13.

5. Methodology

5.1 Experimental design The effect of three factors, intensity, duration and timing of reduced PPFD was experimentally tested on a meadow of seagrass Amphibolis griffithii. The levels within each factor were: intensity – Control or ambient, Moderate and High; duration – 3-, 6- and 9-months; and timing – Post-summer and Post-winter. Each combination of treatments was allocated five replicates, such that n = 120. The intensity was selected to cover the upper range of light reduction encountered during large, commercial dredging operations, and specifically the light reduction experienced during the Geraldton Port Expansion Dredging Operation (Figure 2.2, Geraldton Port Authority, unpublished data). No procedural control for light reduction was used as previous studies have shown that this is not feasible and creates another light reduction treatment (Bulthius 1983, Collier 2006, Mackey et al. 2007). The durations were selected to represent the range of durations in dredging operations along the coast of Western Australia (Michael Mulligan, Geraldton Port Authority pers. comm.). The timing factor was designed to test the effect of different carbohydrate reserves on the response of A. griffithii to light reduction. Previous studies have shown that carbohydrate storage products in the rhizome during winter are unlikely to sustain the plant over low light conditions (Carruthers & Walker 1997). The hypothesis was that a meadow shaded at the end of summer would have greater carbohydrate reserves and hence a greater capacity to cope with light reduction. The recovery of the meadow was followed after removal of the treatments. The impact of PPFD reduction was tested using a fully orthogonal BACI design (Green 1979), whilst the recovery from light reduction was tested using a repeated measures design, sampling within the same plots (Quinn & Keough 2002). The method used to establish the treatments was similar to that used by Gordon et al (1994) and Collier (2006). Each plot was 4.5 m x 3 m and constructed from six, 2 m long cement reinforcing bar (12 mm diameter) driven into the sediment with pole drivers. A PVC (32 mm diameter Class 18) frame was threaded over the reinforcing bar and positioned ~ 1.2 m above the sediment then attached to the reinforcing bar with bolts. The PPFD reduction treatments were created with shade cloth: moderate – 50% shade cloth and high – 80% shade cloth. The cloth was attached to the PVC frame with cable ties (380 x 5 mm) and replaced every 3–6 weeks (Figure 5.1). An effective sampling area of 3 m x 1.5 m (4.5 m2) was chosen to avoid the effects of incident light which encroached into plots from each side by approximately 0.75 m (Collier 2006, Mackey et al. 2007). No plots were constructed for the samples collected before the experiment began. These were collected randomly within the meadow, but outside of the plots. Plot allocation was randomised within these treatments. To assess the impact of PPFD treatments, measurements were made or samples collected at the end of the treatment periods. To assess recovery two approaches were made depending on the sampling duration. For the 3-month plots, samples were collected 3 and 10 months after the shade cloth was removed. The 6- and 9-month samplings were heavily impacted and no leaves were observed in the plots 3 months after the shade cloth was removed. These plots were all sampled in August 2007 such that Post-summer 6-

month plots had 23 months of recovery, Post-summer 9-month plots - 21 months, Post- winter 6-month plots - 17 months and Post-winter 9-month plots - 15 months of recovery. The timing of these sampling events are summarised in Table 5.1.

Figure 5.1: Photographs showing experimental design and sampling methodology. a) Shade screen used to create PPFD reduction treatment. b-e) Attaching shade cloth onto frame with cable ties. f) A single plot and replicate in the experiment. g) Light logger with cleaner unit under shade screen. h) Plot-code attached to frame. i) Repairing frame with underwater drill. j-k) Removing shade cloth. l) Divers sampling in a plot. m) Taking above-ground biomass sample. n) Hole-punching leaves to estimate leaf growth. o) Taking photosynthetic measures with a PAM fluorometer. p-q) Collecting rhizome for physiology measures. r) Boat loaded with old shade cloths.

5.2 Site location The experiment was located in Jurien Bay Marine Park, on the central Western Australian coast in a continuous, monospecific meadow (> 6 ha) of Amphibolis griffithii at 4.5 m water depth, 200 – 300 m NE of Boullanger Island (30°18’34”S, 115°00’26”E – WGS 84) (Figure 5.2).

Figure 5.2: Map showing experimental study site in Jurien Bay, near Boullanger Island.

Table 5.1: Timing of sample collection in this experiment. Duration Post-summer Post-winter Impact Sampling Before (Time 0) 10th March 2005 16th Sept 2005 3 months 14th June 2005 20th Dec 2005 6 months 16th Sept 2005 29th March 2006 9 months 29th Nov 2005 4th July 2006 Recovery sampling 3 – 3 months 7th Oct 2005 28th March 2006 3 – 10 months 18th April 2006 6th November 2006 6 30th August 2007 30th August 2007 9 30th August 2007 30th August 2007

5.3 Environmental parameters measured

5.3.1 Light data ‘Odyssey Dataflow’ submersible incident light sensors were deployed throughout the sampling period to estimate available PPFD reaching the top of the seagrass canopy in each light intensity treatment. Instantaneous PPFD (µmol m-2 s-1) was measured every 10 - 15 minutes, integrated over 1 minute throughout the entire experiment. Cleaner units were deployed with the light loggers to maintain clean sensors (Carruthers et al. 2001), cleaning every 15 minutes. Light data were summarised as total PPFD over the treatment period (% control), average daily PPFD (mol m-2 day-1), average instantaneous PPFD -2 -1 (µmol m s ) and hours of saturating irradiance per day (HSAT) (Dennison & Alberte -2 -1 1982) where HSAT was set at 55 (µmol m s ) (Masini et al. 1995). All light loggers were calibrated against a standard light source.

5.3.2 Water temperature Water temperature data were sourced from Department of Environment and Conservation (unpublished data).

5.4 Biological parameters measured

5.4.1 Field sample collections At the end of each treatment, samples were collected to measure biomass, density, morphology, growth and physiology of the A. griffithii (Table 5.2). Above-ground samples for biomass, density and morphology were pooled from five randomly selected 10 x10 cm units within a 50 x 50 cm quadrat (0.05 m2). Leaf growth was estimated by tagging all leaf clusters on 6 stems using the leaf punch methodology (Short & Duarte 2001). Leaves were punched 1 – 2 weeks before the end of the treatment and then these stems were collected at the same time as the above-ground samples. Six stems with associated below-ground rhizome material were collected separately from within the plot for physiology measures

5.4.2 Seagrass biomass, density and morphology measures The number of stems, clusters and leaves from each above-ground sample were counted to estimate stem, cluster and leaf density. A cluster was defined as a group of leaves separated from the next cluster by visible stem. A leaf was counted if it had emerged from the sheath. One stem was randomly selected from the above-ground biomass sample for additional measures of leaf length and width, internode length and branching frequency. The length and width of the oldest leaf in each cluster and the lengths of the five internodes behind each cluster (most recently produced internodes) were measured. First, second and third order branches were counted to determine branching frequency. The number of leaves per cluster and stem height were counted from the entire sample. Leaves and stems were separated and all algal epiphytes and faunal epiphytes removed by razor blade and tweezers. Each component was dried separately at 60°C for 24 hours and then weighed. All measures (apart from stem height) were recorded in 10 cm height

categories from the base of the stem. The biomass parameters were separated into height categories only for the Post-summer treatments and likewise branching frequency was calculated in only the Post-summer treatments. Maximum, average and 80th percentile canopy heights were calculated from the stem height data in the sample. The average number of leaves per stem and clusters per stem in a plot were calculated from the morphology data. Leaf Area Index (LAI) was calculated from the average leaf area in each sample (m2) multiplied by the leaf density (m-2) in the sample.

5.4.3 Seagrass growth measures The leaf growth from 10-30 tagged clusters at known height categories were measured from each plot. The proportion of clusters that grew was calculated from these 10-30 clusters. Leaf extension was calculated as the sum of all leaves that grew in a cluster (mm leaf cluster-1 day-1). Leaf productivity (g DW m-2 day-1) was estimated from a regression (y = 0.369x) of 316 paired measures of leaf extension (mm leaf cluster-1 day-1) and biomass (g DW) multiplied by the cluster density in the plot. Leaf growth in the recovery period was measured only once using plots shaded for 3-months, 3 months after shade was removed.

5.4.4 Seagrass physiology measures Leaf and rhizome samples for physiology analysis were scraped free of epiphytes, dried and ground in a mill grinder. Only living material was used and leaves were sampled from the upper (40-50 cm) and lower (20-30 cm) canopy. Samples were analysed in a continuous flow isotope ratio mass spectrometry (20-20 IRMS, Europa, Crewe, United Kingdom) for carbon (% DW), nitrogen (% DW) and the atomic ratio of both (δ13C, δ15N). Soluble sugars (% DW) and starch (% DW) were analysed by colorimetric determination (420 nm) with an amylase pre-digest to convert the starch to glucose (Yemm & Willis 1954).

5.4.5 Seagrass photosynthesis – Rapid light curve measures Rapid light curves using the PAM fluorometer (WALZ) were performed on the seagrass leaves from the Post-winter 3-month plots (Day 0, 3, 7, 21, 41, 75 during the impact phase and Day 1, 13, 28, 120 during the recovery phase) and Post-winter 6-month plots (at the end of the impact phase and Day 1 and 47 during the recovery phase). The Rapid Light Curve function was run with light intensities 0, 1, 26, 49, 78, 113, 178, 251, 396, 598 µmol m-2 s-1, with each intensity run for10 seconds. Leaves in the upper (40+ cm) and lower (< 30 cm) canopy were measured. From the rapid light curve data the maximum relative electron transport rate (Max rETR), the saturating light intensity (Ek), Photo-inhibiting light intensity (Ei) and photosynthetic efficiency (α) were calculated following the methods recommended in Ralph and Gademann (2005).

5.5 Data quality All samples were collected using standard procedures by trained divers, and processed using standard procedures by trained research assistants. An independent person checked all electronic data for data entry errors by comparison against original records.

Table 5.2: Parameters measured in the experiment. Biomass & Density Morphology Growth Physiology

Total above-ground biomass2 Leaves per cluster1,2 Cluster growth2 Leaf sugars & starch (g DW m-2) (%) (% DW) Leaf biomass2 Leaves per stem1 Leaf extension1,2 Rhizome sugars and starch (g DW m-2) (mm cluster-1 day-1) (% DW) Stem biomass2 Clusters per stem1 Leaf productivity1, 2 Leaf nitrogen (g DW m-2) (g DW m-2 day-1) (% DW) 15 Algal epiphyte biomass2 Maximum canopy height Leaf ∂ N (g DW m-2) (cm) Faunal epiphyte biomass2 Average canopy height Leaf carbon (g DW m-2) (cm) (% DW) th 13 Leaf density2 80 percentile canopy height Leaf ∂ C (m-2) (cm) Cluster density2 Leaf length1,2 Leaf C:N ratio (m-2) (mm) Stem density Leaf width1,2 Rhizome nitrogen (m-2) (mm) (% DW) Leaf area index Rhizome ∂15N (m2 leaf m-2) Internode length1,2 Rhizome carbon (mm) (% DW) 13 Branching frequency2, 3 Rhizome ∂ C (%) Rhizome C:N ratio

Rapid light curves4 1. Parameter averaged from multiple readings within a plot. 2. Parameter also measured by 10 cm height category. 3. Parameter measured in Post-summer plots only. 4. Parameter measured in Post-winter plots only.

5.6 Statistical analysis

5.6.1 Impact and recovery from PPFD reduction treatments To analyse the impact of light reduction treatments on seagrass parameters a BACI design with used. Data were tested for normality using the Kolmogorov-Smironov goodness of fit test (Zar 1999) and heterogeneity using Cochran’s Test (Cochran 1951) and transformed if necessary. If, after transformations, the data were not normally distributed and the data was unimodal it was assumed that due to the large number of samples the analysis would be robust to deviations from normality (Box 1953, Underwood 1997). With variances heterogeneous after transformation there was an increased risk of a Type 1 error but due to the large, balanced experimental design (18-24 treatments with 5 replicates per treatment) ANOVA is robust to this departure (Underwood 1997). However, the significance level was set to 0.01 in these circumstances as a precaution. Fishers LSD post-hoc tests were carried out if there were significant factors or interactions in the ANOVA. Measurements during the recovery phases were taken repeatedly from within the previously shaded treatment plots. Consequently, to analyse recovery from light reduction treatments, a repeated measures design was used incorporating the impact data

and either 1 or 2 recovery periods, depending on the duration of the shading. The two time periods (Post-summer and Post-winter) were analysed separately. Where more than one measure for a parameter was taken in each plot these were averaged and a single value used in the BACI or Repeated measures ANOVA. If less than 10 clusters were used to estimate growth an average was not calculated, with less than 4 clusters, the number of leaves per cluster was not calculated. Data summarised by canopy height was not analysed for this report, but presented graphically to show trends through the canopy.

5.6.2 Sub-lethal indicators of PPFD reduction Summary statistics were generated for the parameters that we proposed as potential sub- lethal indicators of PPFD reduction stress. The control data were pooled across all treatments and the 1st, 5th, 20th, 50th, 80th, 95th and 99th percentile calculated as well as average, standard deviation, standard error and coefficient of variation (CV – average/standard deviation). A median value was calculated for each PPFD reduction treatment (i.e. each combination of intensity, duration, timing). When there were multiple readings in a plot (i.e. the multiple leaf extension rates measured from within a single plot and then averaged for statistical analysis) these measures were included in the calculation of the median, not just the averages for the plot used in the statistical analysis. The percentile data are presented as these are consistent with the current approaches to environmental quality criteria development in Western Australia. However, development of criteria based on any recommended parameters would require further detailed development.

6. Environmental Responses

6.1 Light (PPFD) The PPFD treatments resulted in a reduction to 13–19% of ambient PPFD (Total irradiance) in the Moderate treatments and 5 –11% in the High treatments (Table 6.1). The degree of light reduction varied depending on the time of year. The Post-summer treatments applied shading during autumn-winter when ambient PPFD was low and this, coupled with the shading treatment, resulted in the lowest absolute PPFD and lowest average instantaneous PPFD in the Post-summer treatments. The same patterns were observed for the hours of saturating irradiance (HSAT), but the reduction relative to the controls was not as great as observed for total irradiance (Table 6.1). The irradiance in the Control plots is close to the range measured in another study on A. griffithii, 5.2 (winter)–36.6 (summer) mol m-2 day-1 (Carruthers & Walker 1997), although higher daily irradiance was observed in this study in Post-winter 3- and 6- months which ran during the summer of 2005/06. Interestingly, impacts due to PPFD reduction were observed in the experimental treatments where the daily PPFD was greater than 5.2 mol m-2 day-1. However, in the study by Carruthers and Walker (1997) 5.2 mol m-2 day-1 was observed in winter whereas these lower values were observed over spring and summer in our study when the water temperature was higher and therefore the PPFD required to saturate photosynthesis is higher (Masini & Manning 1997). Average daily HSAT was also higher in this study than has been previously recorded at another site (Carruthers & Walker 1997).

Canopy PPFD attenuation A. griffithii canopy transmission was on average 25%, e.g. 25% of the light at the top was transmitted to the bottom of the canopy under control conditions (Table 6.2). Both the intensity and duration of PPFD reduction increased the transmission such that the 9- month High treatment had 95% transmission of PPFD (Table 6.2).

6.2 Water temperature Average water temperature ranged from 18.7 to 21.7 °C over the different experimental periods. The maximum average water temperature occurred in the Post-summer 3-month period whilst the minimum occurred in the Post-winter 3-month period (Table 6.3).

Table 6.1: PPFD summary data for all treatments in the experiment determined from in situ PPFD loggers. Numbers in brackets are the% relative to the controls. HSAT intensity was set at 55 µmol m-2 s-1 (Masini & Manning 1997). Timing & Intensity Total Average daily Average Cumulative Average Duration irradiance irradiance instantaneous HSAT HSAT irradiance mol m-2 mol m-2 day-1 µmol m-2 s-1 Hours Hours Post -summer 3 months Control 1 942 (100) 19.0 218 962 (100) 9.52 Moderate 317 (16) 3.1 35 435 (45) 4.31 High 95 (5) 0.9 11 118 (12) 1.17 6 months Control 3258 (100) 16.6 191 1821 (100) 9.34 Moderate 553 (17) 2.8 31 706 (39) 3.62 High 207 (6) 1.0 12 299 (16) 1.53 9 months Control 6690 (100) 25.4 277 2805 (100) 10.09 Moderate 1252 (19) 4.8 52 1361 (49) 4.90 High 64 (10) 2.4 27 590 (21) 2.12 Post-winter 3 months Control 3 996 (100) 41.6 508 1098 (100) 12.20 Moderate 715 (18) 7.4 93 641 (58) 7.13 High 447 (11) 4.7 59 286 (26) 3.18 6 months Control 8 051 (100) 41.3 481 2329 (100) 12.13 Moderate 1 128 (14) 5.8 68 1371 (59) 7.14 High 691 (9) 3.5 39 627 (27) 3.27 9 months Control 9 416 (100) 32.2 383 3215 (100) 11.36 Moderate 1 256 (13) 4.3 51 1803 (56) 6.37 High 784 (8) 2.7 29 803 (25) 2.84

Table 6.2: PPFD attenuation through the seagrass canopy. Duration Treatment Intensity Treatment Canopy Attenuation (% transmission from top to bottom of canopy) Average of all Control 25

3-months PPFD reduction Moderate 25 High 40 6-months PPFD reduction Moderate 60 High 85 9-months PPFD reduction Moderate 90 High 95

Table 6.3: Water temperature expressed as average over the experimental period. Data from Department of Environment and Conservation. Timing and Water Duration temperature (°C) Post -summer 3 months 21.7 6 months 20 9 months 19.6 Post-winter 3 months 18.7 6 months 19.9 9 months 19.8

7. Seagrass Response to PPFD Reduction Treatments

The response of Amphibolis griffithii seagrass meadows to PPFD reduction is presented by groups of parameters: Biomass, Density, Morphology, Growth, Sexual reproduction and Physiology. The main factor of interest is the effect on intensity of PPFD reduction (Moderate and High vs. Control) and how this varies depending on the duration (3-, 6- and 9-months) and timing (Post-summer, Post-winter). All graphs and statistical analyses are presented and discussed with respect to the intensity effect. Firstly, the response expressed as a unit area measure (e.g. biomass, density), or as an average through the canopy (e.g. morphology, growth), or as a measure from 20-30 cm in the canopy (e.g. physiology) is presented graphically and with statistical analysis (Section 7.1-7.6). The responses are then summarised into a ‘Pathway of Response’ to PPFD reduction (Section 7.7).

7.1 Biomass

7.1.1 Total above-ground biomass There was a significant decrease in total above-ground seagrass biomass with PPFD reduction treatments (Table 7.1, Figure 7.1, 7.2, 7.3). In Post-summer treatments total seagrass biomass declined by 45% with 3-months Moderate PPFD reduction and by 55% with High intensity, but only the High intensity treatment was significantly different to the Control (Average total above-ground biomass ± standard error [g DW m-2]: Control – 432 ± 117; Moderate – 243 ± 44; High – 187 ± 42). After 6-months PPFD reduction, biomass continued to decline in the High treatments (to 75%) but remained steady in the Moderate treatment (at 45%). At this time both Moderate and High PPFD reduction treatments were significantly different to the Control, and to each other. After 9-months of PPFD reduction the biomass continued to decline in both the Moderate (to 65%) and High (to 80%) treatments. Again both the Moderate and High treatments were significantly different to the control and to each other. Average total above-ground biomass (g DW m-2) reached minimums after 9-months of reduced PPFD: Moderate – 145 ± 22; High – 90 ± 22 compared to Control – 409 ± 45). There was a slightly different response in the Post-winter treatments (Table 7.1, Figure 7.1, 7.2, 7.3). After 3-months of PPFD reduction there was no effect on the total above- ground seagrass biomass in the Moderate treatment but there was a decline of 60% in the High treatment (Average total above-ground biomass [g DW m-2]: Control – 381 ± 74; Moderate – 413 ± 34; High – 153 ± 7). The High treatment was significantly different to the Control and Moderate at this time. Following 6-months of reduced PPFD both the Moderate and High treatments were significantly lower than the controls (both 60% lower), but not different to each other. After 9-months the biomass continued to decline in both the Moderate treatment (to 65%) and High (to 70%) (Average total above-ground biomass [g DW m-2]: Control – 372 ± 26; Moderate – 126 ± 20; High – 105 ± 23). Moderate and High treatments were significantly different to the Control but not to each other after 9-months.

