The Composition and Variation of Epiphytic Communities on Sydney Seaweeds and the Differing Approaches of Mitigation by Two Species of Macroalga

Home , Balanus

The composition and variation of epiphytic communities on Sydney seaweeds and the differing approaches of mitigation by two species of macroalga

Jacinta Green

A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy

School of Biological Earth and Environmental Sciences Faculty of Science

June 2016 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: Green

First name: Jacinta Other name/s: Kathryn

Abbreviation for degree as given in the University calendar: Ph.D

School: Biological Earth and Environmental Faculty:Science Sciences

Title: The composition and variation of epiphytic communities on Sydney seaweeds and the differing approaches of mitigation by two species of macroalgae

Abstract 350 words maximum: (PLEASE TYPE) Macroalgal epiphytic communities are critical components of marine ecosystems and can drive increased biodiversity. Understanding the epiphytic communities on macroalgae and the mechanisms that algae to mitigate the impact of epiphytes adds to understanding of the evolution and ecology of these communities. The interaction between the communities and the host also informs our understanding of life history traits, growth and resource allocation strategies of the host algae. I assessed the epiphytic communities on a range of macroalgae across several sites around the Sydney, NSW region. The type of algal host was the most significant factor in determining both fouling load and epiphytic community composition. The green alga Caulerpa filiformis J.Agardh and the brown alga Dilophus marginatus J.Agardh were selected for further study. Unexpectedly I recorded a decrease in fouling coverage on both D. marginatus and C. filiformis over the period of this study. A decrease in fouling coverage was correlated with warmer local temperatures for D. marginatus, but the inverse was true for C. filiformis. I found that juvenile sporophyte fronds of the heavily fouled D. marginatus were chemically defended against fouling. The efficacy of these defences was significantly less on extracts from fronds with visible sporangia. The in- situ coverage of epiphytic communities on juvenile and reproductive fronds was reflected in the efficacy of the surface extracts, highlighting the importance of ontogeny. Compounds from juvenile D. marginatus fronds should be further investigated as a source of anti-fouling compounds. C. filiformis is heavily defended against herbivory, but I found little evidence that this alga had any chemical defences against fouling. The strong relationship between damage and fouling and the observation of empty fronds adjacent to abscission zones suggests that epiphyte growth is mitigated by blade abandonment. This is the second recorded case of blade abandonment suggests that C. filiformis may engage in protoplasm resorption to recycle resources. This thesis has demonstrated that in situ fouling is a remarkably poor predictor of the presence of chemical defences. Ontogeny and morphology should be considered in designing bioassays to reveal complex defence strategies.

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Date ……………………………………………...... Acknowledgments

Greg Davis, its done! Thanks for the belief in me Time for the next phase

Angela Moles An inspiring arsekicker Saviour at the end

Paul Adam, mentor Always there, never missing Thanks is not enough

Peter Steinberg, well! Thanks for having me this long And keeping the faith

My medical team I am alive and healthy You got me this far

The skills and lessons Once learnt will not be wasted Science campaigning

Table of Contents

Chapter 1 Introduction 1

Chapter 2 Composition and coverage factors of 15 fouling communities on macroalgae in the Sydney region

Chapter 3 Methodological considerations of 59 settlement assays

Chapter 4 Variation in defence against fouling in the 83 brown alga Dilophus marginatus as a function of age, size, and reproductive status.

Chapter 5 Defence against epibiois in the green alga 123 Caulerpa filiformis

Chapter 6 Discussion 177

Introduction

Figure 1.1: Solieria robusta (Greville) Kylin, and its epibionts. (Image: J.Green).

Introduction

Algal assemblages are critical components of marine communities (Sellheim et al.

2010). They are simultaneously habitats, refuges, nutrient sources and substrates for a myriad of species from amphipods to urchins (Andrew 1993; Duffy and Hay 1991;

Parker et al. 2001; Poore 1994). Sessile members of these communities, attached to the host macroalga, are known as epiphytes (Smith 1996).

Epiphytes can provide valuable ecosystem services in their own right. For example, one of the most common examples, the oyster, can filter and clean the water column

(Ehrich and Harris 2015). Epiphytic communities on macroalga, (which are too soft to host oysters), are also valuable to the wider ecosystem into which they are incorporated. The epiphytes can host their own microbial communities (Toth and Pavia

2002; Verges et al. 2011). They can facilitate the presence of larger members of the ecosystem (Gunnill 1982; Hall and Bell 1988), e.g. by providing food for the small crustaceans that support fish communities (Aumack et al. 2011).

However, whether they have value to their hosts or whether they are detrimental is not so clear cut (Barea-Arco et al. 2001; Brawley and Adey 1981; Bulthuis and

Woelkerling 1983; Dixon et al. 1981; Karez et al. 2000; Nylund et al. 2013; Sand-Jensen

1977; Wahl and Hay 1995). Without the host alga, the epiphytes have limited surfaces on which to establish, but excessive growth can damage or kill the host. The epiphyte/host balance therefore helps shape the larger ecosystem, particularly for habitat-forming species (Connell and Glasby 1999; Dayton 1985; Kennelly 1987).

Introduction

There are potential economic benefits of an increased understanding of the interactions between hosts and their epiphytes. Manmade structures such as fishing nets (Dubost et al. 1996), and ships (Bienen 2004) are under constant pressure from epiphytes. Fouling on ships also aids the dispersal of invasive species (Smale and Childs

2012). There is potential to identify, imitate and exploit the antifouling mechanisms of algae to reduce detrimental fouling in marine systems (Frankovich and Fourqurean

1997; Harms and Anger 1983; Howard and Short 1986; Irlandi et al. 2004; Lebret et al.

2009; Orth and Vanmontfrans 1984; Venugopalan 1990; Wahl 1989; Wahl 2009).

Fouling has been well studied on sea grass hosts (Apostolaki et al. 2011; Balata et al.

2007; Castejon-Silvo and Terrados 2012; Dunn et al. 2008; Frankovich and Fourqurean

1997; Frankovich and Zieman 2005; Giovannetti et al. 2010; Horner 1987; Lavery and

Vanderklift 2002; Nesti et al. 2009; Piazzi et al. 2007; Trautman and Borowitzka 1999).

On seagrasses epiphytes have been identified as playing a positive role, such as nitrogen fixation (Goering and Parker 1972) and protection against desiccation (Orth and Vanmontfrans 1984). Epiphytes also negatively impact seagrasses through shading

(Alcoverro et al. 2004), drag (Andrew and Viejo 1998), and co-consumption (Strong et al. 2009). However, macroalgae have a higher growth rate than do seagrasses, in addition to a different suite of chemical and physical defences, and so pose different challenges for successful epiphytic growth.

Sydney is ideally situated to study epiphytic growth on alga, as it has a large number of bays often characterised by the presence of algal covered rocky reefs (Figure 1.2).

Sydney bays occur along the open coastline as well as within the protected harbours of

Introduction

Port Jackson and Botany Bay. The fringing community of algae (the intertidal and upper sublittoral) in Sydney is dominated by Ecklonia, Sargassum, Dictyotales, and coralline algae and, in recent years by invasive species such as Caulerpa filiformis

J.Agardh (Connell and Irving 2008; Underwood et al. 1991).

Figure 1.2: Alga covered rocky outcrop at Shark Bay, Nielsen Park, Sydney. (Image: J.Green).

The overall aim of this thesis is to add knowledge to our understanding of local Sydney macroalgal epiphytic communities, how they change over time and how much inherent site variation impacts the structure of those communities. Additionally, increasing the knowledge of host response to epiphytic communities has further implications for ecosystem structure and biofouling research. 4

Introduction

The aim of Chapter 2 is to understand temporal and spatial variation in abundance and community structure in of algal epibiont communities in the Sydney region. Propagule load and environmental conditions will, by nature, vary between sites. A single species of alga that grows in multiple locations provides the opportunity to study epiphytic community variation between sites. Quantifying variation in epibiont community composition on multiple algae that co-exist, but exposed to similar environmental stresses and propagule supply, provides valuable data on the role of the host. These data enable the development of testable hypotheses on the role of both environmental variation and the constitutive response by algae to fouling (Arias and

Morales 1969; Arias and Morales 1979; Farnsworth and Ellison 1996; Hewitt et al.

2001; Long and Trussell 2007; Morales and Arias 1977; Morales and Arias 1979; Pereira et al. 2004; Thomas et al. 1987).

The work I describe in Chapter 2 allowed me to identify two very different species of alga for further investigation. Dilophus marginatus J. Agardh, is a short lived brown algae which was significantly more fouled then co-occuring algae. Caulerpa filiformis

J.Agardh is a green alga with relatively low levels of fouling.

In Chapter 3, I critically assess the use of bioassays by quantifying the effect of methodological differences and the potential role of propagule variation. Chemical defences provide one mechanism algae can use to mitigate settlement pressure. To test and isolate effective compounds has long involved the use of settlement assays.

The sophistication of the assays has increased over time – with de Nys et al. (1998) developing a method that targeted ecologically relevant surface compounds, which

Introduction

proved to have a greater similarity between assay results and levels of in-situ fouling

(Nylund et al. 2007), then previously used methods. However the literature contains many reports of seemingly identical bioassays which have yielded contradictory results

(Dahms and Hellio 2009; Holm et al. 2000; Marshall and Steinberg 2014; Raimondi and

Keough 1990).

In Chapter 4, I test surface compounds from D. marginatus for efficacy as anti- settlement compounds against a range of propagules of fouling organisms. I quantify the variation in fouling in situ and the variation in surface compound efficacy within and between seasons. Additionally I quantify the variation in both fouling and surface compound efficacy between life history stages.

In Chapter 5, I test surface compounds from C. filiformis, a species which I expected to be heavily chemically defended due to low level of in-situ rates of fouling and promising results in the literature (Amade and Lemée 1998). As the coenocytic nature of C. filiformis in principle allows for the increased mobility of compounds within the thallus (Amade and Lemée 1998; DeWreede 2006; Meyer and Paul 1992), I also test for within-frond variation of defence compound efficacy and inducible defences.

Our knowledge of epiphytes and the mechanisms by which their presence is mitigated by algal hosts adds to our understanding how our marine ecosystems are structured, and potentially how they may change. The mechanisms used by algae can be potentially be used to resolve problems of direct economic vale to humans and add to our understanding of algal defensive strategies.

Introduction

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Introduction

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Introduction

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Fouling factors

Figure 2.1: Location of sites on the east coast of Australia (Image: Google Earth).

15 Fouling factors

Introduction

Epibiotic communities provide habitat for mobile species, additional surfaces for sessile species and nutrient supply (for both herbivores and the host (Verges et al.

2011). Fouling occurs on any submerged surface, from hard surfaces such as boats and jetties to the soft biological surfaces of seagrasses and seaweeds (Frankovich and

Fourqurean 1997; Harms and Anger 1983; Howard and Short 1986; Irlandi et al. 2004;

Orth and Vanmontfrans 1984; Wahl 2009). Understanding the factors that drive the development of fouling communities adds to our ecological understanding of the marine environment, including possible impacts of changes through time. As fouling on man-made structures (boats, pylons, buoys etc.; (Venugopalan 1990)) is normally considered detrimental understanding the factors may also aid us in developing antifouling technologies.

The composition of fouling communities and the fouling load on macroalgae is a product of propagule supply, growth and survival of fouling organism on the host, grazing of epibiota, abiotic conditions, host response and other factors. In this chapter,

I determine how much of a role is played by the local epiphyte supply (Clark and

Johnston 2005; Clark and Johnston 2009; Lee and Bruno 2009) and by the host algae by quantifying spatial and temporal variation in fouling on five algae in the Sydney region.

Temperature and depth were also examined as two factors which can influence epibiotic load and community structure (Case et al. 2011; Cassidy 2011; Jacobucci et al.

2009; Peters and Breeman 1993; Rohde et al. 2008; Rule and Smith 2007; Shohael et al. 2006).

16 Fouling factors

Spatial variation

Small scale variation in propagule supply and environmental conditions can be gleaned by studying fouling communities on co-existing algae species (Arias and Morales 1969;

Arias and Morales 1979; Farnsworth and Ellison 1996; Morales and Arias 1977;

Morales and Arias 1979). By minimising the variation in propagule supply and environmental conditions any differences in the fouling load are likely to be due to the host response; either to individual fouling species or to the environmental conditions.

Comparing fouling communities on an alga that is present in multiple locations (Long and Trussell 2007; Pereira et al. 2004; Thomas et al. 1987) allows us to further explore if it is the host algal response driving the level of fouling. If the fouling load is consistent across sites (despite invariable differences in environmental conditions, propagule supply, host survival, grazing pressure) then it is possible that the fouling load is in part determined by the host alga response.

Identifying differences in the quantity and composition of fouling communities between different locations provides an opportunity to elucidate the combined effect of all of the abiotic factors that differ between sites, without necessarily knowing what all those abiotic factors are (Hewitt et al. 2001). This tactic has been frequently used in the study of fouling on seagrasses (Balata et al. 2007; Borowitzka et al. 1990; Castejon-

Silvo and Terrados 2012; Lavery and Vanderklift 2002). (Balata et al. 2007) found high variability of fouling communities on the seagrass Posidonia oceanica at both small and large geographic scale, encompassing both similar habitats only a few kilometres apart and distinctly different habitats. Lavery and Vanderklift (2002) found similar variation of fouling communities with geographic separation on Posidonia coriacea and

17 Fouling factors

Amphibolis griffithii. Understanding spatial variation and site specific impacts at the frond level may allow better targeting of potentially interesting species without the need for comprehensive chemical analysis.

Temporal variation

The peak fouling season is generally assumed to be spring and summer (Hellio et al.

2004; Marechal et al. 2004) and levels of fouling in field surveys have often, but not always, been higher in spring/summer (Bellgrove and Aoki 2006).

A spring/summer fouling peak may be related to changes in temperature (Harms and

Anger 1983; Hellio et al. 2004; Shohael et al. 2006) or exposure to daylight (Hellio et al.

2004; Marechal et al. 2004). Several studies of perennial algae have found that chemical defences are more active, or more abundant, in the warmer months (Amade and Lemée 1998; Culioli et al. 2002; Hellio et al. 2004; Marechal et al. 2004). Rather than have more efficacious defences in the warmer months, algae could temporally avoid fouling (and the subsequent cost of producing defences) by growing only when the fouling pressure is low (Marechal et al. 2004). Potentially Dilophus marginatus

JAgardh is one such alga, appearing only in the colder months.

Fouling is often defined as a process of colonisation of surfaces submerged in the marine environment, and as time passes a surface is expected to progressively accumulate a number of fouling organisms (Alcoverro et al. 2004; Jennings and

Steinberg 1997). With the exception of recent work on the chemical mediation of settlement by Delisea pulchra (Greville) Montagne (Steinberg and de Nys 2002;

Steinberg et al. 1998; Steinberg et al. 2002; Steinberg et al. 1997), temporal variation 18 Fouling factors

in fouling communities has traditionally been explored as a function of early colonisers and subsequent competition. Communities are expected to reach a level of stability

(Madin et al. 2009), resulting in an ‘endpoint’ community assemblage (Steinberg and de Nys 2002). To date, the majority of temporal studies have focussed on fouling of hard substrates and seagrasses (Arias and Morales 1979) not on macroalga epibiotic communities.

Abiotic factors

Abiotic factors are the most well studied drivers of fouling (Arias and Morales 1979;

Arrontes 1990; Hellio et al. 2004; Jacobucci et al. 2009; Kocak and Kucuksezgin 2000;

Marechal et al. 2004). Abiotic factors found to be of influence on coverage include temperature (Gillespie and Critchley 1999; Raveendran and Harada 2002; Thom et al.

2003), daylight hours (Marechal et al. 2004), and depth (Amade and Lemée 1998; Head et al. 2004; Madin et al. 2009; Middelboe et al. 2003; Nesti et al. 2009; Piazzi et al.

2002; Rule and Smith 2007).

Here I focus on temperature because temperature has the potential to be one of the biggest drivers in patterns of fouling, as the response of both the fouling organisms

(algal or otherwise) and host algae can be modified by variation in temperature

(Ahlgren 1987). Like most organisms, algae have optimal temperature ranges (Case et al. 2011), and when the temperature varies from the optimum the alga may have reduced growth rates (Cassidy 2011; Peters and Breeman 1993; Uchida et al. 1991), or experience changes in reproduction (Agrawal 2012). Reduced growth rate or an extended growing season may give fouling organisms more time to colonise (Horner

19 Fouling factors

1987; Thom et al. 2003). Temperature changes may also affect the production of primary or secondary defensive chemicals (Shohael et al. 2006). The amount of fouling is generally reduced under colder temperatures (Guo and Yan 1984; Harms and Anger

1983) with limited or no fouling occurring at temperatures below 10.0° C, and maximal levels of fouling generally occurring above 20.0° C (Guo and Yan, 1984). The situation is not as clear for temperate environments such as Sydney

I also focus on depth, as variation in fouling communities due to depth has been found in both large scale experiments that measure differences over tens of metres

(Venugopalan 1990), and in smaller scale experiments that measure differences within metres (Alcoverro et al. 2004; Dubost et al. 1996; Madin et al. 2009; Moring and

Moring 1975). Even smaller scale depth variations may play a role as tips of algae may be more exposed to fouling propagules then the lower area of the fronds (Jennings and Steinberg 1997). Reduced fouling at depth is thought to be related to irradiance levels (Beach et al. 2006), nutrient availability (Beach et al. 2006), and possibly reduced propagule supply (Rohde et al. 2008). Previous research tends to concentrate on faunal communities and hard substrates (Madin et al. 2009; Rule and Smith 2005; Rule and

Smith 2007) and has rarely been explored on algae (Rule and Smith 2007).

D. pulchra grows over a depth range in a number of locations around Sydney, providing an opportunity to test the fouling load at different depths and whether there is an interaction between depth and site.

20 Fouling factors

Aims

My first aim is to quantify the spatial variation of fouling coverage and community composition on a range of species from a range of locations around the Sydney Basin by:

a) Quantifying the level of fouling on co-existing algae which are exposed to the

same general propagule load and similar small scale environmental conditions.

b) Quantifying the level of fouling on algae which are found in multiple locations

which are exposed to differing propagule loads and small scale variation in

environmental conditions.

c) Quantifying the variation in the structure of fouling communities on a given

species of alga from multiple sites.

My second aim is to quantify seasonal and temporal variation of fouling levels on two species of algae.

a) Quantifying the seasonal variation in amount of fouling on two species

of coexisting algae.

b) Quantifying the temporal variation in the amount of fouling over a three

year period.

c) Quantifying the variation in fouling community structures over a three

year period.

