Life History Characteristics of Glassfish, Ambassis Jacksoniensis, Adjacent

Life history characteristics of glassfish, Ambassis jacksoniensis, adjacent

to saltmarsh within a large and

permanently-open estuary

Jack J. McPhee

Doctor of Philosophy (Environmental and Life Sciences)

Supervisors:

Dr Maria Schreider (Environmental and Life Sciences)

Dr Margaret Platell (Environmental and Life Sciences)

“Ambassis jacksoniensis” - Illustrated by Corrine Edwards

ACKNOWLEDGEMENTS

I would like to express my sincerest gratitude to a series of people, without whom this PhD would not have been possible. First and foremost, I would like to give a warm thank you to my two supervisors, Dr Maria

Schreider and Dr Margaret Platell. Maria, your encouragement to push on, not for self-benefit, but for the greater scientific good is a trait that I have valued since your teaching during my undergraduate years. Your motive to work hard in order to seek the truth (Без муки нет науки) is a characteristic that often reminds me why I was inspired to pursue a scientific career to begin with – спасибо! Margaret, your genuinely friendly and inquisitive attitude towards the project, and science in general, is a characteristic that has also shaped me over the years. Your genuine care for the organisms and environments that we study is a continual reminder that such scientific pursuits are not only for the benefit of the scientific community, but are of equal importance to the organisms that we are studying. While Maria’s traditional, clinical, to the point (i.e. “eschew obfuscation”) scientific perspective helped me “cut the fat” during my studies, Margaret has brought the “seasoning,” the fun, the flavour. Margaret, to me you are truly perspicacious in the field of estuarine ecology and I thank you for eliciting me into this world. The two of you are genuinely altruistic scientists in my eyes and I am hugely grateful for all your inspiration. Thank you for being wonderful teachers, colleagues and friends. I would also like to thank the numerous enthusiastic colleagues that assisted me in the often wild and uncomfortable sampling occasions. A special thank you to Jeff Law, Ryan Hefford, Wayne Davis, Stuart Bowers, James Brooker and Daniel

Camilleri. Thank you Gordon Thomson, from Murdoch University, for preparing the gonadal slides for microscopic validation. Thank you Macquarie Geotech for conducting the calorimetric analyses. Thank you

Natalie Moltschaniwskyj, for your guidance with the life history and reproduction chapter. I would also like to thank the University of Newcastle for their provision of funding and resources throughout the duration of the project. Thank you Corrine Edwards for illustrating the cover of my thesis. Lastly, I would like to thank my family and friends (particularly my Mum and Dad, Micaela and to my dogs, Willow, Jessie and Pippa), who have all supported me and kept me sane and motivated throughout the entire process of my PhD. The collection of fish and zooplankton was authorised by the Department of Primary Industries (Permit No. P11/0085-1.0) and UoN

Animal Ethics Committee (Permit No. A-2012-219).

In memory of Dr Kenneth Zimmerman - the man who inspired me to study marine science.

STATEMENT OF ORIGINALITY

This thesis contains no material that has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge, contains no material previously published or written by another person, except where due reference has been made in the text.

______

Jack James McPhee

PUBLICATIONS RESULTING FROM THIS THESIS

The following article has been published by Estuarine, Coastal and Shelf Science and a copy of the publication is attached at the end of this thesis (see Appendix A). Other chapters/components of the research are currently being prepared for submission to various ecological journals for publication.

McPhee, J. J., Platell, M. E., and Schreider, M. J. (2015). Trophic relay and prey switching – A stomach contents and calorimetric investigation of an ambassid fish and their saltmarsh prey. Estuarine, Coastal and Shelf Science, 167, 67-74.

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ABSTRACT

Saltmarsh vegetation, which typically occurs in intertidal areas within estuaries globally, provides an important habitat and feeding ground for estuarine organisms such as crustaceans, gastropods, birds and fish (some of which are of economic importance). Within south-east Australian estuaries, saltmarsh vegetation is both typically bordered by mangroves and tidally inundated three or four times per month during the high tide of the spring tidal cycle (during the day high tide in summer and during the night high tide in winter). In recent decades, saltmarsh vegetation has declined globally due to anthropogenic influence, and in Australia, ‘Coastal Saltmarsh’ is now listed as an Endangered

Ecological Community under the Threatened Species Conservation Act 1995.

This study was conducted within a representative and relatively “unmodified” saltmarsh habitat (Empire Bay Wetland) in a large and permanently open estuary, Brisbane Water Estuary, located in south-eastern Australia. This study, which was conducted at two markedly different times of the year during 2012, examined the general “response” of the estuarine fish (using seine nets) and zooplankton

(using plankton nets) assemblages to tidal inundation, with further emphasis being placed on selected biological and ecological characteristics of the abundant estuarine ambassid, Ambassis jacksoniensis.

Abundances of A. jacksoniensis (mean standard length=37.3 mm, ±0.021 (SE)) and overall fish diversity were greater in nightly winter catches than daily summer catches, which is consistent with previous evidence of important feeding times for estuarine fish (including A. jacksoniensis) upon saltmarsh- derived zooplankton (e.g. crab zoeae released by saltmarsh-dwelling grapsid crabs), during ebb tides that drain saltmarsh following its inundation. Indeed, zooplankton assemblages were dominated by crab zoeae during ebb tides following saltmarsh inundation, while calanoid copepods dominated these assemblages at other times. Moreover, stomach content analyses of A. jacksoniensis showed that crab zoeae were heavily preyed upon during such times, with dietary “switching” to caridean decapods being evident when crab zoeae were not abundantly present within the water column (i.e. during flood tides and during ebb tides that did not follow saltmarsh inundation; as shown within zooplankton assemblages). Despite their high abundance within zooplankton assemblages, calanoid copepods were not preyed upon by A. jacksoniensis, which is likely to reflect the relatively fast escape responses of

iv calanoids to predators. Further, stomach fullnesses of A. jacksoniensis were generally highest during ebb tides on days of saltmarsh inundation, implying that feeding was most marked at these times.

Trophic relay is an ecological model that involves the movement of biomass and energy from vegetation, such as saltmarshes, within estuaries to the open sea via a series of predator-prey relationships. Therefore, the trophic relationship between saltmarsh-dwelling grapsid crabs (which feed on saltmarsh-derived detritus and microphytobenthos), A. jacksoniensis and their predators (which include economically important fish, such as Acanthopagrus australis, Platycephalus fuscus and

Argyrosomus japonicus, provides evidence of partial trophic relay within this system, and thus highlights the ecological and economic importance of saltmarsh within this system. The trophic relationship between A. jacksoniensis and its zooplanktonic prey (e.g. crab zoeae, which is of a red/orange colour) was further investigated, for the first time, by comparisons of the calorimetric contribution of its potential prey (i.e. crab zoeae, and the far paler caridean decapods and calanoid copepods), which found no difference in the energetic densities among such potential prey, suggesting that prey (i.e. zooplankton) abundance and/or prey visibility (due to colour) has a stronger relationship than prey energetic density to the diets of A. jacksoniensis.

The feeding ecology of A. jacksonsiensis was also explored, for the first time, in light of its various life history characteristics (e.g. the seasonality of sex ratios, sexual maturity and somatic/reproductive growth), with links being made between saltmarsh-derived tropic relay and energetic requirements for reproductive purposes. Thus, the gonads of A. jacksoniensis were found to be generally maturing and ripe during summer, while juvenile/inactive and spent gonads were prevalent during winter, consistent with previous evidence that A. jacksoniensis spawn during summer with a lull during winter. The sex ratios of A. jacksoniensis were also heavily female-biased during summer before equalising (to approximately 1:1) during winter, suggesting that male A. jacksoniensis may avoid the shallow sampling locations (seagrass adjacent to the saltmarsh/mangroves) in a strategy to counteract visual predation from fish and birds during daytime (summer) before returning to these waters during the night winter, during a lull in spawning, for important feeding opportunities. Female

A. jacksoniensis, alternatively, may remain in such vulnerable locations due to increased energetic requirements for reproductive purposes (as demonstrated in male vs female somatic/gonadal growth

v analyses). These findings therefore suggest that the seasonal timing of spawning for A. jacksoniensis may be linked to their feeding behaviours (i.e. upon saltmarsh-derived zooplankton), the latter of which is governed by the tidal inundation of saltmarsh vegetation.

As there is a global ecological and economic reliance by fish (particularly A. jacksoniensis) on saltmarshes, which facilitate trophic relay within these systems, it is imperative that management of

Australian estuaries is employed in a manner that appropriately incorporates the conservation of saltmarsh vegetation and thus protects its ecological function within these estuaries.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ...... i STATEMENT OF ORIGINALITY ...... ii PUBLICATIONS RESULTING FROM THIS THESIS ...... iii ABSTRACT ...... iv TABLE OF CONTENTS ...... vii LIST OF TABLES ...... x LIST OF FIGURES ...... xiii CHAPTER ONE GENERAL INTRODUCTION...... 1 1.1 Overview of estuaries ...... 2 1.1.1 Estuaries in a global and an Australian context...... 2 1.1.2 Contribution of estuaries to Australian fishes ...... 4 1.1.3 Characteristics of saltmarshes and mangroves in estuaries...... 5 1.2 Characteristics of estuarine habitats & faunal assemblages...... 8 1.2.1 Overview of fish habitats ...... 8 1.2.2 Fish in saltmarshes and mangroves of Australia ...... 9 1.2.3 Factors affecting estuarine faunas, including zooplankton and fishes ...... 13 1.2.4 Zooplankton assemblages in estuaries ...... 14 1.2.5 Calorimetric content and energetic density of zooplankton and other organisms . 18 1.3 Life history traits of estuarine fishes ...... 21 1.3.1 Sexual reproduction in estuarine fishes ...... 21 1.3.2 Mating systems and spawning times in estuarine fishes ...... 22 1.3.3 Food sources and feeding of estuarine fishes ...... 24 1.4 The Ambassidae...... 28 1.4.1 Reproductive biology of ambassids ...... 29 1.4.2 Life history traits of Ambassis jacksoniensis ...... 31 1.5 Framework of investigation and study aims...... 35 1.6 Outline of thesis...... 37 CHAPTER TWO GENERAL METHODOLOGY ...... 39 2.1 Characteristics of the study region and location ...... 40 2.1.1 The study region - Brisbane Water Estuary ...... 40 2.1.2 The study location - Empire Bay Wetland ...... 43 2.2 Sampling and experimental design ...... 45 2.3 Data collection ...... 46

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2.3.1. Fish sampling and sample treatment...... 46 2.3.2 Zooplankton sampling and sample treatment ...... 48 CHAPTER THREE COMPARISONS OF ESTUARINE FISH ASSEMBLAGES NEAR SALTMARSH...... 49 3.1 Introduction ...... 50 3.2 Methodology ...... 52 3.2.1 Study site, sampling of fish and laboratory procedures...... 52 3.2.2 Statistical analyses ...... 53 3.3 Results ...... 55 3.3.1 Number of fish species and individuals ...... 55 3.3.2 Abundance and diversity of the overall fish assemblage ...... 57 3.3.3 Abundance of Pelates sexlineatus and Ambassis jacksoniensis ...... 59 3.3.4 Comparisons of fish assemblages among seasons, tidal state, month and day ...... 61 3.4 Discussion ...... 64 3.4.1 Total abundances and diversity of the fish assemblage...... 65 3.4.2 Temporal differences in fish abundance and diversity ...... 67 3.4.3 Temporal differences in the species composition of fish assemblages ...... 68 3.4.4 Conclusion ...... 71 CHAPTER FOUR KEY FEATURES OF THE LIFE HISTORY OF GLASSFISH, Ambassis jacksoniensis ... 73 4.1 Introduction ...... 74 4.2 Methodology ...... 76 4.2.1 Study site and sampling of Ambassis jacksoniensis for selected aspects of their reproductive biology ...... 76 4.2.2 Laboratory procedures ...... 77 4.2.3 Statistical analyses ...... 78 4.3 Results ...... 79 4.3.1 Sex ratio and proportion of the different sexes of Ambassis jacksoniensis ...... 79 4.3.2 Sexual maturity stages of Ambassis jacksoniensis ...... 81 4.3.3 Somatic and reproductive condition of Ambassis jacksoniensis...... 83 4.4 Discussion ...... 86 4.4.1 Sex ratios of Ambassis jacksoniensis ...... 86 4.4.2 Ambassis jacksoniensis sexual maturity stages ...... 89 4.4.3 Ambassis jacksoniensis somatic and reproductive conditions ...... 91 4.4.4 Conclusion ...... 91 CHAPTER FIVE COMPARISON OF ESTUARINE ZOOPLANKTON ASSEMBLAGES NEAR SALTMARSH 94 5.1 Introduction ...... 95 5.2 Methodology ...... 97 5.2.1 Study site and sampling of zooplankton...... 97 5.2.2 Laboratory procedures ...... 98

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5.2.3 Statistical analyses ...... 98 5.3 Results ...... 101 5.3.1 Numbers and diversity of the overall zooplankton assemblages ...... 103 5.3.2 Abundances of calanoid copepods and crab zoeae...... 104 5.3.3 Comparisons of zooplankton assemblages among seasons, months, tidal state and days...... 106 5.4 Discussion ...... 108 5.4.1 Tidal and temporal differences in the abundance of crab zoeae ...... 110 5.4.2 Composition of the zooplankton assemblages...... 111 5.4.3 Conclusion ...... 112 CHAPTER SIX TROPHODYNAMIC RELATIONSHIPS BETWEEN Ambassis jacksoniensis AND SALTMARSH-DERIVED CRAB ZOEAE ...... 114 6.1 Introduction ...... 115 6.2 Methodology ...... 118 6.2.1 Study site, sampling of Ambassis jacksoniensis for dietary analyses and zooplankton for bomb calorimetry ...... 118 6.2.2 Laboratory procedures...... 118 6.2.3 Statistical analyses ...... 119 6.3 Results ...... 121 6.3.1 Comparisons of the stomach fullnesses of Ambassis jacksoniensis ...... 124 6.3.2 Dietary composition ...... 126 6.3.3 Calorimetric comparisons...... 128 6.4 Discussion ...... 130 6.4.1 Dietary compositions, prey-switching and seasonality in the feeding of Ambassis jacksoniensis ...... 131 6.4.2 Calorimetry of potential prey and interpretation of feeding patterns ...... 134 6.4.3 Conclusion ...... 135 CHAPTER SEVEN GENERAL DISCUSSION ...... 137 7.1 Overview of key outcomes of the study ...... 138 7.1.1 Fish assemblages…………………………………………………………………………….………………….136 7.1.2 Life history of Ambassis jacksoniensis….………………………………………….………………….138 7.1.3 Zooplankton assemblages…………………………………………………………….…………………….141 7.1.4 Feeding relationships between Ambassis jacksoniensis and crab zoeae……………..143 7.2 General conclusions ...... 150 7.3 Recommendations for future research ...... 151 REFERENCES ...... 155 APPENDIX A ...... 181

LIST OF TABLES

Table 2.1: Mean values (±SE, n=48) of water temperature, salinity, dissolved oxygen and turbity during summer and winter at Empire Bay Wetland, in 2012…………………………………………………..46 Table 3.1: Abundances of each fish species, recorded at flood and ebb tides in summer and winter, and the total numbers and percentage contributions of fish species to the total catches recorded at Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia), in 2012...... 56 Table 3.2: Summary of results for four-factor Analysis of Variance of the abundance of fish in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012.

Significant differences are depicted in bold. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance...... 59 Table 3.4: Summary of results for four-factor Analysis of Variance of the abundance of Pelates sexlineatus in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance...... 60 Table 3.5: Summary of results for four-factor Analysis of Variance of the abundance of Ambassis jacksoniensis in different Seasons, Months nested in Season, Day and Tidal state, at Empire

Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on log(10) transformed; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance. . 60 Table 3.6: Summary of results for four-factor PERMANOVA of the species composition of the fish assemblages in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay

Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (Pseudo-F) pseudo-F ratio test statistic, (P(perm)) permutation significance...... 61 Table 3.7: SIMPER summary: Fish taxa that typify (shaded) and/or distinguish (unshaded) fish assemblages at Empire Bay Wetland in 2012. The ‘*’ denotes that the relative contribution of that fish taxa is greater for the time represented in the vertical column than in the horizontal row, while no ‘*’ denotes that the opposite is true...... 63

Table 4.1: Proportion of different sexes of within samples of Ambassis jacksoniensis from Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in the summer and winter of 2012. ... 80 Table 4.2: Proportion of sexual maturity stages of samples of Ambassis jacksoniensis from Empire Bay Wetland in the summer and winter of 2012...... 82 Table 4.3: Summary of Model II linear regression statistics for log somatic and log reproductive weight vs. log SL relationships for mature female and male Ambassis jacksoniensis sampled at Empire Bay Wetland in summer and winter of 2012 ...... 84 Table 5.1: The numbers of zooplankton taxa separated by tidal state, inundation state (Days 1- 3 vs 4-6) and seasons, and the total numbers of each zooplankton taxa and overall, recorded at Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in 2012...... 101 Table 5.2: The percentage contribution of zooplankton taxa separated by tidal state, inundation state (Days 1-3 vs 4-6) and seasons, and the total numbers of each zooplankton taxa and overall, recorded at Empire Bay Wetland in 2012...... 102 Table 5.3: Summary of results for four-factor Analysis of Variance of the abundance of zooplankton in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance...... 103 Table 5.4: Summary of results for four-factor Analysis of Variance of the diversity of zooplankton taxa in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square- rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance. 104 Table 5.5: Summary of results for four-factor Analysis of Variance for the abundance of calanoid copepods in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland, in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance...... 105 Table 5.6: Summary of results for four-factor Analysis of Variance for the abundance of crab zoeae in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance...... 105 Table 5.7: Summary of results for four-factor PERMANOVA of the taxonomic composition of the zooplankton assemblages in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square- rooted data; (df) degrees of freedom, (MS) mean squares, (Pseudo-F) pseudo-F ratio test statistic, (P(perm)) permutation significance...... 107

Table 6.1: Mean standard length (SL) and weight of Ambassis jacksoniensis at varying seasons, days and tidal states within Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in 2012.123 Table 6.2: Percentage volumetric contributions of dietary categories recorded within Ambassis jacksoniensis stomachs at Empire Bay Wetland in 2012. NB: No saltmarsh inundation occurred on Days 1-3, while saltmarsh inundation did occur on Days 4-6...... 123 Table 6.3: Summary of results for five-factor PERMANOVA of the dietary compositions of Ambassis jacksoniensis in different Seasons, Months nested in Season, Day, Tidal state and Site at Empire Bay Wetland in 2012. Significant differences and interactions are depicted in bold. Analysis conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (Pseudo-F) pseudo-F ratio test statistic, (P(perm)) permutation significance...... 127 Table 6.4: Summary of results for one-factor ANOVA on calorimetric values of potential prey and zooplankton assemblage “types” subjected to bomb calorimetry, derived from Empire Bay Wetland in 2012. (df) degrees of freedom, (MS) mean squares, (F) F-ratio test statistic, (p) significance...... 129

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LIST OF FIGURES

Figure 2.1: Map of Brisbane Water Estuary and Empire Bay Wetland (NSW, Australia). Dot symbols indicates the locations of the sampling sites…...... 62 Figure 3.1: Means (±SE, n=24) of the (a) abundance of fish and (b) number of fish species (diversity) per sample from Empire Bay Wetland, Brisbane Water Estuary (2012), on various Days and Tidal states in each of summer and winter… ...... 57 Figure 3.2: nMDS plot, derived from a “distance among centroids” matrix of different Seasons and Tidal states, which was constructed from a Bray-Curtis similarity matrix using fish species recorded at Empire Bay Wetland in 2012. Symbols represent the combined factor Season x Tidal State...... 62 Figure 4.1: Summary of log-linear analysis showing observed and expected counts of Ambassis jacksoniensis sampled at Empire Bay Wetland, during summer and winter of 2012. “Up” arrows (↑) indicate that counts of such fish sex are statistically higher than expected for that Season, while “down” arrows (↓) indicate that counts of such fish sex are statistically lower than expected for that Season...... 80 Figure 4.2: Summary of log-linear analysis showing expected and observed sexual maturity stages of (a) female and (b) male Ambassis jacksoniensis sampled at Empire Bay Wetland in summer and winter of 2012. “Up” arrows (↑) indicate that counts of such sexual maturity stage are statistically higher than expected for that Season, while “down” arrows (↓) indicate that counts of such sexual maturity stage are statistically lower than expected for that Season...... 83 Figure 4.3: Residual values for mature (a) female and (b) male Ambassis jacksoniensis individuals, that were sampled at Empire Bay Wetland in summer and winter of 2012, from the somatic weight-SL and reproductive weight-SL relationships...... 85 Figure 5.1: Mean abundance (±SE, n=2) of crab zoeae, on various Days and Tidal states at Empire Bay Wetland in 2012. Days not sharing the same letter (e.g. a, b, c) are significantly different. The symbol “*” depicts that mean crab zoeal abundances are greater for ebb than flood Tidal states on such Days………………………………………………………………………………………………………………………………………….104

Figure 5.2: nMDS plot, derived from a “distance among centroids” matrix of different Tidal States and Days, which was constructed from a Bray-Curtis similarity matrix using zooplankton taxa recorded at Empire Bay Wetland, Brisbane Water in 2012. Numbers represent the Days ………………106

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2012. “Up” arrows (↑) indicate that counts of such SF values are statistically higher than expected for that Day, while “down” arrows (↓) indicate that counts of such SF values are statistically lower than expected for that Day...... 124 Figure 6.2: Log-linear regression depicting relationship between stomach fullness categories (SF; marked on x-axis) for Ambassis jacksoniensis, ranging between 0 and 7, over different Tidal states (flood and ebb) and Days (a: 1-3; b: 4-6) at Empire Bay Wetland in 2012. “Up” arrows (↑) indicate that counts of such SF values are statistically higher than expected for that Tidal state and Day, while “down” arrows (↓) indicate that counts of such SF values that are statistically lower than expected for that Tidal state and Day...... 125 Figure 6.3: Log-linear regression depicting relationship between the stomach fullness categories (SF; marked on x-axis) of Ambassis jacksoniensis, ranging between 0 and 7, over Tidal states (flood and ebb) and Seasons (summer and winter) at Empire Bay Wetland in 2012. “Up” arrows (↑) indicate that counts of such SF values are statistically higher than expected for that Tidal state and Season, while “down” arrows (↓) indicate that counts of such SF values are statistically lower than expected for that Tidal state and Season...... 126 Figure 6.4: nMDS plot, derived from a “distance among centroids” matrix of different Tidal States and Days, which was constructed from a Bray-Curtis similarity matrix using volumetric dietary data of Ambassis jacksoniensis recorded at Empire Bay Wetland in 2012. Numbers represent the Day ...... 128 Figure 6.5: Mean (±SE, n=2) calorimetric values of potential prey and zooplankton assemblage “types”, subjected to bomb calorimetry, derived from Empire Bay Wetland. Samples not sharing the same letter (e.g. a, b) are significantly different from each other...... 129

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CHAPTER ONE

GENERAL INTRODUCTION

1.1 Overview of estuaries

1.1.1 Estuaries in a global and an Australian context

The study of estuarine biology, ecology and physico-chemistry is a well-established component of science within the global scientific community, and estuaries have been shown to be of immense ecological importance in linking terrestrial and marine habitats (Nagelkerken et al., 2008). Despite their ecological importance, the highly variable physical forms of estuaries (both globally and within

Australia), has led to great debate over the definition of the term “estuary”, as well as to their defining characteristics and functions (Elliott and and McLusky, 2002; Whitfield, 2005; Hume et al., 2007; Dürr et al., 2011). An internationally accepted definition of an estuary that also incorporates acknowledgment of its biological components was not proposed until the mid-1990’s. This definition, formulated by Perillo (1995), has since been regarded as including all of the various characteristics of estuaries worldwide, including those diverse forms found within Australia.

Perillo (1995) defined an estuary as ‘any semi-closed coastal body of water that extends to the effective limit of tidal influence, within which sea water entering from one or more free connections with the open sea, or any other saline coastal body of water, is significantly diluted with freshwater derived from land drainage, and can sustain euryhaline biological species for either part or the whole of their life cycles.’ This definition, and in particular the notion of the interconnectedness between estuaries and their adjacent habitats (e.g. open seas, rivers), correctly considers that estuarine ecosystems are often influenced by those ecosystems to which they are connected, as recognised by

Dürr et al. (2011). Elliott and Whitfield (2011) acknowledge that although all ecosystems are somewhat interconnected (e.g. via the hydrological cycle), when compared to other types of ecosystems, estuaries have particularly high levels of interconnectedness, especially to riverine and marine ecosystems

(Gómez-Gesteira et al., 2003; Kremer et al., 2010; Potter et al., 2010). Therefore, estuaries are multi-

2 interfaced systems as they have links with terrestrial, freshwater, marine and atmospheric ecosystems

(i.e. estuaries typically have several ecotones; Elliott and Whitfield, 2011).

The extensive coastline of Australia contains a very large number of estuaries, which range from small, intermittently closed coastal lagoons and tidal flats to large, wave or tidally dominated, permanently open estuaries, such as Brisbane Water Estuary (Roy et al., 2001; Potter et al., 2010;

OzCoasts, 2015). Irrespective of estuary type, estuaries can be divided into three broad geomorphic zones, i.e. zones that are fundamentally characterised by their sediment type and location within the estuary. These three zones include: i) the marine tidal delta, which is located towards the mouth of the estuary and is typically comprised of clean quartzose sand and muddy sand; ii) the central mud basin, located within the middle portion of the estuary and comprised of organic-rich mud and sandy mud; iii) and the fluvial delta, located at the highest end of the estuary where freshwater input occurs and comprised of sandy mud and muddy sand (Roy et al., 2001). These zones are also related to the characteristics and extent of natural vegetation along the estuary shoreline, such as tidal marshes and mangroves, which depend on the extent of tidal exchange, their latitude and biogeographical affinities.

These zones, which are common to all estuaries (albeit to a different extent), can be used as the basis for studies examining ecological aspects (e.g. species richness, faunal assemblages) within estuaries, including comparisons among different estuaries or between such estuary zones (Roy et al., 2001). It should be noted that tidal range is also known to affect the physico-chemical (e.g. water salinity and tubidity), geomorphology (e.g. intertidal area), biological (e.g. residence times; see Tweedley et al.,

2016), and thus zonal characteristics of estuaries (i.e. between microtidal, mesotidal and macrotidal water bodies), and thus ecological comparisons among estuaries must consider such potential tidal range influence.

The significance of the contributions by Perillo (1995) and Roy et al. (2001) is that a comprehensive, detailed and specific definition of estuaries, which acknowledges their ecological role, coupled with an understanding of estuarine zonation, has been established in order for future research

3 regarding estuaries to have a common foundation for comparisons and interpretations. For example, sediment morphology in Brisbane Water Estuary, a typical estuary situated in south-east Australia, ranges from i) silica sand within the marine tidal delta, ii) catchment muds within the central mud basin, to iii) silt, mud and sand particles in the fluvial deltas (Roy et al., 2001).

1.1.2 Contribution of estuaries to Australian fishes

It is well established that estuaries act as habitats for numerous Australian fish species during some or all of their lifecycles (for examples see Section 1.2; Blaber, 2000; Tweedley et al., 2014). The abundance and diversity of fishes within estuaries often differs among the estuarine zones (Roy et al.,

2001; Griffiths, 2001; Rotherham and West, 2002). In particular, the morphology of an ocean-estuary interface (i.e. the ‘mouth’ or ‘marine tidal delta’ of an estuary) can influence fish movement between these two aquatic environments (Robinson et al., 1983; Potter and Hyndes, 1994; Hannan and Williams,

1998; Griffiths and West, 1999; Pease, 1999; Potter and Hyndes, 1999). For example, the mouths of estuaries can remain permanently open to the sea, enabling movement of marine fish into estuaries and vice versa (e.g. such as larval terapontids; Neira and Potter, 1994; see also Potter and Hyndes,

1994). Estuarine mouth morphology can also play an important role in the transfer of fish larvae into and out of estuaries during their crucial development periods (Miskiewicz, 1987).

Many estuarine-dependent fishes are of great economic importance to commercial and recreational fisheries in coastal waters (Duke, 1982; Bell et al., 1984; Laegdsgaard and Johnson, 1995).

In order to ensure sustainability of such industries, it is crucial that appropriate management of these fishes, and their ecosystems and habitats, is conducted in a manner that considers the biological and ecological requirements of the fishes’ entire life cycle. Thus, appropriate management of estuaries is imperative for economic and ecological sustainability.

1.1.3 Characteristics of saltmarshes and mangroves in estuaries

Saltmarsh and mangrove vegetation is generally located within the intertidal and supratidal zones of both the tidal and fluvial delta environments of estuaries (West, 1985; Bertness and Ellison,

1987; Bertness, 1991; Connolly et al., 1997). Within the Northern Hemisphere, saltmarshes are generally located in the mid-intertidal zone, with the tall cordgrass (Spartina) typically lying seaward of these saltmarshes, and are nearly always tidally inundated (Nixon and Oviatt, 1973; Bertness and

Ellison, 1987; Bertness, 1991; Connolly et al., 1997; Thomas and Connolly, 2001; Hollingsworth and

Connolly 2006). This contrasts with the characteristics of saltmarshes within Australia, however, which are typically located high within the intertidal zone, with mangroves lying seawards and sometimes transgressing into the saltmarsh proper (Saintilans and Willams, 1999), and which are inundated for shorter and less frequent periods (Hollingsworth and Connolly, 2006; Mazumder et al., 2006a).

Differences between the dominant saltmarsh vegetation between estuaries of the Southern and Northern Hemisphere are also apparent, making it difficult to generalise results from studies conducted in the Northern Hemisphere (Minello et al., 2003). Thus, those of the Northern Hemisphere are comprised mainly of monospecific stands of perennial turfs (Bertness et al., 1992; Pennings and

Callaway, 1992), in which dominant species generally exhibit vegetative zonation as a result of physical gradients associated with elevation and frequency of tidal inundation (Bertness and Ellison, 1987;

Pennings and Callaway, 1992). In contrast, within south-eastern Australia (and elsewhere in the

Southern Hemisphere, e.g. South Africa; Tweedley et al., 2016), the dominant saltmarsh vegetation is typically comprised of succulent shrubs and herbs (Long and Mason, 1983; Adam 1990), which are capable of coping with high salinities and periodic submersion in estuarine water during high spring tides and tidal inundation (Morton et al., 1987).

Despite the differences in the characteristics of saltmarsh in the Northern and Southern

Hemispheres, studies have demonstrated that saltmarsh vegetation typically contributes organic matter (Boschker et al., 1999), recycles nutrients (Schelske and Odum, 1961; Lear and Turner, 1977;

Bunt et al., 1979; Boto and Bunt, 1981; Duke, 1982; Boto et al., 1984; Robertson et al., 1988; Morrisey,

1995), stabilises estuarine sediments (Roy et al., 2001) and filters estuarine and storm water runoff

(Morrisey, 1995). In Australian estuaries, these functions are also carried out by the mangroves that lie seaward of these saltmarshes (Boto and Bun, 1981). Saltmarshes are ecologically important due to their role as a habitat (Robertson et al., 1988) and feeding ground for various aquatic and terrestrial organisms, including fish, crustaceans such as burrowing crabs, gastropod molluscs, a variety of terrestrial arthropods, amphibians, reptiles, birds and mammals (Hyland and Butler, 1988; Freewater et al., 2008). However, the extent of ecological importance of saltmarsh to any fauna will vary with its vegetative composition and characteristics of inundation, as noted earlier.

In the Northern Hemisphere (particularly in Europe and North America), the ecological role of saltmarsh vegetation has been extensively researched, including its significance as a fish habitat and food source (Cain and Dean, 1976; Daiber, 1977; Weinstein, 1979; Weinstein et al., 1980; Weinstein and Brooks, 1983; Talbot and Able, 1984; Costa et al., 1994; Kneib and Wagner, 1994; Costa et al., 1995;

Beck et al., 2001; Vinagre et al., 2011). For instance, high abundances of fish, with relatively full stomachs, have been found within inundated saltmarshes during high spring tides (Cain and Dean,

1976; Daiber, 1977; Weinstein, 1979; Weinstein et al., 1980; Haedrich, 1983; Weinstein and Brooks,

1983; Costa et al., 1994; Costa et al., 1995; Stevens et al., 2006; Krumme et al., 2008). The differences between the vegetative composition and frequency of inundation of these saltmarshes in comparison with saltmarshes in Australia indicate that there are likely to be differences in the ways that fish use these habitats. However, within Australia, there is evidence that fish do enter these habitats and are likely to be feeding during this time (e.g. Hollingsworth and Connolly, 2006; Mazumder et al., 2006a;

Platell and Freewater, 2009; Alderson, 2014).

Economically important fishes are known to use saltmarshes during at least part of their life cycle. For example, sampling of a South Carolinian intertidal saltmarsh found over 22 species (16 families) of larval, juvenile and adult fish, including the economically important Sciaenidae (Leiostomus

6 xanthurus and Micropogon undulates), Mugilidae (Mugil spp.), Ophichthidae (Myrophis punctatus),

Sparidae (Lagodon rhomboides) and Paralichthyidae (Paralichthys spp.; Shenker and Dean, 1979).

Within south-eastern Australia, economically important fish that use saltmarshes include the

Mugilidae (Mugil cephalus, Liza argentea, Myxus elongatus), Sparidae (Acanthopagrus australis),

Sillaginidae (Sillaginodes punctata) and various flatfishes, such as the Platycephalidae (Platycephalus fuscus) and Pleuronectidae (Rhombosolea tapirine; Connolly et al, 1997; Crinall and Hindell, 2004;

Mazumder et al., 2005a). Furthermore, other fish species can be of indirect economic importance, such as the saltmarsh-associated ambassid, Ambassis jacksoniensis (Platell and Freewater, 2009, Mazumder et al., 2006a; McPhee et al., 2015), a small and highly-abundant species that are consumed by larger and economically-important fish species, including A. australis, P. fuscus and the sciaenid Argyrosomus japonicus (SPCC, 1981; Taylor et al., 2006; Mazumder et al., 2006a).

There have been declines worldwide in the coverage of natural vegetation along the shores of estuaries, with, for example, Australian saltmarshes substantially decreasing in area in recent decades as a result of urban development (Hyland and Butler, 1988) and mangrove encroachment (Saintilan and

Williams, 1999; 2000). As a result of the decline of Australian saltmarsh vegetation in recent history,

‘Coastal Saltmarsh’ has now been listed as an Endangered Ecological Community under the Threatened

Species Conservation Act 1995 (DOEACC NSW, 2008). As there is a global ecological and economic reliance on saltmarshes, it is imperative that appropriate management of Australian estuaries is employed in a manner that adequately incorporates the conservation of saltmarsh vegetation and prevention of its further decline. It is essential, therefore, to understand the contribution of a saltmarsh to the estuarine ecosystem and the potential effects of reduction in saltmarsh area on fish populations.

1.2 Characteristics of estuarine habitats & faunal assemblages

1.2.1 Overview of fish habitats

A habitat can essentially be regarded as a typical place (both spatially and temporally) where an organism occurs (Godin, 1997). As individuals of a population are known to occur in restricted areas

(e.g. in the form of home ranges or territories), a habitat might be better defined by emphasising the difference between the actual known place where an organism occurs and the places where an organism is capable of living. For example, the habitat of an estuarine fish could be ‘estuarine water.’

Although more specifically, many species of fish occur only in portions of an estuary (i.e. their habitat is restricted to only part of the estuary) or in multiple estuaries and also the coastal waters that connect them. Further, there may be temporal differences in habitat use (e.g. a day, a tidal cycle, a season; see

Mazumder et al., 2005a; Saintilan et al., 2007; McNeil et al., 2008), and specific uses of habitats (e.g. open water, mud flats, seagrass, mangroves, saltmarsh; Godin, 1997). Therefore, due to the complex nature of itinerant fish (including those that frequent saltmarshes; Mazumder et al., 2005a; Saintilan et al., 2007), defining the habitat of particular species is often difficult.

‘Migration’ refers to the process of habitat selectivity as a result of a regular pattern or change within the environment or habitat (e.g. tidal states: flood and ebb tides, season; Neill, 1984). Thus, as the movements of fish from other parts of the estuary (including mangroves) to saltmarsh were demonstrated by Mazumder et al. (2005a) were a result of high spring tides inundating the saltmarsh

(i.e. a ‘new’ habitat became accessible due to tidal influences), such movement between selected habitats could be considered to be migration. Furthermore, the freshwater ambassid, Ambassis agassiz, migrates from downstream to upstream habitats as a response to changes in river flow (McDowall,

1996; Moffat and Voller, 2002; Lintermans, 2007). In cases where environmental influences (e.g. tides) lead to provisions of “better” or more resources (e.g. prey), which result in fish migration among habitats (e.g. exporting of crab zoeae from saltmarsh-inhabiting grapsid crabs; Mazumder et al., 2006a),

8 such habitat selectivity may be more appropriately regarded as ‘habitat preference;’ either at a specific time (e.g. during saltmarsh inundation times) or in general, but when available (e.g. when saltmarsh is tidally inundated and therefore accessible to estuarine fishes; Johnson, 1980).

Where traditional taxonomic measures of fish communities focus on individual species (see

Elliott and Dewailly, 1995; Mathieson et al., 2000; Elliott et al., 2007), the functional guild approach (see

Elliott et al., 2007; Potter et al., 2015) characterises fish assemblages (i.e. multiple species) using various structural and functional aspects of fish assemblages within estuaries. Thus, the functional guild approach allocates fishes into broad groupings that are based on selected ecological characteristics of fishes including; residency and movement patterns, vertical distribution in the water column, preference of substrate, dietary preferences and reproductive strategies. The functional guild approach considers three broad groupings: 1) the Estuarine Use Functional Group (EUFG) – i.e. how fishes utilise estuaries throughout their life cycle (e.g. residency times and migration patterns); 2) the Feeding Mode

Functional Group (FMFG) – i.e. what are the feeding behaviours of the fishes present; and 3) the

Reproductive Mode Functional Group (RMFG) – i.e. what are the reproductive strategies used by fishes

(Elliott et al., 2007). This approach, which is becoming more prevalent in estuarine ecological studies in recent years; see Elliott et al., 2007; Potter et al., 2015), enables comparisons via the use of guilds, i.e. groups of species that overlap significantly in their niche requirements, exploiting the same class of environmental resources in a similar way (Root, 1967), rather than focussing on individual species, which often differ between environments.

1.2.2 Fish in saltmarshes and mangroves of Australia

On the basis of studies on estuaries in tropical and subtropical Queensland, mangrove vegetation is the most important habitat for estuarine fish (Blaber et al., 1985; Robertson and Duke,

1987; Laegdsgaard and Johnson, 1995). This is consistent with the abundance and diversity of fishes within saltmarsh being lower than in the surrounding vegetation (e.g. seagrass) of a South Australian

9 estuary (Connolly et al., 1997). However, more recent research in temperate south-eastern Australia has indicated that saltmarsh vegetation can be important habitats for fishes in temperate estuaries

(Mazumder et al., 2005a; 2009). The inconsistency of these findings suggest that there is variability in the use of saltmarshes by different fish species and at different geographical locations, which warrants further exploration.