Figure 7.1: Photographs of representative plots of A. griffithii at the end of each PPFD reduction treatment. Plots were shaded at no (Control), Moderate or High intensity for 3, 6 or 9-months, commencing at the end of summer or winter. The significant effect of shading on leaf biomass is obvious thinning of the canopy in the 3-month treatments and the loss of leaves with only stem material remaining in the longer duration treatments.

Figure 7.2: Photographs of stems from representative above-ground biomass samples from plots at the end of each PPFD reduction treatment. Photographs were not taken from the Post-summer 3-month plots and the Post-summer 6-month Moderate is missing.

7.1.2 Leaf biomass Most of the change in total above-ground biomass was due to loss of leaves (Table 7.1, Figure 7.3). The main difference in response between total biomass and leaf biomass was that in the Post-summer treatments both the Moderate and High treatments were significantly different to the controls after 3-months, but there was no significant difference between Moderate and High in all of the durations (3-, 6-, 9-months). The leaf biomass in the Post-winter treatments followed the same pattern as total biomass. After 9- months of High PPFD reduction there were no leaves remaining and only 2 g DW m-2 in the Post-summer Moderate treatment, but 13 g DW m-2 in the Post-winter Moderate treatment.

7.1.3 Stem biomass The stem biomass comprised the remainder of the above-ground biomass (Table 7.1, Figure 7.3). There were some significant impacts due to PPFD reduction. In the Post- summer treatments there was a significant reduction in stem biomass with the 6 and 9- month treatments, but only with High PPFD reduction. In the Post-winter treatments the High plots had a significant reduction in stem biomass after 6-months, but not after 3- and 9-months. With all leaves lost in these plots with High shading, the above-ground biomass attributable to stems was ~ 100 g DW m-2.

7.1.4 Algal epiphyte biomass There was a significant decrease in algal epiphyte biomass with PPFD reduction treatments (Table 7.1, Figure 7.3). In the Post-summer treatments there was a significant reduction with 3-months, 80% decline in the Moderate and 90% decline in the High treatment. The decline continued at 6-months, to 90% in Moderate and 95% in High. However, at 9-months there was a small increase in the amount of algal epiphyte biomass. In the Moderate treatment algal epiphytes were 80% less than the controls and the High treatment, 90%. Overall the algal epiphyte biomass in both PPFD reduction treatments were significantly lower than the controls at all durations, but there was no significant difference between Moderate and High. The minimum algal epiphyte biomass was 12 g DW m-2 (3- and 6-month High treatment average) compared to a maximum of 260 g DW m-2 in the Control (Post-summer 6-month treatment average). The Post-winter treatments showed a slightly different response. After 3-months there was no significant reduction in the Moderate treatment but there was in the High treatment (70%), and the Moderate was significantly different to the High. After 6- months the High treatment was still the only PPFD reduction treatment significantly lower than the Control (80%). The Moderate treatment was at an intermediate level (50%), not significantly different to the Controls or the High treatment. After 9-months both the Moderate (80%) and High (90%) were significantly lower than the Control. The minimum algal epiphyte biomass was 20 g DW m-2 (9-month High treatment average) compared to a maximum of 205 g DW m-2 in the Control (Post-winter 3-month treatment average).

Figure 7.3: Biomass (g DW m-2) of A. griffithii and algal epiphytes following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Total above-ground seagrass biomass; b) Leaf biomass; c) Stem biomass; d) Algal epiphyte biomass. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

The majority of the algal epiphyte biomass was on stems, 68–97% (range of average in control treatments from entire experiment) and the response to PPFD reduction treatments of algal epiphytes on the stem was similar response to total algal epiphyte biomass. Therefore the algal epiphyte biomass was not simply a function of loss of substratum (leaves) (Appendix: Figure 17.1).

7.1.5 Faunal epiphyte biomass The faunal epiphyte biomass was much lower than the algal epiphyte biomass (maximum of 36 g DW m-2 - control average). There was an effect of PPFD reduction on the faunal epiphyte biomass, but only in the Post-summer treatment (Table 7.1, Appendix: Figure 17.2). At 6-months both the Moderate and High treatment were significantly lower than the Control and at 9-months just the High was significantly lower than the control. No effects were detected in the Post-winter treatment.

7.2 Density

7.2.1 Leaf density There was an effect of PPFD reduction treatments on leaf density (Table 7.1, Figure 7.4). The response of leaf density mirrored that of leaf biomass, though the magnitude of change was less. For example in the Post-summer treatments with 3-months PPFD reduction leaf density had declined 45% - Moderate and 65% - High, relative to the Control, whereas leaf biomass showed a 55% and 70% decline in the Moderate and High treatments, respectively. This indicates that PPFD reduction results in a reduction of leaf biomass due to loss of leaves.

7.2.2 Cluster density Cluster density also responded to the PPFD reduction treatments (Table 7.1, Figure 7.4). The response was similar to, but slower than that of leaf density. For example the Post- summer 3-month Moderate treatments were not significantly different to the Controls, although there was a difference in cluster density in the High treatment. By 6-months, however, the cluster density in the Moderate treatments was significantly lower than the controls. These results indicate that the first changes are due to leaf loss from clusters but as reduction of PPFD intensity and duration increases whole clusters are lost.

7.2.3 Stem density Stem density was impacted by the PPFD reduction treatments but the response was not consistent over Post-summer and Post-winter treatments (Table 7.1, Figure 7.4). In Post- summer treatments there was a significant decline in stem density in the High treatments at 6- and 9-months, whereas in the Post-winter treatments stem density was significantly lower in the High treatment at 3-months but no effects were detected at 6- and 9-months .

Figure 7.4: Density (m-2) of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Leaf density; b) Cluster density; c) Stem density. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Table 7.1: Results of statistical analysis to determine significant effects of Timing, Duration and Intensity of PPFD reduction treatments on Amphibolis griffithii seagrass meadow biomass and density parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Parameter df MS F p Parameter df MS F p -2 -2 Above-ground biomass Ln (g DW m ) Leaf Biomass1 (g DW m ) Time (T) 1 0.18 0.94 0.33 Time (T) 1 21.0 1.22 0.27 Duration (D) 3 6.75 35.8 *** Duration (D) 3 519 30.2 *** Intensity (I) 2 9.03 47.9 *** Intensity (I) 2 610 35.4 *** T x D 3 0.48 2.55 0.06 T x D 3 26.4 1.54 0.21 T x I 2 0.11 0.60 0.55 T x I 2 22.4 1.30 0.28 D x I 6 1.90 10.0 *** D x I 6 117 6.78 *** T x D X I 6 0.29 1.52 0.18 T x D X I 6 8.50 0.49 0.81 -2 -2 Stem biomass Ln (g DW m ) Algal epiphyte biomass Sqrt, 1 (g DW m ) Time (T) 1 0.34 1.74 0.19 Time (T) 1 4.74 8.32 ** Duration (D) 3 1.71 8.66 *** Duration (D) 3 6.20 10.9 *** Intensity (I) 2 2.39 12.1 *** Intensity (I) 2 23.0 40.3 *** T x D 3 0.60 3.04 * T x D 3 2.76 4.84 ** T x I 2 0.07 0.37 0.69 T x I 2 0.97 1.70 0.19 D x I 6 0.50 2.52 * D x I 6 4.11 7.21 *** T x D X I 6 0.34 1.71 0.12 T x D X I 6 0.77 1.36 0.24 -2 -2 Faunal epiphyte biomass Ln, 1 (g DW m ) Leaf density1 (m ) Time (T) 1 1.29 1.49 0.22 Time (T) 1 630 0.02 0.89 Duration (D) 3 0.30 0.35 0.79 Duration (D) 3 1.00E+06 28.1 *** Intensity (I) 2 3.68 4.24 * Intensity (I) 2 1.42E+06 39.8 *** T x D 3 0.72 0.82 0.48 T x D 3 1.87E+05 5.25 ** T x I 2 4.16 4.80 * T x I 2 7.30E+04 2.05 0.13 D x I 6 2.21 2.55 * D x I 6 2.98E+05 8.37 *** T x D X I 6 3.50 4.03 ** T x D X I 6 3.16E+04 0.89 0.51 -2 -2 Cluster density1 (m ) Stem density Ln (m ) Time (T) 1 2.24E+03 0.64 0.43 Time (T) 1 1.74 9.85 ** Duration (D) 3 1.19E+05 33.7 *** Duration (D) 3 0.42 2.39 0.07 Intensity (I) 2 1.23E+05 34.8 *** Intensity (I) 2 0.64 3.61 * T x D 3 3.53E+04 10.0 *** T x D 3 0.39 2.23 0.09 T x I 2 7.09E+03 2.01 0.14 T x I 2 0.01 0.05 0.95 D x I 6 2.92E+04 8.27 *** D x I 6 0.29 1.66 0.14 T x D X I 6 4.94E+03 1.40 0.22 T x D X I 6 0.22 1.24 0.29 1. Not homogenous, significance level set to p < 0.01. Ln = Natural log transformed data. Sqrt = Square root transformed data.

7.3 Morphology

7.3.1 Leaves per cluster The number of leaves per cluster showed a significant response to PPFD reduction treatments (Table 7.2, Figure 7.5, Appendix: Figure 17.3). In the Post-summer 3- and 6- month treatments the number of leaves per cluster declined from 3 (Control) to 2 (Moderate and High). At 9-months no or very few clusters were present so it was not possible to calculate this parameter. The Post-winter treatment responded differently. At 3-months there was a reduction in leaves per cluster but only the High PPFD reduction was significantly lower than the Controls. By 6-months, however, both the Moderate and High were significantly lower than the Control.

7.3.2 Leaves per stem The average number of leaves per stem had a similar response as leaf biomass and leaf density showing significant declines with PPFD reduction (Table 7.2, Figure 7.5). After 3-months of Post-summer PPFD reduction there were on average 20 leaves per stem in the Moderate and 13 in the High treatment compared to 34 in the Control. After 9-months of PPFD reduction there was on average 1 leaf per stem in the Moderate and 0 in the High. Compared to the Post-summer treatment the Post-winter plots did not decline as significantly in the 3-month treatments (Average leaves per stem: Control – 37, Moderate – 35, High – 25). However, after 6- and 9-months there were major reductions with only 6 leaves left in the 9-month Moderate and none in the 9-month High.

7.3.3 Clusters per stem The average number of clusters per stem had a similar response as cluster density showing significant declines with PPFD reduction (Table 7.2, Figure 7.5). The number of clusters per stem did not decline as rapidly as the number of leaves per stem.

Figure 7.5: Morphology of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Average leaves per cluster; b) Average leaves per stem; c) Average clusters per stem; d) Canopy height – 80th percentile (cm). Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as not enough leaves were present to determine the measure.

7.3.4 Canopy height The canopy height (80th percentile) was significantly impacted by PPFD reduction, but only after 6-months (Table 7.2, Figure 7.5, Appendix: Figure 17.4). This reflects the initial persistence of stems, which maintained the canopy height and only later became necrotic or abraded at the tips, resulting in the delayed effect on canopy height.

7.3.5 Leaf size There was no significant effect of PPFD reduction on leaf length (based on the largest leaf in a cluster) with 3- and 6-month durations (Table 7.2, Figure 7.6). However, at 9- months in the Post-winter treatment leaf length in the Moderate treatment (36 mm) was significantly lower than the Control (52 mm). There were no leaves in the High treatment to make any measures. Leaf width was significantly lower with PPFD reduction but only after 6-months.

7.3.6 Leaf area index (LAI) The index combining leaf area (length x width) with leaf density declined rapidly and significantly in Post-summer after 3-months PPFD reduction (Table 7.2, Figure 7.6). In Post-winter treatments responses were a little slower in the moderate treatments at 3- months. LAI showed a similar response as leaf density and biomass.

7.3.7 Internode length There was a significant effect of PPFD reduction on internode length, however this response was not consistent across treatments (Table 7.2, Figure 7.6). In the Post-summer 3-month plots average internode length in the High PPFD reduction treatment (7.3 mm) was greater than the Control (5.5 mm), and after 6-months both Moderate (7.3 mm) and High (6.7 mm) were longer than the Control (5.4 mm), but at 9-months there was no significant difference between PPFD intensity treatments, whereas, in the Post-winter plots there was a significant difference after 9-months and the High PPFD reduction treatment (4.2 mm) was significantly lower than the Moderate (5.2 mm) and the Control (5.7 mm).

Figure 7.6: Additional morphology of A. griffithii following PPFD reduction treatments of Timing: Post- summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Average leaf length (mm); b) Average leaf width (mm); c) Leaf area index (m2 leaf m-2); d) Average internode length (mm). Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as not enough leaves were present to determine the measure.

Table 7.2: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass morphology parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Parameter df MS F p Parameter df MS F p Average leaves per cluster1, 2 Median leaves per cluster Time (T) 1 1.10 19.2 *** Time (T) 1 1.41 9.41 ** Duration (D)3 2 7.05 123 *** Duration (D) 3 2 7.73 51.7 *** Intensity (I) 2 5.54 96.6 *** Intensity (I) 2 6.32 42.2 *** T x D 2 1.20 20.8 *** T x D 2 1.40 9.35 *** T x I 2 0.33 5.70 ** T x I 2 0.48 3.23 * D x I 4 2.38 41.5 *** D x I 4 2.66 17.8 *** T x D X I 4 0.17 3.00 * T x D X I 4 0.24 1.61 0.18 Mode leaves per cluster Leaves per stem1, 2 Time (T) 1 1.17 7.62 ** Time (T) 1 600 12.2 ** Duration (D) 3 2 9.94 64.9 *** Duration (D) 3 3.97E+03 80.9 *** Intensity (I) 2 7.49 48.9 *** Intensity (I) 2 5.82E+03 119 *** T x D 2 1.92 12.6 *** T x D 3 121 2.50 0.06 T x I 2 0.33 2.16 0.12 T x I 2 100 2.00 0.13 D x I 4 3.10 20.2 *** D x I 6 1.27E+03 26.0 *** T x D X I 4 0.16 1.04 0.39 T x D X I 6 31 0.60 0.70 Clusters per stem1, 2 Maximum canopy height (cm) Time (T) 1 31.4 5.46 * Time (T) 1 763 12.9 *** Duration (D) 3 447 77.7 *** Duration (D) 3 452 7.62 *** Intensity (I) 2 466 81.0 *** Intensity (I) 2 560 9.44 *** T x D 3 35.9 6.24 ** T x D 3 331 5.59 ** T x I 2 10.8 1.87 0.16 T x I 2 47.1 0.79 0.45 D x I 6 111 19.3 *** D x I 6 148 2.50 * T x D X I 6 4.10 0.71 0.64 T x D X I 6 59.5 1.00 0.43 Average canopy height (cm) 80th percentile canopy height (cm) Time (T) 1 188 7.54 ** Time (T) 1 244 7.09 ** Duration (D) 3 525 21.1 *** Duration (D) 3 316 9.18 *** Intensity (I) 2 309 12.4 *** Intensity (I) 2 523 15.2 *** T x D 3 248 9.96 *** T x D 3 250 7.26 *** T x I 2 13.2 0.53 0.59 T x I 2 31.8 0.92 0.40 D x I 6 99.9 4.01 ** D x I 6 168 4.88 *** T x D X I 6 21.3 0.86 0.53 T x D X I 6 27.0 0.78 0.58 Average leaf length1 (mm) Average leaf width1,2 (mm) Time (T) 1 235 4.06 * Time (T) 1 0.03 0.06 0.80 Duration (D) 2 182 3.14 * Duration (D) 2 3.00 7.82 ** Intensity (I) 2 4.90 0.08 0.92 Intensity (I) 2 1.25 3.27 * T x D 2 362 6.25 ** T x D 2 0.01 0.04 0.97 T x I 2 117 2.02 0.14 T x I 2 1.11 2.90 0.06 D x I 4 60.3 1.04 0.39 D x I 4 1.61 4.19 ** T x D X I 4 63.3 1.09 0.36 T x D X I 4 0.70 1.83 0.13 2 -2 Leaf area index1 (m leaf m sediment) Average internode length (mm) Time (T) 1 1.68 1.02 0.31 Time (T) 1 33.8 33.9 *** Duration (D) 3 44.2 26.9 *** Duration (D) 3 4.74 4.76 ** Intensity (I) 2 62.1 37.7 *** Intensity (I) 2 1.13 1.14 0.32 T x D 3 2.93 1.78 0.15 T x D 3 3.83 3.84 * T x I 2 4.03 2.45 0.09 T x I 2 4.73 4.75 * D x I 6 13.3 8.12 *** D x I 6 1.01 1.02 0.41 T x D X I 6 1.47 0.90 0.50 T x D X I 6 1.51 1.52 0.18 1. Not homogenous, significance level set to p < 0.01. 2. Not normally distributed. 3. Nine month duration treatment not included

7.4 Growth

7.4.1 Leaf productivity There was a significant effect of PPFD reduction on leaf productivity (Table 7.3, Figure 7.7). This was due to some clusters not growing at all and a reduction in leaf extension in the clusters that did grow (Figure 7.7), though the response differed between treatment timing. In the Post-summer treatment after 3-months many of the clusters showed no growth, this did not occur in the Control (0%) but in the Moderate (42%) and High (70%). Leaf extension rates decreased by 85% on average. After 6-months the number of clusters that did not grow reduced; Moderate (24%), High (32%) though there were far fewer clusters and the leaf extension rate remained low. At 9-months there was an almost complete loss of leaves and leaf growth measures could not be calculated. In the Post-winter treatment there was no significant difference in the number of clusters that did not grow after 3-months. However, there was a significant reduction in the leaf extension rate (Moderate – 40%, High - 65%) but it was not as great as was observed in the Post-summer 3-month treatment. However, at 6-months many of the tagged clusters showed no growth (Moderate – 50%, High - 85%) and the leaf extension rate continued to decline in the Moderate plots. The reduction in clusters and leaf growth resulted in a decline in leaf productivity after 3- months, except in the Post-winter 3-month Moderate PPFD reduction treatment, which did not show a significant decline until 6-months (Table 7.3, Figure 7.7).

Table 7.3: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass growth parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Parameter df MS F p Parameter df MS F p -1 -1 Cluster growth1, 2, 3 (% with no growth) Leaf extension1, 3 (mm cluster day ) Time (T) 1 840 6.61 * Time (T) 1 0.25 6.10 * Duration (D) 2 8.78E+03 69.1 *** Duration (D) 2 7.31 182 *** Intensity (I) 2 1.02E+04 80.2 *** Intensity (I) 2 2.41 60.0 *** T x D 2 7.04E+03 55.5 *** T x D 2 3.99 99.6 *** T x I 2 55.0 0.44 0.647 T x I 2 0.00 0.00 0.99 D x I 4 3.58E+03 28.2 *** D x I 4 0.85 21.1 *** T x D X I 4 2.09E+03 16.5 *** T x D X I 4 0.12 3.10 * -2 -1 Leaf productivity Ln, 1 (g DW m day ) Time (T) 1 3.46E-01 18.4 *** Duration (D) 3 4.68E+00 249 *** Intensity (I) 2 4.30E+00 229 *** T x D 3 2.11E+00 112 *** T x I 2 1.20E-01 6.40 ** D x I 6 8.00E-01 42.6 *** T x D X I 6 1.29E-01 6.90 *** 1. Not homogenous, significance level set to p < 0.01. 2. Not normally distributed. 3. Nine month duration treatment not included. Ln = Natural log transformed data.

Figure 7.7: Growth of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post- winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Cluster growth (% with no growth); b) Leaf extension rate (mm cluster-1 day-1); c) Leaf productivity (g DW m-2 day-1). Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as too few leaves were present to determine the measure.

7.4.2 Branching Branching frequency was expressed as the proportion of secondary and tertiary branches relative to the total number of branches. Generally, the branching frequency declined with PPFD reduction, but this did not occur in either intensity treatment until shading had been in place for 6-months (Figure 7.8). The tertiary branching frequency was calculated at the start of the Post-summer and Post- winter PPFD reduction. During the Post-summer PPFD reduction the branches that were tertiary branches ranged from 9 ± 2% (Mar 05) to 3 ± 1% (Jun 05), and during the Post- winter treatments 16 ± 2% (Sept 05) and 8 ± 5% (Nov 05), indicating there was more active branching at the time the Post-winter treatments began.

