Colonial Integration and the Maintenance of Colony Form in Encrusting Bryozoans
Colonial integration and the maintenance of colony form in encrusting bryozoans
Elisa K. Bone
Thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy
November 2006
Department of Zoology The University of Melbourne
Abstract
The form of an organism is often closely linked to its function, and this relationship may be particularly important in organisms where individual form is highly flexible due to the repeated iteration of minute multicellular modules. In many modular taxa, including the bryozoans discussed in this thesis, each module is able to function as largely independent units; an individual module can feed independently, has a separate gut, and has the potential to reproduce. These characteristics mean that the number of modules in a bryozoan colony, and hence its size, is a reasonably accurate measure of the colony’s ability to both capture resources and to produce sexually developed larvae. Size is therefore a more appropriate measure of colony demography than age, the criterion traditionally used for unitary organisms. However, processes that can complicate the demography of modular organisms such as colony damage, fission or fusion also mean that the age structure of the component modules in a colony or fragment remains an important predictor of colony functioning, interacting with the effects of colony size.
Effective colony functioning in a sessile invertebrate necessitates a level of coordination between component zooids; enabling a colony to grow, reproduce, to compete with neighbouring organisms and, in some cases, to develop defensive structures in response to predation threats. In cheilostome bryozoan colonies, individual zooids are extremely compartmentalised, and connections are reduced to tiny pores that enable the transport of nutrients between zooids within a colony. Component zooids may also differ in size, shape and function across colony regions, subsequently affecting their energy requirements and their capacity to both capture resources and transmit nutritious foodstuff to other members of the colony. Changes in colony size through normal growth may also lead to changes in the energy requirements and resource capture rates of individual zooids, since zooid age interacts with the colony’s patterns of module addition and expansion to determine the capacity for growth, reproduction and other functions within an undamaged colony. In Watersipora subtorquata, a common unilaminar cheilostome bryozoan species with very low levels of polymorphism and a regular budding pattern, zooid measurements varied over the course of colony development, and
i feeding rates in colonies of differing sizes indicated that smaller colonies may have higher energy requirements than larger colonies. In addition, over hour-long feeding trials, not all zooids were feeding at any one time. Since the provision of resources to the undeveloped, non-feeding zooids at the edge must be by other feeding zooids in the colony, some transport of resources must be taking place between zooids to meet the colony’s energy requirements.
I examined internal measures of the capacity for inter-zooid resource transfer in 5 species of encrusting bryozoan using scanning electron microscopy (SEM) to visualise the communication pores within the funicular system, and quantified variation in these pores across colony regions in three species. In Watersipora subtorquata and Conopeum seurati, the patterns of pores indicated that resource transfer is strongly directed towards the (growing) colony edge, and this finding correlates well with observations of rapid growth in the field. In the third species, Mucropetraliella ellerii, directional transfer towards the edge appeared weaker, and may be due to the damage history, reproductive state or age of the colony fragments. This result could also be indicative of a lower capacity for growth at the colony edge. Further detailed examination of the communication pores and their formation suggested that the funicular system may be quite flexible in cheilostome bryozoans, and that existing pores may be closed or opened, or the direction of transfer altered, through the formation of new porous connections. Another indicator of the level of integration within a colony is the way a colony responds to external stressors such as the removal of zooids or the close proximity of other organisms. I examined the capacity for recovery in Watersipora subtorquata colonies both after repeated removal of the growing edge and after the removal of groups of zooids from different colony regions in colonies that varied in size, with the explicit aim to assess the potential for nutritious resources to be re-directed after the disruption of colony form. Where the growing edge was removed, colonies showed large decreases in total growth and reproduction compared to undamaged colonies. However, there was some indication that resources may be re-directed towards the colony centre, with the rate of zooid death in the centre of damaged colonies lower than the rate seen in undamaged colonies. Similarly, where colonies were damaged in different colony regions, colony
ii size appeared to interact with the type of damage, and some regeneration contrary to the primary direction of growth was seen in larger colonies.
Where colonies that are genetically similar or otherwise compatible are growing in close proximity, fusion to form a single colony may be a viable response, increasing a colony’s size, which in turn improves its competitive abilities and reproductive capacity. In these cases, we might expect compatible colonies in close proximity to show directional growth towards each other, and incompatible colonies to grow in a way that avoids contact. I did not see any discernable effect on growth direction in W. subtorquata colonies that differed in either their level of apparent relatedness or their size, but stronger effects may have been seen with improved sample sizes and longer experimental periods.
The importance of the growing edge as a resource sink in encrusting bryozoans appears consistent across taxa, but examination of responses to damage in Parasmittina delicatula under field conditions at two locations showed that local conditions and competition can also affect colony recovery, and in this case were more important than the levels of internal colonial integration. This result highlights the importance of considering possible effects of local conditions in detailed examinations of growth and recovery potential across multiple species in modular organisms.
iii
Declaration
This is to certify that:
(i) the thesis comprises only my original work towards the PhD except where otherwise acknowledged (ii) the thesis is less than 100,000 word in length, exclusive of tables, maps, bibliographies and appendices
Elisa Bone
November 2006
iv Acknowledgments
It’s fair to say that almost everybody I have dealt with over the past few years has contributed in some way to this project. The “brians” have become a big part of my life over these years, and the story of their structure and function has been scribbled on countless scraps of paper and serviettes to be explained to anyone that cared to listen. Thank you to you all.
*****
More specifically, I thank my supervisor, Mick Keough, who has shown faith in me throughout, and provided support, encouragement, discussions, advice and the voice of reason. To all the members of the Keough lab, past and present – there are too many of you to mention here, but you know who you are – thank you also for your support, friendship, discussions and help over the years.
Thank you to those who helped me with field work – Claire Bennett, Nathan Knott, Allyson O’Brien, Matt Reardon, David Reid, and to Bec Loughman for reading drafts.
I am very grateful to Joan Clark for teaching me how to use the electron microscope, and then allowing me to use it for hours on end without supervision. Thanks also to Bruce Abaloz for some histology work that provided interesting insights into bryozoan-kelp interactions.
Thanks to everybody in the Department of Zoology, especially my fellow post-graduates, to those who helped find suitable microscopes and equipment for my work, and to Garry Jolly-Rogers, David Macmillan, Mark Padgham and Matt Symonds for interesting discussions that helped to refine my research questions.
A PORES grant from the Melbourne Scholarships Office and a research grant from the Department of Zoology enabled me to travel to the U.S., and many people helped me in securing and completing my exchange at the University of California, Santa Cruz. Thanks to Pete Raimondi, for agreeing to host me in his lab, to Kathleen Donahue for helping organise my visa requirements, and to Betsy Steele for getting me set up in the lab and helping me find suitable equipment. Huge thanks are due to Todd Newberry and Kerstin Wasson, for insightful discussions and encouragement, and for helping me remember that obscure organisms do not necessarily mean obscure questions. Thanks also to Anna, Becky, Hilary and the guys at Segre – Arnold, Jan and Joni.
Finally, many thanks to my friends and family and to my partner, Dave, for everything.
v Table of Contents
1. General Introduction...... 1
2. Colony form and scaling in Watersipora subtorquata...... 9 1. Scaling of zooid size and resource partitioning...... 13 A. Zooid size according to order of development ...... 14 Methods ...... 14 Results ...... 15 Discussion...... 16 B. Role partitioning of colony regions ...... 18 Methods ...... 18 Results ...... 19 Discussion...... 19 2. Patterns of resource capture: roles of colony size and zooid position...... 21 Methods ...... 22 Results ...... 26 Discussion...... 28
3. Internal mechanisms of intra-colony nutrient transfer...... 53 1. Communication pores and pore development ...... 57 A. Communication pores in five species of encrusting bryozoan ...... 58 Methods ...... 58 Results ...... 61 B. Pore development in Watersipora subtorquata...... 63 Methods ...... 64 Results ...... 65 Discussion...... 66 2. Pore patterns across colony regions and implications for colonial integration ...... 66 A. Pore patterns across colony regions in two species with contrasting life histories: Watersipora subtorquata and Mucropetraliella ellerii ...... 66 Methods ...... 68 Results ...... 70 Discussion...... 72 B. Pore patterns across colony regions in Conopeum seurati, and investigation of pore maturation...... 76 Methods ...... 78 Results ...... 79 Discussion...... 81 General Discussion...... 82
4. The influence of local damage on growth patterns in Watersipora subtorquata..... 116 1. Repeated removal of the growing edge...... 119 Methods ...... 120 Results ...... 122 2. Recovery from damage in three patterns: an examination of the flexibility of recovery....124 Methods ...... 125 Results ...... 127 Discussion ...... 128
vi 5. Colony growth and damage recovery in the presence of conspecific colonies ...... 145 1. Fusion in Watersipora subtorquata colonies at two locations ...... 149 A. Colony fusion at Santa Cruz, California: effects of apparent relatedness ...... 150 Methods ...... 150 Results ...... 155 B. Colony pairing and growth Williamstown, Victoria: effect of colony size...... 156 Methods ...... 156 Results ...... 157 2. Damage recovery and colony size in a monoculture of Conopeum seurati ...... 158 Methods ...... 158 Results ...... 160 General Discussion...... 161
6. Damage recovery under field conditions in Parasmittina delicatula ...... 179 Methods ...... 181 Sampling and analysis ...... 183 Results ...... 186 Discussion...... 191
7. General Discussion...... 211
References ...... 221
Note: Figures and tables relevant to each chapter are located at the end of that chapter
vii Chapter 1
General Introduction
The shape and form of an organism is dictated by a complex interplay between its genotype and the environment, and the ways that organisms regulate the development of their component structures have an enormous impact on their continued success. Life history characteristics that may be influenced by an organism’s developmental stage or by their form range from the ability to cope with competition and changes in the environment, to disease resistance, growth and recovery rates, and eventual reproductive capacity (e.g. Wulff 1991; Cocito et al. 2000). These interactions are especially important in organisms whose form is closely linked to their success in the local environment, and are particularly relevant to studies of modular organisms. In modular organisms, the repeated iteration of minute, functionally similar multicellular units allows great flexibility in the final shape, size and form of a colony that is composed of these units (Buss 1985; Lidgard and Jackson 1989; Hughes 2005). Moreover, in many modular species, including the sessile marine invertebrates discussed here, a colony’s size is tightly linked to both its capacity for further growth and its ability to reproduce (e.g. Keough 1986), so that factors that alter the form or size of a colony can profoundly affect its eventual fitness.
While the growth and development of a single module may be equivalent to the somatic growth of a unitary organism, module propagation allows a second level of growth and development. The continued growth of a modular invertebrate colony, for instance, is reliant on the addition of new modules, and in colonies with a single layer of modules, this addition occurs at the colony edge. Since module propagation may not be subject to the same kinds of genetic control mechanisms as somatic growth in a unitary individual, continued growth of a modular colony is, at least potentially, indeterminate (e.g. Jackson 1977; Hughes 1984; Jackson 1985; Jackson and Hughes 1985; Sebens 1987, but see Kim and Lasker 1998). For sessile taxa where the occupation of space is paramount, colony growth by module addition can contribute to the spatial dominance of a single genotype, and enhance a colony’s competitive abilities (e.g. Harvell and Padilla 1990; Fine and
1 Loya 2003). Continued colony growth can therefore hold many advantages for a sessile modular invertebrate, and as a result, colony size is often an important predictor of fitness.
In many modular invertebrates, including bryozoans, the relationship between size and life history variables is particularly strong. In an unspecialised colony, each module has an equal capacity for reproduction and feeding; it therefore follows that every extra module that develops to maturity will increase the colony's reproductive capacity and its ability to gain resources through feeding. A higher capacity for resource acquisition can then enable further colony growth, maintaining the colony’s overall fitness. In addition, modular units within a colonial invertebrate are often functionally similar, and no one module is essential for the continued survival of the colony. This redundancy of function essentially means that processes that would kill a unitary organism may only damage a modular organism (e.g. Highsmith 1982, Lasker 1984; Karlson 1986, 1988; Smith and Hughes 1999). Processes that uniquely affect modular organisms include the removal of component modules through physical damage or shrinkage, the splitting of one colony to form two or more daughter colonies, and the coalescence of two compatible colonies to form a fused chimaera (e.g. Jackson and Winston 1981; Highsmith 1982; Karlson 1986; Wulf 1991). These processes can rapidly change a colony’s size, and thus complicate a simple size- or age-based demographic model. That they can also occur either singly or in a variety of combinations also complicates the history of single genets, and means that a colony’s current size is not always a good predictor of its age (e.g. Hughes and Connell 1987; Hughes et al. 1992). It follows, then, that the demography of modular colonies may be better predicted by a combination of a colony’s current size and its history of damage or fusion interactions (e.g. Hunter 1993; Tanner and Hughes 1996; Linacre and Keough 2003).
Sessile modular invertebrates such as corals, ascidians and bryozoans are prone to disturbance, and may be readily fragmented into smaller daughter colonies or lose modules through physical damage or predation. In some species, fragmentation may be an adaptive response to disturbance, functioning to increase the spatial representation of a genotype in an area (e.g. Bak and Engel 1979; Highsmith 1982; McKinney 1983). A
2 commoner consequence, however, is that the loss of modules incurs shifts in the allocation of resources to the competing needs of maintenance, reproduction and continued colony growth. In all modular organisms, the continued functioning of a colony is reliant on the effective integration and communication between modules within a colony. Integration may be recognised at a number of levels, chiefly structural, behavioural and physiological, and these levels in turn require the development of structural connections, neural networks, and resource-transfer mechanisms. The efficiency of the resource-transfer system, specifically, may be indirectly inferred through the ways that colonies respond to damage or other forms of disturbance (Harvell and Helling 1993; Oren et al. 2001; Fine and Loya 2003). A rapid response that is evident across the whole colony may indicate a high level of integration between modules within a colony, while a localised response may imply that local energy stores are being used in the recovery process (e.g. Meesters et al. 1994). Changes in the ways a colony grows in response to external pressures can also be indicative of the efficiency of resource transfer between units, and thus colonial integration. In the encrusting bryozoan Membranipora membranacea, obstructing colony growth resulted in increased growth on the opposite side to compensate (Harvell and Helling 1993). This response implies that Membranipora is a well-integrated species, reinforced by the rapid formation of defensive spines in the presence of its nudibranch predator (Harvell 1984) or stolons in the presence of competitors (Harvell and Padilla 1990).
Resource-transfer patterns in colonial invertebrates in most cases appear to resemble the source-sink mechanisms seen in clonal plants (e.g. Kaitaniemi and Honkanen 1996; Charpentier et al. 1998; Piqueras 1999), but few studies have unequivocally shown the movement of nutritious resources between colony areas. Studies using radiolabelled foodstuffs are, however, illuminating the specific mechanisms in a number of modular invertebrate taxa. In corals, for example, radiolabelled carbon is transported preferentially to the site of damage repair, at the expense of functions elsewhere in the colony (e.g. Oren et al. 1997b) and predatory snails feed preferentially on these energy sinks (Oren et al. 1998). These directed patterns of resource transfer in many well-studied, widespread modular invertebrates might be closely linked to the form of the circulatory system. In
3 colonial cnidarians and compound ascidians, transfers are facilitated by common coelomic cavities, or by gastrovascular circulatory systems that enable the transfer of resources between colony regions (Carle and Ruppert 1983; Gladfelter 1983; Mackie 1986; Gateño et al. 1998). Further, although resource capture and symbiotic zooxanthellae retention in corals is at the level of the polyp, a reduction in the amount of common energy stores would still be felt colony-wide. In bryozoans, in contrast, resource capture, digestion and excretion are all carried out at the level of the individual module, the zooid. The remains of senescent polypides within zooids are recycled and the resources are often used to fuel the development of another functioning polypide (Palumbi and Jackson 1983). This then leads to the idea that zooids in a bryozoan colony may not be particularly well integrated, a concept that is reinforced by the extreme compartmentalisation of zooids, particularly in the Cheilostomata, as connections between zooids are reduced to tiny pores in many species in this order.
In encrusting unilaminar cheilostome bryozoan colonies, the relationship between modular growth, feeding capacity and reproductive potential, as well as occupation of space, is particularly strong, since a linear increase in the colony perimeter through module addition results in a theoretically exponential increase in colony area (Thorpe 1979; Craig 1995). The standard unit of a bryozoan colony is the feeding zooid, or autozooid. Autozooid shape can vary substantially both within and between species, and often between zooids within the same colony, due to pressures such as minor damage to the colony, fouling by algae or other invertebrates, or irregularities in the substratum. Despite these variations, zooids are predominantly box-like and comprise six walls. Two of these walls are external, one, the surface of the “box”, becoming the frontal surface of the colony and the other the basal surface of the zooid, which is connected to the substratum. The remaining four walls are classified as internal walls, and are either connected to adjacent zooids or, in the case of a newly formed zooid, are exposed to the environment at the colony edge (Boardman and Cheetham 1969; Lidgard and Jackson 1989). Member zooids of bryozoans, as well as extrazooidal parts of a colony, are developed from the expansion and partitioning of the zooecium and the external and internal walls (e.g. Boardman et al. 1973). This mode of development appears to be
4 highly flexible, and has led to a wide variety of zooidal forms and arrangements in the Bryozoa. In cheilostome bryozoans the coelomic space is completely enclosed by these calcified walls, and as a result zooids are much more compartmentalised than in other Bryozoa. With such separation of zooids, the establishment and maintenance of connections is especially important for continued integration between zooids and across whole colonies.
Beklemishev (1964, in Coates and Oliver 1973) identified colony formation as arising from three processes; the weakening of the individuality of zooids, the intensification of the individuality of the colony, and the development of cormidia – functional units within colonies that comprise groups of zooids and polymorphs. The presence of cormidia, therefore, theoretically indicates a high level of colonial individuality, and their development is in this sense an unambiguous indicator of whole-colony functional control. The development of specialised zooids within a colony, or polymorphism, is widespread across bryozoan taxa, despite inherent costs to this process. To explain the high numbers and diversity of polymorphs among bryozoan taxa, Schopf (1973) considered polymorphism to be a trade-off within the colony so that some specialists are evolved at the expense of generalists. Since we may assume specialised polymorphs to be more energetically costly to produce than simple, generalist zooids, and since polymorphs are not contributing to a colony by feeding or reproducing, polymorphism must hold significant fitness advantages to offset these costs, and to be maintained across such a wide range of taxa. Evoking Wilson’s (1971) ergonomic model of caste ratio in social insects, Schopf (1973) asserted that if the need for specialised functions remains constant, selection should favour the maintenance of a physical caste specialised to carry out each task; in effect maintaining a mix of generalists and specialists despite the cost inherent in developing specialists. Hughes and Jackson (1990) investigated levels of polymorphism in bryozoans in tropical environments that were fairly constant, and found that morphological complexity did not predictably increase at low levels of environmental variability, but was instead widespread across taxa in both variable and constant environments. This finding appears to strengthen Schopf’s assertion, and polymorphism may be more relevant to investigations on within-colony integration rather
5 than as an indicator of the role of environmental variability in the evolutions of morphological complexity.