Small fishes were observed to move among seagrass, mangrove and saltmarsh throughout a lunar cycle within a temperate south-eastern Australian estuary (Saintilan et al., 2007). Thus, fish abundances were greatest within seagrass habitats during neap tidal cycles (when saltmarsh was not inundated), but were also relatively high within nearby saltmarsh and mangroves during spring-tidal cycles (i.e. when these habitats were inundated). During the latter times, itinerant fish (including

A. jacksoniensis) were shown to feed on highly abundant zooplankton that is considered to be saltmarsh-derived (Mazumder, 2004; Mazumder et al., 2006a; Hollingsworth and Connolly, 2006;

McPhee et al., 2015).

It should be noted that studies on fish assemblages within creeks directly adjacent to saltmarshes (e.g. Gibbs, 1986; Morton et al., 1987), found similar fish assemblages to those found directly on inundated saltmarshes in Mazumder et al. (2005a and 2006a), which suggests that fish assemblages show commonalities in these two connected habitats. Ambassis jacksoniensis, a common estuarine fish in south-east Australia, are known to feed on saltmarsh-derived prey (such as crab zoeae) from seagrass vegetation adjacent to inundated saltmarsh (McPhee et al., 2015) and, thus, take advantage of saltmarshes from connected habitats when they are accessible.

Saltmarshes, which typically comprise low-lying vegetation on estuarine shorelines, can provide temporary habitats and feeding grounds for itinerant estuarine fish, but only when those saltmarshes become inundated during high, spring tides (e.g. Laffaille et al., 2002; Platell and Freewater, 2009;

Saintilan and Rogers, 2013). Although North American saltmarshes may be inundated up to twice daily, those in Europe (with some exceptions; see Bakker, 2014), South America, South Africa and eastern

Australia are typically inundated during only three to four days of each lunar cycle (e.g. Laffaille et al.,

2001; Thomas and Connolly, 2001; Hollingsworth and Connolly, 2006), which would mean a more limited access by fish. In those estuaries in which saltmarshes are inundated during only the spring-tide cycles, the timing of inundation (i.e. day or night) also varies during the year. For example, on the east coast of Australia, the semi-diurnal spring tides inundate saltmarsh during the day in summer, while such inundation occurs during the night in winter. It should also be recognised that in some estuaries, saltmarsh vegetation may be situated directly landward of mangroves, the latter of which (i.e. mangroves) fringe the shorelines of estuaries and are tidally inundated on every high tide (see Bakker,

2014), and which may lead to interconnectivity of ecological processes (e.g. organism habitat, trophodynamics) between these vegetative zones, particularly during times of tidal inundation

(McPhee et al., 2015).

Despite indications that south-east Australian saltmarshes are important habitats for diverse assemblages of estuarine-dependent fish, it is unreasonable to assume that the specific saltmarsh complex sampled by Mazumder et al. (2005a), Towra Point (Botany Bay), is representative of all south- eastern Australian saltmarshes. Such studies should be repeated in different estuaries and saltmarsh complexes in order to investigate the use of saltmarshes by estuarine fish in a general context.

Mazumder et al. (2005a) focussed on one saltmarsh complex and thus could not assess spatial variability of fish assemblages among different saltmarsh and mangrove locations. The highly diverse fish assemblages observed in Mazumder et al. (2005a) may be due to a combination of the sample site being located within a nature reserve, which was situated near the mouth of the estuary (factors known to influence fish diversity; Hannan and Williams, 1998; Griffiths and West, 1999; Pease, 1999; Paterson and Whitfield, 1996; 2000). On the other hand, Mazumder et al. (2006b) examined the spatial variability of estuarine fish by comparing saltmarsh and mangrove habitats between three various estuary types containing different geomorphic settings (i.e. estuarine zones; not considered in Mazumder et al.,

2005a). The study found some similarities, but also inconsistencies, in fish abundance and diversity among the estuaries. Further, many of the studies on estuarine fish use of saltmarsh in south-eastern

Australia have been conducted in the more or less pristine wetland at Towra Point (e.g. Mazumder et al. 2005a; 2006a; 2006b; Saintilan et al., 2007). In light of this, further research should consider other estuaries and saltmarshes that i) are not located within Botany Bay, and ii) may not be considered pristine. The patterns found in studies conducted at Towra Point should be tested in other estuaries and estuary types (e.g. ones with less protection and/or of less or more urban influence; Underwood,

1997).

The few studies regarding the use of saltmarsh by Australian fishes (e.g. saltmarsh creeks

(Gibbs, 1986; Morton et al., 1987; Davis, 1988); semi-permanent saltmarsh pools (Morton et al., 1988); and inundated saltmarsh flats at high tide (Connolly et al., 1997; Thomas and Connolly, 2001)) pioneered the research that would later attempt to model concepts concerning trophic linkages and dietary interactions between saltmarsh vegetation and estuarine fishes. It was primarily the research of Mazumder et al. (2006a; 2009) that initially assessed the contribution of Australian saltmarsh as a habitat and source of food for Australian estuarine fishes, despite its infrequent inundation, while the research of Mazumder et al. (2005a; 2006b) and Saintilan et al. (2007) further developed the concept of the importance of saltmarsh as a habitat for Australian estuarine fishes. As is often urged by researchers of ecologically valuable environments that are under anthropological threat, these studies argue that further protection (e.g. in the form of legislation and associated policies) and appropriate conservation of Australian saltmarshes is imperative for responsible management of the natural ecological functioning of Australian estuaries.

Habitat use by estuarine fishes (including movement or migration among habitats) may be influenced by ontogenetic, reproductive and/or feeding stages within their life cycles. For example, as some fish may spawn, feed and seek shelter in different habitats (Zimmerman et al., 1984; Rozas and

Minello, 1998; Connolly, 1994; Connolly et al., 1997; Crinall and Hindell, 2004; Mazumder et al., 2005a;

2006a; Hollingsworth and Connolly, 2006; McNeil et al., 2008; Platell and Freewater, 2009; McPhee et al., 2015), these life history cycles can be observed to determine in which habitat, within their habitat

12 range, an organism is likely to be found. Therefore, in terms of conservation of ecologically important habitats, it is important that environmental managers, researchers and policy makers consider the entire life cycles (e.g. spawning and feeding behaviours) of organisms that are ecologically dependent on such habitats, in order to gain a holistic understanding of the ecological requirements of such organisms and the habitats they use.

Vegetated habitats, such as saltmarsh, and which are not always inundated, present particular challenges with sampling of fishes. There has therefore been a wide range of sampling techniques employed to sample fishes in these environments, including fyke, pop and seine nets (e.g. Connolly,

1999; Mazumder et al. 2005a; 2006a; 2006b; McPhee et al., 2015), each of which has the potential to introduce selectivity into any comparisons. Thus, pop nets are used to entrap fish while they are on the saltmarsh, fyke nets catch fish that are exiting the saltmarsh on the ebbing tides, and seine nets are used in the waters just adjacent to saltmarshes (Connolly, 1999; Mazumder et al., 2006b). In this context, however, it is important to note that, within one estuary (Botany Bay), the fish assemblages collected by seine netting at sites that are directly adjacent to inundated saltmarshes (e.g. Gibbs, 1986;

Morton et al., 1987) are very similar to those assemblages that were collected by fyke netting directly within inundated saltmarshes (see e.g. Mazumder et al., 2005a and 2006a), which indicates that either type of net may be used for such sampling.

1.2.3 Factors affecting estuarine faunas, including zooplankton and fishes

Estuarine fauna can be influenced by hydromorphological factors such as sediment, estuary zones, water movements and tidal cycles (Chicharo and Chicharo, 2006; Haines et al., 2006; Wolanski,

2007; Gray and Elliott, 2009; Nicolas et al., 2010). More specifically, estuarine faunal abundance is typically related to habitat availability and autotrophic quality and quantity (i.e. of photosynthesising plants and algae), while faunal diversity is linked to habitat size and diversity (MacIntyre, 1959; Jones et al., 1986), food and shelter abundance (from both natural forces and predators; Blaber and Blaber,

1980; Pollard, 1984; Bell and Worthington, 1992; Gray et al., 1996) and salinities (Jones, 1988). The intermittent availability of habitats for fish, such as when low-lying areas become inundated, or vegetative growth develops (algae or seagrass), can also influence the distribution of fishes at different stages of their life cycle (Saintilan et al., 2007). Thus, when fish use these very temporally-restricted habitats, they are termed “itinerant” (e.g. Mazumder et al., 2006a; Mazumder et al., 2011;

McPhee et al., 2015).

As the general concept of use of estuaries by opportunist fish has been well established on a global scale, it is important that the underlying processes that govern the relationships between itinerant fish and estuaries are researched in detail, and that any characteristics that may typify or differ on any spatial scale (e.g. local, national, global) are understood for appropriate management and conservation of both ecosystems.

1.2.4 Zooplankton assemblages in estuaries

Diverse and highly abundant zooplankton assemblages comprise an important part of estuarine ecosystems and it is known that saltmarshes and mangroves in temperate NSW (Australia) provide habitats for a variety of invertebrates including cnidarians, polychaetes, gastropods, copepods, amphipods and crabs (e.g. Helograpsus haswellianus and Sesarma erythrodactyla; Freewater et al.,

2008; Mazumder et al., 2009). Furthermore, as part of their reproductive behaviour, invertebrate residents of such habitats export large quantities of offspring into the estuary, where they comprise an important part of zooplankton assemblages (Dittel and Epifanio, 1982; Kneib, 1997; Charmantier et al.,

2002; Papadopoulos et al., 2002; Brodie et al., 2007). Although in general zooplankton are consistently abundant, when observed at higher temporal resolutions (e.g. monthly) or across different locations, such export is quite variable, whereby at certain times abundances are much greater than at others

(Christy and Stancyk, 1982; Robertson et al., 1988; Pittman and MacAlpine, 2003). For example, variability in estuarine zooplankton assemblages occurs during ebb tides when saltmarsh-dwelling

14 grapsid crabs release zoeae into the water column following tidal inundation during spring tidal cycles

(Mazumder et al., 2006a; 2009). Such variations have also been attributed to differences in hydrology, tidal height, area of habitat (which is temporally variable and generally determined by tidal height) and species composition, with such effects essentially determining distributions of zooplankton assemblages within estuaries (Lambert and Epifanio, 1982; Hettler, 1989; DiBacco et al., 2001; Weaver and Salmon, 2002; Bloomfield and Gillanders, 2005; Mazumder et al., 2006a).

Differences in zooplankton density and composition can also be found within the same habitat type, but at varying depths in the water column. Areas within estuaries that zooplankton are exported to can also be estimated by the relative vertical positions (i.e. heights) that the zooplankton occupy within the water column (Dittel and Epifanio, 1982; DiBacco et al., 2001; Bretsch and Allen, 2006;

Tilburg et al., 2007). For instance, zooplankton found in the top layers of the water column are typically exported to farther distances from their original point of exportation, while those found in lower layers typically remain closer to their original point of exportation (Dittel and Epifanio, 1982; DiBacco et al.,

2001).

Although much is known about estuarine zooplankton composition and distribution in

European and American estuaries, such findings cannot be easily applied to Australian estuaries due to major differences in vegetation and other estuarine characteristics (see Long and Mason, 1983; Morton et al., 1987; Adam, 1990; Thomas and Connolly, 2001; Minello et al., 2003; Hollingsworth and Connolly,

2006; Mazumder et al., 2006a). Despite this, some similarities exist in compositions of south-eastern

Australian zooplankton assemblages (Mazumder et al., 2006a; 2009) and those found elsewhere

(Pearcy and Richards, 1962; Weinstein, 1979; Rozas and Hackney, 1984; Talbot and Able, 1984; López-

Duarte et al., 2011), whereby during ebb spring tides that follow saltmarsh inundation, crab zoeae are among the dominant taxa.

At Towra Point, Botany Bay (NSW), far greater abundances of zooplankton were found during ebb than flood tides at times of saltmarsh inundation (Mazumder et al., 2006a), which was largely

15 attributed to high densities of saltmarsh-derived crab zoeae being exported from saltmarsh vegetation

(Mazumder et al., 2009). Freewater et al. (2008) found similar patterns in Brisbane Water Estuary further north in NSW. Certain environmental factors do not appear to influence zooplankton assemblages. For example, Robertson et al. (1988) found no relationships between salinity, temperature, mangrove litter and fish predation and zooplankton composition and concentration, suggesting that environmental variables have little effect on zooplankton assemblages, with tidal regimes being the most influential factor (Robertson et al., 1988; Rountree and Able, 2007).

Crabs have planktonic larval phases (of which zoea is the first) and a benthic adult phase in their life cycles (Papadopoulos et al., 2002; Brodie et al., 2007). Saltmarsh-dwelling grapids crabs synchronise the release of larvae (zoeae) to times of saltmarsh inundation (Mazumder et al., 2006a). As a result, of all of the taxa that comprise estuarine zooplankton assemblages, crab zoeae are thought to have the most variable densities (Christy and Stancyk, 1982; Pittman and McAlpine, 2003). Such synchronised mass release of zoeae may be a survival tactic to reduce predation risk and therefore mortality rate of offspring within the population (Kneib and Wagner, 1994; Morgan and Christy, 1995; Morgan, 1996;

Kneib, 1997).

On the semi-diurnal east coast of Australia, spring tides reach higher levels during nighttime in winter, while the opposite is true for summer (i.e. when spring tides reach higher levels during the day).

Moreover, crab zoeal densities (including grapsids) within Australian estuaries are often greatest during nightly high ebb tides in winter months than those during the day or in summer months (Mazumder et al., 2006a). Helograpsus haswellianus, a common Australian saltmarsh crab thought to be one of the main contributors to such high densities of zooplankton (and which feeds primarily upon fine benthic organic material and microphytobenthos within saltmarsh vegetation; Mazumder and Saintilan, 2010;

Alderson et al., 2013), breeds and therefore disperses zoeae during winter months (March-October), which may further explain the high densities of crab zoeae within zooplankton assemblages during winter ebb spring tides (Mazumder and Saintilan, 2003; Mazumder, 2004). Similar patterns were also

16 found for larval release of fiddler crabs in the Northern Hemisphere (Forward, 1987). Crabs may release zoeae during nightly tides of saltmarsh inundation as a strategy to counteract losses from visual predators (Hovel and Morgan, 1997). Furthermore, Mazumder et al. (2006a; 2009) found that the first night of saltmarsh inundation during a spring tide cycle acts as an environmental trigger for zoeal release by saltmarsh-dwelling crabs on subsequent nights of inundation.

The release of crab zoeae into ebb tides of spring tidal cycles allows for their transfer, as zooplankton, to other portions of the estuary/sea for development and recruitment via influx back into the original or different saltmarshes (promoting geographical dispersal of the population; Epifanio et al., 1988; Morgan and Christy, 1995; Morgan, 1996; Charmantier et al., 2002; Mazumder et al., 2006a).

The crab larvae derived from high portions of estuaries (i.e. where saltmarsh is located) are also transferred to lower portions of estuaries (i.e. the mouth; Sandifer, 1975; Dittel and Epifanio et al.,

1982) or sea/adjacent continental shelves for further development (Christy and Stancyk, 1982; for example, fiddler crab larvae within the Delaware River estuary, U.S.A; Epifanio et al., 1988). Crab zoeae are often found in the top layers of the water column during ebb spring tides (DiBacco et al., 2001;

Papadopoulos et al., 2002; Bretsch and Allen, 2006), and it is understandable that zooplankton found in top layers are typically exported to distances far from their original point of exportation (Dittel and

Epifanio, 1982; DiBacco et al., 2001).

Indeed, high abundances of saltmarsh-derived crab zoeae within ebb tide zooplankton assemblages provide an important food source for trophically higher estuarine organisms, such as fish

(Hollingsworth and Connolly, 2006; Mazumder et al., 2006a; Svensson et al. 2007). Periodically high abundances of saltmarsh-derived crab zoeae are well-related to the diets of A. jacksoniensis (Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; McPhee et al., 2015), which in turn are preyed upon by higher, economically important predators in other parts of estuaries (SPCC, 1981; Taylor et al., 2006).

This is an example of ‘trophic relay,’ whereby energetic pathways of organic matter from saltmarshes to open estuaries/seas exist via a series of predator-prey relationships (Kneib, 1997).

1.2.5 Calorimetric content and energetic densities of zooplankton and other organisms

There are two notable methods of determining the calorimetric content and energy densities of organisms. Both of these methods generally measure energy content in calories or joules and energy density in calories or joules per gram. The first method, calorimetry, involves the determination of the energetic content of organic matter and is calculated by measuring the heat of its combustion (Robbins,

1983). Bomb calorimeters that measure the amount of heat released from an oxidised organism are often used for such calorimetric analyses. Proximate composition is an alternative method for measuring energy densities of organisms (Perez, 1994; for examples see Watt and Merrill, 1963;

Sidwell, 1981; Croxall and Prince, 1982, Vlieg 1984; Krzynowek and Murphy, 1987; Gooch et al., 1987).

Calorimetry, as opposed to proximate composition methodology, is considered a far more reliable method of obtaining energetic values of organisms (Wuenschel et al., 2006).

Calorimetric analyses of organisms have the potential to quantify the energetic density/value of a particular prey species to their predators (e.g. Benoit-Bird, 2004; Wuenschel et al., 2006). As stomach content analyses can be used to provide an understanding of ‘what’ a predator eats (e.g. see

Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; Torres-Rojas et al., 2010; McPhee et al.,

2015) and stable isotopes can determine energetic pathways within food webs (i.e. specific source of

‘where’ energy is derived), calorimetric analyses in turn provide information on the energetic value that a predator gains from a particular prey organism (i.e. the ‘how much?’). When used in conjunction with traditional stomach content analyses, calorimetric analyses allow for deeper understandings of trophodynamics as well as construction of bioenergetic models in an ecosystem.

Historically, calorimetric analysis has often been used to investigate energy metabolism of organisms under different environmental conditions. For example, in a study conducted in southern

England, Wang and Widdows (1993) recorded respiratory responses of the estuarine infaunal bivalve,

Abra tenuis, at various oxygen partial pressures in order to determine the effects of hypoxia, normoxia and anoxia, finding an increase in ventilation rates with declining oxygenation of water. Energy

18 metabolism and metabolic depression has also been investigated in other fishes including Anguilla anguilla, Oreochromis mossambicus, Carassius auratus and a marine worm, Sipunculus nudus (van

Ginneken et al., 1996; 1997; 2004; van Ginneken and van den Thillart, 2009). Additionally, Targett

(1979) applied calorimetry to investigate the effects of temperature on the digestive efficiency of the fish, Fundulus heteroclitus.

Another use of calorimetry is to determine the energetic value of an organism or population in order to gain an understanding of its biological and ecological health, condition and functioning. Such condition can be compared among different treatments or at different stages of ontogeny (e.g. seasons of reproduction, feeding or varying water physiochemistry, such as temperature). For example,

Summers (1979) investigated characteristics of life cycle and population ecology of flounder, Platichthys flesus, in the Ythan estuary of Scotland. Among other variables, calorimetric contents of flounder were calculated at various times of the year with respect to spawning seasons in order to compare temporal differences in organism and population condition (Summers, 1979). Calorimetric contents were high during winter prior to spawning, while low in spent fish. Similarly, Gabriel and Armando (1974) determined, via calorimetry, the primary aerial production of various saltmarsh species in St. Louis

Estuary, Mississippi, across seasons.

Such calorimetric information is also capable of providing insights into the potential energetic value of organisms to their potential predators. However, as the energetic density of organisms

(e.g. fish) is influenced by locality (Schultz and Conover, 1997), season (Flath and Diana, 1985) and ontogeny (Deegan, 1986; Sogard and Spencer, 2004), it is important that such effects are taken into consideration when attempting to interpret the health or condition (e.g. via calorimetry) of organisms and their energetic value to potential predators.

Historically, calorimetry has been an under-implemented tool that has only recently become a more widely-used method in ecological research focused on determining potential energetic contributions of certain prey items to predators and their associated role in food webs, trophic

19 dynamics and bioenergetic models (e.g. Miller, 1978; Perez, 1994; Benoit-Bird, 2004; Ciancio and

Pascual, 2006; Wuenschel et al., 2006; Bittar et al., 2012). For example, Perez (1994) used calorimetry to determine the energetic importance of various prey items (20 species of fish and 10 species of cephalopods) to predatory marine mammals, separating them into five groups based on their calorimetric and energetic content. Likewise, energetic values using calorimetric analyses were also obtained for four species of fish and cephalopod that are the known prey of northern fur seals,

Callorhinus ursinus (Miller, 1978).

In conjunction with stomach content analyses, Wassenberg (1990) used calorimetry to investigate the feeding ecology and ontogeny of the prawn, Penaeus esculentus, in Moreton Bay, QLD.

Seeds of the seagrass, Zostera capricorni (common in the diet of juvenile P. esculentus) had high calorimetric values (with an approximate energetic value of crustacean tissue; Thayer et al., 1973; Brey et al., 1988), indicating that Z. capricorni provides an important (energetically), albeit seasonal, food source for juvenile P. esculentus (Wassenberg, 1990). As dietary behaviours vary in energetic intake temporally, such insights can be used to support hypotheses about foraging behaviours of predators.

In a study on the energy requirements, foraging behaviour and energy expenditure of spinner dolphins, Benoit-Bird (2004) used calorimetry to determine the calorimetric content of a suite of known prey species. Similarly, Bittar et al. (2012) examined feeding selective patterns of female ribbonfish,

Trichiurus lepturus L., through calculating the calorimetric values of known prey species. In order to maximise calorimetric uptake, when feeding, T. lepturus selected prey species that provided the highest energy per ingested biomass (Bittar et al., 2012).

Wuenschel et al. (2006) determined the energetic densities of estuarine fish species, Lutjanus griseus and Cynoscion nebulosus, during early life stages, demonstrating the successful application of bomb calorimetry on small individuals (such as small fishes and zooplankton). Similarly, Ciancio and

Pascual (2006) conducted calorimetry on various small organisms from Patagonian freshwater ecosystems, including fish, crustaceans, gastropods, oligochaetes and insects. As expected, fish species

20 contained the highest energetic values with invertebrates being less energetically dense (Ciancio and

Pascual, 2006). This finding is in support of trophodynamic hypotheses, as well as findings based on stomach content analyses, whereby trophically high and intermediate fish typically prey on aquatic invertebrates that are of a relatively lower trophic level (i.e. energetic density of organisms may reflect their trophic level; Kneib, 1997; Hollingsworth and Connolly, 2006; Mazumder et al., 2006b).

To the best of my knowledge, for the purpose of exploring potential energetic contributions within trophodynamic relationships, only a single study has used calorimetry within saltmarshes

(Squiers and Good, 1974) and apart from this, only one other study has used calorimetry within saltmarshes at all (see Gabriel and Armando, 1974). Squiers and Good (1974) determined seasonal patterns in the productivity, calorimetric values and chemical compositions of a North American population of the saltmarsh plant species, Spartina alterniflora. The study found little seasonal variation in calorimetric content of the population and that predators of S. alterniflora consume an amount of total energy that is approximately proportional to the quantity of dry matter intake of this plant (Squiers and Good, 1974). Although this study provided a basis for further energetic quantification of trophodynamic processes within estuaries and saltmarshes, no further studies seemed to have followed this research direction.

1.3 Life history traits of estuarine fishes

1.3.1 Sexual reproduction in estuarine fishes

Sexual reproduction allows for the transfer of genetic information between generations and those genes that are suitable to the relevant environmental conditions, which is determined and evident by continued successful reproduction of offspring throughout generations, is in turn what determines the biological and ecological functioning of that species and its overall population/s (Godin,

1997; Elliot et al., 2007; Potter et al., 2015). Knowledge of fish reproduction is important for

21 understanding the life history and ecological functioning of fish populations at a temporal scale. By determining when fish reproduce, linkages can be made to what fish do at other times (e.g. during times of feeding or migration). For example, Atlantic silverside, Menidia menidia, prioritise spawning occasions to times that least interfere with feeding times (Conover and Kynard, 1984). Alternatively, fish spawning times may also be influenced by times when predation is low (Thresher, 1984). Seasonal spawning periods in some fish may also be influenced by, and coincide with, conditions favourable to growth and survival of the parent (Robertson et al., 1990) or offspring (Ochi, 1986). As an organism’s habitats provide reproductive services (for example food, which provides energy for reproduction;

Mann, 1965), insight into the reproductive requirements of an organism is important when considering ecological resources (e.g. shelter and food) necessary for populations to maintain function and health within an ecosystem. It is therefore important to gain an understanding of the reproductive aspects of any species of interest (for instance one that may be useful as a bioindicator of environmental health and functioning or one that is of anthropological economic or commercial importance), in order to gain a clear and full understanding of the biological life history of the species, its method of continued ecological success and its ecological impact on and role in their environment (e.g. its potential for trophic relay).

1.3.2 Mating systems and spawning times in estuarine fishes

There are many ways in which fish transfer their genetic material to the following generation.

Such methods may more appropriately be referred to as ‘mating systems,’ as described by Godin

(1997). Different mating systems can be defined by various factors, in particular the number of mates that a species successfully reproduces with.

Fish that reproduce with several mates utilise a ‘polygamous’ mating system (Godin, 1997). In the Animal kingdom, polygamy is the ‘basic mating system,’ as it is used by ‘basically all animals’ (Wilson,

1975). The polygamous mating system can be further divided into three forms: ‘polygyny,’ in which

22 males mate with several females, but females only with one male organism; ‘polyandry,’ the reverse of polygyny, in which females reproduce with multiple males, but males only reproduce with one female; and ‘promiscuity,’ in which both sexes reproduce with multiple members of the opposite sex (i.e. where polygyny and polyandry both occur at once).

Alternatively, ‘monogamy’ is the mating system where one animal reproduces with only one member of the opposite sex, and where that member of the opposite sex reproduces only with this animal (i.e. a male/female pair reproduce only with each other during a mating season or their lifecycle;

Godin, 1997). Furthermore, it is possible for individuals within a population to utilise a different, or many different, mating systems to other individuals within the population; reasons for which are complicated and variable even among fishes (Reynolds, 1996).

Reproductive cycles and time of spawning in fishes may be seasonally dependent (e.g. Ochi,

1986; Robertson et al., 1990; Chemineau et al., 2007; Elliot et al., 2007; Lowerre-Barbieri et al., 2011;

Potter et al., 2015) or may occur more or less periodically as a result of other influences (Conover and

Kynard, 1984; Thresher, 1984; Sogard et al., 2008). For example, mating patterns of some fishes are influenced by phases of the moon/tides (e.g. Amphiprion melanopus; Ross, 1978) or time of day (e.g. in nocturnal spawners), while others have more complicated reproductive cycles governed by two or more temporal influences (e.g. daily, monthly and yearly spawnings). Reproductive cycles in fish can be further defined by the frequency of which reproduction occurs during their life cycle. Fish that reproduce multiple times during their life cycle (e.g. monthly or yearly spawners) are referred to as being ‘iteroparous,’ while fish that reproduce just once in their lifecycle may be referred to as being

‘semelparous’ (e.g. some species of the genus Oncorhynchus; Godin, 1997).

1.3.3 Food sources and feeding of estuarine fishes

Numerous international studies suggest that temperate saltmarsh vegetation is an important source of a) food for various species of juvenile and adult estuarine dependent fishes

(Weisberg et al., 1981; Morton et al., 1987; Sumpton and Greenwood, 1990), which promotes trophic relay within the estuary (Kneib, 1997); b) organic matter and nutrients for other estuarine habitats

(Odum 1961; Teal, 1962; Mitsch and Gosselink, 1986; Lefeuvre and Dame, 1994); and c) detritus to adjacent coastal waters (via ebbing tides; Nixon, 1980; Dame et al., 1986). However, due to various physical and ecological differences between Australian estuaries and those found elsewhere in the world, these findings cannot be directly transferred to the south-east Australian context.

Productivity due to zooplankton export (particularly crab zoeae) is higher for tidally inundated saltmarsh vegetation during ebbing spring tidal cycles than mangrove, seagrass and open water

(Mazumder et al., 2009), which demonstrates the importance of saltmarsh vegetation as a producer and reliable source of food for estuarine fishes (an important factor to estuarine trophodynamics;

Kneib, 1997). ‘Trophic relay’ is an ecological model proposed by Kneib (1997), whereby biomass and energy derived from estuarine vegetation is transported by nektonic animal species from upper limits of estuaries (i.e. saltmarsh and mangrove habitats), down to lower reaches and out to open sea via a series of predator-prey relationships within estuarine food webs. For example, energy (i.e. stored in organic matter) is transported from vegetation-derived zooplankton (such as crab zoeae within saltmarshes), to small itinerant fish species (e.g. A. jacksoniensis feeding on crab zoeae), and eventually to larger, often economically important, piscivorous fishes that travel into estuaries at particular times to feed (e.g. A. australis, P. fuscus, A. japonicus; SPCC, 1981; Taylor et al., 2006; Mazumder et al., 2006a; see also Bouillon and Connolly, 2009).

In Mazumder (2004) and Mazumder et al. (2006a), this model was further developed by identifying the important vegetative types that are responsible for initial carbon release, as well as important fish species that transport organic matter up the food web and towards the lower reaches

24 of estuaries (and to marine environments; e.g. crab zoeae released by saltmarsh-dwelling grapsid crabs during ebbing tides after saltmarsh vegetation inundation). Through gut content analyses of itinerant fish exiting saltmarsh during ebb tides, Mazumder et al. (2006a) found high proportions of crab larvae

(zoeae) within the guts of glassfish (A. jacksoniensis), Flat-tail mullet (L. argentea) and Blue-eye

(Pseudomugil signifer), demonstating an important trophic link between the secondary production of saltmarsh inhabitants (i.e. grapsid crabs that feed upon fine benthic organic material and microphytobenthos within saltmarsh vegetation; Mazumder and Saintilan, 2010; Alderson et al., 2013) and opportunistic fish species that feed during ebb tides of the spring tidal cycle. Similar to A. jacksoniensis in south-east Australian estuaries, Smith and Able (1994) found that young of the year

(YOY) mummichogs (F. heteroclitus) contribute to trophic transfer of marsh production to the surrounding estuary. Analyses of volumetric dietary compositions found that three of the twelve fish species (A. jacksoniensis, Atherinosoma microstoma and Redigobius macrostoma), recorded at a saltmarsh site in Brisbane Water Estuary, fed nearly exclusively on crab zoeae during the period of their high abundance during ebbing spring tides (Platell and Freewater, 2009).

However, Mazumder et al. (2006a) and Platell and Freewater (2009) did not include in their studies the gut contents and proportions of crab zoeae from within fish that were caught just prior to the saltmarsh being inundated (i.e. on the flooding tide of the spring tide cycle). It is therefore not possible to determine that the high proportions of crab zoeae found within fish stomachs recorded during ebb tides are due to feeding by estuarine fish on the saltmarsh-derived crab zoeae at that time, rather than prior to tidal inundation.

Hollingsworth and Connolly (2006), on the other hand, found that A. jacksoniensis caught adjacent to saltmarsh after inundation had fuller stomachs with higher proportions of crab zoeae than both those caught adjacent to saltmarsh before inundation and those caught after inundation, but in lower reaches of the estuary where feeding on saltmarsh-derived prey was not possible. By including analyses of gut contents before saltmarsh inundation (as in this study), the high proportions of crab

25 zoeae found in stomachs during ebb tides demonstrates predation by A. jacksoniensis on saltmarsh- derived crab zoeae at this particular time. Further, Hollingsworth and Connolly (2006) considered another logical problem (established by Laffaille et al., 2001). Thus, comparisons of A. jacksoniensis stomach contents caught before and after high tides that did not inundate the saltmarsh (i.e. during high tides of the neap tidal cycle) generally showed no difference (being empty both before and after such neap high tides). Contrary to trophic habits of killifish (F. heteroclitus) in the USA (whereby feeding occurs during high tides whether or not saltmarsh is inundated; Weisberg et al., 1981), the study conducted by Hollingsworth and Connolly (2006) strengthens the concept that inundated saltmarsh vegetation provides an important source of food for estuarine-dependent fishes. Finally, in accordance with the findings of Mazumder (2004), whereby south-east Australian saltmarsh crabs synchronise spawning and larval (zoeal) dispersal with the second night of the spring tidal cycle, with the first night acting as an environmental trigger for second and subsequent night spawning, crab zoeae were found only within the stomachs of fish caught on the second and third nights of saltmarsh inundation

(Hollingsworth and Connolly, 2006).

This pattern of feeding on saltmarsh-derived crab zoeae by A. jacksoniensis during ebb tides following saltmarsh inundation was also observed in the temperate estuary of Patonga Creek,

Hawkesbury River, NSW (McPhee et al., 2015). Ambassis jacksoniensis switched prey from thalassinid larvae (Trypaea australiensis) during ebb tides that did not inundate saltmarsh vegetation to saltmarsh- derived crab zoeae during ebb tides following saltmarsh inundation. The act of switching prey had previously not been observed for this fish species and helped explain their high abundance in estuaries.

The ability of species to “switch prey” (i.e. focus feeding on different food sources in response to temporal and spatial variability in prey availability), is well documented for fish (Murdoch et al., 1975;

Ringler, 1985) and other animals (Suryan et al., 2000; Siddon and Witman, 2004) and is in alignment with general concepts of optimal foraging theory (MacArthur and Pianka, 1966). For example, the freshwater fish, Bidyanus bidyanus, feeds on cladocerans (Daphnia spp.) when they are abundant but

26 switches to calanoid copepods and insects when their preferred prey (cladocerans) becomes limited

(Warburton et al., 1998). Moreover, Spratelloides robustus preys on calanoids for much of the year in nearshore marine waters, but then feeds on benthic prey during winter, when these planktonic crustaceans become scarce (Schafer et al., 2002). This example is consistent with optimal foraging theory whereby feeding (chasing, catching etc.) on a preferred prey may require too much energetic input and thus the organism switches to a less preferred but easier to ingest prey (e.g. which may be, for example, more abundant). Demonstrating adaptive feeding behaviours when prey abundance varies temporally and spatially, guppies (Poecilia reticulata) switch to the most abundant prey at varying times and levels of the water column (Murdoch et al., 1975). Further, feeding behaviour of the crab, Cancer borealis, and its extent of predation on sea urchins (Strongylocentrotus droebachiensis) is dependent on habitat (and shelter), with evidence of prey switching at certain habitats (Siddon and Witman, 2004).

Such switching between different prey types represents an ideal adaptation for optimising the feeding of animals in temporally and spatially variable environments (MacArthur and Pianka 1966; Hughes,

1980).

Other freshwater ambassids are also carnivorous planktivores, with some of these also displaying acts of prey switching. Thus, A. agassizi feed on microcrustaceans and insect larvae

(Medeiros, 2004; Lintermans, 2007), and are mid-water feeders within littoral vegetation (Milton and

Arthington, 1985). Ambassis agrammus are microphagous carnivores that feed mostly within midwater heights, and occasionally benthically, on small crustaceans (e.g. cladocerans, copepods, ostracods and conchostracans) and aquatic insects (e.g. chironomids, baetids and leptocerid larvae;

Bishop et al., 2001). Other studies found that A. agrammus feed on planktivorous crustaceans (Pollard,

1974; Lake, 1978; Sanderson, 1979), with shifts to benthic arthropods in floodplains during wet seasons

(Sanderson, 1979). Similar diets and feeding behaviours have been shown for other ambassids including, Ambassis interrupta (Coates, 1990), Ambassis macleayi (Bishop et al., 2001), Ambassis gymnocephalus, Ambassis natalensis and Ambassis productus (Martin and Blaber, 1983), which are mainly suspension, and occasionally benthic, feeders. Finally, due to well developed dentition and gut

27 morphology (distensible stomach and low relative gut length), A. gymnocephalus, A. natalensis and A. productus may be carnivorous predators (Martin and Blaber, 1984).

Despite the knowledge of the importance of zooplankton to the diets of trophically higher estuarine predators such as fish (Weisberg et al., 1981; Kneib, 1997; Loneragan et al., 1997; Laffaille et al., 2001; Mazumder, 2004; Hollingsworth and Connolly; 2006; Mazumder et al., 2006a; Svensson et al., 2007; Freewater et al., 2008; Platell and Freewater, 2009; McPhee et al., 2015), very little is known about the calorimetric content and energetic contribution of zooplankton to their predators, especially for saltmarsh and mangrove derived zooplankton in south-eastern Australian estuaries and those elsewhere in the Southern Hemisphere (e.g. South Africa). Such information would allow for a quantitative understanding of the energy contributions from the saltmarshes (e.g. how much energy is gained via the predation of zooplankton by fish and how this transfer may affect trophodynamics within the ecosystem), whereas previous knowledge provided only qualitative estimates (e.g. Hollingsworth and Connolly; 2006; Mazumder et al., 2006a; Platell and Freewater, 2009; McPhee et al., 2015).

1.4 The Ambassidae

The fish family Ambassidae (formerly Chandidae and commonly referred to as glassfish) contains 41 species from 8 genera. This diverse and abundant family is also widely distributed and can be found in the Red Sea, the East African coast, Asia and Australasia (Allen, 1982), with examples of freshwater, estuarine and marine species (Bishop et al., 2001). Of these 41 species of ambassids, 13 are known to populate Australian waters (Allen and Burgess, 1990). Most species of Australian ambassids are estuarine, although some species are restricted to freshwater habitats (e.g. A. agassiz in the Murray

Darling Basin; Llewellyn, 2008).

1.4.1 Reproductive biology of ambassids

Studies of the reproductive biology have been conducted on some Australian ambassid species, and much of what is known of their reproduction has been determined using various procedures

(e.g. field observations, aquaria trials etc.). Like many organisms, there are often particular times or seasons in a year that are optimal for ambassids to reproduce and spawn.

Patterns in location and timing of spawning vary among ambassids, with different patterns and strategies being displayed for different species and even different populations of the same species. For example, the gonadal development of Ambassis agrammus, coincides with the onset of rains (late dry

– early wet seasons) in the Northern Territory of Australia (Bishop et al., 2001). Alternatively, in a Papua

New Guinean population of A. agrammus, non-seasonal breeding for the species was observed

(Roberts, 1978). Alternatively, for other freshwater ambassids, Ambassis interrupta (Coates, 1990) and

Ambassis macleayi (Bishop et al., 2001), gonado-somatic indicies (GSI) and gonadal maturity stages remain relatively stable throughout the year, indicative of constant or continual spawning.