21 Fouling factors

My third aim is to determine if warmer temperatures correlate with increases in the fouling coverage on two local algal species.

My fourth aim is determine if the amount of fouling on D. pulchra varies with depth.

22 Fouling factors

Materials and Methods

1) Selection of alga

Five algae commonly found in the Sydney region were selected for survey (Table 1).

The algae selected cover the three main groups of algae: red, green and brown. They include those with secondary metabolites that are known to be effective against herbivory and in one case fouling (D. pulchra) and those with no known chemical defences against herbivory. They have different life histories, e.g. perennials and annuals, appear in a number of locations around Sydney, and grow at a number of depths, thus enabling us to explore a range of possible factors that influence fouling.

Table 2.1: Algae surveyed from around Sydney, Australia and their characteristics of interest to this study Species Type Cell type Life span Chemical defences Depth (Sydney based on against fouling? region (pers. observed obs.)) persistence in the Sydney region Caulerpa green Coenocytic perennial Known against Shallow filiformis (Travizi and herbivory (Amade and Zavodnik Lemée 1998) 2004) Dilophus brown Single nuclear annual Known against Shallow marginatus cells herbivory (Kurata et al. 1988) Dictyopteris brown Single nuclear annual Known against Shallow acrostichoides cells herbivory (Iris et al. 2001) Delisea pulchra red Single nuclear perennial Chemical defences Gradient cells against fouling and herbivory (de Nys et al. 1995; Dworjanyn et al. 2006) Solieria robusta red Coenocytic annual Unknown Deep (Agardh 1842)

23 Fouling factors

2) Sites

The collection locations are dispersed along the coastline of Sydney, New South Wales

(Figure 2.1) and from south to north are: Bare Island (33°59’29.03” S, 151°13’55.92” E),

Long Bay south (33°58’05.72” S, 151°15’16.53” E), Long Bay north, (33°57’59.97” S,

151°15’24.96” E), Clovelly (33°54’53.61” S, 151°16’06.79” E), Bronte (33°54’18.21” S,

151°16’08.75” E), and Shark Bay (Nielsen Park) (33°51’00.08” S, 151°16’03.27” E).

Nielsen Park

Bronte Beach Clovelly

Long Bay

Bare Island 5 km

Figure 2.2: Site locations off Sydney coastline (Australia). (Image: J.Green, base map: Google Earth).

Bare Island, a sheltered site located within Botany Bay, has four of the targeted species: D. pulchra, Solieria robusta (Greville) Kylin, D. marginatus and Caulerpa filiformis J.Agardh. Bare Island is also the only easily accessible site that has the same 24 Fouling factors

alga (D. pulchra) along a depth gradient. Long Bay, a protected bay on the exposed coast, provided two sampling sites: Long Bay south which is one of only two sites in

Sydney in which a population of Dictyopteris acrostichoides (J.Agardh) Bornet could be found, and Long Bay north which has populations of D. pulchra, D. marginatus and

C. filiformis. Clovelly and Bronte are also are sheltered bays on the exposed coast and have persistent populations of C. filiformis. Shark Bay, more commonly referred to as

Nielsen Park, is an inner harbour site that contains populations of D. acrostichoides,

C. filiformis and D. marginatus.

3) Surveys

A series of surveys were undertaken on a seasonal basis (Summer, Autumn, Winter and Spring) over a three year period (2005-2007). A replicate sample at each sampling time and place was composed of an average of five whole thalli haphazardly collected for the small algae and or an average of five haphazardly collected fronds from haphazardly selected thalli each from five haphazardly selected specimens for larger algae. ). Five replicate samples were collected for each algae, at each time at, each place and for D. pulchra only, both small and large D. pulchra thalli were haphazardly collected from both shallow (< 2m) and deep environments (>7m).

Unique geological landmarks at specific depths were established on the initial dive using dive gauges and subsequently referred to when collecting. Samples collected in the sub-tidal region in less than one metre of water are classified as shallow, while samples collected at a depth greater than 7 metres are classified as deep.

25 Fouling factors

4) Algal treatment

Each sample was placed in a labelled zip lock bag, excess water removed, and placed on ice for transport back to the laboratory. Samples were then frozen until they could be further processed. A number of different preservation methods were trialled; freezing maintained both the colour and form of the host algae and the epibionts

(Figure 2.3). Once thawed, each specimen was patted dry with paper towel and wet weight measurements taken using an FX-3200 Electronic Balance d=0.1 g. Each specimen was photographed then dried at 70˚ C for 72 hours and dry weight measurements taken on the same balance.

Adobe Photoshop® V8.0 was used to isolate different types of fouling organisms from different locations on the alga. The type of each fouling organism was noted and categorised into the following algal categories: white encrusting, red encrusting, red flat (erect), red filamentous, brown filamentous, green filamentous and Ulva australis

Linnaeus. Specific instances of the bryozoan Bugula neritina (Linnaeus, 1758) were recorded. Instances of U. australis and B. neritina were singled out as propagules from both species are frequently used in anti-fouling assays.

The images were subsequently analysed using Scion Image for Windows to measure total surface area of each frond and total surface area of fouling (Abramoff et al.

2004). The total surface area of the fouling organism was, in some cases, greater than the surface areas of the host algae, so in these circumstance values greater than 100 % were calculated.

26 Fouling factors

To investigate the relationship between fouling and temperature, samples of

D. marginatus were collected from Bare Island and Nielsen Park and C. filiformis was collected from Nielsen Park, Clovelly, Long Bay and Bronte. Maximum sea temperature

(in the preceeding 30 days) was used as the most biologically relevant temperature measurement, as it relates to maximum host stress (Case et al. 2011). Data for maximum temperatures were sourced from the Office of Environment and Heritage

(NSW Government), from information collected and provided by Manly Hydraulics

Laboratory. Fouling data from a given collection date were aggregated into a single data point.

Figure 2.3: Algal image of a Delisea pulchra frond ready for image processing by Scion Image. (Image: J.Green).

27 Fouling factors

5) Statistical Analyses

C. filiformis samples collected from Clovelly in the Spring of 2005 were excluded from all analyses. The entire C. filiformis population from this date were 100% fouled solely by a red encrusting alga (most likely Melobesia membranacea (Esper) J.V.Lamouroux) which was very rarely found at any other sample date. This set of C. filiformis samples were considered to be affected by an abnormal fouling event that impacted only

C. filiformis. The overall data set was broken down into different analyses because not all algae were present in all places at all sample dates. Analysis of seasonal variation excluded all Spring results for D. marginatus as over the 3 year period D. marginatus was present at only one Spring collection period. Years were described as being a set of seasons rather than calendar year, i.e. Year 1 includes Autumn and Winter from

2005 and Summer 2006. a) Analysis of variation in epibiotic load in the Sydney region

To determine if the epibiotic load is consistent between different species of algae, we used IBM SPSS Statistics v.21 to run a general linear model with species (Caulerpa filiformis, Delisea pulchra, Dictyopteris acrostichoides, Dilophus marginatus and

Solieria robusta) as our predictor variable and fouling load (with coverage of fouling organisms as a percentage of host algal area) as our dependent variable with a significance level of P<0.05. The analysis was conducted independently for two sites,

Bare Island and Nielsen Park. Tukey’s posthoc tests were used to determine the differences in epibiotic load between host species of algae.

28 Fouling factors

To determine if the epibiotic load is consistent between sites around Sydney, we used

IBM SPSS Statistics v.21 to run a general linear model with site (Bare island, Clovelly,

Long Bay, and Nielsen Park) and host algae (Caulerpa filiformis, Delisea pulchra and

Dilophus marginatus) as our predictor variables and fouling load (with coverage of fouling organisms as a percentage of host algal area) as our dependent variable with a significance level of P<0.05. The non-significant interaction was removed. Tukey’s posthoc tests were used to determine the differences in epibiotic load between sites and between host algae.

The seasonal variation of epibiotic load was determined using IBM SPSS Statistics v.21 to run a general linear model with season (Spring, Summer, Autumn and Winter) and host algae (Caulerpa filiformis and Dilophous marginatus) as the predictor variables and fouling load (with coverage of fouling organisms as a percentage of host algal area) as our dependent variable with a significance level of P<0.05. The non-significant interaction was removed, and the algae were analysed independently to determine if there was a seasonal effect.

Longer term variability in fouling was determined using IBM SPSS Statistics v.21 to run a regressions Elapsed time (in months) and host algae (Caulerpa filiformis and

Dilophous marginatus) as the predictor variables and fouling load (with coverage of fouling organisms as a percentage of host algal area). The non-significant interaction was removed.

To determine if the epibiotic load is effected by temperature, IBM SPSS Statistics v.21 was used to run a regression with Temperature (Sea surface temperature) Bare island, 29 Fouling factors

Clovelly, Long Bay, and Nielsen Park) and host algae (Caulerpa filiformis and and

Dilophus marginatus) as our predictor variables and fouling load (with coverage of fouling organisms as a percentage of host algal area) as our dependent variable with a significance level of P<0.05. Algae were then analysed separately due to a significant interaction.

To determine if the epibiotic load is effected by depth, Data were √(푋 + 1) transformed and a general linear model was run using IBM SPSS Statistics v.21 with

Depth (Deep and Shallow) as the predictor variable and fouling load (with coverage of fouling organisms as a percentage of host algal area) as the dependent variable with a significance level of P<0.05. The non-significant interaction was removed. b) Analysis of variation in epibiotic community structure in the Sydney region

Presence/absence of the different categories of fouling types on each frond was used for analysis of the similarity of epibiotic communities between species of algae and between sites. Analysis was undertaken with Primer 6.1.13 & Permanova+ Version

1.0.3 using Jaccard’s index. All replicates were assigned the presence of microfouling in order to take the amount of propagule settlement into consideration as well as propagule community composition. The variation of epibiotic community structure between sites was examined separately for C. filiformis and D. marginatus and was plotted on an MDS plot using S17 Bray Curtis similarity also using Permanova+. The variation in fouling community structure over time was analysed for Nielsen Park. The five replicates for each species of algae from each collection trip were pooled into a single data point for analysis and were plotted on an MDS plot using S7 Jaccard 30 Fouling factors

resemblance. To highlight potential changes in the epibiont supply over time, the MDS plot only differentiates between years, not species or season.

31 Fouling factors

Results 1) Spatial variation across the Sydney region

a) Quantification of fouling loads on co-existing algae

Co-existing algae had substantially different fouling loads. At Nielsen Park fouling loads on different algal hosts were significantly different (P<0.001, F2,86=19.903;

30 Nielsen Park Bare Island

20 c

% fouling

d b b d

0 Caulerpa Delisea Dictyopteris Dilophus Soleria filiformis pulchra acrostichoides marginatus robusta n=50 n=25 n=5 n=72,29 n=5

Figure 2.4). D. marginatus had significantly more fouling (~20%) than D. acrostichoides

(P=0.013) and C. filiformis (P<0.001) which both had ~5% fouling. Similarly at Bare

Island fouling loads differed significantly between species (P<0.001, F2,102=19.303;

32 Fouling factors

30 Nielsen Park Bare Island

20 c

% fouling

d b b d

0 Caulerpa Delisea Dictyopteris Dilophus Soleria filiformis pulchra acrostichoides marginatus robusta n=50 n=25 n=5 n=72,29 n=5

Figure 2.4). D. marginatus had the highest fouling load (~25%), D. pulchra had the lowest fouling load (~3%, P<0.001;

33 Fouling factors

Figure 2.4) while S. robusta had ~7% fouling (P<0.036,

30 Nielsen Park Bare Island

20 c

% fouling

d b b d

0 Caulerpa Delisea Dictyopteris Dilophus Soleria filiformis pulchra acrostichoides marginatus robusta n=50 n=25 n=5 n=72,29 n=5

Figure 2.4).

34 Fouling factors

30 Nielsen Park Bare Island

20 c

% fouling

d b b d

0 Caulerpa Delisea Dictyopteris Dilophus Soleria filiformis pulchra acrostichoides marginatus robusta n=50 n=25 n=5 n=72,29 n=5

Figure 2.4: Quantification of fouling load (area percentage, mean ±se) on Dilophus marginatus, Dictyopteris acrostichoides,and Caulerpa filiformis at Nielsen Park and Dilophus marginatus, Delisea pulchra, and Solieria robusta at Bare Island (Sydney). Significant differences in fouling load at Nielsen Park are indicated by ‘b’ and ‘c’, P<0.001, F2,86=19.903. Significant differences in fouling load at Bare Island are indicated by ‘a’ and ‘d’, P<0.001, F2,102=19.303. P values obtained from separate univariate analyses testing fouling load at each site.

35 Fouling factors

b) Quantification of variation of fouling levels between sites

There was no significant difference in fouling loads between the different sites

(P=0.370, F3,244=1.053; Figure 2.5), but there was significant differences in the fouling load between species with Dilophus marginatus having significantly higher fouling loads then either species of algae, regardless of location (P<0.001, F2,244=28.967; Figure

2.5).

40 Caulerpa filiformis Delisea pulchra Dilophus marginatus

20 a a

% Fouling a

10 b b

b b

0 Bare Island Clovelly Long Bay Nielsen Park

Figure 2.5: Fouling load (area %) on Caulerpa filiformis, Delisea pulchra and Dilophus marginatus at did not vary between Bare Island, Clovelly, Long Bay, or Nielsen Park, Sydney, P=0.370, F3,244=1.053 . P values derived using and ANOVA across all sites and species. Significant difference between species (P<0.001, F2,244=28.967) (from Tukeys post hoc tests) indicated by an ‘a’ and ‘b’.

36 Fouling factors

c) Spatial variation in fouling community structure on Dilophus

marginatus and Caulerpa filiformis.

The fouling communities at Bare Island and Nielsen Park differed in the Autumn of

2005 (P=0.0001), Autumn 2007 (P=0.0001) and Winter 2005 (P=0.0001) but not in

Summer 2006 (P=0.4535), or Autumn 2006 (P=0.348, Figure 2.6).

There were also differences between the epibiont communities on D. marginatus at

Bare Island and Long Bay in Summer 2007 (P=0.035) and Autumn 2007 (P=0.001;

Figure 2.6). In the Winter of 2007 the fouling community at Long Bay was significantly different to both Bare Island and Clovelly (P=0.143, Figure 2.6).

37 Fouling factors

*Autumn 2005, BI & NP, P=0.0001 *Winter 2005, BI & NP, P=0.0001 2D Stress: 0.11 2D Stress: 0.11

Summer 2006, BI & NP, P=0.4535 *Autumn 2006 , BI & NP, P=0.348

2D Stress: 0.06 2D Stress: 0.1

*Winter 2007, BI, C, & *LB P=0.0043 *Summer 2007, BI & LB, P=0.035

2D Stress: 0.06 2D Stress: 0.06

*Autumn 2007, BI, LB, P=0.001 2D Stress: 0.1 Resemblance S17 Bray Curtis similarity for Dilophus marginatus

Legend Clovelly (C) Nielsen Park (NP) Bare Island (BI) Long Bay (LB)

Figure 2.6: MDS plot grouping sampling dates by similarities in the community structure of fouling organisms on Dilophous marginatus. Samples plotted closer together have more similar fouling communities. Dates and sites indicated by “*” are significantly different at P<0.05.

38 Fouling factors

The fouling communities on C. filiformis showed a similar significant variation in structure between sites (Figure 2.7) in the Autumn and Winter of 2005 (P=0.014 and

P=0.0012) and again in the Autumn of 2007 (P=0.0001) and Summer 2007 (P=0.0273).

There were no significant differences in the Summer and Winter of 2006 (P=0.1816 and P=0.1274; Figure 2.7).

39 Fouling factors

*Autumn 2005, C & NP, P=0.014 *Winter 2005, C & NP, P=0.0012 2D Stress: 0 2D Stress: 0.03

Summer 2006, C & NP, P=0.1816 *Autumn 2007, C & NP, P=0.0001 2D Stress: 0.11 2D Stress: 0.03

Winter 2006, C & NP P=0.1274 *Summer 2007, C & NP, P=0.0273 2D Stress: 0 2D Stress: 0.1

Resemblance S17 Bray Curtis similarity for Caulerpa filiformis

Legend Clovelly (C) Nielsen Park (NP)

Figure 2.7: MDS plot grouping sampling dates by similarities in the community structure of fouling organisms on Caulerpa filiformis. Samples plotted closer together have more similar fouling communities. Dates indicated by “*” are significantly different at P<0.05.

40 Fouling factors

2) Temporal variation in fouling

a) Seasonal variation in fouling between seasons

There was a marginal but not significant difference in the fouling load between seasons for both C. filiformis (P=0.063, F3,99=2.510;

30 Caulerpa filiformis, n=50 Dilophus marginatus, n=24

% Fouling

0 Spring Summer Autumn Winter

Figure 2.8) and D. marginatus (P=0.062, F3,111=2.524;

41 Fouling factors

30 Caulerpa filiformis, n=50 Dilophus marginatus, n=24

% Fouling

0 Spring Summer Autumn Winter Figure 2.8). Fouling loads on C. filformis peaked in Summer with the surface area of fouling species around 17% of the surface area of the C. filiformis fronds. The fouling load on D. marginatus peaked in Autumn and was much higher at approximately 20%.

42 Fouling factors

30 Caulerpa filiformis, n=50 Dilophus marginatus, n=24

% Fouling

0 Spring Summer Autumn Winter

Figure 2.8: Mean (±se) % coverage of fouling on Caulerpa filiformis (P=0.063, F3,99=2.510) and Dilophus marginatus (P=0.062, F3,111=2.524) by season. P values from ANOVA.

43 Fouling factors

b) Longer term temporal variation in quantity of fouling load

The fouling load across both species of alga decreased significantly over time (P=0.010,

F1,189=6.722; Figure 2.9). D. marginatus decreased from approximately 30 % to approximately 20 %, while the fouling load on C. filiformis decreased from around 9% to 5%.

100 Caulerpa filiformis, R2 = 0.022 Dilophus marginatus, R2 = 0.048

% Fouling 40

20 y=31.26-0.34x

y=9.54-0.16x 0 0 10 20 30 40 Elapsed Time (months) Figure 2.9: The fouling load on both Caulerpa filiformis and Dilophus marginatus decreased significantly at Nielsen Park over a 40 month period (F1,189=6.722, P=0.010). The fouling load on C. filiformis decreased at slightly faster rate (y=31.26-0.34x) then the fouling load on D. marignatus (y=9.54-0.16x).

44 Fouling factors

c) Variation in the structure of fouling communities over a three year

period at Nielsen Park

The epibiotic communities on C. filiformis and D. marginatus differed significantly from each other in year 1 and year 2 (P=0.001, P=0.001; Table 2.2), but not the third

(P=0.452; Table 2.2). The structure of the epibiotic communities does not consistently vary across all three years (significant interaction P=0.001) suggesting that something other than a common propagule supply is at play.