Where functional roles of modules differ across the colony, and particularly where specialised modules are modified for various roles at the expense of a functioning feeding polypide, the effective transport of resources between feeding autozooids and these non- feeding specialised zooids becomes paramount. As a result, it has been proposed that the level of polymorphism within a colony may be indicative of the level of colonial integration. A colony that has modules occupying different roles must necessarily be able to effectively communicate between these zooids, while the provision of nutritious resources to non-feeding polymorphs also entails a high level of integration. These assumptions seem obvious, but little work has been done on the relationship between apparent levels of colonial integration in bryozoans and the actual capacity for signal transfer between modules within that colony. Where zooids are simple and relatively uniform in shape, size and function, they nonetheless possess neural networks to allow effective communication between modules within a colony, and resource transfer must continue to operate between modules. High levels of integration therefore may not be restricted to species where polymorphism is high; in the absence of significant morphological differentiation between zooids, resource shifts must take place to enable differential functioning within a colony. A thorough investigation of patterns of colonial integration first requires an understanding of the ways colonies are structured, their resource requirements and their capacity for resource acquisition. Second, accurate measurement of an individual colony’s capacity to transfer nutritious resources between zooids within a colony is only possible through an examination of the specific connections between zooids, and in bryozoans, these are components of the funicular system, unique to the phylum (e.g. Bobin 1977; Mukai et al. 1997). In addition, disruptions to a colony’s predicted growth patterns through damage or competition can change the ways resources are allocated towards colony functions, and need to be examined in order to ascertain the level of integration between zooids within a colony.
In this thesis, I explore the relationship between colony growth, damage recovery and structural measures of colonial integration in encrusting bryozoans, using a combination
6 of field experiments and morphological surveys across five species. Since all species used show a general encrusting, unilaminar growth form at the study locations I was able to relatively easily evaluate and compare changes in colony growth patterns and size. In these taxa, growth by module addition occurs at the colony perimeter, and is readily measured. In Chapter 2, I examine the relationship between module number and colony size in Watersipora subtorquata (d’Orbigny, 1852), and evaluate any changes in the size and shape of zooids, and in their feeding rates, across colony regions and in colonies of different sizes. Encrusting unilaminar bryozoans are an ideal group in which to study scaling issues, as they are made up of a single layer of contiguous zooids, and, where the substratum is flat and competition is low, continue to grow as a flat sheet. This, and the apparent retention of zooid size and shape across colony astogeny, has prompted suggestions that these organisms are likely to retain a constant surface area to volume ratio with increasing colony size, and therefore to exhibit geometric isometry, and possibly metabolic isometry (e.g. Jackson 1979b; Hughes and Hughes 1986). Any significant changes in zooid parameters or resource acquisition are likely to show that metabolic isometry is not operating in this species. Any changes in the levels of resource acquisition and resource use across colony regions may also affect the patterns of resource transfer between component modules within a colony. As a consequence, in Chapter 3, I examine internal mechanisms of colonial integration through the analysis of structural components of the funicular system within colonies. I show the general morphology of the communication pores in five species, and conduct further analyses on the number, function and size of pores across colony regions in three of these species; Watersipora subtorquata, Mucropetraliella ellerii (MacGillivray, 1869), and Conopeum seurati (Canu, 1928). Further, I examine pore development in newly settled W. subtorquata colonies, and discuss likely mechanisms of pore formation in C. seurati zooid walls. In Chapters 4 and 5, I examine colony responses to two factors that may influence the growth and form of encrusting species; the removal of modules, and the proximity of other colonies of the same species. In Chapter 4, I evaluate whole-colony responses in W. subtorquata after damage to different colony regions. Since the colony edge is likely to be a strong resource sink, with resources predominantly directed towards this edge, damage to the edge region may have profound consequences for colony growth
7 and reproduction, while damage to other colony regions could disrupt directional nutrient flow. In Chapter 5, I examine growth patterns of W. subtorquata colony pairs in close proximity, and damage recovery in a monoculture of C. seurati colonies. Chapter 6 continues my investigation of damage recovery and colonial integration, but examines these variables under field conditions. In this chapter, I analyse the long-term recovery of Parasmittina delicatula (Busk, 1884) colonies after a single instance of artificial damage that removed modules from different colony regions. This experiment was done using naturally occurring colonies on subtidal pier pilings, and provides a test of the concepts of colonial integration and recovery in the presence of natural variation in environmental conditions, and competitive interactions with other sessile species. Finally, I discuss my findings across all experiments within the General Discussion, Chapter 7.
8 Chapter 2
Colony form and scaling in Watersipora subtorquata
Introduction
One of the main advantages of a modular lifestyle is the relative flexibility of form that may be afforded the integrated reproductive unit, the colony. Unitary organisms have genetically determinate forms, and often physiologically imposed size restrictions, and these limitations also apply to the individual polyps, zooids or modules in a modular invertebrate. However, the repeated iteration of these minute individual modules can allow them to adapt rapidly to varying environmental conditions through changes in the number and arrangement of modules. Modular species exhibit a wide variety of colonial forms, from branched arborescent colonies, unilaminar and bilaminar sheets, through to plate-like, foliose and massive forms. While these forms may be primarily determined through intrinsic mechanisms, they may also be differentially suited to variations in environmental conditions (reviewed in Hughes 2005). For instance, branched forms may be more susceptible to breakage through wave action and storm surges, suiting a calmer environment, massive forms may be more resilient to such damaging processes, and runner-like growth forms may be more able to take advantage of favourable changes in microhabitat conditions (e.g. Jackson 1979b; Karlson et al. 1996; Marshall 2000). Within a species, colonies may also alter their form and patterns of construction to suit local conditions, and these changes can affect a colony’s subsequent growth potential and reproductive output (e.g. Foster 1979; Brazeau and Lasker 1988, 1992; West et al. 1993).
Unitary organisms have genetically determinate forms, and they are also subject to restrictions with respect to size, so that there are limits to what extent a unitary organism can increase in size without physiological and structural problems emerging. Generally, as the mass of an organism increases, its surface area to volume ratio decreases, so that metabolic capacity tends to scale allometrically with increases in individual size. It therefore takes less energy per unit mass to meet the metabolic requirements of larger individuals (e.g. Sebens 1982). Due to these size-related processes, small individuals tend
9 to have higher metabolic rates than larger ones, but in the marine environment, small individuals are also generally more susceptible to processes such as predation, while sessile juveniles are also prone to sedimentation. In addition, prey capture may be more difficult for filter and suspension feeders if their feeding zone is restricted to the boundary layer, or, in the case of ascidians, where small siphon widths and high water viscosity decrease volumetric flow rates and thus restrict the flow of food particles (Sherrard and LaBarbera 2005a, 2005b). Due to these and other processes, juveniles in the benthic marine environment often have disproportionately high mortality rates (e.g. Gosselin and Quian 1997; Hunt and Scheibling 1997). Size is therefore an important factor in determining an individual’s chances of survival, growth and successful reproduction in the marine environment.
It is generally assumed that modular organisms, including colonial invertebrates, escape many of the physical and physiological constraints imposed by volumetric increases in size through the repeated iteration of independently functioning units that are similar in size (e.g. Jackson 1979b). Since colony enlargement and growth in these organisms is by the addition of new, functionally independent modules, rather than the expansion of existing modules, it has been proposed that growth may be, at least theoretically, indeterminate (e.g. Jackson 1985; Hughes and Connell 1987; Sebens 1987). In these organisms, moreover, the addition of largely identical modules can enable the colony to increase in size while maintaining geometric similarity and a constant surface area to volume ratio (e.g. Jackson 1979b; Hughes and Hughes 1986; reviewed in Hughes 2005), suggesting that colony functions may scale isometrically with size. While studies of scaling issues in modular invertebrates have increased in number in recent years, few have found an isometric relationship between body size and metabolic rates, with the exception of Hughes and Hughes (1986), who found no increase in the rate of respiration with increasing body size in the anascan bryozoan Electra pilosa. In contrast, respiration and surface area scaled allometrically with biomass in small colonies of the scleractinian coral Siderastrea siderea (Vollmer and Edmunds 2000), and metabolic rates scaled to the ¾ power in the unilaminate ascidian Botrylloides simodensis in both intact and size- manipulated colonies (Nakaya et al. 2005).
10
Assumptions of isometric increases in size and function in modular invertebrates appear to be particularly relevant to colonies of encrusting, cheilostome bryozoans. In these animals, a dominant growth form on stable substrata is the unilaminar sheet. Growth is by the addition of new modules at the perimeter of the colony, which in many cases, in the absence of competitors, approximates a circle. Vollmer and Edmunds (2000) noted that isometry could only occur where colony biomass is restricted to a single layer of modules with conserved dimensions (see Jackson 1979b). Similarly, Hughes (2005) proposed that such two-dimensional laminar colonies with monomorphic modules might exhibit low physiological interdependency and thus be the most likely candidates for metabolic isometry.
Predictions of isometric scaling in modular species assume not only a constancy of form, but also a level of constancy of function across colonies; essentially, that each unit of mass within a colony metabolises at equivalent rates and contributes equally to colony performance. However, in many modular species, a proportion of colony mass may be taken up by structural components, such as the acellular mesenchymal tissue in coral colonies and the gelatinous matrix in ascidians (e.g. Mackie 1986). Colony biomass may thus be a poor predictor of metabolic capacity in some colonial species. Unlike coral colonies, encrusting bryozoans do not have any acellular mesenchymal tissue in the extramodular spaces, and where growth form is unilaminar, they also lack any basal structural non-feeding zooids that can contribute to an inaccurate measure of effective colony size.
Despite the relatively simple form, an encrusting bryozoan colony may be made up of a number of units with alternate morphologies and different functional roles, which in turn may utilise resources in varying ways. These polymorphs, or kenozooids, can be modified to perform a defensive, structural or solely reproductive role, functions which may place dissimilar metabolic demands on the zooid (Silén 1977). Moreover, Silén (1977) supposed the energy drain on feeding zooids by these non-feeding polymorphs must be considerable, especially in motile polymorphs such as avicularia and vibracula, although he also noted that no attempts had been made to quantify energy consumption in
11 dissimilar zooids across colonies.
We may expect the strongest linear relationships between colony size and scaling components, including metabolic function, in colonies that are composed of mostly size- similar autozooids, and hence have low or non-existent levels of polymorphism. Hughes and Hughes (1986) demonstrated isometric scaling of respiration in the encrusting bryozoan Electra pilosa, a species with monomorphic autozooids and a typically simple, unilaminar two-dimensional growth form. Similarly, Peck and Barnes (2003) noted a direct increase in metabolic costs with increasing organic mass in unilaminar and bilaminar two-dimensional bryozoan species, indicative of an isometric relationship between size and metabolic rate. In both these studies, however, the measure of colony size was ash-free dry mass, which did not consider possible differences in zooid function across colonies.
In addition, despite the apparent uniformity in colony construction across these simple colonies, the means of colony growth may impose differential resource demands on zooids depending on their position within a colony, or on colony size. Hughes and Hughes (1986) noted that the rates of zooid production per individual decreases with increasing colony size, although this variation did not result in changes in colony-wide respiration rates with size. In the encrusting bryozoan Watersipora subtorquata (d’Orbigny), no polymorphism is evident, and larvae are brooded internally within zooids. Importantly, embryo formation is not dependent on the degeneration of the polypide, so a zooid’s feeding capacity is retained throughout embryonic development. Zooid properties such as size and feeding capacity may therefore remain fairly uniform across a colony. W. subtorquata colonies, in the absence of significant crowding or damage, also grow as a flat sheet in the study region of Hobson’s Bay, Victoria, Australia, and as such the surface area and volume values for a colony are closely linked to its two-dimensional area when viewed from above. Importantly, the colony grows by module addition at the colony perimeter so that zooids at this edge are youngest, with an age gradient from youngest to oldest running from the colony edge into the colony centre. The chronologic age of the zooid (indicating the stage of ontogenetic development) and its astogenetic position may both potentially affect colony functioning and morphology
12 (Abbott 1973). Differences in age may influence the ability of colonies to acquire resources, while colony growth results in an increased number of zooids capable of feeding, as well as increased perimeter length. These relationships may influence the likelihood of allometric or isometric scaling with respect to both colony morphology and function. In this chapter, I aim to investigate the assumption of isometric scaling in a modular invertebrate, using morphometric measurements and evaluation of feeding activity in the unilaminar encrusting cheilostome bryozoan W. subtorquata.
First, I investigate whether component modules of small Watersipora subtorquata colonies differ in size through their initial ontogenetic development. The maintenance of a relatively constant zooid size would imply that investment in the addition of modules is constant across the development of the colony. Differences in the size of zooids across the colony may indicate a change in the levels of investment required to develop zooids, while variation in operculum size across colony regions may indicate a change in the resource capture potential of zooids. Operculum size is closely related to the size of the feeding structure, the lophophore, and a larger lophophore can indicate a greater potential to capture resources (e.g. Winston 1977). Second, I consider the patterns of colony construction, reflected in changes in zooid size and relative ratios of feeding and developing areas across colony astogeny, in an evaluation of feeding activity within colonies of different sizes. These measurements should aid in the description of colony construction, and in understanding the effects of colony size on intracolonial resource partitioning in unilaminar modular invertebrates.
1. Scaling of zooid size and resource partitioning
A. Zooid size according to order of development
I examined changes in module size across developmental gradients in two size classes of Watersipora subtorquata colonies.
Methods
13 I used a number of colonies that naturally settled onto 240mm × 240mm transparent PVC plastic sheets of 0.38-mm thickness. These sheets were strengthened and stabilised by backing plates of 6-mm-thick grey PVC, cut to the same size. Four of these plates were arranged in a regular pattern on a large 600mm × 600mm plate of 6-mm-thick grey PVC using stainless steel bolts, and the entire array hung horizontally by ropes from Workshops pier at Williamstown, Victoria. The surface of the plates faced downwards to reduce algal growth and sediment build-up, and a central weight was hung below the plates to prevent excessive movement from wave action and storm surges.
Colonies settled within a two-week period in January 2005, and were therefore subject to similar environmental conditions during their development. This was important, as O’Dea and Okamura (1999) have shown that module size may vary with changes in temperature, and any variation in module size with temperature would confound effects of colony ontogeny or size. I analysed zooid size across colonies in two groups: ‘small’ colonies, and ‘large’ colonies. Since the colonies were settled within the same period of time, I analysed zooid size across small colonies prior to completing the analysis for large colonies.
Within small colonies, I measured the size of each zooid according to their order of development; that is, the order in which they budded off from the ancestral zooid. Secondary zooids bud to flank the primary ancestrula slightly forward of the ancestrula midline (Fig. 1). The fourth zooid generally forms directly forward of the ancestrula, between zooids two and three (Fig. 1a). This initial budding pattern is, however, quite variable in W. subtorquata, and secondary and tertiary buds may form forward or lateral to the ancestrula (Fig. 1b). I was able to discern the actual order of development by repeatedly observing colonies over the course of a few weeks, however after the fifth or sixth bud, the specific order becomes more difficult to determine. In these cases, I adjudged buds to have developed later if they were further from the ancestrula than another bud of similar apparent age. In total, I measured the length and width of 203 zooids across 73 colonies. Some of these zooids were not fully developed, with their operculum ridge incomplete. I excluded these zooids from operculum measurements, and
14 had a total of 189 observations of operculum size across 69 colonies. Zooid numbers in each colony ranged from 1 to 12, and were arranged in up to four zooid rows from the ancestrula. In large colonies (average radius 10.968 mm ± 1.986 (s.e.)), I measured zooid size according to its position in the colony. Colony position was noted by counting the number of zooid rows developed distal to the ancestrula and proximal to the colony edge.
For large colonies, I measured a total of 409 zooids across 6 colonies and the distances from the ancestrula ranged from one to nine zooid rows. In both groups, I measured zooid length as the longest line that bifurcated the operculum from the distal edge to the proximal zooid edge, and the width as the widest point at which a line could be drawn that was perpendicular to this length measurement (see Fig. 2). For newly settled individuals, the total area of a zooid, measured by tracing the edge of that zooid, is strongly correlated with measurements of area as calculated by length by width (M. J. Keough, personal communication), so I was confident that, for these young colonies, length and width measurements alone were sufficiently accurate measures of zooid area. I measured approximate zooid size by multiplying these length and width measurements, and zooid elongation by dividing zooid length by zooid width. I measured operculum length by calculating the length of the line that halved the operculum from its distal to its proximal edge, and operculum width as the maximum length of the line perpendicular to the length measurement (Fig. 2).
Within both groups of colonies (large and small), I analysed zooid characters across increases in order of development, or distance from the growing edge, using analysis of covariance (Quinn and Keough 2002).
Results
Small colonies
Zooid length did not appear to vary with the order of zooid development (Fig. 3a), and was not significantly different across colony development (zooid length = 0.0015 ×
15 2 order of development + 0.6662, r = 0.0025, F1,201 = 0.51, P = 0.476). Similarly, zooid area, estimated from the multiplication of length and width measurements, did not vary significantly with order of development (Fig. 3b, length x width= –0.0017 × order of 2 development + 0.2944, r = 0.0067, F1,201 = 1.352, P = 0.246). Zooids did, however, become slightly more elongated further from the ancestrula (Fig. 4), but the measure of elongation did not change between colonies according to analysis of covariance (Table 1). Operculum area, estimated from length and width measurements, also did not vary according to the order of zooid development (Fig. 5, operculum length × width = 0.0002 2 × order of development + 0.0429, r = 0.004, F1,187 = 0.763, P = 0.384).