Ambassis agrammus seek out and ‘prepare’ spawning sites by ‘cleaning’ chosen patches of weed through ingestion or removal of foreign material (e.g. epiphytes), before males actively defend spawning sites from other fish (Semple, 1985). Furthermore, some ambassids spawn during early mornings with males courting females by pursuing them into vegetation where demersal eggs are deposited and fertilized (Lee, 1975). Some ambassids, such as A. agrammus (Semple, 1985), A. agassiz

(Milton and Arthington, 1985) and A. macleayi (Ivantsoff et al., 1988) attach spherical, transparent eggs to upper surfaces of vegetation within spawning sites (see also Breder and Rosen, 1966). Alternatively,

A. buronesis are midwater spawners that allow eggs to sink to the substrate (Bates, 1935).

Gonads of A. agassiz begin maturing in September (spring), with ripe fish becoming apparent in populations during October and November (Milton and Arthington, 1985). The GSI of A. agassiz are higher during these than non-spawning months (i.e. when gonads are inactive), with male GSI being generally lower than females (Milton and Arthington, 1985). Unlike A. agrammus (Bishop et al., 2001)

29 and A. interrupta (Coates, 1990), which generally spawn throughout the year, A. agassiz are seasonal spawners that are influenced by seasonal rises in water temperature and spawn when water flow is stable (Cadwallader and Backhouse, 1983; Leggett, 1984; Milton and Arthington, 1985; McNeil et al.,

2008). Spawning during pre-flood months promotes egg and larval survival and recruitment, while in later months when offspring become surface schoolers, flooding waters allow for dispersal of the population into downstream areas (Milton and Arthington, 1985). This spawning pattern is similar in the freshwater melanotaeniid, Melanotaenia splendida fluviatilis (Milton and Arthington, 1984).

Aging and length-frequency distribution analyses showed that the majority of a population of

A. agassiz was made up of YOY fish that had spawned during November and December of 2007

(McNeil et al., 2008), while in Milton and Arthington (1985), populations were slower growing and multigenerational (with males and females generally living for two and three years respectively). These conflicting findings demonstrate that life history characteristics of ambassids from different populations of the same species can be variable and therefore generalising across a range of estuaries for any ambassid may not be possible.

Fecundity of freshwater Australian ambassids is well studied for multiple species, including

A. agrammus (Semple, 1985; Bishop et al., 2001), A. macleayi (Bishop et al., 2001), A. interrupta

(Coates, 1990), A. agassiz (Milton and Arthington, 1985) and is highly variable among species. In a pattern that is typical for teleosts (Nikolsky, 1969), the fecundity of A. agrammus (Semple, 1985) and

A. macleayi (Bishop et al., 2001), but not A. agassiz (Milton and Arthington, 1985), is positively related to female length and weight. A similar pattern in size was also related to spawning frequency of A. agrammus, with the smallest and largest females spawning for one night, while large (40-50 mm SL) females spawn for up to four consecutive nights (Semple, 1985). It should be noted that egg sizes of these species differ and thus may reflect differences in fecundity among species (for example, A. macleayi produce large numbers of small eggs; Bishop et al., 2001).

Lengths of first maturity have been studied for A. agrammus (Bishop et al., 2001), A. agassiz

(Milton and Arthington, 1985), A. macleayi (Bishop et al., 2001) and are not highly variable among species (26 – 33 mm SL). Further, lengths of 70 mm SL were reported for 50% maturity of a population of A. interrupta (Coates, 1990). It should be noted, despite suggestions that Ambassis spp. do not generally grow to lengths >80 mm SL (Lake, 1971), A. macleayi has a reported maximum length of 100 mm SL (Pollard, 1974).

1.4.2 Life history traits of Ambassis jacksoniensis

The majority of freshwater ambassids are phytophilic demersal spawners that attach adhesive eggs to vegetation within the water column (see Breder and Rosen, 1966; Milton and Arthington, 1985;

Semple, 1985; Ivantsoff et al., 1988; Bishop et al., 2001), but much less is known about the general spawning strategy of estuarine ambassids. The marine ambassid, Ambassis gymnocephalus, is a midwater spawner, but unlike the freshwater A. buroenisis whose eggs sink to the substrate,

A. gymnocephalus eggs are pelagic (Nair, 1957). The spawning strategy of A. jacksoniensis is unknown.

Ambassis jacksoniensis is a small, schooling (Hurst et al., 2005), short-lived ambassid that reaches sexual maturity within the first year of its life (Mazumder et al., 2009). With a maximum size of

70 mm (Kuiter, 1993), A. jacksoniensis is commonly distributed along the south-eastern coast of QLD and throughout NSW (Munro, 1961; Allen and Burgess, 1990; Allen et al., 2002; Hurst et al., 2005), where it is known to feed primarily on zooplankton, including larvae derived from saltmarsh and mangrove vegetation (see earlier). Known predators of the species include the fish species A. australis,

P. fuscus and A. japonicus (SPCC, 1981; Taylor et al., 2006; Mazumder et al., 2006a), as well as some piscivorous water birds (e.g. Sternula albifrons; NSWDUAP, 2002). However, information on the predators of A. jacksoniensis is based on very limited evidence and remains an area for further research.

Unlike some species of ambassids with populations under survival pressures (e.g. human-induced change to river flow, habitats and ecosystem processes; Cadwallader, 1978; Arthington et al., 1983) in

31 their natural environment (e.g. A. agassiz; see McNeil et. al., 2008), A. jacksoniensis appear resilient and abundant (Mills et al., 2008). Despite limited knowledge of the reproductive aspects of

A. jacksoniensis, two particular studies on the life history of the species provide a strong basis for further research to be built upon.

1.4.2.1 Spawning and larval characteristics of Ambassis jacksoniensis

Miskiewicz (1987) studied spawning characteristics of estuarine fishes in Lake Macquarie, NSW and found two important aspects of the spawning behaviours of A. jacksoniensis, from which another two key pieces of information are evident. First, larvae of A. jacksoniensis were found in high abundances at locations within Swansea Channel, which connects Lake Macquarie to the ocean, with decreasing abundances occurring at locations further into the estuary. A. jacksoniensis (along with

Gerres ovatus) were the dominant taxa at such marine-dominated locations. This indicates that

A. jacksoniensis are likely to be nearshore marine-spawners, and as their typical place of residence is known to be saltmarsh, mangrove and seagrass habitats located within the main body of the estuary

(Mazumder 2004; Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; McPhee et al., 2015), this suggests that A. jacksoniensis migrate or use tidal patterns to disperse from their usual estuarine vegetative habitats to the mouth of the estuary for spawning.

The second major finding was that A. jacksoniensis larvae were caught at these locations between September and July with a peak from February to May, indicating that A. jacksoniensis are multiple, continual spawners throughout most of the year, much like many closely-related species

(e.g. A. interrupta and A. macleayi; Coates, 1990; Bishop et al., 2001). Interestingly, larval abundances dropped during winter months, a period when A. jacksoniensis take dietary advantages of highly abundant zooplankton (particularly crab zoeae) derived from saltmarshes within estuaries (Mazumder et al, 2006a; Hollingsworth and Connolly, 2006; McPhee et al., 2015). It could be hypothesised that

A. jacksoniensis decrease spawning effort (at the mouths of estuaries) during winter, as crucial

32 saltmarsh and/or mangrove-derived food is available well inside the estuary and away from the mouth

(i.e. they cannot be at both places at the same time, so A. jacksoniensis ‘stop’ spawning during winter to feed). It is possible that the high energetic input of the zooplankton during winter provides

A. jacksoniensis with energy stores for high spawning effort during the rest of the year (Mann, 1965).

Knowledge of the energetic content of A. jacksoniensis prey, and hence possible energetic contribution of prey to A. jacksoniensis, would indicate whether indeed A. jacksoniensis could benefit from feeding on saltmarsh-derived zooplankton, and would provide insight into the ecology and life history of A. jacksoniensis, as well as general estuarine ecological characteristics and trophodynamic relationships concerning saltmarshes.

Miskiewicz (1987) also examined the larval characteristics of the closely related and co- occurring Ambassis marianus. Unlike the nearshore marine-spawning A. jacksoniensis, A. marianus were generally estuarine spawners with larvae being recorded throughout the estuary. Similar to A. jacksoniensis, larvae of A. marianus were found throughout the year, with a decrease during winter

(Miskiewicz, 1987).

1.4.2.2 Genetic structure of Ambassis jacksoniensis

Marine fish populations are generally shallowly structured (i.e. populations do not greatly differ genetically over geographical distances; Bilton et al., 2002) and at scales where structure is present

(usually in cases of mutation-genetic drift equilibrium), such structure is generally a result of ‘isolation by distance,’ by which a positive relationship between geographic distance and genetic distance occurs

(Wright, 1943; Slatkin and Maddison, 1990). On the other hand, freshwater fish populations are generally strongly structured (i.e. populations differ greatly genetically even between relatively geographically-close populations; Wong et al., 2004).

Mills et al. (2008) examined genetic structuring of A. jacksoniensis and A. marianus populations on the south-east coast of Australia through the use of ATP8 gene of mitochondrial DNA. Mills et al.

(2008) aimed to determine whether an intermediate generalised ‘estuarine’ genetic structure could exist for estuarine fish populations of species such as A. jacksoniensis and A. marianus, as had been previously suggested for other estuarine fishes (e.g. Farrington et al., 2000; Dawson et al., 2002; Cabral et al., 2003; Watts and Johnson, 2004; Doukakis et al., 2005). Analysis of haplotype frequencies of populations sampled from Tin Can Bay (QLD) and Kempsey (NSW) demonstrated that A. jacksoniensis and A. marianus had high levels of haplotype diversity (more typical of marine fishes than estuarine or freshwater fishes) with star-like phylogenies. However, as such diversity was not partitioned according to the geographic scale of the study, these findings were indicative of panmictic populations

(i.e. diversity was genetically homogenous on a geographical scale), which suggests the capability of high dispersal between estuaries for these species, with little or no barriers for genetic flow (Mills et al.,

2008). Further, despite a lack of knowledge of larval phase durations of these estuarine species, these findings suggest they may be long, which would allow for high geographic dispersal and the resulting panmixia (Mills et al., 2008).

High diversity and star-like phylogenies suggest that these species have had, by geological standards, a relatively recent population expansion, whereby there have been enough generations for evolutionary changes from the ancestral haplotypes, but not enough for genetic drift and selection to

‘kill off’ uncommon haplotypes (Mills et al., 2008; see also Posada and Crandall, 2001). Thus, the population expansion has led to what might be mistaken for panmixia (Mills et al., 2008). A. jacksoniensis and A. marianus populations were not genetically isolated by distance, as hypothesised

(i.e. equilibrium was not present), which may be due to high dispersal capabilities and fairly high connectivity (compared to fishes from freshwater environments) that exists between populations for these species (i.e. geographic genetic flow may be occurring at a greater rate than genetic divergence, which may counteract any indication of ‘isolation by distance’; see also Purcell et al., 2006).

While Miskiewicz (1987) concluded that the highly abundant A. jacksoniensis are nearshore marine-spawners (see earlier), Mills et al. (2008) provide evidence to indicate high, likely larval, dispersal capabilities between populations and estuaries of their known distribution. Such findings help explain the high abundance and wide geographical distribution of A. jacksoniensis along the south- eastern coast of Australia, as well as provide further evidence of their reproductive behaviour as near shore marine-spawners. This evidence of high abundance and distribution is relevant as A. jacksoniensis are known to be an important trophic link between saltmarsh-derived zooplankton (Mazumder et al.,

2006a; Hollingsworth and Connolly, 2006; McPhee et al., 2015) and trophically higher, economically important fishes (SPCC, 1981; Taylor et al., 2006; Mazumder et al., 2006a), and are therefore important in the trophic functioning of Australian estuarine ecosystems.

1.5 Framework of investigation and study aims

The above literature review provides a summary of the current research conducted on estuarine, and particularly saltmarsh, ecology, both within Australia and globally. Within this review, emphasis has been placed on the role of saltmarsh in providing habitats and sources of food for those dependent on saltmarsh vegetation, as there is much evidence to highlight the importance of saltmarsh to the life cycles of estuarine organisms. As saltmarsh vegetation is only tidally inundated on three or four days per month within south-east Australia, in contrast to other saltmarshes in many other estuaries worldwide, this study seeks to determine what effect, if any, such temporal provision of an inundated saltmarsh habitat has on the fish assemblages of a representative saltmarsh in south-eastern

Australia. Further, as the literature has demonstrated the significant exportation of crustacean larvae

(e.g. crab zoeae) from inundated saltmarsh into the zooplankton during ebb tides, this study will investigate the dietary interactions between such temporally-variable zooplankton and the diets of a

35 common estuarine ambassid, Ambassis jacksoniensis, in an attempt to assess the extent of any trophic relay from saltmarsh vegetation to estuarine fish via predator/prey relationships.

The focus of the study is to assess the importance of saltmarsh to estuarine biota using a representative and relatively “unmodified” saltmarsh habitat (Empire Bay Wetland) in a large and permanently-open estuary (Brisbane Water Estuary), located in south-eastern Australia, as a model habitat. This study, which was conducted at two markedly different times of the year, examines the general response of the estuarine fish and zooplankton assemblages to the infrequent inundation of saltmarsh at Empire Bay Wetland, with further emphasis being placed on selected biological and ecological characteristics of Ambassis jacksoniensis, a small fish that is highly abundant in estuarine saltmarshes and is known to prey upon zooplankton. The nature of the dietary relationship between this ambassid and its zooplanktonic prey, which may include saltmarsh-derived zooplankton, will be further enhanced by an innovative assessment of the potential calorimetric contributions of such prey to their potential predators.

This study, which was conducted in open waters directly adjacent to the saltmarsh at Empire

Bay Wetland, aims to:

1) Quantify the abundance, diversity and composition of fish assemblages, taking into account

tidal states (e.g. flood/ebb tides), presence of saltmarsh inundation and time of year (e.g.

summer and winter).

2) Explore the relative abundances of the most common species, including A. jacksoniensis within

those fish assemblages, and quantify any response to tidal state, presence of saltmarsh

inundation and time of year.

3) Assess sex ratios, sexual maturity stages and gonadal development of A. jacksoniensis at two

markedly different times of the year, in order to help determine the population structure and

reproductive biology of this species.

4) Quantify the abundance, diversity and composition of zooplankton assemblages, taking into

account tidal states, presence of saltmarsh inundation and time of year.

5) Explore the relative abundances of saltmarsh-derived zooplankton (crab zoeae) and other

abundant taxonomic groups within those zooplankton assemblages, and quantify any response

to tidal state, presence of saltmarsh inundation and time of year.

6) Using stomach content analyses, to determine the extent of feeding and the dietary

compositions of A. jacksoniensis and examine any differences between tidal states, presence

of saltmarsh inundation and time of year.

7) To ascertain the potential energetic contribution (calorimetric value) of potential zooplankton

prey to the diets of A. jacksoniensis to understand the bases of the ingestion of different types

of prey by this ambassid.

1.6 Outline of thesis

This thesis is comprised of seven main chapters. Chapter One outlines those concepts that are central to each chapter and contains the study rationale, framework for the study and the study aims.

Chapter Two contains the general methodology of the study, including details of the study site (Empire

Bay Wetland), main experimental design (for Chapters Three - Six) and general details of the sampling/laboratory procedures and statistical analyses used in subsequent chapters. Chapter Three describes the fish assemblages of Empire Bay Wetland and compares them among factors such as tidal state, day/inundation state and season in order to gain insight into the role of saltmarsh vegetation as a habitat for estuarine fishes. Chapter Four investigates the life history of the abundant estuarine fish,

A. jacksoniensis, including reproductive traits in two different seasons, in order to provide context on its reproductive cycle in relation to its feeding patterns at the study site (Chapter Six). Chapter Five describes the general zooplankton assemblages of Empire Bay Wetland and compares them between

37 tidal states, days/inundation states and seasons in order to gain insight into the role of saltmarsh vegetation as an exporter of zooplankton to other estuarine areas. Chapter Six investigates the diets of

A. jacksoniensis and compares them between times when this fish species either does, or does not have access to saltmarsh-derived zooplankton, as well as between seasons and tidal states. Chapter Six also quantifies the calorimetric content of zooplankton and compares them to a common prey item of

A. jacksoniensis (crab zoeae). Chapter Seven consolidates the major findings of the study in order to give an overall view of the biology and ecology of A. jacksoniensis and the role of saltmarsh as a crucial ecological resource for estuarine organisms.

CHAPTER TWO

GENERAL METHODOLOGY

2.1 Characteristics of the study region and location

2.1.1 The study region - Brisbane Water Estuary

The study was undertaken at one location, Empire Bay Wetland (~33°29’25”S, 151°21’19”E), which is located within the study region of Brisbane Water Estuary in south-eastern Australia

(Figure 2.1). Brisbane Water Estuary is a permanently-open estuary, which lies approximately

50 km north of Sydney (NSW) and opens into northern Broken Bay, into which the large Hawkesbury

Estuary also opens. The tidal range of this estuary is approximately 1 m AHD and, like other eastern

Australian estuaries, are semi-diurnal, i.e. occur twice daily, but with one of those daily high tides being typically higher than the other (Ford et al., 2006). During spring tides, these high tidal levels can exceed

1.8 m AHD, which is sufficient to inundate the saltmarshes along its shoreline once during each 24 h period for the typically three-day long spring tides.

Brisbane Water Estuary is a tidally-dominated estuary with a narrow (~150 m wide) entrance channel that separates into several basins or water bodies at distances of 6-8 km inland (Ford et al.,

2006). The basins within the estuary, which may also be linked by narrow channels, can reach water depths of 5-6 m (Cardno, 2008). There are several major tributary creeks including Ettalong Creek, Woy

Woy Creek, Corrumbine Creek, Upper and Lower Narara Creek, Upper and Lower Erina Creek and

Kincumber Creek, the latter of which supplies freshwater output for the study location of Empire Bay

Wetland (Cardno, 2008).

Brisbane Water Estuary has an estimated total catchment area of 165 km2 and falls under the

NSW State Government, Hunter Central Rivers Catchment Management Authority (Cardno, 2008).

Much of the foreshore of this estuary has been urbanised although there are still portions of foreshore that are reserves and National Park (Cardno, 2008). Much of the remainder of the catchment is comprised of both urbanised development and National Park. This urbanisation of Brisbane Water

Estuary has led to this estuary being classified as being “extensively modified” (see OzCoasts, 2015).

As a result of rising sea levels, Brisbane Water Estuary was formed through the drowning of an ancient river valley in relatively recent geological time (Cardno, 2008). The geology of the catchment is based on Hawkesbury Sandstone and the Narrabeen Group Terrigal Formation (see Ward, 1972).

Furthermore, Quaternary Alluvium is present over the majority of the Woy Woy-Umina Peninsula (an urbanised peninsula located on its south-western banks), which consists of gravel, silt and clay (Benson,

1986). With respect to the zones established by Roy et al. (2001), the sediment morphology of the estuary substrata ranges from i) silica sand within the marine tidal delta, to ii) catchment muds within the central mud basin, and ultimately iii) silt, mud and sand particles in the fluvial deltas.

Brisbane Water Estuary has a warm-temperate climate with rainfall being typically higher during summer months (Murphy, 1993). Mean rainfall ranges from 68.5 mm in September to 152.3 mm in February, while mean air temperatures range from 27.6°C in January to 4.6°C in July (Bureau of

Meteorology, 2012). During summer months, wind directions are usually south/south-easterlies (with a tendency for onshore north-easterly winds in afternoons), while winter wind directions are typically south/south-westerlies (Murphy, 1993).

There are known areas of saltmarsh and mangrove vegetation in Brisbane Water Estuary, with saltmarsh vegetation being predominantly situated within the intertidal zone landward of the mangrove forest (typical of saltmarsh/mangrove complexes in south-eastern Australia; Bakker, 2014).

Although these intertidal habitats have declined in recent history (Cardno, 2008; see also Chapman and

Roberts, 2004), there are still substantial stands at a few locations within the estuary, such as at Erina

Creek Wetland, Cockle Bay Nature Reserve and including the study location of Empire Bay Wetland.

Despite their relatively small coverage, these areas of saltmarsh in Brisbane Water Estuary are considered as ‘regionally significant’ (Laegdsgaard, 2002; Cardno, 2010).

Figure 2.1: Map of Brisbane Water Estuary and Empire Bay Wetland (NSW, Australia). Dot symbols indicates the locations of the sampling sites.

The subtidal habitats of Brisbane Water Estuary comprise relatively large amounts of Zostera mulleri subsp. capricorni in the intertidal zone and dense stands of Posidonia australis immediately adjacent in subtidal waters (Cardno, 2008). During 2001 and 2002, the mean water temperatures in the basins of Brisbane Water Estuary ranged from 14.6-24.8 C,̊ mean salinities ranged from 2.1-36.1, mean pH from 7.0-8.6, mean turbidity from 0.9-46.0 NTU, and mean dissolved oxygen concentrations from

0.03-8.7 mg/L (Oceanics, 2003). During 2001, 2002, 2006 and 2007, in these same locations, the mean concentrations of total Nitrogen ranged from 0.1-18.0 mg/L, while mean concentrations of total

Phosphorous ranged from 0.01-0.18 mg/L (Oceanics, 2003), with the conclusion being made that

Brisbane Water Estuary is functioning reasonably well ecologically, considering its extent of foreshore development (Cardno, 2008). Recreational activities in Brisbane Water Estuary include boating (power boating, sailing, paddle etc.), swimming, fish, wind surfing, diving (SCUBA and snorkeling) and kite surfing, while commercial activities include oyster farming, boat tours, boat charters and ferry operations (Root, 2005).

2.1.2 The study location - Empire Bay Wetland

The study location of Empire Bay Wetland is located on the south-eastern shore of Brisbane

Water Estuary, approximately 4-5 km from the entrance (Figure 2.1) and is therefore subject to substantial tidal influence. The extensive saltmarsh at this site is inundated on a lunar basis, during spring tides that exceed 1.8 m AHD, with such high tides occurring during the day in summer and in the night-time during winter (pers. ob.; see also Platell and Freewater, 2009).

The soil landscape of saltmarshes at Empire Bay Wetland is predominantly estuarine

“Mangrove Creek” (see Fig. 3.5 in Cardno, 2008), which have a high acidic sulphate potential, are highly saline and sodic, have low soil fertility and contain highly organic soil materials (Murphy, 1993). The surrounding land use in this area is suburban, with access to the saltmarsh restricted to off-road vehicles, such as bicycles and trail bikes.

The vegetative composition of Empire Bay Wetland is primarily classified as a mixture of

Saltmarsh/Grassland and Estuarine Mangrove Scrub (Cardno, 2008). As south-eastern Australian saltmarshes occur at higher elevations than mangroves, the saltmarsh at Empire Bay Wetland becomes tidally inundated less frequently than the mangroves (Wilson and Whittaker, 1995) and occurs on comparatively drier soils that possess a greater salinity range (Chapman, 1974). High spring tides

(>1.8-9 m AHD) generally inundate the entire saltmarsh community (Platell and Freewater, 2009), however in summer, saltmarshes are known to entirely ‘dry out’ (NPWS, 2009; Platell and Freewater,

2009).

Typical of south-eastern Australia, saltmarsh at Empire Bay Wetland is characterised by

Sarcocornia quinqueflora, Sporobolus virginicus and Triglochin striatum in the low marsh (at lower elevations and closer to the water body and mangrove vegetation), while Juncus kraussii and Suaeda australis are abundant in the high marsh (at higher elevations and closer to terrestrial flora; Harty, 1994;

Roberts and Sainty, 2005; Cardno, 2008). Casuarina glauca is also located around the edges of Empire

Bay Wetland (i.e. as part of the terrestrial flora; Cardno, 2008). The mangrove community of Empire

Bay Wetland consists of Avicennia marina var. australasica and Aegiceras corniculatum (Cardno, 2008;

NPWS, 2009). Within the adjacent subtidal portion of Empire Bay Wetland, Zostera capricorni is present, while Posidonia australis and Halophila spp. are also present, but to a lesser extent (Cardno,

2008). Intertidally, the substratum largely consists of bare sand/mud-flats, algae and mangrove pneumatophores.

Within the saltmarsh/mangrove habitat at Empire Bay Wetland, a range of aquatic and terrestrial fauna has been recorded, including arthropods (burrowing crab species such as Sesarma erythrodactyla, Helograpsus haswellianus and Heloecius cordiformis, various insects and spiders), molluscs (gastropods such as Salinator solida, Ophicardelus ornatus, Bembicium auratum, Littorina scabra, and Assiminea tasmanica), and various fish and bird species (see Cardno, 2008). Although

Roberts and Sainty (2005) stated that the low marsh of Empire Bay Wetland was in ‘good’ condition,

44 they have classified the wetland as ‘disturbed’ due to previous impacts from bicycle tracks in recent years.

2.2 Sampling and experimental design

Sampling for fish and zooplankton was carried out at two sites within the study location of

Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) at two different tidal states (flood and ebb tides) on six sequential days in each of two months during both summer and in winter in 2012. The two sites were located over seagrass and directly adjacent to the mangrove/saltmarsh complex of the study, and were < 1 m in depth. The sampling on six sequential days was aimed at capturing any differences related to saltmarsh inundation, with Days 1-3 having tidal heights of < 1.8 m AHD (and thus no saltmarsh inundation) and Days 4-6 representing those times when saltmarshes were inundated, i.e. when spring tides exceeded 1.8 m AHD. During summer, saltmarshes were inundated during the day-time high tide while in winter such inundation occurred during the night-time high tide.

The main experimental design contained five factors: Season (summer and winter); Month

[nested in season] - January, February [summer 2012], June and July [winter 2012]; Day - 1-6 (Days 1-

3: no inundation and Days 4-6: saltmarsh inundation); Tidal State (flood and ebb); and Site (1 and 2).

Sampling for fish and zooplankton therefore occurred 96 times in 2012, representing six sequential days in two months in each of two seasons, carried out on both flood and ebb tides on each sampling occasion.

For the fish and zooplankton assemblage data (i.e. Chapters Three and Five respectively), the factor Site was not included in the design as sites were considered as replicates. However, site was considered in the case of the dietary data for A. jacksoniensis, in which substantial numbers of fish

(i.e. replicates) were able to be collected at the two sites. Sites were located approximately 300 m apart to ensure independence of the samples.

Due to limitations in time and other logistical (e.g. safety) issues associated with sampling multiple replicates within a saltmarsh environment during tidal inundation (Connolly, 1999), higher spatial replication was unachievable in the present study.

2.3 Data collection

A hand-held YEO-KAL water meter was used to measure water quality variables such as water temperature (°C), salinity, dissolved oxygen concentration (mg/L) and turbidity (NTU) in the middle of the water column on each sampling occasion at Empire Bay Wetland. These data showed that salinities were typically close to or at seawater (28.9-33.6) and water temperatures ranged from 13.9-25.9°C, being lowest in winter and greatest in summer (Table 2.1). The dissolved oxygen concentration was relatively high, i.e. 4.6-8.1 mg/L, and the turbidity was typically low (0.3-33.3 NTU) during the study period (Table 2.1).

Table 2.1: Mean values (±SE, n=48) of water temperature, salinity, dissolved oxygen and turbity during summer and winter at Empire Bay Wetland, in 2012.

Summer Winter Temperature (°C) 24.206 (0.21) 15.677 (0.22) Salinity 30.523 (0.35) 32.225 (0.18) Dissolved oxygen (mg/L) 6.462 (0.22) 6.456 (0.12) Turbidity(NTU) 3.555 (0.72) 1.675 (0.74)

2.3.1. Fish sampling and sample treatment

Fish were sampled at the two sites in Empire Bay Wetland using a seine net of 20 m length,

2 m height, 2 m cod-end, and 8 mm mesh size. This net was deployed by hand, with a person that initially stood adjacent to a boat that was anchored on the side of each site that was “upstream” of the tidal flow, while holding the lead and float line of one end of the seine net in each hand. The seine net was then walked parallel (and with) the tidal flow, with the remaining net being fed out from the

46 anchored boat, until its full length was reached on the opposite side of the site. Once the net was fully extended, it was then hauled in a circular direction, keeping maximum distance from the boat and walking against the tide before meeting back up with the boat (see McPhee et al., 2015). Thus, 250-350 m2 of the channel was sampled for each site (seine net haul). The hauling process was performed once at each site.

Tide charts provided the date and time of commencement of sampling for each particular ‘day’

(i.e. the first seine haul during the flood tide), although seine net hauls were sometimes collected after midnight (up until ~2am of the following day) due to the timing of high tides (during winter). Flood tide sampling took place approximately one hour prior to saltmarsh inundation, while ebb sampling took place during the time between when the tide began to ebb until the saltmarsh was no longer inundated.

These procedures were only performed on days when the saltmarsh became inundated between flood and ebb tidal states (i.e. days 4-6). On days when the high tide did not inundate the saltmarsh (i.e. days

1-3), flood sampling took place approximately one hour prior to the high tide slack water, while ebb sampling took place during the ebbing of the tide.

Fish were removed from the seine net and placed immediately into an ice-slurry to be euthanased (Hollingsworth and Connolly, 2006; Platell and Freewater, 2009). All other animals

(e.g. crustaceans, cephalopods etc.) were removed from the net and released back into the water unharmed.

Fish were subsequently frozen, stored at -15°C and then thawed for laboratory analysis of fish assemblages (further details in Chapter Three) and the reproductive traits of A. jacksoniensis (further details in Chapter Four). For examination of the diets of A. jacksoniensis, individuals were subsequently preserved in 10% formaldehyde (with seawater) for at least one month to ensure adequate fixation of body tissues and transferred to 70% ethanol (Chapter Six).

Further details of the laboratory procedures and all details of the statistical analyses undertaken for the separate chapters are provided in Sections 3.2, 4.2 and 6.2.

2.3.2 Zooplankton sampling and sample treatment

Zooplankton were sampled using a plankton net, containing a 250 μm mesh and with a 950 mm long body and a 300 mm opening. Using markers on the shoreline as a guide, the net was towed along a 100 m transect at each of the two sampling sites at the study location, being deployed at a standard distance (2 m) behind the boat and approximately 15 cm below the surface of the water,

(Sulkin et al., 1980; Epifanio et al., 1984; DiBacco et al., 2001; Papadopoulos et al., 2002; Bretsch and

Allen, 2006; Ford et al., 2006; Freewater et al., 2008), and to enable direct comparisons to be made with the results of Freewater et al. (2008), which was conducted near saltmarsh environments in the study estuary. The boat speed was kept to approximately 1-2 knots during plankton tows, to minimise any disturbance to the surface layers of the water or seagrass vegetation. NB Although flowmeters were deployed the blades were often observed to be stationary while towing (pers. ob.), and resultant readings were highly variable. For this reason, distance along the shoreline was considered to be a more reliable indicator.

The net and sample was thoroughly rinsed into labelled containers (700 mL), with 5% formaldehyde for preservation. All sampling equipment was thoroughly washed after each tow in order to prevent cross contamination between samples. All plankton samples were subsequently transferred to 70% ethanol for at least one week, prior to subsampling and measurement of the zooplankton assemblages.

For the zooplankton that were obtained for calorimetric comparisons (in Chapter Six), additional tows were undertaken at each site at the same times as other zooplankton sampling using the same procedure as above, but with these samples being frozen prior to bomb calorimetry being undertaken (Section 6.2.3.2).

The detailed descriptions of laboratory procedures and statistical analyses are provided in

Section 5.2. The collection of fish and zooplankton was authorised by the Department of Primary

Industries (Permit No. P11/0085-1.0) and UoN Animal Ethics Committee (Permit No. A-2012-219).

CHAPTER THREE

COMPARISONS OF ESTUARINE FISH ASSEMBLAGES NEAR

SALTMARSH

3.1 Introduction

Although the habitats provided by mangrove and seagrass vegetation are considered crucial for fishes in estuaries (Blaber et al., 1985; Robertson and Duke, 1987; Laegdsgaard and Johnson, 1995;

Heck et al., 2003), saltmarsh is typically viewed as being less important as it contains fewer habitat- providing creeks and pans due to its less frequent tidal inundation (Connolly et al., 1997). However, studies have shown that saltmarshes can provide important habitats for fish (Hettler, 1989; Peterson and Turner, 1994; Irlandi and Crawford, 1997; Thomas and Connolly, 2001; Mazumder et al., 2005a;

Hollingsworth and Connolly, 2006; Mazumder et al., 2006a; 2009). Thus, densities of fish are often greater within saltmarsh than adjacent mangrove vegetation within estuaries (e.g. Botany Bay, south- eastern Australia) and fish assemblages also substantially differ between these two habitats (Mazumder et al., 2005a). Fish have also been shown to move from over seagrass to saltmarsh and mangroves when those latter two habitats became available at high tides (Saintilan et al., 2007).

It is considered that fish use saltmarsh habitats as shelter from predators (Connolly et al., 1997;

Rozas and Minnello, 1998; Crinall and Hindell, 2004) and/or sources of abundant food

(Zimmerman et al., 1984; Connolly, 1994). The denseness of the vegetation is considered to hinder predation by both avian and piscine predators (Rozas and Odum, 1988). Within Australia, certain fish species, including representatives of the Ambassidae (Ambassis jacksoniensis), Sparidae

(Acanthropagrus australis and Rhabdosargus sarba), Gobiidae (Redigobius macrostoma and

Mugilogobius platynotus) and Tetraodontidae (Tetracenos hamiltoni), feed on invertebrate prey (which is abundant within saltmarsh habitats) during times when it is inundated (Mazumder, 2004; Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; Platell and Freewater, 2009; McPhee et al., 2015).

The wide range of sampling techniques employed to sample fishes in saltmarshes, which includes fyke, pop and seine nets (e.g. Connolly, 1999; Mazumder et al. 2005a; 2005b; 2006a; 2006b;

Platell and Freewater, 2009; McPhee et al., 2015), has the potential to confound any comparisons

50 between studies. However, it is noteworthy that the fish assemblages collected by seine netting at sites that are directly adjacent to inundated saltmarshes (e.g. Gibbs, 1986; Morton et al., 1987) were very similar to those assemblages that were collected by fyke netting directly within those same inundated saltmarshes in Botany Bay (Mazumder et al., 2005b; 2006a). This implies that seine nets, which can be effectively deployed in the waters directly adjacent to saltmarshes during both flood and ebb tides, would be central to understanding whether there are any differences in the fish assemblages at different tidal states during spring tidal cycles when saltmarshes become inundated. Moreover, these net types can be easily deployed at different times of the day and during the year.

Seasonal differences in the densities of fish species and fish assemblages within saltmarshes are also commonly recorded within estuaries (Morton et al., 1987; Rountree and Able, 1993; Thomas and Connolly, 2001; Crinall and Hindell, 2004), which suggests that estuarine fishes migrate among vegetative habitats (e.g. saltmarsh, mangrove, seagrass) at different times of the year. Similarly,

Ambassis agassizi migrates from downstream to upstream habitats as a response to seasonal changes in river flow, leading to changes in its densities at any particular location (McDowall, 1996; Moffat and

Voller, 2002; Lintermans, 2007, McNeil et al., 2008). Seasonal changes in fish assemblages are typically most marked between the warmest and coolest times of the year. For example, fish assemblages in a mangrove environment in Puerto Rico demonstrated highly significant differences between rainy and dry seasons (Rooker and Dennis, 1991), while fish assemblages differed seasonally between summer/autumn and winter/spring in a south-west Australian estuary (Loneragan and Potter, 1990).

The above examples demonstrate that estuarine fish use (and may migrate among) different habitats within estuaries to fulfil various biological needs (e.g. shelter and feeding). In terms of estuarine conservation and management, a thorough understanding of the fish species (and thus assemblages) using these habitats is important. This is particularly important in the case of saltmarsh, which is likely to play an important role for at least some estuarine fish species and the areas of which have declined in recent years (Hyland and Butler, 1988; Saintilan and Williams, 1999; 2000; Chapman and Roberts,

2004). Due to the limited access of saltmarsh vegetation to fishes, it is imperative that any such study of fish assemblages at saltmarsh habitats considers any temporal variability (e.g. distinction between flood and ebb tides, times of saltmarsh inundation and season) to gain a full understanding of saltmarsh use by estuarine fishes.

The first aim of the study was to quantify the abundance, number of species and the species composition of fish assemblages within an area that is directly adjacent to a representative saltmarsh habitat and is located within the basin of a temperate estuary in south-eastern Australia (Empire Bay

Wetland, Brisbane Water Estuary, NSW), taking into account two tidal states (e.g. flood/ebb tides), presence of saltmarsh inundation and seasons. A second aim was to explore the relative abundances of two species, i.e. Pelates sexlineatus and A. jacksoniensis, that were found to be very abundant within those fish assemblages (see Results), with respect to the above factors.

It is hypothesised that: (1) There will be differences in the abundance, number of species and composition of fish assemblages at times of saltmarsh inundation and non-inundation, as well as during flood and ebb tides, which will reflect certain fish species (including A. jacksoniensis) moving into waters near saltmarsh and then ultimately onto the saltmarsh during the times of inundation and (2) The fish assemblages will differ between summer and winter, reflecting any seasonal movements by certain fish species.

3.2 Methodology

3.2.1 Study site, sampling of fish and laboratory procedures

Fish were sampled, using a 20 m long seine net, at two sites in Empire Bay Wetland (Brisbane

Water Estuary, NSW, Australia), using the full experimental design as the basis for sample collection

(see Chapter Two for the detailed description of the study site, experimental design and sampling methods).

All fish were euthanased in an ice-slurry following capture and stored frozen at -15 °C until subsequent laboratory examinations. Fish were thawed prior to being identified to species, using the taxonomic keys provided by Allen et al. (2002) and Gomon et al. (2008). The total number of individuals of each species in each sample was recorded.

3.2.2 Statistical analyses

Data for the overall fish abundance and number of species (i.e. total number of species per sample), and of the species composition of the fish assemblages, were analysed using a combination of univariate and multivariate analyses. These analyses were based on the following design: Season, 2 levels (Summer, Winter), fixed, orthogonal; Month, 2 levels (January, February, June, July), random, nested in Season; Day, six levels (1, 2, 3, 4, 5, 6; which took into account the states of saltmarsh inundation (inundated (1-3) and non-inundated (4-6)), fixed, orthogonal; Tidal state, two levels (flood, ebb), fixed, orthogonal; n = 2.

3.2.2.1 Univariate analyses

Analyses of Variance (ANOVA; based on a four-way design: Season, Month nested within

Season, Day and Tidal State, at  = 0.05) were used to determine whether there were any significant differences in the overall fish assemblage, using data for the total number of fish and the total number of fish species. Further four-factor ANOVAs were also performed on the total numbers of two fish species that were particularly abundant within the samples (i.e. Pelates sexlineatus and Ambassis jacksoniensis).

Homogeneity of variances were examined using residual plots and if data were heterogeneous, log(10) transformation was applied prior to rechecking residual plots. Where significant differences (=

0.05) were found between the levels of main fixed factors (i.e. Day), Tukey’s HSD post-hoc pairwise

53 tests were conducted to determine which levels of the factors caused a significant difference within the main effect. All univariate plots depict significant fixed factors and interactions only (i.e. variability among random or non-significant fixed factors and interactions are not displayed; see Jackson and

Brashers, 1994; Reinard, 2006; McPhee et al., 2015). Univariate analyses were conducted using the statistical package SPSS (Version 21 – SPSS, 2012).