The three species of algae sampled did not display a consistent response to seasons.

For example, there were significant differences between the epibiont communities on

D. marginatus and C. filiformis in Winter (P=0.01, Table 2.2), but not in Summer

(P=0.378, Table 2.2) or Autumn (P=0.299, Table 2.2).

Examining each alga separately, we find that there were no differences in the epibiotic communities found on C. filiformis between seasons (Autumn-Winter, P=0.371;

Autumn-Summer, P=0.799; Winter-Summer, P=0.289; Table 2.3). We do find seasonal differences in the epibiotic communities on D. marginatus between Autumn and

Winter (P=0.001, Table 2.3) and Autumn and Summer (P=0.001, Table 2.3) and

Summer and Winter (P=0.047, Table 2.3).

The main driver in the differences between the epibiotic communities was primarily a function of which species of algae was the host, explaining 23.34% of the differences.

Variation from year to year explained 12.49% of the differences and seasonal variation explained only 4.22% of the variation.

45 Fouling factors

Resemblance: S7 Jaccard 2D Stress: 0.11 Year Year 1 Year 2 Year 3

Figure 2.10: Permanova MDS plot grouping sampling dates by similarities in the community structure of fouling organisms on Dilophous marginatus and Caulerpa filiformis. Sampling dates plotted closer together have more similar fouling communities.

Table 2.2: Summary of P-values from pairwise tests for differences from the MDS analysis on the fouling communities between Caulerpa filiformis and Dilophus marginatus. Between Years Year 1 Year 2 Year 3 C. filiformis D. marginatus 0.001 0.001 0.452

Between Seasons Year 1 Year 2 Year 3 Autumn, Winter 0.001 0.11 0.001 Autumn, Summer 0.001 0.889 0.001 Winter, Summer 0.001 0.033 >0.999

Autumn Winter Summer C. filiformis D. marginatus 0.299 0.001 0.378

46 Fouling factors

Table 2.3: Summary of P-values from pairwise tests for differences from the MDS analysis on the fouling communities of Caulerpa filiformis and Dilophus marginatus between collection dates. Between Years C. filiformis D. marginatus Year 1, Year 2 0.052 0.001 Year 1, Year 3 0.001 0.001 Year 2, Year 3 0.001 n/a

Between Seasons Autumn, Winter 0.371 0.001 Autumn, Summer 0.799 0.001 Winter, Summer 0.289 0.047

47 Fouling factors

3) Fouling and temperature

There were marginally non-significant relationships between both species of algae and maximum sea surface temperature (Figure 2.11). The fouling load on C. filiformis was marginally positive (P=0.056, F1,23=4.096; Figure 2.11) while the fouling load on

D. marginatus was marginally negative (P=0.063, F1,20=3.931; Figure 2.11). The relationship between temperature and fouling load accounted for over 16% of the variation in the fouling load for both C. filiformis (R2=0.163) and D. marginatus

(R2=0.179; Figure 2.11).

60 Caulerpa filiformis, R2=0.163 Dilophus marginatus, R2=0.179

% Fouling

y = 78 + -2.33x

y = 23.45 + 1.26x 10

0 19 20 21 22 23 24 25 26 27

Maximum Temperature (°celsius)

Figure 2.11: The decreasing relationship between fouling and temperature on Caulerpa filiformis (P=0.056, F1,23=4.096) and the increasing relationship between fouling and temperature on Dilophus marginatus (P=0.063, F1,20=3.931) at Nielsen Park.

48 Fouling factors

4) Variation in fouling at depth across two sites.

I found no significant difference in the amount of fouling on D. pulchra as a function of depth (P=0.337, F1,40=0.947; Figure 2.12) or between sites (P=0.572, F1,40=0.324; Figure

2.12). The fouling load on shallow plants at both sites was highly variable, while the fouling load at depth showed little variability.

40 Bare Island Long Bay

% Fouling 20

0 Deep Shallow

Figure 2.12: The comparison of fouling loads on Delisea pulchra at two different depths (above 2 metres and below 7 metres) at two different locations (Bare Island and Long Bay) Sydney. There was no difference due to depth (P=0.337, F1,40=0.947) or between sites (P=0.572, F1,40=0.324).

49 Fouling factors

Discussion

The variation among hosts appears to be the primary driver for fouling load for two very different species of algae, C. filiformis and D. marginatus. The two species had significantly different levels of fouling to each other. But, the fouling load within species was similar across sites. Each site, at any given time, would have a propagule supply different to other sites, which was reflected in the variation in the composition of the epibiotic communities. Fouling load was consistent to each species despite variation in community structure, therefore host response not propagule supply is determinative of fouling load. However, the structure of the epibiotic community may still be a function of propagule supply.

Host response was considered to be a potential driver for fouling load for the green alga C. filiformis. I expected a comparatively low and consistent level of fouling.

Efficacious chemical defences against herbivory are ubiquitous among the Order

Caulerpales (Amade and Lemée 1998; Dobretsov et al. 2006; Mahendran et al. 1979;

Meyer and Paul 1992; Nielsen et al. 1982; Paul and Fenical 1986). As a persistent alga, r-K theory would suggest that C. filiformis with a relatively long life span could grow slower and invest more in defences (Pianka 1970). If the defence strategy for

C. filiformis was more complex than suggested by r-K theory, being coenocytic

(DeWreede 2006) it would have the ability to relocate chemical defences to the regions of the thallus most vulnerable or under attack. C. filiformis also had levels of fouling similar to the heavily defended D. pulchra (Steinberg and de Nys 2002;

Steinberg et al. 1998).

50 Fouling factors

The fouling load on D. marginatus was much higher than the fouling load on any of the co-existing macro-algae studied. Therefore, the expectation for D. marginatus to have a strong host response to fouling load was therefore not high. While D. marginatus

(and more generally, the Order Dictyotaea) produces defensive dipterpenes (Kurata et al. 1988; Ravi and Wells 1982; Siamopoulou et al. 2004), the role of dipterpenes in defence against fouling as opposed to herbivory is not clear (Nylund et al. 2007). The short, fast growth cycle of D. marginatus is also suggestive of an r type plant where resources are more likely to be channelled into growth rather than defence (Pianka

1970).

Critically my results show that the apparent absence or presence of fouling load is not, as often considered, indicative of a host mediated response to fouling (Paul et al.

2014). An absence of fouling load can be related to a number of anti-fouling measures.

The algae may produce chemical defences (e.g. Delisea pulchra, (Dworjanyn et al.

2006) or may discard fronds once they are fouled (Littler and Littler 1999). Fouled fronds may also simply be more susceptible to frond breakage (Andrew and Viejo

1998) due to increased drag or attract grazers which keep the epiphyte load down

(Dudley 1992).

Temperature could also be a factor in the amount of fouling carried by the host. My results whilst not significant at α=0.05 show a possible trend in fouling load over a limited temperature range. The potential trend of fouling load on D. marginatus is for a decrease in fouling load in warmer temperatures. D. marginatus is rarely present in the warmer months. If warmer temperatures do play a role in decreasing the fouling

51 Fouling factors

load on C. filiformis, then the cold season growth cycle of D. marginatus is not due to it temporally avoiding a Summer pulse in supply of fouling propagules (Hoffmann 1987).

There was an unexpected decrease in the fouling load on both C. filiformis and

D. marginatus over the period of my study. At an individual frond level, species with a longer persistence in the environment such as C. filiformis would be expected to accumulate fouling species over time as frond exposure to propagules is extended (Lee and Bruno 2009). In this study however, with haphazardly selected fronds from haphazardly selected clumps, variation at the individual frond level should not be detectable, and if it was, it would result in an increase in fouling over time, not the reverse. D. marginatus has a distinct growing season, with clumps only appearing once the water has cooled down. D. marginatus is rarely present year around (King and

Farrant 1987; Phillips 1992). The specimens I sampled across the 40 months are from a number of different generations. The decrease in fouling load, therefore, is generational.

Not only was the temporal decline in fouling load for both species unexpected, the rate of decline was similar. Given the differing natures of D. marginatus and

C. filiformis as outlined above I did not expect to find any similarities in their response to epibionts. By testing across multiple species, over multiple sites, over multiple years, my results suggest that small and medium scale environmental factors are not likely to be drivers of this trend. The obvious larger scale environmental factor to explore is climate change. Climate change may be impacting the host response or the propagule behaviour. Alternatively, if the presence of epibiotic communities is detrimental to the

52 Fouling factors

host survival, a longer study may determine if evolutionary pressures are playing a role

(Arrontes 1999; Schmitt et al. 1995). My work opens up a number of interesting research opportunities. Exploring the potential for a link between temperature and fouling loads will not only increase our knowledge of the individual plant response today, but may offer insight into how these algae species will respond to climate change in the future. It would be advisable to resample the population to confirm the trend is continuing, to incorporate changes in water chemistry into any fouling surveys and to undertake manipulative experiments.

Dilophus marginatus carries a higher fouling load than co-existing algae

My survey of five species of algae showed one species, D. marginatus, had significantly higher levels of fouling than other co-occurring species (C. filiformis, S. robusta,

D. acrostichoides, and D. pulchra). Initially the higher fouling load and short life cycle seem to infer that D. marginatus is a would direct resources towards fast growth

(Pianka 1970). Epibiotic communities are not necessarily all bad for a host alga. Co- consumption (Karez et al. 2000; Strong et al. 2009) and nutrient availability (Gross et al. 2003) are just two possible benefits of hosting epiphytes. Yet my results suggest there is mitigation of the fouling load by D. marginatus with an asymptotic limit of around 20-25 %. This dichotomy suggests the possibility for a complex defence strategy and is explored in Chapter 4.

Seasonal variation in fouling load

The notion of a general summertime ‘fouling pressure peak’ has been under pressure for some time (Bellgrove and Aoki 2006). My results, whilst not significant at α=0.05 %,

53 Fouling factors

suggest that if there are temporal peaks in fouling load, then the peak is species specific, not universal. Even if there are identifiable species level peaks they should not be related to an imposed seasonal construct. The notion of four climatic seasons in

Australia is under challenge (Entwisle 2014) and it would be far better to measure fouling load against quantifiable factors such as temperature (Harms and Anger 1983;

Hellio et al. 2004; Shohael et al. 2006) or daylight exposure (Hellio et al. 2004;

Marechal et al. 2004). Nevertheless my results were suggestive of both an Autumn and

Summer peak in fouling loads which is worth exploring.

I have identified potential support for seasonal variation in the efficacy of algal antifouling strategies (Chapter 4, this thesis). I have also identified one species of alga with the potential for a complex defence strategy D. marginatus (Chapter 4, this thesis) and another species, C. filiformis (Chapter 5, this thesis) which has high potential for being a source of chemical defences against fouling.

54 Fouling factors

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62 Methods

Introduction

Fouling of marine surfaces is a global problem that has attracted research for decades

(Lebret et al. 2009). Settlement assays, in which propagules (larvae, spores) of marine organisms are exposed to varying surfaces or conditions, have been a common tool to study variation in settlement or the effectiveness of anti-fouling technologies (Bryan et al.

1997; Dahms et al. 2004; Fletcher 1989). In the latter case such assays have been used to test inhibition of settlement for a wide range of organisms (Briand 2009; Dahms and Hellio

2009) including bacterial biofilms (Bryan et al. 1997; Dahms et al. 2004; Dobretsov et al.

2006; Egan et al. 2001; Marechal et al. 2004; Rao et al. 2007), barnacle cyprids (Lau et al.

2003; Marechal et al. 2004), the reattachment of young juvenile mussels (da Gama et al.

2008), and algal spores or gametes (Dobretsov et al. 2006; Dworjanyn et al. 2006; Egan et al. 2001; Gribben et al. 2006),and for a range of surfaces.

Despite the importance and widespread use of settlement assays, there is often variation in results of these assays (Holm et al. 2000; Marshall and Steinberg 2014; Raimondi and

Keough 1990) and some of this variation could be due to differences in methodologies or experimental conditions used in different experiments. A comprehensive review by

Dahms and Hellio (2009) identified a number of methodological differences and factors that should be considered when interpreting the outcomes of settlement assay. For example, potentially important abiotic differences among studies include the type of

59 Methods

container or settlement dish used, the duration of the assay, the time of year (or season) in which the assay is done (Jarrett 2003) the conditions in which the propagules are kept, and light availability or consistency (Egan et al. 2001; Nylund et al. 2007). Potentially important biotic factors include the number of propagules per dish (Bryan et al. 1997;

Callow et al. 1997; Dahms et al. 2004; Elbourne et al. 2008), as many invertebrate larvae will settle gregariously (Bacchetti De Gregoris et al. 2012), and the size, age or condition of the larvae (Gribben et al. 2006; Marshall and Steinberg 2014).

Behaviour of propagules under differing conditions is well understood for some propagule taxa (Magin et al. 2010; O'Connor and Richardson 1998) including Ulva sp. (Ulvaceae,

Linnaeus) (Carl et al. 2014; Finlay et al. 2002; Finlay et al. 2008; Imchen 2012; Walker et al.

2005) and Polysiphonia sp. (Rhodomelaceae) (Dworjanyn et al. 2006; Fletcher 1979).

Given the importance of surface characteristics – texture, wettability, colour (Finlay et al.

2008; Scardino et al. 2003; Walker et al. 2005) to settlement, the choice of settlement dish for assays is very important. A number of different types and sizes of settlement dishes have been used in settlement assays (Dahms and Hellio 2009). Glass and plastic dishes and polystyrene (plastic), differ in their texture, wettability and other factors (Roberts et al. 1991) and have been used interchangeably in assays using B. neritina (Bryan et al.

1997; Dahms et al. 2004; Davis and Wright 1990; Gribben et al. 2006; Qi et al. 2008;

Wendt and Woollacott 1999) .

60 Methods

Many organisms respond to light, and are either negatively or positively phototactic

(Fletcher 1989), and this can vary over the course of their life history. This attribute is often used to induce propagule release from adults (Bryan et al. 1997; Dahms et al. 2004;

Egan et al. 2001), separate gametes and zoospores (Fletcher 1989), or actively select the most active spores (Egan et al. 2001). The absence of light may also facilitate even settlement of spores across the settlement plate (Bryan et al. 1997; Egan et al. 2001;

Nylund et al. 2007).

The amount of time propagules are given to settle can impact on results. Toonen and Tyre

(2007) suggest that the ability to delay metamorphosis will increase substrate selectivity, and that a given larvae will display differing selectivity as the time to metamorphose decreases. Gribben et al. (2006) found that older (bryozoans) larvae of both Watersipora subtorquata and B. neritina settle at much higher rates than younger larvae. The desperate larvae hypothesis (Elkin and Marshall 2007; Marshall and Keough 2003;

Pechenik et al. 1993; Swanson et al. 2007; Toonen and Tyre 2007; Wendt 1998) relies on either there being an absence of food (for feeding larvae) or for non-feeding larvae to exhaust their resources for selectivity to decrease (Elkin and Marshall 2007). The amount of time propagules are left before quantifying the results of settlement assays may therefore be critical.

The light conditions that propagules are left in also varies between studies (Bryan et al.

(1997) and Nylund et al. (2007), Elbourne et al. (2008) and Phang et al. (2009), Fletcher

(1989)and Callow et al. (1997)) and even within the same institution (Egan et al.

61 Methods

(2001),and Nylund et al. (2007)). Phototaxis is relied upon to initiate propagule release in a number of taxa including B neritina, but the behaviour of the larvae can vary between positive and negative phototaxis (Pires and Woollacott 1997). Propagules of

Bugula neritina in anti-fouling assays are often kept in the dark for a short period of time, e.g one hour (Dahms et al. 2004; Dobretsov and Qian 2006; Gribben et al. 2006) prior to counting, which avoids issues of older larvae settling despite the presence of anti-fouling compounds (Gribben et al. 2006). However, changes in phototactic behaviour (and thus the impact of light) of the larvae takes place over a much longer timeframe (Wendt and

Woollacott 1999) and studies have often left larvae to settle for up to 24 (Davis and

Wright 1990; Schmitt et al. 1995; Walters et al. 1996). In at least one study, Qi et al. (2008) did not specified if the larvae were kept in the dark at all. Settlement behaviour over time has been quantified (Schmitt et al. 1998) but only on a linear scale and without light variation. Interactions between time to settle and exposure to light are unknown for

B. neritina. An understanding of the impact of the methodological issues is currently missing from the literature for B. neritina.

Seasonal variation in production of anti-fouling defences is well documented and is often attributed to an increase in fouling over the warmer months. However, the possibility that epibionts themselves may seasonally vary their behaviour in response to increased levels of antifouling compounds is rarely considered (Qvarfordt et al. 2006). Seasonal variation in settlement success for all three propagule taxa (B. neritina, Ulva australis Linnaeus and

Polysiphonia sp.) needs to be determined.

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Understanding the variation that may occur due to methodological differences is important for the assays I conducted in this thesis, and may help explain the variation in results in prior studies and help guide future studies. In this chapter following methodological considerations I address are 1) the type of settlement container (glass or plastic) used in B. neritina assays and the effect of including a the solvent, DCM 2) the interaction between light exposure and time for B. neritina propagules to settle and 3) seasonal variation in the behaviour of B. neritina, Ulva australis Linnaeus and Polysiphonia sp. propagules over a year in a range of solvents.

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Materials and Methods

1) General protocols

a) Bugula neritina assays

Bugula neritina colonies were collected from underneath moored boats from Rose Bay,

Sydney (33°52'17.62"S, 151°15'33.41"E) or from Careel Bay Marina, Avalon

(33°37'13.44"S, 151°19'23.34"E). Colonies were transferred directly into light-proof containers filled with filtered seawater for transport. Colonies were then stored for a minimum of 24 hours in a dark 15˚C constant temperature room with aeration. To harvest propagules from the colonies, individual colonies were removed from the dark and placed into individual beakers filled with fresh filtered sea water and exposed to strong light. This was undertaken as close to natural sunrise as possible. Preliminary experiments in my laboratory had determined more reliable propagule release at this time (pers. obs). As propagules were released from the colonies, they were collected with a pipette and used in assays immediately. All dishes were placed in a 15˚C constant temperature room. All dishes were covered and subsequent to the light exposure experiment left for two hours before the dishes were removed and percentage settlement counted.

b) Ulva lactuca assays

Methods for Ulva australis generally follow Nylund et al. (2007). Fertile U. australis fronds were collected early in the morning, in the week preceding the full moon when they are most likely to be fertile (Heydt 2011), from the rock platform at Clovelly Bay, Sydney

(33°54'53.63"S, 151°16'14.49"E). Fertile fronds were identified by a darker brown band at

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the leading edge of the frond (Heydt 2011). The U. australis fronds had excess water shaken off and transferred into zip lock bags and placed on ice in a dark container for transport. The fronds were then placed in the fridge until use later the same day.