Large colonies
In large colonies, zooid length appeared to increase in rows away from the ancestrula (Fig. 6a). Analysis of covariance showed a significant difference in the length of zooids both between colonies and across zooid regions (Table 1). Similarly, analysis of covariance showed a significant difference in zooid width between colonies, but not with increasing distance from the ancestrula (Table 1). Zooid area, estimated from the length and width measurements, increased significantly both with increasing distance from the ancestrula (Fig. 7a, Table 1) and between colonies (Table 1), while zooids were also more elongated further away from the ancestrula (Fig. 7b, elongation = 1.817 × distance 2 from ancestrula + 0.108, r = 0.186, F1,407 = 92.927, P = <0.001). Operculum size increased slightly with increasing distance from the ancestrula, and although distance explained only around 22% of the variation in operculum size, the difference was 2 significant (Fig. 8; operculum size = 0.0027 × distance + 0.037, r = 0.222, F1,407 = 115.943, P = <0.0001).
Discussion
The size of the initial ancestrula was highly variable across colonies in this experiment Ancestrula size in W. subtorquata is strongly correlated with the size and inherent energy reserves and fitness of the settling larva (Marshall 2003; Marshall and Keough 2003).
16 After some initial variation, zooid size did not change between the first few zooids developed from the ancestrula, as analysed in small colonies. Across a broader spectrum of distances, however, in larger colonies, zooid length, zooid size (length by width), and operculum size (length by width) increased further from the ancestrula, and most measurements in large colonies also varied significantly between colonies. This indicates that individual colony development may be regulated to increase relative zooid size as the colony grows. As a colony grows, the angle between developing zooids decreases to enable further module addition in a circular colony. Zooid elongation may be a consequence of this narrowing of zooids, although Okamura and Partridge (1999) demonstrated in Membranipora membranacea that elongation of zooid form in this species was linked to environmental water flow speeds. In low flow environments, zooids were quite elongated, but they became less elongate as flow speeds increased, and zooid shape became more irregular. It is possible that the elongation seen in zooids within W. subtorquata colonies is a result of changes in flow rates during the course of colony development, although this appears unlikely. A more likely explanation is that elongation and zooid enlargement with increasing distance from the ancestrula is a result of the geometric restrictions close to the ancestrula, and the zooid budding pattern.
New zooids bud from the distal end of the ancestrula, and subsequent zooids bud forwards of these secondary zooids. Since the colony must eventually become circular, a certain amount of compression of the very inner zooids needs to take place so that the direction of growth can continue outwards, around and eventually behind the ancestrula. Silén (1977) noted this region as the ‘zone of astogenetic change’, and subsequent zooid addition as astogenetic repetition. As a result, we may expect that the larger, more elongate zooids further from the ancestrula represent the standard or “normal” state of zooid geometry within colonies, and within this species. Nevertheless, a longer zooid would still necessitate either an enlargement and elongation of the polypide within the zooecium, or an elongation in the musculature that attaches the polypide to the zooecial walls, but the costs of these changes are not known. Thus, this increase in zooid size may require additional investment in tissue development as a colony grows, and a consequent increase in resource acquisition.
17
Because the number of feeding zooids increase as a colony grows by module addition, this additional feeding capacity may be sufficient to meet these demands. In addition, operculum size appears to increase with distance from the ancestrula, and, as operculum size is closely linked to lophophore and mouth size in many bryozoans (Winston 1977), these changes in operculum size could indicate higher ingestion rates and feeding capacity. Thus, the resource demands in large colonies may be adequately covered by these changes, however to confirm this assumption, I needed to examine both the partitioning of roles and patterns of feeding within colonies.
B. Role partitioning of colony regions
As new modules are added to a colony, the feeding capacity of that colony increases. Lophophore extension and the action of pumping water through the lophophores, as the only visible source of activity, was assumed to be the major source of energetic costs to bryozoans (Peck and Barnes 2003). Although eversion and retraction of the lophophore requires activity of the parietal muscles, it is also likely that other sources of activity pose significant energetic costs in bryozoans. These processes may include the development of new colony area, damage repair, and the development of gametes and embryos. Growth occurs at the perimeter, which in turn increases with colony size, so the growth of new colony area may place significant resource demands on colonies, which need to be met by existing feeding zooids. I examined the relative areas of colonies that are devoted to both feeding and to further colony development, by calculating and comparing the area occupied by feeding autozooids to the remaining, non-feeding developing area situated at the colony perimeter.
Methods
For these measurements, I used colonies from the previous analysis and a number of additional colonies from a range of sizes that had settled naturally on clear PVC sheeting deployed at Workshops pier, Williamstown. I photographed each colony using an
18 Olympus C-5050 digital camera mounted on an Olympus Z-40 binocular dissector microscope, and calculated, using Image J software, the total area of the colony that was composed of feeding zooids, and the total area composed of developing or non-feeding zooids. The zooid walls are very distinct in W. subtorquata, and fully-developed zooids have a dark operculum and distinct dark ridges from the operculum down the lateral walls. The polypide is also often visible through the frontal wall in young colonies. Therefore, I had no trouble delineating the margins between zooids, or distinguishing between feeding and developing zooids. I plotted colony size, measured as the number of zooids, against the ratio of feeding to non-feeding area, and the amount of non-feeding area per feeding zooid. I analysed the relationships between factors using least-squares linear regression, performed with Systat 11.
Results
There was a close, significant linear relationship between the number of zooids in a colony and the total area of that colony (Fig. 9a, Table 2), and a similarly tight relationship with colony area and the area occupied by feeding zooids (Fig. 9b, Table 2). While the ratio of feeding to non-feeding area, contrary to predictions, declined with an increase in the number of zooids in a colony (Fig. 10), this relationship was largely driven by the massive variation in ratios of feeding to non-feeding area in very small colonies. I also plotted the amount of non-feeding area per feeding zooid against the number of feeding zooids, and while this again produced a negative relationship (Fig. 11), the variation was not significant (Table 2).
Discussion
In very small colonies, the ratio of feeding to non-feeding area was highly variable, a pattern that may be expected since, in a newly settled colony of 1–2 zooids, where new buds are initially forming, the feeding zooids may occupy most of the colony area, whereas when these buds are almost fully developed, their area may be equal to or greater than the area occupied by the feeding zooids. A clearer result was that the amount of non-
19 feeding, developing colony area per feeding zooid within colonies decreased with increasing colony size.
A possible consequence of this decrease is that fewer developing zooids need to be supplied with nutrients for their continued development by adjacent feeding zooids. Hart (2001) showed that the continued growth at the colony edge was most likely fuelled by nutrient input from zooids just behind the edge in this species, so with increased colony growth, the older, more central zooids cease to direct resources to this mode of colony growth. This increased independence of zooids may enable the colony to direct more of its acquired resources towards further growth of the colony, or may indeed allow the individual zooids to direct resources into reproduction. Further, a colony that has ceased directing the majority of its resources towards growth may be able to better respond to threats that include competition, predation, disease and disturbance.
In large colonies, zooid size and operculum size increased with increasing distance from the ancestrula. Since feeding area increases linearly with colony area, we may expect that the resource capture potential and energetic demands of colonies are scaled isometrically with colony size. However, a perfect isometric relationship between colony size and feeding area would yield a slope of 1, and the slope in this case is less than 1, meaning that as colony area increases, feeding area also increases, but not to the same extent. I saw a huge variation in the ratio of feeding colony area to developing colony area when colonies were small (under around 10–15 zooids), whereas in larger colonies, this relationship appeared to plateau.
The amount of non-feeding area per feeding zooid declines with colony size, such that the relative resource demands placed on each feeding zooid is lessened, and with increasing colony size, these demands may be distributed across the colony. Furthermore, although I was dealing with two groups of colonies that were very different in size, the ‘large’ colonies are by no means in the upper range of colony size in W. subtorquata; most of these colonies had not commenced reproduction and showed limited zooid senescence in the central regions, and much larger colonies are found in natural
20 populations. Under these conditions, it appears likely that the ratios of feeding area to developing area will decrease further as colony perimeter increases, and that the demand for resources to be invested in growth will subsequently decline. This could lead to either a reduction in feeding activity to account for the decreased demand, or to a surplus of nutrient stores. In effect, there may be a crucial tipping point at which resource demands for individual zooids decline to the point where supply outstrips demand. This may correspond to the point in colony astogeny where growth of new colony area begins to decline, and could indicate a shift in resource allocation towards, potentially, reproduction. Evaluating the partitioning of roles within colonies and correlating these with patterns of reproductive onset and embryo production would be an ideal next step to pursue in this line of enquiry.
If small colonies have additional resource demands due to investment in developing colony regions, these may manifest as increases in the resource capture rates. To investigate this premise, I conducted a small feeding experiment, using 15 colonies of varying sizes.
2. Patterns of resource capture: roles of colony size and zooid position
In this section, I used a small-scale feeding trial to investigate responses to resource availability in colonies of differing sizes. Given the differences in apparent role partitioning with changes in colony size seen in the previous section, feeding rates may differ over colony size to account for variable resource demands. My specific predictions were that: if metabolic demands scale linearly with colony size (isometric scaling), we should see no change in the proportion of zooids feeding at any one time with increasing colony size, and no change in the individual rate of feeding with colony size. On the other hand, if small colonies have higher metabolic demands than large colonies (allometric scaling), I would expect to see a linear decrease in the proportion of zooids feeding at any one time with an increase in colony size, and a decrease in feeding rates of individual zooids with increasing colony size. In addition, I expected to see a more rapid response to the presence of algal food in smaller colonies, which I took to be reflective of their level
21 of ‘desperation’ in terms of resource acquisition. Further, if zooids near the colony edge are more important to future colony growth, these zooids should feed more often than those in the colony centre, and at a higher rate. Therefore, my third prediction is that feeding rate and total feeding time should increase with a decreasing distance from the colony edge.
Methods
Colony preparation
Artificial substrata comprising four 240 mm × 240 mm clear PVC sheets of 0.38 mm thickness bolted to a 600 mm × 600 mm backing plate, were hung by ropes at Workshops pier, Williamstown in October 2005, with the faces of the plates facing down to prevent sediment build-up. I checked these plates periodically for natural settlement during September–October 2005, monitored any settlers that were present and cleared competing organisms from the plate at regular intervals. On November 7, I brought the plates back into the lab, and chose a number of colonies for analysis on the basis of their size range and apparent health. Sixteen colonies from a range of sizes were used in the feeding experiments. I cut around the colonies to remove them from the larger plastic sheeting, and affixed them to glass microscope slides (26 mm × 75 mm) using a small quantity of silicone sealant. I then placed the slides in a plastic slide rack, and immersed this in a flow-through seawater system within a light-tight box. The seawater was filtered to 1µm to prevent any potential prey items entering the box. I starved the colonies in this way for a minimum of 48 hours, after which I commenced feeding trials, with the first trials completed on November 9. Since I could not complete experiments on all 16 colonies within the one day, I intermittently returned unused colonies to the field between trials so that they could resume normal feeding, before bringing them back to the lab, starving them again and completing more trials. My timeline of experiments was:
9/11 all colonies starved 48 hours, colonies 1–5 in feeding trials 11/11 unused colonies returned to field
22 20–23/11 starvation 23/11 feeding trials on colonies 6–12 24/11 unused colonies returned to field 30/11 remaining colonies brought back to lab 30/11–2/12 starvation 2/12 feeding trials on colonies 13–16
For all colonies, I recorded the number of zooids, colony area in mm2, and the area occupied by feeding zooids. These measurements were calculated by analysing digital images of colonies using Image J software as per the method in the previous section. From these base measurements, I calculated the ratio of feeding to non-feeding area and the amount of non-feeding area per feeding zooid, and compared these values to those found in the previous section. All colonies used appeared healthy, did not have substantial damage, and were regular in their growth pattern, with an average circularity measure of 0.931 (± s.e. of 0.011), where a value of 1 indicates a perfect circle. Healthy colonies approximate a circular shape, while colonies that have been damaged or their growth obstructed tend to have more elongate or irregular forms, so this circularity index was further evidence that colonies were healthy and growing in a regular fashion.
Algal food
The uniflagellate protozoan Tetraselmis suecica Kylin (Prasinophyceae) was used as an algal food in the experiment (CSIRO Microalgae Supply strain CS-187). T. suecica has high lipid content, and is a commonly used, highly successful food in aquaculture operations (e.g. Patiño-Suárez et al. 2004). It has also been used successfully as a food in previous bryozoan feeding studies (Riisgård and Goldson, 1997; Riisgård and Manriquez 1997) At a volume of approximately 300 µm3, with a cell diameter ranging from 6.9 to 10.7 µm, T. suecica is an appropriately sized food for Watersipora subtorquata, whose zooids have an operculum width of around 200–250 µm, and a lophophore size likely to be similar to that of other species in the genus with similar-sized zooids, such as Watersipora subovoidea, which has an average lophophore diameter of 660 µm. In prior feeding trials, W. subtorquata colonies were responsive to the presence of T. suecica, and successfully ingested cells. Moreover, T. suecica, with a high chlorophyll a content and a
23 very strong green colour, is visible in the digestive tract of W. subtorquata post-ingestion (E. K. Bone, personal observation), and I was therefore able to confirm ingestion in the feeding trials by noting the presence of this green substance within the digestive tract.
Concentration of algal cells
I balanced the volume of filtered seawater and algal mixture such that the final concentrations of chlorophyll a were within the ranges generally found at Williamstown. Murray and Parslow (1999), using data obtained in the Port Phillip Bay Environmental Study (Harris et al. 1996), predicted the range of chlorophyll a values found in Williamstown in summer to be between 2 and 6 micrograms per litre. In experiments
-1 using Rhinomonas sp., around 800 Rhinomonas cells per ml is equivalent to 1 µgl of chlorophyll a (Riisgård and Goldson 1997). Rhinomonas sp. is a species very similar to T. suecica, both in size and chlorophyll a content. The concentration of T. suecica cells in
the supplied culture was, based on previous growth work, in the range 5.00 × 105 to 1.4 × 106 mL–1 (C. Johnston (CSIRO), personal communication). Working on this as an example, I calculated that 1ml of algal concentrate per 470 ml of filtered seawater would have a chlorophyll a content equivalent to around 2 µg L–1, the mean summer value for Williamstown waters. While these calculations are necessarily an estimate, I was confident that the chlorophyll a concentration in the feeding trials would at least be within the range found in natural conditions. I wanted the algal concentration to be reflective of natural conditions, so that any feeding activity could be regarded as similar to what would be happening in the field. Feeding activity has been shown to vary with algal concentration (Riisgård and Manríquez 1997), but I was interested in obtaining a relative value of feeding activity, rather than maximum feeding efficiency and so maintained these fairly low concentrations through the feeding trials.
Video procedure and analysis
Upon return to the lab, colonies were starved by placing them in a light-tight box within a flow-through seawater system where the incoming flow was directed through a
24 sequence of 20, 5, and 1-µm filters. Before analysis, the colonies were placed in a microscope slide rack within a small plastic container filled with this filtered seawater (FSW). For video analysis, I placed the target colony in a larger beaker of FSW on the stage of a Zeiss Stemi 2000-C binocular dissector with a Sony CCD-RIS video attachment, and used TDK SC analogue videotape to record feeding activity over time. After allowing the colonies to acclimatise for a few minutes, I added a small amount of Tetraselmis suecica concentrate to the FSW within the beaker at the beginning of the feeding trial, using the time of addition as time zero. The amount of FSW was constant at 200ml, while I used between 0.2mL and 0.8mL of T. suecica concentrate, equivalent to
between 2 and 8 µgL–1 cholorophyll a. Video data for a total of 14 colonies were suitable for analysis, since a video tape error distorted much of the recording for colony 12, and colony 3 had been damaged in the field.
Over a period of 60 minutes, I monitored feeding activity in zooids across colonies. As well as lophophore protrusion, I also observed what I refer to as ‘partial opening’. In this behaviour, the zooid operculum flips up and the polypide is partially protruded, without any extension of the tentacles. This behaviour often preceded full lophophore protrusion and feeding, and was common in zooids. While the function of partial operculum opening is not known, I recorded it as zooid activity along with full lophophore protrusion.
In smaller colonies, for the first 30 min, I recorded the activity of each zooid, as well as its position within the colony relative to the ancestrula and the colony edge. I recorded when a zooid partially opened its operculum, when the lophophore was fully extended, and when the lophophore retracted. Over the final 30 min, I simply recorded the number of zooids that were active (either partially open or feeding) per minute. In larger colonies, the activity of all zooids was more difficult to ascertain, therefore I used the second measurement of colony activity over the entire 60 min.
I made more detailed measurements of feeding activity in smaller colonies, or where few zooids were feeding. For each zooid, feeding time was measured as the total amount of time that the lophophore was extended from the zooecium. I recorded single feeding
25 times (i.e. time elapsed between lophophore extrusion and retraction) and combined these to form the total feeding times over the first 30 min of taping. I measured the amount of tentacle flicking in feeding zooids whose lophophore was both clearly visible, and had been extruded for more than two consecutive minutes, recording the number of tentacle flicks in a minute at five minute intervals. I counted both individual tentacle flicks and collective tentacle flicks (see Winston 1977; Riisgård and Manríquez 1997 for descriptions) and interpreted both to indicate an urgency of feeding, and a high need to secure particle capture. I was able to accurately observe tentacle-flicking rates in five colonies, with the number of lophophores observed ranging from one to eight. The total number of minute-long observations per colony ranged from 6 to 17, with an average of 13.6 observations.
Summary data were obtained for each colony by calculating the time to first activity and first feed, the maximum percentage of zooids that were feeding at any time within the experimental period, and the total time spent feeding as a proportion of the total time. In addition, I recorded the position of the zooid that was primarily active, with reference to the colony edge. I predicted that zooids closer to the edge would be more active due to the higher demands of growth at the edge. However, since colonies varied in size and the sample size was very small, this measure would not be an accurate indicator of the importance of the edge to feeding activity, therefore I excluded it from my analyses.
Results
Colonies used for the feeding trial adhered to the general patterns of colony size and feeding area found in the previous section. The ratio of feeding to non-feeding area increased with colony size (Fig. 12a, Table 3), and the amount of non-feeding area per feeding zooid declined with colony size (Fig. 12b), resembling a reverse logarithmic relationship, and this decline was significant (Table 3).