Figure 7.8: Branching frequency of A. griffithii following Post-summer PPFD reduction treatments Duration: 3-, 6-, 9-months and Intensity: Control, Moderate, High. Average with standard error bars.

7.5 Sexual reproduction Sexual reproduction was assessed by viviparous seedling density in March 2005 (Post- summer Time 0), June 2005 (Post-summer 3-months) and March 2006 (Post winter 6- months) when seedlings were present. Seedlings were observed in all treatments with 3- months PPFD reduction but were observed only in the Control plots after 6-months PPFD reduction (Figure 7.9).

Figure 7.9: Seedling production in A. griffithii following PPFD reduction treatments of Timing: Post- summer, Post-winter Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Seedling density (m-2); b) Seedling per stem. Average with standard error bars.

7.6 Physiology

7.6.1 Carbohydrates The main carbohydrate in Amphibolis griffithii was soluble sugars, accounting for up to 20% of dry weight (DW) in the rhizome and 15% DW in the leaves on average (from Control data). Starch in the rhizome was on average 2% of DW and in the leaves 3% DW. There was a significant effect of PPFD reduction on all forms of carbohydrates but the response depended on the type of carbohydrate and the treatment (Table 7.4, Figure 7.10). In Post-summer treatments there was a significant reduction in rhizome sugars after 3-months down to 5% DW and this was maintained throughout 6- and 9-months . Rhizome starch, leaf sugars and leaf starch declined after 6-months PPFD reduction relative to the controls. In the Post-winter treatments rhizome sugar was significantly lower in the 3-month PPFD reduction treatments (Moderate – 16% DW, High – 13% DW) compared to the Control (20% DW), but the concentrations were higher than the Post-summer 3-month treatments. Rhizome sugar continued to decline at 6-months and reached minimums of 6% DW (Moderate) and 4% DW (High) at 9-months. Rhizome starch showed variable patterns with duration and intensity, whereas leaf sugar and starch declined after 3- months of PPFD reduction in both the Moderate and High treatments.

Table 7.4: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass carbohydrate parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Parameter df MS F p Parameter df MS F p Rhizome sugars Ln (% DW) Rhizome starch (% DW) Time (T) 1 5.09 147 *** Time (T) 1 2.25 23.9 *** Duration (D) 2 1.63 47.3 *** Duration (D) 2 2.33 24.7 *** Intensity (I) 2 9.54 276 *** Intensity (I) 2 0.28 3.02 0.05 T x D 2 0.68 19.6 *** T x D 2 0.84 8.95 *** T x I 2 0.43 12.6 *** T x I 2 1.24 13.1 *** D x I 4 0.54 15.6 *** D x I 4 1.53 16.2 *** T x D X I 4 0.13 3.80 ** T x D X I 4 0.02 0.17 0.95 Leaf sugars - 3-months duration Ln (% DW) Leaf sugars - 6-months duration Ln (% DW) Time (T) 1 1.14 44.4 *** Time (T) 1 0.80 1.14 0.30 Intensity (I) 2 0.97 37.8 *** Intensity (I) 1 40.8 57.8 *** T x I 2 0.95 37.1 *** T x I 1 2.66 3.76 0.07 Leaf starch - 3-months duration (% DW) Leaf starch - 6-months duration (% DW) Time (T) 1 0.13 1.54 0.23 Time (T) 1 0.02 0.06 0.81 Intensity (I) 2 0.71 8.79 ** Intensity (I) 1 4.37 18.0 ** T x I 2 0.49 6.06 ** T x I 1 0.01 0.02 0.89 Not homogenous, significance level set to p < 0.01. 3. Nine month duration treatment not included. Ln = Natural log transformed data.

Figure 7.10: Carbohydrate content (% DW) of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Rhizome soluble sugars; b) Rhizome starch; c) Leaf soluble sugars; d) Leaf starch. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as not enough leaves were present to determine the measure.

7.6.2 Leaf nutrient content Leaf carbon content was significantly affected by PPFD reduction treatments, however, the response was not consistent with intensity, duration or timing (Table 7.5, Figure 7.11). Leaf nitrogen was significantly higher in the Post-summer 6-month High PPFD reduction treatment (2.1% DW) compared to the Control (1.8% DW) and both the Post- winter Moderate and High had greater leaf nitrogen content at 3-, 6- and 9-months (e.g. 3-months Control: 0.9%, Moderate: 1.4%, High – 1.4% DW) (Table 7.5, Figure 7.11). The leaf C:N ratio followed a similar pattern to leaf nitrogen (Table 7.5, Figure 7.11). At 9-months there was an almost complete loss of leaves and physiology measures could not be determined in all treatments.

Figure 7.11: Leaf nutrient content (% DW) of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Carbon; b) Nitrogen; c) Carbon:nitrogen ratio. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as not enough leaves were present to determine the measure.

7.6.3 Rhizome nutrient content Rhizome carbon was not affected by PPFD reduction (Table 7.5, Figure 7.12). Treatment averages ranged from 37.5 – 42% DW. There were significant differences in rhizome nitrogen with PPFD reduction treatments, however, the response was not consistent with duration or timing (Table 7.5, Figure 7.12). In the Post-summer, 6-month High treatment rhizome nitrogen was significantly higher than the Control (0.69 vs. 0.55% DW). In the Post-winter treatments rhizome nitrogen was significantly higher than the controls at 3- months and both Moderate and High were higher at 9-months. The rhizome C:N ratio followed a similar pattern to rhizome nitrogen (Table 7.5, Figure 7.12).

Figure 7.12: Rhizome nutrient content (% DW) of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Carbon; b) Nitrogen; c) Carbon:nitrogen ratio. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

7.6.4 Carbon and nitrogen isotopes There was no effect of PPFD reduction treatments on leaf and rhizome ∂13C and rhizome ∂15N (Table 7.5, Figure 7.13). However, there was a significant effect on leaf ∂15N and this was consistent across both timings and all durations (Table 7.5, Figure 7.13). Leaf ∂15N was approximately 30% lower in the Post-summer PPFD reduction treatments compared to the Control, but in the Post-winter treatments the percent reduction increased with duration and intensity (eg 3-month Moderate – 15%, High – 25%, 6- month Moderate – 25%, High – 50%).

Figure 7.13: Carbon and nitrogen stable isotope ratio (δ13C, δ15N) of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Rhizome δ15N; b) Leaf δ15N; c) Rhizome δ13C; d) Leaf δ13C. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as no leaves were present to determine the measure.

Table 7.5: Results of statistical analysis to determine effect of Timing, Duration and Intensity of PPFD reduction treatments on A. griffithii seagrass nutrient parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Parameter df MS F p Parameter df MS F p Leaf nitrogen1, 3 (% DW) Rhizome nitrogen1 (% DW) Time (T) 1 2.22 71.1 *** Time (T) 1 0.02 1.53 0.22 Duration (D) 1 1.71 54.7 *** Duration (D) 2 0.26 26.3 *** Intensity (I) 2 1.04 33.5 *** Intensity (I) 2 0.07 6.75 ** T x D 1 0.03 0.80 0.38 T x D 2 0.04 4.28 * T x I 2 0.60 19.2 *** T x I 2 0.00 0.35 0.70 D x I 2 0.10 3.04 0.06 D x I 4 0.03 2.67 * T x D X I 2 0.01 0.25 0.78 T x D X I 4 0.02 1.73 0.15 15 15 Leaf ∂ N3 Rhizome ∂ N Time (T) 1 0.04 0.26 0.61 Time (T) 1 5.84 19.1 *** Duration (D) 1 3.72 27.2 *** Duration (D) 2 4.65 15.2 *** Intensity (I) 2 7.29 53.3 *** Intensity (I) 2 0.06 0.20 0.82 T x D 1 4.95 36.2 *** T x D 2 0.63 2.06 0.13 T x I 2 0.30 2.16 0.13 T x I 2 0.78 2.54 0.09 D x I 2 0.42 3.08 0.05 D x I 4 0.38 1.23 0.31 T x D X I 2 0.03 0.19 0.82 T x D X I 4 0.62 2.02 0.10 Leaf carbon3 (% DW) Rhizome carbon1 (% DW) Time (T) 1 21.7 16.5 *** Time (T) 1 9.43 2.71 0.10 Duration (D) 1 32.8 25.0 *** Duration (D) 2 6.93 1.99 0.14 Intensity (I) 2 2.96 2.25 0.12 Intensity (I) 2 13.8 3.98 * T x D 1 3.61 2.75 0.10 T x D 2 21.0 6.03 ** T x I 2 3.52 2.68 0.08 T x I 2 2.75 0.79 0.46 D x I 2 2.70 2.05 0.14 D x I 4 2.80 0.81 0.53 T x D X I 2 8.84 6.73 ** T x D X I 4 1.81 0.52 0.72 13 13 Leaf ∂ C3 Rhizome ∂ C Time (T) 1 6.21 2.11 0.15 Time (T) 1 27.1 21.7 *** Duration (D) 1 19.8 6.74 * Duration (D) 2 0.27 0.22 0.80 Intensity (I) 2 1.69 0.57 0.57 Intensity (I) 2 0.09 0.07 0.93 T x D 1 14.0 4.76 * T x D 2 7.85 6.29 ** T x I 2 0.30 0.10 0.90 T x I 2 0.65 0.52 0.60 D x I 2 1.94 0.66 0.52 D x I 4 0.91 0.73 0.58 T x D X I 2 2.77 0.94 0.40 T x D X I 4 1.11 0.89 0.48 Leaf C:N1, 3 Rhizome C:N Time (T) 1 0.70 60.3 *** Time (T) 1 922 3.59 0.06 Duration (D) 1 0.58 50.2 *** Duration (D) 2 6.47E+03 25.2 *** Intensity (I) 2 0.53 45.5 *** Intensity (I) 2 3.90E+03 15.2 *** T x D 1 0.02 1.26 0.27 T x D 2 1.96E+03 7.61 ** T x I 2 0.38 32.3 *** T x I 2 702 2.73 0.07 D x I 2 0.01 0.77 0.47 D x I 4 578 2.25 0.07 T x D X I 2 0.02 1.51 0.23 T x D X I 4 482 1.87 0.12 1. Not homogenous, significance level set to p < 0.01. 3. Nine month duration treatment not included.

7.7 Impact – response pathway The results demonstrate response pathways of Amphibolis griffithii meadows to increased intensity and duration of light reduction over two different time periods, Post-summer and Post-winter (Figure 7.14). These pathways vary depending on the time of year. In this section only the parameters that showed a consistent response to PPFD reduction at a particular time (Post-winter or Post-summer), when (3-, 6- or 9-months) the significant difference occurred, and whether it occurred with Moderate and/or High treatments are presented. Parameters that did not show a significant effect of intensity or did not show a consistent response to PPFD reduction over a time (Post-summer or Post-winter) are not discussed.

7.7.1 Post-summer After 3-months, Moderate PPFD reduction in the Post-summer treatments showed morphological, growth and physiological changes, including a loss of leaves (leaf biomass, density, leaves stem-1, leaves cluster-1) and reductions in leaf growth (clusters 15 that grew, leaf extension, leaf productivity), rhizome sugars and leaf ∂ N (Figure 7.14). Algal epiphyte biomass also declined. These parameters also decreased in the High PPFD reduction treatment, but in addition there was a reduction in leaf clusters (cluster density, cluster stem-1) and increased internode length. After 6-months in the Moderate treatments these same responses were observed: a reduction in leaf clusters (cluster density, cluster stem-1) and increased internode length, as well as a reduced canopy height and reduced leaf carbohydrates and rhizome starch. In the High treatments and 6-months duration, there was also a loss of stems (stem biomass, density), reduced leaf width and increased leaf nitrogen content. At 9-months no additional responses were detected.

7.7.2 Post-winter After 3-months, Moderate PPFD reduction in the Post-winter treatments showed a reduction in leaf growth (leaf extension rate) but no change in the proportion of clusters that were growing or the leaf productivity (Figure 7.14). Some physiological changes were also observed including a reduction in leaf sugars and starch, an increase in leaf nitrogen with a resultant decrease in the leaf C:N ratio and a decrease in leaf ∂15N. No morphological changes were observed. However, in the High PPFD reduction treatment there were morphological changes such as loss of leaves (leaf biomass, leaf and cluster density, clusters stem-1, leaves stem-1, leaves cluster-1) and reduced leaf width, as well as a reduction in growth (leaf productivity) and physiological changes – reduced rhizome sugars. The algal epiphyte biomass also declined. After 6-months in the Moderate PPFD reduction treatment the same responses that occurred in the 3-month High were observed, except there was no decline in algal epiphyte biomass. In addition there was a reduction in canopy height and the proportion of clusters that grew declined. In the 6-month High PPFD reduction treatment no further changes were noted. Unlike the Post-summer treatments, there were additional changes after 9-months. In the Moderate treatment leaf length and width declined as well as algal epiphyte biomass, and in the High treatment there was a decrease in internode length.

7.7.3 Impact – response pathway summary Generally it is predicted that plants initially respond to stress through physiological adjustments, and if the stress continues or increases in intensity then morphological adjustments occur. If these adjustments are inadequate for maintenance of the plants’ energy balance, or if the stress continues or intensifies, then plant death will eventually occur (Waycott et al. 2005). In this experiment the intensity of stress (i.e. reduction in total PPFD and lower hours of saturating irradiance) was greater in the Post-summer 3- and 6-month treatments i.e. over the autumn and winter months with respect to Post- winter i.e. over the spring and summer months (see Table 7.1). These treatments therefore cover a continuum of stress, which is represented in the x-axis of Figure 7.15. From the variables which responded to the various treatments (Figure 7.14), it is apparent that as the intensity of stress (Moderate vs. High PPFD reduction) and the duration of stress (3-, 6-, 9-months) increased the initial response was physiological adjustment (Post-winter 3-month Moderate), followed by morphological adjustment (Post-summer 3- month Moderate and High, Post-winter 3-month High), which continued through the 6- and 9-months (Figure 7.15). Thus, the plants are responding in accordance with accepted pathways of response, though in this case the minimum stress applied in the Post-summer treatments (Moderate shading for 3-months) appears to have exceeded the capacity of the plant to cope through physiological responses alone.

Figure 7.14: Response pathway showing morphological, growth and physiological responses of A. griffithii meadows following PPFD reduction treatments of Timing (Post-summer, Post-winter), Duration (3-, 6-, 9-months) and Intensity (Moderate, High). Only those parameters that showed a significant difference from the control and had a consistent response within Timing treatment are shown.

Figure 7.15: Response of A. griffithii to increased intensity of stress (PPFD reduction) in the 3-month PPFD reduction treatments.

8. Canopy Response to 3-months PPFD Reduction Post- summer

The response to PPFD reduction at different heights through the canopy was examined in the 3-month Post-summer treatments. This approach was used to explore if the response to PPFD reduction was dependent upon the height within the canopy. Where responses are canopy-height dependent, this would need to be taken into account when monitoring seagrass condition or in the development of sub-lethal indicators. Previous studies (Mackey et al. 2007) indicated that some parameters only responded to PPFD reductions at some canopy heights, likely reflecting the complex variation in light availability through canopies and how this changes as canopies thin due to leaf loss. The data is plotted to demonstrate the trends through the canopy, but statistical analyses are not presented.

8.1 Biomass and density

Figure 8.1: Total seagrass, leaf and algal epiphyte biomass (g DW m-2) and cluster and leaf density (m-2) of A. griffithii at 10 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments Post-summer with Intensity: Control, Moderate, High factors. Average with standard error bars. All canopy heights are heights above sediment.

In the 3-month PPFD reduction treatments the largest amount of above-ground seagrass biomass was lost from 30-60cm above the sediment (Figure 8.1). This was reflected in both the leaf biomass and density. However, the most clusters were lost from 40-50 cm above the sediment. Algal epiphyte biomass was lost throughout the canopy, except in the lowest 20 cm near the sediment (Figure 8.1).

8.2 Morphology

Figure 8.2: Leaf length, width, leaves per cluster and internode length of A. griffithii at 10 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments Post-summer with Intensity: Control, Moderate, High. Average with standard error bars. All canopy heights are heights above sediment.

Leaf length and width increased with increasing canopy height in controls and treatments, and there was no obvious impact of PPFD reduction (Figure 8.2). The average number of leaves per cluster also increased with increasing canopy height, but the PPFD reduction treatments had less leaves per cluster than the controls throughout the canopy. In contrast, the average internode length in treatments was higher than the controls, but only between 40-60 cm above the sediment (Figure 8.2).

8.3 Growth

Figure 8.3: Cluster growth, leaf extension and productivity of A. griffithii at 10 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments Post-summer with Intensity: Control, Moderate, High. Average with standard error bars. All canopy heights are heights above sediment.

The proportion of clusters that grew varied through the canopy in the treatments and with intensity of PPFD reduction (Figure 8.3). On average greater than 80% of the clusters did not grow in the lower 20-30 cm of the canopy, ~ 70% in the 30-50 cm and 20% in the 50- 60 cm category in the High treatment. In the Moderate treatment 55% of the clusters did not grow in the lower 20-30 cm of the canopy and 35-45% in the 30-60 cm range. Leaf extension rates also varied through the canopy in the Control treatment, increasing up to a maximum in the 40-50 cm height category (Figure 8.3). With PPFD reduction leaf extension rates were impacted throughout the canopy. Similar patterns were observed in leaf productivity estimates (Figure 8.3).

8.4 Physiology

8.4.1 Carbohydrates

Figure 8.4: Leaf sugar and starch of A. griffithii at 20-30 and 40-50 cm height categories through the seagrass canopy after 3-months of PPFD reduction treatments Post-summer & Post-winter with Intensity: Control, Moderate, High. Average with standard error bars. All canopy heights are heights above sediment.

There were no obvious differences in leaf soluble sugar and starch concentration at different heights in the seagrass canopy for Control and Moderate PPFD treatments (Figure 8.4). However, the leaf starch concentrations were greater in the Lower canopy in the High PPFD reduction treatment, Post-winter. The leaf starch and sugar concentrations were also significantly lower in the Post-winter PPFD reduction treatments relative to the Controls (Figure 8.4).

8.4.2 Nutrient content and nitrogen and carbon isotopes

Figure 8.5: Leaf ∂15N, Leaf ∂13C, Leaf N & C at 20-30 and 40-50 cm height categories through the A. griffithii seagrass canopy after 3-months of PPFD reduction treatments Post-summer with Intensity: Control, Moderate, High. Average with standard error bars. All canopy heights are heights above sediment.

There were no obvious differences in the leaf ∂15N at different heights in the canopy, but the PPFD reduction treatments were consistently lower than Controls (Figure 8.5). In contrast, leaf nitrogen content did vary with canopy height, with more nitrogen in the leaves in the Upper canopy in the PPFD reduction treatments compared to the Control (Figure 8.5). Leaf ∂13C tended to be more negative in the lower part of the canopy across all treatments, though there was no obvious PPFD reduction effect (Figure 8.5). Leaf carbon content was lower in the Lower canopy compared to the Upper canopy, but only in the Control treatment (Figure 8.5).

8.4.3 Photosynthetic responses Photosynthetic responses to PPFD reduction were only assessed in the Post-winter treatments in the Upper canopy (40+ cm) and the Lower canopy (< 30 cm). No statistical analyses are included in this report; the data is graphed to show the trends (Figure 8.6). There were differences in the photosynthetic characteristics between the Upper and Lower canopy. Generally, the Maximum rETR, Saturating and Photo-inhibition light intensities were higher in the Upper canopy compared to the Lower canopy, whilst the Photosynthetic efficiency was greater in the lower canopy. These differences are characteristic of a response to lower light levels at the bottom of the canopy. In Control plots there was on average a 75% reduction in PPFD from the top of the canopy to the bottom of the canopy (Table 6.2). There was an effect of PPFD reduction on photosynthetic characteristics and this was consistent across the two canopy heights, however, the Upper canopy showed greater deviation from the control compared to the Lower canopy (Figure 8.6). With PPFD reduction the Maximum rETR, Saturating (Ek) and Photo-inhibition (Ei) light intensities were lower than controls, whilst the Photosynthetic efficiency (α) was greater than controls (Figure 8.6). There were few and small differences between the Moderate and High treatments, except that photosynthetic efficiency was on average higher in the Moderate treatment at 3- and 6-months. With increased duration of PPFD reduction the deviation from the control tended to be greater. These photosynthetic responses are again consistent with plants adapting to reduced PPFD.