3.2.2.2 Multivariate analyses

The species composition data for the fish assemblages (i.e. number of individuals of each fish species in each sample) were analysed using the various subroutines in the statistical package PRIMER v6 with the PERMANOVA+ add on (Clarke and Gorley, 2006; Anderson et al., 2008). The fish assemblage data were first square-root transformed and the Bray-Curtis measure used to create a similarity matrix that formed the basis of significance testing via Permutational Analyses of Variance (PERMANOVA), using Type III sums of squares (Anderson, 2001; Anderson et al. 2008). PERMANOVA was based on a four-way fully-crossed design (Season, Month nested within Season, Day and Tidal State) at  = 0.05 and, when p > 0.25 for any interactions, those interaction effects were pooled before PERMANOVAs were reanalysed (as recommended by Winer et al., 1991; see Results). A test for homogeneity of multivariate dispersions (PERMDISP) was done prior to PERMANOVA to determine whether there were any differences in dispersions within groups.

When PERMANOVA showed significant differences, pairwise post-hoc tests were carried out to determine specific patterns of differences among the levels within those factors. The above results were visually depicted using a non-metric multi-dimensional scaling (nMDS) ordination plot, derived from a ‘distances among centroids’ matrix constructed using the original Bray-Curtis similarity matrix

(see above; Clarke and Gorley, 2006).

One-way Similarity Percentages analyses (SIMPER; Clarke, 1993; Clarke and Gorley, 2006) were then used to identify the fish species that typified the fish assemblage of each a priori group and/or those that were responsible for distinguishing between the fish assemblage in each pair of groups.

3.3 Results

3.3.1 Number of fish species and individuals

A total of 7218 fish, representing 17 species and 15 different families of fish, were recorded at

Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) during 2012 (Table 3.1). Pelates sexlineatus and Ambassis jacksoniensis were the most abundant species, contributing 32.9 and 28.9% to the total catches, respectively. Mugilogobius platynotus and Rhabdosargus sarba were the next most abundant, making up 13.9 and 7.1%, respectively, of the total fish catch. The remaining 13 species each contributed no more than 4% to the total catches (Table 3.1).

Pelates sexlineatus were more abundant during winter (1866) than summer (509), and were also more abundant during ebb (1377) than flood (998) tides (Table 3.1). Ambassis jacksoniensis were similarly abundant during winter (1034) and summer (1049), and were also approximately equally abundant during ebb (969) and flood (1114) tides. However, their numbers were substantially greater during the summer flood (727) and winter ebb (647) than during summer ebb (322) and winter flood

(387; Table 3.1). When the total catch of 7218 fish was adjusted to an area, based on the 96 samples and a seine net sample area that ranged between 250 and 350m2, the mean total density of fish ranged between 0.21 and 0.30 m-2.

Table 3.1: Abundances of each fish species, recorded at flood and ebb tides in summer and winter, and the total numbers and percentage contributions of fish species to the total catches recorded at Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia), in 2012.

Number of fish Summer Winter Total Percentage Family Genus/species Common name Flood Ebb Flood Ebb Numbers Contribution Terapontidae Pelates sexlineatus Six-lined Trumpeter 245 264 753 1113 2375 32.9 Ambassidae Ambassis jacksoniensis Port Jackson Glassfish 727 322 387 647 2083 28.9 Gobidae Mugilogobius platynotus Mangrove Goby 88 119 480 319 1006 13.9 Sparidae Rhabdosargus sarba Tarwhine 218 211 39 45 513 7.1 Kyphosidae Girella tricuspidata Luderick 35 71 36 115 257 3.6 Tetrarogidae Centropogon australis Fortescue 5 20 115 106 246 3.4 Gerreidae Gerres subfasciatus Common Silverbiddy 2 7 85 150 244 3.4 Atherinidae Atherinosoma microstoma Small-mouthed Hardyhead 47 8 87 30 172 2.4 Monacanthidae Meuschenia freycineti Six-spine Leatherjacket 28 34 8 14 84 1.2 Ambassidae Ambassis marianus Estuary Glassfish 40 40 1 2 83 1.2 Tetraodontidae Tetractenos glaber Smooth Toad 0 1 17 31 49 0.7 Mugilidae Liza argentea Flat-tail Mullet 0 21 3 20 44 0.6 Hemiramphidae Hyporhamphus regularis River Garfish 1 5 3 15 24 0.3 Monacanthidae Monacanthus chinensis Fan-bellied Leatherjacket 5 6 5 7 23 0.3 Clinidae Heteroclinus perspicillatus Common Weedfish 1 1 2 5 9 0.1 Belonidae Tylosurus gavialoides Stout Longtom 1 2 0 0 3 <0.1 Enoplosidae Enoplosus armatus Old Wife 1 1 1 0 3 <0.1

Total 1444 1133 2022 2619 7218

3.3.2 Abundance and number of species of the overall fish assemblage

Four-factor ANOVA showed that fish were significantly more abundant overall during winter than in summer (p = 0.024; Tables 3.2). This reflects both the total (Table 3.1) and the mean (Fig 3.1a) catches of fish being substantially greater in winter than summer. Fish abundance also differed between Days, although inconsistently among Months, i.e. there was a significant interaction between Day and the random factor of Month (p = 0.046; Table 3.2).

ANOVA demonstrated that the fish assemblages were significantly more diverse during winter than summer (p = 0.037; Table 3.3). This difference was small, however, with samples in winter only comprising an average of just over one fish species more than the samples in summer (i.e. 6.3 and 7.6 species respectively; Fig. 3.1b).

Table 3.2: Summary of results for four-factor Analysis of Variance of the abundance of fish in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation df MS F P Season (Se) 1 1.255 39.9 0.024 Month (M) [nested in Se] 2 0.031 0.2 0.796 Day (D) 5 0.063 0.5 0.791 Tidal State (T) 1 0.014 0.3 0.620 Se x D 5 0.160 1.2 0.378 Se x T 1 0.387 9.1 0.094 D x M[Se] 10 0.134 3.1 0.046 T x M[Se] 2 0.042 1.0 0.411 D x T 5 0.055 1.3 0.356 Se x D x T 5 0.074 0.7 0.220 D x T x M[Se] 10 0.044 1.1 0.406

Residual 48 0.041

140 (a) 120

100

Mean number of fish sample Mean per of number 20

0 Summer Winter Season

9 (b) 8

2 Mean number of fish fish sample species Mean of pernumber 1

0 Summer Winter Season

Figure 3.1: Means (±SE, n=24) of the (a) abundance of fish and (b) number of fish species per sample from Empire Bay Wetland, Brisbane Water Estuary (2012), on various Days and Tidal states in each of summer and winter.

Table 3.3: Summary of results for four-factor Analysis of Variance of the number of species of fish in different Seasons, Months nested in Season, Day and Tidal state at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation df MS F p Season (Se) 1 0.157 25.4 0.037 Month (M) [nested in Se] 2 0.006 0.3 0.783 Day (D) 5 0.004 0.2 0.961 Tidal State (T) 1 0.068 6.5 0.126 Se x D 5 0.029 1.2 0.370 Se x T 1 0.066 6.2 0.130 D x M[Se] 10 0.024 2.3 0.101 T x M[Se] 2 0.011 1.0 0.391 D x T 5 0.015 1.5 0.287 Se x D x T 5 0.003 0.3 0.884 D x T x M[Se] 10 0.010 1.1 0.361

Residual 48 0.009

3.3.3 Abundance of Pelates sexlineatus and Ambassis jacksoniensis

ANOVA did not detect a significant difference in the abundances of P. sexlineatus, with only the factor of Season being close to significance (p =0.064; Table 3.4). The mean (and SE) of catches per sample for this species ranged between 0 (±0) and 115 (±38) at Empire Bay

Wetland.

The abundances of A. jacksoniensis were shown by ANOVA to differ between Days and

Tidal states inconsistently among Months (p < 0.001), i.e. there was a significant three-way interaction between Day and Tidal state and the random factor of Month (Table 3.5). The mean

(and SE) catches per sample of this ambassid ranged between 11 (±1) and 64 (±12) at Empire

Bay Wetland.

Table 3.4: Summary of results for four-factor Analysis of Variance of the abundance of Pelates sexlineatus in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation df MS F P Season (Se) 1 5.13 14.2 0.064 Month (M) [nested in Se] 2 0.36 2.1 0.381 Day (D) 5 0.19 0.7 0.657 Tidal State (T) 1 0.50 4.0 0.182 Se x D 5 0.71 2.4 0.108 Se x T 1 0.27 2.2 0.279 D x M[Se] 10 0.29 1.2 0.380 T x M[Se] 2 0.12 0.5 0.608 D x T 5 0.17 0.7 0.634 Se x D x T 5 0.28 1.2 0.388 D x T x M[Se] 10 0.24 1.1 0.417

Residual 48 0.23

Table 3.5: Summary of results for four-factor Analysis of Variance of the abundance of Ambassis jacksoniensis in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on log(10) transformed; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation df MS F P Season (Se) 1 0.01 0.2 0.703 Month (M) [nested in Se] 2 0.06 0.5 0.711 Day (D) 5 0.07 1.0 0.447 Tidal State (T) 1 0.02 0.1 0.754 Se x D 5 0.07 1.1 0.451 Se x T 1 1.17 7.4 0.113 D x M[Se] 10 0.07 0.6 0.781 T x M[Se] 2 0.16 1.4 0.285 D x T 5 0.05 0.4 0.833 Se x D x T 5 0.03 0.2 0.936 D x T x M[Se] 10 0.11 4.8 <0.001

Residual 48 0.02

3.3.4 Comparisons of fish assemblages among seasons, tidal state, month and day

Although PERMANOVA showed that fish assemblages varied between Tidal states

(flood and ebb tides), the pattern of this difference varied between summer and winter (i.e. a two-way interaction between Season and Tidal state was significant; p = 0.001; Table 3.6).

Pairwise tests showed that the fish assemblages significantly differed between the flood and ebb Tidal states in both summer (p = 0.002) and winter (p = 0.013). Pairwise tests also showed that the fish assemblages did not significantly differ between summer and winter during either flood (p = 0.336) or ebb Tidal states (p = 0.343). The patterns of differences in the fish assemblages among Days were inconsistent at different Months, as shown by an interaction between Day and the random factor of Month (Table 3.6).

Table 3.6: Summary of results for four-factor PERMANOVA of the species composition of the fish assemblages in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on log(10) transformed data; (df) degrees of freedom, (MS) mean squares, (Pseudo-F) pseudo-F ratio test statistic, (P(perm)) permutation significance.

Source of variation df MS Pseudo-F P(perm) Season (Se) 1 22825.0 18.4 0.353 Day (D) 5 757.1 0.9 0.642 Tidal State (T) 1 1383.5 2.3 0.029 Month (M) [nested in Se] 2 1240.5 2.1 0.024 Se x D 5 1057.0 1.2 0.291 Se x T 1 2502.4 4.3 0.001 D x T 5 894.3 1.2 0.241 M[Se] x D 10 874.7 1.5 0.012 Pooled: M[Se] x D x T + Se x D x T 15 720.3 1.2 0.102 Pooled: Res + M[Se]xT 50 582.3

On the nMDS ordination plot, the points for the fish assemblages during summer formed a distinct group, separate from those for the fish assemblages in winter (Fig. 3.2). There is a separation between flood and ebb Tidal state points within each Season on that plot

(Fig. 3.2). PERMDISP showed no differences in multivariate dispersion (F = 2.87; p = 0.18), so all patterns could be attributed to the differences in assemblage composition.

Figure 3.1: nMDS plot, derived from a “distance among centroids” matrix of different Seasons and Tidal states, which was constructed from a Bray-Curtis similarity matrix using fish species recorded at Empire Bay Wetland in 2012. Symbols represent the combined factor Season x Tidal State.

SIMPER showed that three species (A. jacksoniensis, P. sexlineatus and Mugilogobius platynotus) typified fish assemblages across all Tidal states and Seasons (Table 3.7). SIMPER was then used to determine which species help explain the overall difference between the two

Seasons (within each Tidal state) and two Tidal states (within each Seasons), before identifying

62 those species that show different patterns of abundance between the two Tidal states in summer and winter. The seasonal differences in the abundances of A. jacksoniensis varied with

Tidal state, with this ambassid being relatively more abundant in the flood tides in summer than winter, while it was relatively more abundant in the ebb tides of winter than summer.

Table 3.7: SIMPER summary: Fish taxa that typify (shaded) and/or distinguish (unshaded) fish assemblages at Empire Bay Wetland in 2012. The ‘*’ denotes that the relative contribution of that fish taxa is greater for the time represented in the vertical column than in the horizontal row, while no ‘*’ denotes that the opposite is true. ‘NA’ denotes that no fish taxa distinguished the fish assemblages.

Summer Flood Summer Ebb Winter Flood Winter Ebb Summer A. jacksoniensis Flood R. sarba P. sexlineatus M. platynotus

Summer A. jacksoniensis* A. jacksoniensis Ebb G. tricuspidata P. sexlineatus A. marianus R. sarba M. platynotus

Winter P. sexlineatus NA A. jacksoniensis Flood M. platynotus M. platynotus R. sarba* P. sexlineatus A. jacksoniensis* C. australis C. australis

Winter NA P. sexlineatus P. sexlineatus P. sexlineatus Ebb M. platynotus G. tricuspidata A. jacksoniensis R. sarba* A. jacksoniensis M. platynotus C. australis A. microstoma* C. australis A. jacksoniensis

For both flood and ebb tides, relatively greater catches of P. sexlineatus, M. platynotus and Centropogon australis distinguished the catches in winter from those in summer, while the opposite was true for R. sarba, i.e. it was relatively more important in summer than winter

(Table 3.7).

SIMPER also showed that Girella tricuspidata was present in relatively greater amounts in the ebb than flood tides in both seasons (Table 3.7). Ambassis jacksoniensis was relatively more important in the flood than ebb tide assemblages during only summer, while its congener

(Ambassis marianus) was relatively more important in the ebb than flood tide in this season. In winter, A. jacksoniensis and P. sexlineatus were both relatively more important in the ebb than flood tides (Table 3.7).

3.4 Discussion

This study has provided a detailed description of the fish assemblages that are located directly adjacent to a representative and relatively unmodified saltmarsh habitat (Empire Bay

Wetland) in a large and permanently open estuary (Brisbane Water Estuary) in south-eastern

Australia. Contrary to the hypothesis of the study, these fish assemblages showed no overall response to the infrequent inundation of this saltmarsh habitat during two times of year

(summer and winter). Thus, neither the overall fish abundance nor diversity of this fish assemblage showed a marked response to saltmarsh inundation (Days 1-3 vs 4-6), which implies that this saltmarsh is of limited importance in broadly structuring the fish assemblages in waters that are directly adjacent to this relatively unmodified saltmarsh. However, it should be recognised that, even though there was not an assemblage-wide response, it was evident that the catches of the second most abundant species, Ambassis jacksoniensis, were at least partly related to both inundation (i.e. days within the lunar cycle) and tidal state (i.e. flood and ebb tides). These broad trends will now be discussed in more detail.

3.4.1 Total abundances and number of species of the fish assemblage

The 7218 individuals that were recorded in the present study, when adjusted to a unit area using the likely dimensions of the seine net haul, ranged between i.e. 0.21 and 0.30 m-2, and were thus lower than in comparable studies of other Australian saltmarshes habitats, with

Thomas and Connolly (2001) and Mazumder et al. (2005a) reporting densities of up to 0.46 and

0.56 m-2, respectively. However, these differences are more likely to reflect differences in sampling techniques than a real difference between the saltmarshes in those studies. Thus, the present study used seine nets, which enabled a precise comparison to be made of fish assemblages both before (flood) and after (ebb) saltmarsh inundation, while the other two studies used buoyant pop nets that captured fish that were directly over the saltmarsh. In this context, the lower densities in the present study are consistent with the relatively lower abundances of fish in seine than pop nets that were recorded in the same vegetated habitats

(seagrass) in the Port River Estuary, South Australia (Connolly, 1994a; see also Rozas and

Minello, 1997).

It should be recognised that although the densities of fish are relatively low, the number of species was similar in the present study compared to other Australian saltmarshes.

Thus, the 17 species recorded at Empire Bay Wetland was only slightly less than the 23 species recorded for a subtropical Australian saltmarsh (Thomas and Connolly, 2001), and was also comparable to other temperate saltmarshes in Australia (e.g. Gibbs, 1986; Connolly et al.,

1997; Mazumder et al., 2005a), where 11, 2 species and 16 species were found respectively.

Similarly, in two Californian estuaries, eight and four fish species were captured from tidally inundated saltmarshes (Desmond et al., 2000), and highlights the low diversity and high dominance by a few species that is common to estuaries in the area (Horn and Allen, 1985), elsewhere in the Northern Hemisphere (Subrahmanyam et al., 1976; Shenker and Dean, 1979;

Talbot and Able, 1984; Cattrijsse et al., 1994; Kneib and Wagner, 1994) and in Australia (Gibbs,

1986; Morton et al. 1987; Davis, 1988; Thomas and Connolly, 2001).

The suite of fish species recorded at Empire Bay Wetland was relatively restricted, with over three quarters of the overall fish catches being dominated by only three species, i.e.

Pelates sexlineatus (Terapontidae), Ambassis jacksoniensis (Ambassidae) and Mugilogobius playynotus (Gobiidae). The first of these species is known to be abundant in the study region of Brisbane Water Estuary (Sanchez‐Jerez et al., 2002; Alderson et al., 2013), but has not previously been suggested to be associated with saltmarsh (see Mazumder et al., 2005a;

2005b; 2006b; Alderson, 2014). It is more likely that this population of terapontid (which consisted of relatively small individuals that are likely to represent juveniles), occur over the seagrass meadows that are adjacent to saltmarsh at Empire Bay Wetland (Sanchez‐Jerez et al.,

2002), and are unlikely to leave these habitats to enter saltmarsh. Mugilogobious platynotus and A. jacksoniensis have been previously found at saltmarsh locations within Brisbane Water

Estuary (see Alderson, 2014), however the latter species contribution to the total catches in the present study was lower than in the more modified environment of Towra Bay, i.e. 28.9 vs

43.0%, respectively (Mazumder et al., 2005a). Such a difference is mostly likely due to the much greater catches of P. sexlineatus and M. platynotus in the present study, which were respectively non-representative (i.e. not captured) and low in abundance at Towra Point

(Mazumder et al., 2005a). Further, the less abundant, but economically important, small-sized

Girella tricuspidata (Kyphosidae) and Liza argentea (Mugilidae) captured in the present study have also been previously sampled at saltmarsh locations within Brisbane Water Estuary

(Alderson, 2014), while small-sized Rhabdosargus sarba (Sparidae) have been found in saltmarshes (Mazumder et al., 2005a) and seagrass habitats in NSW (Gray, McElligott and

Chick, 1996).

3.4.2 Temporal differences in fish abundance and number of species

Fish abundance and number of species (i.e. total number of fish and total number of fish species per sample) were greater in the nightly winter catches than daily summer catches, regardless of whether saltmarsh vegetation was inundated or not (i.e. irrespective of day). This seasonal difference contrasts with the low winter catches of fish species that were recorded adjacent to saltmarsh in a nearby estuary (Mazumder et al., 2005a). Furthermore, Laegdsgaard and Johnson (1995) found fish abundance and species richness to be greatest during summer and lowest during winter, while other studies also found fish abundances to be highest during summer (Robertson and Duke, 1987; Laegdsgaard and Johnson, 1995). The night-time winter, over day-time summer, dominance of fish abundance and number of species at Empire Bay

Wetland may be explained by high abundances of saltmarsh derived zooplankton (e.g. crab zoeae) known to be important in the diets of estuarine fish during such times (see Chapters

Five and Six; Hollingsworth and Connolly, 2006; Mazumder et al., 2006a). The above differences indicate variability, and non-generality, among estuaries in the seasonality of fish abundance and number of species in saltmarsh areas.

The catches of the highly abundant A. jacksoniensis demonstrated a significant interaction with Day (i.e. state of inundation), Tidal state (i.e. flood and ebb) and with the random factor of Month. Although interpretations of significant differences and interactions with random factors can be unreliable (see Jackson and Brashers, 1994; Reinard, 2006; McPhee et al., 2015), the implication that there is a difference in the abundances of A. jacksoniensis with respect to Tidal state and saltmarsh inundation is supported by Saintilan et al. (2007), who demonstrated that the catches of fish and this ambassid decreased in seagrass habitats during times of saltmarsh inundation. Further, although the highly abundant P. sexlineatus were found in similar numbers across all factors, P. sexlineatus are a relatively small-sized fish known to be

67 highly abundant within estuaries, particularly within seagrass vegetation (e.g. Connolly, 1994b;

Gray, McElligott and Chick, 1996; Sanchez‐Jerez et al., 2002).

The similarity demonstrated by the total fish abundances and number of species of fishes with respect to tidal state and day of the spring tidal cycle implies that the fish assemblages overall are not showing a marked response to the inundation of saltmarsh at

Empire Bay Wetland. This lack of a pattern between times of saltmarsh inundation and non- inundation may indicate that the proximity to saltmarsh has no influence on the fish assemblages overall. In this context, however, it is noteworthy that some species do show a stronger “response” to inundation of saltmarsh (see Section 3.4.3). It may also be that, due to the extensive tidal mixing at the study site, there is a general reliance of fishes on these habitats

(e.g. for shelter and/or feeding), regardless of the tidal height and its effect on saltmarsh inundation (i.e. days) at Empire Bay Wetland.

3.4.3 Temporal differences in the species composition of fish assemblages

Although the fish assemblages overall did not differ with respect to tidal state or day

(and thus saltmarsh inundation) at Empire Bay Wetland, the species composition of these assemblages did differ between flood and ebb tides, and to different extents during both the summer and winter. This difference between fish assemblages with tidal state was also recorded by Wilson and Sheaves (2001) in a sandy estuarine habitat of Ross River, North

Queensland, Australia, who suggested that omnivorous fishes may move into intertidal estuarine areas on flooding tides to prey on available intertidal organisms.

In the present study, one species (Girella tricuspidata) was always more abundant on the ebb than flood tides, implying that this fish may migrate closer to the saltmarsh and thus capitalise on either the habitat or feeding resources provided by Empire Bay Wetland. Juvenile

G. tricuspidata in nearby Botany Bay have been shown to have strong habitat preferences for

68 inundated mangrove vegetation after initial settlement phases in seagrass (Bell et al., 1984), while other research using buoyant pop nets in the same estuary indicate movement of this species among seagrass, mangrove and saltmarsh habitats (Saintilan et al., 2007). Girella tricuspidata may also be found in higher abundances during ebb than flood tides as a result of migration for feeding opportunities upon newly available inundated vegetation (e.g. algae;

Anderson, 1986; Raubenheimer, 2005) or exported prey (Raubenheimer, 2005).

Ambassis jacksoniensis distinguished assemblages, both seasonally and tidally, by being more abundant in summer flood vs ebb and winter ebb vs flood assemblages. This is consistent with the seasonal differences in abundances of another species of ambassid

(Ambassis agassizi; McNeil et al., 2008). In the case of A. jacksoniensis being more abundant in the winter ebb than flood tides, the former is the time when this species has been recorded to extensively feed on saltmarsh-derived zooplankton (Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; McPhee et al., 2015), owing to mass zoeal release by a few species of crabs

(e.g. Helograpsus haswellianus) at this time (Mazumder and Saintilan, 2003; Mazumder et al.

2006a; 2008; Mazumder and Saintilan, 2010; Alderson et al., 2013). This possibility is also explored in Chapters Five and Six of this thesis. It is also relevant that, within these same catches, P. sexlineatus also showed the same temporal trend as A. jacksoniensis, suggesting that there may be a common supporting mechanism, such as both of these fish species feeding on crab zoeae. In this respect, it is therefore relevant that the juveniles of P. sexlineatus feed extensively on seagrass-associated zooplankton within Brisbane Water Estuary (Sanchez‐Jerez et al., 2002), and thus may also be able to feed on crab zoeae. Interestingly, the closely-related ambassid, A. marianus, although far less abundant than A. jacksoniensis, was also relatively more important in the ebb than flood tide in summer and this pattern may also be linked to feeding opportunities by such ambassids upon prey derived from recently tidally-inundated vegetation (Pollard, 1974; Lake, 1978; Sanderson, 1979; Martin and Blaber, 1983; Milton and

Arthington, 1985; Coates, 1990; Bishop et al., 2001; Medeiros, 2004; Mazumder et al., 2006a;

Hollingsworth and Connolly, 2006; Lintermans, 2007; McPhee et al., 2015).

The overall seasonal differences within the fish assemblages were related to three species (P. sexlineatus, M. platynotus and C. australis) being more abundant in the winter than summer, in both flood and ebb tides, while the recreationally important R. sarba was more abundant in the summer than winter in both tidal states. This is consistent with the seasonal differences that have been previously recorded for the species composition of saltmarsh fish assemblages worldwide (e.g. Morton et al., 1987; Rountree and Able, 1993; Thomas and

Connolly, 2001; Crinall and Hindell, 2004). Other than for feeding-related reasons (see earlier and Chapter Six), such seasonal differences in fish assemblages may also be related to environmental variations (e.g. salinity, dissolved oxygen; see Gelwick et al., 2001; Akin et al.,

2005) or reproduction and recruitment/migration effects (see Rountree and Able, 1993;

Ribeiro et al., 2006). It is however noteworthy that salinity and dissolved oxygen levels at

Empire Bay Wetland were similar between summer and winter sampling occasions (see

Chapter Two). Such lack of environmental variation may rule out the possibility that the seasonal differences in estuarine fish assemblages found in the current study were the result of environmental variations (see Gelwick et al., 2001; Akin et al., 2005), which therefore strengthens the possibility of the feeding related reason proposed earlier. Further, as small fish are known to consume more food at times of higher than lower temperatures (Fonds et al.,

1992), it should be noted that water temperatures recorded at Empire Bay Wetland were higher during summer than winter (see Chapter Two), which may also explain the increased abundances of A. jacksoniensis found during summer (see also Chapter Six).

3.4.4 Conclusion

The present study, which used seine nets in waters directly adjacent to saltmarshes at

Empire Bay Wetland, Brisbane Water Estuary, recorded lower fish densities than other comparable Australian studies, which is most likely to be attributed to the choice of sampling equipment. Although this choice may therefore seem sub-optimal, it is relevant that this sampling method can be deployed in those waters in both tidal states and with adequate temporal replication, unlike the buoyant pop nets or fyke nets used in other studies (Rozas and

Minello, 1997; Thomas and Connolly, 2001; Mazumder et al., 2005a; 2005b).

The abundance of fish (particularly A. jacksoniensis) and the fish diversity was greater in nightly winter catches than daily summer catches, regardless of the effect of saltmarsh inundation. This is relevant as zooplankton (such as crab zoeae) exported from ebb tides following saltmarsh inundation has previously been shown to be greater during nightly winter inundation events than at other times (see Chapter Five). In turn, such zooplankton can represent important food sources for the itinerant fish that enter saltmarsh (including A. jacksoniensis). These important feeding times may explain higher abundances of fish during nightly winter ebb tides in the present study. However, as different trends have also been reported in other studies, it should be noted that variability in the seasonality of fish abundance and diversity in saltmarsh areas does occur among estuaries, reflecting environmental differences among those estuaries.

Saltmarsh inundation essentially had no effect on fish assemblages overall, in terms of their abundance or diversity, although there was some indication that the abundance of

A. jacksoniensis was related to tidal state (which was inconsistent between seasons). This study is a reminder of the potential contribution of saltmarsh habitats to the life history needs of estuarine fish (such as A. jacksoniensis), including shelter (both for juvenile and adult fish) and sources of food and foraging areas. These elements that saltmarsh habitats provide for

71 estuarine fish are investigated in later chapters using the glassfish, A. jacksoniensis, and its predation on saltmarsh-derived crab zoeae, as an example of ecological dependence of estuarine fish on saltmarsh vegetation.

CHAPTER FOUR

KEY FEATURES OF THE LIFE HISTORY OF GLASSFISH,

Ambassis jacksoniensis

4.1 Introduction

The timing of fish reproductive events, which is often restricted to relatively short periods, such as within one season during a year, is important for understanding the life history and ecological functioning of fish populations (Copp and Peňáz, 1988). By determining when fish reproduce, linkages can be made to their behaviours at other times (e.g. during times of feeding or migration). For example, Atlantic silverside (Menidia menidia) prioritise spawning occasions to times that least interfere with feeding times (Conover and Kynard, 1984).

Alternatively, fish spawning times may also occur during those times when predation is relatively low (Thresher, 1984), for example when egg-predators are inactive or likely to be satiated by prior feeding (Robertson, 1991). Spawning times in some fishes may also be influenced by, and coincide with, conditions favourable to the parent (Robertson et al., 1990) or offspring growth and survival (e.g. when food is abundant or when predation pressures are low; Ochi, 1986). As an organism’s habitat provides reproductive services (for example food, which provides energy for reproduction; Mann, 1965), insight into the reproductive requirements of an organism is important when considering the ecological resources (e.g. food or shelter) necessary for populations to maintain function and health within an ecosystem.

Reproductive cycles in some fishes may not be seasonally dependent, but may occur more, or less, periodically as a result of other influences. For example, mating of some fishes may be influenced by moon/tidal phases (e.g. the iteroparous Amphiprion melanopus; Ross,

1978) or time of day (e.g. in nocturnal spawners), while other fishes have more complicated reproductive cycles governed by many temporal influences (e.g. daily, monthly and yearly spawnings; Milton and Arthington, 1985; Coates, 1990; Bishop et al., 2001).

Ambassis jacksoniensis, which is a small, schooling (Hurst et al., 2005), relatively short- lived ambassid, is one of the most abundant fish species occurring within estuaries of south- eastern Australia. It is thought to reach sexual maturity within one year (Mazumder et al.,

2009). It has been suggested, based on the high abundances of the larvae of A. jacksoniensis within the channel of a large south-eastern Australian estuary (Lake Macquarie, NSW) and low larval abundances elsewhere in this estuary, that A. jacksoniensis are nearshore spawners in waters that are essentially marine (Miskiewicz, 1987). As this species is known to be abundant in vegetated habitats (e.g. saltmarsh, mangroves and seagrass) within the basins of estuaries in south-eastern Australia (Gray and McElligot, 1996; Mazumder, 2004; Hollingsworth and

Connolly 2006; Mazumder et al., 2006a; McPhee et al., 2015) this may imply that A. jacksoniensis may migrate to estuary mouths to spawn.

Based on the consistently high larval abundances of A. jacksoniensis during the year in the entrance channel of Lake Macquarie (see above), Miskiewicz (1987) also concluded that this ambassid reproduces throughout the year, albeit with a decrease in winter. It is possible that this may be related to the fact that A. jacksoniensis are known to feed on the highly abundant saltmarsh-derived crab zoeae that are particularly abundant during winter in south- eastern Australian estuaries (Hollingsworth and Connolly, 2006; Mazumder et al., 2006a;

McPhee et al., 2015).

Patterns in the location and timing of spawning are known to vary both within and among different species of the Ambassidae. For example, intraspecific differences were observed for Ambassis agrammus, in which the gonadal development of a population in the

Australian Northern Territory coincided with wet seasons (Bishop et al., 2001) while, in a Papua

New Guinean population just to the north and with a similar climate, non-seasonal breeding was observed (Roberts, 1978). Alternatively, for the freshwater Ambassis macleayi and

Ambassis interrupta, both the gonadosomatic indices and gonadal maturity stages remained

75 relatively stable throughout a year, indicative of constant spawning (Coates, 1990; Bishop et al., 2001). Furthermore, Milton and Arthington (1985) demonstrated freshwater Ambassis agassiz to be seasonal breeders influenced by seasonal rising in water temperatures (see also

Cadwallader and Backhouse, 1983; Leggett, 1984; Llewellyn, 2008; McNeil et al., 2008). There is presently no direct information on the spawning strategies (i.e. location and timing of spawning) of any estuarine ambassids, including the highly abundant Ambassis jacksoniensis.

In order to help elucidate the likely role of saltmarsh habitats in the life history of

A. jacksoniensis that are found in waters directly adjacent to saltmarshes, this study aims to obtain data on selected aspects of the reproductive biology and ecology of this abundant ambassid within the waters of Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) at two contrasting times of the year (summer and winter). It is hypothesised that this ambassid will undergo seasonal spawning within this temperate environment, which will be reflected in

(1) differences in the proportions of sexual maturity stages of both female and male A. jacksoniensis between summer and winter; (2) differences in the amount of somatic energy invested in gonadal development by both females and males in summer and winter; and (3) differences in sex ratios between summer and winter.

4.2 Methodology

4.2.1 Study site and sampling of Ambassis jacksoniensis for selected aspects of their reproductive biology

Individuals of Ambassis jacksoniensis (as part of the overall fish assemblages; see

Chapter Three) were sampled, using a 20 m long seine net, at two sites in Empire Bay Wetland

(Brisbane Water Estuary, NSW, Australia), using the full experimental design as the basis for

76 sample collection (see Chapter Two for the detailed description of the study site, experimental design and sampling methods).

Fish were euthanased in an ice-slurry following capture and stored frozen at -15 °C until subsequent laboratory examinations. Fish were thawed prior to identification, with ten

A. jacksonsiensis being haphazardly removed from each seine net haul and set aside for subsequent analyses of selected aspects of their reproductive biology. Individuals were then preserved in 10% formaldehyde for 24-48 h before transferring to 70% ethanol for further preservation for at least a week prior to examination. For each individual, the standard length

(SL) was measured to the nearest 0.1 mm and its wet weight determined to the nearest

0.0001 g.

4.2.2 Laboratory procedures

Each A. jacksoniensis was dissected, their gonads macroscopically sexed (as female, male or unsexable) and then removed using fine forceps. Using a dissecting microscope, a sexual maturity stage was then assigned to each gonad using the following stages: I (juvenile),

II (inactive), III (maturing), IV (ripe) and V (spent; see Blackburn, 1950; Beumer, 1979; Milton and Arthington, 1985). As stages I and II were essentially indistinguishable for A. jacksoniensis, they were combined into one category for statistical analyses (i.e. four stages were used – see below). Gonads were weighed to the nearest 0.0001 g.

Gonads of randomly-selected A. jacksoniensis from each macroscopically-assigned sexual maturity stage of each sex were sent to an external laboratory (Murdoch University) where the mid-regions of each gonad were embedded in paraffin wax, sectioned (5 μm) and stained (haemotoxylin and eosin) before being prepared into microscope slides for microscopic histological validation of macroscopically-assigned sexing and identification of sexual maturity stages using a compound microscope.

4.2.3 Statistical analyses

Sizes (standard length; SL) at which 50% of male and female A. jacksoniensis had reached sexual maturity (i.e. a sexual maturity stage of at least III) were determined using gonadal stages in order to determine the median size of fish that are capable of contributing offspring to the population through reproduction.

Sex ratios and sexual maturity stages in A. jacksoniensis populations at Empire Bay

Wetland during times of saltmarsh inundation (i.e. potential “important feeding times”) and saltmarsh non-inundation were explored using log linear analyses. These log-linear analyses (

= 0.05) used the following designs:

Sex ratio:

Season (2 levels – summer, winter); Day (6 levels - 1, 2, 3, 4, 5, 6; which took into

account states of saltmarsh inundation (inundated (1-3) and non-inundated (4-6)));

Tidal State (2 levels – flood, ebb); Sex (2 levels – female, male).

Sexual maturity stages:

Season (2 levels – summer, winter); Day (6 levels - 1, 2, 3, 4, 5, 6; which took into

account states of saltmarsh inundation (inundated (1-3) and non-inundated (4-6)));

Tidal State (2 levels – flood, ebb); Sexual maturity stage (4 levels – I/II, III, IV, V).

Univariate analyses were conducted using the statistical package IBM SPSS (Version

21 – SPSS, 2012).

Measures of somatic condition were calculated using somatic weight-SL geometric mean linear regression (Model II) equations for female and male A. jacksoniensis separately using log(10) transformed data, from which residuals were obtained for each fish (see Pecl and

Moltschaniwskyj, 2006). Residuals are the difference between a fish’s observed weight and the

78 weight that is predicted by the regression equation. Residuals were standardised by dividing each value by the standard deviation of the predicted values. Thus, a fish that is heavier for its length than predicted by the regression equation (i.e. a positive residual) can be considered as being in better somatic condition than one that is lighter for its length than predicted (i.e. a negative residual). Residuals were also calculated from reproductive (i.e. gonad) weight-SL regressions for female and male fish separately.

Associations between the somatic and reproductive conditions were then tested for each sex by correlating residuals from somatic weight-SL and reproductive weight-SL regressions against each other (see Pecl and Moltschaniwskyj, 2006). Regression analyses were conducted on sexually-mature fish only and also excluded ‘spent’ fish (i.e. stage V), as their gonad weights were low (close to zero). Therefore, regressions were conducted on

A. jacksoniensis that were maturing or ripe (i.e. stages III and IV).

4.3 Results

4.3.1 Sex ratio and proportion of the different sexes of Ambassis jacksoniensis

Of the 960 Ambassis jacksoniensis that were sampled at Empire Bay Wetland, females contributed 678 (~70.6%) to that total, of which 417 (~43.4%) and 261 (~27.2%) were sampled in summer and winter, respectively. Males contributed a total of 282 (~29.4%), of which 63

(~6.6%) and 219 (~22.8%) were sampled in summer and winter, respectively (Table 4.1).

Table 4.1: Proportion of different sexes of samples of Ambassis jacksoniensis from Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in the summer and winter of 2012.

Season Female Male Summer 0.43 0.07 Winter 0.27 0.23

Sex ratios of A. jacksoniensis were shown to not be consistent between the summer and winter (2 = 22.498, d.f. = 10, p = 0.013). This was attributed to counts of females being higher than expected (under the statistical null hypothesis) during summer and lower than expected during winter, while the opposite was found for males (Fig. 4.1).

450  400 350

300  250 

Count 200 Female 150 Male

100  50 0 Summer Winter Season

Figure 4.1: Summary of log-linear analysis showing observed and expected counts of Ambassis jacksoniensis sampled at Empire Bay Wetland, during summer and winter of 2012. “Up” arrows (↑) indicate that counts of such fish sex are statistically higher than expected for that Season, while “down” arrows (↓) indicate that counts of such fish sex are statistically lower than expected for that Season.

4.3.2 Sexual maturity stages of Ambassis jacksoniensis

The size (SL) at which 50% (median) of female and male A. jacksoniensis had reached sexual maturity (i.e. the minimum size at which 50% of the sampled A. jacksoniensis had a sexual maturity stage of at least III) was 38 mm (Fig. 4.2). Of the 678 female A. jacksoniensis sampled in summer and winter, a total of 29 and 56 had sexual maturity stages of I and II

(juvenile and inactive), while 129 and 15 were stage III (maturing), 259 and 2 were stage IV

(ripe), and 0 and 188 were stage V (spent), during summer and winter respectively (Table 4.2).