Feeding activity varied across colonies. In some colonies, lophophore protrusion was extremely rapid, occurring just after the addition of the algal culture, while in others, no
26 lophophore protrusion occurred at any time in the one-hour period. However, in all colonies, some partial opening of the opercula took place, indicating that zooids are at least active. In general, this partial opening of the opercula preceded the protrusion of lophophores for feeding, with the one exception of a very small colony that fed immediately upon the addition of algae. I therefore took instances of partial operculum opening as a possible indicator that zooids were detecting the presence of algae in the water.
In the five colonies in which I observed tentacle flicking, the rate of flicks (both individual and collective) per minute ranged from 11.06 to 22.03, with an average of 14.537 ± 1.998 (s.e.). There appeared to be no difference in flicking rate per zooid either within colonies, or across colonies of different sizes, although the sample size was very small.
One pattern I observed was that, where the partial opening of opercula was followed by full lophophore protrusion and feeding, the number of zooids that were feeding gradually increased, and the number of partially opened zooids showed a subsequent gradual decline. Eventually the number of partially open zooids declined to zero, while the proportion of zooids that were feeding reached a plateau. I described this pattern as one of a responsive colony, and two examples from colonies of different sizes are shown in Fig. 13. In a small colony, the plateau was reached at around 75% of zooids, while in a larger colony the plateau was at around 50% of zooids (Fig. 13). A contrasting pattern involved low numbers of opercula that were partially open, or feeding at any one time, and no general pattern of increase in either form of zooid activity over time. Examples of such unresponsive small and large colonies are shown in Fig. 14.
Across all colonies, there was no difference in the time taken to the first zooid activity (Table 3) and, although the time to first feed appeared to decrease with the number of zooids (Fig. 15a), several small colonies with very rapid reaction times affected the analysis, and the difference was not significant with zooid number (Fig. 15a, Table 3). The total percentage of experimental time in which zooids were feeding appeared to
27 decline as colonies increased in size from a few zooids to around 150 zooids, but larger colonies also had high rates of feeding (Fig. 15b), and this change in feeding times was not significant (Table 3). The maximum number of zooids that were feeding at any one time appeared to increase with colony size (Fig. 16a), and these numbers, when calculated as a percentage of the total number of zooids within a colony, declined with colony size (Fig. 16b), although this pattern was not significant (Table 3). It is worth noting, however, that very high percentages of feeding zooids, exceeding 70%, were only seen in very small colonies composed of fewer than 20 zooids.
Discussion
Feeding activity
Colonies used in the feeding experiment demonstrated an increase in the ratio of feeding to non-feeding area with increasing colony size, and the amount of non-feeding area per feeding zooid showed a concomitant decline as colonies became larger. These patterns suggest that as colony size increases, the demand for resources to be invested in further colony growth decreases, and small colonies may in fact have higher resource demands per zooid than larger colonies. These findings strengthen my initial predictions that smaller colonies would need to feed more per zooid, and more overall, in order to meet these energetic demands.
Overall trends in feeding activity were difficult to identify due to the small sample size, however it appeared that very small colonies, of less than 20 zooids, were more likely to feed within the allotted 60-minute time period, were quicker to respond to the presence of food, and generally had a higher percentage of zooids within the colony that were feeding at any one time. These attributes declined as colonies increased in size, although very large colonies also showed short response times and were feeding for a large proportion of the experimental period.
In addition to resource demands, a number of environmental factors may affect feeding
28 activity in bryozoans, including flow rates and algal concentrations, while colony architecture and the placement of lophophores may influence feeding efficiency. The feeding action of lophophores, characterised by the beating of cilia on the tentacles, creates water movement that can aid feeding by directing particles towards the mouth, and a reversal of the cilia movements, specifically, can concentrate the flow of particles and increase feeding efficiency (Strathmann 1982). Bryozoans are therefore recognised as active feeders, distinct from more passive filter feeders (e.g. Winston 1977, 1979). Where a complete canopy of lophophores is formed, however, and the colony is large, the presence of many such ‘feeding pumps’ can cause interference and resource depletion in the proximity of lophophores (e.g. Winston 1979; Okamura 1984; Grünbaum 1995; Kim and Lasker 1998). Many species alleviate this problem by ceasing feeding activity in a small area or positioning lophophore crowns in such a way as to create small gaps in the canopy, or ‘chimneys’, where outgoing currents may be expelled away from the colony surface (e.g. Lidgard 1981; Grünbaum 1997; Eckman and Okamura 1998; von Dassow 2005). Where colonies are small, we would not expect lophophore interference to be a limiting factor in feeding rates, but in large colonies, it may be quite high, reducing feeding efficiency (e.g. Grünbaum 1995). The percentage of zooids that were feeding only approached 100 in a few very small colonies, and in large colonies, when feeding did occur, the feeding zooids tended to be clumped in small patches in the mid-region of the colony, rather than at the edge or towards the centre (data not shown). This clumping of zooids may thus be a way of maximising the strength of feeding currents while minimising the number of zooids that are feeding, and thus using energy, at any one time.
The concentration of algal food can alter feeding rates in marine bryozoans. Higher concentrations are consistent with high rates of feeding and particle clearance (Riisgård and Goldson 1997) and were associated with reduced polypide lifespans in Electra pilosa (Bayer et al. 1994). While I kept the algal concentration relatively constant for each colony, I used a single addition of algae at the beginning of the experiment, so concentration of algal cells in the mixture may have been depleted over time. Given previous findings of low feeding rates with low algal concentration, we might expect this depletion to reduce feeding rates over the course of the experiment. However, the general
29 pattern for responsive colonies was an increase in feeding rates over time, not a decrease, and one zooid within a small colony fed rapidly to the point of gorging, with algae observed leaking from the base of the zooid, possibly due to a perforated gut and ruptured zooid wall. These observations suggest that, despite the relatively low algal concentrations, colonies were not food limited, and differences in feeding rates were more likely to be driven by individual differences between colonies.
Flow rates can also affect feeding efficiency in bryozoan species, and Okamura (1985) found that both small and large colonies increased their particle clearance rates in higher flow environments. However, as my experiments were conducted in still water, most particle mixing would most likely result from the activity of the lophophores themselves, although some microturbulence may have been evident through, for example, the water being heated by the light source, through the movements of surface air currents, and so on. Upon lophophore eversion, the beating of numerous cilia creates currents act to direct water flow towards the lophophore, increasing water movement and consequently the efficiency of particle delivery to these lophophores, and subsequent particle ingestion. Due to these effects, we may expect large colonies to be more efficient feeders, if a significant number of lophophores are open. Given the advantages of a high flow regime for particle delivery rates to the lophophores, it was interesting to observe that in many colonies, zooids did not feed in high numbers across the feeding trials. It may be that the benefits of feeding are lower than the expected energy gain from consuming that resource, if ingestion rates are not high in all zooids that are open and feeding. Although I did not look at clearance rates per zooid, it is possible that a small clutch of zooids in a large colony may have a comparable ingestion rate to zooids in a small colony. Large colonies may thus be able to utilise fewer zooids to meet their resource demands, and this may be due to their increased efficiency through current generation, their lower resource demands, or both. Small colonies, in contrast, may have higher ingestion rates per zooid; Best and Thorpe (2002) showed that, in an abundance of food, small colonies are able to create stronger ciliary currents to improve particle delivery to the mouths and Okamura and Doolan (1993) showed that small colonies respond more strongly to increases in flow than large colonies, possibly through an increase in their rate of cilia beating, although
30 larger colonies always had higher clearance rates.
Although high feeding rates in very small colonies may be accounted for by their high resource demands, similar activity in very large colonies could be a result of the many zooids that are able to respond to the food stimulus and subsequently transmit a signal to neighbouring zooids to respond. Alternatively, larger colonies, although not visibly reproductive at the time of the feeding trials, may be directing resources to the development of embryos, and thus may have higher requirements than medium-sized colonies. Very large colonies may also be able to maintain high levels of flow around feeding lophophores, increasing rates of feeding, and this improved water movement can help increase particle ingestion rate per zooid, so that even if fewer zooids feed, the total rate may be the same. In contrast, the high metabolic demands of small colonies appeared to force rapid feeding in a high proportion of zooids in most cases, however in one case, only a single zooid out of five within a small colony fed throughout the entire feeding trial.
Easy visualisation of the algal food in the gut passage of the bryozoan zooids allowed me to determine that inter-zooidal transportation of food is occurring between feeding and non-feeding zooids in many cases. A striking example occurred in the small colony composed of five zooids, where only one zooid had fed throughout the trial. After around half an hour, a green substance was detected in adjacent autozooids. This indicates that this one feeding zooid is transporting an unidentified, but likely nutrient-rich, algal matter to other zooids within the colony that, while capable of feeding, did not feed. In this case, individual resource capture may thus be more energetically expensive than transporting resources between zooids using internal mechanisms.
Summary - Scaling of colony morphology and function
Despite isometric scaling of module number with colony size in W. subtorquata subtorquata and strong linear relationships evident between whole-colony size and the number of zooids within colonies, allometric scaling was suggested through changes in
31 the relative areas of regions with different functions, and in asymmetries in feeding activity across colonies of different sizes. While module number was strongly linked to colony size, the relative amount of non-feeding area decreased with colony size. This suggests that the resource demands per zooid may be lowered as colony size increases, and in turn that an allometric relationship exists between colony size and metabolic demands. While I did not evaluate total metabolic rates of colonies across size gradients, feeding trials showed that very small colonies, with corresponding large areas of non- feeding, developing tissue, were quicker to respond to feeding stimuli, and showed higher rates of feeding than larger colonies. However, when colonies became very large, response times to the presence of food and overall feeding rates again increased, reflecting increased metabolic demands due to the possible imminent onset of reproduction, a well-developed signalling system between zooids in the presence of an algal food, or better particle delivery to tentacles through increased numbers of feeding zooids.
Zooids within a colony may differ in their capacity to capture and utilise resources as a function of maturity, zooid position within a colony, or of colony size. These factors interact to determine the potential of each zooid to feed and reproduce, and contribute to overall colony growth and fitness. I observed the transport of a significant amount of algae between functional autozooids, confirming that resource sharing may occur even when the recipient zooid is capable of independent resource capture. Resource sharing in this way may be a more important source of nutrition in both feeding and non-feeding zooids than previously thought, and may also be a significant source of energy consumption. In the following chapter, I examine the specific internal structures that facilitate the internal transport of captured resources between zooids within a colony, to enable functions such as the development of new zooids, regeneration of damaged areas, and reproduction.
32 a) b) c) 3 1 1 3 4 2 4 2 2 4 3 3 1
Figure 1. Examples of initial budding patterns in Watersipora subtorquata. The typical budding pattern (a) is characterised by the second (2) and third (3) zooids budding in positions flanking the ancestrula (noted with a 1), with their opercula distal and slightly to the side of the ancestral operculum. The fourth zooid then develops in the space between the second and third zooids, and is positioned directly distal to the ancestrula. Alternate budding patterns include (b) the third zooid developing forward of the ancestrula, rather than in a flanking position. This pattern may be continued with the development of the fourth zooid between the space provided by the second and third zooid, again directly forward, rather than flanking, the ancestrula (c). Alternative budding patterns may arise through changes in the microhabitat, for example crowding by new settlers, or through internal changes to the zooids from which new buds form, and the specific causes of changes in budding patterns have not been defined in this species.
Figure 2. Measurement of zooid dimensions in Watersipora subtorquata. Zooid length was measured as the maximum length of a line that bisects the operculum, runs down the length of the zooid and stops at the border with another zooid (solid yellow line), while zooid width was measured as the maximum width of the zooid measured perpendicular to the zooid length measurement (white dashed line). Operculum length, similarly, was measured as the maximum length of the line bifurcating the operculum from distal to proximal edges (solid white line), and width as the maximum length of the line perpendicular to the length measurement (yellow dotted line). These measurement standards allowed unambiguous measurement even in zooids with irregular growth.
34 a)
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Figure 3. Zooid length (a) and zooid length × zooid width (b) according to their order of development in small colonies of Watersipora subtorquata. Zooid length and width were measured according to the criteria described in Figure 1.
35
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Figure 4. Zooid elongation (zooid length/width) according to the order of development in small Watersipora subtorquata colonies. The total number of colonies is seventy-three.
0.08
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Figure 5. Operculum length × width in zooids in small colonies of Watersipora subtorquata, according to the order of development. a)
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Figure 6. Zooid length measured across all colonies (a) and for each individual colony (b) with increasing distance from the ancestrula in large Watersipora subtorquata colonies. The number of colonies analysed was six.
38
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Figure 7. Zooid size (zooid length × width) (a) and zooid elongation (zooid length divided by width) (b) with increasing distance from the ancestrula in large Watersipora subtorquata colonies. The total number of colonies is six.
39
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Figure 8. Operculum length ×width in zooids at increasing distances from the primary ancestrula in large Watersipora subtorquata colonies. The total number of colonies analysed is six. a) 10000
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Figure 9. Relationships between zooid number and colony area (a) and between colony area and feeding area (b) in colonies of Watersipora subtorquata.
41
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Figure 10. Ratios of feeding (area occupied by autozooids) to non-feeding (developing) area according to zooid numbers in colonies of Watersipora subtorquata.
42
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Figure 11. The amount of non-feeding area per feeding zooid against the number of feeding zooids in Watersipora subtorquata colonies.
43 a) 3.5
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Figure 12. The ratio of feeding to non-feeding area (a) and the amount of non-feeding area per feeding zooid (b) according to the number of zooids in a colony, in Watersipora subtorquata colonies used in the feeding experiment.
44 100 a) 90
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Figure 13. Examples of ‘responsive’ feeding activity over time in (a) a small Watersipora subtorquata colony composed of 17 zooids and (b) a medium sized colony composed of 54 zooids. I defined a responsive pattern as one where zooid partial openings are followed by a gradual increase in fully open, feeding zooids, and a subsequent decrease in partially open zooids as the colony reaches a plateau of feeding capacity. Note the rapid response time in the small colony and the relatively long response time in the larger colony. 45 100 a) 90
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Figure 14. Examples of ‘unresponsive’ feeding activity over time in (a) a small Watersipora subtorquata colony composed of 12 zooids and (b) a large colony composed of 116 zooids. Note that there is no substantial increase in the number of zooids that are fully open and feeding over the duration of the experiment. This may indicate a delayed response to the presence of algal food.
46
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Figure 15. Time to first feed (a) and the percent of total time spent feeding (b) according to the number of zooids in feeding colonies of Watersipora subtorquata.
47 a)
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Figure 16. The maximum number of zooids that fed (a) and the maximum percentage of zooids within a colony that fed (b) according to colony size in Watersipora subtorquata colonies in the feeding experiment.
48 Table 1. Analyses of covariance for a range of zooid measurements in small colonies (zooid length) and large colonies (zooid length, width and length × width). Colony is a fixed factor and either the order of development in small colonies, or the distance in from the ancestrula in larger colonies, is a covariate. Significant effects are highlighted in bold.
Small colonies a) Zooid length Source of variation df MS F P Colony 5 0.008 1.120 0.351 Order of development 1 0.011 1.526 0.218 Residual 196 0.007 b) Zooid elongation Colony 5 0.096 1.727 0.130 Order of development 1 0.354 6.371 0.012 Residual 196 0.056 Large colonies a) Zooid length Colony 5 0.412 35.385 <0.001 Distance from ancestrula 1 4.481 385.025 <0.001 Residual 402 0.012 b) Zooid width Colony 5 0.109 38.314 <0.001 Distance from ancestrula 1 0.010 3.480 0.063 Residual 402 0.003 c) Zooid length × width Colony 5 0.304 113.774 <0.001 Distance from ancestrula 1 0.858 321.598 <0.001 Residual 402 0.003
49 Table 2. Summary of linear regression analyses on colony morphology and scaling patterns of feeding and non-feeding areas in Watersipora subtorquata feeding experiment. In all analyses, the number of colonies analysed is 60.
Co- X-variable Y-variable Intercept r2 F P efficient
Colony construction
Number of Colony area 149.37 –113.93 0.895 493.091 <0.001 zooids (mm2)
Colony area Feeding area 0.644 -23.739 0.998 29358.65 <0.001 (mm2) (mm2)
Role partitioning
Ratio of Number of feeding to 1.531 0.007 0.008 0.464 0.498 zooids non-feeding area Number of Non-feeding feeding area per 51.137 0.003 0.000002 0.0001 0.991 zooids feeding zooid
50 Table 3. Summary of linear regression analyses on both colony morphology and feeding patterns in the Watersipora subtorquata feeding experiment. In all analyses, the number of colonies analysed is 14. Significant effects are highlighted in bold.
Co- X-variable Y-variable Intercept r2 F P efficient Colony construction
Ratio of Number of feeding to 0.0165 1.2385 0.5469 10.864 0.009 zooids non-feeding area Non-feeding Number of area per –1.399 136.06 0.438 7.028 0.026 zooids feeding zooid
Feeding activity
Number of Time to first –0.445 222.077 0.007 0.081 0.781 zooids activity Number of Time to first 1.822 940.941 0.012 0.121 0.735 zooids feed Number of Total % time 0.003 49.362 1.66 × 10–5 0.0002 0.989 zooids feeding Number of Maximum % 43.782 –0.221 0.171 2.269 0.160 zooids zooids feeding
51 Chapter 3
Internal mechanisms of intra-colony nutrient transfer
Introduction
The colonial lifestyle typical of modular organisms means that a degree of co-ordination between modules is required for effective colony functioning, including growth by the development of new modules, damage repair, co-ordination of defence functions, feeding, gamete release, and the development of sexually produced larvae. Co-ordination and integration within a modular colony can be recognised at three levels; physical, behavioural and physiological. Behavioural responses to immediate threats, for example the presence of a predator or a physical disturbance, require co-ordination via the nervous system or conducting epithelia to escape or to retract vulnerable feeding structures (e.g. Shapiro 1992), while responses to more sustained threats or to colony damage requires a higher level of physiological integration to effect life history changes. For example, damage is a critical modifier of growth and reproduction in modular marine invertebrates, and since no one module is essential to the survival of the colony, colonies may recover from disturbance events and processes that cause damage. Damage can increase the risks of mortality or further fragmentation, induce reproduction or decrease reproductive output, and these effects have been shown for a number of clonal plants (e.g. Ruohomäki et al. 1997; Piqueras 1999) and modular invertebrates, especially tropical cnidarians (e.g., Highsmith 1982; Lasker 1984; Karlson 1986, 1988; Chadwick and Loya 1990; Smith and Hughes 1999). These effects may be a consequence of the alteration of the age structure of modules within a colony following damage, or changes in the way colonies allocate resources among colony regions, made necessary by the regeneration process.