Figure 8.6: Photosynthetic measures of A. griffithii leaves in the upper and lower canopy derived from Rapid Light Curves. A) Maximum relative electron transport rate (rETR) upper canopy, B) Photosynthetic efficiency (α), C) Saturating light intensity (Ek) and D) Photo-inhibition light intensity (Ei). Average with standard error bars. nd indicates no data for that duration and/or intensity as not enough leaves were present to determine the measure.

The photosynthetic responses to PPFD reduction were measured in more detail (0, 3, 7, 21, 41, 75 days impact, and 1, 12, 29 and 121 days recovery) in the Post-winter 3-month plots. Each parameter will be discussed independently (Figure 8.7, 8.8). In the Upper canopy the Maximum rETR was lower in the High treatment relative to the Control 3 days after the PPFD reduction treatment was imposed, and remained lower than the Controls throughout the treatment (Figure 8.7). The Moderate treatment showed a different response, and remained similar to the Control for 21 days, declined to intermediate levels between the Control and High treatments after 41 days and at the end of the PPFD reduction treatment was similar to the High treatment. Over the recovery phase the Max rETR in both PPFD reduction treatments recovered to Control conditions after 29 days. In the Lower canopy the High treatment followed a similar response as was observed in the Upper canopy but the response in Moderate treatments was slightly different. After 3 days the Max rETR was lower than the Controls but similar to the High, by 7 days the Moderate treatment was at an intermediate level and then at the end of the PPFD reduction treatment was similar to the High treatment. The recovery response was similar to the Upper canopy. The photosynthetic efficiency (α) also varied over the duration of the experiment (Figure 8.7). In the Upper canopy, α was higher in the High treatment compared to the Control after 3 days and higher in the Moderate treatment after 7 days. By 41 days there was no difference in α between the High and the Control, but the Moderate remained higher than the Controls throughout the PPFD reduction period. Over the recovery phase there appeared to be no difference in α with PPFD reduction treatment. The differences between the Control and PPFD reduction treatments were more variable in the lower canopy. The Saturating and Photo-inhibition light intensities were generally higher in the Controls relative to the Moderate and High treatments after 3 days of PPFD reduction in the Upper canopy and after 21-41 days in the Lower canopy (Figure 8.8). Within a day of re- exposure to ambient PPFD there were no obvious differences in the Saturating and Photo-inhibition intensities between the Control and treatments.

Figure 8.7: Photosynthetic measures of A. griffithii leaves derived from Rapid Light Curves. 1A) Maximum relative electron transport rate (rETR) upper canopy and 1B) lower canopy. 2A) Photosynthetic efficiency (α) upper canopy and 2B) lower canopy. Average with standard error bars. Shaded block is impact phase and white block is recovery phase.

Figure 8.8: Photosynthetic measures of A. griffithii leaves derived from Rapid Light Curves. 1A) Saturating light intensity (Ek) upper canopy and 1B) lower canopy. 2A) Photo-inhibition light intensity (Ei) upper canopy and 2B) lower canopy. Average with standard error bars. Shaded block is impact phase and white block is recovery phase.

9. Recovery From 3-month PPFD Reduction Treatments

Samples were collected to assess the recovery of plots exposed to 3-months PPFD reduction, after 3 and 10 months re-exposure to ambient light. Recovery of a parameter was defined as when the impact treatment was not significantly different to the control. The principal aim of this part of the experiment was to quantify the time required for the Amphibolis griffithii meadow to recover.

9.1 Biomass

9.1.1 Seagrass biomass After 10 months re-exposure to ambient PPFD the total above-ground biomass and leaf biomass in the impact treatments (Moderate and High) were no longer significantly different to the Control in both the Post-summer and Post-winter plots (Table 9.1, Figure 9.1, 9.2). Of significance was the response of the Post-winter, moderately shaded plots. During the shading period these plants showed no loss of leaf or total above-ground biomass (Table 9.1, Figure 9.1, 9.2). Despite this, the total above-ground and leaf seagrass biomass in this treatment declined to a level where it was significantly less than the Control (p < 0.05 for Total above-ground biomass), but after 10 months it had increased again to the same level as the Controls.

9.1.2 Algal epiphyte biomass The algal epiphyte biomass increased over the recovery period, although the pattern of recovery varied depending on the timing (Post-summer or Post-winter) (Table 9.1, Figure 9.1). The Post-summer Moderate treatment was still significantly lower than the Control after 3 months recovery, however, by 10 months both Moderate and High were no longer different to the Control. In contrast, the algal epiphyte biomass in the Post-winter treatment recovered after 3-months in both intensity treatments.

Figure 9.1: Photographs of plots over the recovery period for 3-month treatment both Post-summer and Post-winter. No photographs for Post-winter 10 month recovery.

Figure 9.2: Biomass (g DW m-2) of A. griffithii and algal epiphytes following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Total above-ground seagrass biomass; b) Leaf biomass; c) Stem biomass; d) Algal epiphyte biomass. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Recovery Duration. Average with standard error bars.

Figure 9.3: Density (m-2) of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Leaf density; b) Cluster density; c) Stem density. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

9.2 Density In the Post-summer treatment, leaf density was not significantly different to the controls at 10 months (Table 9.1, Figure 9.3). The average cluster density had increased but was still significantly different to the controls at 10 months. In contrast the Post-winter treatment showed no significant difference between leaf and cluster density after 3 months recovery. As was observed with the biomass data, leaf density in the Moderate treatment declined over the first 3 months recovery period (Figure 9.3).

Table 9.1: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass meadow biomass and density parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Post-summer Post-winter Parameter df MS F p Parameter df MS F p -2 -2 Above-ground biomass Ln (g DW m ) Above-ground biomass (g DW m ) Intensity (I) 2 1.53 7.05 ** Intensity (I) 2 175 7.19 ** Recovery (R) 2 0.82 5.12 * Recovery (R) 2 162 11.6 *** R x I 4 0.37 2.30 0.09 R x I 4 93.1 6.63 ** -2 -2 Leaf Biomass Ln (g DW m ) Leaf Biomass Ln (g DW m ) Intensity (I) 2 3.65 15.3 *** Intensity (I) 2 0.92 6.63 * Recovery (R) 2 1.19 6.32 ** Recovery (R) 2 0.85 10.2 ** R x I 4 0.65 3.48 * R x I 4 0.68 8.14 *** -2 -2 Stem biomass (g DW m ) Stem biomass (g DW m ) Intensity (I) 2 22.6 2.49 0.12 Intensity (I) 2 36.7 4.66 * Recovery (R) 2 26.8 3.85 * Recovery (R) 2 38.4 10.7 *** R x I 4 12.7 1.83 0.16 R x I 4 16.1 4.45 ** -2 -2 Algal epiphyte biomass Sqrt,(g DW m ) Algal epiphyte biomass (g DW m ) Intensity (I) 2 1.60 2.24 0.15 Intensity (I) 2 80.6 3.49 0.06 Recovery (R) 2 10.3 21.4 *** Recovery (R) 2 465 15.4 *** R x I 4 2.70 5.61 ** R x I 4 18.2 0.6 0.67 -2 -2 Faunal epiphyte biomass Ln (g DW m ) Faunal epiphyte biomass Ln (g DW m ) Intensity (I) 2 1.99 1.38 0.29 Intensity (I) 2 0.58 2.26 0.15 Recovery (R) 2 0.20 0.19 0.83 Recovery (R) 2 0.49 2.02 0.15 R x I 4 1.22 1.17 0.35 R x I 4 0.60 2.47 0.07 -2 -2 Leaf density Ln (m ) Leaf density Ln (m ) Intensity (I) 2 2.75 16.9 *** Intensity (I) 2 1.19 8.02 ** Recovery (R) 2 1.02 5.38 * Recovery (R) 2 0.74 9.65 ** R x I 4 0.49 2.60 0.06 R x I 4 0.40 5.24 ** -2 -2 Cluster density (m ) Cluster density Ln (m ) Intensity (I) 2 2.34E+04 12.6 ** Intensity (I) 2 0.82 6.76 * Recovery (R) 2 4.75E+03 2.09 0.14 Recovery (R) 2 1.62 23.0 *** R x I 4 3.47E+03 1.53 0.22 R x I 4 0.38 5.44 ** -2 -2 Stem density (m ) Stem density Ln (m ) Intensity (I) 2 44.9 1.32 0.30 Intensity (I) 2 0.61 11.2 ** Recovery (R) 2 138 5.79 ** Recovery (R) 2 1.49 19.3 *** R x I 4 112 4.70 ** R x I 4 0.14 1.85 0.15 Ln = Natural log transformed data. Sqrt = Square root transformed data.

9.3 Morphology The number of leaves per cluster increased from 2 to 3 leaves, back to the same as the Controls, within the first 3 months in both the Post-summer and Post-winter plots (Table 9.2, Figure 9.4). In contrast, the average leaves per stem and clusters per stem did not recover after 10 months in the Post-summer treatment, but in the Post-winter treatment recovery occurred after 3 months (Table 9.2, Figure 9.4). The leaf size showed a significant but inconsistent response over the recovery period in the Post-summer treatment (Table 9.2, Figure 9.5). After 3-months recovery the leaf length and width was smaller in the Moderate treatment compared to the Control, despite no significant difference detected at the end of the 3-months PPFD reduction. By 10 months there was no difference in the leaf length but in the Moderate treatment leaves were wider than those in the Controls. Leaf area index recovered to control conditions after 10 months (Table 9.2, Figure 9.5). In the Post-winter treatment there was no difference in leaf length and width at the end of 3-months PPFD reduction and over the recovery period (Table 9.2, Figure 9.5). Leaf area index of the High treatment recovered after 3 months (Table 9.2, Figure 9.5). The internode length was significantly longer in the High PPFD reduction treatment after 3 months but recovered within 3 months re-exposure to ambient PPFD (Table 9.2, Figure 9.5). No differences were detected in the internode length of the Post-winter treatment at the end of the impact period and over the recovery period (Table 9.2, Figure 9.5).

Figure 9.4: Morphology of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Average leaves per cluster; b) Average leaves per stem; c) Average clusters per stem; d) Canopy height – 80th percentile (cm). Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Figure 9.5: Additional morphology of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Average leaf length (mm); b) Average leaf width (mm); c) Leaf area index (m2 leaf m-2); d) Average internode length (mm). Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Table 9.2: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass morphology parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Post-summer Post-winter Parameter df MS F p Parameter df MS F p Average leaves per cluster Average leaves per cluster Intensity (I) 2 0.59 6.32 * Intensity (I) 2 0.10 4.8 * Recovery (R) 2 3.10 56.4 *** Recovery (R) 2 1.17 126 *** R x I 4 0.53 9.72 *** R x I 4 0.06 6.30 ** Mode leaves per cluster Mode leaves per cluster Intensity (I) 2 0.51 2.17 0.16 Intensity (I) 2 0.36 4.57 * Recovery (R) 2 3.91 31.2 *** Recovery (R) 2 0.69 5.39 * R x I 4 0.84 6.71 ** R x I 4 0.22 1.74 0.17 Leaves per stem Leaves per stem Intensity (I) 2 954 14.4 ** Intensity (I) 2 311 2.46 0.13 Recovery (R) 2 40.2 0.91 0.42 Recovery (R) 2 224 1.92 0.17 R x I 4 103 2.33 0.08 R x I 4 106 0.91 0.48 Clusters per stem Clusters per stem Intensity (I) 2 70.9 11.1 ** Intensity (I) 2 8.40 0.90 0.43 Recovery (R) 2 6.94 1.32 0.29 Recovery (R) 2 0.88 0.10 0.90 R x I 4 11.3 2.15 0.11 R x I 4 10.8 1.26 0.31 Maximum canopy height (cm) Maximum canopy height (cm) Intensity (I) 2 83 1.46 0.27 Intensity (I) 2 145 1.64 0.24 Recovery (R) 2 131 2.76 0.08 Recovery (R) 2 122 3.71 * R x I 4 37.6 0.80 0.54 R x I 4 33.1 1.00 0.43 Average canopy height (cm) Average canopy height (cm) Intensity (I) 2 128 3.64 0.06 Intensity (I) 2 0.80 0.01 0.99 Recovery (R) 2 144 3.38 0.05 Recovery (R) 2 408 13.9 *** R x I 4 19.1 0.45 0.77 R x I 4 65.1 2.21 0.10 80th percentile canopy height (cm) 80th percentile canopy height (cm) Intensity (I) 2 94.0 2.25 0.15 Intensity (I) 2 25.8 0.39 0.68 Recovery (R) 2 121 3.20 0.06 Recovery (R) 2 149 12.7 *** R x I 4 9.50 0.25 0.91 R x I 4 26.9 2.28 0.09 Average leaf length (mm)1 Average leaf length (mm) Intensity (I) 2 92.5 6.24 ** Intensity (I) 2 60.6 2.57 0.12 Recovery (R) 2 832 25.6 *** Recovery (R) 2 16.8 0.77 0.47 R x I 4 80.1 2.46 0.07 R x I 4 24.9 1.15 0.36 Average leaf width (mm)1,2 Average leaf width (mm) Intensity (I) 2 0.20 1.49 0.26 Intensity (I) 2 0.16 0.49 0.62 Recovery (R) 2 1.27 9.22 ** Recovery (R) 2 0.39 1.45 0.26 R x I 4 0.79 5.74 ** R x I 4 0.39 1.43 0.25 2 -2 2 -2 Leaf area index1 (m leaf m sediment) Leaf area index1 (m leaf m sediment) Intensity (I) 2 14.3 16.9 *** Intensity (I) 2 5.15 4.69 * Recovery (R) 2 5.27 6.94 ** Recovery (R) 2 4.41 10.2 ** R x I 4 3.28 4.32 ** R x I 4 2.09 4.84 ** Average internode length (mm) Average internode length (mm) Intensity (I) 2 2.16 2.55 0.12 Intensity (I) 2 0.11 0.12 0.89 Recovery (R) 2 1.39 2.13 0.14 Recovery (R) 2 3.52 3.98 * R x I 4 1.63 2.51 0.07 R x I 4 1.45 1.64 0.20 1. Not homogenous, significance level set to p < 0.01. 2. Not normally distributed

9.4 Growth Growth was estimated only after 3 months recovery, not at 10 months recovery. There was no significant difference between PPFD reduction treatments in cluster growth (proportion of clusters that grew) after 3 months re-exposure to ambient light. The leaf extension rate increased in both the Post-summer and Post-winter impact treatments. In the Post-summer treatments the Moderate and High were growing faster than the Controls. In the Post-winter treatment however, growth rates increased but they were still significantly lower than the controls. Leaf productivity recovered in the Post-winter treatments after 3 months (Table 9.3, Figure 9.6).

Table 9.3: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass growth parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Post-summer Post-winter Parameter df MS F p Parameter df MS F p -1 -1 -1 -1 Leaf extension (mm cluster day ) Leaf extension (mm cluster day ) Intensity (I) 2 0.20 10.1 ** Intensity (I) 2 0.52 29.8 *** Recovery (R) 1 2.34 146 *** Recovery (R) 1 2.05 109 *** R x I 2 0.72 44.6 *** R x I 2 0.10 5.10 * -2 -1 -2 -1 Leaf productivity Ln (g DW m day ) Leaf productivity Ln (g DW m day ) Intensity (I) 2 17.7 34.0 *** Intensity (I) 2 4.20 15.9 *** Recovery (R) 1 21.7 53.7 *** Recovery (R) 1 0.26 1.01 0.33 R x I 2 5.55 13.7 ** R x I 2 2.84 11.1 ** Ln = Natural log transformed data.

Figure 9.6: Growth of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Cluster growth (% with no growth); b) Leaf extension rate (mm cluster-1 day-1); c) Leaf productivity (g DW m-2 day-1). Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration.

9.5 Sexual reproduction No seedlings were observed over the recovery period of the Post-summer treatments (June – April 06). In the Post-winter treatments seedlings were observed after 3 months recovery (April 06) but only in the Control plots, after 10 months recovery (Nov 06) in the Moderate treatments and not at all in the High treatments (Figure 9.7).

Figure 9.7: Seedling production in A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Seedling density (m-2); b) Seedling per stem. Average with standard error bars.

9.6 Physiology

9.6.1 Carbohydrates Rhizome soluble sugars recovered after 3 months in both the Post-summer and Post- winter treatments (Table 9.4, Figure 9.10). Rhizome starch was significantly higher in the Post-winter PPFD reduction treatment after 3-months but there were no significant differences after 3 months recovery (Table 9.4, Figure 9.10). The response of leaf soluble sugars and starch varied between Post-summer and Post-winter treatments (Table 9.4, Figure 9.10). There was no difference in leaf soluble sugars in the Post-summer plots after 3-months PPFD reduction but after 10 months recovery the Moderate treatment was significantly higher than the control. Leaf starches also showed no significant difference after 3-months PPFD reduction, but after 3 months recovery the Moderate treatment was significantly lower than the control and after 10 months recovery both the Moderate and High were higher than the Control. In contrast, in the Post-winter plots leaf soluble sugars and starch was significantly lower in the impact treatment after 3-months PPFD reduction but recovered after 3 months re-exposure to ambient light.

9.6.2 Nutrient content No differences in leaf carbon, nitrogen and C:N ratio were observed after the impact and recovery phase in the Post-summer treatment (Table 9.5, Figure 9.11). However, in the Post-winter treatment there were some significant differences after 3-months PPFD reduction and these recovered after 3 months re-exposure to ambient PPFD (Table 9.5, Figure 9.11). Similar patterns occurred with the rhizome carbon, nitrogen and C:N ratio (Table 9.5, Figure 9.12).

9.6.3 Nitrogen and carbon isotopes Leaf δ15N recovered after 3 months in the Post-summer and Post-winter treatments (Table 9.5, Figure 9.13). In contrast the response of rhizome δ15N was different in the Post-summer and Post-winter treatments. Rhizome δ15N was significantly lower in the Post-summer PPFD reduction treatment after 3-months impact and did not recover over the 10 months recovery period (Table 9.5, Figure 9.13), whereas no differences in rhizome δ15N were detected after the impact and recovery phase in the Post-winter plots (Table 9.5, Figure 9.13). Leaf and rhizome δ13C did not differ after the impact and recovery phase in all treatments (Table 9.5, Figure 9.13).