Of the 282 male A. jacksoniensis sampled in summer and winter, a total of 15 and 23 had sexual maturity stages of I and II (juvenile and inactive), while 23 and 27 were stage III (maturing), 24 and 3 were stage IV (ripe), and 1 and 166 were stage V (spent), during summer and winter respectively (Table 4.2).

0.9 0.8

0.7 0.6

0.5 0.4

Proportionmature 0.3 0.2 0.1

0 29 34 39 44 49 54 Size (SL) of A. jacksoniensis (mm)

Figure 4.2: The size (SL) at which 50% (median) of A. jacksoniensis had reached sexual maturity.

Table 4.2: Proportion of sexual maturity stages of samples of Ambassis jacksoniensis from Empire Bay Wetland in the summer and winter of 2012.

I – II III IV V Sex Season (Juveniles/Inactive) (Maturing) (Ripe) (Spent) Male Summer 0.05 0.08 0.09 0.00 Winter 0.08 0.10 0.01 0.59 Female Summer 0.04 0.19 0.38 0.00 Winter 0.08 0.02 0.00 0.28

The proportion of female sexual maturity stages were not consistent between summer and winter seasons (2 = 186.862, d.f. = 3, p < 0.001; Fig. 4.3a), which was attributed to proportions of stage I-II and V (juveniles/inactive and spent) fish being lower than expected during summer and higher than expected during winter, while stage III and IV (maturing and ripe) fish were higher than expected during summer and lower than expected during winter.

Similarly, the proportions of male sexual maturity stages were also inconsistent between seasons (2 = 21.676, d.f. = 3, p < 0.001; Fig. 4.3b), where proportions of stage I-II, III and IV

(juveniles/inactive, maturing and ripe) fish being higher than expected during summer and stage IV (ripe) fish being lower than expected during winter. Reciprocally, proportions of male stage V (spent) fish were lower than expected during summer and higher than expected during winter (Fig. 4.3b).

In general, high proportions of stage III and IV (maturing and ripe) A. jacksoniensis were found during summer for both sexes, while high proportions of stage I-II and V

(juveniles/inactive and spent) A. jacksoniensis were found during winter for both sexes.

300 (a)  250

200 

150 

Count Summer 100 Winter  50     0 I - II III IV V Sexual maturity stage

300 (b) 250

200  150

Count Summer 100 Winter

50      0 I - II III IV V Sexual maturity stage

Figure 4.3: Summary of log-linear analysis showing expected and observed sexual maturity stages of (a) female and (b) male Ambassis jacksoniensis sampled at Empire Bay Wetland in summer and winter of 2012. “Up” arrows (↑) indicate that counts of such sexual maturity stage are statistically higher than expected for that Season, while “down” arrows (↓) indicate that counts of such sexual maturity stage are statistically lower than expected for that Season.

4.3.3 Somatic and reproductive condition of Ambassis jacksoniensis

The increase in total reproductive weight with length (SL) for A. jacksoniensis was approximately 18% faster in females than males (as shown by the respective slope for female and male reproductive weight; Table 4.3). Most of the variability in reproductive weight was

83 explained by fish length (SL; ~86% and ~84% for males and females respectively; Table 4.3).

Females demonstrated an increase in reproductive weight with SL that was 24% faster than that of somatic weight with SL, while in males, this increase was 21% (Table 4.3).

Table 4.3: Summary of Model II linear regression statistics for log somatic and log reproductive weight vs. log SL relationships for mature female and male Ambassis jacksoniensis sampled at Empire Bay Wetland in summer and winter of 2012

Relationship n Slope 95% C.I. of slope Intercept r2 Female Somatic weight 405 2.411 2.296 - 2.525 -3.788 0.809 Reproductive weight 405 2.978 2.848 - 3.107 -5.784 0.836 Male Somatic weight 76 2.088 1.842 - 2.335 -3.303 0.794 Reproductive weight 76 2.518 2.284 - 2.751 -5.263 0.862

There was considerable variation among both male and female individuals in the relationship between somatic weight-SL residuals and reproductive weight-SL residuals

(Fig. 4.4 a;b). For female A. jacksoniensis, there was a positive correlation between somatic weight-SL residuals and reproductive weight-SL residuals (r = 0.36, n = 405, p < 0.0001), i.e. females in greater somatic condition were in better reproductive condition (Fig. 4.4a).

However, no association between somatic weight-SL residuals and reproductive weight-SL residuals were found for male A. jacksoniensis (r = -0.07, n = 76, p = 0.5652), i.e. males in greater somatic condition were not necessarily in greater reproductive condition (Fig. 4.4b).

8 (a) 6

SL SL residuals 2 -

0 -5 -3 -1 1 3 5 -2

-4

Reproductive weight Reproductive -6

-8 Somatic weight-SL residuals

8 (b) 6

SL SL residuals 2 -

0 -5 -3 -1 1 3 5 -2

-4

Reproductive weight Reproductive -6

-8 Somatic weight-SL residuals

Figure 4.4: Residual values for mature (a) female and (b) male Ambassis jacksoniensis individuals, that were sampled at Empire Bay Wetland in summer and winter of 2012, from the somatic weight-SL and reproductive weight-SL relationships.

4.4 Discussion

4.4.1 Sex ratios of Ambassis jacksoniensis

Within Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia), the sex ratio of

A. jacksoniensis was heavily biased towards females (i.e. approximately a 7:3 female to male ratio). Although this overall ratio was typical of the summer months, January and February, the ratio equalised to an approximate 1:1 during winter months, June and July, where male A. jacksoniensis contributed similar numbers to samples as females. One possible explanation for such differences during the summer is provided for by the authors of studies on two other ambassids, Parambassis siamensis in Taiwan (Chen and Kuo, 2009) and Parambassis ranga in

Bangladesh (Mortuza et al., 1996) and Japan (Ishikawa and Tachihara, 2012), where it was suggested that males were distributed in more offshore areas than females, and thus were unable to be sampled by nearshore capture methods. Alternatively, a female bias may indeed be the true sex ratio in the population of these species (as in P. ranga in southern Japan;

Ishikawa and Tachihara, 2012). However, studies on other Australian ambassids (A. agassizii;

Milton and Arthington, 1985) and Papua New Guinean (A. interrupta; Coates, 1990) found no bias in sex ratios, while examples of male-biased sex ratios that are both season and habitat dependent have also been found in Australia (A. macleayi and A. agrammus; Bishop et al.,

2001). For the latter species, localised movements within the water body, which is possibly related to reproductive cycles, may influence any bias in sex ratios (Bishop et al., 2001). Thus, sex ratio appears to be variable among ambassids both within Australia and internationally.

One aim of the study was to determine specifically which A. jacksoniensis populations

(e.g. with regards to sex ratio and sexual maturity stages) are utilising saltmarsh, and its surrounding habitats at Empire Bay Wetland. Therefore, due to the seasonal difference in saltmarsh inundation times (i.e. summer-day and winter-night inundation), it is difficult to

86 determine whether differences among populations between seasons can be attributed to seasonality, the time of sampling (i.e. light-dependence) or a combination of both. Thus, any conclusions drawn from the study must emphasise the potential influence of either or both of these factors on populations of A. jacksoniensis, as sex ratio and sexual maturity stages of A. jacksoniensis may not be entirely tidally governed due to the high mobility of these itinerant fish.

There are several potential reasons for what may be a light-dependent sex ratio in populations of A. jacksoniensis at Empire Bay Wetland (i.e. day vs night populations). Firstly, male A. jacksoniensis may be underrepresented in samples during days as they are situated at different areas to the sampling “sites” (shallow seagrass areas adjacent to mangrove/saltmarsh complexes) during the time of sampling. For example, as sampling was conducted during the day during summer, high proportions of males may be situated in deeper, less accessible portions of the water body (see Ishikawa and Tachihara, 2012) in order to avoid predation by visual predators at shallow seagrass habitats during daylight (e.g. water birds, piscivorous fish such as Acanthopagrus australis, Platycephalus fuscus and Argyrosomus japonicus; SPCC, 1981;

Taylor et al., 2006; Mazumder et al., 2006a). In another example in Panama, the loricariid,

Ancistrus spinosus, counteract predators (e.g. piscivorous birds) by avoiding areas of shallow water (Power, 1984).

Thus, during winter when saltmarsh inundation occurs at night, male A. jacksoniensis may be found in equal proportions to females at sampling locations because they feed there when they are less susceptible to visual predation. It is thus noteworthy that inundated saltmarsh during night-time winter spring tides provides an important food source for

A. jacksoniensis in another estuary (Mazumder et al., 2006a; McPhee et al., 2015).

Simultaneously, female A. jacksoniensis may risk being present at the shallower sampling locations as their requirement to feed upon saltmarsh, mangrove and/or seagrass-derived food

87 sources outweigh the risk of predation. Such increased requirement for females to feed at these times is likely governed by their increased energetic requirements for reproductive purposes (i.e. in order to achieve Stage IV sexual maturity stages; Mann, 1965).

Apart from having light-dependent sex ratios, A. jacksoniensis sex ratio may also (or alternatively) be seasonally dependent in populations at Empire Bay Wetland. For example, as

A. jacksoniensis have been shown to likely be multiple nearshore spawners throughout most of the year, that prefer more marine waters for such spawning (Miskiewicz 1987); a high proportion of male A. jacksoniensis may be migrating to other parts of the larger study region

(Brisbane Water Estuary), such as the mouth of the estuary, during spawning times (i.e. summer). The sex ratios then return to approximately 1:1 during winter, when a decrease in A. jacksoniensis spawning occurs, as found in the present study (see Section 4.5.2 below) and in

Miskiewicz (1987). Furthermore, this possible male migration of A. jacksoniensis to the estuarine mouth may also be occurring simultaneously to the theory that males are not found at shallow areas during daylight to avoid visual predators (with a return to such areas at night during winter to feed on saltmarsh-derived food sources; Mazumder et al., 2006a). It should be noted that the present study shows winter as an important time for A. jacksoniensis feeding on saltmarsh-derived crab zoeae following inundation (albeit only slightly less important than summer – see Chapter Six). Further, previous studies have also shown that winter is the most important feeding opportunity for A. jacksoniensis, as grapsid crabs release high abundances of zoeae from saltmarsh during nightly inundation as a strategy to counteract visual predators

(such as A. jacksoniensis; Morgan and Christy, 1995; Hovel and Morgan, 1997; Mazumder et al.,

2006a; Hollingsworth and Connolly, 2006). Thus, male A. jacksoniensis may return to saltmarsh locations during nightly, winter saltmarsh inundation times in order to feed, during a lull in spawning effort (Miskiewicz, 1987).

4.4.2 Ambassis jacksoniensis sexual maturity stages

Populations of A. jacksoniensis at Empire Bay Wetland generally had maturing and ripe gonads during summer months, January and February, while juvenile/inactive and spent gonads dominated populations during winter months, June and July. These findings support the conclusion that A. jacksoniensis spawn throughout summer months with a lull during winter, which is consistent with that observed previously for the same species in a slightly more northern environment (Lake Macquarie), based on the presence of their larvae within the zooplankton (Miskiewicz, 1987). Such multiple spawning by A. jacksoniensis may explain their high abundances in estuaries along the south-east coast of Australia (Saintilan, 2009) and may be linked with the important feeding opportunities provided by saltmarsh-derived crab zoeae and other zooplankton (Conover, 1984; Mazumder et al., 2006a; Hollingsworth and Connolly,

2006; McPhee et al., 2015). For instance, as A. jacksoniensis were found to feed extensively during summer saltmarsh inundation events at the study site (see Chapter Six), they are likely to be in a better condition (and thus more likely to be successful) for spawning than during winter events, when feeding was lower in general (i.e. as a result of reduced energetic gain from feeding; see Deady and Fives, 1995; Denny and Schiel, 2001).

Alternatively, Mazumder et al. (2006a) found, via stomach content analyses, that

A. jacksoniensis fed more in winter than summer during saltmarsh inundation events in nearby

Botany Bay. This seasonal feeding difference was attributed to greater availabilities of the dominant A. jacksoniensis prey, saltmarsh-derived crab zoeae, that are released in higher quantities during nightly winter saltmarsh inundation events (Morgan and Christy, 1995; Hovel and Morgan, 1997). If this seasonal feeding behaviour is the norm for A. jacksoniensis, then it is possible that such a lull in spawning during winter is a result of A. jacksoniensis prioritising feeding during these important saltmarsh inundation times in order to maximise somatic and reproductive condition for later times when spawning may take precedence over feeding

(i.e. summer; see also Conover and Kynard, 1984). This is especially relevant if A. jacksoniensis migrate to the mouth of estuaries (away from saltmarsh vegetation) in order to spawn, as previously suggested (Miskiewicz, 1987; Mills et al., 2008). It should also be noted that reproductive activity can lead to decreased feeding intensity in some fishes during spawning seasons that at other, non-spawning seasons, feed more intensely in order to accumulate fat/energy reserves for spawning times (Deady and Fives, 1995; Denny and Schiel, 2001).

Therefore, as it has been suggested that grapsid crabs are known to prioritise zoeal release for nightly winter saltmarsh inundation events (Morgan and Christy, 1995; Hovel and Morgan,

1997), it is possible that saltmarsh-derived crab zoeal release (which is ultimately governed by the influence of tidal inundation of saltmarsh; Mazumder et al., 2008) may indirectly influence the spawning behaviour, and seasonality, of A. jacksoniensis.

As feeding strategies to maximise somatic and reproductive condition for spawning is not clear-cut or general for A. jacksoniensis (as for example, Mazumder et al. (2006a) found winter to be the most important feeding season, while the present study (Chapter Six) alternatively found summer to be the most important), it is possible that other factors may explain or contribute to the spawning behaviour and seasonality of A. jacksoniensis. For example, Australian studies have linked seasonal spawning in freshwater ambassids to rising water temperatures (Milton and Arthington, 1985) and rainfall (Bishop et al., 2001), indicating that seasonality in the spawning of these ambassids also coincides with warmer seasons (as found in summer for A. jacksoniensis in the present study; NB the present study also found a marked difference in water temperatures at Empire Bay Wetland, i.e. 14-15 C̊ in winter vs 25-

26 C̊ in summer – see Chapter Two). Likewise, in Japan, the freshwater P. ranga, that have not been observed to feed in saltmarshes, are similarly near-continual spawners with a decline in winter corresponding with a decrease in water temperatures and day length (Ishikawa and

Tachihara, 2012).

4.4.3 Ambassis jacksoniensis somatic and reproductive conditions

This study showed that the female A. jacksoniensis demonstrated faster growth than males and this was also true for the increase in reproductive/gonadal weight with length (SL).

Similarly, Ishikawa and Tachihara (2012) found that lengths (and growth rates) of another ambassid, Parambassis ranga, were generally greater in females than males. This is thought to be typical for female fishes, that are known to allocate more energy for reproductive purposes

(Mann, 1965), and that also possess larger and heavier gonads (ovaries) than males (testes).

However, previous studies on growth of ambassids have shown that males of A. macleayi have faster growth and maximum lengths than females, while A. agassizii displayed no difference in growth between sexes (Milton and Arthington, 1985; Bishop et al., 2001), indicating variability in the reproductive growth among ambassids.

Female A. jacksoniensis demonstrated an increase in reproductive weight with length

(SL) faster than that of somatic weight with length (SL), and a positive association was found between somatic weight-SL residuals and reproductive weight-SL residuals for females, which indicates that female A. jacksoniensis in greater somatic condition were also in greater reproductive condition. The emphasis that A. jacksoniensis place on reproductive growth demonstrates their high spawning capabilities, which may also explain their high abundances

(Saintilan, 2009), wide distribution (Mills et al., 2008), near continual and multiple spawning

(Miskiewicz, 1987) and opportunistic lifestyle within south-eastern Australian estuaries in general (Winemiller and Rose, 1992; see also Ishikawa and Tachihara, 2012).

4.4.4 Conclusion

This study explored, for the first time, various life history characteristics of the highly abundant, A. jacksoniensis, with a focus on seasonal and tidal differences in sex ratio, sexual maturity and somatic/reproductive growth. The present study found a heavily female-biased

91 sex ratio for A. jacksoniensis at Empire Bay Wetland during summer (i.e. January and February) that equalised (to approximately 1:1) during winter (i.e. June and July), and which may be a result of light and/or seasonal influence on male A. jacksoniensis that migrate during summer to areas of the estuary that were not sampled in the study (e.g. deeper and less vulnerable waters; Power, 1984; Bishop et al., 2001; Ishikawa and Tachihara, 2012), or the estuarine mouth for spawning (Miskiewicz, 1987), before returning to Empire Bay Wetland during winter in order to feed on important saltmarsh-derived zooplankton (Mazumder et al., 2006a; McPhee et al., 2015). Simultaneously, female A. jacksoniensis may risk being present at the shallower sampling locations due to increased energetic requirements for reproductive purposes (Mann,

1965). Sex ratio appears to be variable among ambassids both within Australia and internationally, and due to potentially confounding seasonal and day/night-time inundation of south-east Australian saltmarshes, generalisations about the sex ratios of A. jacksoniensis may not be possible.

The study also found that the gonads of A. jacksoniensis at Empire Bay Wetland were generally maturing and ripe during summer (i.e. January and February), while juvenile/inactive and spent during winter months (i.e. June and July), supporting previous evidence that A. jacksoniensis spawn during summer months with a lull during winter (Miskiewicz, 1987) and potentially explaining the high abundance in south-east Australian estuaries (Saintilan, 2009).

Such reproductive timing of A. jacksoniensis may also be linked to important feeding events during saltmarsh inundation (Mazumder et al., 2006a; Hollingsworth and Connolly, 2006;

McPhee et al., 2015), whereby such feeding provides the energetic requirements for reproduction (Mann, 1965) and may ultimately indirectly influence the spawning behaviour, and seasonality, of A. jacksoniensis. However, as seasonal feeding behaviours of A. jacksoniensis appear to be inconsistent among estuaries, and studies (e.g. Mazumder et al.,

2006a vs the current study – Chapter Six), it is possible that factors unrelated to feeding (such as water temperature; Milton and Arthington, 1985; Bishop et al., 2001; Ishikawa and

Tachihara, 2012), or offspring survival and recruitment (Lambert and Ware, 1984), may alternatively, or simultaneously, be influencing the life history (e.g. reproductive cycles) of A. jacksoniensis.

Finally, the study also demonstrated that female A. jacksoniensis had faster growth and increases in reproductive/gonadal weight with length than males, as female fish allocate more energy for reproductive purposes (Mann, 1965). However, previous studies on growth of ambassids have shown conflicting trends (e.g. P. ranga (Ishikawa and Tachihara, 2012),

A. macleayi (Milton and Arthington, 1985) and A. agassizii (Bishop et al., 2001)), indicating variability and non-generality in the reproductive growth among ambassids.

In general, the present study has demonstrated the high reproductive and multiple- spawning capabilities of A. jacksoniensis (Miskiewicz, 1987), which may explain their high abundance (Saintilan, 2009) and wide distribution (Mills et al., 2008) in south-east Australian estuaries. Such life history traits have been linked to known important feeding on saltmarsh- derived zooplankton (such as crab zoeae), which likely contributes to energetic requirements for these reproductive purposes (Mann, 1965), and in turn highlights the importance of saltmarsh habitats for life cycles of estuarine fish specifically and estuarine ecology in general.

CHAPTER FIVE

COMPARISONS OF ESTUARINE ZOOPLANKTON

ASSEMBLAGES NEAR SALTMARSH

5.1 Introduction

The diverse and abundant zooplankton assemblages that are present in estuarine ecosystems, and particularly those of the mesoplankton, provide crucial trophic links between primary producers, such as phytoplankton, and higher-level consumers like fish and crustaceans, which may eventually be preyed upon by economically important species at a higher trophic level (Kneib, 1997; 2000; Nagelkerken, 2009). The majority of individuals that comprise the mesoplankton includes taxa that complete their life cycle within the zooplankton, including various copepods (calanoids, harpacticoids and cyclopoids), other small crustaceans, and representatives of other phyla (such as chaetognaths and ctenophorans), many of which feature in estuaries worldwide (Collins and Williams, 1981; Fulton, 1984; 1984b; Gifford and

Dagg, 1988; Purcell et al., 2001; Guo et al., 2002).

A significant component of the mesoplankton, however, consists of larval forms of marine and estuarine invertebrates, including cnidarians, polychaetes, gastropods, amphipods and crabs (Rozas and Minello, 1998; Freewater et al., 2008; Mazumder et al., 2009). These planktonic larvae are typically derived from their benthic adults, many of which inhabit surrounding estuarine vegetation such as saltmarshes and mangroves. For example, in the case of grapsid crabs (e.g. Helograpsus haswellianus and Sesarma erythrodactyla), whose adults occur within saltmarsh vegetation (Mazumder and Saintilan, 2010; Alderson et al., 2013), these crustaceans export large quantities of offspring (as larvae in zoeal stages) into nearby estuarine waters (Dittel and Epifanio, 1982; Kneib, 1997; Charmantier et al., 2002; Papadopoulos, 2002;

Brodie et al., 2007; Mazumder et al., 2009).

Due to the physical complexity of estuaries (e.g. tidal regimes and morphology), zooplankton assemblages often vary temporally (e.g. tidally and seasonally) and with location or habitat type (Robertson et al., 1988; Velho et al., 1999; Plourde et al., 2002; Bloomfield and

Gillanders, 2005; Marques et al., 2006). In the case of season, it has been demonstrated that different taxa can dominate estuarine zooplankton assemblages during different seasons

(Hoffmeyer, 2004; Tan et al., 2004), and this has also been related to zooplankton biomass and grazing (see Froneman, 2001). Variations in zooplankton assemblages have also been attributed to differences in hydrology, tidal height, area of habitat (which is temporally variable and generally determined by tidal height) with such effects essentially determining distributions of zooplankton assemblages within estuaries as a result of larval dispersal from riparian vegetation (Lambert and Epifanio, 1982; Hettler, 1989; DiBacco et al., 2001; Weaver and Salmon, 2002; Bloomfield and Gillanders, 2005; Mazumder et al., 2006a).

Specifically, south-east Australian saltmarshes are known sources of high densities of crab zoeae and mollusc larvae (i.e. through exportation), as well as sinks for amphipods and copepods (which are among the dominant taxa in zooplankton assemblages during flood tides;

Freewater et al., 2008; Mazumder et al., 2008). Similar trends exist for mangroves, although lower densities of zooplankton are generally exported (Mazumder, 2004; Mazumder et al.,

2008). It has been suggested that such synchronised mass release of larvae may be a survival tactic to reduce predation risk and therefore mortality rate of offspring within the population

(Kneib and Wagner, 1994; Morgan and Christy, 1995; Morgan, 1996; Kneib, 1997).

There are seasonal differences in the timing of saltmarsh inundation in south-eastern

Australian saltmarshes, with the high spring tides that inundate saltmarsh occurring once per

24 hours and during the day-time high tides in summer, but during night-time high tides in winter (see Chapter Two). The reported release of relatively greater amounts of zoeae by the adults of grapsid crabs during the winter ebb tides (at night) than the summer ebb tides (during the day) is considered to be a strategy to counteract visual predators, such as teleosts (Morgan and Christy, 1995; Mazumder et al., 2006a; see also Chapter Six).

This study aims to quantify the abundance, diversity and the taxonomic composition of zooplankton assemblages within an area that is directly adjacent to a representative saltmarsh habitat and is located within the basin of a temperate estuary in south-eastern

Australia (Empire Bay Wetland, Brisbane Water Estuary, NSW), taking into account any differences between seasons, presence of saltmarsh inundation and tidal states. A second aim was to explore the relative abundances, with respect to the above factors, of two taxa, i.e. calanoid copepods and crab zoeae, that were found to be very abundant within those zooplankton assemblages (see Results).

It is hypothesised that: (1) There will be differences in the abundance, diversity and taxonomic composition of zooplankton assemblages and major taxa at times of saltmarsh inundation and non-inundation, as well as during flood and ebb tides as a result of crab zoeal export during ebb tides, (2) Crab zoeae will be in greater abundance on ebb tides during saltmarsh inundation periods, but more so in winter than summer and (3) The zooplankton assemblages will differ between summer and winter, reflecting differential reproduction by zooplanktonic species.

5.2 Methodology

5.2.1 Study site and sampling of zooplankton

Zooplankton were sampled, using a 250 um mesh plankton net with a 300 mm opening at two sites in Empire Bay Wetland, using the full experimental design as the basis for sample collection (see Chapter Two for the detailed description of the study site, experimental design and sampling methods).

Each plankton tow was dragged over a distance of 100 m using markers randomly placed on the shoreline as a guide. The net and zooplankton sample was thoroughly rinsed into containers, and preserved using 5% formaldehyde until subsequent laboratory examinations.

The net was thoroughly washed after each tow in order to prevent cross contamination between samples.

5.2.2 Laboratory procedures

Zooplankton samples were transferred to 70% ethanol at a standardised volume of

100 mL for at least one week. Using a pipette, five 1 mL sub-samples were removed from each sample and placed on a glass petri dish for subsequent examination (see Freewater et al.,

2008). Samples were mixed by stirring for five seconds between the removal of each sub- sample. The pipette was rinsed after each use in order to prevent cross contamination between sub-samples. A dissecting microscope was used to identify the zooplankton taxa in each sub- sample using the taxonomic keys provided by Ritz et al. (2003) and Poore (2004). Zooplankton were identified to the lowest practicable taxon and then enumerated.

The number of taxa in each sub-sample were collated to provide the number of different taxa in the five sub-samples overall and used to represent the number of taxa in the entire sample. The numbers for the individual taxa in each sub-sample were meaned and then adjusted upwards to correspond to the 100 mL of each sample, i.e. multiplied by 20.

5.2.3 Statistical analyses

Data for the overall zooplankton abundance and number of taxa (i.e. total number of taxa in each sample), and of the taxonimic composition of the zooplankton assemblages were analysed using a combination of univariate and multivariate analyses. These analyses were

98 based on the following design: Season, 2 levels (Summer, Winter), fixed, orthogonal; Month, 2 levels (January, February, June, July), random, nested in Season; Day, six levels (1, 2, 3, 4, 5, 6; which took into account the states of saltmarsh inundation (inundated (1-3) and non-inundated

(4-6)), fixed, orthogonal; Tidal state, two levels (flood, ebb), fixed, orthogonal; n = 2.

5.2.3.1 Univariate analyses

Analyses of variance (ANOVA; based on a four-way design: Season, Month within

Season, Day and Tidal State, at  = 0.05) were used to determine whether there were any significant differences in the overall zooplankton assemblage, using data for the total number of zooplankton and the total number of zooplankton taxa. Further four-factor ANOVAs were also performed on the total numbers of two zooplankton taxa that were particularly abundant within the samples (i.e. calanoid copepods and crab zoeae).

Homogeneity of variances were examined using residual plots and if data were heterogeneous, square-root transformation was applied prior to rechecking residual plots.

Where significant differences (= 0.05) were found between the levels of main fixed factors

(i.e. Day), Tukey’s HSD post-hoc pairwise tests were conducted to determine which levels of the factors caused a significant difference within the main effect. Univariate analyses were conducted using the statistical package SPSS (Version 21 – SPSS, 2012).

5.2.3.2 Multivariate analyses

The species composition data for the zooplankton assemblages (i.e. number of individuals of each zooplankton taxa in each sample) were analysed using the various subroutines in the statistical package PRIMER v6 with the PERMANOVA+ add on (Clarke and

Gorley, 2006; Anderson et al., 2008). The zooplankton assemblage data were first square-root

99 transformed and the Bray-Curtis measure used to create a similarity matrix that formed the basis of significance testing via Permutational Analyses of Variance (PERMANOVA), using Type

III sums of squares (Anderson, 2001; Anderson et al. 2008). PERMANOVA was based on a four- way fully-crossed design (Season, Month within Season, Day and Tidal State) at  = 0.05 and, when p > 0.25 for any interactions, those interaction effects were pooled before PERMANOVAs were reanalysed (as recommended by Winer et al., 1991; see Results). A test for homogeneity of multivariate dispersions (PERMDISP) was done prior to PERMANOVA to determine whether there were any differences in dispersions within groups.

When PERMANOVA showed significant differences, pairwise post-hoc tests were carried out to determine specific patterns of differences among the levels within those factors.

The above results were visually depicted using a non-metric multi-dimensional scaling (nMDS) ordination plot, derived from a ‘distances among centroids’ matrix constructed using the original Bray-Curtis similarity matrix (see above; Clarke and Gorley, 2006).

One-way Similarity Percentages analyses (SIMPER; Clarke, 1993; Clarke and Gorley,

2006) were then used to identify the zooplankton taxa that typified the zooplankton assemblage of each a priori group and/or those that were responsible for distinguishing between the zooplankton assemblage in each pair of groups. All univariate and multivariate plots depict significant fixed factors and interactions only (i.e. variability among random or non- significant fixed factors and interactions are not displayed; see Jackson and Brasher, 1994;

Reinard, 2006; McPhee et al., 2015).

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5.3 Results

Ten main zooplankton taxa and a total of 7618 individuals were recorded at Empire

Bay Wetland (Brisbane Water Estuary, NSW, Australia) in 2012, with calanoid copepods being the most abundant (56.1%), followed by crab zoeae (23.2%; Tables 5.1 and 5.2). Calanoid copepods were always the most numerous of the zooplankton in the samples during flood tides, contributing 36.6 – 83.0% to all zooplankton. However, their percentage contributions were often lower in the ebb tides in summer, but not winter. In contrast, crab zoeae tended to be more important in the ebb than flood tides, which was particularly marked during days 4-6, i.e. following saltmarsh inundation (Tables 5.1 and 5.2). Total numbers of zooplankton were greater in all flood than ebb tides except for Days 4-6 during winter (Table 5.1). With zooplankton assemblages being sampled using a plankton net with 300 mm opening over a 100 m transect, the numbers of all zooplankton expressed as a density ranged from 331 to 729 individuals/m-3, with an overall mean of 561 individuals/m-3.

Table 5.1: The numbers of zooplankton taxa separated by tidal state, inundation state (Days 1-3 vs 4-6) and seasons, and the total numbers of each zooplankton taxa and overall, recorded at Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in 2012.

Summer Winter Days 1-3 Days 4-6 Days 1-3 Days 4-6 Taxa Flood Ebb Flood Ebb Flood Ebb Flood Ebb Total Calanoida 258 142 749 150 975 638 627 731 4270 Crab zoeae 154 210 187 639 41 159 68 307 1765 Harpacticoida 25 26 27 29 89 136 58 123 513 Copepod remnants 106 67 45 58 37 36 48 52 449 Cnidaria 114 65 35 51 6 14 4 3 292 Caridea larvae 41 42 19 46 26 80 10 21 285 Teleost larvae 2 5 8 9 0 0 0 0 24 Polychaeta larvae 1 1 1 1 0 5 2 1 12 Gastropoda larvae 3 3 0 0 0 0 0 0 6 Insecta 0 1 0 1 0 0 0 0 2 Total 704 562 1071 984 1174 1068 817 1238 7618

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Table 5.2: The percentage contribution of zooplankton taxa separated by tidal state, inundation state (Days 1-3 vs 4-6) and seasons, and the total numbers of each zooplankton taxa and overall, recorded at Empire Bay Wetland in 2012.

Summer Winter Days 1-3 Days 4-6 Days 1-3 Days 4-6 Taxa Flood Ebb Flood Ebb Flood Ebb Flood Ebb Total Calanoida 36.6 25.3 69.9 15.2 83.0 59.7 76.7 59.0 56.1 Crab zoeae 21.9 37.4 17.5 64.9 3.5 14.9 8.3 24.8 23.2 Harpacticoida 3.6 4.6 2.5 2.9 7.6 12.7 7.1 9.9 6.7 Copepod remnants 15.1 11.9 4.2 5.9 3.2 3.4 5.9 4.2 5.9 Cnidaria 16.2 11.6 3.3 5.2 0.5 1.3 0.5 0.2 3.8 Caridea larvae 5.8 7.5 1.8 4.7 2.2 7.5 1.2 1.7 3.7 Teleost larvae 0.3 0.9 0.7 0.9 - - - - 0.3 Polychaeta larvae 0.1 0.2 0.1 0.1 - 0.5 0.2 0.1 0.2 Gastropoda larvae 0.4 0.5 ------0.1 Insecta - 0.2 - 0.1 - - - - 0.0

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5.3.1 Abundance and number of taxa of the overall zooplankton assemblages

ANOVA detected no significant differences among any of the four factors for either the total numbers of zooplankton nor the number of taxa of the zooplankton at Empire Bay

Wetland (Tables 5.3 and 5.4). The mean (and SE) abundances of zooplankton sampled from plankton tows (n=5 sub-samples) ranged between 1.4 (±0.5) and 77.2 (±3.7), while the mean number of taxa of the zooplankton showed a narrow range of between 1.2 (±0.2) and 5.2 (±0.2).

Table 5.3: Summary of results for four-factor Analysis of Variance of the abundance of zooplankton in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation df MS F P Season (Se) 1 3.213 0.1 0.793 Month (M) [nested in [Se] 2 2.339 2.9 0.239 Day (D) 5 1.002 0.2 0.953 Tidal State (T) 1 0.026 0.03 0.878 Se x D 5 2.744 0.6 0.727 Se x T 1 0.690 0.8 0.467 D x M[Se] 10 4.880 2.6 0.071 T x M[Se] 2 0.872 0.5 0.638 D x T 5 3.876 2.1 0.150 Se x D x T 5 3.853 2.1 0.152 D x T x M[Se} 10 1.855 2.0 0.053

Residual 48

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Table 5.4: Summary of results for four-factor Analysis of Variance of the number of taxa of zooplankton in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation df MS F p Season (Se) 1 0.227 0.1 0.788 Month (M) [nested in [Se] 2 0.058 0.5 0.711 Day (D) 5 0.022 0.3 0.920 Tidal State (T) 1 0.082 5.5 0.144 Se x D 5 0.033 0.4 0.832 Se x T 1 0.075 5.0 0.155 D x M[Se] 10 0.262 2.6 0.071 T x M[Se] 2 0.015 0.8 0.474 D x T 5 0.548 2.1 0.150 Se x D x T 5 0.545 2.1 0.152 D x T x M[Se} 10 0.019 0.4 0.915

Residual 48

5.3.2 Abundances of calanoid copepods and crab zoeae

For calanoid copeods, ANOVA detected no significant differences among any of the four factors (Table 5.5), and the mean (and SE) abundances of these crustaceans ranged between 0 (±0) and 48 (±3.5). However, ANOVA did show that the abundance of crab zoeae differed between Tidal states inconsistently among Days (i.e. a Day*Tidal State interaction was significant; p = 0.025; Table 5.6). Post-hoc tests (Tukey HSD) showed that crab zoeal abundances were significantly greater during ebb than flood tides on Days 1, 4, 5 and 6 (i.e. primarily on days of saltmarsh inundation; Fig. 5.1).

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Table 5.5: Summary of results for four-factor Analysis of Variance for the abundance of calanoid copepods in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland, in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation Df MS F p Season (Se) 1 39.763 1.7 0.324 Month (M) [nested in [Se] 2 23.594 6.0 0.057 Day (D) 5 1.315 0.3 0.908 Tidal State (T) 1 6.219 4.2 0.178 Se x D 5 2.758 0.6 0.697 Se x T 1 4.425 3.0 0.227 D x M[Se] 10 4.543 2.3 0.120 T x M[Se] 2 1.494 0.7 0.514 D x T 5 2.084 1.0 0.469 Se x D x T 5 5.863 2.8 0.079 D x T x M[Se} 10 1.467 1.4 0.184

Residual 48

Table 5.6: Summary of results for four-factor Analysis of Variance for the abundance of crab zoeae in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (F) F ratio test statistic, (p) significance.

Source of variation Df MS F p Season (Se) 1 10.7 4.5 0.166 Month (M) [nested in [Se] 2 2.3 2.9 0.232 Day (D) 5 1.8 3.7 0.038 Tidal State (T) 1 22.6 31.9 0.030 Se x D 5 0.5 0.8 0.575 Se x T 1 0.7 1.0 0.423 D x M[Se] 10 0.5 1.3 0.269 T x M[Se] 2 0.7 1.8 0.172 D x T 5 1.1 2.8 0.025 Se x D x T 5 0.588 1.5 0.275 D x T x M[Se} 10 0.393 1.1 0.410 Residual 48 0.392

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12 b, c * a, b, c * c *

8 a * a, b a, b 6

Abundance of crab crab of zoeaeAbundance 4

0 Flood Ebb Flood Ebb Flood Ebb Flood Ebb Flood Ebb Flood Ebb 1 2 3 4 5 6 Saltmarsh not inundated Saltmarsh inundated Day and Tidal state

Figure 5.1: Mean abundance (±SE, n=2) of crab zoeae, on various Days and Tidal states at Empire Bay Wetland in 2012. Days not sharing the same letter (e.g. a, b, c) are significantly different. The symbol “*” depicts that mean crab zoeal abundances are greater for ebb than flood Tidal states on such Days.

5.3.3 Comparisons of zooplankton assemblages among seasons, months, tidal state and days

PERMANOVA showed that, although zooplankton assemblages varied between Tidal states (flood and ebb tides), the pattern of this difference varied between Days (i.e. a Day x

Tidal State interaction was significant; p = 0.036; Table 5.7). However, post-hoc pairwise tests failed to detect any significant differences at p < 0.05. The use of nMDS ordination demonstrated that the zooplankton assemblages of Empire Bay Wetland for all but one (Day

2) of the samples for ebb tides were located on the right of the plot, while those for flood tide points were grouped together on the left of that plot (Fig. 5.2). PERMDISP showed no differences in multivariate dispersion (F = 3.12; p = 0.13), so all patterns could be attributed to the differences in assemblage composition.

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Table 5.7: Summary of results for four-factor PERMANOVA of the taxonomic composition of the zooplankton assemblages in different Seasons, Months nested in Season, Day and Tidal state, at Empire Bay Wetland in 2012. Significant differences are depicted in bold. Analyses were conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (Pseudo- F) pseudo-F ratio test statistic, (P(perm)) permutation significance.

Source of variation Df MS Pseudo-F P(perm) Season (Se) 1 12389 0.8 1.000 Day (D) 5 667 0.6 0.835 Tidal State (T) 1 6182 8.2 0.048 Month (M) [nested in Se] 2 14599 39.6 0.001 Se x D 5 934 0.9 0.582 Se x T 1 182 0.2 0.399 D x T 5 995 2.1 0.036 M[Se] x D 10 1038 2.8 0.001 Mo[Se] x T 2 749 2.0 0.053 Se x D x T 5 759 1.6 0.122 Mo[Se] x D x T 10 461 1.3 0.149 Residual 48 17681 368.4

However, generally and most clearly, zooplankton assemblages differed between the two Tidal states (p = 0.048; Table 5.7). There was also significant variability in zooplankton assemblages among the random factor of Month (nested in Season; p = 0.001) and the interaction between Month (nested in Season) and Day (p = 0.001; Table 5.7).