The development of zooids or polyps in a modular invertebrate, both at the original growing edge and at the site of damage repair, requires energy; because the new modules are not yet able to feed, this energy must be transported to the growing area from feeding zooids nearby (Mackie 1986; McKinney and Jackson 1989). In clonal plants, the speed
52 and efficiency of the regeneration process is mediated by the flow of metabolites from undamaged, functioning areas (sources), to the damaged areas, which act as resource sinks (Ruohomäki et al. 1997). Source-sink mechanisms have also been invoked for various invertebrate taxa and may be instrumental in determining both regeneration processes and patterns of predation. For example, Oren et al. (1997) demonstrated a net movement of carbon products towards regenerating areas, and in undamaged colonies, corallivore snails fed preferentially on colony margins where new growth was occurring (Oren et al. 1998). New and regenerating areas therefore appeared to operate as resource sinks, and work investigating the effects of damage in the encrusting bryozoan Membranipora membranacea suggests that regeneration in this species may also be governed by a source-sink mechanism (Harvell and Helling 1993).
Resource sharing has been proposed as a measure of true colonies (Mackie 1986), as opposed to aggregations of individuals, and is important in the co-ordination of colony growth, maintenance, defence and reproduction. In particular, with increasing levels of specialisation of modules within colonies, the need for the intracolonial translocation of metabolites and other materials becomes paramount. In plants, physiological integration is effected with relative ease by means of the common vascular system while in modular invertebrates such as colonial cnidarians and compound ascidians, these functions are facilitated by common coelomic cavities, or by gastrovascular circulatory systems that enables the transfer of resources between colony regions (Carle and Ruppert 1983; Gladfelter 1983; Mackie 1986; Gateño et al. 1998).
In the cheilostome bryozoans, the situation is much more complex. Nutrient transfer between regions within and between zooids is facilitated by a network of epithelial tissue termed the funiculus, hollow branching strands of which run from the gut and are distributed throughout the zooid (Carle and Ruppert 1983; Mukai et al. 1997). In cheilostome colonies, individual zooids are extremely compartmentalised and connected to adjacent zooids only by minute communication pores in the zooid’s proximal, distal and lateral walls (see Fig. 1). Funicular tissue is closely associated with these connective pores, thus providing a means of nutrient transport between zooids. While the nutrient
53 transport system in the Bryozoa has been described as analogous to the blood vascular system (Carle and Ruppert 1983; Ruppert and Carle 1983), its specific structure and function are unique. Although there have been a relatively small number of studies concerned with the funicular system and its workings, they have been detailed in their descriptions, and have provided insights into the function of this mode of nutrient transport.
In recent years, knowledge of the components of the funicular system across cheilostome bryozoan taxa has increased, through comprehensive descriptive studies by Silén (1944), Lutaud (1961, 1983), Banta (1969) and Bobin (1971, 1977) that elucidate the general morphology of and cell types contained within the communication organs. However, few studies to date have examined the ecological implications of such a system in a functioning colony, or quantified variation in the morphology of the system within a single colony, between colonies within a species, or across colonies of different species. With such high levels of compartmentalisation between zooids within a colony, one might assume that individual zooids are relatively independent in their nutrient acquisition, reproduction and so on. In fact, in colonies where zooids are unspecialised, each zooid may feed and has the potential to reproduce, and as such may be self- sustaining to a certain degree. However, the presence of non-feeding, specialised zooids, or non-feeding growing or damaged regions, for example, all necessitate the transport of nutrients between colony regions, and as such, an efficient nutrient transfer system is required.
Boardman and Cheetham (1973) recognised six measures of colonial integration in bryozoans, incorporating zooid wall types, interzooidal connections, morphological variation between zooids in a colony, and the position of heterozooids. McKinney and Jackson (1989) also noted increases in colonial rather than modular functions and the development of specialised zooids as important indicators of colonial integration, and stressed the importance of colony form, noting that encrusting bryozoans generally had poor structural integration and inferior functional integration in comparison to rigidly erect or free-living forms. Perhaps the most widely used and applicable measure of
54 colonial integration is the degree of polymorphism, or specialisation of modules within a colony. Colonies that are composed of many different polymorphs specialised for different functions are necessarily more integrated than those where zooids are more uniform, since these polymorphs are usually non-feeding and must obtain nutrients from elsewhere in the colony (Coates and Jackson 1985). The behavioural coordination of these modules also becomes more important with increasing specialisation. The encrusting bryozoans used in the present study are, for ease of comparison, generally unilaminar in form and, with the exceptions of reproductive polymorphs, are monomorphic. Given this apparent uniformity within colonies, and lack of complexity of form, one might assume that these colonies would exhibit a low level of physiological interdependency and integration (Hughes 2005) and may be effectively independent modules, with an increase in metabolic rates approaching an isometric relationship (Chapter 2, this volume). However, within this group of rather unspecialised colonies, we may see a range of effective levels of integration that vary with the particular life history characteristics of each species. One might argue, for example, high levels of integration are needed for high growth rates and responding to or repairing damage. In addition, in rapidly changing environments, where different parts of a colony can be exposed to varying microhabitat conditions, high levels of integration may be equally essential for re-allocating resources from feeding areas to non-feeding areas, or to reproductive zooids within a colony. Differing rates of nutrient acquisition by zooids in different colony regions may also lead to the need for high levels of physiological integration between modules (Chapter 2, this volume). Fine-scale differences in effective levels of integration between colonies that are essentially uniform in their structure and colony formation may, then, at least partly explain different patterns of growth, reproduction and damage repair across superficially similar species. During this chapter, I describe and compare the morphology of skeletal components of the funicular system in a number of bryozoan species with a unilaminar encrusting growth form. I examine the utility of these components for assessing the growth capacities of different species, through the description of changes in pore type, number and size across colony ages and colony regions. I then discuss the ecological implications of these patterns, with reference to the potential for the colony to transport nutrients between colony areas.
55
1. Communication pores and pore development
A. Communication pores in five species of encrusting bryozoan
I examined the general structure of communication pores in colonies of five species of bryozoan with an encrusting growth form; Watersipora subtorquata (d’Orbigny, 1852), Mucropetraliella ellerii (MacGillivray, 1869), Membranipora membranacea (Linnaeus, 1767), Conopeum seurati (Canu, 1928) and Parasmittina delicatula (Busk, 1884). All species exhibit primarily unilaminar growth, however W. subtorquata may form multilaminar foliose colonies in some cases, and P. delicatula may also show multilaminar growth, and frontal budding to form large mounds. In all cases, the colonies examined in this study showed zooidal budding and unilaminar colony form. In addition, W. subtorquata, M. ellerii, M. membranacea and C. seurati all exhibit growth where zooids are arranged in quincunx; therefore, after uninterrupted growth, each zooid will have six neighbouring zooids that may receive or supply nutrients to the target zooid (see Fig. 1). In P. delicatula, zooids appear to be arranged in quincunx (see Fig. 7a), however due to the highly calcified and complex frontal wall, this pattern is more difficult to distinguish. In species where zooids are arranged in quincunx, pores along one lateral wall serve to both receive and transmit nutrients. In lateral walls, the mural pore plates, alternatively termed septulae or pore chambers by other authors, form as double-layered calcium carbonate hemispheres, and may contain a number of pores. Branched strands of funicular tissue are associated with the communication pores, which are plugged with complexes of special cells that aid in the transport of material through the cells of the funiculus (Bobin 1977; Mukai et al. 1997). Significantly, material can only be transmitted one way through the pore via these special cells, and in all species with quincuncial zooid arrangement studied to date, this polarity in function is reflected by the specific morphology of the pore plates. The special cells are positioned so that their nucleated lobes are positioned on one side of the partition between the zooids. Typically this is the side of the donor or proximal zooid that initiates the formation of connective pores. This proximal side becomes concave as the nucleated lobes enlarge with maturity,
56 while on the distal side a ring of calcium carbonate (the “annulus”) builds up around the position of the developing pore plate. This process results in a hemispherical structure; formed by the two layers of calcium carbonate originating from each of the two adjacent zooids. The pore chambers, or plates, therefore appear convex when viewed from the side of the donor zooid (the “outgoing” side, or abannular side) and concave on the side of the receiving zooid (the incoming side, or annular side) (after Banta 1969, in Bobin 1977; Mukai et al. 1997). This pattern has been shown to hold true for a number of cheilostome bryozoans with quincuncial arrangement, including Watersipora nigra (Banta 1969).
Given similar arrangement of zooids in the species examined, I expected the morphologies of pore plates to be fairly similar between species, and that zooids would contain annular, or ‘incoming’ pore plates on lateral walls within the proximal zooid half, while the lateral walls on distal halves of zooids would contain abannular, ‘outgoing’ pore plates.
Methods
Study species
Watersipora subtorquata. Watersipora subtorquata is a common cosmopolitan encrusting cheilostome bryozoan (F. Watersiporidae). Zooid sizes are quite large, in the order of 600–800 µm in length and 300–500 µm in width (see Chapter 2). It has a fully calcified frontal wall without avicularia, and grows by zooidal budding at the colony periphery. After internal fertilisation, embryos are brooded within the maternal zooid, and are released as large, lecithotrophic larvae. In the study area of northern Port Phillip Bay, Victoria, Australia, it settles on a variety of substrata, including pier pilings and mussel shells.
Mucropetraliella ellerii. Mucropetraliella ellerii (Macgillivray), F. Petraliellidae, is a species endemic to southern Australia. It is a fairly heavily calcified species, and its fully calcified frontal wall is more complex than in W. subtorquata, with a small
57 avicularian complex present (Cook and Bock 2002; Tillbrook and Cook 2004). It is also a brooding species, with ovicells external to the maternal zooid. A characteristic feature is the development of rhizomes for substratum attachment, allowing the colony to grow and settle on a number of irregular and mobile substrata (Klemke 1993; Tillbrook and Cook 2004).
Membranipora membranacea. Membranipora membranacea (Linnaeus), F. Membraniporidae, is a common, cosmopolitan anascan bryozoan that produces planktotrophic cyphonautes larvae, and is a common component of fouling assemblages world wide. The frontal wall in this species and others in the family comprises a thin cuticular membrane on the frontal surface of zooids. Zooids are fairly regular in shape, and lack heavy calcified structures such as ovicells or avicularia, although defensive spines may be develop rapidly after exposure to particular species of predatory nudibranch molluscs (see Harvell 1984; 1992).
Conopeum seurati. Conopeum seurati (Canu, 1928), F. Membraniporidae, is another common cosmopolitan species in the same family as Membranipora. Like Membranipora, the frontal wall is a thin cuticular membrane, and the colony produces planktotrophic larvae that are released early in development and are not brooded within. Zooids within a colony are fairly simple, with no ovicells or avicularia, although spines are often present at the distal ends of zooids. These are presumably used for defence, although I could find no studies examining their development.
Parasmittina delicatula. Parasmittina delicatula (Busk, 1884); synonym Smittina unispinosa (Bock 2005) is an encrusting cheilostome bryozoan in the family Smittinidae. It is a brooding bryozoan species, and the sexually-produced larvae develop in calcified ovicells associated with maternal zooids until maturity. In general, P. delicatula has a very heavily calcified, complex frontal wall, and numerous avicularia are found on the surface of colonies. P. delicatula colonies appear to be fairly long-lived, and reproduction typically only occurs when colonies are very large, when larvae are produced in high numbers (M. J. Keough, personal communication). Growth experiments conducted over
58 12 months (Chapter 6) support these assertions, with some colonies containing thousands of developing embryos at any one time.
Colony collection
I allowed colonies of Watersipora subtorquata and Conopeum seurati to settle naturally on artificial substrata consisting of 4 sheets of transparent PVC sheeting of 0.38-mm thickness, fastened to a 240 × 240mm square 6-mm-thick grey PVC backing plate for stability, using stainless steel screws. I then hung the resulting panel of artificial substrata by ropes at approximately 2 m below the low water mark at Workshops Pier, Williamstown, Victoria. M. ellerii, M. membranacea and P. delicatula colonies were all obtained from on or under Queenscliff pier. I obtained M. ellerii colonies by collecting mature colonies from pier pilings and gently prising colonies off the substratum, M. membranacea colonies from blades of the common kelp Ecklonia radiata, carefully peeling colony fragments off the kelp blade using a scalpel, and P. delicatula colony fragments by scraping off colonies growing on pier pilings using a paint scraper.
Scanning Electron Microscopy (General Methods)
I used similar methods to visualise the connective pores in each section of this chapter, which are described as follows. I examined patterns of pores and pore plates using scanning electron microscopy (SEM). Colonies were prepared for SEM by first immersing colonies or colony fragments in a 5% sodium hypochlorite solution until all living tissue was dissolved, leaving only the calcium carbonate skeleton. For some colonies, this process took under one hour, while other colonies, especially those with brooded larvae present, required repeated applications of the solution. Once all living tissue was dissolved, the remaining calcium carbonate skeleton was rinsed and dried. I then dissected the colonies to reveal the area of interest; in W. subtorquata, M. ellerii and P. delicatula colonies, this also entailed the removal of the frontal wall, while in M. membranacea, and particularly in C. seurati, I needed to pare back the cuticular frontal membrane which was often only partially dissolved after the sodium hypochlorite
59 treatment. Following the dissections, I cleaned the specimens of loose material, fixed them to SEM stubs using superglue and sputter coated them in gold for microscopic analysis using a Polaron E5000 sputter coating unit. Where these methods vary, I have specified the changes within the relevant section.
Within lateral walls of all six species, I inspected communication pore plates (septa) for the annular or abannular morphology as described in Banta (1969) and Chaney (1983), and as depicted in Figures 1 and 2.
Results
Figures 3 to 6 show examples of pore plate morphology in C. seurati, M. membranacea, M. ellerii and W. subtorquata, respectively. In these species, the morphologies of the septa within lateral walls resemble those described by Banta, with septa comprising an annular structure surrounding clusters of pores. The numbers of pores varied according to species, but a pore number of between 4 and 8 was found in most instances. In C. seurati (Figs 3, 16), pores were arranged in between 1 and 4 plates across lateral walls, and the number and direction of pore plates appeared to depend on the zooid’s position within the colony (see Part B, this chapter). Within each pore plate, the number of pores averaged at around 6 pores (see Fig. 3b), but ranged from 4 up to 9 in some cases. A heavy annular ring surrounded pore clusters within proximal zooid halves, while the abannular plates were typically flatter, rather than strictly hemispherical, in structure (Fig. 3b). I also noted in many cases the presence of small spines projecting from the zooid wall into the body cavity (e.g. Fig. 3b); the function of these spines is unclear. On transverse walls, pores were typically scattered across the lower region of the wall (Fig. 3a) and not arranged in definite plates such as those found on lateral walls. In Membranipora, pore plates on lateral walls numbered from around 4 to 6 (Fig. 4a), while pores within each pore plate numbered upwards of 5 in most cases (Fig. 4b). Shapiro (1992) also found pore numbers of 4 or more on lateral isocontact plates in this species. In Membranipora, I did not find as clear distinctions between annular and abannular plates pore plates within the lateral wall as in other species I examined, and there appeared no difference between plates in
60 the distal and proximal halves of zooids (Fig. 3a). This may be typical of M. membranacea colonies, since pore clusters on transverse walls appeared very morphologically similar to those on lateral walls (Fig. 3a). The similarity in structure across plate positions and types may also be a function of the age and condition of colonies that I used for analysis. In M. ellerii, the numbers of pore plates on lateral walls numbered from one to four, and pore numbers per plate were typically fairly low, with between two and six pores per plate. Further description of pore patterns in M. ellerii can be found in Part B, this chapter. In W. subtorquata, the number of pore plates on lateral walls ranged from one to six, with a typical number being four. The numbers of pores within these plates usually numbered upwards of 5, and in some cases were as high as 12, although young colonies appeared to have fewer pores per plate (see next section). Annular and abannular pore plates were readily distinguished due to the presence of heavy annular calcium carbonate rings around plates in the proximal halves of zooids (Fig. 6bi), while abannular pore plates, like those of M. ellerii, were fairly flattened in structure and lacked the annular ring (Fig. 6bii). Similarly, further descriptions of pore patterns in W. subtorquata are found in Part B of this chapter.
In these four species, pore plates with annular structures were found in the proximal regions of zooids, while those with an abannular structure were found in distal regions, conforming to the general trends in encrusting bryozoans, and suggesting that the bulk of nutrient movement is generally towards the growing colony edge. However, in the final species, P. delicatula, pores did not appear to be arranged in distinct clusters, either in the transverse or lateral walls. Pores instead occurred singly, and were typically arranged in a row on lateral walls, while on transverse walls their distribution was more scattered (Fig. 7b, c). The zooid walls were also more irregular and rough than in the other species, while zooid arrangement, although appearing to be in quincunx when viewed from the frontal wall (Fig. 7a) often proved to be more erratic at the level of the internal walls.
B. Pore development in Watersipora subtorquata
During examination of communication pores across colonies, I noticed subtle differences
61 in their structure that are likely to be reflective of their mode of development. Some pores appeared to be partially formed, or re-plugged with material, while in other cases shallow depressions had formed in the wall that had the appearance of a developing pore. Silén (1944) proposed that pore development is effected by the sequential dissolution of the calcium carbonate walls, and found that, in Electra pilosa, the development of pore plates is completed only after calcification of the entire lateral wall. To this end, a ring of calcium carbonate forms on lateral wall of the donor zooid. This calcified ring enlarges and grows inwards to enclose a hemispherical space between itself and the lateral wall. He speculated that, as the plate grows and the funicular connections develop, part of the lateral wall enclosed by the mature pore plate dissolves to form the communication pores.