To harvest the propagules, U. australis fronds were placed in autoclaved filtered sea water

(FSW) at one end of long transparent container. A light source was placed at the other end of the container, and a pipette was used to collect propagules as they streamed towards the light. Harvested propagules were then placed in a small beaker containing FSW and agitated to prevent settlement. Harvesting continued until propagules had changed the colour of the FSW from clear to a pale cloudy green upon visual observation. One ml of this propagule suspension was added, with 4 ml of FSW, to each assay dish. Assay dishes were placed in a 15˚C constant temperature room and left for 6 days. An inverted microscope was used to count attached propagules within a viewing frame. Five haphazardly selected viewing frames were averaged from each dish to arrive at an estimate of fouling intensity.

c) Polysiphonia sp. assays

Methods for harvesting and utilising Polysiphonia sp. in these assays generally follow

Nylund et al. (2007). Polysiphonia sp. occurs commonly as an epiphyte on large, subtidal, red and brown algae. Red and brown algae on the rock platform at Clovelly Bay, Sydney

(33°54'53.63"S, 151°16'14.49"E) were searched and selected for suspected Polysiphonia sp. thalli. Excess water was shaken off and the algae placed on ice in dark containers for transport , and refrigerated until required later the same day. A dissecting microscope was

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then used to identify and isolate Polysiphonia sp. fronds. Carpospores (from female gametophytes) were collected with a pipette as they were released from the fronds and immediately used in assays. All dishes were placed in a 15˚C constant temperature room for 24 hours when they were removed and percentage of spores attached to the base of the petri dishes was calculated.

2) Settlement rates of Bugula neritina propagules in glass and polystyrene petri dishes coated with or without dicholoromethane

Approximately 20 B. neritina were added to ten replicates of each of the four treatment dishes. Each treatment consisted of autoclaved 9 mm2 petri dishes of either glass or polystyrene filled with 4 ml of filtered autoclaved sea water (FSW). In addition to the effects of dishes made from different materials, the effect of dichloromethane (DCM), which is often used as a solvent in assays of chemical settlement inhibitors, was also tested. Half of both the glass and polystyrene dishes were coated with 5 % DCM, left to evaporate on a shaker table, and then filled with 4 ml of FSW before use.

3) Response of Bugula neritina larvae to intermittent light during settlement

Approximately 20 B. neritina were added each to 9 mm2 plastic petri dish with 4 ml of filtered autoclaved sea water. Dishes were then randomly assigned as either ‘control’ or

‘light exposed’. All dishes were placed in a 15˚C constant temperature room. All dishes were covered and left for one hour. After one hour all ‘light exposed’ dishes were uncovered, while the ‘control’ dishes were left covered. Starting at the one hour mark and every ten minutes afterwards five each of both control and light exposed dishes were

66 Methods

removed and percentage settlement counted. The experiment was halted after two hours except for ten remaining dishes which were left covered for a total of four hours.

4) Seasonal response of Bugula neritina, Ulva lactuca and Polysiphonia sp. propagule settlement to a variety of solvents.

Propagules used in settlement assays are often tested against extracts from a range of algae species. Different solvents have been tested with each species of algae to determine optimum solvent (and immersion time) for each species of alga (Nylund et al. 2007). While one would expect 100% of the solvent to evaporate prior to the propagules being introduced to the assay dishes, the variation in settlement between differing of solvent controls suggests that this is not so. Assays were conducted on a range of solvents that mimic the controls.

Treatment dishes for all assays were prepared by coating 9 mm plastic petri dishes and left to evaporate on a shaker table before use, the solvents used were DCM, Ethanol, and

Hexane. These solvents are used in antifouling assays to strip potential anti-fouling chemicals from the surface of different algae (Nylund et al. 2007). Autoclaved filtered sea water (FSW) was used as control dishes. B. neritina, Polysiphonia sp. and U. australis were collected in the summer and winter of 2008 and tested against each of the solvents. The

B. neritina and U. australis assays were opportunistically repeated one spring, but due to quantity of propagules available, I was only able to run 2 solvent trials, DCM and FSW.

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5) Statistical Analyses

To determine if the settlement of Bugula neritina propagules was consistent between types of different surface textures as exhibited by glass and plastic petri dishes we used

IBM SPSS Statistics v.21 to run a general linear model with dish type (glass or plastic) and treatment (solvent or filtered autoclaved sea water (FSW)) as our predictor variables and percentage successful settlement as our dependent variable with a significance level of

P<0.05. The non-significant interaction was removed.

The effect of exposing Bugula neritina propagules to light during the initial 2 hours of an incubation period was analysed using IBM SPSS Statistics v.21 to run a general linear model with minutes (uncovered after an initial 1 hr in darkness in 10 minute increments) as the predictor variable and percentage successful settlement as our dependent variable with a significance level of P<0.05. As there was a significant interaction, each time increment was analysed independently from each other.

To determine if epibiotic propagules (larvae) have a consistent response across seasons, the % settlement of propagules (our dependent variable) harvested across different seasons (Summer, Winter and Spring) our predictor variable was analysed using a general linear model through IBM SPSS Statistics v.21. Each target species (Bugula neritina,

Polysiphonia sp. and Ulva australis) was examined independently for each of the common solvents (filtered autoclaved seawater (FSW), dichloromethane (DCM), ethanol and

Hexane).

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Results

1) Response of Bugula neritina to variation in surface texture (glass versus

plastic)

There is a significant difference in the settlement between dish types. The glass surface significantly reduces the amount of settlement (P=0.015, F1,39=6.497,

50 Glass Plastic n=10

b b

% Settlement 20 a

a 10

0 5% DCM FSW Control

Figure 3.1) for both the control dishes and dishes treated with 5 % DCM.

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50 Glass Plastic n=10

b b

% Settlement 20 a

a 10

0 5% DCM FSW Control

Figure 3.1: Settlement of Bugula neritina propagules in filtered sea water in glass and plastic petri dishes that had being coated with 5 % DCM. Significant differences (at P<0.05) are indicated by differing letters (“a” and “b”).

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a) Propagule behaviour in response to light exposure

Any exposure to light during the first 2 hours of the incubation period significantly reduced the rate of Bugula neritina propagule settlement, by approximately half (P<0.001,

F1,70=17.065; Figure 3.2). This response varied significantly over time (P=0.020, F6,70=2.754;

Figure 3.2), with consistent differences between covered and uncovered dishes that had been uncovered for up to 50 minutes after the initial 60 minutes of dark. The variation in response largely disappeared after a total incubation time of 120 minutes, i.e. 60 minutes in the dark, then 60 minutes in the light. After 4 hours, settlement reached an average of almost 100% in both treatments.

100

Covered Uncovered 80 n = 10

a 60

a a

% Settlement 40

20 b b b

0 0 10 20 30 40 50 60 (P=0.118) (P=0.007) (P=0.101) (P=0.033) (P=0.180) (P=0.001) (P=0.912)

Minutes uncovered after an initial 1 hr in darkness

Figure 3.2: Settlement of Bugula neritina propagules in plastic petri dishes response to an increasing light exposure duration.

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2) Variation in propagule behaviour

a) Bugula neritina propagules

There was a significantly higher level of settlement of summer propagules compared to winter propagules of B. neritina in Ethanol treated dishes (P=0.002, F1,20=12.441). In filtered seawater spring propagule settlement was significantly lower (P<0.001,

F2,45=14.773) than summer propagule settlement (Tukeys P<0.001) and winter (Tukeys

P=0.003) settlement. There was a marginally non-significant difference in propagule behaviour between summer and winter settlement (Tukeys P=0.064) in hexane coated dishes or in the DCM coated dishes.

Summer Winter Spring 100 n=10

a a a

% Settlement 40

0 FSW DCM Ethanol Hexane Figure 3.3: Variation in the settlement of Bugula neritina propagules in plastic petri dishes coated with either dichloromethane (DCM), ethanol or hexane controlled against settlement in filtered sea water (FSW) between seasons.

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b) Polysiphonia sp. propagules

There were higher levels of settlement of Polysiphonia sp. propagules in the winter then in summer when settling on DCM (P=0.028, F1,20=5.734, Figure 3.4) but not in filtered seawater, ethanol or on hexane coated surfaces.

100 Summer Winter

80 b

a 60

% Settlement

0 FSW DCM Ethanol Hexane

Figure 3.4: Variation in the settlement of Polysiphonia sp. propagules in plastic petri dishes coated with either dichloromethane (DCM) and ethanol controlled against settlement in filtered sea water (FSW).

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c) Ulva australis propagules

The settlement of U. australis propagules in seawater was significantly higher in winter then in spring (P=0.001, F1,20=15.974, Figure 3.4) in filtered seawater. However, there was no significant difference between summer and winter settlement on DCM or Ethanol coated surfaces.

16 a Summer 14 Winter Spring

% Settlement b 6

0 FSW DCM Ethanol Figure 3.5:Variation in the settlement of Ulva australis propagules in plastic petri dishes coated with either dichloromethane (DCM) and ethanol controlled against settlement in filtered sea water (FSW).

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Discussion

Behaviour of Bugula neritina can be affected by light and texture

My results show that exposure to light alters the settlement process of B. neritina propagules. Alteration in settlement process due to light exposure could be an advantage for the population fitness of B. neritina as it allows the larvae to potentially avoid areas exposed to excessive intermittent light which are not conducive for the adult colonies

(Roberts et al. 1991). The overwhelming disadvantage of phototactic behaviour for the individual larvae is that it increases the time the larva is in the water column. An increase in in the time spent in the water column has a range of secondary impacts. In the water column, the larva is privy to increases in predator exposure time, increase in the chance of being swept away from suitable habitat (Wendt 2000) and can also impact on future fecundity (Wendt 1998). Additionally, the larva has a limited amount of stored energy, which at least 20% of is required for successful settlement (Jaeckle 1994).

Understanding the role that light can play in the settlement process (not only for Bugula neritina, but potentially any propagule used in a fouling assay) is critical to ensure that assay results are due to the activity of anti-fouling compounds, and not an artefact induced by propagule behaviour or an effect of larval age (Gribben et al. 2006).

Understanding this behaviour could also explain variation in B. neritina assay results, particularly when the propagules have been incubated in darkness for less than two hours

(Dahms et al. 2004; Dobretsov and Qian 2006). If propagules have been incubated for less than two hours, handling procedures may allow intermittent light to impact the behaviour

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of the propagules. Assays using B. neritina as a fouling propagule should allow two hour of incubation time in the dark before counting. Waiting until the light effect disappears should result in increased consistency of B. neritina settlement response.

My results also show that the propagules of Bugula neritina have a much higher settlement rate (more than double) when introduced into plastic petri dishes, than when introduced into glass petri dishes. Although not surprising as propagules from many species show a similar preference (Magin et al. 2010; O'Connor and Richardson 1998).

Petri dishes not only differ in compound (e.g. glass or plastic), but also texture of the dish and wettability (Roberts et al. 1991). The impact of surface texture on settlement is used as an anti-fouling technique (e.g. molluscs. (Scardino et al. 2003). And surface variation has been shown to impact the settlement of a range of species including hydroids

((Ronowicz et al. 2008), algae ((Sidharthan et al. 2004) and oysters (Whitman et al. 1998).

It is therefore important that the surface preference for your target fouling species is understood and taken into consideration when reviewing or synthesising results from the past literature (Wendt and Woollacott 1999). Surface texture of the petri dish is also important to take into consideration when planning experimental design. If you are attempting to deter settlement with chemicals, using glass petri dishes which also deter settlement may decrease the possibility of discerning an effect.

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Fouling propagules demonstrate seasonal variation in settlement response

All three species of propagule exhibited variation in settlement response on different common solvents across different seasons. The solvents were evaporated as part of the methodological process, so I expected no effect on the settlement of propagules between the treatment dishes. Whether some of the solvent is remaining coated onto the bottom of the petri dish, or the solvent is reacting with the plastic petri dish, this effect of the solvent needs to be controlled for.

Furthermore all three of my species: B. neritina, Polysiphonia sp. and U. australis exhibited variation in propagule settlement in at least one of the treatment dishes. Winter propagules of both B. neritina and U. australis had increased levels of settlement in filtered sea water. Further work understanding what is occurring with the different solvents and settlements of propagules over multiple replicate seasons should be undertaken, and until then, results derived from experiments conducted within a single season should be discussed with caution.

My results show that quantifying the impacts of the methodological decisions is critical in understanding conflicts and limitations in the literature as well as designing better more reliable experiments into the future. Specifically there are distinct choices, that depend on the question you are trying to answer, that should be made between selecting glass or plastic experimental dishes; that B. neritina should be incubated for two hours to avoid effects of light; you may need multiple controls for an assay; and experiments should be replicated across multiple seasons and replicate years to tease out variation in propagule

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behaviour. This chapter shows that considerations of methodological impacts on experimental design can be crucial.

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82 Dilophus marginatus

Figure 4.1: Dilophus marginatus fronds (Image: J.Green)

83 Dilophus marginatus

Introduction

There are millions of microorganisms in every litre of seawater (Acinas et al. 2004), as well a diversity of propagules (larvae, spores) from benthic eukaryotes which require a settlement surface in order to complete their life cycle . Any submerged surface is thus under constant attack from propagules of potential fouling organisms. These propagules, when they settle and grow on other marine organisms, can have strong detrimental effects on their hosts (Tegner and Dayton 1987, Duffy 1990, Jones 1992,

Duffy and Hay 2000, Karez et al. 2000, da Gama et al. 2008b, Newcombe and Taylor

2010, Verges et al. 2011). This constant pressure from fouling thus exerts a strong selection pressure on potential hosts to evolve strategies against fouling for survival.

While some marine organisms develop symbioses with fouling organisms (e.g. decorator crabs; Stachowiwicz and Hay (1999)), antifouling strategies for most marine organisms involve avoiding, reducing or actively deterring fouling. These include physical (e.g. surface texture and sloughing), biological (e.g. facilitating specific bacterial communities) and/or chemical strategies (Wahl 1989, Steinberg et al. 1997,

Steinberg and de Nys 2002, Scardino and de Nys 2004, Wahl 2009, Stengel et al. 2011).

Regarding the latter, a range of anti-fouling compounds including furanones, and dipterpenes have been identified from marine organisms including algae (Amade and

Lemée 1998, Steinberg et al. 1998, Dworjanyn et al. 2006, Bianco et al. 2009).

Understanding the defence strategy of a given organism helps understanding of the ecology and evolution of these organisms (Turley et al. 2013) and more broadly informs understanding of life history traits, growth strategies, and resource allocation

(Herms and Mattson 1992, Pavia et al. 2002). Having information about the defence

84 Dilophus marginatus

strategy can also aid in bioprospecting, particularly where the goal is to identify and exploit those defences to develop products which can be used to prevent or limit fouling (Nylund et al. 2013). There is an ongoing search for effective compounds which have low environmental impact and that can be synthesised for commercial use (Wahl

1989). Bioprospecting for novel antifouling compounds form natural sources typically involves the use of bioassays (Fletcher 1989, Briand 2009, Dahms and Hellio 2009), which provide a reasonably quick method of determining if a particular species has chemicals that deter the settlement of fouling propagules. There are many cases, however, where the results from one bioassay directly contradict the results of others

(Briand 2009). A seagrass or alga with a more complex chemical defence strategy may result in variation in the production of antifouling metabolites, and thus explain some of the variation in results of bioassays. This may facilitate the discovery of additional, previously undetected anti-fouling compounds. There are several theories (r-K strategy theory and optimal defence theory) in the literature that link defence strategy with life history traits. If defence strategy can be closely linked to life history traits, selection of seagrass/algae or other hosts for bioassays can be improved.

The efficacy of surface chemistry extracts from the brown alga Dilophus marginatus against a range of fouling propagules.

Many species of Dilophus marginatus J.Agardh (Figure 4.1) contain compounds such as diterpenes (Ravi and Wells 1982) which deter herbivores both in the laboratory and in the field (Barbosa et al. 2007). Diterpenes also deter the settlement of larvae of herbivores (Kurata et al. 1988) and have anti-microbial effects (Reichel and Borowitzka

85 Dilophus marginatus

1984). However, despite the high potential for antifouling compounds in this species,

D. marginatus typically had higher levels of fouling than other surrounding algae in my field surveys (Chapter 2, this thesis), and the effect of surface extracts from local

D. marginatus against fouling propagules is inconsistent. Gribben et al. (2006) found that extracts from D. marginatus deterred the settlement of freshly hatched

Bugula neritina (Linnaeus, 1758) and Watersipora subtorquata (d'Orbigny, 1852) larvae, but not older propagules, while Nylund et al. (2007) found no impact on the settlement of the common local foulers Polysiphonia sp. and U. australis at natural or double the natural surface concentration.

In Chapter 3 (this thesis), I quantified the impact of a number of methodological decisions which may have impacted on the outcomes of previous studies.

Does the efficacy of surface inhibitors against fouling from D. marginatus correlate with in-situ levels of fouling through time?

The amount of fouling on an alga should on average change over time (Fletcher and

Callow 1992, Holm et al. 1997, Hanamura 2000). New tissue, with little time to be exposed to fouling propagules should be free of fouling until propagules have time to settle. Older tissue will have been exposed to potential fouling propagules for a far greater period of time. However, the relationship between fouling and frond age is not necessarily linear. There could be many other factors impacting the fouling rates on a given alga. For example, temperature and depth may affect the abundance and composition of the fouling community (Ahlgren 1987, Jennings and Steinberg 1997,

Head et al. 2004, Rule and Smith 2007, Jacobucci et al. 2009). 86 Dilophus marginatus

Fouling may also vary due to the presence of defensive anti-fouling chemicals, the efficacy or quantity of which can vary over time (Hellio et al. 2004). There may be seasonal variation in the production of defensive compounds (de Nys et al. 1995,

Steinberg et al. 1998, Culioli et al. 2002, Hellio et al. 2004). There is also seasonal variation in the efficacy of the compounds themselves (Cronin and Hay 1996, Amsler

2001, Haavisto et al. 2010). Chemical defences can accrete over time, resulting in a heavily defended older frond (Yan et al. 2015).

Antifouling effects may also vary if compounds are inducible as opposed to constitutive. Inducible defences are defences that are produced in response to an attack (Pavia and Toth 2000, Toth and Pavia 2000, Arnold and Targett 2003) and are mostly known for defences against herbivory. Inducible defences have not yet being explored with respect to fouling. Induced defence theory is popular because chemical defences are thought to be costly, and therefore considered not likely to be inherent or persistent (Arnold and Targett 2003). This would only be true if the primary purpose of the compounds was defence (Herms and Mattson 1992, Arnold and Targett 2003).