Table 9.4: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass carbohydrate parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Post-summer Post-winter Parameter df MS F p Parameter df MS F p Rhizome sugars (% DW) Rhizome sugars (% DW) Intensity (I) 2 87.9 6.33 * Intensity (I) 2 10.4 1.15 0.35 Recovery (R) 2 565 51.3 *** Recovery (R) 2 113 9.75 ** R x I 4 38.5 3.49 * R x I 4 35.9 3.09 * Rhizome starch (% DW) Rhizome starch (% DW) Intensity (I) 2 0.01 0.11 0.89 Intensity (I) 2 0.20 1.26 0.32 Recovery (R) 2 4.54 48.5 *** Recovery (R) 2 0.66 7.90 ** R x I 4 0.06 0.65 0.63 R x I 4 0.41 4.93 ** Leaf sugars (% DW) Leaf sugars Ln (% DW) Intensity (I) 2 8.30 4.10 * Intensity (I) 2 0.72 20.0 *** Recovery (R) 2 147 131 *** Recovery (R) 2 0.29 8.18 ** R x I 4 6.20 5.60 ** R x I 4 0.65 18.5 *** Leaf starch (% DW) Leaf starch (% DW) Intensity (I) 2 0.69 6.66 * Intensity (I) 2 0.54 1.53 0.26 Recovery (R) 2 5.05 24.2 *** Recovery (R) 2 1.42 7.81 ** R x I 4 2.28 10.9 *** R x I 4 0.47 2.59 0.06 Ln – Natural log transformed

Figure 9.10: Carbohydrate content (% DW) of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Rhizome soluble sugars; b) Rhizome starch; c) Leaf soluble sugars; d) Leaf starch. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Figure 9.11: Leaf nutrient content (% DW) of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Carbon; b) Nitrogen; c) Carbon:nitrogen ratio. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Figure 9.12: Rhizome nutrient content (% DW) of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Carbon; b) Nitrogen; c) Carbon:nitrogen ratio. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Figure 9.13: Carbon and nitrogen stable isotope ratio (δ13C, δ15N) of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Rhizome δ15N; b) Leaf δ15N; c) Rhizome δ13C; d) Leaf δ13C. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

Table 9.5: Results of statistical analysis to determine effect of Intensity of PPFD 3-month reduction treatments over the recovery period on A. griffithii seagrass nutrient parameters. *** = p < 0.001, ** = p < 0.01 & > 0.01, * = p < 0.05 & > 0.01. Post-summer Post-winter Parameter df MS F p Parameter df MS F p Leaf nitrogen (% DW) Leaf nitrogen (% DW) Intensity (I) 2 0.02 1.12 0.36 Intensity (I) 2 0.21 18.4 *** Recovery (R) 2 1.56 73.3 *** Recovery (R) 2 0.07 6.51 ** R x I 4 0.02 0.94 0.46 R x I 4 0.17 15.5 *** Rhizome nitrogen (% DW) Rhizome nitrogen (% DW) Intensity (I) 2 0.00 0.84 0.45 Intensity (I) 2 0.01 1.57 0.25 Recovery (R) 2 0.22 49.9 *** Recovery (R) 2 0.02 8.60 ** R x I 4 0.01 1.17 0.35 R x I 4 0.02 8.32 *** Leaf ∂15N Leaf ∂15N Intensity (I) 2 0.51 2.85 0.10 Intensity (I) 2 0.47 3.49 0.06 Recovery (R) 2 2.50 23.1 *** Recovery (R) 2 0.04 0.75 0.48 R x I 4 0.58 5.31 * R x I 4 0.35 6.03 ** Rhizome ∂15N Rhizome ∂15N Intensity (I) 2 1.12 5.53 * Intensity (I) 2 0.05 0.26 0.77 Recovery (R) 2 0.18 1.43 0.26 Recovery (R) 2 1.44 13.0 *** R x I 4 0.28 2.25 0.09 R x I 4 0.21 1.92 0.14 Leaf carbon (% DW) Leaf carbon (% DW) Intensity (I) 2 0.92 0.77 0.48 Intensity (I) 2 0.31 0.42 0.66 Recovery (R) 2 2.79 1.72 0.20 Recovery (R) 2 13.6 12.5 *** R x I 4 2.91 1.80 0.16 R x I 4 3.44 3.16 * Rhizome carbon (% DW) Rhizome carbon (% DW) Intensity (I) 2 2.34 1.40 0.28 Intensity (I) 2 2.32 0.59 0.57 Recovery (R) 2 12.3 7.26 ** Recovery (R) 2 59.5 13.4 *** R x I 4 3.2 1.88 0.15 R x I 4 2.21 0.50 0.74 Leaf ∂13C Leaf ∂13C Intensity (I) 2 0.06 1.26 0.32 Intensity (I) 2 1.00 0.36 0.70 Recovery (R) 2 0.93 11.1 *** Recovery (R) 2 20.8 3.95 * R x I 4 0.08 0.94 0.46 R x I 4 4.16 0.79 0.54 Rhizome ∂13C Rhizome ∂13C Intensity (I) 2 0.16 0.37 0.70 Intensity (I) 2 0.39 0.52 0.61 Recovery (R) 2 3.56 2.47 0.11 Recovery (R) 2 0.11 0.17 0.84 R x I 4 1.07 0.74 0.57 R x I 4 1.44 2.30 0.09 Leaf C:N Ln Leaf C:N Ln Intensity (I) 2 0.01 0.34 0.72 Intensity (I) 2 0.18 18.8 *** Recovery (R) 2 1.01 76.8 *** Recovery (R) 2 0.02 2.73 0.08 R x I 4 0.02 1.34 0.28 R x I 4 0.16 23.3 *** Rhizome C:N Ln Rhizome C:N Intensity (I) 2 0.02 0.64 0.54 Intensity (I) 2 1.10E+03 1.57 0.25 Recovery (R) 2 1.83 63.9 *** Recovery (R) 2 4.31E+03 11.9 *** R x I 4 0.04 1.52 0.23 R x I 4 2.28E+03 6.32 ** Ln = Natural log transformed

9.7 Recovery - response pathway of A. griffithii after 3-months of PPFD reduction The results of this experiment have indicated a recovery response pathway of Amphibolis griffithii meadows following 3-months of PPFD reduction with two intensities (Moderate, High) and timings (Post-summer, Post-winter) (Table 9.6). These recovery pathways varied with time of year and also the intensity of PPFD reduction. We focus on the parameters that showed a significant change over the recovery period at a certain timing (Post-winter or Post-summer), when that significant difference occurred either at 3 or 10 months, and whether it occurred with Moderate and/or High treatments. Parameters that did not show a significant effect of intensity are not discussed. Recovery is defined as when the treatments are not significantly different to controls.

9.7.1 Post-summer After 3-months PPFD reduction and a subsequent 3 months re-exposure to ambient PPFD a number of morphology, growth and physiology parameters recovered (i.e. they were no longer significantly different to Control treatments) (Table 9.6). These included an increase in the number of leaves per cluster back to an average of 3, cluster production, leaf extension and areal leaf productivity, but areal productivity only in the Moderate treatment. Leaf extension rates were greater in the plots recovering from shading than in the Controls. Rhizome sugars and leaf ∂15N also increased, returning to the same as the Controls. A number of parameters were impacted only under High intensity shading and recovered after 3 months re-exposure to ambient PPFD, these were internode length and leaf carbon, which both decreased back to control conditions. Algal epiphyte biomass was impacted after 3-months PPFD reduction but recovery occurred only in the High intensity treatment after 3 months. Some parameters were not impacted after three months light reduction but declined 3 months into the recovery period. These included cluster density and cluster stem-1 in the Moderate treatment (Table 9.6). After 10 months of re-exposure to ambient light, some of the biomass and density parameters had recovered, as well as additional growth and physiology parameters (Table 9.6). Above-ground biomass (total and leaf), leaf density and the leaf area index recovered to control conditions. However, the number of leaf clusters (density, clusters stem-1) and the number of leaves per stem were still lower after 10 months, although they were on a trajectory back to control conditions. Leaf starch (Moderate and High) and soluble sugars (Moderate only) increased to concentrations greater than the control after 10 months. Algal epiphyte biomass also recovered in the Moderate treatment, lagging behind the High treatment which recovered after 3 months.

Table 9.6: Summary of recovery response-pathway variables following 3 months shading post-summer and post-winter with 3 and 10 months re-exposure to ambient light. Green indicates no significant difference between treatment and control, red indicates reduction relative to control and yellow indicates an increase relative to control. nd = no data, M= moderate intensity shading, H = high intensity shading.

Parameters Timing: Post-summer Timing: Post-winter Impact Recovery Recovery Impact Recovery Recovery 3 mo 3 mo 10 mo 3 mo 3 mo 10 mo M H M H M H M H M H M H Physiology Leaf sugar ↑ ↓ ↓ Leaf starch ↑ ↑ ↓ ↓ Leaf nitrogen ↑ ↑ Leaf ∂15N ↓ ↓ ↓ ↓ Leaf C:N ↓ ↓ Rhizome sugars ↓ ↓ ↓ Rhizome starch ↑ ↑ Growth Leaf extension ↓ ↓ ↑ ↑ nd nd ↓ ↓ ↓ ↓ nd nd Leaf cluster growth ↓ ↓ nd nd nd nd Leaf productivity ↓ ↓ ↓ nd nd ↓ nd nd Morphology, biomass & density Leaf biomass ↓ ↓ ↓ ↓ ↓ ↓ ↓ Leaf density ↓ ↓ ↓ ↓ ↓ Leaves stem-1 ↓ ↓ ↓ ↓ ↓ ↓ ↓ Leaves cluster-1 ↓ ↓ ↓ Cluster density ↓ ↓ ↓ ↓ ↓ ↓ Cluster stem-1 ↓ ↓ ↓ ↓ ↓ ↓ Leaf width ↓ ↓ Leaf length Internode length ↑ ↑ ↑ Algal epiphyte biomass ↓ ↓ ↓ ↓

9.7.2 Post-winter The Post-winter treatment was not as significantly impacted after 3-months PPFD reduction as the Post-summer treatment, and generally showed a faster recovery (Table 9.6). For example, a number of biomass, density, morphology, growth and physiology parameters were only impacted in the High treatment after 3-months PPFD, and many of these recovered to Control values after 3 months re-exposure to ambient PPFD, including leaf measures (leaves cluster-1, leaves and clusters stem-1, leaf and cluster density, leaf area index, leaf productivity, leaf C:N); rhizome soluble sugars; and algal epiphyte biomass. Parameters that were impacted in both the Moderate and High 3-month PPFD reduction treatments also showed recovery with 3 months re-exposure to ambient light. These were physiological parameters such as leaf ∂15N, leaf C:N and leaf soluble sugars and starch, which increased back to the control state, and leaf nitrogen and rhizome starch, which decreased back to control conditions. After 10 months recovery the above-ground biomass (total and leaf) was the same as the controls. Interestingly, the biomass in the Moderate intensity treatment declined over the first 3 months of re-exposure to ambient PPFD, despite showing no significant difference from the controls at the end of the 3- month PPFD reduction. The mechanism for this reduction is unknown, although it has been observed in other shading studies (e.g. Malta et al. 2006). A similar response was not apparent with the High shading treatment, suggesting that different processes were operating between the two intensity treatments during the recovery period.

9.7.3 Recovery – response pathway summary Response pathways in the in the recovery phase were similar to the impact phase (Section 7.7). The physiology measure of leaf ∂15N and the morphology measure of leaves per cluster were consistently the first parameters to respond. Variability in the PPFD reduction impacts was also reflected in the recovery period. Recovery in the Post-summer treatments that were exposed to a greater PPFD reduction (Table 6.1) was generally slower and more variable. For example, leaf density took 10 months to recover in the Post-summer plots but only 3 months in the Post-winter. This probably represents an interaction of the ambient condition and the plant’s annual growth cycle. Other parameters such as cluster density, clusters and leaves stem-1 showed no recovery, whereas in the Post-winter there was full recovery after 3 months. Cluster production through branching was also affected. To produce more clusters from the existing clusters on a stem, branching needs to occur. As branching tends to peak annually in spring, with minimums in winter (Section 7.4.2), the timing of recovery and the number of clusters left on a stem will influence the time taken for the cluster density and cluster stem-1 to recover. Growth parameters also varied depending on the timing of the impact and recovery, but the response pattern was reversed. Leaf extension did recover after 3 months in the Post-summer but did not recover in the Post-winter treatments. Leaf sugars and starch declined in the Post-summer Moderate treatment 3 months into recovery but they increased in the Post-winter treatments. These responses may reflect a different mechanism of recovery. The Post-summer treatments appear to invest energy into growth and formation of new leaves, and not storing sugars and starch in the leaves, whereas the Post-winter treatments were storing sugars and starches in the leaves rather than growing.

10. Recovery From 6-month PPFD Reduction Treatments

10.1 Biomass and density Plots that were shaded at the end of summer for 6 months showed no recovery after almost two years re-exposure to ambient PPFD (Figure 10.1, 10.2, 10.3, 10.4). In fact, total above-ground biomass was lower after 2 years re-exposure to ambient PPFD than at the end of the shading treatment due to loss of stems and the complete absence of leaves. Algal epiphyte biomass also showed no recovery. Recovery in the Post-winter plots that had been shaded for 6 months varied with the intensity of shading. The High treatments did not recover but the Moderate treatments maintained a similar leaf biomass and density to that observed at the end of the 6-month impact treatment, though the stem biomass declined. The remaining stems were about half the size of those in the Control plots, around 30 cm high. These stems were either new stem recruits or older stems where the top portion of the stem had broken off. Algal epiphyte biomass in the Moderate intensity recovered to the Control state (Figure 10.2, 10.3, 10.4). The number of stems with leaves was counted in permanent quadrats at the end of the PPFD reduction treatment and then on two occasions over the recovery period (Figure 10.4, 10.5). The height of the stems was also recorded to give an indication of the type of recovery, with shorter stems indicating recovery from new shoots or seedlings and taller stems indicating a recovery response from existing shoots or growth of young stems. In the Post-summer 6-month Moderate and High treatments there was loss of stems from all height categories, except in the shortest (0-10 cm) height category where there was an increase of stems after 7.5 months recovery. These data are consistent with stem recruitment, though the new stems did not persist 17 months into the recovery period (Figure 10.5). In contrast, the Post-winter Moderate & High treatments had an increase of stems with leaves in the 10-30 cm height category ~ 14 months into the recovery period, indicating recruitment of new stems or persistence of recently recruited stems (Figure 10.5). However, stems were lost from all other height categories and the meadow structure was quite different to Controls (Figure 10.1). At the final sampling, two seedlings were observed in the recovery plots.

Figure 10.1: Photographs of 6-month plots at the end of the impact treatment and then in August 2007, 23 months (Post-summer) and 17 months (Post-winter) following re-exposure to ambient PPFD.

Figure 10.2: Biomass (g DW m-2) of A. griffithii following recovery from 6-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Total above-ground seagrass biomass; b) Leaf biomass; c) Stem biomass; d) Algal epiphyte biomass. Recovery duration varies with treatment. Average with standard error bars.

Figure 10.3: Density (m-2) of A. griffithii following recovery from 6-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Leaf density; b) Cluster density; c) Stem density. Recovery duration varies with treatment. Average with standard error bars.

Figure 10.4: Canopy height – 80th percentile (cm) of A. griffithii following recovery from 6-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. Recovery duration varies with treatment. Average with standard error bars.

Figure 10.5: Stems with leaves expressed as% of number of stems with leaves at end of PPFD reduction treatments and categorised into stem heights for the Post-summer (PS) and Post-winter (PW) 6-month plots of Moderate and High intensity at 1) the end of the impact period, 2) 7.5(PS) or 5.5 (PW) months recovery and 3) 17(PS) or 13.5 (PW) months recovery. Data has been scaled such that at Time 1, 0 = number of stems with leaves at the end of the PPFD reduction treatment in a permanent quadrat and at Time 2 and 3, positive values indicate increases in the number of stems with leaves whilst negative values indicate loss of stems with leaves and 0 is no change.

11. Recovery From 9-month PPFD Reduction Treatments

11.1 Biomass and density The recovery patterns observed in the plots shaded for 9-months were similar to those observed in the 6-month plots, though recovery was followed for a shorter duration (Figure 11.1, 11.2, 11.3, 11.4). Post- summer 9-month PPFD reduction treatments with 21 months re-exposure to ambient PPFD showed no recovery, and biomass continued to decline. The Post-winter 9-month High treatment showed a similar response. The Moderate treatment, however, maintained biomass and leaf density and were similar to values at the end of the 9-month impact (Figure 11.2, 11.3, 11.4).

Figure 11.1: Photographs of 9-month plots at the end of the impact treatment and then in August 2007, 21 months (Post-summer) and 15 months (Post-winter) following re-exposure to ambient PPFD.

Figure 11.2: Biomass (g DW m-2) of A. griffithii and algal epiphytes following recovery from 9-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Total above-ground seagrass biomass; b) Leaf biomass; c) Stem biomass; d) Algal epiphyte biomass. Recovery duration varies with treatment. Average with standard error bars.

Figure 11.3: Density (m-2) of A. griffithii following recovery from 9-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Leaf density; b) Cluster density; c) Stem density. Recovery duration varies with treatment. Average with standard error bars.

Figure 11.4: Canopy height – 80th percentile (cm) of A. griffithii following recovery from 6-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. Recovery duration varies with treatment. Average with standard error bars.

In the Post-summer 9-month High PPFD reduction treatments there was complete loss of stems with leaves, and no change over the recovery period. Some stems persisted in the Moderate treatment, in the 20-30 cm height category over the recovery period, however, stems were lost from other height categories (Figure 11.5). In the Post-winter treatments stems were lost from 20-40 cm in the Moderate, and 30+ cm category in the High, with either no change in the Moderate in the lower height categories or an increase in stems with leaves in the 0-30 cm height category of the High treatment (Figure 11.5).

100

Figure 11.5: Counts of stem with leaves categorised into stem heights for the Post-summer (PS) and Post- winter (PW) 9-month plots of Moderate and High intensity at 1) the end of the impact period, 2) 7.5 (PS) and 4 (PW) months and 3) 17 (PS) and 14 (PW) months. No data in the Post-winter high as no stems remained at the end of the shading period.

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12. Sub-lethal Indicators

12.1 Sub-lethal indicators of PPFD reduction As Amphibolis griffithii plots shaded for 6 or 9 months showed little recovery within 1-2 years, the focus for sub-lethal indicators was based on the 3-month results. This increased the focus on variables that respond within timeframes that permit subsequent recovery. Additionally, we defined ideal sub-lethal indicators as those that showed consistent responses across times and with increased intensity and duration of PPFD reduction, and which were highly responsive, that is significant differences between the impacted treatment (Moderate, High) and Controls were apparent at 3 months. The following table lists all biomass, density, morphology, growth and physiology parameters that were measured and the response relative to the Control (Table 12.1). Green indicates no significant difference between the Control and both impact treatments, orange indicates only one treatment had a significant difference and red indicates that both the Moderate and High were significantly different to the Control. Based on this summary, two parameters were significantly different to the control at 3- months in both Moderate and High PPFD reduction treatments: Leaf extension rate and Leaf ∂15N. There were a number of parameters that showed a significant response at 3- months for both start times (Post-summer and Post-winter), but this was confined to only 1 PPFD reduction treatment on at least one or both of those occasions. These included total above-ground seagrass, leaf and algal epiphyte biomass; leaf and cluster density; leaves per cluster, leaves per stem and clusters per stem, leaf area index, leaf productivity and rhizome sugars.

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Table 12.1: Response of all parameters measured to different timings, durations and intensity of PPFD reduction. Red = both High and Moderate were significantly different to the Control, Orange = only one PPFD reduction treatment was different to the Control, and Green = not significantly different to Control. ‘–‘ = no data from this time period. Timing Post-summer Post-winter Duration (months) 3 6 9 3 6 9 Biomass Total above-ground High √ √ High √ √ Leaf √ √ √ High √ √ Stem x High High High x √ Algal epiphyte √ √ √ High High √ Faunal epiphyte x √ High x x x Density Leaf √ √ √ High √ √ Cluster High √ √ High √ √ Stem x High High High x x Morphology Average leaves per cluster √ √ - High √ - Mode leaves per cluster √ √ - High √ - Median leaves per cluster √ √ - High √ - Leaves per stem √ √ √ High √ √ Clusters per stem High √ √ High √ √

Maximum canopy height x √ √ High x High Average canopy height x √ x High √ High 80th percentile canopy height x √ √ x √ High Average leaf length x x - x x - Average leaf width x High - x High - Leaf area index √ √ √ High √ √ Average internode length High √ x x x High Growth Cluster growth √ √ - x √ - Leaf extension √ √ - √ √ - Leaf productivity √ √ √ High √ √ Physiology Rhizome sugars √ √ √ High √ √ Rhizome starch x √ √ √ High - Leaf sugars x √ - √ √ - Leaf starch x √ - √ √ - Leaf nitrogen x High - √ √ - Rhizome nitrogen x High x High x √ Leaf ∂15N √ √ - √ √ - Rhizome ∂15N x x x x x x Leaf carbon High High - High Mod - Rhizome carbon x x x x x x Leaf ∂13C x x - x x - Rhizome ∂13C x x x x x x Leaf C:N x High - √ √ -

Rhizome C:N x High x High x √

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12.2 Sub-lethal indicators of recovery from PPFD reduction Potential sub-lethal indicators of recovery were based on the 3-month treatments. An ideal sub-lethal indicator would respond consistently both across start times (Post- summer, Post-winter), intensity (Moderate, High) and in the direction of the response. Only one parameter, leaf ∂15N, responded to both intensity treatments after 3-months and recovered after 3 months (Table 12.2). Three parameters, leaves cluster-1, rhizome sugars and leaf carbon were impacted with 3-months PPFD reduction but not always with both intensity treatments, and recovered after 3 months. Leaves cluster-1 and rhizome sugars both increased to control conditions, whereas leaf carbon decreased in the Post-summer treatment and increased in the Post-winter treatment to reach control conditions. Total above-ground seagrass and leaf biomass recovered after 10 months in both the Post- summer and Post-winter treatments.