Similarly, Banta (1969) suggested that, while transverse communication pores were formed concordant with the formation of the transverse wall, communication pores in lateral walls formed subsequent to lateral wall formation. However, contrary to Silén’s analysis, he proposed that the septa remained a thin uncalcified membrane until the completion of the pore plate, after which time it dissolves. But, throughout this chapter, I analyse communication pores in zooid walls after thorough dissolution of any living tissue. Any uncalcified membranes present would also be destroyed by this process, therefore this explanation appears incorrect, and more in keeping with Boardman and Cheetham’s (1969) assertion that the walls of the zooecium calcify early in zooid ontogeny. In addition, I have observed lateral walls that are either completely free of pores, or possess only one type of pore, depending on the position of adjacent zooids (this chapter, part B). That the communication pores can be formed subsequent to lateral wall development also uncovers the possibility that the formation of these pores is not determinate, but is a flexible process governed by the particular nutrient requirements of that area of the colony. In this instance, one might expect pores across all colony regions to vary in their development stage if within-colony patterns in nutrient acquisition and requirements had changed subsequent to lateral wall formation.
Methods
62 I examined the pores on lateral walls in young, immature colonies of the encrusting bryozoan Watersipora subtorquata. Colonies had been settled from larvae in the lab on the same day, and were all 14 days old. A total of six colonies were appropriate for analysis, and the sizes of these colonies ranged from 3 to 33 zooids, meaning that the growth rates differed dramatically from colony to colony. Because of this potentially problematic situation, I used the position within the colony as a proxy for zooid maturity. Within each colony, I examined one to four pore plates, and noted their position within the colony relative to the growing edge. I also noted the apparent developmental stage of each pore within the plates and the area of the pore measured. I described the different stages as:
• Stage 1: primary development, the pore is a shallow depression in the zooid wall, with no clear passage through the wall • Stage 2: secondary pore development, almost fully formed, but residual material partially or fully plugs pore opening • Stage 3: fully developed pore, with a clear passage through both walls
Fig. 8 shows examples of each stage of development. I prepared and examined colonies using the general SEM methods described in Part A, section 1. For all pores, I measured pore size using Image J image analysis software. I measured the size of pores at Stage 1 by tracing around the perimeter of the entire depression, and those at Stage 2 by tracing around the perimeter of the pore, including any plugs or crumbs in the size analysis. I predicted that septa on zooids of increasing age, indicated by the increasing distance from the edge, would have a higher proportion of pores that had completed their development, and were thus at stage 3, while those closer to the edge would still be developing and have more pores at stages 1 and 2. In addition, I predicted that, with increasing zooid maturity, the sizes of mature, stage 3 pores would increase.
Results
The proportion of pores at each stage differed according to position within the colony
63 (Fig. 9). At three zooid rows from the edge, all pores were at stage 3 and had completed development, while at two zooid rows from the edge, pores were relatively evenly spread between stages 1 and 3, with between 20 and 40% of pores at each development stage. Just behind the growing edge, at one zooid row back, around 80% of pores were at stage 1, while 20% were at stage 2. At the very edge, where zooids were youngest, almost 60% of pores were at the first developmental stage, a smaller proportion were at stage 2, and less than 20% were at the final developmental stage. The sizes of pores differed
significantly according to their development stage (Fig. 9, one-way ANOVA: F2,46 = 3.643, P = 0.034), with stage 1 pores largest, followed by stage 3 (fully developed pores), while stage 2 pores, that were partly formed or partly plugged with calcium carbonate, were the smallest pores. The sizes of pores also varied according to development stage, and relative to the growing edge (Fig. 10b), although within each stage, pore size appeared relatively consistent. Analysis of covariance subsequently revealed a significant effect of size, but not of the relative distance from the edge (Table 1).
Discussion
The general pattern of pores at the designated stages appeared to reflect the maturity of zooids within the colonies, as indicated by the distance from the growing edge. At the furthest distance from the growing edge only fully developed pores were present, consistent with my predictions of zooid maturation, while at the next closest point to the edge all types of pores were present, and just behind the growing edge only the immature stages were present, with most pores at the first development stage. However, at the very edge, pores of all three types were present, contradicting this general pattern. Since the history of all colonies was known, this finding is not due to any effects of damage or module removal, resulting in discrepancies in zooid age at equivalent positions within several colonies. However, as previously noted, colonies varied widely in sizes after fourteen days, and as such growth rates would have been significantly different between colonies. A colony that grows slower may in fact have zooids at the colony edge that are comparable in age to more central zooids within a rapidly-growing colony. Thus, edge zooids in a slow-growing colony may be more mature than zooids proximal to the edge in
64 a fast-growing colony. All colonies from which pores at the colony edge were measured were only 3–6 zooids in size at 14 days, compared to the colonies whose pore sizes were measured at one to three zooid rows back from the edge, which were from 6 to 33 zooids in size. This suggests that variations in growth rates may be a valid reason for the presence of mature, stage 3 zooids at the very edges of colonies. Pore size according to stage also varied, with stage 1 pores being largest. Since stage 1 pores are measured as depressions in the zooid wall, their sizes may not be accurate indicators of the final pore size. Stage 2 pores were the smallest, likely due to their incomplete development, while stage 3 pores were of intermediate size.
2. Pore patterns across colony regions and implications for colonial integration
A. Pore patterns across colony regions in two species with contrasting life histories; Watersipora subtorquata and Mucropetraliella ellerii
Here I present surveys of pore sizes and types on lateral zooid walls across colony regions in two common encrusting cheilostome bryozoans. In these colonies, new growth occurs sequentially at the colony’s perimeter. Most Recent species, including the two in this study, exhibit growth by zooidal budding, where new zooids bud distally to existing zooids at the colony margin. Transverse walls develop subsequent to the bud, sequentially separating the developing bud into new, feeding, autozooids (McKinney and Jackson 1989). As previously stated in Part A, in encrusting species with quincuncial zooid arrangement, pore chambers form as double-layered calcium carbonate hemispheres, plugged by funicular tissue that exerts a polarity on the direction of nutrient movement (Bobin 1977, Mukai et al. 1997). This polarity is reflected in the morphology of the pore plates, with the presence of an annulus, a heavily calcified ring surrounding the cluster of pores, indicating an “incoming” plate on the side of the receiver zooid, and the absence of an annulus or a distinct convex structure to the pore plate indicating that it functions as an “outgoing” plate, and is located on a receiver zooid (Banta 1969; Bobin 1977; Mukai et al. 1997). Banta (1969) showed this pattern to be true in a number of cheilostome bryozoans with quincuncial arrangement, and I have also confirmed these
65 patterns in four of five species studied in Part A or this chapter. Confirmation that the funicular system functions in this way has been problematic to date, but Bobin (1971) showed movement of lipids through the rosette complex from a degenerating zooid to a stolon in Bowerbankia. Although Bowerbankia is a ctenostome species, radio-labelling studies have confirmed that nutrient transfer is primarily directed towards the growing edge in young colonies of the cheilostome species Membranipora membranacea (Miles et al. 1995; Best and Thorpe 2002).
With these structural and functional aspects of the system in mind, I sought to examine whether the patterns of pore plates and pores, being morphological characters of the funicular system, reflect patterns of growth in two encrusting bryozoan species, Watersipora subtorquata and Mucropetraliella ellerii. My specific predictions are two- fold: firstly, if the morphological features of pore plates reflect their function as implied by previous histological and microscopic evidence, we should see no convex, ‘outgoing’ pore plates at the extreme edge of the colony. Secondly, the patterns of pores and pore plates should reflect our knowledge of the fragmentation patterns of the two species, in that the species with a known higher level of growth should have a higher capacity to direct nutrients towards continued growth at the colony edge, and this ability should be apparent in the numbers and types of pores present at different regions of the colony.
Methods
Colony characteristics and collection methods
Watersipora subtorquata. Watersipora subtorquata has very high natural growth rates, and fairly high incidences of natural fragmentation. The high growth rates of colonies means that there is a distinct age gradient across the colony from the proximal region to the distal growing edge, and these changes in age have significant consequences for a colony’s ability to re-grow and recover from damage. For example, Hart (2001) showed that fragments formed from older, central parts of colonies had slower recovery rates than those formed from the young growing edge, and feeding zooids a few rows
66 back from the edge appear to be primarily responsible for further growth of that edge. In addition, the amount of growing tissue in smaller colonies is proportionately higher than in larger colonies (Chapter 2, this volume), meaning that small colonies may be required to direct more nutrients towards this growing area. The ratio of feeding to non-feeding area decreases as colony size increases, meaning that as colonies grow, the level of investment in further colony growth is reduced, freeing up potential energy stores for the initiation of reproduction (Chapter 2). These characteristics suggest that small colonies of W. subtorquata may be directing a large store of its nutrients, gained from feeding by zooids close to the edge, towards further growth. As a result, I would predict that, in this species, there will be a high number of abannular, ‘outgoing’ pores with increasing proximity to the colony edge.
Colonies were collected from artificial settlement plates deployed at Williamstown, near Melbourne, Victoria. All colonies were monitored after settlement to ensure that they were undamaged at the time of collection. A total of 7 colonies were used in the SEM analysis, with sizes ranging from 5 to 62.5mm2, at an average size of 29.286 mm2. All colonies were reproductively immature, reinforcing our prediction that nutrients will be strongly directed towards further colony growth in this species.
Mucropetraliella ellerii. Settlement of Mucropetraliella ellerii colonies in the lab proved unsuccessful, so experimental colonies were collected from St. Leonard’s pier in southern Victoria, and carefully removed from their solitary ascidian substratum. A total of nine colonies were used in the analysis, with sizes ranging from 43.75 to 212.5mm2, and a mean size of 86.806 mm2. Most colonies were reproductively active, with eight of nine colonies producing from 1 to 63 embryos, at a mean of 24.55 embryos per colony. Due to the nature of the collection, the damage history of the colonies was unknown.
Scanning electron microscopy (SEM)
The patterns of pores and pore plates within lateral zooid walls were examined using scanning electron microscopy (SEM) according to the general methods described in Part
67 A. Fig. 5 shows examples of images generated from the SEM process in M. ellerii, including zooid walls and pore plates, while Fig. 11 shows examples using W. subtorquata.
Survey design and statistical analysis
The frontal walls on each colony were, as far as possible, dissected away to reveal the lateral internal walls of zooids in a sequential line from primary ancestrula to growing edge. In some cases, this process resulted in damage to parts of a colony or the accumulation of fine particles of calcium carbonate that often occluded pores or plates. These and any other areas where visualisation of pores was ambiguous, such as areas where the preparation was incomplete, were excluded from SEM analysis. For each zooid that was deemed suitable for analysis, its position relative both to the ancestrula and to the edge were noted. The number and type of pore plates were noted along one lateral wall, as well as the number of pores within each plate, and the sizes of all clearly visible pores were measured using SigmaScan 2.0. For both species, patterns of pore numbers and direction across colony regions were analysed using analysis of covariance, as were patterns in pore sizes according to pore direction, across colony regions. Separate linear regression analyses were carried out for incoming and outgoing plates and pore numbers, while differences in overall pore numbers and average pore sizes over all positions were analysed using analysis of variance. At each position away from the colony edge, the sizes of outgoing and incoming pores were compared using t-tests. Since the colonies of the two species were not of equivalent age or damage history, the specific patterns of pores and pore sizes were not compared by species; at any rate, we might expect the two species to exhibit differences in the nature of the funicular system not related to life history. These differences were noted, but not analysed statistically.
Results
Watersipora subtorquata
68 Number and direction of plates and pores. In Watersipora subtorquata, the numbers of plates differed significantly both with the type of plate and with the position within the colony relative to the growing edge (Fig. 12a, Table 2a), whilst the numbers of pores of the two different types also showed significant variation across colony regions, and varied according to type (Fig. 12b, Table 2b). To assess whether this variation in pore numbers is due to a changing number of pore plates, I compared plate and pore numbers per zooid for both incoming and outgoing pore plates. Analysed separately, the number of incoming plates per zooid remained fairly stable across the five zooid rows from the growing edge, and the distance from the edge only explained 0.12% of variation in incoming plate numbers (incoming plates = 2.789 – 0.012 × distance from edge, F1,42 = 0.049, P = 0.824, r2 = 0.00119). Incoming pores, similarly, remained fairly steady, with the distance from the edge only explaining 0.9% of the variation in numbers (incoming 2 pores =14.059 – 0.461 × distance from edge, F1,42 = 0.386, P = 0.538, r = 0.0091). In contrast, the distance from the edge explained a significant amount of variation in numbers of outgoing plates (outgoing plates = 1.118 – 0.588 × distance from edge, F1,42 = 22.909, P<0.001, r2 = 0.353) and outgoing pores (outgoing pores = 7.118 – 2.922 × 2 distance from edge, F1,42 = 12.577, P<0.001, r = 0.230). These results appear to be due to the drop in outgoing plates and pores at the extreme colony edge. Overall, there were more incoming pores per zooid than outgoing pores (paired t-test; t1,42 = 2.774, P = 0.008).
Pore size. A total of 143 pores across the seven colonies were deemed clearly visible enough for analysis of size using SigmaScan 2.0. At all zooid positions where they were present, outgoing pores were larger in size than incoming pores (Fig 13a). The size of pores varied significantly with the type of pore but not with the distance from the growing edge (Fig. 13a, Table 3). Overall, outgoing pores were significantly larger than incoming pores (one-way ANOVA, F1,140 = 25.29, P<0.001, Fig.13b).
Mucropetraliella ellerii
Number and direction of plates and pores. A total of 68 zooids containing 225
69 pore plates were examined across the nine colonies used in the analysis. In Mucropetraliella ellerii, incoming plate numbers were variable across the colony regions, but remained fairly steady at around eight per zooid, while outgoing plates dropped to zero at the colony, but again were reasonable steady at around eight plates at other positions in the colony (Fig. 14a). The number of outgoing pores dropped to zero at the colony edge, whereas incoming pores remained at a reasonably steady level (Fig. 14b). Plate numbers differed significantly both according to the type of plate and the distance from the growing edge (Table 4a), while pore numbers varied significantly with their distance from the edge, but not according to pore type (Table 4b, Fig. 14b). Analysed separately, the distance from the growing edge again did not significantly explain variation in the numbers of incoming plates (incoming plates = 2.660 + 0.013 × distance 2 from edge, F1,52 = 0.054, P = 0.818, r = 0.001), or pores (incoming pores = 2.882 2 +246.882 × distance from edge, F1,52 = 0.594, P = 0.444, r = 0.011), but did explain a significant amount of the variation in number of outgoing plates (outgoing plates = 1.691 2 – 0.259 × distance from edge, F1,52 = 17.210, P<0.001, r = 0.249) and outgoing pores 2 (outgoing pores = 6.344 – 0.757 × distance from edge, F1,52 = 13.316, P = 0.001, r = 0.204). Again, these results appear to be driven by the drop in outgoing plate and pore numbers at the colony edge. In M. ellerii, the overall numbers of incoming and outgoing pores per zooid were not significantly different (paired t-test, t1,66 = 0.124, P = 0.902).
Pore size. A total of 134 pores across the nine colonies were clearly visible enough for size analysis using SigmaScan 2.0. Across colony regions, analysis of covariance (Quinn and Keough 2002) showed that the type of pore had a significant effect on the size of that pore, while the position in the colony did not (Fig. 15a, Table 5). Overall, pore size differed significantly according to type in M. ellerii, but in this species, in contrast to W. subtorquata, incoming pores were, overall, significantly larger than outgoing pores (one-way ANOVA, F1,130 =12.757, P<0.001, Fig. 15b).
Discussion
The patterns of pore plates and types across both W. subtorquata and M. ellerii appeared
70 fairly similar in many respects, with, in particular, no instance of an abannular, or ‘outgoing’ pore plate at the extreme colony margin in either species. Since there are no developing zooids at the colony margin, there is subsequently no need for nutrient transport beyond this edge, rendering an ‘outgoing’ porous connection redundant. As a consequence, it confirms histological evidence obtained by Banta (1969), Bobin (1977) and other investigators that the morphologies of the pores and pore plates reflect their function with respect to the direction of nutrient transfer throughout the colony. Given this is the case, we may presume with some confidence that the descriptions of pore functions are accurately reflected in their morphology, and begin to build up a picture of the capacity for resource transfer in the two species.
The two species exhibited subtle differences in the specific morphology of the pore plates, with M. ellerii having more heavily calcified annular rims and more pronounced curvature in than W. subtorquata. Since M. ellerii colonies were larger, this result could be driven by the increased maturity of zooids and porous connections. However, morphological differences between species are not unexpected, and these differences need not lead to differences in function. Banta (1969) examined communication pores in 10 species of cheilostome bryozoans, concluding that the general morphology of those pores and pore plates was similar, and that most differences are seen in the number of pores and relative calcification of the pore chambers. This appears to hold true for the two study species, as both exhibited remarkably similar overall patterns in pore and plate numbers.
In W. subtorquata, pore plate numbers were reasonably steady across colony regions, with both incoming and outgoing plates numbering around 2–3 per zooid across colony regions, with the exception of the growing edge, where outgoing plates dropped to zero, while incoming plates remained steady. Since pore numbers are partially dependent on the number of plates, the patterns of the numbers of pores per zooid mirrored the pattern of plates, with, again, outgoing pores dropping to zero at the colony edge. Similarly, in M. ellerii, numbers of both incoming and outgoing pores and plates remained fairly stable across colony regions, with the exception of a drop at the extreme colony edge, where no
71 developing zooids exist, and another drop towards the central region of the colony. This suggests that the central region of colonies in M. ellerii may be limiting the inter-zooid transfer of nutrients, thereby contributing little towards further growth. That the numbers of porous connections can change throughout colony development suggests that the funicular system is relatively flexible. Outgoing plate and pore numbers decline at both the colony edge, and towards the colony centre, suggesting that, as the distance from the edge increases, the colony is directing fewer resources to growth. Additionally, since central zooids were originally edge zooids, presumably connections may be either added during development, as in the case of outgoing pore plates, or reduced, which may explain the differences in plate and pore numbers across colony regions. That this system is flexible, capable of change according to developmental stage or damage history, seems clear, and warrants further investigation.