Many effective defences are known to have a range of functions (Christophersen 1985,

Bennett and Wallsgrove 1994, Tringali 1997, Rogers et al. 2000, Amsler et al. 2001, Da

Gama et al. 2002, Hammami et al. 2010).

If inducible defenses against fouling are present, then we would expect to see variation in the efficacy of anti-fouling compounds in response to variation in fouling, for example by season. The notion of a fouling ‘season’, with increased summer fouling pressure, is widespread (Harms and Anger 1983, Jarrett 2003, Hellio et al. 2004,

87 Dilophus marginatus

Satheesh and Godwin Wesley 2008). More generally an increase in the supply of fouling propagules at specific times of the year could also lead to a variation in the quantity of fouling on a given species (Bellgrove and Aoki 2006, Clark and Johnston

2009). Seasonal fouling pressure may also result in host algae temporally avoiding fouling propagules by growing in the colder months when the fouling pressure is low.

Correlating the efficacy of surface compounds in laboratory experiments with in-situ levels of fouling with has had increased focus in recent years (Nylund et al. 2007), but it has been limited to a single point in time.

Can the efficacy of surface chemistry be linked to reproductive status?

Optimal defense theory assumes that chemical defences are expensive to produce

(Herms and Mattson 1992), and there is some evidence for this for macroalgae

(Jormalainen et al. 2003, Dworjanyn et al. 2006). This results in the prediction that algae should protect those tissues that are most valuable for future fitness. Valuable tissue is often considered to be young growing tissue, reproductive tissue, or attachment points (Steinberg 1984, VanDam et al. 1996, Hyvarinen et al. 2000, Pavia et al. 2002, Asplund et al. 2010). It has been demonstrated that algae can preferentially produce defensive compounds in different regions of the algal thallus (Pavia et al.

2002, Fairhead et al. 2005, Macaya et al. 2005, Connan et al. 2006).

The aims of this chapter are to a) quantify the efficacy of extracts from the surface of

D. marginatus fronds against a range of local fouling propagules under my methodological regime, b) to compare temporal changes in the efficacy of surface extracts from D. marginatus with the trajectory of in-situ fouling over time, and c) test 88 Dilophus marginatus

if fronds with reproductive structures will have more efficacious surface extracts as they could be considered more valuable to the survival of D. marginatus.

Materials and Methods

1) Study organism - Dilophus marginatus

The brown macroalga D. marginatus from the Order Dictyotales, Family Dictyotaceae occurs in the lower intertidal and subtidal rocky shores in regions of temperate

Australia (Phillips 2000). D. marginatus has a short, single season life cycle which enables easy field collection of new sporophyte fronds and is commonly found in almost every bay on Sydney’s coastline over the colder months (pers. obs.). The reproductive stage of the D. marginatus sporophyte is easily distinguishable with sporangia developing only in undulate thallus cavities. (Phillips 2000). D. marginatus grows from an apical meristem.

2) The efficacy of surface extracts Dilophus marginatus against a range of fouling propagules

The potential for inhibition against a range of fouling organisms by surface extracts from D. marginatus was investigated in the winter of 2007. Replicate fronds from

D. marginatus were collected from Bare Island (33°59’30.80” S, 151°13’53.60” E) from individual plants or clumps. Ten fronds were used for each treatment for each fouling propagule. The fouling propagules used in assays were from B. neritina,

Polysiphonia sp. and U. australis (as described in Chapter 3, this thesis). Each frond was randomly assigned to one of the following treatments: seawater control, solvent 89 Dilophus marginatus

control, extracts at natural surface concentrations (1x), and double the natural surface concentration (2x). To calculate natural surface concentration, a comprehensive surface area per weight table was generated. Each frond was weighed and recorded.

Once the surface chemicals from the entire frond had been extracted, a proportion of the solvent calculated to contain the surface extracts from an area equivalent to the size of the petri dish was added to 9 cm2 polystyrene petri dishes and shaken until evaporated. Surface chemicals were extracted by immersion in 6% Dichloromethane

(DCM) for 40 sec. Immersion time was experimentally determined with natural surface concentration determined to be achieved when the maximum amount of non-polar metabolites were extracted by the solvent before cells began lysing (as determined by

Nylund et al. (2007)). Extracts were added to. 4 ml of filtered autoclaved seawater

(FSW) were added to each of the dishes with the propagules. B. neritina settlement was counted after 2 hours in the dark (Chapter 3, this thesis).

3) Does the efficacy of surface inhibitors positively correlate with in-situ levels of fouling through time?

To correlate the efficacy of surface chemistry with in-situ levels of fouling I needed to monitor the level of fouling over time. Due to the fragility of D. marginatus fronds, in- situ monitoring is technically difficult. I proxied the passage of time in two ways:

1) Sample fronds across the growing season. The fronds collected early season will all be new fronds (less than one month old), whilst the fronds collected later in the season will have an increasing proportion of older fronds as well as some new fronds.

2) Because the fronds of D. marginatus do not branch I use frond size as a proxy for age. 90 Dilophus marginatus

Weekly reconnaissance of known D. marginatus habitat on snorkel commenced at

Bare Island in April 2007. Clumps of fronds, each larger than an outstretched hand, started to appear at the onset of winter. Replicate samples were then collected every week from Bare Island for six weeks. Each replicate sample consisted of five haphazardly collected fronds from each of five haphazardly sampled algal clumps.

Algae were collected in cliplock bags with surrounding water. Samples were taken back to the university where they were dried and frozen until they could be digitally photographed and percentage fouling analysed using Scion Image J (Chapter 2, this thesis). Each replicate datum was an average of the five fronds from a single algal clump.

Settlement of Polysiphonia sp. spores on surface extracts from early versus late season

D. marginatus fronds was done as describe above and in Chapter 3 (this thesis).

Settlement of spores in the filtered autoclaved sea water (FSW) control and the treatment dishes was calculated as a percentage of settlement in the solvent control dishes as the use of a solvent had been determined to impact on the settlement rate of Polysiphonia sp. propagules (Figure 3.4, Chapter 3, this thesis).

To determine whether surface extract activity correlated with levels of fouling across the duration of the growing seasons, I ran another bioassay using additional fronds collected at start and finish of the sampling regime. The bioassay was conducted as above using U. australis as it is one of the most common fouling propagules in the region (as described in Chapter 3, this thesis).

91 Dilophus marginatus

4) Seasonal variation in surface extract efficacy

I took advantage of an unusual but chance finding of some summer growing

D. marginatus to determine if D. marginatus if extracts were more efficacious in the warmer summer months. The summer fronds were collected in the summer of 2008 from Nielsen Park. Settlement in the summer assay was compared to the settlement on extracts from randomly selected fronds in the Winter 2007. It is impossible to determine the number of U. australis propagules introduced into the assay. To control for variation in the number of propagules between the assays, the settlement percentage of U. australis is calculated against the total settlement in the FSW control

(not shown). Propagules of U. australis had previously shown no variation in settlement due to solvent, but a seasonal response in FSW (Figure 3.5, Chapter 3, this thesis). Therefore FSW was considered the most appropriate control.

5) Does the efficacy of surface deterrents correlate with reproductive status?

In the winter of 2007 individual fronds from haphazardly selected clumps of

D. marginatus were collected from Bare Island. Fronds were then classified as having no sporangia visible, or with visible reproductive structures. Surface chemicals were extracted as outlined Chapter 3 (this thesis). Assays using U. australis (as described in

Chapter 3, this thesis) were conducted at natural, double, quadruple and octuple natural concentrations, there were not enough fronds available late season to replicate all concentrations. Fronds from D. marginatus collected over a three-year period for the surveys discussed in Chapter 2, (this thesis) were classified as either

92 Dilophus marginatus

juvenile (pre-reproductive) or adult (reproductive structures visible) and total fouling biomass (measured using area) quantified using Scion Image J (Chapter 2, this thesis).

Percentage of area fouled or fouling coverage was determined by the relationship between the surface area of the fouling organisms to the surface area of the host. In some cases the surface area of the fouling organism is greater than the surface area of the host algae therefore the percentage fouled is greater than 100%.

Entire fronds with or without reproductive structures were tested in the Ulva assays

Isolating just the reproductive areas of the fronds may have led to cross contamination by within tissue compounds leaked when the tissue is cut. The general contents within the cell might be inhibitory in their own right (Nylund et al. 2007).

When determining settlement rates for U. australis on extracts from D. marginatus, the number of settled propagules per dish is the average of 5 haphazardly selected fields of view under an inverted microscope. Actual settlement of the treatments and the controls is recorded. To determine settlement of U. australis on early and late season D. marginatus extracts I needed to control for variation in the number of propagules introduced into the assays. Therefore the settlement percentage of

U. australis in treatment dishes is calculated against the total settlement in the FSW control.

6) Statistical Analyses

Unless otherwise specified, IBM SPSS Statistics v.21 was used to analyse all data in this chapter. Data was tested for normality of distributions before Univariate Analysis of

93 Dilophus marginatus

Variance with an alpha of 0.05 was used to determine significance. Data for each fouling propagule were examined individually. Where there were significant differences between groups, Tukey’s posthoc tests were used to determine where the differences lay.

To determine the effect of surface extracts from Dilophus marginatus on the settlement of fouling organism, a general linear model was run with treatment

(seawater control, solvent control, natural concentration of surface extract and double natural concentrate of surface extract) as the predictor variable and successful settlement of propagule as the dependent variable. The three fouling species, Bugula neritina, Polysiphonia sp. and Ulva australis were analysed individually.

Linear regression was used to quantify change in the amount of fouling coverage over time. Weeks elapsed (from first noticing significant growth of Dilophous marginatus) was the predictor variable, with increments of one week for six weeks. Fouling load

(with coverage of fouling organisms as a percentage of host algal area) was our dependent variable.

A linear regression was also used to determine if there was a relationship between fouling coverage and frond size. . Frond size (our predictor variable) was log transformed before analysis. Fouling load (with coverage of fouling organisms as a percentage of host algal area) was our dependent variable.

To determine if the rate of defensive compounds increases the longer Dilophus marginatus persists in the environment, we used a general linear model with

94 Dilophus marginatus

treatment (seawater control, solvent control, extracts at natural concentration, extracts at double natural concentration) and season (early and late) as the predictor variables, and successful settlement of fouling propagules as the dependent variable.

For Bugula neritina¸settlement is the percentage of successfully attached larvae, while for Ulva australis propagules, settlement is an average settlement, Each treatment was analysed individually as there was significant interactions between treatments and season.

To determine if propagules settle with the same success rate all year round, a general linear model was run with treatment (seawater control, solvent control, extracts at natural concentration, extracts at double natural concentration) and season (summer and winter) as the predictor variables, and successful settlement of fouling propagules as the dependent variable.

To determine if settlement of fouling propagules is not affected by reproductive status, a general linear model was run with treatment (extracts at natural concentration, extracts at double natural concentration) and reproductive status (small non- reproductive fronds, large reproductive fronds) as the predictor variables and average successful settlement of Ulva australis propagules as the dependent variable. The non- significant interaction term was removed from the analysis.

To quantify if fouling loads are not affected by reproductive status, a general linear model was run with reproductive status (non-reproductive fronds and reproductive fronds) as the predictor variable and percentage fouled as the dependent variable.

95 Dilophus marginatus

Results

1) Effect of Dilophus marginatus surface extracts on a range of fouling

propagules

Extracts of D. marginatus significantly inhibited settlement of Polysiphonia sp.

propagules (P=0.004, F3,38=5.351; Figure 4.2) with ~ 20 % less settlement on the

treatment dishes relative to settlement on the solvent control. Doubling the

concentration of the surface extracts from D. marginatus did not increase the

inhibitory effect as there was no difference due to concentration of the extracts. In

contrast, U. australis propagules had enhanced settlement on dishes coated with

D. marginatus extracts (P=0.001, F3,38=6.481; Figure 4.2) when compared to the

solvent and fresh water controls. Again, extract concentration made no difference

to settlement (P=0.999, Figure 4.2). There was no significant effect of

D. marginatus extracts on the settlement of B. neritina propagules (P=0.350,

F3,38=1.132; Figure 4.2).

96 Dilophus marginatus

100 100

a a 80 80

Seawater

b ) b Solvent 1x 60 2x 60

Ulva australis

(

Proagules settled

% Settled 40 40

b b 20 20 a a

0 0 Bugula neritina Polysiphonia sp. Ulva australis Figure 4.2: Settlement of Bugula neritina and Polysiphonia sp. (% settled) and Ulva (number) propagules in seawater, solvent control, natural concentration (1x) and double natural concentration (2x) of surface extracts from Dilophus marginatus fronds. Significant differences between treatments indicated by ‘a’ and ‘b’ for Polysiphonia sp. (P=0.004, F3,38=5.351) and Ulva australis (P=0.001, F=3,386.481). There was no effect of the extracts on the propagules of Bugula neritina (P=0.350, F3,38=1.132).

97 Dilophus marginatus

2) Inhibition by surface extracts vs. in-situ levels of fouling over time

a) Temporal changes in fouling

There was no significant increase in the amount of visible fouling throughout the

2 growing season (P=0.083, F1,143=3.038, R = 0.021, n = 143; Figure 4.3).

180 R 2 = 0.021

160

140

120

100

% fouling 60

1 2 3 4 5 6

Weeks elapsed Figure 4.3: Percentage of fouling (area) on Dilophus marginatus fronds over a 6 week period in the winter of 2007 2 (P=0.083, F1,143=3.038, R = 0.021, n = 143).

98 Dilophus marginatus

b) Relationship between fouling and frond size

There was no significant correlation between frond size and the percentage of fouling

2 on fronds from D. marginatus (P=0.194, F1,144=1.706, n=143, R =0.0152; Figure 4.4).

180

160

140

120

100

% fouling

2.0 2.5 3.0 3.5 Ln Frond size (cm 2) Figure 4.4: Percentage fouling (area) on Dilophus marginatus fronds of different sizes (log transformed), P=0.194, 2 F1,144=1.706, n=143, R =0.0152.

99 Dilophus marginatus

c) Settlement of Polysiphonia sp. propagules on surface extracts from

early and late season Dilophus marginatus

Settlement of Polysiphonia sp. propagules on double the natural concentration of surface extracts from D. marginatus fronds differed significantly between fronds collected from early in the season (6.83% of settlement on solvent controls) and fronds collected later in the season (71.8% of settlement on solvent controls; P<0.001,

F1,18=51.783; Figure 4.5). There was no difference between the settlement of

Polysiphonia sp. propagules between extracts from early and late season fronds in response to natural concentration of surface extracts.

120 Early Late

100

80 b

Mean % Settlement 40

0 Dilophus marginatus Dilophus marginatus FSW x1 x2

Figure 4.5: Settlement of Polysiphonia sp. propagules on natural and double concentrations of surface chemicals extracted from early and late season Dilophus marginatus fronds, calculated as a percentage of settlement on the Dichloralmethane (Solvent) control. “a” indicates treatment that was significantly different P<0.001, F1,18=51.783.

100 Dilophus marginatus

d) Settlement of Ulva lactuca propagules on surface extracts from early

and late season Dilophus marginatus

Settlement of U. australis propagules on extracts from early vs. late season

D. marginatus fronds was significantly different, both at single (natural) concentrations

(P<0.001, F1,18=23.977; Figure 4.6) and double natural surface concentrations (P<0.001,

F1,19=72.435; Figure 4.6). In both concentrations, settlement of propagules on extracts from late season D. marginatus fronds was two to three times higher than settlement on early season extracts.

The extracts from both early season and late season D. marginatus fronds were also significantly different from the solvent controls. Settlement on extracts from early season fronds was significantly less ( P<0.001, F5,50=9.827; Figure 4.6) with on average

50 % settlement compared to approximately 75 % settlement on the solvent controls.

Settlement on extracts from late season fronds were significantly higher (~30 %) than the solvent controls (P=0.001, F3,38=6.481; Figure 4.6).

101 Dilophus marginatus

140 b b

120

100 Early Season Late Season

60 a

FSW settlement rates)

% settlement (compared to

a 20

0 FSW Solvent Natural Double Quadruple Octuple

Extract concentrations Figure 4.6: Increased settlement of Ulva australis propagules on natural (P<0.001, F1,18=23.977) and double (P<0.001, F1,19=72.435) concentrations of surface chemicals extracted from late season Dilophus marginatus as compared to settlement on extracts from early season D. marginatus. Also compared to controls of filtered and autoclaved sea water (FSW) and dichloralmethane (solvent) control. Additional assays at quadruple and octuple levels of Dilophus marginatus surface extracts from early season fronds were also done.

102 Dilophus marginatus

e) Settlement of Ulva lactuca propagules on surface extracts from summer

and winter growing Dilophus marginatus fronds

Propagules from U. australis settled far more readily in response to surface extracts from D. marginatus collected in winter then in response to surface extracts from s fronds collected in summer. In winter settlement in extract dishes was 20 % of the settlement in FSW dishes (set to 100% not shown), which was significantly higher than the 4-8 % settlement seen on the summer extract dishes (P<0.001, F1,54=140.970;

Figure 4.7). Doubling the concentration of the extract did not result in a significant difference in either summer or winter (P=0.168, F1,54=1.954; Figure 4.7).

Summer 100 Winter

% propagules settled 40

0 1x 2x FSW Ulva australis Control

Figure 4.7: Settlement (mean +- se) of Ulva australis propagules on natural and double concentrations of surface chemicals (P=0.168, F1,54=1.954) extracted from summer and winter (P<0.001, F1,54=140.970) growing Dilophus marginatus fronds as a percentage of settlement in filtered and autoclaved sea water (FSW).

103 Dilophus marginatus

3) The effect of reproductive status on deterrence of fouling propagules.

a) Ulva lactuca assay on small non-reproductive and large reproductive

Dilophus marginatus fronds

The presence of reproductive structures on fronds of D. marginatus significantly affected settlement of U. australis propagules relative to non-reproductive fronds

(P=0.003, F1,84=9.299; Figure 4.8). Extracts from small, non-reproductive fronds averaged less than half the settlement of U. australis propagules than extracts from the large reproductive fronds. There was no increase in the efficacy of the extracts when the concentration was doubled.

Non-reproductive Reproductive

60 b

a a

Propagules settled

0 1x 2x Concentration Figure 4.8: Increased settlement of Ulva australis propagules on extracts on large Dilophus marginatus containing reproductive structures (P=0.003, F1,84=9.299) when compared to settlement on extracts from small non- reproductive fronds.