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Table 12.2: Recovery of all parameters from 3-months PPFD reduction Post-summer and Post-winter. Red = both High and Moderate were significantly different to the Control, Orange = only one PPFD reduction treatment was different to the Control, and Green = not significantly different to Control. R1 = 3 months recovery, R2 = 10 months recovery. ‘–‘ = no data from this time period. Timing Post-summer Post-winter Duration (months) 3 R1-3 R2-10 3 R1-3 R2-10 Biomass Total above-ground High √ x High √ x Leaf √ √ x High √ x Stem x x x High x x Algal epiphyte √ Mod x High x x Faunal epiphyte x x x x x x Density Leaf √ √ x High x x Cluster High √ √ High x x Stem x x Mod High High x Morphology Average leaves per cluster √ x x High x x Mode leaves per cluster √ x x High x Mod Median leaves per cluster √ x x High x x Leaves per stem √ √ √ High x x Clusters per stem High √ √ High x x Maximum canopy height x x x High x x Average canopy height x x x High x x 80th percentile canopy height x x x x x x Average leaf length x Mod x x x x Average leaf width x Mod Mod x x x Leaf area index √ √ x High x x Average internode length High x x x x x Growth Cluster growth √ x - x x - Leaf extension √ √ * - √ √ - Leaf productivity √ High - High x - Physiology Rhizome sugars √ x x High x x Rhizome starch x x x √ x x Leaf sugars x x Mod √ x x Leaf starch x Mod √ √ x x Leaf nitrogen x x x √ x x Rhizome nitrogen x x x High x x Leaf ∂15N √ x x √ x x Rhizome ∂15N √ √ √ x x x Leaf carbon High x x High x x Rhizome carbon x x x x x x Leaf ∂13C x x x x x x Rhizome ∂13C x x x x x x Leaf C:N x x x √ x x Rhizome C:N x x x High Mod x

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12.3 Summary statistics of possible sub-lethal indicators Based on section 12.1 a number of possible sub-lethal indicators of PPFD reduction have been recommended due to their consistent response across PPFD reduction timing and intensity treatments. To then incorporate these parameters into monitoring or compliance programmes it will be necessary to have an understanding of the distribution of data in a natural setting versus an impacted setting. To represent the natural seagrass conditions we present summary statistics of the Control data from all treatments (Before, Impact and Recovery; n is variable), and the median value from the impacted treatments separated into the different timing, duration and intensity combinations (n=5). The median of the impacted treatments is also expressed as the percentile value from the Control data set. This data has been separated into groups according to the number of measures per plot e.g. leaf biomass where there was one measure per plot and leaves cluster-1 where there was more than one measure per plot. The complete dataset has been included in this analysis as the number of replicates is much higher and more variable than parameters were there was only one measure per plot.

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Table 12.3: Summary statistics of parameters proposed as useful sub-lethal indicators. Control data is from all Before, Impact and Recovery treatments across all times periods whereas the Impact treatments are expressed for each timing, duration and intensity combination. Number in brackets is the percentile value from the control dataset for the median value of the impacted dataset. Total Leaf Algal Leaf density Cluster Leaf area Rhizome Leaf Leaves Leaves Leaves Clusters Clusters Leaf seagrass biomass epiphyte density index sugars ∂15N per per per per stem per stem extension biomass biomass cluster stem stem with with leaves leaves CONTROL DATA 1st 143 74 24 4138 1243 1.2 9.8 2.5 1 21 23 7 8 0.16 5th 184 91 35 4458 1480 1.4 10.0 2.9 1 23 26 7 8 0.40 20th 245 134 94 6608 1952 2.2 13.1 3.4 2 27 31 7 8 0.66 50th 378 197 168 10000 3220 3.2 17.3 3.7 3 34 38 11 12 0.92 80th 482 263 253 13532 4548 4.3 20.8 4.1 4 43 47 14 15 1.33 95th 614 328 418 18370 5804 5.7 23.7 4.4 4 54 58 17 18 2.30 99th 815 478 495 23438 7964 7.3 26.1 5.0 5 75 75 20 23 3.26

Mean 388 205 183 10408 3358 3.3 17.3 3.7 3.03 36.4 39.4 11.6 12.6 1.06 Stdev 152 87 110 4343 1509 1.4 4.4 0.5 0.92 10.7 10.8 3.3 3.4 0.59 Se 17 10 12 489 170 0.2 0.6 0.1 0.01 1.2 1.2 0.4 0.4 0.02 Count 79 79 79 79 79 79 50 50 7737 79 79 79 79 1552 CV 39 42 60 42 45 42 25 14 30 29 27 28 27 56

IMPACT DATA (Duration & Intensity) Timing 3 Mod 208 (9) 101 (9) 19 (<1) 5420 (10) 2580 (35) 1.79 (12) 5.6 (<1) 2.6 (2) 2658 (20) 0.1187 21 (1) 24 (2) 10 (35) 11 (35) (<1) Post- 3 High 160 (3) 63 (<1) 9 (<1) 3920 (<1) 1940 (20) 1.16 (<1) 4.9 (<1) 2.7 (3) 2496 (20) 0.0945 17 (<1) 18 (<1) 8 (10) 10 (26) (<1) summer 6 Mod 226 (11) 39 (<1) 25 (1) 2200 (<1) 1080 (<1) 0.66 (<1) 5.0 (<1) 2.9 (5) 2224 (20) 6 (<1) 9 (<1) 3 (<1) 4 (<1) 0.2954 (2) 6 High 98 (<1) 7 (<1) 12 (<1) 440 (<1) 240 (<1) 0.13 (<1) 5.2 (<1) 2.5 (1) 260 (20) 3 (<1) 5 (<1) 1 (<1) 3 (<1) 0.1742 (1) 9 Mod 154 (3) 0 (<1) 54 (10) 0 (<1) 0 (<1) 0.25 (<1) 4.3 (<1) nd 313 (50) 0 (<1) 4 (<1) 0 (<1) 1 (<1) 1.1546 (71) 9 High 96 (<1) 0 (<1) 20 (<1) 0 (<1) 0 (<1) 0.02 (<1) 3.5 (<1) nd 32 (50) 0 (<1) 1 (<1) 0 (<1) 1 (<1) 0.948 (55) Timing 3 Mod 407 (58) 228 (65) 159 (45) 13500 (80) 4960 (86) 4.05 (75) 17.2 (48) 3.6 (40) 31314 (50) 0.55146 40 (70) 42 (66) 14 (81) 15 (79) (12) Post- 3 High 152 (3) 66 (<1) 77 (14) 4520 (5) 1920 (5) 1.71 (10) 12.9 (18) 3.2 (15) 2497 (20) 21 (1) 26 (5) 9 (22) 11 (35) 0.20129 (2) winter 6 Mod 142 (<1) 34 (<1) 58 (12) 2440 (<1) 1180 (<1) 0.60 (<1) 10.2 (6) 2.5 (1) 2279 (20) 12 (<1) 18 (4) 5 (<1) 8 (2) 0.2053 (2) 6 High 145 (2) 10 (<1) 29 (2) 920 (<1) 480 (<1) 0.19 (<1) 5.5 (<1) 1.8 (<1) 2126 (20) 4 (<1) 7 (<1) 2 (<1) 3 (<1) 1.0411 (62) 9 Mod 102 (<1) 11 (<1) 32 (4) 1020 (<1) 440 (<1) 0.16 (<1) 5.9 (<1) 2.0 (<1) 2124 (20) 6 (<1) 9 (<1) 2 (<1) 4 (<1) 0.5068 (10) 9 High 98 (<1) 0 (<1) 15 (<1) 0 (<1) 0 (<1) 0.13 (<1) 3.8 (<1) nd 26 (20) 0 (<1) 3 (<1) 0 (<1) 2 (<1) nd

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12.4 Potential indicators of sub-lethal reduction in light availability

12.4.1 Leaf ∂15N Leaf ∂15N was one of only two parameters where both the Moderate and High treatment were significantly different to the Control in both Post-summer and Post-winter 3-month PPFD reduction treatments (Figure 7.13). Leaf ∂15N did vary at different times of the year in Control plots, so additional reference site data would be needed to verify use of the parameter. There was no variation in leaf ∂15N with canopy height so the location in the leaves where the samples are taken for analysis could be flexible. This measure was one of the least variable parameters (CV = 14), generally the median value in impacted plots was around the 5th percentile of control plots. However, at certain times of the year, such as December when the Post-winter 3-month samples were collected, the median value of the impacted sites was at the 15th (High) and 40th (Moderate) percentile of the Control data (Table 12.3). As sewerage can elevate and agricultural nutrient pollution can lower the ∂15N signal (Jones et al. 2001) implementation of this parameter as a sub-lethal indicator would need to consider the sources of nitrogen to site. Epiphytes must be removed from leaf samples, the leaves dried and ground and then analysed in a mass spectrophotometer following standard methods. Generally 2 mg of dried ground plant material is required. As this parameter also recovered after 3 months re-exposure to ambient PPFD it may be useful for the detection of the removal of PPFD stress in the short term. This variable has rarely been measured in seagrasses in relation to change in light availability. Given this, it is important to validate that the changes are consistent with a light-induced mechanism. Analogous studies in terrestrial angiosperms indicate similar responses of leaf ∂15N to reduced light availability and have demonstrated the mechanism. Changes in the ways nitrogen is allocated in plants can alter leaf ∂15N (Stock & Evans 2006). A large proportion of leaf N is invested in the photosynthetic apparatus (Evans 1989b), but the nature of that allocation varies according to light conditions. Plants grown in low light invest more N in light harvesting (Evans & Poorter 2001), particularly the light harvesting antennae and pigments, to maximise carbon gains, while in high light environments investment in electron carriers and Calvin cycle enzymes maximises carbon gain (Evans 1989a). Relative to the bulk ∂15N of cells, proteins are enriched whereas chlorophyll, lipids, amino sugars and alkaloids are depleted (Werner & Schmidt 2002). Therefore, an investment of more nitrogen into chlorophyll and other light capturing pigments under reduced light conditions is likely to produce a more negative ∂15N value. Although we did not measure pigments in the leaves, Mackey et al (2007) showed increased chlorophyll in leaves following intense reduction of light availability. The response of ∂15N to shading is, therefore, consistent with a light-induced mechanism, occurring early in the cause-effect pathway of light reduction in seagrass ecosystems.

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12.4.2 Leaf extension rate Leaf extension rate was the other parameter where both the Moderate and High treatment were significantly different to the Control in both Post-summer and Post-winter 3-month PPFD reduction treatments (Figure 7.7). This parameter also varied at different times of the year in Control plots, so use of this parameter would also need to be in comparison to a reference site for validation. With the variation in leaf extension throughout the canopy, e.g. the greatest growth rates were observed from 40-50 cm height in the control plots (Figure 8.3), selection of clusters for tagging would need to be standardised. Leaf extension rate was one of the most variable parameters (CV = 56), generally the median value in impacted plots was less than the 3rd percentile of Control data, however the median value in the Post-winter 3-month Moderate PPFD reduction treatment was the 12th percentile of Control data (Table 12.3). In the current study a minimum of 10 and a maximum of 30 measures were made in a plot and the average used in statistical analysis. Due to the high variation of leaf extension rates within the canopy height of the plant and also the plant response of stopping growth in clusters under PPFD reduction stress, it is recommended that appropriate replication should be used. To estimate leaf extension by the hole punch method, approximately two weeks is needed between tagging and harvesting. As the recovery time for return to the control state varied, with 3 months in the Post-summer treatments but not the Post-winter treatments, so it may not be a reliable parameter to use to monitor recovery. Leaf productivity was also identified as a potential sub-lethal indicator, but this parameter was not significantly different to the Control with all 3-month treatments (Figure 7.7). It had similar characteristics as leaf extension rate with variation over the year and with canopy height (Figure 8.3). However, as leaf productivity can be calculated from the leaf extension rate data by measuring the dry weight of new growth and multiplying by the cluster density, its use with other measures may be worthwhile for summarising meadow health.

12.4.3 Leaves per cluster The number of leaves per cluster in the impacted treatments was significantly different to the controls after 3-months PPFD reduction, except in the Post-winter Moderate treatment, where it was at an intermediate value between the Control and High treatment (Figure 7.5, Appendix: Figure 17.3). It also was reasonably consistent over the year, ranging from an average of 2.7 ± 0.1 (Dec 05) to 3.5 ± 0.1 (Sept 05) leaves per cluster. The higher number of leaves per cluster is likely to occur during the active branching time, and before the internodes elongate to separate the clusters. As the number of leaves per cluster is reasonably consistent and constrained by the architecture and growth patterns of the plant, it may be one of the only parameters that could be used without comparison to a reference site, however adequate site-specific data would be needed to validate this decision. As one of the first morphological impacts from PPFD reduction is the loss of a single leaf from a cluster (Figure 7.14), leaving on average 2 leaves per cluster, an appropriate number of replicates must be taken to detect this change. The number of leaves per cluster also increases with height in the canopy, from an average of 2 at 0-10 cm up to 3.2 in the upper canopy, therefore this should be taken into consideration when collecting measurements (Figure 8.2). Leaves per cluster was a

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moderately variable parameter (CV = 30), ranging from 1-5 in control plots. The median value of the impacted plots was the 20th percentile of the Control data, except for the Post-winter Moderate, which had the same median as the Control data. The number of leaves per cluster can be expressed based on the mean, median or mode value (Appendix: Figure 17.3). The mean and the mode were the most sensitive for detecting differences between Control and impacted plots (Appendix: Figure 17.3), so the methodology of data summary for multiple readings taken in a plot needs consideration. Mode may be the most useful summarisation for leaves cluster-1, as this is a correct biological representation, as only 1-5 leaves are present in a cluster. Leaves per cluster also recovered within 3 months in all treatments. This measure was taken from all leaf clusters harvested from a quadrat but the count could be taken in situ without requiring destructive sampling.

12.4.4 Leaves per stem The number of leaves per stem is an integrated measure of the number of clusters per stem and number of leaves per cluster. At 3-months PPFD reduction this measure was significantly different to the Control in all treatments, except the Post-winter Moderate (Figure 7.5). The number of leaves per stem did show some variation over the year, so comparison with a reference site would be useful. The number of leaves per stem can be expressed in two ways; based on all stems in the sample, including those stems without leaves, or only on stems that have leaves. However, as this affects the summary statistics the implications need to be considered (Table 12.3). Including stems with no leaves will reduce the mean and median, so it may be easier to detect differences from a reference site. A sampling plan might only target stems with leaves, and this approach is still likely to detect differences with reference data. Both methods of summary have similar variation (CV = 29 or 27) and the median of impacted plots has a similar percentile value in the control data (less or equal to the1st percentile for leaves per stem including stems with no leaves, or less or equal to the 5th percentile for leaves per stems where only the stems with leaves are included). Clusters per stem had a similar response to leaves per cluster, however the impact only occurred with High intensity PPFD reduction. Also the loss of clusters is further along the response pathway of Amphibolis griffithii where, with longer durations or greater intensities of PPFD reduction the clusters stop growing, and all remaining leaves in the cluster fall off. This is not as sensitive an indicator as leaves per stem and may only be consistent when the plants have declined beyond their ability to recover in the short term.

12.4.5 Leaf biomass and density Leaf biomass and density showed very similar responses to PPFD reduction in this experiment (Figure 7.3, 7.4). Leaf biomass and density was reasonably consistent across the year, but due to likely variations with site, these measures should be used by comparison with reference sites. Both parameters vary with canopy height, with the greatest biomass and density generally from 30-50 cm from the base, and this is where the majority of leaves are lost during PPFD reduction (Figure 8.1). This should be considered when using this parameter as a sub-lethal indicator. Leaf biomass and density are moderately variable parameters (CV = 40) and the impacted plots (3-months) were

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generally less than the 10th percentile of Control data. These measures would require destructive sampling and consideration should be given to the clumped distribution of stems when designing the collection methodology. Leaf density would be less time consuming to measure than leaf biomass as biomass measures would require removal of algal epiphytes and drying. Total above-ground biomass had a similar response to leaf biomass but was not as sensitive to PPFD reduction due to the slow response of stems. Therefore leaf biomass is recommended as an indicator over total above-ground seagrass biomass. Cluster density also had a similar response to leaf density, although it tended to take longer and a greater intensity of PPFD reduction to have a significant effect, therefore leaf density would be recommended over cluster density. Leaf area index was calculated by multiplying leaf density by the average leaf area. It had a very similar response as leaf density to PPFD reduction treatments and had similar summary statistics (Figure 7.6, Table 12.3). To generate this parameter more measurements must be taken such as leaf length by width.

12.4.6 Rhizome sugars Rhizome sugars were significantly impacted by PPFD reduction, except in the Post- winter Moderate treatment, which was at an intermediate level between the Control and High treatments (Figure 7.10). There were variations over the year so measurements would need to be compared to a reference site. Rhizome sugars was one of the least variable parameters (CV = 25), but the median value of the impacted treatments in relation to the control dataset varied with timing and also intensity in the Post-winter treatment (Table 12.3). In the Post-summer treatment the median value of the Moderate and High treatment was <1st percentile of the control data. However, the median value of the Moderate treatment was similar to the median of the control data whereas the median of the High treatment was the 18th percentile of the control data. This variation in timing highlights the need to have paired reference site data to compare it to. Samples for sugars must be collected, dried, ground and chemically analysed, generally with a minimum of 200 mg DW of material.

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12.4.7 Algal epiphyte biomass Algal epiphyte biomass was the final parameter that was significantly different to the controls after 3-months PPFD reduction, except for the Post-winter Moderate treatment (Figure 7.3). This parameter is highly variable over the year, so it is important to compare measures with a reference site. Algal epiphyte biomass also varies with canopy height, with the greatest biomass in 40-60cm (Figure 8.1), so this should be considered when using this parameter. Algal epiphyte biomass was the most variable of all potential sub- lethal indicators (CV = 60). As with rhizome sugars the median value of the impacted treatments varied with timing, such that in all Post-summer treatments the median was <1st percentile of control data, whereas in the Post-winter treatments the median was the 45th (Moderate) and 14th (High) percentile of the control data. Generally it is considered that algal epiphytes are faster at responding to PPFD reduction than seagrass as they have fewer energy stores to cope with the stress. However, this experiment has demonstrated that some seagrass parameters were more consistent in response to the type of PPFD reduction imposed on them.

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13. Management Application of the Research Results

Underlying the rationale for this research project were nine information gaps that limit the capacity to understand, predict and manage the impacts of reduced light availability on benthic primary producer habitats. These gaps were previously listed in the original research proposal, and are:

Environmental Modeling 1. What are the physiological requirements of key habitat forming primary producers and their environmental tolerances to varying levels of light and suspended sediment;

Impact prediction 2. What are the levels of susceptibility of the dominant benthic communities (which are most sensitive and should be avoided); 3. What are the relative levels of resilience of the dominant benthic communities (which are least sensitive and can withstand greater levels of pressure than others); 4. What drives the susceptibility/resilience (why does this occur, is it linked to energy storage capacity, photosynthetic efficiency); 5. Does resilience/susceptibility vary with season (used to time dredging to minimize impact);

Impact management 6. What are the primary and secondary indicators of sub-lethal stress in the dominant benthic communities (useful for monitoring program design); 7. What levels of pressure (e.g. light reduction) are associated with the primary and secondary indicators of stress (i.e. cause-effect relationships) (useful for establishing alert and action criteria); 8. What is the duration of pressure that is tolerable before the primary and secondary indicators of stress are exceeded (temporal component of alert and action management regimes and criteria to recommence dredging after management intervention); 9. What are the pathways of recovery and how long does it take (post development monitoring and project closure plans)?

The research program was designed to address a number of information gaps for the seagrass Amphibolis griffithii. The following sections outline the key management-related findings and conclusions that can be drawn from the research program on A. griffithii in the context of these generic gaps.

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A. griffithii was chosen as it was one of the main seagrass species impacted by a large, turbid plume that was generated by the Geraldton Port Enhancement project in 2002-03. This plume was more extensive (spatially and temporally) than expected, and at sites monitored within the plume 72-100% cover of seagrass was lost. A. griffithii is a meadow-forming seagrass that can survive in a variety of conditions, and grows most commonly down to 12 m, but has been observed to depths of 48 m (Ducker et al. 1977, Carruthers et al. 2007). Unlike other common meadow forming species such as Posidonia sinuosa with strap-like leaves, A. griffithii has a vertical branching stem with terminal leaf clusters and a higher proportion of the biomass is above-ground (Cambridge 1999). Energy stores in the rhizome are lower than Posidonia and are unlikely to support the plant under severe light limitation during winter (Carruthers & Walker 1997).