In W. subtorquata, outgoing pores were larger in size than incoming pores at all positions in the colony where they were present, and at distances of 2–3 zooid rows from the edge, outgoing pores are significantly larger, indicating there is a greater amount of funicular tissue, and thus a greater capacity for nutrient transfer, towards the colony edge. Field measures of growth and regeneration in this species have suggested that the primary contribution to colony growth is made by the rows of zooids immediately behind the growing edge (Hart 2001), and an increase in the numbers and sizes of outgoing pore plates behind the edge appears to confirm this observation at the level of the funicular system. In M. ellerii, I saw few differences in the sizes of incoming and outgoing pores at different colony locations, while in W. subtorquata, outgoing pores were always larger than incoming pores. These results suggest that, in colonies of this age at least, M. ellerii colonies are not allocating as many resources towards further growth as W. subtorquata. Overall, incoming pores were larger than outgoing pores, again suggesting that the centre of the colony is contributing little to further colony growth. This assumption, based on the pattern of pores, reflects the lower growth rates seen in natural populations of this species by Klemke (1993). In addition, fragmentation experiments examining growth rates of fragments taken from the young colony edge and the older colony centre did not show significant differences between these two colony regions (Klemke 1993).
72
A significant possible source of error in this analysis is that in the M. ellerii analysis, older colonies, whose damage history and therefore modular age structure was unknown, were used. Furthermore, their reproductive status can influence the degree of nutrient transfer within a colony, especially with respect to the growing edge .We might not expect a reproductive zooid, for example, to donate its resources towards the growth of neighbours, but rather towards the growth of the developing embryo. It is possible that energy stores, instead of being allocated to growth, were instead diverted to reproductive needs, thus reducing the degree of nutrient transfer towards that edge, explaining the pattern of fewer pores towards the edge. Although no statistical analyses are shown for comparative data of pores in the two species, W. subtorquata zooids had, on average, more pores per zooid, while in M. ellerii, pore sizes were larger. These characteristics may reflect the greater need for young W. subtorquata colonies to make connections to aid in further growth, while in M. ellerii, larger pore sizes may simply reflect the maturity of zooids in these colonies. In previous studies involving both species, colonies composed of older zooids tend to have lower growth rates than those composed of young zooids, with the latter better able to regenerate new tissue after damage (Klemke 1993; Hart 2001), while other cheilostome bryozoans show decreased feeding and greater tissue degeneration in older zooids (Palumbi and Jackson 1983). The ages of zooids within colonies thus appears to be a likely source of discrepancies in pore sizes and number between the two species in this study. However, without a comparison of colonies of equivalent age, it is unknown whether these differences are species-specific or a product of colony age, and these differences prevent unambiguous interpretations and comparison of the data in the two species. Using colonies of equivalent age in further comparisons of nutrient transport capacities across bryozoan species would be advantageous.
Despite the limitations posed by the inconsistencies in colony age and size, particularly in the M. ellerii analysis, this study incorporates knowledge of the ecological characteristics and damage responses of different extant species into analysis, and represents an important step forward in the understanding of the internal nutrient transfer system in bryozoans. Moreover, the variation in pore numbers combined with the consistency in
73 plate numbers, as well as patterns of calcification on the zooid walls and basal area suggest that this system is flexible to changes in the environmental and internal conditions of the colony. In many cases, communication pores appeared to be partially plugged with calcium carbonate, while shallow depressions that appeared likely to become a full pore were often observed on pore plates in samples of both species. However, because electron microscopy is necessarily destructive, these observations cannot be conclusive, and explicit details of the changes in the skeletal structure of these funicular connections throughout colony development are lost. Further work involving confocal microscopy or fluorescent tracers, is required to elucidate the potential for flexibility within the funicular system, as an essentially unique system of nutrient transfer among organisms.
B. Pore patterns across colony regions in Conopeum seurati, and investigation of pore maturation
In this section, I examine the pattern of pore plates and pores across colony region in a third species, the encrusting anascan cheilostome Conopeum seurati. In addition, I also propose a mechanism of pore development, and analyse patterns of pores and relative pore sizes across colony regions as potential evidence of these developmental patterns. We have seen in this chapter that the porous connections that aid intra-zooidal nutrient transfer in encrusting cheilostome bryozoans may be flexible; that is, it is likely that pores can form, expand, close up or be re-formed, and that these actions may result from intrazooidal characteristics such as zooid maturation, or from changes in the interzooidal nutrient needs in a colony. Moreover, using knowledge of pore development patterns in the close examination of existing pores may provide information on the approximate age of that pore.
Histological evidence has suggested that the formation of a pore is initiated by the zooids from which the nutrients will eventually flow; the ‘outgoing’ or abannular side of the pore plate. This zooid may be known as the donor zooid, whilst the other zooid in the process may be known as the recipient. Formation of a functional pore necessitates a
74 contiguous line of funicular tissue running through the calcium carbonate wall of both the donor zooid and the recipient zooid. Bobin (1971) detailed the structure of the rosette cell complex within the communication pores, and noted that only the rosettes of the distal half of the donor zooid belong to it, while in the proximal half, the receiving part of the zooid contains the rosette complexes of the adjacent donor zooid. This suggests that the donor zooid makes the initial connection with the receiver zooid, and may also control the relative strength of that connection.
The special cells embedded within the opening of the communication pore are characterised by having distinctly nucleated lobes, which form only at one end of the cell (see Fig. 18). The rosette complex, made up of a number of these special cells at the position of the nucleated lobes, is formed on the side of the recipient zooid, or the ‘incoming’ side. The formation of the special cells in the annular, incoming region of the pore plate must necessarily be subsequent to the formation of a contiguous connection between the adjacent zooids; that is, pores must be formed before the special cells can mature. The cells, and therefore the lobes, might be expected to enlarge upon maturity. Since the special cells completely plug the pores within the zooid walls, we might expect that, on enlargement of the nucleated lobes, the pore on the ‘incoming’ or annular side of the pore plate to become larger with maturity, and that the presence of nuclei would cause a disproportionate increase in the size of pores on this, the incoming side, relative to those on the outgoing, donor or abannular side of the pore plate, where nuclei are not present. In effect, I predict that, due to the process of pore formation, the relative sizes of pores on each of the outgoing and incoming sides are indicative of pore maturity. Moreover, due to the polarity generated by the position of the nucleated lobes, we may expect that the relative sizes of pores on either side of the zooid wall are also valid indicators of the strength of nutrient movement through that wall.
Here I describe the relative sizes of pores measured from both sides of the zooid wall in an encrusting bryozoan with quincuncial zooid arrangement. I used a single colony of the encrusting cheilostome bryozoan Conopeum seurati and analysed pore sizes across colony regions from near the ancestrula to just behind the growing edge, as for W.
75 subtorquata and M. ellerii colonies in the previous section. Owing to the pattern of growth in encrusting species, and given no substantial previous damage to the colony, the zooids at the colony margin will be the youngest, while those towards the centre will be older. In addition, I measured the sizes of individual pores within plates viewed from both the incoming and outgoing sides of the zooid wall at three positions across the colony; four, three and two zooid rows proximal to the growing edge.
Given known patterns of colony growth and communication pore development in encrusting species, my specific predictions were:
1. If the morphology of the pores accurately reflects their function, we should see only incoming pore morphologies in the proximal halves of zooids, and only outgoing pore morphologies in the distal halves of zooids 2. If growth is favoured in a colony of this size, the number of outgoing pores should increase in zooids close to the colony edge 3. That pores within zooids at the edge of the colony will exhibit size ratios of incoming size to outgoing size that are less than that found in zooids further towards the centre of the colony, reflecting differences in pore age in the two regions.
Methods
Colony preparation
I allowed colonies of Conopeum seurati allowed to settle naturally on a 240-mm square of 0.38-mm thick clear PVC plastic, fastened to a 240-mm square, 6-mm thick grey PVC backing plate for stability, using stainless steel screws. I secured this backing plate to a larger plate, and hung the apparatus at around 2 m below the low water mark at Workshops Pier, Williamstown, Victoria. I chose a single colony for analysis, and the thin plastic substratum made it possible for the colony to be removed from the plate for SEM analysis by cutting a hole in the plastic around the colony and removing that piece.
76 Scanning electron microscopy
I examined pores within lateral zooid walls using scanning electron microscopy (SEM) according to the general methods described in Part A.
Sampling and analysis
I mapped out a fixed transect from three zooids from the ancestrula to one zooid before the colony margin. Zooids closer to the ancestrula and the colony edge were unfit for analysis due to crumbling of the zooid walls at the edge and the persistence of remains of the cuticular membrane in zooids close to the ancestrula. I therefore analysed pores within zooids at six positions in the colony, within a total of eleven zooids. To investigate whether relative pore sizes on either side of the wall differ across colony regions, I analysed selected pore chambers where both sides of the zooid walls were clearly visible and noted the size of each pore measured from both sides. This process was repeated at three sites along the initial transect; at four, three and two zooid rows proximal to the growing edge of the colony. At four zooids proximal to the edge, I measured the areas of all pores contained within one pore plate in the lateral wall. At three zooids proximal to the edge, two comparisons were made; one between the areas of pores on opposite sides of the transverse wall, and one between pore sizes within one pore plate on opposite sides of the lateral wall between zooids. At two zooid rows proximal to the growing edge, pores within one pore plate were compared on opposite sides of the lateral wall between zooids.
Results
Pore numbers and pore sizes
The numbers of pores per zooid of both types varied across colony regions (Fig. 17a). Incoming pores outnumbered outgoing pores at six zooids back from the growing edge, but at five rows back numbers were almost equal, while closer than four rows to the
77 growing edge, there were more outgoing pores than incoming pores per zooid. The number of outgoing pores increased dramatically at one zooid row back from the edge, while incoming pores remained at a steady level similar to that found at six rows from the edge. The sizes of these pores also varied according to type and position (Fig. 17b) but here the pattern was more apparent. Outgoing pores were all around the same size, at around 100 µm2 in area, while incoming pore size varied widely across colony regions. At five zooid rows back from the colony edge, incoming pores were more than twice as large as outgoing pores. Similarly, at four and two rows from the edge, incoming pores were substantially larger than outgoing pores. However, at three rows and one row back from the growing edge, incoming pore sizes were smaller on average than outgoing pore sizes. Contrary to predictions, the ratio of incoming pore size to outgoing pore size did not appear to increase with increasing distance from the colony edge.
Comparisons of pore sizes
Comparison 1: Four zooid rows proximal to edge (Lateral wall). At a distance of four zooid rows behind the growing edge, the communication pores, when viewed from the incoming side of the wall, were significantly larger than the same pores viewed
from the outgoing side of the wall (Fig. 19, n = 7; paired t-test, t6 = 4.147, P = 0.003)
Comparison 2: Three zooid rows proximal to edge (Transverse wall). At three zooid rows proximal to the growing edge, pores on the transverse were different in size when viewed from either side of the wall (Fig. 20). Pores viewed from the incoming side of the wall were significantly larger than when the same pore was viewed from the outgoing side of the wall (n =32; paired t-test, t31 = 2.192, P = 0.036).
Comparison 3: Three zooids rows proximal to edge (Lateral wall). At three zooid rows proximal to the growing edge, pores on a single pore plate were viewed from either side of the lateral zooid wall (Fig. 21). When viewed from the incoming side of the wall, pores were significantly larger than when the same pores were viewed from the
outgoing side of the wall (Fig. 22, n = 10; paired t-test, t9 = 12.090, P<0.001).
78
Comparison 4: Two zooid rows proximal to edge (Lateral wall). At a distance of two zooid rows proximal to the edge, the development of a continuous pore connection through the double-layered lateral zooid wall is incomplete. As seen in Fig. 23, the abannular, outgoing pore chambers have completed development, but pores have not yet opened up within the opposite side of the zooid wall.
Discussion
The number of pores per zooid appeared to change across colony regions, although I saw large differences between incoming and outgoing pores only at positions adjacent to the growing edge. At these positions, outgoing pores significantly outnumbered incoming pores, suggesting that there are high levels of nutrient transfer towards the growing edge, and at a more central position, the number of incoming pores outnumbered outgoing pores. This large investment in the growing edge mirrors the pattern found in the fast- growing Watersipora subtorquata (this chapter, part B 1) and again suggests that the colony is investing much of its nutritious stores towards colony growth. However, the strength of this association is limited by the low sample sizes of zooids taken at many positions within the colony, and since I only examined one colony, no generalisations can be made with respect to other C. seurati colonies. This preliminary investigation, however, provides promising evidence for the use of porous connections to examine the strength of intra-colony resource transfer in a third cheilostome species.
Although analysis of the relative sizes of pores viewed from either side of the zooid wall was necessarily limited by the number of clearly prepared pore plates, as the distance from the growing edge increases, the difference between the sizes of pores when viewed from the two sides of the wall also increases, The incoming sides of pores were larger than outgoing sides of pores at both four and three zooids proximal to the colony edge. This pattern was apparent in pores within both the transverse and lateral zooid walls, although the difference in pore sizes was largest in the lateral walls. This is consistent with Banta’s (1969) assertion that while the transverse pores form in unison with the
79 formation of the transverse wall, the lateral pores form subsequent to the lateral walls. Furthermore, at a distance of two zooid rows proximal to the growing edge, while the outgoing pores were fully developed, the incoming connections on the opposite side of the wall had barely begun to develop. I found other evidence of this phenomenon in this species, with cases of divergent levels of development in the same pore connection when viewed from opposite sides of the wall; always involving the full development of abannular plates and the lagging development of annular plates. This evidence further supports the view that the donor zooid initiates pore development.
The discrepancies in pore size between the outgoing and incoming sides of the wall may also reflect the relative maturity of the pores at each position within the colony. Zooids near to the colony centre are, in the absence of damage, older than those close to the colony edge, and as such contain porous connections that are more mature than those at the colony edge. Research by Banta (1969) and other workers has suggested that the nucleated lobes of the special cells embedded within the incoming sides of pore plates enlarge with increasing levels of cell maturity. Enlargement of these nucleated lobes must be accommodated by a concomitant enlargement of the pore opening within the calcium carbonate wall, and the evidence presented appears to support this notion. The relative ratio of pores size from the incoming and outgoing sides of walls can therefore be an indicator of the level of maturity of that pore.
General Discussion
The morphology of the communication septa and pores within lateral zooid walls in encrusting unilaminar bryozoans appears to be relatively consistent across species, with a similar abannular and annular side being apparent in most cases. This is consistent with the findings of Banta (1969), who, in analysing colonies from ten different species, found most variation in the number and arrangement of pore plates and pores, rather than in the specific morphology of each pore plate.
Notable exceptions to these patterns are the apparently undifferentiated pores seen in very
80 young colonies (this chapter, part 1B) and the ‘fusion pore plates’ as described by Shapiro (1992). In very young colonies of Watersipora subtorquata, many pore plates had similar morphologies to the typical ‘incoming’ annular plate found in more mature colonies. However, the location and arrangement of these plates appears confusing in light of the known and predicted functions of the different morphologies. For example, in one case, the ancestrula of a small W. subtorquata colony of three zooids in total had no outgoing plates on either zooid wall, with all plates having this apparent annular incoming morphology when viewed from the internal wall of the ancestrula. This is in contrast to predictions of nutrient transfer being primarily ‘outwards’, or towards the growing edge, in bryozoans. One scenario is that these plates are undifferentiated, and, with morphologies that are inconsistent with both the ‘typical’ annular and abannular forms found in other areas of the colony, enable nutrient transfer in either direction. This may be reflective of the flexibility of nutrient transfer when colonies are young, enabling transfer between the ancestrula and the next developed zooid in either direction, depending on the specific needs of each zooid within the colony. As we saw in Chapter 2, the development of colonies takes place with the sequential development of new zooids distal to the ancestrula on both sides. It is feasible to anticipate a situation where both the ancestrula and the second developed zooid, although both independently feeding, would require supplementary nutrient input to fuel the development of additional zooids distal to their zooid margins. However, this possibility, that the pore essentially functions as a two-way valve, with nutrient transport able to be effected in either region, is unsubstantiated, and requires further study to look at the actual rates and directions of nutrient transfer between zooids.
Shapiro (1992), when analysing pore plates that formed following the fusion of compatible individuals that were behaviourally co-ordinated, noted that these allocontact plates took the form of two hemispheres placed base to base, rather than the single hemisphere with the perforated dome typical of lateral pore plates, and apparent in borders between incompatible colonies (isocontact plates), and in Chapter 5 of this volume, fusion borders between colonies of Conopeum seurati often contained pore plates similar to the isocontact plates reported by Shapiro. The apparently two-way septa
81 found in fused compatible colonies is a full calcium carbonate dome, essentially a double abannular plate, a very different form to the predominantly annular septa forms seen in immature W. subtorquata colonies, seemingly indicating that they could not function as a fusion pore plate would. However, Chaney (1983) found what he termed ‘fusion septula’ between fused colonies of Thalamoporella californica. These connections superficially resembled the annular pore plates found on lateral walls, but were more irregular in their shape and spacing along the wall (Chaney 1983). If the function of these pores was to act as a two-way connection between colonies, we might expect that pores in young colonies with comparable morphology to function in a similar way. Alternatively, the apparently annular morphology of these septa may simply be a result of the immaturity of the inter- zooid connection, and not represent any functional differences between septa within young zooids and those in more mature colonies.
The patterns of pores and pore plates across colony regions has the potential to be a powerful indicator of the capacity for nutrient transfer within a colony, as evidenced by the preliminary studies carried out on W. subtorquata, M. ellerii and C. seurati colonies. In all cases, the morphology of the septa reflected the apparent function, validating previous histological and morphological studies. No outgoing, abannular pore plates were found at the very edge in any species, but, in lightly calcified species the number of these outgoing pores was very high just behind the growing edge. This appears to indicate a disproportionate level of nutrient transport through the funicular system to this growing edge, a pattern supported by radiolabelling studies by Best and Thorpe (1985) and Miles et al. (1995). In contrast, zooids at more central positions within colonies had reduced capacity to transport nutrients in the direction of the edge, through the existence of fewer outgoing connections. This finding appears consistent with the general pattern of ageing within colonies; central zooids are older, and may have a lower rate of feeding (but see Chapter 2), and thus smaller reserves of energy to distribute to adjacent zooids. In addition, a central position in the colony may also coincide with the onset of reproduction in a zooid, meaning that any energy stores accumulated through feeding might be expected to be retained to fuel the development of eggs, in the case of C. seurati or a brooding embryo, as in W. subtorquata and M. ellerii. Alternative explanations for the
82 discrepancy in pore numbers and sizes across colony regions may arise through simple differences in pore number with zooid maturity. In part 1B of this chapter, very young Watersipora subtorquata colonies generally had low numbers of pores. This pattern might be retained over colony development, so that, as the colony grows, the inner zooids (or the oldest zooids) continue to have low pore numbers, whereas the outer zooids have increased numbers of pores, possibly owing to the higher number of adjacent zooids to which connections need to be formed. Although this might not preclude the possibility that pores can re-form or be closed according to demand, it is a simpler explanation, and warrants further detailed investigation.