104 Dilophus marginatus

b) Percentage fouling on reproductive and non-reproductive Dilophus

marginatus fronds

The percentage of the total in-situ fouling community was significantly greater on

D. marginatus fronds with reproductive structures compared to those without

(P<0.001, F1,773=86.598; Figure 4.9). The surface area of fouling organisms on non- reproductive fronds was equivalent to approximately 5 % of the D. marginatus frond surface area, while fronds with visible reproductive structures averaged fouling to the equivalent of 20 % of frond surface area.

% fouled

0 Non-reproductive Reproductive fronds fronds Figure 4.9: Increased percentage of fouling coverage on reproductive Dilophus marginatus when compared to non-reproductive fronds (P<0.001, F1,773=86.598).

105 Dilophus marginatus

Discussion

Reproductive status matters

Historically, researchers have randomly or haphazardly selected replicates of marine algae for use in biosassays without regard to reproductive status (Nylund et al. 2007,

Briand 2009, Dahms and Hellio 2009). My results clearly show significant and substantial differences in the efficacy of surface compounds between pre reproductive and reproductive or post reproductive fronds. These differences have not been accounted for in standard bioassays conducted with randomly selected fronds. This could explain the at least some inconsistencies in the literature with regards to assays conducted from a range of marine plants (as reviewed by Briand (2009)). Significant trends in fouling patterns were obscured when the assays were conducted with no regard to reproductive status. The natural tendency to put aside results that are not significant and instantly definitive should be resisted (Bradley and Gupta 1997). Rather than discarding research into species with less promising results (for the purposes of bioprospecting for anti-fouling compounds) a research program that incorporates consideration of life history traits could reveal a much more complex and informative defensive strategy. Such knowledge can add to our understanding of defence theory.

Reproductive status has not only been ignored in settlement assays, it has also been ignored when quantifying in-situ fouling. There are a number of existing studies that document the amount of in-situ fouling, either by comparing fouling levels (between seasons, sites, latitudes etc.; Lavery and Vanderklift (2002), Thom et al. (2003) and

Pardi et al. (2006)) or by correlating laboratory assay results with efficacy of the

106 Dilophus marginatus

surface compounds in the field (Nylund et al. 2007). The significant differences I found

(~15 %) in the amount of fouling on pre reproductive and reproductive fronds, irrespective of the frond size, demonstrates the necessity to take reproductive status into consideration when quantifying in-situ fouling.

My results open an obvious and important avenue for future work. Is reproductive state only important to D. marginatus? I suggest that it would be advisable to consider reproductive status in bioassays and marine surveys for any marine plants, at least until we have been able to quantify the generality of my findings. Additionally, as I have demonstrated reproductive status is important in one case, previous studies which do not take reproductive status into consideration may need to be revisited. In light of this finding, many species of marine algae which have been discarded as sources of effective anti-fouling compounds might be worth reconsideration (Bradley and Gupta 1997).

My work shows the reproductive status of the frond has a stronger correlation with fouling than does frond size. Thus, my use of frond size to proxy age should not be used as a precedent. The triggering of the production of reproductive structures and associated changes in defences may not necessarily be age related (Agrawal 2012).

Concentration matters

Compounds from D. marginatus, were not always effective at deterring settlement at natural concentrations for some fouling species. Where the compounds were effective, doubling the natural concentration often increased the effect. This questions whether

107 Dilophus marginatus

there is any point to declaring that a species has chemical defences if they are not effective at natural concentrations. Apart from the now understood need to take reproductive status into consideration we also need to take the calculation of ‘natural’ concentration and the manner in which D. marginatus exists in the real world.

The calculation of ‘natural’ concentration is not precise. Fronds are weighed before the extraction of non-polar surface compounds. A percentage of the extract that is considered equivalent (on a surface area to weight calculation) to the size of the assay dish is then used (Nylund et al. 2007). This crude process ignores any changes to the density of the algal tissue over time. It also ignores the likelihood that reproduction is not binary but a process from a highly defended state to a less defended but reproductive state. Additionally, if defences are induced, what we are estimating as the natural concentration may be a background level. Defences may be induced at a higher concentration. Therefore natural concentration should be considered ‘in the ballpark of’ rather than an exact calculation or as a conservative estimate.

D. marginatus clumps with multiple fronds attaching to the substrate at a single point.

Fouling often bonds the fronds together, i.e. it is therefore rare to observe a frond on its own (pers. obs.). If anti-fouling compounds leach into the surrounding environment

(as settlement cues are known to do; Swanson et al. (2007)), it is possible that a propagule could encounter the anti-fouling compounds in the concentrations greater then produced by a single frond. Producing compounds at a lesser rate required to deter settlement from fouling propagules and relying on the proximity of surface chemistry of adjacent fronds may be a way for the algae to limit production of

108 Dilophus marginatus

defensive compounds. Testing at a higher concentration ensures that any effect of defensive compounds is detected in spite of variation of compound concentration between fronds.

Seasonal variation in epiphytic defence

Extracts from D. marginatus fronds that grew in the summer months had a much higher level of effectiveness against the settlement of U. australis propagules then extracts from winter growing fronds (Figure 4.7). Higher defences in the summer months could be in response to higher levels of epiphytic coverage (Chapter 2, this thesis). Therefore it is possible that local winter growing algae, like D. marginatus, could be temporally avoiding higher fouling loads. If temporal avoidance of fouling is part of the defence strategy, then the need for an additional chemical defence strategy would be lessened. D. marginatus has been recorded as being present all year round in some locations (Allender and Kraft 1983). If this is true for Sydney then there are a number of potential avenues to explore to understand my results. The increased fouling load in the warmer months may simply overload the fronds to the point that the presence of D. marginatus populations is simply unnoticed. The increased efficacious of compounds in the summer months is a direct induced response to seasonal fouling pressure. Increased resource allocation to production of defences in the warmer months is traded at the expense of growth, therefore D. marginatus fronds are too small to be easily observed. D. marginatus may temporally avoid the summer fouling load for its reproductive stage.

109 Dilophus marginatus

Can we link life history traits with the presence of chemical defences

Two broad defence theories, r-K and optimal defence theory, might help explain my findings in this chapter (Pianka 1970, Herms and Mattson 1992). r-K theory defines species as ‘r’ types – with a fast short life cycle, where investment in growth ensures that any damage to a plant is easily outpaced by new growth (Coley 1988). The opposite ‘K’ types are heavily defended slow growers (Coley 1988). Both growth and defence could be limited by resources availability (Coley et al. 1985). Terrestrially, r-K theory makes sense and we can easily pinpoint species that demonstrate ‘r’ traits, such as the grasses and also species that demonstrate ‘K’ traits, such as the cacti. At first glance, D. marginatus, with its short annual season and rapid growth (Phillips 1992) and much higher levels of fouling then co-existing algae (Chapter 2, this thesis) could be expected to have little or no chemical defences. However, my results show that

D. marginatus has a complex defensive strategy. During its juvenile phase,

D. marginatus is heavily defended, therefore r-K theory would suggest that the juvenile growth rates are restricted. Further studies on resource allocation would be required to confirm this, but it must be done at each life stage.

Optimal defence theory postulates that levels of defences will vary with more valuable areas more heavily protected (McKey 1979, Rhoades 1979). Tissues that contribute more to plant fitness or are more prone to predation are predicted to be better defended (McKey 1979, Rhoades 1979). Since fitness ultimately depends on reproductive success, it is often reasoned that organisms should be under strong selection to allocate resources to the protection of reproductive tissue against potential consumers (e.g., Steinberg (1984), Hyvarinen et al. (2000), Ohnmeiss and

110 Dilophus marginatus

Baldwin (2000), but see Pavia et al. (2002)). Optimal defences may also protect a plant only when needed, with a physical attack triggering (inducing) the production of defences. There are many documented cases of an increase in the production of defences (Pavia and Brock 2000, Pavia and Toth 2000, Rothausler et al. 2005, Svensson et al. 2007) after an herbivorous attack (Lüder and Clayton 2004, Halm et al. 2011), or damage (Pavia and Brock 2000, Gomez and Huovinen 2010), or in response to the saliva of grazing snails (Coleman et al. 2007b). Induced defences are more likely to result in defensive chemicals being present in older fronds when individual plants are more likely to have been exposed to potential threats.

By incorporating reproductive status into my experiments I have been able to progress our understanding of the efficacy of anti-fouling compounds from D. marginatus from a collection of unclear and contradictory results, to showing a clear link between in- situ fouling levels, the efficacy of its chemical defences, and its reproductive status. It is worthwhile to take reproductive status into consideration when exploring defensive strategies, whether in the marine or terrestrial environment.

111 Dilophus marginatus

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Figure 5.1: Caulerpa filiformis, in an intertidal rock pool, Tamarama Beach, Sydney (Image: J.Green)

123 Caulerpa filiformis

Introduction

Caulerpa filiformis J.Agardh is a green alga found in the intertidal and subtidal zones in the Sydney region (Glasby et al. 2015). A number of observations suggest C. filiformis is defended against epibionts, with chemical deterrents likely playing a role. For example, when compared to surrounding algae it is often the least fouled algae in the community (Chapter 2, this thesis). The Order to which it belongs, the Caulerpales, is well known for its secondary chemistry, e.g., caulerpenyne, caulerpin and related compounds, which are strong deterrents of herbivory (Paul and Fenical 1986, Amy et al. 2006). The genus is generally considered to have chemical defences against fouling

(Amade and Lemée 1998, Dobretsov et al. 2006a, Dobretsov et al. 2006b), but bioassays of anti-fouling compounds in Caulerpa spp. have to date focused on the effects of extracts of whole thalli rather than on more ecologically appropriate surface extracts (de Nys et al. (1998) although see Nylund et al. (2007)).

C. filiformis in particular has been shown to have effective anti-fouling surface compounds against some fouling organisms has been demonstrated by Nylund et al.

(2007). Being coenocytic C. filiformis has the potential to move compounds quickly around the thallus which leads to the potential for complex defence strategies

(Dyrynda 1986). The likelihood of C. filiformis of having effective compounds is therefore considered high, and with my discovery that blanket screening assays can mask complex defence strategies, my overarching aim of this chapter is to test the potential for complex defences in C. filiformis.

Targeted assays, which select areas, types of fronds or life history stages, have the potential to uncover complex defensive patterns and to determine if the efficacy of

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defensive compounds varies as a function of choice. The efficacy of chemical defenses may vary temporally as the plant ages (Chapter 4, this thesis), between parts of the thallus (Fairhead et al. 2005) in response to an external event (Cronin and Hay 1996,

Pavia and Toth 2000, Lavery and Vanderklift 2002, Hemmi et al. 2004, Fairhead et al.

2005, Rothausler et al. 2005, Connan et al. 2006, Macaya and Thiel 2008). My first aim is to establish a baseline in line with other studies that use haphazardly selected fronds as opposed to fronds deliberately targeted to uncover complexities.

Secondary metabolites can be produced as cells are formed throughout the entire life cycle or in response to an external event (Kurz and Constabel 1998). They can accumulate over time (Hou et al. 2010) which would result in higher concentrations of anti-settlement compounds in older fronds. My second aim is to test whether or not

C. filiformis accumulates defensive compounds over time. As a proxy for age I test the settlement of propagules from fouling species between a) small and large fronds (I assume larger fronds are older) and b) primary and secondary blades (Figure 5.2). If compounds do accumulate in increasing concentration over time (Borum 1987,

Cebrian et al. 1999, Hou et al. 2010) and they are inhibitory against fouling propagules, there should be less settlement on larger fronds compared to the smaller fronds and less settlement on the primary blades as opposed to the secondary blades.

Many algae vary in the quantity of secondary metabolites found across the thallus

(Fairhead et al. 2005, Connan et al. 2006). Being coenocyctic C. filiformis may be able to direct compounds to specific areas of its thallus (DeWreede 2006). Variation in chemical defences along the thallus may be optimal for algae with tips and densely packed basal regions having differing levels of exposure to suspended propagules

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(Keough 1986, Jennings and Steinberg 1997), as is the case for C. filiformis (pers. obs.).

The location of caulerpenyne and caulerpin, which deter herbivores, varies within the tissue (Meyer and Paul 1992, Amade and Lemée 1998) but we do not know if this extends to compounds on the surface. Thus my third aim is to test whether extracts from the surface of C. filiformis tips are more efficacious than compounds from the base of the fronds.

Coenocytic algae such as C. filiformis, can direct resources throughout the tissue which enables the algae to quickly heal physical damage (DeWreede 2006). Physical damage may also be a trigger for the production of defences (Lowell et al. 1991, Cronin and

Hay 1996, Pavia and Toth 2000, Jormalainen et al. 2003, Toth and Pavia 2006, Coleman et al. 2007a, Verges et al. 2008, Haavisto et al. 2010) known as induced or inducible defences. Conversely, physical damage to algal tissue can decrease defences (Dixon et al. 1981, Toth and Pavia 2006, Campbell et al. 2011). While there is an extensive literature on inducible chemical defences against herbivores, this has not been explored for defences againts fouling. My fourth aim is to test if extracts from fronds that have been recently damaged have increased efficacy in preventing settlement of propagules from fouling species.

The composition and cover of epiphytic communities on macroalgae varies throughout the year (Arias and Morales 1979, Arrontes 1990, Satheesh and Godwin Wesley 2008).

The variation in these communities is often thought to be a consequence of variation in the supply of fouling propagules (Grosberg and Levitan 1992, Bellgrove et al. 2004,

Clark and Johnston 2005, Bellgrove and Aoki 2006, Lee and Bruno 2009). There is also evidence that the efficacy of anti-fouling compounds varies throughout the year

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(Culioli et al. 2002, Hellio et al. 2004, Marechal et al. 2004). The marine literature and similar work in the terrestrial environment on plant defences suggests two possible scenarios. First, the presence of epiphytic propagules may trigger or ‘induce’ (Lindroth and Batzli 1986, Paul and Vanalstyne 1992, Cronin and Hay 1996, Pavia and Toth 2000,

Hemmi et al. 2004, Macaya and Thiel 2008) the production of anti-fouling compounds.

Second, algae may have evolved seasonal variation in efficacy of anti-fouling compounds in response to seasonal variation in fouling pressure (Hellio et al. 2004). In either scenario peaks of anti-fouling activity would be targeting increased fouling pressure. Previous studies of seasonal variation in algal extracts have generally found the extracts to be more inhibitory against settlement of organisms in warmer months

(Amade and Lemée 1998, Culioli et al. 2002, Hellio et al. 2004). This is also true for defences against herbivores (Bolser and Hay 1996, Amade and Lemée 1998). There are a number of algae that will induce defences compounds once targeted by herbivores

(Paul and Vanalstyne 1992, Cronin and Hay 1996, Pavia and Toth 2000, Hemmi et al.

2004, Rothausler et al. 2005, Toth et al. 2007, Macaya and Thiel 2008). My fifth aim is to determine if defences from C. filiformis are more effective against fouling organisms in the summer months.

In summary, in this chapter I will test variation in defences of C. filiformis against epiphytes in series of assays by 1) establishing a baseline using a simple settlement assay, 2) testing if C. filiformis fronds accumulate anti-fouling compounds over time, 3) testing if the efficacy of extracts from different regions of C. filiformis differs, 4) testing for inducible defences against fouling and finally, 5) testing if C. filiformis has higher level of defences in response to a summer fouling peak.

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Materials and Methods

1) Propagule harvesting and general assay technique.

Methods for propagule harvesting and for settlement assays follow Chapter 3 (this thesis). Larvae of Bugula neritina (Linnaeus, 1758) were obtained from colonies collected by hand from underneath haphazardly selected boats moored in Careel Bay

Marina, Avalon (33°37'13.44"S, 151°19'23.34"E) and kept overnight in filtered water.

Ulva australis Linnaeus gametes and Polysiphonia Greville sp. spores were harvested from fronds collected at Clovelly Bay, Sydney (33°54'53.63"S, 151°16'14.49"E). The fronds were collected, shaken dry and kept refrigerated at 4 ° C. Light exposure was used to trigger propagule release for all three species. The fouling species used in each assay were determined by the extent to which propagules could be obtained during the time the assays were run.

Assay dishes were prepared by agitating pieces of C. filiformis in 5 % dichloromethane

(DCM) for 30 seconds, generally follow methods developed by Nylund et al. (2007) and fully explained in Chapter 4 (this thesis). This solvent mixture was then added to plastic petri dishes before evaporating on a shaker table. Propagules were then added with 4 ml of filtered autoclaved seawater (FSW) to the treatment petri dishes, solvent control dishes, and FSW control dishes and kept in a constant temperature room at 19 ° C.

Percentage settlement of newly settled zooids of B. neritina were counted after 2 hours in the dark. Germlings of Polysiphonia sp. were counted after 24 hours and

U. australis after 6 days of a 15/9 light/dark cycle.

128 Caulerpa filiformis

Where pieces of frond where wounded, either by separation of a secondary frond from a primary fronds, or by cutting with a razor blade, the fronds were kept on damp paper until the wound was visible healed and there was no leakage of internal tissue.

2) Baseline settlement of fouling propagules against extracts from the surface of Caulerpa filifomis fronds

In the winter of 2007 fronds from C. filiformis were collected from Clovelly Bay. Single fronds (Figure 5.2) were collected from individual clumps. A frond was randomly assigned to either natural concentration (1x) of C filiformis surface compounds and double the concentration (2x) until there were 10 replicate fronds per treatment.

Surface chemicals from the fronds were extracted as outlined above. Additional control dishes of seawater or seawater plus were prepared. Assays using propagules of

B. neritina, Polysiphonia sp. and U. australis were conducted following the protocols described in Chapter 3 (this thesis).

3) Variation in the anti-fouling efficacy of surface compounds from Caulerpa filiformis as a function of size and age

C. filiformis grows in clumps and reproduces both vegetatively (via stolons and rhizoids; (Khou et al. 2007) and sexually. The majority of fronds a clump can be classified as ‘simple’ (Figure 5.2, pers. obs.), however in some cases, secondary (and also tertiary) fronds arise from the primary frond (Figure 5.2). The presence of secondary and tertiary fronds allows us to put frond age in a chronological order. The ability to age a frond is important as the need for defences against epiphytes can vary through time.

129 Caulerpa filiformis

If C. filiformis has induced defences, then older fronds have had more time to be exposed to a triggering event (Edwards 1983, Rohde et al. 2004). Older fronds can have changes in resource allocation (Alcoverro et al. 2004). Larger fronds (while likely, but not necessarily, to be older than smaller fronds) can have increased access to resources (Coley et al. 1985).

Fronds were collected from Clovelly Bay were weighed and classified as small (<400 mg) or large (>800 mg). Similar to 1) there were 10 replicates for each treatment.