13.1 Environmental modeling

13.1.1 Information Gap 1

What are the physiological requirements of A. griffithii and its environmental tolerances to varying levels of light and suspended sediment? The project provides information on the physiological light requirements and tolerances of one key habitat forming primary producer, Amphibolis griffithii. For A. griffithii, all light reduction treatments had a significant effect on plants, with flow-on effects to structural and functional attributes of the habitat. The light reductions imposed in this study were deliberately severe; even the ‘moderate’ reductions were in the order of at least 80% reduction in PPFD compared to ambient. Therefore, this study does not provide the minimum light threshold levels. Other work has provided physiological light and temperature requirements for A. griffithii (Masini & Manning 1997). What is clear from the current study is that reductions of PPFD in the order of 80% for three months or more will result in losses of leaf biomass in the order of 75%, though, if shading is limited to three months or less, recovery can be expected over periods in the order of 10 months. Longer durations or greater intensities of shading increase the impact and decrease the capacity for recovery. Reductions in PPFD of the order of 80% of ambient PPFD for six months results in loss of almost all leaf biomass with no detectable recovery for periods in the order of two years. The responses, provided separately for plants shaded at different times of year, are summarised in Figure 13.1. These experimental light reductions are similar to reductions observed during the Geraldton Port Enhancement project. As continuous light logging was not carried out during the dredging operation and there were no before-dredging light data, the only comparison we can make with our experimental data are for the daily instantaneous light measures. One measurement per day was taken at a number of sites around the dredging area (see Figure 2.2 for location of sites) for several months during the dredging operation. We compared the instantaneous light measures from March 2003, when the turbid plumes were first noted extending further than was predicted. At that time bottom PPFD averaged 40 µE m-2 s-1 (each site averaged over time and then the sites averaged), with a range of 22 – 71 µE m-2 s-1 and the range of minima at sites being 0.01 – 37 µE m-2

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s-1. These conditions fall within the range where impacts were observed in our experiments (average instantaneous irradiance 27 – 93 µE m-2 s-1, see Table 6.1). In a dredging campaign, the changes in light availability are driven by changes in TSS load. How then, do the sorts of experimental light reductions induced here relate to changes in TSS load? Due to the complex nature of suspended particles, TSS-light attenuation coefficient (LAC) relationships need to be derived on a site specific basis, which we have not done. However, recent experimental studies establishing the relationship between light attenuation and TSS for sediments from seagrass habitats in Albany (Ecologia 2007) provide some perspective on the sorts of changes in TSS required to the light reduction we created in our experiment. As the sediments for Albany are not the same as those in Jurien Bay, this example is indicative only. Light reduction treatments in our study generated LACs of up to 0.37 m-1 in shading treatments with 79- 87% light reduction (moderate), and up to 0.47 m-1 in shading treatments with 89-95% light reduction (high). Based on Ecologia (2007), to achieve this sort of LAC in Albany would have required a TSS concentrations of 16 mg L-1 in moderate treatments and 21 mg L-1 in high shading treatments. Background levels in a similar region in Jurien Bay are 1-3 mg L-1 TSS, with light attenuation coefficients of 0.12 m-1 (Bancroft 2005).

Figure 13.1: Effect of reduced light (PPFD as% of the ambient PPFD at the surface of the canopy) on the leaf biomass of Amphibolis griffithii, for shading after summer (Post-summer, commencing March) and shading after winter (Post-winter, commencing September).

Since ambient light intensity is temporally highly variable, it may be more meaningful to express light requirements in absolute terms rather than as proportions of surface or ambient PPFD. The PPFD levels provided here are relative to ambient (i.e.% reductions) but have also been provided as absolute amounts of quanta (Table 6.1). Translating the above effects into absolute amounts of PPFD, plants receiving less than 715 mol m-2 of PPFD or 7.4 mol m-2 d-1 over a 3-month period may be expected to show the severe impacts observed in the experiments.

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It may also be useful to consider light reductions in terms of the amount of time the plants experience intensities above those required to saturate photosynthesis (HSAT). The diagrams in Figure 13.1 have been re-formatted to show leaf loss as a function of average HSAT (Figure 13.2). This reveals, again, the seasonal difference in response, but also that duration of shading is crucial to the effect on A. griffithii. For example, plants receiving on average ~ 4-5 hours of HSAT per day will show about a 40% loss of leaf biomass, and potential for recovery if that light climate is experienced for 3 months. However, if the same average light climate is experienced for 6 months or 9 months then 80 to 99% leaf loss can be expected with no recovery for at least 2 years. Clearly, observable impacts will occur at higher absolute PPFDs over this period, but this study did not examine those more moderate reductions.

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Figure 13.3: The relationship between the cumulative hours of difference between the hours of saturating irradiance of control plots versus shaded plots with the leaf biomass of A. griffithii expressed as a percentage of the control. Red lines indicate the points from which there was no recovery observed. The data also includes results from another shading experiment on A. griffithii (Mackey et al. 2007).

The combined effects of the intensity and duration on the cumulative stress response of A. griffithii to light reduction can be assessed through the cumulative deviation from HSAT over the duration of the experiment, rather than the average HSAT (Figure 13.3). To do this the leaf biomass (as a percentage of the control) was plotted against the summed daily deviation of each treatment HSAT from the control HSAT (x axis of Figure 13.3). Data from Mackey et al (2007) are also included, where the HSAT and leaf biomass response was measured over 40 days to include a shorter-term data point. This cumulative stress response shows that with increasing light reduction (intensity and duration) there is an exponential increase in the loss of leaf biomass. Importantly, when A. griffithii experienced 845 hours less of saturating irradiance than in control conditions, then no recovery of the seagrass meadow was observed over a two-year period.

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The tolerance of A. griffithii to reductions in PPFD is affected not only by the duration of the reduction but also the timing. Plants shaded for three months over the autumn-winter period (post-summer) had greater loss of leaf biomass and other impacts than those shaded over the spring-summer period (post-winter). Similarly, the recovery following cessation of shading was affected by the timing of the shading. This temporal variation in responses and recovery cannot be fully explained but does coincide with significant differences in ambient light availability and water temperature. For example, plants shaded after summer had lower light availability in any given treatment than those shaded after winter (317 vs. 715 mol m-2 of PPFD over the first three months of shading). Some of the differences in plant responses to shading at different times of year would be due to these differences in ambient PPFD availability. In addition, there is likely to have been an effect of seasonal variation in temperature. Masini and Manning (1997) previously showed that the photosynthetic light requirements of A. griffithii were significantly affected by temperature over the range 13-21°C, with higher compensating irradiance (Ec), saturating irradiance (Ek) and maximum photosynthetic rate (Pmax) at higher temperatures. The average temperature experienced by plants over the first three months of shading in the post-summer (21.7 °C) and post-winter (18.7°C) shading periods covered a significant portion of this temperature range and the responses of plants were consistent with the photo-physiological responses observed by Masini and Manning (1997), that is plants shaded directly after summer (autumn/winter period) showed a more severe response, possibly due to the higher light requirements to meet respiratory demands over this period. The outcomes of the research indicate that the physiological requirements and tolerances of A. griffithii to light reduction are complex, likely involving interactions with temperature and therefore temporally variable. Efforts to model the response of the species to changes in light climate need to factor in the time of year and particularly the temperature and changes in ambient light.

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Impact on ecological function and provision of ecosystem services theory Human impacts such as a dredging event can result in a loss of ecosystem function and services during the impact phase, and following cessation of impact until full recovery is achieved. Thus, in assessing impacts, the period of loss of ecological function can also be taken into account, a period that extends beyond the point when the stress (i.e. presence of a dredge plume) is removed. This period of lost ecosystem function can extend over prolonged periods depending on the severity of impact and rate and extent of recovery. Ideally, where an impact on an ecosystem is unavoidable, it would be managed in a way to minimise affect on the ecosystem and the timescale of loss of function (Figure 13.4). Figure 13.4 demonstrates three hypothetical impacts. The y-axis indicates the severity of the impact and the x-axis the duration of the impact. Any elevation above zero on the y- axis indicates a loss of function relative to a reference or pre-impact condition. The loss of function during the impact and recovery period is equivalent to the area under the line. The loss can be relatively small during the impact phases followed by rapid recovery, yielding a low cumulative loss of function (green line). The yellow and red lines indicate similar magnitudes of initial impact with slow recovery (yellow) or no recovery (red), representing cases of prolonged or permanent loss of ecological function and ecosystem services, sustained well after removal of the initial impact. Clearly, it is desirable for the design of a dredging programme to approach the green case rather than the red.

Figure 13.4: Hypothetical scenarios demonstrating loss of function in an ecosystem with different extent and duration of impact and recovery. The green line has the least loss of function and the red line the most, where loss of function is the area under the line.

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Loss of function in A. griffithii shading experiments Loss of ecological function in A. griffithii seagrass meadows was defined as the loss of leaf biomass relative to the control (Figure 13.5). Generally after three months of shading (blue line) there was a loss of function but following removal of shading the line approaches or reaches zero by 10 months, indicating the return of ecological function relative to the control. Hence, there was a loss of function over a 13 month period. In contrast, when plants were shaded for six or nine months there was generally no indication of the line approaching zero following cessation of shading, indicating ongoing loss of function for over 30 months. The one exception was the Post-winter Moderate treatments where function was returning after 23 months.

Figure 13.5: Timescales of loss of ecological function (expressed as the loss of leaf biomass relative to the control) over the duration of the Impact and recovery phase. Different coloured lines indicate different durations of shading. The dotted lines indicate when shading was removed and the plants re-exposed to ambient light. The numbers in brackets indicate the amount of light reduction each treatment received.

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Cause-effect pathways that link PPFD availability to seagrass community health. The data provided from this study confirm that the pathway of effect of shading on A. griffithii is fundamentally one of a carbon-deficit. These pathways are described in Section 7.7 and summarized below. As available light is reduced, plants enter an initial phase of physiological response to maintain a positive carbon balance, reflected in changes to the measurable aspects of photochemistry (rETR curves). During spring- summer, these physiological adaptations may be sufficient to offset moderate reductions in light availability. However, prolonged, more severe or similar reductions at other times of the year may exceed the capacity for physiological adaptation and the plants begin to demonstrate morphological changes. Primarily plants shed leaves, which have a high respiratory demand. Plants first lose individual leaves from clusters and then whole clusters are lost. This simultaneously reduces the demand for carbon and the attenuation of light through the now thinned canopy, increasing the light availability to the remaining leaves. The morphological changes resulting from severe light reductions remove both seagrass and epiphytic algal biomass from the seagrass canopy. Both these components have important roles in providing structurally complex habitat and food resources for the seagrass-associated faunal community. As part of a separate project, the implications of these changes for fauna and tropho-dynamics are being examined. It is sufficient to state here that the severity of changes to habitat structure noted in this study have dramatic consequences for the abundance, biomass and productivity of the macroinvertebrate fauna in A. griffithii meadows, which in turn will have consequences for higher trophic levels. The results from the current study will permit the ongoing study into the responses of seagrass-associated fauna to changes in meadow structure and function, and together these studies will provide data to inform development and implementation of models to predict potential trophic consequences of light-induced changes to seagrass meadows.

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Summary

• Large-scale dredging projects cause intense (acute and chronic) and widespread impacts on benthic light climate due to the light attenuating effects of sediments liberated to the water column by dredging. Light dependent benthic organisms such as seagrasses are particularly susceptible to deterioration in the quality and quantity of light they receive. A. griffithii has been found here to be susceptible to the intensities and durations of light reduction that could be expected to occur during large-scale dredging operations in WA.

• All of the shading treatments in our experimental study had severe affects on A. griffithii and the minimum light requirements will be significantly higher than those reported in this study.

• Total irradiances over 3-9 months, averaged daily and average instantaneous PPFDs, as well as hours of saturating light intensity corresponding to the treatments that induced impacts on A. griffithii are provided in Table 6.1 of this report.

-2 • In meadows receiving as much as 715 mol m over 3 months (equivalent to 82% shading or 18% of ambient light), severe loss of plant biomass and alteration of meadow structure occurs, and there is a strong likelihood of significant recovery over a subsequent 10-month period.

-2 • In meadows receiving as much as 1128 mol m over 6 months (equivalent to 83% shading or 17% of ambient light), almost complete loss of A. griffithii can be expected with little prospect of recovery over at least the subsequent two years.

• These tolerances of A. griffithii to light stress will alter with time of year. Plants are likely to tolerate high (though not extreme) shading more effectively over the spring period, but may then show a delayed onset of impacts, even after shading has been completely removed.

• As the responses of A. griffithii meadows to light reduction were influenced by the time of year that light stress was imposed, efforts to model plant responses should incorporate temporal variability related to, at least, the ambient light and temperature conditions.

• Algal epiphyte biomass, a source of food and habitat for other organisms was also impacted by shading.

• The changes in seagrass morphology and algal biomass caused by shading have important consequences for the habitat- and food-provision roles of A. griffithii meadows. These changes result in significant loss of macroinvertebrate abundance, biomass and productivity, with likely adverse consequences for higher trophic levels.

• The loss of ecological function associated with light reduction extends significantly beyond the period of shading. Even in cases where seagrass showed the capacity to recover, elements of the ecological function of the habitat were lost for up to 13 months. In more severe cases, ecological function was still severely impacted almost two years after shading ceased.

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13.2 Impact prediction

13.2.1 Information Gap 2 & 3

What are the relative levels of susceptibility and resilience of Amphibolis griffithii communities to imposed light stress? This project was the intended first phase of a research programme into the ecophysiology of benthic primary producers; it addressed a single primary producer habitat – seagrass meadows dominated by Amphibolis griffithii. As such, the study provides the necessary data for A. griffithii to be ranked against other benthic primary producer habitats should those data be collected in the future.

How does the resilience of Amphibolis griffithii compare with other seagrass communities? Notwithstanding the above, the current project has revealed an important finding regarding the presumed relative sensitivity and resilience of A. griffithii to shading. Standard functional-form models of seagrasses place A. griffithii towards the centre of the continuum of seagrasses resilience and colonization potential. Based on its eco- physiological attributes, A. griffithii has been presumed to be relatively resilient to disturbance and a potential recoloniser (Walker et al. 1999). Relative to ‘large’ seagrass species such as Posidonia spp, species of Amphibolis are expected to show greater susceptibility to disturbance, due to their lower rhizome reserves, but a greater potential to recover. Recent studies by Collier and co-workers (Collier 2006) have described the response of P. sinuosa to intensities and durations of shading similar to some of those imposed on A. griffithii in this study. At comparable depths to this study, P. sinuosa showed an 84% decline in shoot density over three months of high intensity shading (<10% of ambient PPFD), increasing to 94% decline after seven months, equating to 60% loss of above ground biomass. Following seven months of moderate shading (28% of ambient PPFD) P. sinuosa showed significant recovery after 13 months, but there was no recovery in the heavily shaded plots. In comparison, the current study indicates that A. griffithii does not show any enhanced capacity for recovery from light deprivation impacts. This is supported in Figure 13.6 where P. sinuosa (Collier 2006) and A. griffithii (this study) shaded for six months have similar loss of ecological function curves, where loss of function was calculated as the loss of leaf biomass relative to the control.

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Figure 13.6: A comparison of the loss of function estimate in the experimental Amphibolis griffithii seagrass meadows after 6 months of light reduction PW (post-winter) and PS (post-summer) with the loss of function estimate for Posidonia sinuosa based on Collier (2006). The dotted lines indicate when shading was removed.

Monitoring of seagrasses following widespread losses in Geraldton further corroborates the finding that A. griffithii may take years to recover form dredging-related light reductions. Monitoring of impacted meadows in Champion Bay (CSIRO 2007) detected some but not complete recovery (increase in A. griffithii cover toward pre-dredging conditions) 24-36 months after the impact (see Figure 2.2). The experimental plots shaded for 6-9 months (<20% ambient PPFD) showed no recovery (increase in leaf biomass relative to controls) 15-23 months after the shading was removed. Therefore, the recovery time-scales observed in post-impact monitoring at Geraldton and in this experiment are similar. Similarly, when the loss of ecological function observed in the experimental study is compared with the loss observed post-dredging at Geraldton, the results are comparable (Figure 13.7), giving more assurance that the experimental results are indicative of the responses that can be expected under a large-scale dredging programme. The above findings challenge any presumption that A. griffithii is a species with a capacity for rapid recovery following severe disturbance. This is an important factor to be taken into account when considering the likely impacts of dredging on the species and the timescale required for a complete return of ecological function.

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Figure 13.7: A comparison of the loss of function estimate in the experimental Amphibolis griffithii seagrass meadows after 9 months of light reduction with the loss of function estimate at a number of sites exposed to a turbid plume from dredging during the Geraldton Port Enhancement Project. The dotted lines indicate when shading was removed or dredging stopped.

Which seagrass parameters show the clearest adverse responses and/or consistent trends with respect to reduced PAR?

What information do the results provide on selecting robust indicators of Amphibolis community health for adoption by managers?

To properly protect and conserve key elements of the marine environment, indicators applied in monitoring programs should ideally show rapid response, be sub-lethal, able to be measured using non-destructive techniques and linked to cause-effect pathways associated with stressors of concern. Seagrass health monitoring programmes currently running in Western Australia reaffirm the importance and relevance of these key principles (Lavery & McMahon 2007). The findings of this study point to an array of sub-lethal indicators of A. griffithii health that show promise for environmental monitoring and management application, based on the underlying assumption that the health of this ecosystem is dependent on the health of the species itself. Table 12.1 of this report summarises the research finding on seagrass parameters showing consistent, adverse responses to reduced light availability. The parameters are categorised by the consistency of response across different intensities of shading and at different times of shading. Of all the variables measured, 28 showed a response to reduced light availability, at least one combination of intensity, duration and timing of shading. Parameters that show most promise for management applications are those that respond quickly (by three months), at both times of year and to moderate or high levels of shading. Fourteen parameters met these general requirements, and of those, only two (leaf extension rate and leaf ∂15N) had responses that were consistent at both times of year and to moderate levels of light reduction. The sub-set of fourteen parameters is an appropriate set of parameters from which predictive and monitoring variables can be

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further developed, with the final choice being dependent on aims and practicalities associated with the particular application. Summary statistics are provided for fourteen variables in the form of percentile distributions for each of those variables measured in unshaded treatments. Other seagrass monitoring programmes currently running in Western Australia have emphasised the value in using parameters which respond rapidly, can be measured non-destructively, and which can confidently be placed on the cause-effect pathway of ecosystem change. Leaf ∂15N, while attractive, requires further investigation with respect to the mechanism that drives change in the variable. Leaf extension rate is a consistently responsive variable, but care needs to be taken to standardise the canopy height at which it is sampled. The number of leaves per cluster is an attractive variable for use in monitoring since it meets all of the desirable characteristics, though the small effect size, a change from three to two leaves per cluster, necessitates careful measurement. The number of clusters per stem is another easy to measure variable, but is closer to the lethal end of the response pathway than the number of leaves per cluster. Leaf biomass and density, and epiphytic algal biomass respond rapidly to light reduction but require destructive sampling. Finally, rhizome sugars were significantly affected by reductions in light availability, but showed sufficient inconsistency over times and intensities of treatments that its application may prove problematic. A more thorough consideration of the value of these parameters as indicators of ecosystem health is provided in Section 12.4.

13.2.2 Information Gap 4

What drives the susceptibility/resilience (why does this occur, is it linked to energy storage capacity, photosynthetic efficiency)? This research has examined a wide range of variables in order to test the underlying assumption that changes in A. griffithii condition, in response to reduced light availability, are driven by reduced carbon fixation leading to a carbon deficit which, if prolonged, cannot be offset by drawing on stored carbohydrate reserves. This ultimately results in the plant moving through a sequence of physiological and then morphological changes, which have the effect of reducing carbon fixation requirements and increasing light availability to the remaining photosynthetic tissue. However, even these morphological changes are insufficient to sustain the plants under extreme or prolonged shading. These pathways of effect are summarised in Section 7.7, and the presumed pathway of recovery responses in Section 9.7. The research confirms that the assumptions about the pathways of response are valid, but has added a much greater level of detail to that understanding. We can now characterise the relative position of different variables along the cause-effect pathway and can reconcile these with fundamental principles of plant eco-physiology. We can also characterise the differences in responses at different times of year and again, these can be explained in ways consistent with our understanding of plant physiology. This improved knowledge has underpinned the discussion of potential parameters for use in the monitoring of A. griffithii habitats.