SIMPER showed that calanoid copepods, crab zoeae and harpacticoid copepods typified flood tides, while crab zoeae and calanoid copepods typified ebb tides. Crab zoeae contributed most to the difference between Tidal states by being relatively more abundant during ebb tides, while calanoid copepods were relatively more abundant during flood tides.

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5.4 Discussion

The zooplankton assemblages at Empire Bay Wetland (Brisbane Water Estuary, NSW,

Australia) were dominated by calanoid copepods, which were shown to be by far the most abundant zooplankton taxa, contributing an average of ~56% to the total numbers. Such dominance of the zooplankton at Empire Bay Wetland by these crustaceans is paralleled by the dominance and high contribution (>90%) of calanoid copepods to zooplankton elsewhere in

Australia (Kimmerer and McKinnon, 1985; Duggan et al., 2008), North America (Heinle, 1966;

Lawrence et al., 2004), Europe (Collins and Williams, 1981; Baretta and Malschaert, 1988) and

South Africa (Jerling and Wooldridge, 1991), and reflects a global dominance of the zooplankton by this group in estuaries. The only other major taxa to make a substantial contribution were the crab zoeae, i.e. 23% to the total numbers.

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The taxa that were recorded can be assigned to either the holoplankton (calanoid and harpacticoid copepods and cnidarians), collectively contributing up to ~62.5% of all zooplankton, and also to the meroplankton (most particularly crab zoeae but also the larvae of polychaetes, gastropods, carids and teleosts), which also made an appreciable contribution, i.e. ~27.5%, to the total numbers of zooplankton. The relative contributions of such meroplankton is likely to be enhanced by contributions from the nearby saltmarsh habitats, in which grapsid crabs and gastropods are known to be abundant, via larval release into the estuarine water column during ebbing tides that follow saltmarsh inundation (see later;

Mazumder et al., 2008).

The number of taxa of the zooplankton did not differ with any of the four factors in this study, which may reflect the relatively coarse level of identification undertaken in the samples from Empire Bay Wetland (in which zooplankton were divided into 10 different taxonomic categories). However, such patterns have also been shown for zooplankton in another estuary that used a finer taxonomic level (see Hwang et al., 2010), and in a comparable study in a nearby estuary (Botany Bay), in which zooplankton were divided into just five taxonomic categories (Mazumder et al., 2008). The lack of difference for the number of taxa of the zooplankton assemblage is paralleled with there also being no significant differences for both the abundance of all zooplankton and of the calanoid copepods, most probably reflecting the lack of seasonality within those calanoid copepods, which make such an overwhelming contribution to the zooplankton assemblages at the study site. For example, populations of calanoid copepods have shown seasonal stability under varying levels of salinity (e.g. tidally influenced) and nitrogen loads/chlorophyll levels (Lawrence et al., 2004), which demonstrates their capability to remain in similar abundances under varying environmental conditions. It is thus noteworthy that water quality sampled in the current study at Empire Bay Wetland (see

Chapter Two) showed a slight salinity increase (i.e. variation) from ~30.5 during summer to

~32.0 during winter, while water temperature and turbidity similarly varied among seasons.

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Such variations in the water quality of Empire Bay Wetland among seasons may explain the seasonal stability of the zooplankton at the study site, where no temporal differences were found in number of zooplankton taxa or abundance – i.e. fluctuations in water quality levels such as salinity and temperature may not impact these aspects of the zooplankton assemblage.

5.4.1 Tidal and temporal differences in the abundance of crab zoeae

The abundances of crab zoeae varied with tidal state (i.e. food and ebb tides), but also with whether or not the saltmarsh was inundated (i.e. days). Thus, although the numbers of crab zoeae were always greater in the ebb than flood tides during each sampling occasion, this difference was significant (and markedly so) only when the saltmarsh had become inundated

(with the exception of day 1; see later). This finding lends support to the model that saltmarsh- dwelling grapsid crabs release high numbers of zoeae into ebb tides following saltmarsh inundation (Mazumder et al., 2008). In the present study, however, the extent of the difference with respect to the tidal state during saltmarsh inundation is markedly less than in a comparable study (Mazumder et al., 2006a; 2008). This could reflect the relatively

“unmodified” state of Empire Bay Wetland in comparison to the levee channels that were sampled at Towra Point (Mazumder et al., 2006a; 2008), and which may also result in a

“dilution” of the zooplankton due to the location of the study site within Brisbane Water

Estuary (i.e. having many basins and rivers upstream during ebbing tides that may be capable of mixing ebbing tidal water into the general tidal channel within which sampling was undertaken in the present study).

Although significant amounts of crab zoeae were found in the ebb tides on day 1, it is relevant that even though the saltmarsh was not inundated at that time, the tidal levels were increasing as the spring tide approached, with the result that a greater area of the fringing mangrove vegetation would have been inundated. It is therefore possible that the grapsid crabs

110 that also dwell in mangroves showed a comparable pattern of zoeal release following such inundation. Such grapsids may include the same or similar species (e.g. Heloecius cordiformis,

Parasesarma erythrodactyla and Paragrapsus laevis; Mazumder and Saintilan, 2003) that can co-occur in saltmarsh and mangrove vegetation in south-eastern Australia (Hovel and Morgan,

1997; see also McPhee et al., 2015).

There was no significant difference in the abundance of crab zoeae between summer and winter in the present study. Thus, although seasonal differences in such abundances have been previously shown (e.g. Christy and Stancyk, 1982; Mazumder, 2004), this was not found to be the case at Empire Bay Wetland. For example, the release of crab zoeae is thought to be greater during winter (Mazumder, 2004), when saltmarsh inundation occurs at night, as a survival strategy to minimise larval mortality by visual predators (such as A. jacksoniensis;

Green and Anderson, 1973; Hovel and Morgan 1997). However reciprocally, crab zoeal export has also been shown to be greatest during summer, rather than winter, within ebb tides during spring tidal cycles in a South Carolinan estuary (Christy and Stancyk, 1982). This inconsistency among studies may be attributed to the fact that crab zoeae are thought to have highly variable densities among different tidal phases, seasons and locations (Christy and Stancyk, 1982;

Pittman and McAlpine, 2003).

5.4.2 Composition of the zooplankton assemblages

The taxonomic composition of the zooplankton assemblages was strongly related to tidal state, mostly due to the crab zoeae being more abundant on the ebb tides and particularly during days of saltmarsh inundation, which is consistent with their release into ebbing tides during times of such inundation (see earlier). It is also relevant that benthic harpacticoid copepods were contributing to zooplankton on the flood more than ebb tides (as found within a North American estuary; see Fleeger et al., 1984), and which may be related to

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“active emergence” behaviours during flood tides (i.e. from benthic habitats into the zooplankton; see Palmer, 1988; Armonies 1988; 1989; Walters and Bell, 1994) as a transportation strategy to other portions of the estuary (Guinea, 1997; see also Warwick and

Gee, 1984). Calanoid copepods were highly abundant during both flood and ebb tides, and this reflects their known high abundance in estuarine zooplankton assemblages (see earlier;

Heinle, 1966; Collins and Williams, 1981; Kimmerer and McKinnon, 1985; Baretta and

Malschaert, 1988; Jerling and Wooldridge, 1991; Lawrence et al., 2004; Duggan et al., 2008).

Interestingly, the remaining taxa that were found to contribute to the zooplankton (i.e. cnidarians, insects, carids, teleosts, polychaetes and gastropods) did not typify either flood or ebb tidal states, nor did they contribute to any significant differences in the zooplankton assemblages. Given that zooplankton assemblages were sampled across a variety of environmental conditions in the present study (e.g. tidal states, seasons), it is noteworthy that

Robertson et al. (1988) found no relationships between salinity, temperature, mangrove litter or fish predation with zooplankton composition or concentration, suggesting that such environmental variables may have little effect on certain zooplankton taxa such as cnidarians, insects, carids, teleosts, polychaetes and gastropods (Robertson et al., 1988; see also Raposa and Roman, 2003; Rountree and Able, 2007).

5.4.3 Conclusion

The present study did not discern an effect of either time of year, tidal state (i.e. flood and ebb) or saltmarsh-inundation (i.e. days) on the overall zooplankton abundance, which was attributed to the consistently high abundances of the calanoid copepods that represent the holoplankton (and which were therefore not derived from saltmarshes) at Empire Bay Wetland.

Although the crab zoeae were also relatively abundant, they were found mainly in ebb tide samples and particularly following saltmarsh inundation, which supported the model proposed

112 by Mazumder et al. (2008), whereby saltmarsh-dwelling grapsid crabs release high numbers of zoeae into ebb tides following saltmarsh inundation.

When examining the samples from ebb tides that inundated saltmarsh, crab zoeae were shown to be similarly abundant during both summer and winter at Empire Bay Wetland.

This is in direct contrast to the varying conclusions of other works on the same and different species (e.g. Christy and Stancyk, 1982; Mazumder, 2004), which implies that any generalities on the seasonality of saltmarsh-derived crab zoeal release may not be possible. One point of agreement between the various studies, however, is that tidal regimes (e.g. ebbing tides following saltmarsh inundation) are the most influential factor for crab zoeal release (see

Robertson et al., 1988; Raposa and Roman, 2003; Rountree and Able, 2007).

Saltmarsh-derived crab zoeae, that comprise an important part of zooplankton assemblages at Empire Bay Wetland, in turn provide an important food source for estuarine fish (including A. jacksoniensis; Mazumder et al., 2006; Hollingsworth and Connolly, 2006;

Platell and Freewater, 2009; McPhee et al., 2015) and which in turn supports estuary function and may also contribute to trophic relay (Kneib, 1997; 2000; Nagelkerken, 2009). In light of this, the present study highlights the importance of appropriate conservation and management of saltmarsh vegetation in order to maintain the natural health and functioning of estuaries.

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CHAPTER SIX

TROPHODYNAMIC RELATIONSHIPS BETWEEN

Ambassis jacksoniensis AND

SALTMARSH-DERIVED CRAB ZOEAE

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6.1 Introduction

‘Trophic relay’ is an ecological model in which the biomass and energy obtained by organisms feeding on and within estuarine vegetation, is transported by nektonic predators such as fish and mobile crustaceans, from the upper limits of estuaries down to their lower reaches and then out to the open sea (Kneib, 1997; 2000; Nagelkerken, 2009). For example, energy (i.e. stored in organic matter) is transported from zooplankton (such as zoeae released from the saltmarsh grapsid crabs; e.g. Helograpsus haswellianus that feed upon fine benthic organic material and microphytobenthos within saltmarsh vegetation; Mazumder and

Saintilan, 2010; Alderson et al., 2013), to small itinerant fish species (e.g. Ambassis jacksoniensis feeding on crab zoeae), and eventually to larger piscivorous fishes that travel into estuaries at particular times to feed (e.g. Acanthopagrus australis, Platycephalus fuscus,

Argyrosomus japonicus; SPCC, 1981; Taylor et al., 2006; Mazumder et al., 2006a). These larger piscivores, which freely move between the ocean and estuary and which provide the mechanism for transporting this energy and biomass to the open ocean, are often also of economic importance (SPCC, 1981; Deegan, 1993; Taylor et al., 2006; Bouillon and Connolly,

2009).

The energy and nutrient cycling provided by trophic relay is fundamental for the functioning of ecosystems that are geographically widespread, and promotes connectivity between those environments. Other examples of trophic relay have been found both in

Australia (Vance et al., 1998; Bouillon and Connolly, 2009) and internationally (Fry et al., 1999;

Adnan et al., 2002). For example, in a demonstration of trophic relay, Fry et al. (1999) used stable isotope analyses to trace the diets and movements of pink shrimp (Farfantepenaeus duorarum) from estuarine habitats (e.g. seagrass and mangrove vegetation) to open ocean environments on the south-western Florida shelf. The concept of trophic relay has important implications for the functioning of such estuarine ecosystems and, based on assessments of

115 predator-prey relationships via stomach-content and stable isotope analyses in Australian estuaries (Vance et al., 1998; Fry et al., 1999; Adnan et al., 2002; Melville and Connolly, 2003), is likely to be a feature of those estuaries.

Although the process and extent of trophic relay is driven by predator-prey relationships, it would be affected by water movements within an estuarine system, which, in turn, are influenced by factors such as the volumes of freshwater flow, characteristics of the estuarine mouth and tidal movements (Kneib, 1997). In the context of tidal movement, the fauna within the saltmarsh vegetation that often fringes the shoreline of estuaries provides an important food source for estuarine fish when tidally inundated and would therefore promote trophic relay within such estuaries (Weisberg et al., 1981; Morton et al., 1987; Sumpton and

Greenwood, 1990; West and Zedler, 2000; Laffaille et al., 2002; Platell and Freewater, 2009).

However, the extent of any potential trophic relay via saltmarshes will be affected by the frequency of tidal inundation, which varies geographically (Laffaille et al., 2001; Thomas and

Connolly, 2001; Costa et al., 2003; Hollingsworth and Connolly, 2006; Daleo et al., 2009; Bakker,

2014). Any extent of trophic relay will also be affected by potential contributions of biomass and energy from fringing mangroves, such as those in estuaries of South America and south- eastern Australia (Wilson and Whittaker, 1995).

Any temporal and spatial variability in prey availability (such as zoeal release by saltmarsh-dwelling crabs), can provide an opportunity for “switching” of prey by estuarine predators (Murdoch et al., 1975; Ringler, 1985; Suryan et al., 2000; Siddon and Witman, 2004).

For example, a small estuarine fish (Ambassis jacksoniensis) that is abundant in estuaries of south-eastern Australia (Saintilan, 2009), switched feeding from thalassinid larvae to crab zoeae in a saltmarsh environment when those zoeae became abundant following saltmarsh inundation (McPhee et al., 2015). Knowledge of prey-switching has implications for trophic relay and can also lead to insights into the feeding strategies used by various species

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(i.e. preferential vs opportunistic feeding; sensu Wassenberg, 1990), and may be linked to a maximisation of energetic gain among different available prey (e.g. Bittar et al., 2012). In the latter case, it is possible to determine, via calorimetry, the energetic density of different prey types and thus the potential energetic value of those prey to their likely predators

(e.g. Robbins, 1983; Benoit-Bird, 2004; Wuenschel et al., 2006).

Despite the importance of zooplankton to the diets of estuarine predators, including both adult and their larval stages (Weisberg et al., 1981; Kneib, 1997; Loneragan et al., 1997;

Laffaille et al., 2001; Hollingsworth and Connolly; 2006; Mazumder et al., 2006; Svensson et al.,

2007; Platell and Freewater, 2009), little is known of the potential energetic contribution of different zooplankton prey to their predators, and particularly for those in estuaries, including saltmarshes. The collation of such information, along with an understanding of the dietary composition of key predators, would allow for quantitative assessment of the potential energetic transfer within this type of environment.

This study aims to unravel the possible trophic pathways of the ambassid

A. jacksoniensis within a large estuary and explore the potential contribution of saltmarsh to trophic relay within this estuary. The specific objectives are to 1) determine the stomach fullnesses and dietary compositions, using stomach-content analyses, of A. jacksoniensis, focussing on any differences between seasons, tidal states (i.e. flood vs ebb) and taking into account whether or not the saltmarsh is inundated, and 2) ascertain the potential energetic contribution (calorimetric value) of possible zooplankton prey to the diets of A. jacksoniensis.

With respect to the first objective, it is firstly hypothesised that the dietary compositions of

A. jacksoniensis differs between varying tidal states and seasons (summer and winter). It is also predicted that saltmarsh-derived crab zoeae contribute greatly to diets of A. jacksoniensis at times when they are present in zooplankton (i.e. ebbing tides after saltmarsh inundation).

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6.2 Methodology

6.2.1 Study site, sampling of Ambassis jacksoniensis for dietary analyses and zooplankton for bomb calorimetry

Individuals of Ambassis jacksoniensis (as part of the overall fish assemblages; see

Chapter Three) were sampled, using a 20 m long seine net, at two sites in Empire Bay Wetland

(Brisbane Water Estuary, NSW, Australia), using the full experimental design as the basis for sample collection (see Chapter Two for the detailed description of the study site, experimental design and sampling methods).

Fish were euthanased in an ice-slurry following capture and stored frozen at -15 °C until subsequent laboratory examinations. Fish were thawed prior to identification, with ten

A. jacksonsiensis being haphazardly removed from each seine net haul and set aside for subsequent dietary analyses. Individuals were then preserved in 10% formaldehyde for one month and stored in 70% ethanol prior to those dietary analyses, with any shrinkage in body size (standard length; SL) being <1% (McPhee, unpubl. data).

The potential zooplankton prey of A. jacksoniensis, to assess for energetic content via bomb calorimetry, were obtained on the same sampling occasions, using the same plankton net and procedures that were used to obtain the zooplankton assemblages (see Section 2.3.2).

Additional zooplankton samples (i.e. plankton tows) were also undertaken at such times in order to obtain enough biomass to satisfy bomb calorimetric analyses (see later). Samples were frozen in the field and later thawed prior to being separated for bomb calorimetry.

6.2.2 Laboratory procedures

Each A. jacksoniensis was measured (SL, to the nearest 0.1 mm) and weighed (to the nearest 0.00001 g). Under a dissecting microscope, its stomach was removed, cut open and

118 given an ordinal score category for fullness ranging from 0 (empty) to 10 (100% full). Any ingested prey was identified to the lowest possible taxon and the percentage volumetric contribution (%V) of each dietary item to the overall volume of the stomach contents was visually estimated (sensu Platell and Freewater, 2009; McPhee et al., 2015). Five main dietary groups were identified, consisting of caridean decapods, crab zoeae, amphipods, sergestid decapods and algae.

Using a dissecting microscope, the zooplankton were identified into (1) Three potential prey “types” (i.e. crab zoeae, caridean decapods and calanoid copepods) and (2) Four zooplankton assemblage “types” (i.e. the overall flood and ebb zooplankton assemblages, both when the saltmarsh was not inundated (Days 1-3) and when it was inundated (Days 4-6)).

Samples of the same “type” were then amalgamated and two replicate samples of each prey and zooplankton assemblage “type” were subjected to calorimetry. The number of replicates was therefore low as a consequence of the small size of such prey, the total mass required for an individual sample and the extensive time required to sort and obtain such samples.

6.2.3 Statistical analyses

6.2.3.1 Stomach fullnesses and dietary compositions

Stomach fullnesses were analysed using a three factor (Season, Day, Tidal state) log- linear analysis ( = 0.05) by placing the stomach fullness values into 8 categories (0, 1, 2, 3, 4,

5, 6 and 7; NB: No stomach fullness exceeded 7 on the 0 to 10 scale). Sites and Months were pooled due to small numbers in one or more categories of stomach fullness at the two Sites in the two Months sampled of each Season.

The dietary composition data for the stomach contents of A. jacksoniensis were analysed using the various subroutines in the statistical package PRIMER v6 with the

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PERMANOVA+ add on (Clarke and Gorley, 2006; Anderson et al., 2008). The mean percentage volumetric contributions of the five dietary items to the stomach contents of each

A. jacksoniensis were square-root transformed and used to construct a Bray-Curtis similarity matrix. The dietary data for this matrix comprised a five-way fully-crossed design as follows:

Season (fixed, orthogonal), Month (random, nested in Season), Day (fixed, orthogonal), Tidal state (fixed, orthogonal) and Site (random, orthogonal). A permutational multivariate analysis of variance (PERMANOVA; Anderson, 2001; Anderson et al., 2008), using Type III sums of squares ( = 0.05), was conducted to determine whether the diets of A. jacksoniensis differed among the different Seasons, Months, Days, Tidal states and Sites and whether there were any interactions between those factors. A test for homogeneity of multivariate dispersions

(PERMDISP) was done prior to PERMANOVA to ascertain that there were no differences in dispersions within groups. Factors were pooled where possible (i.e. when p > 0.25 as recommended by Winer et al., 1991).

When PERMANOVA showed significant differences, pairwise post-hoc tests were carried out to determine specific patterns of differences among the levels within those factors

(groups). One-way Similarity Percentages analyses (SIMPER; Clarke, 1993; Clarke and Gorley,

2006) were then used to identify the dietary items that typified the dietary composition of each a priori group and/or those that were responsible for distinguishing between the dietary compositions in each pair of groups. The above results were visually depicted using a non- metric multi-dimensional scaling (nMDS) ordination plot, derived from a ‘distances among centroids’ matrix constructed using the original Bray-Curtis similarity matrix (see above; Clarke and Gorley, 2006). All univariate and multivariate plots depict significant fixed factors and interactions only (i.e. variability among random or non-significant fixed factors and interactions are not displayed; see Jackson and Brasher, 1994; Reinard, 2006; McPhee et al., 2015).

Univariate analyses were conducted using the statistical package SPSS (Version 21 – SPSS,

2012).

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6.2.3.2 Calorimetric measurements

Samples were oven dried at 60-80°C for 24-72 h and ground to powder (Ciancio and

Pascual, 2006). Dried samples were weighed (to 0.001g) and sent to an external laboratory

(Macquarie Geotech in Australia), where they were burned at 30 atm of oxygen in a bomb calorimeter to determine their calorimetric content. This was achieved by comparisons of the sample after burning with that of the initial dry weight. Calorimetric values were expressed in cal g-1 (dry weight).

The calorimetric values of the potential prey and zooplankton assemblage “types” were then statistically compared using a one-way ANOVA ( = 0.05) in SPSS (Version 21 – SPSS,

2012). Homogeneity of variances was examined prior to the analyses using residual plots and if data were heterogeneous, square-root transformation was applied before rechecking residual plots. Where significant differences ( = 0.05) were found between levels, post-hoc pairwise Tukey’s HSD tests were conducted to determine which levels were different from one another.

6.3 Results

The standard lengths (SL) of Ambassis jacksoniensis had a narrow range, i.e. 36.2 -

38.2 mm, with a mean of 37.3 mm (n = 960, SE = 0.021 mm; Table 6.1) at Empire Bay Wetland

(Brisbane Water Estuary, NSW, Australia). The wet weights of A. jacksoniensis were also small, ranging between ~0.9 g and 1.0 g and with a mean of 0.96 g (n = 960, SE = 0.002 g). Both the mean standard lengths and mean wet weights of A. jacksoniensis were very consistent between the two Seasons and Tidal states and whether or not the saltmarsh was inundated (i.e. Days 1-

3; no inundation; Days 4-6 saltmarsh inundation; Table 6.1).

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The stomach fullnesses of all A. jacksoniensis ranged from 0 (empty) to 7 (70% full).

Five dietary categories were recorded within the stomachs of A. jacksoniensis with caridean decapods making the greatest contribution (27.3%) to the overall dietary volume, and moderate, but similar, contributions by the other four dietary categories (crab zoeae, amphipods, sergestid decapods and algae; 16.8-19.8% (Table 6.2). Crab zoeae were never found in stomachs during flood tides, with the sole exception of winter during times of saltmarsh inundation (i.e. Days 4-6), where they made up 3.6% of the overall stomach volume.

During ebb tides that did not inundate the saltmarsh (Days 1-3), crab zoeae made up 39.4% and 4.9% of the stomach contents in summer and winter, respectively (Table 6.2). However, the contributions of crab zeoae were even greater on ebb tides during times of saltmarsh inundation (Days 4-6), and particularly so during the winter, with these larvae comprising ~45% of the stomach contents in both summer and winter. Overall, volumetric contributions of crab zoeae to stomachs were far greater during ebb tides than flood tides, especially on days of saltmarsh inundation (i.e. Days 4-6; Table 6.2).

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Table 6.1: Mean standard length (SL) and weight of Ambassis jacksoniensis at varying seasons, days and tidal states within Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in 2012.

Summer Winter Both seasons Mean St. Dev Days 1-3 Days 4-6 Days 1-3 Days 4-6 Days 1-3 Days 4-6 Variable Flood Ebb Flood Ebb Flood Ebb Flood Ebb Flood Ebb Flood Ebb SL (mm) 37.3 36.2 36.6 37.0 38.2 37.4 37.7 37.8 37.8 36.8 37.2 37.4 37.28 0.66 Weight (g) 1.0 0.9 1.0 1.0 1.0 0.9 1.0 0.9 1.0 0.9 1.0 1.0 0.96 0.05

Table 6.2: Percentage volumetric contributions of dietary categories recorded within Ambassis jacksoniensis stomachs at Empire Bay Wetland in 2012. NB: No saltmarsh inundation occurred on Days 1-3, while saltmarsh inundation did occur on Days 4-6.

Percentage Contribution Summer Winter Both seasons Days 1-3 Days 4-6 Days 1-3 Days 4-6 Days 1-3 Days 4-6 Percentage Dietary category Flood Ebb Flood Ebb Flood Ebb Flood Ebb Flood Ebb Flood Ebb contribution Caridea 73.5 22.0 58.0 3.8 43.9 20.2 4.1 4.6 65.2 21.0 18.5 4.3 27.3 Crab zoeae 0.0 39.4 0.0 44.6 0.0 4.9 3.6 45.6 0.0 19.3 2.7 45.2 16.8 Amphipoda 0.0 0.0 3.2 0.0 56.1 25.8 46.2 19.0 15.7 15.0 34.7 11.6 19.2 Sergestidae 0.0 0.0 0.0 0.0 0.0 46.4 41.5 16.7 0.0 27.0 30.4 10.2 16.9 Algae 26.5 38.6 38.9 51.5 0.0 2.7 4.6 14.1 19.1 17.8 13.7 28.7 19.8

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6.3.1 Comparisons of the stomach fullnesses of Ambassis jacksoniensis

Log-linear regression showed that the frequency of stomach fullness values were not consistent between Days in the two Seasons (2 = 118.237, d.f. = 35, p < 0.001). This was due to both the numbers of low stomach fullness values (i.e. 0-2) being higher than expected (under the statistical null hypothesis), and the number of high stomach fullness values being lower than expected, on Days

1-3 (i.e. when saltmarsh was not inundated), while the opposite was generally the case for Days 4-6

(i.e. when saltmarsh was inundated), for both Seasons (with slightly greater stomach fullness values during summer, particularly on Day 5; Fig. 6.1).

70  60  Summer 50 Winter

40  

30  

Number of Numberfish   20  

10     0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Saltmarsh not inundated Saltmarsh inundated

Figure 6.1: Log-linear regression depicting the relationship between stomach fullness categories (SF; marked on x-axis) of Ambassis jacksoniensis, ranging between 0 and 7, over subsequent Days (1-6) for summer and winter at Empire Bay Wetland (Brisbane Water Estuary, NSW, Australia) in 2012. “Up” arrows (↑) indicate that counts of such SF values are statistically higher than expected for that Day, while “down” arrows (↓) indicate that counts of such SF values are statistically lower than expected for that Day.

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Stomach fullness values were also inconsistent between Tidal states among Days (2 = 56.275, d.f. = 35, p = 0.013), which was generally due to these values being lower than expected during flood tides and higher than expected during ebb tides (Figs. 6.2a,b). A seasonal difference was also detected for stomach fullnesses during ebb tides (i.e. stomach fullnesses were inconsistent between Tidal states across Seasons; 2 = 47.867, d.f. = 7, p < 0.001), in which stomach fullness values were higher during summer ebb than winter ebb tides (Fig. 6.3).

60 (a)  50

30  

20  Number of Numberfish 10  0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Flood Ebb Flood Ebb Flood Ebb Day 1 Day 2 Day 3

60 (b)

50    40

20  Number ofNumber fish    10  0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Flood Ebb Flood Ebb Flood Ebb Day 4 Day 5 Day 6

Figure 6.2: Log-linear regression depicting relationship between stomach fullness categories (SF; marked on x-axis) for Ambassis jacksoniensis, ranging between 0 and 7, over different Tidal states (flood and ebb) and Days (a: 1-3; b: 4-6) at Empire Bay Wetland in 2012. “Up” arrows (↑) indicate that counts of such SF values are statistically higher than expected for that Tidal state and Day, while “down” arrows (↓) indicate that counts of such SF values that are statistically lower than expected for that Tidal state and Day.

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200  180 160 140 120 100   80  Number of Numberfish 60  40    20     0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Flood Ebb Flood Ebb Summer Winter

Figure 6.3: Log-linear regression depicting relationship between the stomach fullness categories (SF; marked on x-axis) of Ambassis jacksoniensis, ranging between 0 and 7, over Tidal states (flood and ebb) and Seasons (summer and winter) at Empire Bay Wetland in 2012. “Up” arrows (↑) indicate that counts of such SF values are statistically higher than expected for that Tidal state and Season, while “down” arrows (↓) indicate that counts of such SF values are statistically lower than expected for that Tidal state and Season.

6.3.2 Dietary composition

The dietary compositions of A. jacksoniensis were shown by PERMANOVA to differ among Tidal states (i.e. between flood and ebb tides; p = 0.033) and among Days (p = 0.028) and that there was no interaction between these factors (Table 6.3). Pairwise post-hoc comparisons of Days, using

PERMANOVA, showed that dietary compositions differed between Days 2 and 6 (p = 0.05).

The use of nMDS ordination demonstrated that the dietary compositions of A. jacksoniensis during flood tides formed a distinct group that was separate from the ebb tide points on the plot

(Fig. 6.4). Furthermore, during ebb tides, the points for Days 1-3 (when saltmarsh is not inundated) lay separate to those for Days 4-6 (when saltmarsh is inundated). At ebb tides on Days 4-6, there was also a greater spread of points than at other times (Fig. 6.4), however PERMDISP showed no differences in multivariate dispersion (F = 2.98; p = 0.16).

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Table 6.3: Summary of results for five-factor PERMANOVA of the dietary compositions of Ambassis jacksoniensis in different Seasons, Months nested in Season, Day, Tidal state and Site at Empire Bay Wetland in 2012. Significant differences and interactions are depicted in bold. Analysis conducted on square-rooted data; (df) degrees of freedom, (MS) mean squares, (Pseudo-F) pseudo-F ratio test statistic, (P(perm)) permutation significance.

Source of variation df MS Pseudo-F P(perm) Season (Se) 1 99298 3.2 0.140 Day (D) 5 20224 2.1 0.028 Tidal State (T) 1 196000 25.1 0.033 Site (Si) 1 785 0.2 0.760 Month (M) [nested in Se] 2 29068 7.7 0.061

Two-way interactions Se X D 5 12941 1.3 0.214 Se X T 1 50402 6.5 0.093 Se X Si 1 3557 0.9 0.509 D X T 5 11133 1.5 0.190 D X Si 5 776 0.5 0.947 T X Si 1 413 0.3 0.662 M[Se] X D 10 9988 4.6 0.001 M[Se] X T 2 7450 4.7 0.145 M[Se] X Si 2 3763 3.3 0.002

Three-way interactions Se X D X T 5 8896 1.1 0.384 Se X D X Si 5 1300 0.6 0.808 Se X T X Si 1 560 0.4 0.645 D X T X Si 5 846 0.4 0.938 M[Se] X D X T 10 8196 4.1 0.003 M[Se] X D X Si 10 2174 1.9 0.006 M[Se] X T X Si 2 1588 1.4 0.210

Four-way interactions Se X D X T X Si 5 1611 0.8 0.618 M[Se] X D X T X Si 10 2004 1.8 0.011 Residual 864 1129

SIMPER showed that caridean decapods both typified and distinguished the dietary compositions of A. jacksoniensis on Days 1-3 (when saltmarsh was not inundated) from those dietary compositions when the saltmarsh was inundated (Days 4-6), when crab zoeae both typified and distinguished those dietary compositions on Days 4-6. SIMPER also showed that greater amounts of

127 caridean decapods were ingested by A. jacksoniensis during flood than ebb tides, while the ingestion of crab zoeae and algae occurred to a greater extent on ebb tides.

Figure 6.4: nMDS plot, derived from a “distance among centroids” matrix of different Tidal States and Days, which was constructed from a Bray-Curtis similarity matrix using volumetric dietary data of Ambassis jacksoniensis recorded at Empire Bay Wetland in 2012. Numbers represent the Day.

6.3.3 Calorimetric comparisons

The calorimetric values of the seven zooplankton “types” were shown by ANOVA to be significantly different (Table 6.4). Post-hoc Tukey’s HSD test highlighted that the calorimetric values of the three potential prey “types” (i.e. crab zoeae, caridean decapods and calanoid copepods), which ranged from 566.6 to 574.3 cal g-1 did not significantly differ from one another (Fig. 6.5). Likewise, the calorimetric values of the four overall zooplankton assemblage “types” (i.e. Day 1-3 flood, Day 1-3 ebb,

Day 4-6 flood and Day 4-6 ebb), which ranged from 217.5-323.4 cal g-1, also did not significantly differ from one another (Fig. 6.5). However, Tukey’s HSD comparisons demonstrate that the three potential

128 prey “types” had significantly greater calorimetric values than the four overall zooplankton assemblage

“types." (Figure 6.5)

Table 6.4: Summary of results for one-factor ANOVA on calorimetric values of potential prey and zooplankton assemblage “types”, subjected to bomb calorimetry, derived from Empire Bay Wetland in 2012. (df) degrees of freedom, (MS) mean squares, (F) F-ratio test statistic, (p) significance.

Source of variation df MS F p Zooplankton treatment 6 295.412 11.176 0.001

Residual 7 26.433

700 a a a 600

500

400 1 - b b b cal/g 300 b 200

100

0 Calanoida Caridae Crab zoeae Day 1-3 Day 1-3 Ebb Day 4-6 Day 4-6 Ebb Calanoida Caridea Crab zoeae Day 1-3 Day 1-3 Ebb Day 4-6 Day 4-6 Ebb Flood Flood

Figure 6.5: Mean (±SE, n=2) calorimetric values of potential prey and zooplankton assemblage “types”, subjected to bomb calorimetry, derived from Empire Bay Wetland. Samples not sharing the same letter (e.g. a, b) are significantly different from each other.

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6.4 Discussion

This study has demonstrated that A. jacksoniensis “switches” feeding from caridean decapods to saltmarsh-derived crab zoeae after inundation of saltmarsh at Empire Bay Wetland (Brisbane Water

Estuary, NSW, Australia) during the spring tides that occurred in summer and winter of 2012. As this ambassid is very abundant within this estuary (and also other estuaries in the same region; Mazumder et al., 2006a; Platell and Freewater, 2009) it is well placed, therefore, to contribute to any potential trophic relay, via such prey-switching, from large estuaries to their adjacent marine waters.

A very restricted size range (36-38 mm SL) of A. jacksoniensis was recorded in the present study.

Although this is far smaller than the total length (TL) of 67 mm recorded for this species at a different site in the same estuary (Platell and Freewater, 2009; McPhee et al., 2015), and thus implying that only younger individuals were obtained in this study, it was clear, based on internal examination, that these fish comprised both females and males, including some that had reached maturity (see Chapter Four).

It should be noted that there is a difference of approximately 10 mm between the standard (SL) and total length (TL) of A. jacksonsiensis (McPhee, unpubl. data), i.e. on average, fish in the present study therefore ranged from ~46 to 48 mm TL. It is also noteworthy that due to the restricted size of A. jacksoniensis sampled at Empire Bay Wetland, it is unlikely that there were any marked differences related to the prey biomass ingested among A. jacksoniensis.

This study clearly demonstrated that, on the basis of stomach fullnesses, A. jacksoniensis fed to a far greater extent on ebb than flood tides, irrespective of whether the saltmarsh habitat was inundated. This marked tidal difference in feeding, which is likely to be related to food availability (see later), is also recorded for the same species in a nearby south-eastern Australian estuary (McPhee et al.,

2015), as well as another saltmarsh-inhabiting fish species (Fundulus heteroclitus) in eastern North

America (Weisberg et al., 1981).

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Ambassis jacksonsiensis fed mainly on planktonic crustaceans, with caridean decapods being of greater importance overall than crab zoeae, sergestid decapods and planktonic amphipods. This focus on small prey is consistent with other freshwater ambassids, such as Ambassis agassizi and

Ambassis agrammus, which feed on microcrustaceans (e.g. cladocerans, copepods, ostracods and conchostracans) and insect larvae (e.g. dipteran, ephemeroptan and trichopteran larvae; Pollard, 1974;

Sanderson, 1979; Bishop et al., 2001; Medeiros, 2004; Lintermans, 2007). Although the algae consumed in moderate amounts by A. jacksoniensis are likely to be benthic, which is consistent with this species and other species (A. agrammus) also feeding on benthic food (Bishop et al., 2001;

Platell and Freewater, 2009), it could also have been suspended in the water column by tidal action during the spring tidal levels of more than 1.8 m AHD recorded in the present study.

6.4.1 Dietary compositions, prey-switching and seasonality in the feeding of Ambassis jacksoniensis

Although zooplanktonic crustaceans always comprised the main prey of A. jacksoniensis, the volumetric contributions of caridean decapods and crab zoeae differed between flood and ebb tides and/or whether or not saltmarsh was tidally inundated. Thus, caridean decapods were ingested mainly on both flood tides throughout the spring-tide cycle and on ebb tides during days when the saltmarsh was not inundated. The importance of caridean decapods within the diets probably reflects their high abundances in the water column during this time (Bernard, 2009; see also Chapter Five). However, it should be noted that such feeding on caridean decapods occurred when another common prey type

(i.e. crab zoeae) was not found (nor expected to be present; Hovel and Morgan, 1997; Mazumder et al., 2006a) within the water column (see Chapter Five).

Crab zoeae were ingested in far greater amounts when A. jacksoniensis fed during ebb tides following saltmarsh inundation, implying that saltmarsh is the likely source of such zoeae, which is consistent with directly comparable studies (Hollingsworth and Connolly, 2006; Mazumder et al.,

2006a; Platell and Freewater, 2009; McPhee et al., 2015). It is therefore proposed that A. jacksoniensis

131 switched from feeding on caridean decapods to crab zoeae, following the inundation of saltmarsh during spring-tide cycles that led to subsequent zoeal release by saltmarsh-dwelling grapsid crabs during tides that ebbed from saltmarshes.

Ambassis jacksoniensis were also found to feed on crab zoeae at times other than ebb tides following saltmarsh inundation (i.e. when they were not expected to be available within zooplankton assemblages at Empire Bay Wetland). Notably, crab zoeae were similarly found within zooplankton assemblages sampled via plankton net during ebb tides that did not inundate saltmarsh (see

Chapter Five). This contrasts with a complete absence of crab zoeae in A. jacksoniensis diets at such times in Coombabah estuary (subtropical Queensland) in Hollingsworth and Connolly (2006). It is likely that crab zoeae ingested by A. jacksoniensis at such times in the present study were derived from the same or similar species of grapsid crab that are known to occur in mangrove forests in eastern Australia

(Hovel and Morgan, 1997; McPhee et al., 2015). Although crab larvae could be clearly identified at the larval stage (e.g. zoeae), identification of crab larvae to level of species was not achieved due to difficulties in species identification of zoeal stages (Flores et al., 2003).