Surface chemicals were extracted as outlined above, and assays using B. neritina and

U. australis propagules were done.

Fronds with intact secondary blades (Figure 5.2) were collected from individual clumps and randomly assigned to each treatment. The secondary blades were separated from the primary blades and the either the primary or the secondary blade was selected for treatment. The blade was left between pieces of damp paper towel until the injury had healed ~30 sec. The other blade was discarded. Surface chemicals were then extracted from each blade type as outlined above. There were 10 replicates for each treatment.

Assays were conducted using Polysiphonia sp. and U. australis propagules.

130 Caulerpa filiformis

Primary Primary Secondary Secondary

Tertiary

Forked Abscisson zone with healthy tissue above Single Branched

Figure 5.2: Caulerpa filiformis frondclassification (Image: J.Green)

4) Intra –thallus variation in the anti-fouling efficacy of surface compounds from Caulerpa filiformis

In the winter of 2007 large fronds of C. filiformis were collected from Clovelly Bay.

Fronds were randomly assigned to each treatment. Rather than full frond immersion and agitation in solvent the frond was weighed and a portion (equivalent to a surface area of 9 cm2) of either the tip or the base was repeatedly dipped into the solvent and vigorously shaken following de Nys et al. (1998). Only the tip or the base was used from a single frond. This assay was conducted using B. neritina.

5) Inducible defences

To test for the possibility of inducible defences 4 single fronds were haphazardly collected from each of 10 haphazardly selected spatially separated clumps each from both Nielsen Park and Clovelly. Clumps selected were spatially separated to minimise

131 Caulerpa filiformis

the possibility that the clumps were vegetatively part of the same plant, although it is possible that different clumps had been formed from fragments from the same parent

(Khou et al. 2007). Each frond was randomly assigned to one of the following treatments: cropped, uncropped, solvent control and FSW control. Those fronds assigned to the ‘cropped’ treatment were cut (mid-frond) with autoclaved razor blades. The clipped fronds were placed into FSW and left for several minutes until visible internal leakage ceased, i.e. the wound was healed (pers. obs.). All fronds from all treatments then had the surface chemicals extracted (as outlined in Chapter 4, this thesis) and used in assays with B. neritina and Polysiphonia sp. (as described in Chapter

3, this thesis) were conducted.

Additionally, a survey of the in-situ relationship between visually damaged fronds (a frond that was either missing a tip or a segment from the frond) and fouling was undertaken to glean whether defensive compounds are induced. 518 fronds were collected from 103 clumps of C. filiformis across 3 sites and 4 years. The presence of visible damage was noted and the percentage of fouling algae as a percentage of frond area was calculated using the photographic method described in Chapter 2 (this thesis).

6) Temporal variation in defences

Fronds from C. filiformis were collected in the winter of 2007 and the summer of 2008.

Assays using Polysiphonia Greville sp. and Ulva australis Linnaeus (as described in

Chapter 3, this thesis) were conducted. Propagule availability dictated this combination of assay and season. The Polysiphonia sp. and U. australis assays were

132 Caulerpa filiformis

also conducted in FSW and on 5% furanone extracted (in this lab) from Delisea pulchra

(Greville) Montagne as a control as a known anti-foulant. Furanone is a known ant- fouling compound (de Nys et al. 1995, Dworjanyn et al. 2006) and is used a control to test whether or not the response of the propagules changes independently of the compound.

7) Statistical Analyses

IBM SPSS Statistics v.21 was used for all data analysis with significance level set at

P<0.05. All data were analysed using a general linear model. Where more than one factor was tested and significant interactions were found, the factors were examined individually and Tukey’s posthoc test used for pairwise comparisons for significant differences within that factor.

In testing for the baseline settlement of propagules our predictor variable was

Treatment (concentration of extract – natural (1x) or double (2x) the natural concentration, solvent control and seawater control). The dependent variable was the percentage successful settlement of Bugula neritina larvae and Polysiphonia sp. progaules. Settlement of Ulva australis propagules was adjusted to be a percentage of the solvent control.

Assays comparing temporal variation had two predictor variables treatment (extract concentration (1x and 2x) plus solvent and FSW controls) and frond size (small or large) as the dependent variable. There was no significant interaction between frond size and treatment for the settlement of either Bugula neritina or Ulva australis

133 Caulerpa filiformis

To determine there is a variation in algae response between primary and secondary blades, a general linear model was run with treatment (seawater, solvent control, extracts from primary blades, and extracts from secondary blades) as the predictor variable, and settlement (for Ulva australis, the number of propagules settled and for

Polysiphonia sp. the percentage of successfully settled propagules) as the dependent variable. There was no significant interaction for either U. australis or Polysiphonia sp.

To determine if there is intra-algal variation a general linear model with thepredictor variable treatment (solvent, FSW control, basal region of the blade, tip of the blade) and a dependent variable of settlement (percentage successful settlement of Bugula neritina larvae).

To determine if damage can induce the production of defences a general linear model was run with treatment (solvent control, FSW control, extracts from cropped fronds, extracts from uncropped fronds) as the predictor variable, and settlement (percentage successful settlement for propagules of Polysiphonia sp. and Bugula neritina larvae, and mean settlement of Ulva australis propagules) as the dependent variable. Analysis of each fouling species was conducted independently.

To determine if there was a relationship between damage and fouling the fronds were sorted into four categories: unfouled and undamaged, fouled but not damaged, damaged but not fouled, and both fouled and damaged the percentage of each category was calculated. A general linear model was then used to determine if there was a relationship between the predictor variable (damage or not damaged) and our

134 Caulerpa filiformis

dependent variable (surface area of fouling organisms as a percentage of the surface area of the frond.

And finally to determine if there is a relationship between seasonal changes and defence production, a general linear model was run. Treatment (solvent control, FSW control, single concentration of surface extract and double the concentration of surface extract) and season (Summer and Winter) were the predictor variables and settlement (the mean settlement of Ulva australis, and a percentage of successful settlement of fouling propagules for Polysiphonia sp. and Bugula neritina) was the dependent variable. The model was run separately for each of the three fouling species; U. australis, Polysiphonia sp., and B. neritina, as well as a Furnanone control.

There were no significant interactions, so the interaction terms were removed from the analysis.

135 Caulerpa filiformis

Results

1) Baseline effect of a range of surface extracts on the settlement of a range of fouling propagules

Surface extracts of Caulerpa filiformis had no significant effect on settlement of propagules of B. neritina (P=0.579, F3,85=0.660; Figure 5.3), Polysiphonia sp. (P=0.927,

F3,58=0.153; Figure 5.3) or U. australis (P=0.614, F3,58=0.605; Figure 5.3). Settlement in dishes containing seawater only, evaporated solvent +seawater, evaporated solvent + seawater +natural concentration and evaporated solvent + seawater +double concentration of C. filiformis extracts did not differ. Settlement rates in the assays were normal or high for these assays, with average settlement for B. neritina 63% and

78% for Polysiphonia sp. (Figure 5.3). The settlement of U. australis propagules is adjusted to a percentage settlement as compared to the solvent control set at 100%.

136 Caulerpa filiformis

140

120 Seawater Solvent 1x 2x 100

% settlement

0 Bugula Polysiphonia Ulva neritina sp. australis

Figure 5.3: Settlement of Bugula neritina larvae (P=0.579, F3,85=0.660), Polysiphonia sp. propagules (P=0.927, F3,58=0.153) and Ulva australis propagules (P=0.614, F3,58=0.605) in petri dishes containing filtered sea water (FSW) , evaporated solvent (dicholormethane (DCM)) or natural (1x) and double (2x) concentrations of surface extracts from Caulerpa filiformis .

137 Caulerpa filiformis

2) Variation in anti-fouling efficacy as a function of the age or size of fronds

There was no significant difference in the settlement of B. neritina propagules on surface extracts from small versus large fronds of C. filiformis (P=0.273, F1,63=1.225;

Figure 5.4). Doubling the concentration of surface extract also had no significant effect on settlement rates (P=0.506, F1,63=0.449; Figure 5.4).

80 Seawater Solvent 1x 2x 60

% settlement

0 Controls Small fronds Large fronds

Figure 5.4: Mean settlement (±se) of Bugula neritina propagules on surface extracts from small and large (P=0.273, F1,63=1.225) Caulerpa filiformis fronds at natural and double concentration (P=0.506, F1,63=0.449). Control on filtered seawater and the solvent (dichloromethane, DCM).

138 Caulerpa filiformis

There was also no significant difference in the settlement of U. australis propagules on surface extracts from small versus large fronds of C. filiformis (P=0.634, F1,50=0.229;

Figure 5.5). Doubling the concentration of surface extract had no significant effect on settlement (P=0.290, F1,50=1.146; Figure 5.5).

10 Small fronds Large fronds

Propagules settled 4

0 1x 2x

Concentration

Figure 5.5: Mean settlement (±se) of Ulva australis propagules on surface extracts from small and large (P=0.634, F1,50=0.229) Caulerpa filiformis fronds at natural and double concentration (P=0.290, F1,50=1.146). Controls (not shown) on filtered seawater and the solvent (dichloromethane, DCM).

139 Caulerpa filiformis

There was no significant difference in the settlement of U. australis propagules on surface extracts from primary vs. secondary C. filiformis fronds (P=0.728, F1,49=0.122;

Figure 5.6).

Propagules settled 10

0 FSW Solvent Primary Secondary blade blade

Figure 5.6: Mean settlement (±se) of Ulva australis propagules on surface extracts from primary and secondary Caulerpa filiformis fronds (P=0.728, F1,49=0.122). Control on filtered seawater and the solvent (dichloromethane, DCM).

140 Caulerpa filiformis

There was no significant difference in the settlement of Polysiphonia sp. propagules on surface extracts from primary vs. secondary C. filiformis fronds (P=0.968, F1,47=0.002;

Figure 5.7), even when concentrations were doubled (data not shown, P=0.423

F2,47=0.880; Figure 5.7).

100

% Settlement 40

0 FSW Solvent Primary Secondary blades blades

Figure 5.7: Mean settlement (±se) of Polysiphonia sp. propagules on surface extracts from primary and secondary Caulerpa filiformis fronds at natural and half concentration (P=0.968, F1,47=0.002). Controls in filtered seawater and the solvent (dichloromethane, DCM).

141 Caulerpa filiformis

3) Intra-algal variation in efficacy of anti-fouling compounds

There was no significant difference in settlement of B. neritina propagules in response to surface extracts from the tips vs. the bases of fronds of C. filiformis, or relative to filtered seawater and the solvent control (P=0.086, F3,40=2.374; Figure 5.8).

% Settlement

0 FSW Solvent Bases Tips

Figure 5.8: Settlement of Bugula neritina propagules on extracts from the tips and bases of Caulerpa filiformis blades as well as a filtered seawater and solvent (dichloromethane, DCM) control (P=0.086, F3,40=2.374).

142 Caulerpa filiformis

4) Inducible defences

a) Testing for inducible defences using cropped and uncropped Caulerpa

filiformis fronds

There was no variation in the effect of surface chemical extracts following physical damage of the fronds on the settlement of B. neritina (P=0.230, F3,58=1.480, Figure

5.9), Polysiphonia sp. propagules (P=0.323, F3,60=1.188, Figure 5.9) or U. australis

(P=0.773, F4,48=0.449, Figure 5.9).

100

Bugula neritina

sp.) 80 Polysiphonia sp. Ulva australis

% Settlement

B. neritina, Polysiphonia

(

) 20

Settlement

Ulva australis

( 0 Cropped Uncropped Solvent FSW

Figure 5.9: Settlement of propagules of Bugula neritina (P=0.230, F3,58=1.480), Polysiphonia sp. (P=0.323, F3,60=1.188),and Ulva australis (P=0.773, F4,48=0.449) on extracts between cropped and uncropped Caulerpa filiformis (mean+-se). Settlement of propagules on solvent (dichloromethane, DCM) and filtered seawater

143 Caulerpa filiformis

b) The relationship between damage and fouling on Caulerpa filiformis

The majority (90.35%) of surveyed C. filiformis fronds had visible physical damage while less than half had visible fouling (Figure 5.10). A fouled frond was much more likely to have damage with only a small percentage (2.82%) of fouled fronds not exhibiting any damage. While visible fouling only appeared on 48.5% of damaged fronds damaged fronds had significantly more fouling (P=0.006, F1,518=7.765; Figure

5.10) than undamaged fronds.

46.5% 14 43.8%

damaged = 90.35% 40 fouled = 47.87% 12 n = 518

10 30

% fronds

20 % fouling 6

4 10 8.3%

2 1.35% 0 unfouled fouled unfouled fouled & & & & 0 undamaged undamaged damaged damaged undamaged damaged

Figure 5.10: Proportion of fouled and/or damaged Caulerpa filiforms fronds and the increased coverage of fouling surface area on damaged as opposed to undamaged fronds (+-se) (P=0.006, F1,518=7.765).

144 Caulerpa filiformis

5) Seasonal variation in the efficacy of surface extracts against fouling organisms.

The settlement rate of Polysiphonia sp. spores on extracts from C. filifomis fronds was higher in winter (average ~79%) than in summer (average ~60%; P=0.049, F1,78=4.014;

Figure 5.11) but doubling the concentration of the extract made no different (P=0.249,

F4,78=1.382). There were no differences in the settlement of Polysiphonia sp. on the furanone (P=0.908, F1,56=0.014), suggesting that the spore response was similar across the two times. Doubling the concentration of furnanone did significantly reduce the amount of settlement (17% vs 4%; (P<0.001, F2,56=49.048; Figure 5.11) across both seasons.

Similarly, there was also significantly more settlement of U. australis gametes on the winter extracts from C. filiformis (P<0.001, F1,121=55.583; Figure 5.11) but again doubling concentration made no difference (P=0.065, F2,121=2.798). Furanone significantly deterred settlement of U. australis gametes. In the summer only 2 of the

10 furnanone ‘natural’ concentration dishes had any settlement at all, both 20%. In the

‘double natural’ concentration and both winter treatments, there was no successful settlement of U. australis gametes at all.

There was no significant difference in the settlement of B. neritina propagules against winter vs. summer C. filiformis extracts (P=0.798, F1,105=0.066; Figure 5.11). There were not enough propagules in either season to run furanone controls.

145 Caulerpa filiformis

80 Summer b Winter

a a

% propagules settled c c 20 b b a d a d 0.2,0 0,0 0 1x 2x 1x 2x 1x 2x 1x 2x 1x 2x Ulva australis Furanone control Polysiphonia sp. Furanone control Bugula neritina

Figure 5.11: Variation in settlement of Ulva australis (P<0.001, F1,121=55.583), Polysiphonia sp. (P=0.049, F1,78=4.014) propagules and Bugula neritina larva (P=0.798, F1,105=0.066) between winter vs. summer surface extracts from Caulerpa filiformis (mean +-se) at natural (1x) and double the natural (2x) concentration. % settlement for each propagules was adjusted to a filtered seawater control (not shown). Significant differences in settlement between seasons is noted by an ‘a, b’ combination. Significant differences in settlement due to extract concentration shown by a ‘c, d’ combination. There was 0 settlement of U. australis propagules on furanone at double concentration and settlement in only 2 of the 10 replicates at single concentration. Settlement of Polysiphonia sp. propagules on furanone did not vary between seasons, but was impacted by concentration (P<0.001, F2,56=49.048).

146 Caulerpa filiformis

Discussion

Despite previous literature suggesting Caulerpa filiformis was heavily defended against epiphytes (Nielsen et al. 1982, Paul and Fenical 1986, Amade and Lemée 1998, Nylund et al. 2007) I found little supporting evidence for chemical defences against fouling.

My initial baseline assay (Figure 5.3) found that extracts from haphazardly selected

C. filiformis fronds did not significantly inhibit the settlement of propagules from the local fouling organisms Polysiphonia sp., U. australis and B. neritina. Whilst this was unexpected (Amade and Lemée 1998, Nylund et al. 2007) and contradicted previous results from our lab (Nylund et al. 2007) assays of haphazardly selected fronds can mask complex defences (Chapter 4, this thesis). But assays, in which fronds are selected on a trait (whether it be size, frond type, frond region, damage, or season) also failed to show any significant effect of C. filiformis frond extracts.

Chemical defences of algae can be expected to vary over time for any number of reasons: age of the frond (Borum 1987), induced defences (Edwards 1983, Rohde et al.

2004), resource availability (Coley et al. 1985), resource partitioning, (Alcoverro et al.

2004), evolved defence strategies (Coley 1986, Brown 1988, Herms and Mattson 1992,

Pfister 1992, Paul et al. 2014), Chapter 4, this thesis) etc. I found no evidence that the efficacy of chemical defences from C. filiformis fronds varied as a function of the age or size or portion of the frond. There was no significant difference between settlement on extracts from smaller vs. larger fronds, nor between extracts from primary (older) and secondary blades (Figures 5.4-5.7). I found no evidence that defences varied between different parts of the frond (Figures 5.7-5.88), nor that defences could be triggered by physical damage to the frond (Figure 5.9).

147 Caulerpa filiformis

It was therefore surprising to discover that there did appear to be a significant difference in the efficacy of surface extracts between seasons (Figure 5.11). Both

U. australis and Polysiphonia sp. settled less in summer. However, in Chapter 3 (Figure

5.5, Chapter 3, this thesis) I found that the propagules themselves can seasonally vary their behaviour. In particular, Polysiphonia sp. settled at a higher rate in winter on dishes treated with DCM, the same solvent used to extract surface compounds from

C. filiformis fronds. U. australis also demonstrated higher settlement in winter (Figure

5.6, Chapter 3, this thesis). The use of furanone, a compound with demonstrated anti- settling ability (de Nys et al. 1995, Steinberg et al. 1998, Dworjanyn et al. 2006) should have been able to be used as a control for seasonal variation in propagule behaviour.

But furanone was so effective against the settlement of U. australis gametes that any seasonal variation of propagules in the furanone dishes is undetectable. With only one seasonal replicate these results are not determinative, but do leave open the possibility is that seasonal variation in settlement is due to variation in propagule behaviour, not due to chemical defences.

The use of furanone, whilst indeterminate in quantifying seasonal variation in behaviour by U. australis gametes, does confirm that I was running the assays correctly it also demonstrated a positive relationship between concentration and efficacy of defence (Figure 5.11). There was no such relationship between concentration of compounds extracted from C. filiformis and settlement deterrence in any of the assays

I conducted. The evidence for chemical defences is therefore slight.