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13.2.3 Information Gap 5

Does resilience/susceptibility vary with season (used to time dredging to minimize impact)? Time of year clearly affects the susceptibility of A. griffithii to shading and to its subsequent recovery. Importantly, however, the nature of this affect is contrary to that which was anticipated. Previously, it was assumed that plants would have their minimum carbohydrate reserves at the end of winter and maximum reserves at the end of summer, following a full season of growth under high light conditions. This was demonstrated in the study with rhizome carbohydrates on average 20% DW at the end of summer and 14% DW at the end of winter. It was then expected that plants would have their maximum capacity to withstand shading at the end of summer, when they could draw on a large storage reserve. Contrary to this expectation, plants displayed a more rapid response to shading imposed at the end summer than at the end of winter. The ambient light levels are lower at the end of summer (autumn) compared to the end of winter (spring), so that a similar proportion of light reduction results in a lower absolute amount of light. Plants shaded at the end of winter showed no impact from shading, with up to about 80% reduction of light relative to ambient. A plausible explanation for this is that those plants were entering a period of increasing ambient light intensities and over the shading period received more than double the PPFD of plants subjected to the same percent reduction in ambient at the end of summer. Thus, the crucial information for managers is the absolute PPFD that plants will receive, rather than the anticipated percent reduction of ambient light, and this will vary with time of year. A second important finding was that the timing of a shading event might cause a delayed impact. While our moderate intensity of shading imposed at the end of winter had no detectable effect on seagrass biomass after three months, there was a dramatic decline in biomass after six months, to levels comparable to plants shaded at the end of summer. Thus the effects of timing on seagrass response appear only to apply to relatively short- term shading events. Again, this probably relates to the absolute amount of light received over a shading period. The longer the shading period, the more similar the absolute amounts of light received by plants shaded at different times due to a longer integration over seasons. For example, plants shaded at our moderate intensity for nine months had about 1 250 mol m-2 of irradiance irrespective of whether this was post-summer of post- winter. The third important finding in relation to timing was the consequences for recovery. While plants shaded for three months at moderate shading intensities after winter showed no detectable loss of biomass, they had a significant loss of leaf biomass in the three months following the removal of shading. In contrast, plants shaded at the end of summer had a loss of leaf biomass during shading but showed no subsequent loss in the 3 months following removal of shading. The loss of biomass following removal of shading in the former case has been reported in other species of seagrass (Malta et al. 2006), providing increased confidence that this is not an experimental artefact. While the mechanism behind this affect is not clear, it is quite possibly related to the sudden increase in light following removal of shading, which may cause photo-oxidative damage to previously dark-adapted plant tissue. The end of shading corresponded with the period of highest

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ambient light intensities, January-April. It may also relate to the growth characteristics of the plants at that time. We noted that maximum branching in control plants occurred during spring (16%), with intermediate levels in summer and autumn (8%) and minimums in winter (3%). The period of declining biomass during the recovery period was summer, so it is possible that the combination of reduced light, followed by photo- oxidative stress at a time of naturally high growth demands to support branching exceeded the tolerances of the plants, resulting in a loss of biomass despite the removal of shading. Clearly, then, the potential impact and rate of recovery will be affected by the timing of any dredging-related reductions in light availability. This temporal variation appears to be a function of both ambient conditions and the seasonal changes in plant biology. With respect to ambient conditions, light intensity and the daily period of saturating light intensity vary throughout the year. Similarly, temperature varies and both these factors influence the photochemistry of the plant. During spring-summer plants have higher light availability but also require more light to meet their carbon demands (Masini & Manning 1997). Therefore, the significance of a given increase in light attenuation coefficients due to dredging or other activities needs to be assessed in terms of the absolute PPFDs at the seagrass canopy and how those PPFDs compare with the requirements of the plant at the mean temperature for that period. Similarly, increased requirements at some times of year may be related to the increased branching frequency that occurs on a seasonal basis. It is important to also note, however, that the strongest effects of timing of light reduction occurred in those plots subjected to the moderate intensities of shading. At the highest intensities of shading, plants responded similarly in both post-summer and post-winter shading periods. Thus, once light reductions become severe, the interactive effects of plant eco-physiology and ambient conditions become less relevant.

13.3 Impact management

13.3.1 Information Gaps 6 & 7

What are the primary and secondary indicators of sub-lethal stress in the dominant benthic communities (useful for monitoring program design); and

What levels of pressure (e.g. light reduction) are associated with the primary and secondary indicators of stress (i.e. cause effect relationships) (useful for establishing alert and action criteria); The indicators of sub-lethal stress were largely addressed in the preceding paragraphs of this section (13.2.1) and in Section 12. The project has clearly identified a number of variables that respond rapidly (i.e. within three months) to moderate levels of light reduction. Earlier studies on A. griffithii (Mackey et al. 2007) examined a smaller set of parameters but at greater frequency (monthly over three months). That earlier work indicates that some of the variables shown here to respond within three months also responded as quickly as one month (e.g. leaves per cluster, leaf extension and rhizome sugars). This provides confidence that the variables we recommend for consideration as

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‘early warning’ indicators of sub-lethal stress on the basis of the current study are likely to respond at significantly shorter timescales than the 3 month interval used here. There has been a widespread hope that physiological attributes of plants may provide indications of sub-lethal stress ahead of morphological changes, which frequently involve the loss of plant biomass. Of the variables we have identified as having potential for application as indicators of light-related stress, two of the most consistent and sensitive were the ∂15N value of leaf tissue and leaf extension rates, both ‘physiological’ attributes that respond early in the cause-effect pathway and most likely prior to any morphological changes. While this offers some of the strongest evidence yet that physiological parameters might be applied in monitoring, further characterization of the responses of these variables will need to be undertaken before applying them as the basis of criteria in monitoring and management frameworks. Summarising the discussion in Section 12, 28 variables respond to reductions in light availability, 14 showing consistent and early responses, eight of which are highlighted for potential application in seagrass monitoring programmes. Each of the parameters we have drawn particular attention to has positive and negative aspects with respect to their potential application as indicators of seagrass ecosystem condition (Section 12.4 and summarized below, Table 13.1). Summary statistics for the parameters are presented in Table 12.3. In keeping with the approaches outlined in ANZECC (2000) and the State Environmental (Cockburn Sound) Policy (Govt of WA 2005), the summary statistics are provided as percentile distributions for the parameters in control plots, the median value in shaded plots and its corresponding percentile value in the control dataset.

13.3.2 Information Gap 8

What is the duration of pressure that is tolerable before the primary and secondary indicators of stress are exceeded (temporal component of alert and action management regimes and criteria to recommence dredging after management intervention). As detailed in the previous discussion and summarized in Figure 13.2, three months of shading at the moderate intensity exceeded the tolerance of A. griffithii. In most instances, the response was apparent within three months of imposed stress. The exception was post-winter shading when three months did not have an immediate effect on the plants, though a severe effect was noticed once the shading was removed. It is important to note that the ‘moderate’ intensity shading imposed in this study was, in fact, a severe level of shading. During the post-summer (autumn-winter) shading period the three months of moderate shading reduced the total irradiance received at the canopy surface to only 14% of ambient, and reduced the hours of saturating light intensities to 45% of ambient. In the post-winter shading period (spring-summer), total irradiance was reduced to 18% of ambient and hours of saturating irradiance to 58% of ambient. These are, in themselves, severe reductions in light availability and it is important to view the severity and timescale of the plant responses in light of the severity of the treatments.

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Notwithstanding the above, the light reduction treatments imposed in the study are comparable to those observed in large, commercial dredging operations in the region as was the minimum measurement period, three months (e.g. CSIRO 2007). Visual observations of the plots during the experiments indicated that impacts were realised much earlier than the first sampling occasion at three months. A significant pilot study (Mackey et al. 2007) used similar level of high intensity shading and demonstrated significant, detectable effects within one month. Therefore, it can be concluded that at the intensities of light reduction stress imposed in the study, A. griffithii will show responses between one and three months after shading is imposed and possibly earlier. While the study did not examine less severe light reductions, the rapidity and magnitude of changes observed in plants subjected to the treatments used, and the lack of recovery in most of those treatments, suggest that the changes in light climate summarized in Table 6.1 far exceeded the tolerances of A. griffithii at most times of year. It can be concluded, therefore, that less severe reduction in PPFD will result in loss of seagrass biomass, though it is not possible to predict the ‘no effect’ threshold.

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Table 13.1 Summary of the plant and algal parameters in Amphibolis griffithii habitats which were responsive to shading treatments within 3 months, showed high levels of consistency in the response and have the greatest potential for development as indicators of plant and ecosystem condition in relation to light reductions.

Parameter Type Advantages Uncertainties or other comments Leaf tissue ∂15N Physiological Highly consistent response across all levels of shading & timing of Controls showed temporal variation so would require comparison shading and throughout canopy heights. against ‘reference’ data Low degree of variation and impacted sites showed a large effect Other environmental factors can change the ∂15N signal and would size relative to controls. need to be considered. Recovered after 3 months exposure to ambient PPFD so may be Processing of the samples for analysis is time consuming and useful at detecting the cessation of light reduction. relatively costly Leaf extension rate Physiological Highly consistent response across all levels of shading & timing of Controls showed temporal variation so would require comparison shading and throughout canopy heights. against ‘reference’ data

Varies depending on location in the seagrass canopy, so consistency in the sampling height would be required. Requires a high degree of replication or pooling of individual leaves within sampling locations. Time consuming; requiring repeat visits to the sampling site and significant expertise in leaf and cluster marking. Leaves per cluster Morphological Showed a generally consistent and rapid responds to shading. The absolute effect size is generally small and so an adequate sample size is required to provide confidence in observed changes. Relatively little variability in control plots over a full annual cycle, thus it is one of the few parameters that might be used without Increases with height in the canopy, so consistency in the sampling comparison to a reference site. height would be required. Recovers rapidly (within 3 months) so may be useful in recovery Can be expressed as a mean, median or mode, with mean and mode monitoring. the most sensitive at detecting effects. Mode may be a useful statistic and is a meaningful representation for this whole integer variable.

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Table 13.1 (con’t). Summary of the plant and algal parameters in Amphibolis griffithii habitats which were responsive to shading treatments within 3 months, showed high levels of consistency in the response and have the greatest potential for development as indicators of plant and ecosystem condition in relation to light reductions. Parameter Type Advantages Uncertainties or other comments Leaves per stem Morphological Showed a generally consistent and rapid response to shading and Controls showed temporal variation so would require comparison integrates the number of clusters per stem and number of leaves per against ‘reference’ data. cluster. Can be expressed as a function of all stems or of stems with leaves. The effect size is large relative to controls, making it a relatively The choice will depend on the objective of the monitoring and degree sensitive indicator. of sensitivity required (see section 12.4). Easy to measure in laboratory Earlier along the pathway of effect than clusters per stem. While possible to measure non-destructively this may be difficult in the field, and is more likely to require destructive sampling and subsequent analysis in the laboratory, with associated costs. Leaf biomass/density Morphological Moderate effect size relative to controls makes these relatively Reasonably consistent across the year, but due to likely variations sensitive indicators of reduced light availability. among sites, these measures should be used by comparison with reference sites. Leaf density is less time consuming to measure than leaf biomass. Varies depending on location in the seagrass canopy, so consistency Leaf biomass strongly reflects changes in total above-ground in the sampling height would be required. biomass but shows greater effect size, and leaf density reflects cluster density, but responds more rapidly. Requires destructive sampling and significant processing time in the laboratory. Leaf density would be less time consuming. Leaf density also strongly reflected changes in Leaf Area Index. When sampling, consideration should be given to the clumped distribution of stems.

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Table 13.1 (con’t). Summary of the plant and algal parameters in Amphibolis griffithii habitats which were responsive to shading treatments within 3 months, showed high levels of consistency in the response and have the greatest potential for development as indicators of plant and ecosystem condition in relation to light reductions. Parameter Type Advantages Uncertainties or other comments Rhizome sugars Physiological Rhizome sugars generally responded to light reduction in most Controls showed temporal variation so would require comparison treatments but on occasions may be insensitive. against ‘reference’ data. Response effect size may be small for moderate shading, making this a potentially insensitive variable for shading effects less severe than the quite large levels imposed in the study. Variation in effects due to the timing of PPFD reduction indicates the need to have paired reference site data. The collection, processing and analysis of samples for carbohydrates is time consuming and, potentially, costly. Algal epiphyte biomass Morphological Responsive to PPFD reduction, though this is variable for more Controls showed temporal variation so would require comparison moderate intensities of light reduction at some times of year. against ‘reference’ data. Fast response to PPFD reduction as they have relatively small Varies depending on height in seagrass canopy, requiring consistency energy stores to cope with the stress. in the sampling height. Inconsistency in responses at some times of year. Requires destructive sampling and labour intensive processing.

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13.3.3 Information Gap 9

What are the pathways of recovery and how long does it take (post development monitoring and project closure plans). The pathways of recovery are described in Section 9.7. In the Post-winter treatments (i.e. shaded during spring-early summer) physiological and morphological parameters generally showed a rapid recovery, approaching or reaching control levels within 3 months and with almost complete recovery of all parameters by 10 months. This probably reflects the relatively high ambient light conditions during both the shade period and the recovery period, and is consistent with the plants having sufficient remaining photosynthetic material and carbohydrate reserves to take advantage of the improved light conditions and initiate rapid growth at a period of naturally high branching rates. Thus the combination of severity of impact, ambient light intensities during the recovery period and the plants’ natural growth phase coincided to allow a relatively rapid recovery. As noted earlier, the biomass in the Moderate intensity treatment declined over the first 3 months of recovery conditions, possibly reflecting damage to the photosynthetic apparatus (e.g. Malta et al. 2006). This clearly indicates the potential for different recovery pathways following shading at different times of year. We observed no recovery in those plants shaded for 6 or 9 months at either moderate or high intensities. The common characteristic of all these treatments was that leaf biomass fell to below 25% of the control. It is possible, therefore, that a threshold in leaf loss dictates the potential for subsequent recovery, probably by limiting the photosynthetic capability and meristems for branching and initiation of new growth.

13.3.4 Information Gap New

Can the frequency of light reductions affect the impact on and recovery of Amphibolis griffithii? One aspect of dredging operation that is relatively easy to manage is the timing of the activity. This raises the question of whether three intervals of one month dredging campaigns, separated by, for example, one month of no activity, would produce the same or less effect as a single three month campaign. This information gap was not identified as a focus of the research and the study was not designed to address this question. Intuitively, repeated, short light reduction events interspersed with periods of ambient conditions would more likely allow the plants to recover depleted carbohydrate reserves and keep them within the ‘physiological’ pathway of impact rather than moving into the ‘morphological’ pathway with its attendant loss of biomass and photosynthetic capacity. This would clearly result from the increased total PPFD during the impact stage of a 6 month ‘interval dredging’ campaign compared to the 3 month impact stage of an uninterrupted dredging operation. For example, using the PPFD data gathered during the first 3 months of the post-summer shading period, a plant shaded in the moderate intensity treatments would receive 317 mol m-2 of PPFD, 16% of ambient, while a plant subjected to 1 month of shading followed by 1 month of recovery and a second month of

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shading would receive 857 mol m-2 over the same period, 44% of ambient. Whether this is sufficient to prevent morphological changes is unknown. At the same time, a number of other factors may offset some of the benefits intuitively ascribed to more frequent but shorter duration dredging. For example, the residual effect of dredge plumes on water clarity would need to be considered. Similarly, the unexpected and highly contrasting recovery dynamics of plants shaded for 3 months after winter and after summer underlies the complex interactions of biological phases of plants and ambient environmental conditions that influence recovery. Similarly, we have no data to assess the capacity of A. griffithii to withstand multiple, repeated shading events, and whether the physiological response pathways are capable of repeatedly coping with high intensity light reduction. However, the demonstrated potential of the plant to recover from moderate shading suggests that this would a fruitful area of enquiry when contemplating the design of dredging operations.

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14. References

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15. Publications and Presentations From This Research

Papers Mackey P, Collier C, Lavery P (2007) Effects of experimental reduction of light availability on the seagrass Amphibolis griffithii. Mar Ecol-Prog Ser 342:117-126

Honours theses Barwick H (2006) The effects of light reduction treatments on mobile epifauna in an Amphibolis griffithii (Black) den Hartog seagrass ecosystem. Edith Cowan University. 72 pp. Mackey P (2004) Effects of temporary PAR reduction on the seagrass Amphibolis griffithii (Black) den Hartog. Honours, Edith Cowan University. 55 pp.

Interim Reports Lavery P, McMahon K (2006). Ecophysiology of benthic primary producers. pp154-168. In: Keesing JK, Heine JN (Eds). Strategic Research Fund for the Marine Environment Final Report . Volume 1: the SRFME initiative and collaborative linkages program. 260 pp. Strategic Research Fund for the Marine Environment, CSIRO, Australia.

Presentations McMahon K, Lavery P (2006). Response and recovery of seagrass, algae & fauna to light reduction in an Amphibolis griffithii meadow. SRFME Symposium, Perth, March 2006. Lavery P, McMahon K, Collier C (2006). Seagrass ecosystems of SW Australia. Centre for Advanced Studies (CEAB), Blanes, Spain, June 2006. Lavery P, McMahon K (2006). Effects of light reduction on seagrass ecosystems and their trophic implications. Botany Institution, Stockholm University, Sweden, June 2006. McMahon K, Lavery P (2006). Resilience of seagrass systems to dredging: Perspectives from temperate and tropical regions. Australian Marine Science Association 44th Annual Conference & Society of Wetland Scientists 27th International Conference, Cairns, Australia July 2006. McMahon K, Lavery P (2006). Dredging in seagrass systems: A case study with the Amphibolis griffithii. International Seagrass Biology Workshop VII. Zanzibar, Tanzania, September 2006. Lavery P, McMahon K (2007). Ecophysiology of Benthic Primary Producers. SRFME Symposium, Perth, March 2007 Gartner A, Lavery P, McMahon K, Brearley A, Barwick H (2007). Trophic implications of seagrass habitat disturbance from reduced light. Australian Marine Science Association, Melbourne, July 2007. McMahon K, Lavery P, Mulligan M (2007). Effects of timing, duration and intensity of dredging on an Amphibolis seagrass ecosystem – impact and recovery. Australian Marine Science Association, Melbourne, July 2007. Lavery P, McMahon K (2007). Managing the effects of dredging on seagrass ecosystems – effects and indicators of light reduction. Centre for Ecosystem Management Seminar Series, Joondalup, September 2007. McMahon K, Lavery P (2007). Effects of timing, duration and intensity of dredging on an Amphibolis seagrass ecosystem – impact and recovery. Jurien Bay Marine Park Advisory Committee Meeting, October 2007. McMahon K, Lavery P, Mulligan M, Brearley A, Gartner A & Barwick H (2007) Indicators of the indirect effects of dredging in a temperate seagrass ecosystem, Western Australia. Estuarine Research Federation Conference, Providence, Rhode Island, USA, November 2007.

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16. Graphic Appendix

Figure 16.1: Algal epiphyte biomass (g DW m-2) on A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Algae on leaf, b) Algae on stem. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

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Figure 16.2: Faunal epiphyte biomass (g DW m-2) on A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

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Figure 16.3: Leaf cluster morphology of A. griffithii following PPFD reduction treatments of Timing: Post-summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Average leaves per cluster; b) Median leaves per cluster; c) Mode leaves per clusters. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars. nd indicates no data for that duration and/or intensity as not enough leaves were present to determine the measure.

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Figure 16.4: Canopy height (cm) of A. griffithii following PPFD reduction treatments of Timing: Post- summer, Post-winter; Duration: 3-, 6-, 9-months; and Intensity: Control, Moderate, High. a) Canopy height – 80th percentile; b) Maximum canopy height; c) Average canopy height. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

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Figure 16.5: Leaf cluster morphology of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Average leaves per cluster; b) Median leaves per cluster; c) Mode leaves per clusters. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

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Figure 16.6: Canopy height (cm) of A. griffithii following recovery from 3-months of PPFD reduction treatments with Timing: Post-summer, Post-winter and Intensity: Control, Moderate, High factors. a) Canopy height – 80th percentile; b) Maximum canopy height; c) Average canopy height. Letters indicate significant differences between PPFD reduction treatments (Intensity) at a particular Timing and Duration. Average with standard error bars.

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