Although such prey switching is largely consistent with other studies on the feeding of

A. jacksoniensis (Hollingsworth and Connolly, 2006; Mazumder et al., 2006a; Platell and Freewater,

2009; McPhee et al., 2015), there are some notable differences. Thus, in this study, A. jacksoniensis did not feed exclusively on crab zoeae following saltmarsh inundation, but also ingested algae and other crustaceans, unlike in a subtropical Australian estuary (Coombabah Lake), where it fed only on crab zoeae after the saltmarsh was inundated (Hollingsworth and Connolly, 2006). Moreover,

A. jacksoniensis fed on caridean decapods, as well as sergestid decapods and amphipods during the times that the saltmarsh was not inundated, which contrasts with the finding that A. jacksoniensis essentially fasted at non-inundation times in that other subtropical Australian estuary (Hollingsworth and Connolly, 2006). Finally, McPhee et al. (2015) showed that thalassinid larvae, rather than caridean decapods, were ingested by A. jacksoniensis during ebb tides when saltmarsh was not inundated in the

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Hawkesbury River estuary, that lies 15 km to the south of the estuary in the present study. It is likely that these differences in prey switching by A. jacksoniensis reflect differences in prey availability in the different environments, which means that any examination of prey switching in this species or other opportunistic feeders should ideally account for any such environmental differences between localities.

The switching of prey by A. jacksonsiensis from caridean decapods to crab zoeae following saltmarsh inundation, implies that such inundation either directly influences the availability of carideans within the water column or, more likely, that another food source (i.e. crab zoeae) becomes available and “preferable” as prey (or whose available abundance becomes greater than that of carideans, if

A. jacksoniensis fed opportunistically). As the stomach fullnesses of A. jacksoniensis were greater following inundation of saltmarsh than when the saltmarsh was not tidally inundated, crab zoeae apparently became more abundant than caridean decapods and/or were ingested more readily by

A. jacksoniensis. In the present study, zooplankton assemblage samples showed that crab zoeal abundances were higher during ebb tides than flood tides, particularly during times of saltmarsh inundation (see Chapter Five). The above trend in stomach fullness is consistent with other studies on the dietary compositions of this species (Hollingsworth and Connolly, 2006; Mazumder et al., 2006a;

Platell and Freewater, 2009, McPhee et al., 2015). This has also been recorded for the diets of other fish species such as Fundulus grandis in North American saltmarshes and Dicentrarchus labrax and Liza ramada in European saltmarshes, which reflected a switch to saltmarsh-derived food sources when they became available (Rozas and LeSalle, 1990; Laffaille et al., 2001; 2002).

It is also noteworthy that A. jacksoniensis did not feed on calanoid copepods, despite their high abundance in zooplankton assemblages (see Chapter Five). Despite the high abundance of calanoid copepods within estuaries globally (Heinle, 1966; Collins and Williams, 1981; Kimmerer and

McKinnon, 1985; Baretta and Malschaert, 1988; Jerling and Wooldridge, 1991; Lawrence et al., 2004

Duggan et al., 2008), their lack of importance in the diets of A. jacksoniensis has also been demonstrated in comparable studies in south-east Australia (e.g. Hollingsworth and Connolly, 2006;

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Mazumder et al., 2006a; Platell and Freewater, 2009; McPhee et al., 2015), which suggests the possibility that A. jacksoniensis are “preferential feeders” or otherwise disinclined to prey upon calanoid copepods (e.g. due to the fast escape responses of calanoids to their predators and the associated energy requirements needed for their capture, and/or perhaps their less obvious colouration (see later); see Pichlová-Ptáčníková and Vanderploeg, 2011). This may also be a result of differing sampling procedures between the two studies for these two different communities

(zooplankton vs fish), which may show some bias when comparing the two studies, i.e., zooplankton assemblages were only sampled from the surficial layer of the water column (where calanoid copepods are often abundant; see Fulton, 1984), while fish were sampled from all depths.

The seasonal difference in stomach fullnesses (which were greater in summer than in winter) was not accompanied by a seasonal difference in the dietary compositions of A. jacksoniensis, suggesting that it feeds on the same prey at these times of the year but ingests greater amounts of prey in summer. This contrasts with the observation that stomach fullnesses of A. jacksoniensis were greater during winter in an estuary (Botany Bay) further south (Mazumder et al., 2006a), which was linked to the greater amount of zoeae released by grapsids during night-time ebb tides. However, as saltmarsh inundation, and thus crab zoeal release, occurs during the day during summer, the pronounced feeding by A. jacksoniensis during summer in the present study may be a result of enhanced feeding activity via visual predation during daylight (Green and Anderson, 1973; Hovel and Morgan, 1997). Such differences highlight the variability of feeding patterns by A. jacksoniensis in different estuarine environments.

6.4.2 Calorimetry of potential prey and interpretation of feeding patterns

This study explored, for the first time, energetic values of Australian estuarine zooplankton and their potential calorimetric contribution to predators (such as A. jacksoniensis), with the view that, when a predator switches prey, this may reflect a greater calorimetric value for that prey, which in turn

134 maximises energy intake per unit effort, as suggested by optimal foraging theory. However, the “prey- switching” by A. jacksoniensis between caridean decapods and crab zoeae was not found to be related to differences in the calorimetric values of these prey, which were similar to each other and another potential prey “type” (calanoid copepods), ranging between 567 to 574 cal g-1. This similarity probably reflects the close relationship (crustaceans) and functional similarity of these taxa in terms of body size, food resources and habitats occupied (Mazumder et al., 2006a; 2008). It is therefore likely that the prey-switching of A. jacksoniensis reflects greater abundances of the preferred prey (i.e. crab zoeae;

Mazumder et al., 2006a; 2008), prey patchiness (e.g. at times of crab zoeal release; Mazumder et al.,

2006a) or other forms of prey behaviour that may make them easier to catch and/or more desirable to the predator (e.g. higher visibility or their orange/red hue; see Green and Anderson, 1973; Hovel and

Morgan, 1997).

Interestingly, the energetic values for these three potential prey “types” were also significantly greater than that for the overall zooplankton assemblage “types,” which implies that A. jacksoniensis is targeting more energetically dense prey, i.e. crustaceans rather than gastropods and cnidarians

(Chapter Five). For example, freshwater Patagonian gastropods had less than half the calorimetric value of crustaceans in the same system, i.e. 1143 vs 2950-3994 cal g-1, respectively (Ciancio and Pascual,

2006).

6.4.3 Conclusion

The present study has demonstrated that A. jacksoniensis, by “switching” to feeding on the zoeae derived from grapsid crabs, with those crabs feeding on detritus and plant material within the saltmarshes of Empire Bay Wetland (Brisbane Water Estuary), is in a strong position to contribute to any trophic relay within this system. Such trophic relay would be furthered by A. jacksoniensis leaving the estuary for adjacent marine waters, which has been recorded for this species in other estuaries in this geographical region (Hadwen and Arthington, 2007). As A. jacksoniensis, in turn, can be ingested

135 by larger predatory fishes such as Platycephalus fuscus, Acanthopagrus australis and Argyrosomus japonicas (which are of economic importance; SPCC, 1981; Baker and Sheaves, 2006; Mazumder et al.,

2006a; Taylor et al., 2006) and piscivorous water birds such as Sterna albifrons (NSWDUAP, 2002) that can move between both estuarine and marine waters (Pollock, 1982; NSWDUAP, 2002; Gray and

Barnes, 2008; Silberschneider and Gray, 2008), any trophic relay is able to be extended from this estuary to the adjacent marine environment.

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CHAPTER SEVEN

GENERAL DISCUSSION

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7.1 Overview of key outcomes of the study

7.1.1 Fish assemblages

The data presented in this thesis has demonstrated that, for a representative saltmarsh (Empire

Bay wetland) in a large south-eastern Australian estuary (Brisbane Water Estuary in NSW), the abundances of fish (and particularly Ambassis jacksoniensis) and number of species of the fish assemblage were greatest during nightly winter catches compared to daily summer catches, irrespective of whether or not the saltmarsh was tidally inundated (see Chapter Three). As many of the species (including A. jacksoniensis, Pelates sexlineatus and Girella tricuspidata) recorded within these fish assemblages are zooplanktivores (Raubenheimer, 2005; Sanchez‐Jerez et al., 2002; McPhee et al.,

2015), this nightly winter peak in fish abundance and number of species could be related to the high densities of zooplankton crab zoeae exported by mangrove and saltmarsh-dwelling grapsid crabs during ebb tides at such times (i.e. during night-time winter particularly following saltmarsh inundation;

Mazumder et al., 2009; but see also Chapter Five), which are, in turn, a known important food source for estuarine fish (including the visual predator, A. jacksoniensis; Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; Platell and Freewater, 2009; McPhee et al., 2015; see also Chapter Six). However, as different patterns in the abundances of fish and the number of species of fish assemblages have also been reported in comparable studies (see Robertson and Duke, 1987; Laegdsgaard and Johnson, 1995), it should be noted that seasonality of fish abundance and number of species in saltmarsh areas is one of many examples (see also below) of ecological variability that occurs among estuaries both globally and within south-east Australia.

In the present study, saltmarsh inundation essentially had no observed effect on either fish abundance (including A. jacksoniensis), number of species nor the composition of the fish assemblages

(see Chapter Three), which contradicts those reported for saltmarsh habitats both within this estuary and elsewhere in Australia (e.g. Connolly et al., 1997; Saintilan et al., 2007; Alderson, 2014). The lack of

138 consistency among studies regarding the effect of saltmarsh inundation on fish assemblages, abundance and number of species also highlights the ecological variability of saltmarsh habitats among estuaries, even within the same geographical area, and is a demonstration of the dangers of making generalisations from local studies.

As trends in fish assemblages, abundance and number of species at saltmarsh locations are often viewed as being influenced by times of high densities of zooplankton export (i.e. night-time winter ebb tides; Mazumder et al., 2006a), it is noteworthy that the effect of newly-inundated saltmarsh vegetation may be of less direct importance to zooplanktivorous fish diets than tidal state (i.e. flood/ebb) or season, as estuarine fish are able to feed at locations not directly within saltmarsh (e.g. adjacent seagrass habitats; McPhee et al., 2015; see also Chapter Six) and from sources that do not require saltmarsh inundation (e.g. mangrove vegetation). Therefore, as the present (Chapter Six) and previous studies (Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; Platell and Freewater,

2009; McPhee et al., 2015) show that the influence of saltmarsh inundation (and its export of highly abundant crab zoeae) is important to the diets of fish (such as A. jacksoniensis), the current research suggests that estuarine fish assemblages, abundances and number of species are more strongly influenced by season, diel (i.e. day and night) and tidal state (i.e. flood and ebb) than the effect of

‘saltmarsh inundation,’ and these trends are likely driven by feeding, shelter-seeking and reproductive behaviours of the various fishes. It is thus also possible that the movements of estuarine fish may also be the result of a behavioural locomotive response to specific environmental stimuli (Krebs and

Kacelnik, 1991), for example flooding tides, due to their potential to provide sheltered habitat and prey availabilities (i.e. feeding opportunities).

As some fish seek shelter, feed and spawn in different habitats (Zimmerman et al., 1984; Rozas and Minello, 1998; Connolly, 1994a; Connolly et al., 1997; Crinall and Hindell, 2004; Mazumder et al.,

2005a; 2006a; Hollingsworth and Connolly, 2006; McNeil et al., 2008; Platell and Freewater, 2009;

McPhee et al., 2015), habitat use by estuarine fishes (including movement or migration among habitats)

139 may be influenced by ontogenetic, reproductive and/or feeding stages within their life cycles, which should be explored to determine which habitat an organism is likely to be utilising at any given time.

Therefore, in terms of conservation of ecologically important habitats, it is important that environmental managers, researchers and policy makers consider the entire life cycles (e.g. spawning and feeding behaviours) of organisms that are ecologically dependent on such habitats, in order to gain a holistic understanding of the ecological requirements for such organisms and the habitats they use.

7.1.2 Life history of Ambassis jacksoniensis

This research explored sex ratios of A. jacksoniensis for the first time and found a heavily female-biased sex ratio during summer that approximately equalised to 1:1 during winter. This pattern was attributed to light and/or seasonal influences on male A. jacksoniensis that may migrate during summer to other areas, such as deeper waters, for shelter from predation (see Power, 1984; Bishop et al., 2001; Ishikawa and Tachihara, 2012), or, alternatively, the estuarine mouth to spawn (see

Miskiewicz, 1987), before their return to Empire Bay Wetland during winter (when there is a lull in spawning; Miskiewicz, 1987; see Chapter Four and below), in order to feed on the saltmarsh-derived zooplankton (e.g. crab zoeae) known to be abundant at these times (Mazumder et al., 2006a; McPhee et al., 2015; but see also Chapters Five and Six). Simultaneously, at such times, female A. jacksoniensis may risk remaining present at the shallower, and more vulnerable, feeding locations that were sampled

(i.e. seagrass, mangrove and saltmarsh vegetation at Empire Bay Wetland) due to increased energetic requirements for reproductive purposes (Mann, 1965), which accounts for their dominance in sex ratios during summer. It is, however, noteworthy that sex ratio is variable among species of ambassids both within Australia and internationally (see Milton and Arthington, 1985; Coates, 1990; Mortuza et al.,

1996; Bishop et al., 2001; Chen and Kuo, 2009; Ishikawa and Tachihara, 2012), and thus generalisations about the sex ratios of A. jacksoniensis (i.e. among estuaries) may not be reasonable until the conduction of comparable studies on the species in other areas.

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This study also explored, for the first time, somatic/reproductive growth of A. jacksoniensis

(including the size at which 50% of female and male A. jacksoniensis reached sexual maturity), and demonstrated that females have faster growth, and increase in reproductive/gonadal weight with length to a greater extent than males (see Chapter Four). This trend may similarly be attributed to the requirement for female fish to allocate more energy to reproductive purposes (Mann, 1965), and which could be related to their dominance within populations of A. jacksoniensis at Empire Bay Wetland during summer (i.e. female A. jacksoniensis prioritise feeding opportunities, over shelter, to allocate more energy for reproduction). However, previous studies on ambassids have shown conflicting trends between sexes for growth (e.g. Milton and Arthington, 1985; Bishop et al., 2001; Ishikawa and

Tachihara, 2012), which precludes any generalisations for this family.

The gonads of A. jacksoniensis were recorded as being generally maturing and ripe during summer (indicative of spawning during summer), and juvenile/inactive and spent during winter

(indicative of a lull in spawning). This supports previous evidence that A. jacksoniensis are highly reproductive, multiple spawners throughout summer months, with a lull during winter (Miskiewicz,

1987), with such multiple spawning considered to occur at estuarine mouths (see Miskiewicz, 1987).

These features help explain the high abundance of A. jacksoniensis (Saintilan, 2009), as well as its wide distribution throughout south-east Australian estuaries (Mills et al., 2008). Moreover, by determining when fish reproduce, linkages can be made to their behaviours at other times (e.g. during times of feeding or migration). For example, Atlantic silverside, Menidia menidia, prioritise spawning occasions to times that least interfere with feeding times (Conover and Kynard, 1984).

It is thus possible that patterns in the timing of spawning for A. jacksoniensis may be linked to the important feeding events known for this species during nightly winter saltmarsh inundation in other estuaries (Mazumder et al., 2006a; Hollingsworth and Connolly, 2006; McPhee et al., 2015), where such feeding provides the energetic requirements for reproduction (Mann, 1965; Deady and Fives, 1995;

Denny and Schiel, 2001). Therefore, the abiotic function of spring tides (and its association to saltmarsh

141 inundation) may indirectly govern the spawning behaviour and seasonality of A. jacksoniensis via the temporally sparse feeding opportunities from saltmarsh vegetation (and its inhabitants) during the ebbing spring tides that follow its inundation (see Chapter Six). It is thus relevant that reproductive cycles and spawning times for many fishes are seasonally dependent (e.g. Ochi, 1986; Robertson et al.,

1990), or may occur more or less periodically as a result of other influences (Conover and Kynard, 1984;

Thresher, 1984), such as time of day (e.g. in nocturnal spawners) or moon/tidal phases (e.g. Amphiprion melanopus; Ross, 1978), the latter of which governs saltmarsh inundation in south-east Australia. It is noteworthy, however, that as feeding behaviours of A. jacksoniensis are inconsistent among estuaries and studies (see later), factors unrelated to feeding (such as water temperature; see Milton and

Arthington, 1985; Bishop et al., 2001; Ishikawa and Tachihara, 2012), or offspring survival and recruitment (see Lambert and Ware, 1984), may alternatively, or simultaneously, be influencing the life history (e.g. reproductive cycles) of A. jacksoniensis.

The findings in the current research are a reminder of the potential (and geographically variable) contribution of saltmarsh (i.e. Empire Bay Wetland) as a habitat and provision to the life history needs of estuarine fish (such as A. jacksoniensis). Such provisions include shelter, sources of food and foraging areas (for both juvenile and adult fish), which are essential for the life cycles

(including reproductive and recruitment aspects; Mann, 1965) of estuarine fishes. Estuarine fishes using such provisions, can in turn contribute to ‘trophic relay’ (Kneib, 1997; 2000; Nagelkerken, 2009;

McPhee et al., 2015), via their role as prey to trophically higher species, some of which are of economic importance (see SPCC, 1981; Taylor et al., 2006), and are thus an important component of estuarine ecology. In general, A. jacksoniensis are a highly-reproductive fish species that are likely to obtain the energetic requirements for multiple spawning by feeding upon high abundances of saltmarsh-derived zooplankton (such as grapsid crab zoeae) during spring tide inundation events. As itinerant estuarine fish (such as the highly abundant A. jacksoniensis) are in turn preyed upon by trophically higher organisms (some of which are economically important; SPCC, 1981; Taylor et al., 2006), and therefore contribute to trophic relay (Kneib, 1997; 2000; Nagelkerken, 2009; McPhee et al., 2015), such reliance

142 on saltmarsh habitats thus highlights the importance of A. jacksoniensis, grapsid crabs and saltmarsh vegetation to the natural health and functioning of estuarine and adjacent ecosystems.

7.1.3 Zooplankton assemblages

Sampling of the zooplankton assemblages showed that calanoid copepods were the most abundant taxa within zooplankton assemblages at Empire Bay Wetland (see Chapter Five), and this dominance has been recorded in estuaries within Australia and elsewhere (Kimmerer and McKinnon,

1985; Duggan et al., 2008) and elsewhere (Heinle, 1966; Collins and Williams, 1981; Baretta and

Malschaert, 1988; Jerling and Wooldridge, 1991; Lawrence et al., 2004). The high abundance of calanoid copepods was most pronounced during flood tides, with such patterns being apparently unaffected by the occurrence of saltmarsh inundation. Nevertheless, crab zoeae were also highly abundant in general and were the dominant zooplankton organisms during ebb tides, particularly following saltmarsh inundation. This is in support of the model proposed by Mazumder et al. (2009), whereby saltmarsh-dwelling grapsid crabs release high numbers of larvae (zoeae) into ebb tides following saltmarsh inundation during the spring tidal cycle.

In contrast to previous findings (e.g. Mazumder et al., 2009; Freewater et al., 2009), the current research found that overall zooplankton abundance (but not specifically crab zoeal abundance) was unaffected by neither tidal state (flood and ebb) nor the presence of saltmarsh inundation, and this trend was attributed to the continuously high numbers of calanoid copepods that were likely derived from non-saltmarsh sources (such as seagrass), and thus present within the zooplankton at all times. It is thus noteworthy that populations of calanoid copepods have shown seasonal stability under varying levels of both salinity (e.g. tidally influenced) and nitrogen loads/chlorophyll levels (Lawrence et al.,

2004), which demonstrates their capability to sustain their abundances under varying environmental conditions.

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Although previous studies (e.g. in Botany Bay; Mazumder et al., 2006a) demonstrated that crab zoeae were released in high numbers during nightly winter ebb tides following saltmarsh inundation

(which was attributed to a survival strategy to counteract predation by visual predators; Green and

Anderson, 1973; Hovel and Morgan 1997), the current research found that season/time of saltmarsh inundation did not affect crab zoeal (or other zooplankton) abundance at Empire Bay Wetland. This suggests that tidal regimes (i.e. the inundation of the saltmarsh vegetation and the following ebbing tides that drain it) were more influential than seasonal differences to saltmarsh-dwelling crab zoeal release. Notably, Robertson et al. (1988) found no relationships for zooplankton composition and concentration with salinity, temperature, mangrove litter or fish predation, suggesting that environmental variables have little effect on zooplankton assemblages, with tidal regimes being the most influential factor (Robertson et al., 1988; Rountree and Able, 2007). It is also noteworthy that, of all the organisms that comprise estuarine zooplankton assemblages, crab zoeae are thought to have the most variable densities (Christy and Stancyk, 1982; Pittman and McAlpine, 2003). The inconsistency of the current findings with previous studies (i.e. effect of tidal state/saltmarsh inundation on zooplankton abundance and seasonality of crab zoeal abundance; see Mazumder et al., 2009;

Freewater et al., 2009) further stresses the need to be cautious when generalising results among estuaries within south-east Australia.

In general, the research demonstrates that saltmarsh-derived crab zoeae comprise an important part of the zooplankton assemblages at Empire Bay Wetland, particularly during ebb tides following saltmarsh inundation. With crab zoeae being a crucial food source for itinerant estuarine fish

(e.g. A. jacksoniensis; Hollingsworth and Connolly, 2006; Mazumder et al., 2006a; Platell and Freewater,

2009; McPhee et al., 2015; see also Chapter Six), this highlights the importance of saltmarsh vegetation as a habitat for saltmarsh-dwelling invertebrates (e.g. grapsid crabs) that contribute to trophic relay and play an important role in estuarine ecosystems.

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7.1.4 Feeding relationships between Ambassis jacksoniensis and crab zoeae

This study confirmed that the very abundant A. jacksoniensis feed on saltmarsh-derived crab zoeae at Empire Bay Wetland, particularly during ebbing tides that follow saltmarsh inundation (when crab zoeae also dominated zooplankton assemblages; see Chapter Five). The feeding patterns found for A. jacksoniensis in the present study are consistent with findings for saltmarshes in the same (i.e.

Brisbane Water Estuary), nearby and other Australian estuaries (Hollingsworth and Connolly, 2006;

Mazumder et al., 2006a; Platell and Freewater, 2009; McPhee et al., 2015). Interestingly, A. jacksoniensis fed greatly on caridean decapods at times when crab zoeae were not abundant or expected to be found within zooplankton assemblages (i.e. during flood tides and during ebb tides that did not inundate saltmarsh), and thus “switched” prey to crab zoeae when they were available to feed on. A similar feeding pattern was found for A. jacksoniensis in a nearby estuary where diets switched from thalassinid larvae to crab zoeae following saltmarsh inundation (McPhee et al., 2015). Feeding patterns, in which fish switch to feeding on saltmarsh-derived food sources when they become available, have also been found in North American and European saltmarshes (Rozas and LeSalle, 1990;

Laffaille et al., 2001; 2002). Further, based on stomach fullnesses, A. jacksoniensis fed to a far greater extent on ebb than flood tides, irrespective of whether the saltmarsh habitat was inundated, and this pattern has also been observed for the same species in a nearby south-eastern Australian estuary

(McPhee et al., 2015), as well as for another saltmarsh-inhabiting fish species (Fundulus heteroclitus) in eastern North America (Weisberg et al., 1981). In general, despite recurring evidence among south- east Australian estuaries that A. jacksoniensis feed on saltmarsh-derived crab zoeae (see Hollingsworth and Connolly, 2006; Mazumder et al., 2006a; Platell and Freewater, 2009; McPhee et al., 2015), there are still notable differences in their specific diets both within and among estuaries. It is thus likely that these differences in prey-switching and diets of A. jacksoniensis reflect differences in prey availability in the different environments and geographical locations, which means that any conclusions about the specific feeding behaviours, or life history traits, of A. jacksoniensis in different environments should be made carefully.

145

Ambassis jacksoniensis fed predominantly on small, planktonic crustaceans (e.g. crab zoeae and caridean decapods), similar to other freshwater ambassids, such as Ambassis agassizi and Ambassis agrammus, that feed on microcrustaceans (e.g. cladocerans, copepods, ostracods and conchostracans), insect larvae (e.g. dipteran, ephemeroptan and trichopteran larvae (Pollard, 1974; Sanderson, 1979;

Bishop et al., 2001; Medeiros, 2004; Lintermans, 2007). As A. jacksoniensis feed on saltmarsh-derived crab zoeae (the latter of which similarly use saltmarsh vegetation as a habitat and source of food), and are, in turn, also preyed upon by trophically higher organisms (some of which are of economic importance; SPCC, 1981; Taylor et al., 2006), this fish species is well placed, therefore, to contribute to any potential trophic relay (Kneib, 1997; 2000; Nagelkerken, 2009), via such prey-switching, from estuaries to their adjacent marine waters. It is noteworthy that as A. jacksoniensis are multiple spawners (Miskiewicz, 1987; see also Chapter Four), and are therefore in frequent possession of ripe, summer gonads, this species’ provision as an energy source for predators (which contribute to trophic relay), is further supported by the fact that their gonads are likely often calorimetrically high. For example, calorimetric values of flounder, Platichthys flesus (in the Ythan estuary of Scotland), were higher prior to, compared to after, spawning times (Summers, 1979), indicating that ripe fish prior to spawning would provide an energetically higher food source to predators than spent fish after spawning.

The seasonal differences in stomach fullnesses, which consisted of greater values in summer than in winter (similarly seen for other fish species, e.g. Notolabrus fucicola (Denny and Schiel, 2001),

N. celidotus (see Jones, 1980; 1984)), were not accompanied by a seasonal difference in the dietary compositions of A. jacksoniensis, suggesting that it feeds on the same prey at these times of the year but ingests greater amounts of prey during summer. This finding is in direct contrast to previous studies, which showed that stomach fullnesses of A. jacksoniensis are greater in winter (e.g. in Botany Bay further south; Mazumder et al., 2006a), and which was attributed to greater zoeal release by grapsids during night-time ebb tides (i.e. winter) as a strategy to counteract visual predators such as

A. jacksoniensis. It is thus noteworthy that the current research (see Chapter Five) found that season

146 did not affect zooplankton abundance (including crab zoeae), with tidal state (flood/ebb) and saltmarsh inundation being the most influential factors. Therefore, as seasonality did not govern crab zoeal abundance at Empire Bay Wetland, it is reasonable that A. jacksoniensis fed more greatly on saltmarsh- derived crab zoeae during daylight (i.e. summer) ebb tides following saltmarsh inundation than during comparable night-time ebb tides (i.e. winter), as they are likely to be visual predators (see Green and

Anderson, 1973; Hovel and Morgan, 1997). Additionally, pronounced day-time feeding by

A. jacksoniensis may also be attributed to patterns in prey activity (Kingett and Choat, 1981; Jones

1988), seasonal differences in water temperatures (Denny and Schiel, 2001; note that the present study found a marked difference in water temperatures at Empire Bay Wetland, i.e. 14-15 C̊ in winter vs 25-

26 C̊ in summer) and/or reproductive activity. The reproductive activity for some fishes can lead to decreased feeding intensity during spawning seasons that at other, non-spawning seasons, feed more intensely in order to accumulate fat/energy reserves for spawning times (Deady and Fives, 1995; Denny and Schiel, 2001; see also below), and this may indeed be a reproductive/feeding behaviour of

A. jacksoniensis at Empire Bay Wetland.

The research also explored, for the first time, energetic values of Australian estuarine zooplankton and their potential calorimetric contribution to predators (such as A. jacksoniensis), with the view that, when a predator switches prey, this may reflect a greater calorimetric value for that prey, which in turn maximises energy intake per unit effort, as suggested by optimal foraging theory.

However, the “prey-switching” by A. jacksoniensis between caridean decapods and crab zoeae was unrelated to the calorimetric values of these prey, which were similar to each other and another potential prey “type” (calanoid copepods; ranging between 567 and 574 cal g-1). This calorimetric similarity has been attributed to the close relationship (crustaceans) and functional similarity of these taxa in terms of body size, food resources and habitats occupied (Mazumder et al., 2006a; 2009). The prey-switching of A. jacksoniensis has thus been attributed to greater abundances of the preferred prey during ebbing spring tides (i.e. crab zoeae; Mazumder et al., 2006a; 2009; see also Chapter Five), prey patchiness (e.g. at times of crab zoeal release; Mazumder et al., 2006a) or other forms of prey

147 behaviour that may make them easier to catch and/or more desirable to the predator (e.g. higher visibility or their red/orange hue, which is supported by their increased feeding during daylight/summer due to visual predation; see above; Green and Anderson, 1973; Hovel and Morgan, 1997). Furthermore, highly abundant taxa (such as crab zoeae) are often generally the main source of energy within ecosystems and can ultimately govern assemblage structures (Gentry and Dodson, 1987).

Overall, calorimetric values of estuarine zooplankton in this study were lower than those of similar organisms from other ecological systems (e.g. Patagonian freshwater crustaceans in Ciancio and

Pascual (2006)). To the best of my knowledge, no analogous studies on calorimetry of estuarine zooplankton have been conducted, and it is difficult to determine why calorimetric values in the present study are comparably so low. The reasons may include differences in energy transfer among organisms between systems, intrinsic differences in the ecology (and thus energy assimilation and transfer) of these different organisms or systems (for examples see Hairston and Hairston, 1993), or life stages of these organisms (i.e. I analysed larval stages while Ciancio and Pascual (2006) analysed adult stage crustaceans). For example, crustacean calorimetric densities can vary ontogenetically throughout life stages as a result of reproduction and development (e.g. Mysis relicta; Hakala, 1979). Crustacean calorimetric densities also vary with sex (Hakala, 1979), a variable not considered (nor possible) in the present study. Calorimetric values of the same crustacean species from different locations and studies have also been shown to be variable, where for example values of Mysis mixta range between approximately 3609 and 4924 cal/g (e.g. Rumohr et al., 1987; Wiktor and Szaniawska, 1988; Gorokhova and Hansson, 2000).

From a broader, estuarine trophodynamic perspective, it is also possible that predatory fishes in systems with calorimetrically higher crustaceans (e.g. freshwater Patagonian systems) may eat lower abundances of such prey (i.e. have lower stomach fullnesses) and thus ingest similar amounts of calories compared to predatory fishes at Empire Bay Wetland that may eat higher abundances of zooplankton

(and that have comparatively higher stomach fullnesses). Research into such differences, as well as

148 energy assimilation by zooplankton predators (e.g. A. jacksoniensis), is required in order to gain a more complete understanding of energy transfer within these systems. Investigations of potential specific nutritional contributions of prey to predators (e.g. determination of percentages of protein, fat and carbohydrates within body tissues of organisms via proximate composition) may provide further insights into predatory gains among different prey.

It is also significant that the A. jacksoniensis shown to feed and contribute to trophic relay at

Empire Bay Wetland were of a small size and young age (compared to other individuals sampled in another study elsewhere in Brisbane Water Estuary; see Platell and Freewater, 2009). This implies that

A. jacksoniensis (which reach sexual maturity within the first year of its life; Mazumder et al., 2008) that rely on and relay saltmarsh-derived energy (i.e. organic matter via trophic relay) at Empire Bay Wetland, are the same individuals that are reproductively contributing to the population (as demonstrated by the current research; see Chapter Four). As some fish prioritise spawning occasions to times that least interfere with feeding times (e.g. Atlantic silverside, Menidia menidia; Conover and Kynard, 1984), and such feeding fulfils energetic requirements for reproductive purposes via the accumulation of fat/energy reserves (Mann, 1965; Deady and Fives, 1995; Denny and Schiel, 2001), it is possible that the reproductive timing (i.e. spawning) of A. jacksoniensis is governed by the important feeding on saltmarsh-derived zooplankton (i.e. crab zoeae) within ebbing tides that drain saltmarsh vegetation during the spring tidal cycle. This theory is supported by evidence that A. jacksoniensis in another estuary predominantly feed upon saltmarsh-derived crab zoeae during winter saltmarsh inundation events (Mazumder et al., 2006a), which was at a time (i.e. winter) where a lull in spawning is known for the species (Miskiewicz, 1987; see also Chapter Four). It is therefore relevant that in the present study,

A. jacksoniensis were more abundant in night-time winter fish assemblages than day-time summer assemblages (see Chapter Three), and that at both times, pronounced feeding upon saltmarsh-derived crab zoeae was evident (see Chapter Six).

149

Feeding times of some fish have been linked to strategies to maximise offspring growth and survival (e.g. when food is abundant or when predation pressures are low; Ochi, 1986). Therefore, it could also be hypothesised that spawning seasons of A. jacksoniensis (i.e. summer in the present study

– Chapter Four; see also Miskiewicz, 1987) may coincide with their preferred feeding times (i.e. also summer in the present study) as a strategy to allow A. jacksoniensis larvae to similarly use visual predatory behaviours to feed on high abundances of saltmarsh-derived zooplankton during day-time ebbing spring tides. Thus, whether populations of A. jacksoniensis feed more greatly during night-time winter (e.g. Mazumder et al, 2006a) or day-time summer (e.g. the present study) spring tidal cycles, the timing of reproductive spawning for the species may in either case be governed by tidally controlled inundation of saltmarsh, and its associated provision of a food source. Further, the timing of feeding

(and indirectly, spawning), for A. jacksoniensis, may similarly be the result of a behavioural locomotive response to specific environmental stimuli (e.g. flooding spring tides that result in high prey availabilities; Krebs and Kacelnik, 1991). These links (and their associated hypotheses) between the feeding and reproductive traits of A. jacksoniensis (both of which suggest the governing of spawning times by tidally controlled saltmarsh feeding), highlights the importance of saltmarsh habitats (including their role as a food source) to the sustainability and overall ecological functioning of estuaries and their adjacent marine waters.

7.2 General conclusions

The present research has therefore demonstrated that A. jacksoniensis, by “switching” to feeding on the zoeae derived from grapsid crabs, with those crabs living within and feeding on fine benthic organic material and microphytobenthos within the saltmarshes of Empire Bay Wetland,

Brisbane Water Estuary (Mazumder and Saintilan, 2010; Alderson et al., 2013), is in a strong position to contribute to any trophic relay within this system. Such trophic relay would be furthered by A.

150 jacksoniensis leaving the estuary for adjacent marine waters, which has been recorded for this species in other estuaries in this geographical region (Hadwen and Arthington, 2007). As A. jacksoniensis, in turn, can be ingested by larger predatory fishes (some of which are of economic importance) such as

Platycephalus fuscus, Acanthopagrus australis and Argyrosomus japonicas (SPCC, 1981; Baker and

Sheaves, 2006; Mazumder et al., 2006a; Taylor et al., 2006) and piscivorous water birds such as Sterna albifrons (NSWDUAP, 2002) that can move between both estuarine and marine waters (Pollock, 1982;

NSWDUAP, 2002; Gray and Barnes, 2008; Silberschneider and Gray, 2008), any trophic relay would be expanded from this estuary to the adjacent marine environment.

Moreover, as the A. jacksoniensis that are contributing to trophic relay within and beyond

Empire Bay Wetland (i.e. via their dietary association with saltmarsh-derived prey), are also reproductively contributing to their large population (the timing of which may be governed by such tidally controlled saltmarsh feeding), this research, therefore, further highlights the importance of saltmarsh vegetation to the ecology of south-east Australian estuaries. As saltmarsh vegetation is in decline both globally and in Australia (Saintilan and Williams, 1999; Adam, 2009; Saintilan and Rogers,

2013), and its loss as a habitat can severely affect the organisms and ecosystems that rely on them

(e.g. crab zoeae and A. jacksoniensis; see also Saintilan and Williams, 1999; Laegdsgaard, 2006), the current research strongly demonstrates that appropriate conservation and management of Australian saltmarshes (such as Empire Bay Wetland) is imperative in order to maintain the natural health and functioning of estuaries.

7.3 Recommendations for future research

The research presented within this thesis contributes to our current understanding of the role of south-east Australian saltmarsh vegetation as a habitat and food source that facilitates (and potentially governs) trophic relay and reproductive output from grapsid crabs and fish, which provides

151 productive and naturally functioning estuarine ecosystems. However, due to the complexity of these systems (including the non-generality of many of its processes), several gaps in knowledge still remain before a complete understanding of its functioning can be obtained.

The current study sampled both at times of saltmarsh inundation and non-inundation for all of its main chapters in order to test the theory that differences in samples at Empire Bay Wetland between flood and ebb tides may have simply been a function of high tides rather than any relationship to saltmarsh inundation. However, due to the semi-diurnal location of the study and its presence of two high tides per day, only one of these high tides were sampled per day as just one typically inundated saltmarsh vegetation on any given day (i.e. the day-time high tide during summer and the night-time high tide during winter). In order to disentangle the confounding factor of season and day/night high tide (including its inundation of saltmarsh), further research can compare samples from both high tides on any given day.

The current study supports prior evidence that A. jacksoniensis are highly abundant (Mazumder et al., 2006a; Platell and Freewater, 2009), multiple-spawners (with a lull during winter; Miskiewicz,

1987; Mills et al., 2008), however prior studies also suggest they are near-shore marine-spawners

(Miskiewicz, 1987; Mills et al., 2008). As A. jacksoniensis are known to reside in saltmarsh, mangrove and seagrass habitats (located in the main body of estuaries; Mazumder, 2004; Mazumder et al., 2006a;

Hollingsworth and Connolly 2006; McPhee et al., 2015) the current and prior (e.g. Miskiewicz, 1987) studies on the life history and reproduction of A. jacksoniensis suggest they may migrate to estuary mouths to spawn. Thus, future research on migration patterns of A. jacksoniensis from wetlands (i.e. saltmarsh, mangrove and seagrass habitats) to estuarine mouths to spawn, may elucidate our understanding of linkages between feeding and reproductive behaviours of the species (especially during summer when spawning appears more prevalent and sex ratios were female biased; Miskiewicz,

1987). Further, the study only obtained sampled of a very narrow narrow size range (36-38 mm SL) of

A. jacksoniensis. Future studies that sample A. jacksoniensis of size ranges beyond those sampled within

152 the study, as well as other fish species present at the study site and elsewhere in the estuary, would allow for more general insight into the life history and diets of the fish fauna at Empire Bay Wetland.

The present study also demonstrated that A. jacksoniensis feed on saltmarsh-derived zooplankton, which was important for growth and allocation of energy for reproductive purposes

(Mann, 1965; Deady and Fives, 1995; Denny and Schiel, 2001). As the timing of feeding behaviours for fish have been linked to strategies to maximise offspring growth and survival (e.g. when food is abundant or when predation pressures are low; Ochi, 1986), it could alternatively be hypothesised that spawning seasons (i.e. summer in the present study) may coincide with preferred feeding times

(i.e. summer in the present study) as a strategy to allow A. jacksoniensis larvae to similarly use visual predatory behaviours to feed on high abundances of saltmarsh-derived zooplankton. Future studies on the diets of A. jacksoniensis larval offspring (including feeding behaviours and any seasonal effects associated with this) can be considered in order to test hypotheses regarding increased spawning of the species during times of high zooplankton abundance (i.e. during summer), as a strategy to increase offspring growth rates (see also Lambert 1984). Further, investigation into comparisons between the life-history patterns and food consumption of A. jacksoniensis would allow for linkages to be made between the reproductive cycles and diets of the species.