However, I did find a significant and interesting relationship between damage and fouling which may help explain how C. filiformis mitigates settlement pressure. The

148 Caulerpa filiformis

relationship between damage and fouling is strong. Almost every frond I sampled had some level of damage (Figure 5.10). Those fronds that were undamaged were also more likely to have no visible fouling (Figure 5.10). It was rare to find a frond that was fouled but had no associated damage. I classified the damage into two categories: tip damage and frond damage. Damage at the tip of the frond was typified by a visible abscission scar. Given the morphology of a C. filiformis frond there is no reliable way of extrapolating the original length of the frond prior to the breakage occurring. Where the entire tip of the frond had been discarded the visible scar obscures any physical characteristics which could identify the mechanism. Thus fronds that may have been heavily fouled at the tip may now appear to have no visible fouling. Like Cummings and

Williamson (2008) I found tip damage where the entire top of the frond was missing to be the most common (unpublished data).

Where damage is located along the length of the blade it can be identified as a bite mark (Cummings and Williamson 2008) or is associated with epiphytes (pers. obs.). I observed multiple fronds with an abscission zone around either side of epiphytic growth was clearly visible. Additionally, I observed multiple occasions where an abscission zone was forming across the width of the frond (Figure 5.12).

149 Caulerpa filiformis

Figure 5.12: Caulerpa filformis blade with possible abandonment of several fouled blades in progress. (Image: J. Green)

This frond (Figure 5.12) strongly suggests that C. filiformis can use its ability to self heal

(DeWreede 2006) to discard damaged fronds and that damage caused by fouling can be one of the mechanisms for this to occur. Blade abandonment provides a mechanism for sloughing of fouled fronds with little impact on the remaining frond segment (Littler and Littler 1999). Additionally, if still healthy, abandoned blades can facilitate population growth as they can successfully establish new colonies (Herren et al. 2006, Khou et al. 2007).

Khou et al. (2007) found frond fragments in abundance along the Sydney coastline. The majority of fronds were not intact, indicating that they had been generated by breakages somewhere along the length of the original fronds. Khou et al. (2007) also

150 Caulerpa filiformis

demonstrated that all types of fragments had the ability to start a new colony.

Breakages in fronds could be caused by grazing (Trowbridge 1993, Clements and

Choat 1997, Cummings and Williamson 2008), other physical damage such as wave stress (Andrew and Viejo 1998), boat propellers and increased drag (Anderson and

Martone 2014) due to epiphyte load or, as I propose, by deliberate discarding of the fouled fronds (Littler and Littler 1999).

The ability of C. filiformis to discard fronds opens up many interesting research areas.

C. filiformis may recycle the protoplasm (Littler and Littler 1999) and included resources. Healthy discarded fronds maybe the genesis for new colonies (Khou et al.

2007) and a mechanism for population distribution. The ability to discard fronds may not be limited to the green algae, there are red and brown algae that are also coenocytic or siphonous (Littler and Littler 1999, DeWreede 2006).

151 Caulerpa filiformis

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175 Caulerpa filiformis

176 Discussion

The aim of this thesis was to assess the interactions between macroalgal hosts and epiphytic communities from the Sydney region. I did this in three ways. First, I assessed the variation in epiphytic community composition and coverage in the Sydney region.

Second, I tested the response of two species of algae. Third, I quantified the impacts of methodology on the results of the type of bioassays that are commonly used to determine whether macroalgae possess anti-fouling compounds on the surface of their fronds. Macroalgal host was the main factor determining both fouling load and epiphytic community composition. In situ fouling load had remarkably little relationship with the likelihood of a chemical mechanism to deter fouling. Caulerpa filiformis J.Agardh, a green alga, appears to have no chemical mechanism to deter fouling. One possible defence strategy suggested by the physicality of damaged fronds is that it may discard them, a possibility that may have far reaching implications for algal defence strategy theory in general or may be limited to siphonous algae.

Ontogeny proved to be the key in understanding the defence strategy of

Dilophus marginatus J.Agardh, having implications for ongoing research into antifouling compounds.

Patterns of epiphytes

Algal communities are critical components in the marine environment. They play host to a range of organisms that use them as substrate, nutrient sources, shelter and

177 Introduction

protection, or generally as habitat . Sessile organisms such as algae, bryozoans, fungi, and diatoms that grow on larger algae and seagrasses are known as epiphytes.

Composition of epiphytic communities initially depends on the successful settlement of propagules from epiphyte species. Thus the life history of epiphytes influences the patterns of epiphytic communities (as discussed by Wahl and Mark (1999)). The epiphyte needs to be in the same location (Belegratis et al. 1999), depth (Head et al.

2004, Korpinen et al. 2007, Rohde et al. 2008), and reproducing at the right time of year (Raberg et al. 2005) to be successful. The epiphytes may also have specific settlement cues (chemistry, surface texture; (Bers and Wahl (2004), Magin et al.

(2010), and Salta et al. (2010)) colour (Hodson et al. 2000, Swain et al. 2006, Shine et al.) and need to have a life cycle that completes within the life cycle of the host.

The epiphytic community in my study consisted primarily of algal epiphytes, although there were occurrences of the bryozoans Bugula neritina (Linnaeus, 1758) and

Melobesia membranacea (Esper) J.V.Lamouroux). In one instance, in the Spring of

2005, the entire population of C. filiformis at Clovelly in the bay had been overgrown by M. membranacea. While M. membranacea colonies were occasionally found on host algae in other sampling sessions, in this instance the bryozoan was dominant, often entirely obscuring the entire C. filiformis frond. The population of C. filiformis collapsed soon after and it was two years before the population in Clovelly Bay started recovering (pers. obs.). The influence of local site conditions had some impact on the structure of the communities, but the overriding driver of the structure of the epiphytic communities was the variation among hosts.

178 Discussion

Like Nylund et al. (2007), I did not find significantly less fouling on the heavily defended

Delisea pulchra (Greville) Montagne. The low levels of epiphytic coverage on D. pulchra are attributed to the anti-settlement effect of furanones (de Nys et al. 1998). But algal may protect against epiphytic settlement via physical mechanisms as well (Littler and

Littler 1999, Bers and Wahl 2004, Magin et al. 2010). So low levels of epiphytic coverage, like I found on C. filiformis are not necessarily indicative of a chemical defence strategy. Conversely, high levels of fouling, as I found on Dilophus marginatus

J.Agardh did not indicate lack of any defences against settlement of epiphytic propagules rather they indicated a complex ontogenetically related defence strategy.

The quantity of fouling on Dictyopteris acrostichoides (J.Agardh) Bornet and Solieria robusta (Greville) Kylin was also not significantly different to that of C. filiformis and

D. pulchra. The similar fouling load on these algae suggests that each of these species also has a successful mitigation mechanism, chemical or otherwise.

Seasonal variation in the composition of epiphytic communities is relatively well studied. Most of the previous work has focused on correlations with variation in temperature and/or daylight length as well as propagule supply (Arias and Morales

1979, Harms and Anger 1983, Hepburn et al. 2006). I found only marginally significant differences in the fouling load between seasons with possibly a higher epiphytic load in the summer months.

The variation in epiphytic coverage may be related to seasonal variation in the efficacy of anti-fouling compounds (Morales and Arias 1977, Arias and Morales 1979, Morales and Arias 1979, Arrontes 1990, Culioli et al. 2002, Hellio et al. 2004, Marechal et al.

179 Introduction

2004). I did find that compounds extracted from both D. marginatus and C. filiformis were more efficacious in the summer. An increase in the efficacy of anti-fouling compounds in the warmer summer months has been linked to an increase in fouling pressure in those months (Marechal et al. 2004).

However, there is limited research if the propagules themselves vary their behaviour

(Jarrett 2003) between seasons. All three of my propagule taxa had a seasonal variation of settlement success in at least one scenario. In particular, a higher percentage of Polysiphonia harvested in Winter successfully settled in solvent only dishes as well as the dishes with extracts from C. filiformis suggesting that the seasonal variation I found in extracts from C. filiformis, may be an artefact of seasonal variation of successful settlement of Polysiphonia sp. Propagules to be used in bioassays need to be screened for seasonal variation in settlement success or the possibility needs to be controlled for.

Ecological impacts of epiphytes

Epiphytes can be detrimental to their host by attracting epifauna such as mesograzers that will damage the host (Tegner and Dayton 1987, Duffy 1990, Newcombe and

Taylor 2010). Larger herbivores may also be attracted by the epibionts and while grazing on the epibionts may increase consumption of the host alga termed ‘co- consumption’ (Karez et al. 2000, da Gama et al. 2008, Verges et al. 2011). Thus even if larger herbivores do not directly prefer the host as a nutrient source they may cause associational damage to the algae reducing its fitness (Jones 1992, Duffy and Hay

2000). Epiphytes can also damage their host algae by reducing the host metabolic rate

180 Discussion

through shading (Bulthuis and Woelkerling 1983, Beach et al. 2003, Dunn et al. 2008) blade loss (Dixon et al. 1981), nutrient competition (Karez et al. 2000, Dunn et al.

2008), reduced growth rates (Dodds 1991, Nylund et al. 2013), reduced photosynthesis

(Sand-Jensen 1977).

While much of the previous work has focussed on the negative effects of fouling host

(Tegner and Dayton 1987, Duffy 1990, Newcombe and Taylor 2010) , epiphytes can also benefit their hosts by deterring herbivores (Karez et al. 2000, Barea-Arco et al.

2001), associational resistance (Wahl and Hay 1995), nitrogen fixation (Goering and

Parker 1972), or resource transfer.

The algae may also successfully tolerate the presence of epibionts to achieve coexistence (Barea-Arco et al. 2001). The deliberate discard of damaged fronds (Littler and Littler 1999), which is potentially the strategy I found for C. filiformis, can result in an increase in the number of fronds, while other algae may benefit from the increase in nutrients due to the breakdown of epiphytes (Harlin 1973). The damage to hosts caused by epiphytes can benefit the ecosystem contributing to habitat turnover – maintaining or increasing species diversity (Hay 1986).

Not only do epiphytes colonise soft biotic substrates such as algae and seagrasses, but also artificial structures. Fouling of artificial structures is costly (Lebret et al. 2009) and has large direct impacts across a number of industries including pearling, aquaculture, and shipping. Understanding the mechanisms already evolved by marine organisms may enable us to find solutions that we can mimic, adapt, reproduce or exploit for our purposes (Clare 1996, Bhadury and Wright 2004, Mayer and Hamann 2004, Ralston 181 Introduction

and Swain 2009, Magin et al. 2010, Qian et al. 2010). I found, in the case of

D. marginatus, the life history stage of the alga plays a big role in determining the presence of effective anti-settlement compounds. Incorporating ontogeny into the screening process for new compounds may provide more efficient in identifying potential compounds.

How macroalgae deter settlement of epiphytes

The host can mitigate the pressure from epiphytes. The host can have physical properties that are unattractive for epibiont settlement (Dyrynda 1986, Andersson et al. 1999, Scardino et al. 2003, Magin et al. 2010, Choi and Kim 2012) or a physical response to the presence of an epibiont e.g. a deliberate discard of damage fronds

(Littler and Littler 1999). Third parties such as grazers may reduce the epiphytic load

(Brawley and Adey 1981b, Brawley and Adey 1981a, Bronmark 1985, Coen 1988,

Dodds 1991, Dudley 1992, Strong et al. 2009, Newcombe and Taylor 2010, Aumack et al. 2011). The host can also defend against epiphytic settlement using surface associated bacteria (Dobretsov and Qian 2006, Rao et al. 2007), and by compounds produced by the bacteria (Egan et al. 2001).

An alga itself may produce chemical defenses that deter settlement and indeed a broad range of chemical defences have been identified (Sieburth and Conover 1965,

Davis et al. 1989, Clare 1996, Hay 1996, Steinberg et al. 1997, Amade and Lemée 1998, de Nys et al. 1998, Steinberg et al. 1998, Amsler et al. 2001, Steinberg et al. 2001,

Steinberg and de Nys 2002, Steinberg et al. 2002, Bhadury and Wright 2004, Fusetani

2004, Amsler and Fairhead 2006, Dobretsov et al. 2006, Dworjanyn et al. 2006, Volk

182 Discussion

and Furkert 2006, Barbosa et al. 2007, Medeiros et al. 2007, Amsler et al. 2009, Bianco et al. 2009, Cabrita et al. 2010).

Bioassay reliability

The mechanisms by which marine organisms control fouling include physical

(Andersson et al. 1999, Littler and Littler 1999, Bers and Wahl 2004) and chemical traits (de Nys et al. 1995, Clare 1996). To explore chemical deterrents against fouling with the aim of using them in an applied fashion, extracts from macroalgae (or other organisms) are often screened for the ability to deter settlement using laboratory bioassays (Rittschof et al. 1992, Da Gama et al. 2002, Briand 2009). Such laboratory based bioassays are conducted both on broad extracts and specific compounds

(Dahms and Hellio 2009) and have, over time, increased in complexity and are continually being refined (de Nys et al. 1998). These refinements have improved the correlation between results from bioassays and what we see in the field (Nylund et al.

2007). But bioassay results are still often inconclusive and inconsistent across different assays and with field results. Among other factors, inconsistencies in bioassay results can be the result of an incomplete understanding of the behaviour (or variation of settlement success) of propagules chosen for the assay or differing methods or both

(Rittschof et al. 1984, Wassnig and Southgate 2012). Incorporating the understanding I bring to the settlement behaviour of B. neritina will result in more reliable results into the future.

Two of my assay species, U. australis and Polysiphonia are commonly used in settlement assays and the behaviour of their propagules is well understood. The third,

183 Introduction

B. neritina, we know is light sensitive (Lynch 1952, Wendt 1998, Wendt and Woollacott

1999, Wendt 2000), and is found as an epibiont on local Sydney algae (Chapter 2, this thesis). Colonies of B. neritina are also often found on the bottom of boats in intermittent light conditions (pers. obs.) suggesting that propagules may have a mechanism to avoid settling under full light conditions. I found that propagules from

B. neritina were very sensitive to light exposure during the first few hours. While the

B. neritina propagules appeared (under a microscope) to be attached to the surface of the petri dish, disturbance by light exposure proved otherwise. My thesis has contributed to understanding the settlement behaviour of Bugula neritina (Linnaeus,

1758) in intermittent light conditions.

One possibility is that B. neritina has a reversible as well as an irreversible settlement stage (Thorson 1964, Dodou et al. 2011). Settlement can be considered as a process with a number of stages, in which propagules first move towards the surface (Tamburri et al. 1992, Wieczorek and Todd 1998), then often test the surface for suitability

(Pascual and Zampatti 1995), attach, and then (for animal larvae) finally metamorphose (Burke 1983). Distinct reversible and irreversible settlement stages have been identified for a number of species (Müller et al. 1976, Wahl 1989) including bacteria (Hinsa et al. 2003) and barnacles (Rittschof et al. 1984). If B. neritina does have a reversible settlement stage, it opens up the possibility that reversible settlement is more widespread then currently considered.

184 Discussion

The importance of host life histories

Understanding plant defensive strategies enables us to understand interactions between species, drivers of community composition and potential impacts of changes in the environment (Hay 1986). Terrestrial research has generated several theories regarding defence strategies that are underpinned by the notion that internal plant resources are limited, and once used for defences are unable to be allocated to other functions (Herms and Mattson 1992). This applies to both physical and chemical defences. A plant or alga may evolve a strategy to trade-off investment in one function for another (Herms and Mattson 1992). A plant (or alga) can reduce the demand for resources by producing defences only where and when necessary, that is, by optimally allocating defences. Such optimal defence theory (ODT) predicts that within an organism, it will defend regions that have the highest fitness value (Rhoades and Cates

1974). By producing defences through the juvenile phase, D. marginatus is defending the fronds until they reach the stage where they can add to the population. ODT may be able to be applied to population fitness as well as individual fitness.

Some defences may be switched on only when required (the induced defence model

(IDM; (Karban and Baldwin 1997)). D. marginatus potentially demonstrates the opposite, a switching off of defence production when no longer necessary. Extracts from juvenile fronds with no visible sporangia, were very effective at deterring the settlement of U. australis whereas extracts from fronds with visible sporangia were not. Re-examining actual fouling load by distinguishing between fronds with and without visible sporangia resulted in a distinct difference in fouling load. My work with

D. marginatus builds on the recently modelled understanding of the role ontogeny

185 Introduction

plays in resource allocation and defence strategy, whereby the production of defences is not at the expense of growth (Paul et al. 2014).

The concentration of furnanone was found to vary with life history stage (Wright et al.

2000)

Blade abandonment

My results suggest that C. filiformis could be the second green alga to abandon fouled fronds as a mechanism to control epiphytic growth. The first, Avrainvillea longicaulis

(Littler and Littler 1999), not only abandons fouled fronds, it recycles the protoplasm for rapid growth of new blades. The morphology of damaged C. filiformis fronds

(Figure 5.12, Chapter 5, this thesis) strongly suggests that the internal cytoplasm is gone from the frond. The protoplasm could be lost to the environment, or like

A. longicaulis resorbed by the healthy tissue. Given the ability of C. filiformis to heal

(DeWreede 2006) it would be unlikely that damage to the frond would result in leakage of the entire store of protoplasm. Indeed stimulated damage in the laboratory through cutting of the fronds results in the frond sealing protoplasm leaks within seconds (pers. obs.). But resorption of cytoplasm and senescence of the frond is not the only possibility. In some cases healthy frond tissue was observed above and below abscission zones (Figure 5.2, Chapter 5, this thesis). Once abscission is complete, a healthy fragment may remain. A healthy fragment could be dispersed and become the genesis for a new C. filiformis colony (Khou et al. 2007). Therefore damage resulting in fragments may be beneficial for the spread the population as a whole, while being a disadvantage to the individual plant (Vroom et al. 2005).

186 Discussion

Blade abandonment may be widespread among algae generally or more specifically restricted to the siphonous or coenocytic algae, for which there is increased freedom of movement of cytoplasm within algal fronds. Both A. longicaulis and C. filiformis are considered to be siphonous. Lacking cells walls may be advantageous in quickly moving compounds around the frond to facilitate wound healing (DeWreede 2006) allowing fronds to be to discarded with minimal damage to the remainder of the algae. The possibility for blade abandonment and recycling of cytoplasm to be widespread amongst algae opens up new areas of research in resource allocation and defence theory.

187 Introduction

Conclusion

Defences against epiphytes in macroalgae are complex. Defences can be classified as chemical or physical (Harlin and Lindbergh 1977, Hay and Fenical 1992, Littler and

Littler 1999) in nature. My work has highlighted that chemical defences, so prevalent in defence against herbivory (Stachowicz and Hay 1999, Amsler and Fairhead 2006,

Amsler et al. 2009), are not necessarily indicative that defensive compounds are used to also mitigate epiphyte settlement. In situ epiphyte coverage should not be used as a predictor of the presence of a defence strategy rather understanding of defences should be integrated into an overall understanding of the life history of the organism.

Bioassays need to take into account various factors including seasonal variation in settlement success of propagules, surface preference, and light conditions.

188 Discussion

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