Further, as sex ratios in the current study were female biased during summer, several plausible alternative locations for the unaccounted male A. jacksoniensis were considered (e.g. deeper, less vulnerable water or the estuarine mouth for spawning), however one other possible (albeit less plausible) location was not provided; within the shallow waters of the inundated saltmarsh. Although this possible explanation could be confirmed via sampling of A. jacksoniensis using fyke nets within the saltmarsh vegetation at Empire Bay Wetland, it is unlikely based on the findings of the current study, as a female biased sex ratio was still evident in summer during times of no saltmarsh inundation (i.e. at times when the saltmarsh vegetation was an unavailable habitat for fish). NB: sampling of fish by fyke net within saltmarsh was not conducted in the current study as comparisons of tidal states between

153 days of saltmarsh inundation and non-inundation were a major focus of the research (which would not be possible via fyke net sampling within the saltmarsh vegetation).

Known predators of A. jacksoniensis include fish species Acanthopagrus australis, Platycephalus fuscus and Argyrosomus japonicus (SPCC, 1981; Taylor et al., 2006; Mazumder et al., 2006a), as well as some piscivorous water birds (e.g. Sternula albifrons; NSWDUAP, 2002), however information on the predators of A. jacksoniensis is based on very limited evidence and remains an area for further research.

Investigation into the dietary compositions of these predatory species, other piscivorous fishes and the zooplankton, as well as the potential calorimetric contribution of A. jacksoniensis to their predators, would provide further insight into the role of A. jacksoniensis and other fauna in the facilitation of trophic relay within south-east Australian estuaries. Such insights would also be highlighted via the use stable isotope and fatty acid analyses, which could provide information on the sources and sinks involved in trophic relay between estuarine habitats and predators within Brisbane Water Estuary.

154

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APPENDIX A

Trophic relay and prey switching - a stomach contents and

calorimetric investigation of

an ambassid fish and their saltmarsh prey

Jack J. McPheeA,C*, Margaret E. PlatellA, and Maria J. SchreiderA.

ASchool of Environmental and Life Sciences, University of Newcastle, PO Box 127, Ourimbah, New South Wales, 2258

CCorresponding author. Email: [email protected]

Running head: Trophic relay and prey switching in an ambassid fish

181

Estuarine, Coastal and Shelf Science 167 (2015) 67e74

Contents lists available at ScienceDirect

Estuarine, Coastal and Shelf Science

journal homepage: www.elsevier.com/locate/ecss

Trophic relay and prey switching e A stomach contents and calorimetric investigation of an ambassid ﬁsh and their saltmarsh prey

* Jack J. McPhee , Margaret E. Platell, Maria J. Schreider

School of Environmental and Life Sciences, University of Newcastle, PO Box 127, Ourimbah, New South Wales 2258, Australia article info abstract

Article history: Trophic relay is an ecological model that involves the movement of biomass and energy from vegetation, Received 2 October 2014 such as saltmarshes, within estuaries to the open sea via a series of predator-prey relationships. Any Received in revised form potential for trophic relay is therefore affected by water movements within an estuary and by the ability 30 June 2015 of a predator to “switch” prey in response to fluctuating abundances of those prey. Saltmarsh-dwelling Accepted 12 July 2015 grapsid crabs, which feed on saltmarsh-derived detritus and microphytobenthos, release zoeae into Available online 14 July 2015 ebbing tides that inundate saltmarshes during spring-tide cycles within tidally-dominated estuaries, such as Brisbane Water Estuary, therefore providing an opportunity to examine whether prey-switching Keywords: fi Dietary compositions and/or trophic relay may occur in sh that feed on those zoeae (such as the highly abundant estuarine Coastal wetlands ambassid, Ambassis jacksoniensis). This model was examined by sampling A. jacksoniensis near salt- Mangroves marshes in a large, temperate south-eastern Australian estuary during flood and ebb tides on days of Estuaries saltmarsh inundation and non-inundation over four spring-tide events in 2012. Stomach fullnesses of Australia A. jacksoniensis were generally highest during ebb tides on days of saltmarsh inundation, implying that feeding was most marked at these times. Caridean decapods dominated diets during flood tides and on days of no saltmarsh inundation, while crab zoeae dominated diets during ebb tides and on days of inundation, suggesting that, when saltmarsh-derived zoeae became abundant, A. jacksoniensis switched to feeding on those prey. Three potential zooplankton prey (calanoid copepods, caridean decapods and crab zoeae) did not differ calorimetrically, indicating that switching of prey by A. jacksoniensis is not directly related to their preying on energetically greater prey, but reflects opportunistic feeding on more abundant and/or less elusive prey. As A. jacksoniensis is able to switch prey from estuarine caridean decapods to saltmarsh-derived crab zoeae, this very abundant ambassid would be well-placed to promote any trophic relay, via further water movements or other predator-prey relationships, to the adjacent marine environment. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction et al., 1998; Fry et al., 1999; Adnan et al., 2002; Melville and Connolly, 2003), is likely to be a feature of those estuaries. ‘Trophic relay’ is an ecological model in which the biomass and Although the process and extent of trophic relay is driven by energy obtained by organisms feeding on and within estuarine predator-prey relationships, it would be affected by water move- vegetation, is transported by nektonic predators such as fish and ments within an estuarine system, which, in turn, are influenced by mobile crustaceans, from the upper limits of estuaries down to factors such as the volumes of freshwater flow, characteristics of their lower reaches and then out to the open sea (Kneib,1997, 2000; the estuarine mouth and tidal movements (Kneib, 1997). In the Nagelkerken, 2009). The concept of trophic relay has important context of tidal movement, the fauna within the saltmarsh vege- implications for the functioning of such estuarine ecosystems and, tation that often fringes the shoreline of estuaries provides an based on assessments of predator-prey relationships via stomach- important food source for estuarine fish when tidally inundated content and stable isotope analyses in Australian estuaries (Vance and would therefore promote trophic relay within such estuaries (Weisberg et al., 1981; Morton et al., 1987; Sumpton and Greenwood, 1990; West and Zedler, 2000; Laffaille et al., 2002; Platell and Freewater, 2009). However, the extent of any potential * Corresponding author. E-mail address: [email protected] (J.J. McPhee). trophic relay via saltmarshes will be affected by the frequency of http://dx.doi.org/10.1016/j.ecss.2015.07.008 0272-7714/© 2015 Elsevier Ltd. All rights reserved. 68 J.J. McPhee et al. / Estuarine, Coastal and Shelf Science 167 (2015) 67e74 tidal inundation, which varies geographically. For example, salt- fullnesses and dietary compositions, using stomach-content ana- marshes in northern America are typically inundated up to twice lyses, of A. jacksoniensis, focussing on any differences between daily, while those in Europe (with some exceptions; see Bakker, seasons, tidal states (i.e. flood vs ebb) and taking into account 2014), South America and eastern Australia, the last of which are whether or not the saltmarsh is inundated, and 2) ascertain the often bordered by mangroves (Wilson and Whittaker, 1995), are potential energetic contribution (calorimetric value) of possible typically inundated during only three to four days of each lunar zooplankton prey to the diets of A. jacksoniensis. With respect to the cycle, i.e. during the spring-tide cycle (e.g. Laffaille et al., 2001; first objective, it is firstly hypothesised that the dietary composi- Thomas and Connolly, 2001; Costa et al., 2003; Hollingsworth tions of A. jacksoniensis differ between varying tidal states and and Connolly, 2006; Daleo et al., 2009). seasons (summer and winter). It is also predicted that saltmarsh- Variations in the timing of inundation of saltmarshes (i.e. day derived crab zoeae contribute greatly to diets of A. jacksoniensis or night) during the year in Europe and Australia, also have im- at times when they are present in zooplankton (i.e. ebbing tides plications for any trophic relay driven by prey derived from those after saltmarsh inundation). environments. For example, on the east coast of Australia, the semi-diurnal spring tides inundate saltmarsh during the day in 2. Materials and methods summer, while such indundation occurs during the night in winter. In estuaries of this region during winter, a saltmarsh 2.1. Study site and environmental variables grapsid crab (Helograpsus haswellianus) that feeds primarily upon fine benthic organic material and microphytobenthos within Empire Bay Wetland (~332902500S, 1512101900E) represents a saltmarsh vegetation (Mazumder and Saintilan, 2010; Alderson saltmarsh habitat that is situated approximately 4e5 km from the et al., 2013), releases zoeae into nightly ebb tides following salt- entrance of the permanently-open Brisbane Water Estuary, on the marsh inundation, with zoeae becoming particularly abundant temperate, south-eastern coast of Australia. Brisbane Water Estuary within adjacent zooplankton assemblages (Mazumder and is a tidally-dominated shallow inlet, with a narrow entrance Saintilan, 2003; Hollingsworth and Connolly, 2006; Mazumder (~150 m wide) and a main tidal channel that separates into several et al., 2006), and thus promoting any potential for trophic relay. basins/water bodies at distances of 6e8 km inland, and has Furthermore, the release of zoeae from other common saltmarsh- maximum water depths of 5e6 m in the main water body (Ford dwelling grapsids, such as Heloecius cordiformis, Parasesarma et al., 2006; Cardno Lawson Treloar, 2008). The tidal range of erythrodactyla and Paragrapsus laevis, from saltmarshes following Brisbane Water Estuary is approximately 1 m during most of the tidal inundation during winter and at other times of the year year and, like other estuaries in eastern Australia, such tides are (Mazumder et al., 2006, 2009) would also be likely to promote semi-diurnal (Ford et al., 2006). The spring-tidal cycle in this trophic relay during those times. environment, which includes tidal heights > ca 1.8 m Australian Any temporal and spatial variability in prey availability, such as Height Datum (AHD), typically occurs over three or more consec- zoeal release by crabs into the zooplankton, can provide an op- utive days or nights in each lunar month, and results in the inun- portunity for “switching” of prey by estuarine predators (Murdoch dation of saltmarsh within Empire Bay Wetland where the study is et al., 1975; Ringler, 1985; Suryan et al., 2000; Siddon and Witman, conducted. Thus, the highest tides (and therefore saltmarsh inun- 2004). For example, McPhee et al. (2015) found that, in autumn, the dation) occurred during the day in summer and at night during small ambassid Ambassis jacksoniensis, which is particularly abun- winter. dant in estuaries of south-eastern Australia (Saintilan, 2009), The catchment of Brisbane Water Estuary has a total area of switched feeding from thalassinid larvae to crab zoeae in a salt- 165 km2 with land uses that range from urban to semi-rural, and marsh environment when those zoeae became abundant following is managed by the New South Wales state body, the Hunter saltmarsh inundation. Knowledge of prey-switching has implica- Central Rivers Catchment Management Authority (Cardno tions for trophic relay and can also lead to insights into the feeding Lawson Treloar, 2008). Much of the foreshore of this estuary strategies used by various species (i.e. preferential vs opportunistic has been urbanised, although there are still portions of foreshore feeding; sensu Wassenberg, 1990), and may be linked to a max- that are reserves and National Parks, which includes the Empire imisation of energetic gain among different available prey (e.g. Bay Wetland study site (Cardno Lawson Treloar, 2008). Fresh- Bittar et al., 2012). In the latter case, it is possible to determine, via water output is derived from the nearby Kincumber Creek, which calorimetry, the energetic density of different prey types and thus lies ca 4 km away (Cardno Lawson Treloar, 2008), as well as Erina the potential energetic value of those prey to their likely predators Creek and Narara Creek. (e.g. Robbins, 1983; Benoit-Bird, 2004; Wuenschel et al., 2006). Typical of south-eastern Australian saltmarshes, mangroves Despite the importance of zooplankton to the diets of estua- (Avicennia marina var. australasica and Aegiceras corniculatum) lie rine predators, including both adult and their larval stages between the open water and the saltmarsh at Empire Bay Wetland (Weisberg et al., 1981; Kneib, 1997; Loneragan et al., 1997; (Cardno Lawson Treloar, 2008; NPWS, 2009). Low-lying saltmarsh Laffaille et al., 2001; Hollingsworth and Connolly, 2006; plants such as Sarcocornia quinqueflora, Sporobolus virginicus and Mazumder et al., 2006; Svensson et al., 2007; Platell and Triglochin striatum are prevalent in the low marsh, while taller Freewater, 2009), little is known of the potential energetic plants (Juncus kraussii and Suaeda australis) are abundant in the contribution of different zooplankton prey to their predators, and high marsh (Harty, 1994; Roberts and Sainty, 2005; Cardno Lawson particularly for those in estuaries, including saltmarshes. The Treloar, 2008; NPWS, 2009). Intertidally, the substratum at Empire collation of such information, along with understanding of the Bay Wetland consists largely of bare sand/mud-flats and mangrove dietary composition of key predators, would allow for quantita- pneumatophores and, within the adjacent subtidal area, the tive assessment of the potential energetic transfer within this eelgrass Zostera mulleri subsp. capricorni is prevalent, while two type of environment. other seagrasses (Posidonia australis and Halophila spp.) are also This study aims to unravel the possible trophic pathways of the present (Cardno Lawson Treloar, 2008). ambassid A. jacksoniensis within a large estuary and explore the On each sampling occasion, the water temperature, salinity, potential contribution of saltmarsh, which is tidally inundated at dissolved oxygen concentration and turbidity were measured in only certain times of the year, to any trophic relay within this es- the middle of the water column at the two sampling sites using a tuary. The specific objectives are to 1) determine the stomach handheld Yeo-Kal 611 water quality meter. J.J. McPhee et al. / Estuarine, Coastal and Shelf Science 167 (2015) 67e74 69

2.2. Field sampling and laboratory processing factors. A test for homogeneity of multivariate dispersions (PERMDISP) was done prior to PERMANOVA to ascertain that there A mixed model sampling design was used to test for any dif- were no differences in dispersions within groups. Factors were ferences in the stomach fullness and dietary composition of pooled where possible (i.e. when p > 0.25 as recommended by A. jacksoniensis among Seasons (summer and winter), Months Winer et al., 1991). When PERMANOVA showed significant differ- (nested in seasons), Days (1e6) e which took into account the ences, pairwise post-hoc tests were carried out to determine spe- states of saltmarsh inundation (inundated (1e3) and non- cific patterns of differences among the levels within those factors inundated (4e6)), Tidal states (flood and ebb) and Sites (two). (groups). One-way Similarity Percentages analyses (SIMPER; Samples of A. jacksoniensis were therefore obtained from Empire Clarke, 1993; Clarke and Gorley, 2006) were then used to identify Bay Wetland in January and February (summer) and June and July the dietary items that typified the dietary composition of each a (winter) in 2012 at two tidal states (flood and ebb) on six consec- priori group and/or those that were responsible for distinguishing utive days, the first three of which represented the non-inundated between the dietary compositions in each pair of groups. The above saltmarsh and the latter three representing the inundated salt- results were visually depicted using a non-metric multi-dimen- marsh in all spring-tide events. Months were randomly chosen to sional scaling (nMDS) ordination plot, derived from a ‘distances incorporate replication within Seasons. Samples of fish were among centroids’ matrix constructed using the original BrayeCurtis collected at two sites in the study location, which were located similarity matrix (see above, Clarke and Gorley, 2006). All univar- approximately 300 m apart to ensure independence of the samples iate and multivariate plots depict significant fixed factors and in- of fish. teractions only (i.e. variability among random or non-significant Individuals of A. jacksoniensis were collected using a seine net, fixed factors and interactions are not displayed). which was 20 m long with a 2 m cod-end, 2 m in height and had a mesh size of 8 mm. On each sampling occasion, this seine net was 2.3.2. Calorimetric measurements deployed by hand into the open water immediately adjacent to the The potential prey of A. jacksoniensis were obtained using a edge of the saltmarsh habitat. One such haul was carried out at each plankton net, comprising a 250 mm mesh with a 950 mm long body sampling site, with a total of four seines carried out during each day and a 300 mm opening, which was deployed on the same sampling (in summer) and night (in winter) at the two sites on each tidal days as in the sampling design for Ambassis jacksonsiensis. Samples state. Flood tide sampling occurred ca 1 h prior to high tide slack were obtained by towing the net within the water column and water, while ebb tide sampling occurred soon after the tide was directly adjacent to the shoreline at Empire Bay Wetland, at a observed to be receding. standard distance (2 m) alongside the boat and approximately Ten A. jacksoniensis were haphazardly removed from each seine 15 cm below the surface of the water, at a speed of approximately net haul and euthanased in an ice-slurry. Individuals were pre- 1e2 knots (Epifanio et al., 1984; DiBacco et al., 2001; Bretsch and served in 10% formaldehyde for one month and stored in 70% Allen, 2006; Ford et al., 2006). Samples were frozen in the field ethanol prior to laboratory examination, with any shrinkage in and later thawed and, using a dissecting microscope, identified into standard length (SL) being <1% (McPhee, unpubl. data). three potential prey “types” (i.e. calanoid copepods, caridean Each A. jacksoniensis was measured (SL, to the nearest 0.1 mm) decapods and crab zoeae) and four zooplankton assemblage and weighed (to the nearest 0.1 g). Under a dissecting microscope, “types” (i.e. the overall flood and ebb zooplankton assemblages, its stomach was removed, cut open and given an ordinal score both when the saltmarsh was not inundated (Days 1e3) and when category for fullness ranging from 0 (empty) to 10 (100% full). Any it was inundated (Days 4e6)). Samples of the same “type” were ingested prey was identified to the lowest possible taxon and the then amalgamated and two replicate samples of each prey and percentage volumetric contribution (%V) of each dietary item to the zooplankton assemblage “type” were subjected to calorimetry. The overall volume of the stomach contents was visually estimated number of replicates was therefore low as a consequence of the (sensu Platell and Freewater, 2009; McPhee et al., 2015). Five main small size of such prey, the total mass required for an individual dietary groups were identified, consisting of caridean decapods, sample and the extensive time required to sort and obtain such crab zoeae, amphipods, sergestid decapods and algae. samples. Samples were oven dried at 60e80 C for 24e72 h and ground to 2.3. Data analyses powder (Ciancio and Pascual, 2006). Dried samples were weighed (to 0.001 g) and sent to an external laboratory (Macquarie Geotech 2.3.1. Stomach fullnesses and dietary compositions in Australia), where they were burned at 30 atm of oxygen in a Stomach fullnesses were analysed using a three factor (Season, bomb calorimeter to determine their calorimetric content by Day, Tidal state) log-linear analysis by placing the stomach fullness reweighing of the sample and comparisons with the initial dry values into 8 categories (0, 1, 2, 3, 4, 5, 6 and 7; NB: No stomach weight. Calorimetric values were expressed in cal g 1 (dry weight). fullness exceeded 7 on the 0 to 10 scale). Sites and months were The calorimetric values of the potential prey and zooplankton pooled due to small numbers in one or more categories of stomach assemblage “types” were then statistically compared using a one- fullness at the two sites in the two months sampled of each season. way ANOVA in SPSS (Version 21 - SPSS (2012)). Homogeneity of The mean percentage volumetric contributions of the five di- variances was examined using residual plots and if data were etary items to the stomach contents of each A. jacksoniensis were heterogeneous, square-root transformation was applied prior to square-root transformed and used to construct a BrayeCurtis rechecking residual plots. Where significant differences (at a ¼ 5%) similarity matrix. The dietary data for this matrix comprised a five- were found between levels, post-hoc pairwise Tukey's HSD tests way fully-crossed design as follows: Season (fixed, orthogonal), were conducted to determine which levels were different from one Month (random, nested in Season), Day (fixed, orthogonal), Tidal another. state (fixed, orthogonal) and Site (random, orthogonal). A permutational multivariate analysis of variance (PERMANOVA; Anderson, 3. Results 2001; Anderson et al., 2008), using Type III sums of squares, was conducted to determine whether the diets of A. jacksoniensis During 2012, water temperatures at Empire Bay Wetland were differed among the different seasons, months, days, tidal states and around 14e15 C in winter and 25e26 C in summer, while salinities sites and whether there were any interactions between those lay between 28.9 and 33.6 in both seasons. Dissolved oxygen 70 J.J. McPhee et al. / Estuarine, Coastal and Shelf Science 167 (2015) 67e74

concentrations ranged between 4.6 and 8.1 mg L 1 and turbidity between 0.3 and 33.3 NTU. On each sampling occasion, these environmental measures varied little between the ebb and ﬂood tides. The sizes of A. jacksoniensis ranged between 36.2 and 38.2 mm SL, with a mean of 37.3 mm (n ¼ 960, SE ¼ 0.021 mm). The mean wet weights ranged between ~0.9 g and 1.0 g, with a mean of 0.96 g (n ¼ 960, SE ¼ 0.002 g). The stomach fullnesses of all A. jacksoniensis ranged from 0 (empty) to 7 (70% full). Caridean decapods made a greater overall contribution to the total dietary volume, i.e. 27.3%, than the other four dietary items, i.e. crab zoeae, amphipods and sergestid decapods and algae, which had similar and moderate contributions ranging between 16.8 and 19.8%.

3.1. Stomach fullnesses

Log-linear regression showed that the frequency of stomach fullness values were not consistent between Days in the two Sea- sons (c2 ¼ 118.237, df ¼ 35, p < 0.001). This was due to both the numbers of low stomach fullness values (i.e. 0e2) being higher than expected, and the number of high stomach fullness values being lower than expected, on Days 1e3 (i.e. when saltmarsh was not inundated), while the opposite was generally the case for Days 4e6 (i.e. when saltmarsh was inundated), for both Seasons (with slightly greater stomach fullness values during summer, particu- Fig. 2. Log-linear regression depicting relationship between stomach fullness cate- larly on Day 5; Fig. 1). gories (SF; marked on x-axis) for Ambassis jacksoniensis, ranging between 0 and 7, over different Tidal states (flood and ebb) and Days (a: 1e3; b: 4e6). “Up” arrows indicate Stomach fullness values were also inconsistent between Tidal that counts of such SF values are statistically higher than expected for that Tidal state 2 states among Days (c ¼ 56.275, df ¼ 35, p ¼ 0.013), which was and Day, while “down” arrows indicate that counts of such SF values that are statis- generally due to these values being lower than expected during tically lower than expected for that Tidal state and Day. flood tides and higher than expected during ebb tides (Fig. 2a; b). A seasonal difference was also detected for stomach fullnesses during ebb tides (i.e. stomach fullnesses were inconsistent between Tidal was no interaction between these factors (Table 1). Pairwise post- states across Seasons; c2 ¼ 47.867, df ¼ 7, p < 0.001), in which hoc comparisons of Days, using PERMANOVA, showed that di- ¼ stomach fullness values were higher during summer ebb than etary compositions differed between Days 2 and 6 (p 0.05). winter ebb tides (Fig. 3). The use of nMDS ordination demonstrated that the dietary compositions of A. jacksoniensis during flood tides formed a distinct group separate from the ebb tide points on the plot (Fig. 4). 3.2. Dietary compositions Furthermore, during ebb tides, the points for Days 1e3 (when saltmarsh is not inundated) lay separate to those for Days 4e6 PERMANOVA showed that the dietary compositions of (when saltmarsh is inundated). At ebb tides on Days 4e6, there was A. jacksoniensis differed among Tidal states, i.e. between flood and also a greater spread of points than at other times (Fig. 4), however ebb tides (p ¼ 0.033) and among Days (p ¼ 0.028) and that there PERMDISP showed no differences in multivariate dispersion (F ¼ 2.98; p ¼ 0.16).

Fig. 1. Log-linear regression depicting the relationship between stomach fullness Fig. 3. Log-linear regression depicting relationship between the stomach fullness categories (SF; marked on x-axis) of Ambassis jacksoniensis, ranging between 0 and 7, categories (SF; marked on x-axis) of Ambassis jacksoniensis, ranging between 0 and 7, over subsequent Days (1e6) for summer and winter. “Up” arrows indicate that counts over Tidal states (ﬂood and ebb) and Seasons (summer and winter). “Up” arrows of such SF values are statistically higher than expected for that Day, while “down” indicate that counts of such SF values are statistically higher than expected for that arrows indicate that counts of such SF values are statistically lower than expected for Tidal state and Season, while “down” arrows indicate that counts of such SF values are that Day. statistically lower than expected for that Tidal state and Season. J.J. McPhee et al. / Estuarine, Coastal and Shelf Science 167 (2015) 67e74 71

Table 1 Table 2 Summary of results for five-factor PERMANOVA of the dietary compositions of Summary of results for one-factor ANOVA on calorimetric values of potential prey Ambassis jacksoniensis in different Seasons, Months nested in Season, Day, Tidal state and zooplankton assemblage “types” from Empire Bay Wetland, Brisbane Water and Site at Empire Bay Wetland, Brisbane Water, NSW in 2012. Significant differ- (2012), subjected to bomb calorimetry, (df) degrees of freedom, (MS) mean squares, ences and interactions are depicted in bold. Analysis conducted on square-rooted (F) F ratio test statistic, (p) significance. data; (df) degrees of freedom, (MS) mean squares, (Pseudo-F) pseudo-F ratio test statistic, (P(perm)) permutation significance. Source df MS F p Zooplankton treatment 6 295.412 11.176 0.001 Source df MS Pseudo-F P(perm) Residuals 7 26.433 Season (Se) 1 99,298 3.159 0.140 Day (D) 5 20,224 2.081 0.028 Tidal State (T) 1 196,000 25.132 0.033 3.3. Calorimetric comparisons Site (Si) 1 785 0.209 0.760 Month (M) [nested in Se] 2 29,068 7.726 0.061 Two-way interactions ANOVA showed that calorimetric values of the seven Se D 5 12,941 1.339 0.214 zooplankton “types” differed significantly (Table 2). Post-hoc Se T 1 50,402 6.491 0.093 Tukey's test, however, showed that the calorimetric values of the Se Si 1 3557 0.945 0.509 “ ” D T 5 11,133 1.453 0.190 three potential prey types (calanoid copepods, caridean decapods 1 D Si 5 776 0.358 0.947 and crab zoeae), which ranged from 566.6 to 574.3 cal g (Fig. 5), T Si 1 413 0.260 0.662 did not significantly differ from one another. Likewise, the calori- M[Se] £ D 10 9988 4.594 0.001 metric values of the four overall zooplankton assemblage “types” M[Se] T 2 7450 4.691 0.145 e fl e e fl e £ (i.e. Day 1 3 ood, Day 1 3 ebb, Day 4 6 ood and Day 4 6 ebb), M[Se] Si 2 3763 3.333 0.002 1 Three-way interactions which ranged from 217.5 to 323.4 cal g (Fig. 5), also did not Se D T 5 8896 1.111 0.384 significantly differ from one another. However, Tukey's compari- Se D Si 5 1300 0.598 0.808 sons did demonstrate that the three potential prey “types” had Se T Si 1 560 0.352 0.645 significantly greater calorimetric values than the four overall D T Si 5 846 0.422 0.938 “ ” M[Se] £ D £ T 10 8196 4.090 0.003 zooplankton assemblage types . M[Se] £ D £ Si 10 2174 1.926 0.006 M[Se T Si 2 1588 1.407 0.210 4. Discussion Four-way interactions Se D T Si 5 1611 0.804 0.618 fi M[Se] £ D £ T £ Si 10 2004 1.775 0.011 This study has demonstrated that the sh A. jacksoniensis Residual 864 1129 “switches” feeding from caridean decapods to saltmarsh-derived crab zoeae after inundation of a saltmarsh habitat (Empire Bay Wetland) during spring tides within a tidally-dominated estuary in south-eastern Australia (Brisbane Water Estuary). As this ambassid SIMPER showed that caridean decapods both typified and is very abundant within this estuary (and also other estuaries in the distinguished the dietary compositions of A. jacksoniensis on Days same region; Mazumder et al., 2006; Platell and Freewater, 2009)it 1e4, and that crab zoeae, in turn, both typified and distinguished is well placed, therefore, to contribute to any potential for trophic those dietary compositions on Days 4e6. SIMPER also showed that relay, via such prey-switching, from large estuaries to their adjacent greater amounts of carideans were ingested by A. jacksoniensis marine waters. during flood than ebb tides, while the ingestion of crab zoeae and A restricted size range of A. jacksoniensis was recorded in this algae occurred to a greater extent on ebb tides. present study, i.e. 36e38 mm SL. Although this is far smaller than the total length (TL) of 67 mm recorded for this species at a different site in the same estuary (Platell and Freewater, 2009; McPhee et al., 2015), and thus implying that only younger individuals were obtained in this study, it was clear, based on internal

Fig. 4. nMDS plot, derived from a “distance among centroids” matrix of different Tidal Fig. 5. Mean (±SE, n ¼ 2) calorimetric values of potential prey and zooplankton States and Days, which was constructed from a BrayeCurtis similarity matrix using assemblage “types” from Empire Bay Wetland, Brisbane Water (2012), subjected to volumetric dietary data of Ambassis jacksoniensis recorded at Empire Bay Wetland, bomb calorimetry. Samples not sharing the same letter (e.g. a, b) are significantly Brisbane Water in 2012. Numbers represent the Day. different from each other. 72 J.J. McPhee et al. / Estuarine, Coastal and Shelf Science 167 (2015) 67e74 examination, that these fish comprised both females and males, inundated (Hollingsworth and Connolly, 2006). Moreover, including some that had reached maturity (McPhee, unpubl. data). A. jacksoniensis fed on caridean, as well as sergestid decapods and It should be noted there is a difference of approximately 10 mm amphipods during the times that the saltmarsh was not inundated, between the standard (SL) and total length (TL) of A. jacksonsiensis which contrasts with the finding that the fish essentially fasted at (McPhee, unpubl. data), i.e. on average, fish in the present study non-inundation times in that other subtropical Australian estuary therefore ranged from ~46 to 48 mm TL. (Hollingsworth and Connolly, 2006). Finally, McPhee et al. (2015) This study clearly demonstrated that, on the basis of stomach showed that thalassinid larvae, rather than caridean decapods, fullnesses, A. jacksoniensis fed to a far greater extent on ebb than were ingested by A. jacksoniensis during ebb tides when saltmarsh flood tides, irrespective of whether the saltmarsh habitat was was not inundated in the Hawkesbury river estuary, that lies 15 km inundated. This marked tidal difference in feeding, which is likely to the south of the estuary in the present study. It is likely that these to be related to food availability (see later), is also recorded for the differences in prey-switching by A. jacksoniensis reflect differences same species in a nearby south-eastern Australian estuary (McPhee in prey availability in the different environments, which means that et al., 2015), as well as another saltmarsh-inhabiting fish species any examination of prey switching in this species or other oppor- (Fundulus heteroclitus) in eastern North America (Weisberg et al., tunistic feeders should ideally account for any such environmental 1981). differences between localities. A. jacksonsiensis fed mainly on planktonic crustaceans, with The switching of prey by A. jacksonsiensis from caridean deca- caridean decapods being of greater importance than crab zoeae, pods to crab zoeae following saltmarsh inundation, implies that sergestid decapods and planktonic amphipods. This focus on small such inundation either directly influences the availability of car- prey is consistent with other freshwater ambassids, such as ideans within the water column or, more likely, that another food Ambassis agassizi and Ambassis agrammus, which feed on micro- source (i.e. crab zoeae) becomes available and “preferable” as prey crustaceans (e.g. cladocerans, copepods, ostracods and con- (or whose available abundance become greater than that of car- chostracans) and insect larvae (e.g. dipteran, ephemeroptan ideans, if A. jacksonsiensis fed opportunistically). As the stomach (Baetidae) and trichopteran (Leptoceridae) larvae; Pollard, 1974, fullnesses of A. jacksoniensis were greater following inundation of Sanderson, 1979; Bishop et al., 2001; Medeiros, 2004; Lintermans, saltmarsh than when the saltmarsh was not tidally inundated, crab 2007). Although the algae consumed in moderate amounts by zoeae apparently became more abundant than caridean decapods A. jacksoniensis is likely to be benthic, which is consistent with this and/or were ingested more readily by A. jacksoniensis. This trend in species and other species (A. agrammus) also feeding on benthic stomach fullness is consistent with other studies on the dietary food (Bishop et al., 2001; Platell and Freewater, 2009), it could also compositions of this species (Hollingsworth and Connolly, 2006; have been suspended in the water column by tidal action during Mazumder et al., 2006; Platell and Freewater, 2009; McPhee the spring tidal levels >1.8 m recorded in the present study. et al., 2015). This has also been recorded for the diets of other fish species such as Fundulus grandis in North American salt- 4.1. Dietary compositions, prey-switching and seasonality in the marshes and Dicentrarchus labrax and Liza ramada in European feeding of A. jacksoniensis saltmarshes, which reflected a switch to saltmarsh-derived food sources when they became available (Rozas and LeSalle, 1990; Although zooplanktonic crustaceans comprised the main prey Laffaille et al., 2001, 2002). of A. jacksoniensis, the volumetric contributions of caridean deca- The seasonal difference in stomach fullnesses (which were pods and crab zoeae differed between flood and ebb tides and/or greater in summer than in winter) was not accompanied by a whether or not saltmarsh was tidally inundated. Thus, caridean seasonal difference in the dietary compositions of A. jacksoniensis, decapods were ingested mainly on both flood tides throughout the suggesting that it feeds on the same prey at these times of the year spring-tide cycle and on ebb tides during days when the saltmarsh but ingests greater amounts of prey in summer. This contrasts with was not inundated. The importance of caridean decapods within the observation that stomach fullnesses of A. jacksoniensis were the diets probably reflects high abundances in the water column greater during winter in an estuary (Botany Bay) further south during this time (Bernard, 2009; McPhee, unpubl. data). However, it (Mazumder et al. 2006), which was linked to the greater amount of should be noted that such feeding on caridean decapods occurred zoeae released by grapsids during night-time ebb tides that was when another common prey type, i.e. crab zoeae, was not expected considered an adaption by those grapsides that would overcome to be present within the water column (Hovel and Morgan, 1997; any adverse effects on such prey by visual predators, including Mazumder et al., 2006). A. jacksoniensis. Such differences highlight the variability of feeding Crab zoeae were ingested in far greater amounts when patterns by A. jacksoniensis in different estuarine environments. A. jacksoniensis fed during ebb tides following saltmarsh inundation, implying that saltmarsh is the likely source of such zoeae, 4.2. Calorimetry of potential prey and interpretation of feeding which is consistent with directly comparable studies patterns (Hollingsworth and Connolly, 2006; Mazumder et al., 2006; Platell and Freewater, 2009; McPhee et al., 2015). It is therefore proposed This study explored, for the first time, energetic values of that A. jacksoniensis switched from feeding on caridean decapods to Australian estuarine zooplankton and their potential calorimetric crab zoeae, following the inundation of saltmarsh during spring- contribution to predators (such as A. jacksoniensis), with the view tide cycles that led to subsequent zoeal release by saltmarsh- that, when a predator switches prey, this may reflect a greater dwelling grapsid crabs during tides that ebbed from saltmarshes. calorimetric value for that prey, which in turn maximises energy Although such prey-switching is largely consistent with other intake per unit effort, as suggested by optimal foraging theory. studies on the feeding of A. jacksoniensis (Hollingsworth and However, the “prey-switching” by A. jacksoniensis between car- Connolly, 2006; Mazumder et al., 2006; Platell and Freewater, idean decapods and crab zoeae was not found to be related to 2009; McPhee et al., 2015), there are some notable differences. In differences in the calorimetric values of these prey, which were our study, A. jacksoniensis did not feed exclusively on crab zoeae shown to be statistically similar to each other and another potential following saltmarsh inundation, but also ingested algae and other prey “type” (calanoid copepods), ranging between 567 and crustaceans, unlike in a subtropical Australian estuary (Coombabah 574 cal g 1. This similarity probably reflects the close relationship Lake), where it fed only on crab zoeae after the saltmarsh was (crustaceans) and functional similarity of these taxa in terms of J.J. McPhee et al. / Estuarine, Coastal and Shelf Science 167 (2015) 67e74 73 body size, food resources and habitats occupied (Mazumder et al., in shallow estuarine nurseries. Mar. Ecology-Progress Ser. 323, 75e82. 2006, 2009). It is therefore likely that the prey-switching of Bakker, J.P., 2014. Ecology of Salt Marshes: 40 Years of Research in the Wadden Sea. Wadden Academy, The Netherlands. 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Supervising Scientist (1997)). Report 145. It is noteworthy that the energetic values for these three po- Bittar, V.T., Awabdi, D.R., Tonini, W.C.T., Vidal Jr., M.V., Beneditto, D., Madeira, A.P., tential prey “types” were also significantly greater than that for the 2012. Feeding preference of adult females of ribbonfish Trichiurus lepturus “ ” through prey proximate-composition and caloric values. Neotropical Ichthyol. overall zooplankton assemblage types , which implies that 10, 197e203. A. jacksoniensis is targeting more energetically dense prey, i.e. Bretsch, K., Allen, D.M., 2006. Tidal migrations of nekton in salt marsh intertidal crustaceans rather than gastropods and cnidarians (McPhee, creeks. Estuaries Coasts 29, 474e486. unpub. data). For example, freshwater Patagonian gastropods had Cardno Lawson Treloar, 2008. Brisbane Water Estuary Processes Study. Prepared for Gosford City Council and Department of Environment and Climate Change. less than half the calorimetric value of crustaceans in the same Ciancio, J., Pascual, M., 2006. Energy density of freshwater Patagonian organisms. system, i.e. 1143 vs 2950e3994 cal g 1, respectively (Ciancio and Ecol. Austral 16, 91e94. Pascual, 2006). Clarke, K.R., 1993. Non-parametric multivariate analyses of changes in community structure. Aust. J. Ecol. 18, 117e143. Clarke, K., Gorley, R., 2006. PRIMER V6. User Manual/Tutorial. PRIMER-E, Plymouth, 5. Conclusion UK. Costa, C.S., Marangoni, J.C., Azevedo, A.M., 2003. Plant zonation in irregularly flooded salt marshes: relative importance of stress tolerance and biological The present study has demonstrated that A. jacksoniensis,by interactions. J. Ecol. 91, 951e965. ~ “switching” to feeding on the zoeae derived from grapsid crabs, Daleo, P., Silliman, B., Alberti, J., Escapa, M., Canepuccia, A., Pena, N., Iribarne, O., 2009. Grazer facilitation of fungal infection and the control of plant growth in with those crabs feeding on detritus and plant material within the south-western Atlantic salt marshes. J. Ecol. 97, 781e787. saltmarshes of a large estuary in south-eastern Australia (Brisbane DiBacco, C., Sutton, D., McConnico, L., 2001. Vertical migration behavior and hori- Water Estuary), is in a strong position to contribute to any trophic zontal distribution of brachyuran larvae in a low-inflow estuary: implications e relay within this system. Such trophic relay would be furthered by for bay-ocean exchange. Mar. Ecology-Progress Ser. 217, 191 206. Epifanio, C.E., Valenti, C.C., Pembroke, A.E., 1984. Dispersal and recruitment of blue A. jacksoniensis leaving the estuary for adjacent marine waters, crab larvae in Delaware Bay, USA. Estuar. Coast. Shelf Sci. 18, 1e12. which has been recorded for this species in other estuaries in this Ford, J., Fowler, A., Suthers, I., 2006. 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