While pore patterns provided a fair reflection of the capacity of different colony regions to transport nutrients between zooids, I sought additional information on the maturation of individual pores by comparing the sizes of pores from both the abannular and annular sides of the zooid wall in zooids at different colony positions. While there wasn’t a clear progression in the differences in sizes with increasing zooid age, in older zooids, pores were larger on the incoming face than the outgoing, and in very young zooids, outgoing porous connections were present, and the corresponding incoming connection had yet to form. Analysis of pore size ratios is potentially a powerful tool for investigating ages of individual zooids where the colony has been fragmented or damaged, thus altering the ages of zooids within the colony, and, in tandem with analysis of pore patterns across colony regions, can impart much information on levels of colonial integration.
83 annular abannular face face transverse wall annulus
lateral wall
Figure 1. Outline of a zooid within a colony, showing the relative location of pore chambers (septa) within both transverse and lateral walls in a generalised encrusting cheilostome bryozoan with quincuncial arrangement of zooids. Arrows represent the polarity of pores within the septa, while the large arrow at the top of the diagram represents the direction of the growing edge. Adapted from Chaney (1983).
84
A 500µm
ancestrula az L
T az dz pore plates (septa) within lateral zooid wall
100µm B annular face of septum
abannular face of septum
ancestrula
annulus az
az dz
Figure 2. Light micrograph from the underside of a live colony of Watersipora subtorquata, comprised of three autozooids, showing the location of communication pore plates, or septa, within lateral walls. (A) An overview of the colony, showing the central ancestrula, two fully developed autozooids (az) and one developing zooid (dz). T and L indicate transverse and lateral walls respectively. The location of two pore plates within the lateral wall is circled. (B) A close up view of the pore plates, showing the hemispherical structure of each septum, comprised of the annular and abannular faces, and the heavily calcified annulus. Arrows indicate the direction of nutrient transfer through the septa.
85
A 50µm
B 20µm
Figure 3. Scanning electron micrograph of interior-walled communication pores in Conopeum seurati. (A) Pores in the distal transverse wall of zooid, showing lack of clustering. (B) Pores arranged in pore plates (septa) on the interior lateral wall of zooid. The left cluster is an example of an abannular, or ‘outgoing’ pore plate. Note the convex morphology and the six pores contained within the pore plate. The right cluster is an example of an annular, ‘incoming’ pore plate. Note the concave morphology and the heavily calicified ring (annulus) that partially obscures two of the five pores.
86
A 250µm
B 30µm
Figure 4. Inter-zooid communication plates (septa) in Membranipora membranacea. (A) Overview of frontal colony surface, showing the quincuncial arrangement of zooids. Note the presence of septa on internal lateral walls. (B) Close-up view of pore plates, showing clusters of pores within each plate.
87
A 1000µm
B 250µm
proximal half of zooid distal half of zooid (receiving nutrients) (transmitting nutrients)
C 50µm 50µm
(i) (ii)
Figure 5. Scanning electron microscopy (SEM) analysis in Mucropetraliella ellerii. (A) colony with the frontal removed to reveal the internal walls and pore plates. (B) detail of the lateral wall of one zooid, showing the morphological variation between pore plates in the proximal half of the zooid and those in the distal half of the zooid. (C) details of the two types of pores plates: (i) incoming pore plate, (ii) outgoing pore plate.
88
A 300µm
B 20µm 20µm
(i) (ii)
Figure 6. Communication pores within lateral interior walls of Watersipora subtorquata. (A) Overview of lateral interior wall in a Watersipora colony fragment. Adjacent zooids have been dissected away to reveal the communication pores, clustered in pore plates, on the interior walls of zooids. (B) Detail of communication pore plates; (i) incoming, or annular, pore plate. Note the heavily calcified ring, the annulus, surrounding the six communication pores; (ii) Outgoing, or abannular, pore plate. The function of the pore plate is indicated by the lack of an annulus surrounding the pores. Note the four clearly visible pores within the plate, and the shallow depression near the top of the pore plate, indicating possibly a developing pore, or one that is being refilled with calcium carbonate.
89 A 200µm
B 200µm
C
100µm
Figure 7. Colony outline (A) and communication pores within transverse (B) and lateral (C) walls of the encrusting cheilostome bryozoan Parasmittina delicatula. Arrows indicate the position of specific pores. Note the striking differences in morphology between pores in this species and those in other species examined to date, particularly the absence of any obvious clustering of pores onto pore plates, and the absence of the annular calcium carbonate ring around pores. 90
A 15µm
1
3 2
B 10µm
Figure 8. Examples of pores at each stage of development in two-week-old colonies of Watersipora subtorquata. (A) Pore plate containing pores at all stages of development; numbers indicate the pore’s development stage. (B) Pore plate containing two pores, both at stage 3 of development.
91
Development stage
Stage 1
Stage 2
Stage 3 1.00
0.80 Proportion of pores at each 0.60 stage
0.40
0.20
0.00 -3 –2 –1 0 Distance from edge (zooids)
Figure 9. Proportion of pores at each development stage according to the relative position within the colony in young colonies of Watersipora subtorquata. Stage 1 indicates a shallow depression forming in the colony wall; stage 2 is a partially formed or partially plugged pore, while stage 3 is a fully formed pore.
92 a) 180 160
140
Pore area 120 2 (µm ) 100 80 60 40 20 0
300 b) Development stage
Stage 1
200 Stage 2
Pore area Stage 3 (µm2)
100
0
–4 –3 –2 –1 0 Distance from edge (zooids)
Figure 10. Relative sizes of pores in young colonies of Watersipora subtorquata at a) each stage of development, across colony positions. Stage 1 indicates a shallow depression forming in the colony wall; stage 2 is a partially formed or partially plugged pore, while stage 3 is a fully formed pore. b) sizes of pores according to stage and relative to the growing edge of the colony. Bars show mean values, ± one standard error.
93
A 250µm
proximal half of zooid (receiving distal half of zooid (transmitting nutrients)
colony edge
B 50µm
50µm (i) (ii)
Figure 11. Scanning electron microscopy (SEM) in Watersipora subtorquata. (A) View of lateral wall in an autozooid of Watersipora, showing differences in morphology of pore plates in the proximal and distal regions of the zooid. Note the presence of partial plugs in some pores. (B) Close up views of two types of pore plates: (i) incoming plate, (ii) outgoing plate. Note the lack of an annulus around pore within the outgoing pore plate.
94 5 a) 4 Average number of plates per 3 zooid
2
1 Incoming plates Outgoing plates
0 -5 -4 -3 -2 -1 0 Distance from growing edge (zooids)
35
b) 30
25 Average number 20 of pores per zooid 15
10
5 Incoming pores Outgoing 0 pores -5 -4 -3 -2 -1 0 Distance from growing edge (zooids)
Figure 12. Pore patterns across colony regions in Watersipora subtorquata; (a) numbers of incoming and outgoing pore plates per zooid, according to the zooids position relative to the growing edge, (b) numbers of incoming and outgoing pores per zooid, relative to the growing edge. Data points are mean values, ± one standard error.
95 20000 a)
15000 Average pore size (µm2) 10000
Incoming pores Outgoing pores 5000
0 -4 -3 -2 -1 0 Distance from colony edge (zooids)
12000 b)
10000
Average 8000 pore size 2 (µm ) 6000
4000 Incoming pores Outgoing pores 2000
0
Figure 13. Pore sizes in Watersipora subtorquata; (a) sizes of both incoming and outgoing pores at different positions across colony regions, relative to the growing edge, and (b) overall average sizes of incoming and outgoing pores in W. subtorquata. Bars show mean values, ± one standard error.
96
5 a)
4 Number of plates per zooid 3
2 Incoming pore plates 1 Outgoing pore plates
0 -9 -8 -7 -6 -5 -4 -3 -2 -1 0
16 b)
12 Number of pores per zooid 8
4 Incoming pores Outgoing pores
0 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 Distance from the growing edge (zooids)
Figure 14. Patterns of pore numbers across colony regions in Mucropetraliella ellerii. (a) shows the number of pores per zooid, grouped by pore type, and (b) shows the number of plates per zooid, of both incoming and outgoing types. Data presented are mean values at each point from the edge, ± standard error.
97 100000 a) 80000 Average pore size 2 (µm ) 60000 * no data available for incoming pores at this point
40000 *
20000
0 -6 -5 -4 -3 -2 -1 0 distance from growing edge (zooids)
100000 b)
80000 Pore size (µm2) 60000
40000 Incoming pores
20000 Outgoing pores
0
Figure 15. Sizes of communication pores in Mucropetraliella ellerii: a) sizes of incoming and outgoing pores in zooids at different positions relative to the growing edge. Note that there is no data for incoming pores at one zooid back from the growing edge, due to insufficient numbers of pores suitable for analysis; b) overall differences in sizes between incoming pores and outgoing pores. Bars show mean values, ± standard error.
98 A 250µm
15µm B
Figure 16. SEM analysis in Conopeum seurati. (A) Overview of part of a colony, showing the lack of calcification on the frontal wall, the regular arrangement of zooids, and the beginnings of spine formation at the margins of many zooids. Irregularities in the basal wall are due to the roughening of the plastic substrate prior to settlement. (B) The two pore morphologies with lateral zooid walls in Conopeum: the left side of the diagram, corresponding to the proximal region of a single zooid, shows an abannular or outgoing pore plate, comprised of six communication pores, while the right side of the diagram shows an annular or incoming pore plate, with five pores visible. Note the ring of heavy calcification, or annulus, surrounding the pores.
99
70 a)
60
Number of pores 50 per zooid 40
30 Incoming pores Outgoing pores 20
10 -7 -6 -5 -4 -3 -2 -1 0 Distance from edge (zooids) 600
500 b) Pore area 400 (µm2)
300 Incoming pores 200 Outgoing pores
100
0 -7 -6 -5 -4 -3 -2 -1 0 Distance from edge (zooids)
Figure 17. Numbers (a) and sizes (b) of both incoming and outgoing pores at different distances from the colony edge in Conopeum seurati. Pores were measured on one colony across a single transect, running from three zooids from the ancestrula to one zooid behind the growing edge. Values shown are mean values, ± one standard error.
100
donor zooid
lateral wall “outgoing” pore opening
“incoming” pore opening special cell
receiving zooid
funicular strand
Figure 18. Diagram of a generalised communication pore, showing the close association with strands of the funiculus, and the special cells that plug the pore opening. Note the position of the nuclei within the special cells, on the side of the receiver zooid. While pore development is first initiated on the side of the donor zooid, further development may result in the enlargement of the nucleated lobes of these special cells, resulting in the expansion of the incoming pore opening. Adapted from Mackie (1986).
101
800
700
600 Pore size (µm2) 500 400
300
200 Incoming side 100 Outgoing side
0
Figure 19. Comparison 1. Comparison of pore sizes on opposite sides of the lateral zooid wall at a position of 5 zooid rows distal to the ancestrula and 4 zooid rows proximal to the edge of the colony in Conopeum seurati. Sizes are the relative sizes of the same pores on each side of the wall, averaged across all pores present on that plate. The total number of pores is seven. Bars are mean values, ± one standard error.
102
150
100 Pore size (µm2) Incoming side 50 Outgoing side
0
Figure 20. Comparison 2. Comparison of pore sizes on opposite sides of the transverse zooid wall at a position of 4 zooid rows distal to the ancestrula and 3 zooid rows proximal to the edge of the colony in Conopeum seurati. Sizes are the relative sizes of the same pores on each side of the wall, averaged across all pores present on that plate. The total number of pores is thirty-two. Bars show mean values, ± one standard error.
103
A 10µm 1 3 2
4
8 9 10 5
6 7
B 10µm 1 2 X 3 X 4
10 9 8 5
6 7
Figure 21. Pore plates viewed from both sides of the zooid walls in Conopeum seurati. (A) plate viewed from annular, incoming side of the wall, located in the distal region of zooid 3, at 4 zooids back from the colony edge. (B) same plate viewed from the abannular, outgoing side of the wall, located in the proximal region of zooid 7, at 3 zooids away from the colony edge. Each number corresponds to a single pore, viewed from opposite sides of the wall in the two diagrams. Crosses indicate that the pore was not visible from the opposite side.
104
70
60
50 Pore size 40 (µm2) Incoming side 30 Outgoing side 20
10
0
Figure 22. Comparison 3. Comparison of pore sizes on opposite sides of the lateral zooid wall at a position of 4 zooid rows distal to the ancestrula and 3 zooid rows proximal to the edge of the colony in Conopeum seurati. Sizes are the relative sizes of the same pores on each side of the wall, averaged across all pores present on that plate. Bars show mean values, ± one standard error. The total number of pores is ten.
105 A 10µm 20µm
(i) (ii)
B 50µm 30µm
(i) (ii)
Figure 23. Pore morphologies from opposite sides of the zooid wall in Conopeum seurati. (A) Pore plates in the distal portion of zooid 5, at 2 zooids proximal to the colony edge: (i) outgoing pore plate 1; (ii) outgoing pore plate 2. (B) View from the corresponding side of the adjacent zooid wall, in the proximal region of zooid 8, at 1 zooid proximal to the colony edge; (i) midsection of proximal wall, opposite outgoing pore plate 1 pictured in A(i). Note the slight bulging of the zooid wall, indicating that the pore plate is in early development. However, the characteristic annular calcium carbonate ring is yet to develop. B(ii) region of proximal wall opposite site of outgoing pore plate 2 pictured in A(ii). Again, note the bulging of the zooid wall and the lack of annular formation, indicating it is at a similar stage of development to that in B(i).
106
Table 1. Analysis of covariance of pore sizes in young colonies of Watersipora subtorquata. according to development stage at different positions from the colony edge. Significant effects are highlighted in bold. Squared multiple R = 0.103.
Source of variation df Mean-squares F P Pore development stage 2 20382.759 3.922 0.027 Distance from edge 1 3659.145 0.704 0.406 Residual 45 5196.861
107
Table 2. Analysis of covariance of plate and pore numbers on lateral walls of Watersipora subtorquata colonies. (a) number of pore plates per zooid according to plate type and relative to the growing edge. Squared multiple R = 0.297. (b) number of pores according to type and relative to the distance from the growing edge. Squared multiple R = 0.161. Significant effects are highlighted in bold. a) Number of plates per zooid according to plate type and distance to colony edge Source of variation df Mean-squares F P Plate direction 1 14.727 21.914 <0.001 Distance from colony edge 1 9.422 14.019 <0.001 Residual 85 0.672 b) Number of pores per zooid according to pore type and distance to colony edge Source of variation df Mean-squares F P Pore direction 1 272.544 8.700 0.004 Distance from edge 1 225.263 7.191 0.009 Residual 84 31.326
108
Table 3. Analysis of covariance of pore sizes according to pore direction and the distance from the growing edge in Watersipora subtorquata. Significant effects are highlighted in bold. Squared multiple R = 0.159
Source of variation df Mean-squares F P Pore direction 1 208569000 22.575 <0.001 Distance from edge 1 8890677.9 0.962 0.328 Residual 139 9238900.4
109 Table 4. (a) Analysis of covariance of plate numbers and direction in Mucropetraliella ellerii at different positions from the colony edge. Significant effects are highlighted in bold. Squared multiple R = 0.122. (b) Analysis of covariance of pore numbers and direction in Mucropetraliella ellerii at different positions from the colony edge. Significant effects are highlighted in bold. Squared multiple R = 0.059. a) Number of plates per zooid according to type, relative to the growing edge Source of variation df Mean-squares F P Plate direction 1 4.664 7.348 0.008 Distance from edge 1 6.933 10.922 0.001 Residual 131 0.635 b) Number of pores per zooid according to type, relative to the growing edge Source of variation df Mean-squares F P Pore direction 1 0.119 0.017 0.896 Distance from edge 1 56.857 8.182 0.005 Residual 131 6.949
110
Table 5. Analysis of covariance of pore sizes and directions in Mucropetraliella ellerii at different positions from the colony edge. Significant effects are highlighted in bold. Squared multiple R = 0.103.
Source of variation df Mean-squares F P Pore direction 1 590956000 14.035 <0.001 Distance from edge 1 83309200 1.979 0.162 Residual 129 42105000
111
Table 6. Analysis of covariance of (a) number of pores per zooid according to pore type and relative to the growing edge (squared multiple R = 0.491), and (b) sizes of pores according to type and relative to the distance from the growing edge (squared multiple R = 0.069), on lateral walls of Conopeum seurati. Significant effects are highlighted in bold. a) Number of pores per zooid according to pore type and distance to colony edge Source of variation df Mean-squares F P Pore type 1 748.167 9.323 0.006 Distance to edge 1 880.503 10.973 0.003 Residual 21 80.246 b) Pore sizes according to pore type and distance to the colony edge Source of variation df Mean-squares F P Pore type 1 66125.144 7.010 0.009 Distance to edge 1 44986.596 4.769 0.030 Residual 218 9433.043
112 Chapter 4
The influence of local damage on growth patterns in Watersipora subtorquata
Introduction
Disturbance is common to all environments, and can affect ecosystem functioning through removing species vulnerable to disturbance, or by altering the life histories of individual organisms through physical damage. On marine hard substrata, where colonial organisms such as bryozoans, ascidians and corals often dominate, and space is limited, disturbance can remove individuals and clear space for growth of other colonies or settlement of new recruits (e.g. Ayling 1981; Sebens and Thorne 1985). Disturbance may also act on a more local scale, removing parts of a single colony. Modular organisms are seen as resistant to disturbance that removes part of a colony, since no module is essential for colony survival and thus module removal does not necessarily kill colonies (e.g. Vuorisalo and Tuomi 1986). However, this physical damage and partial colony mortality can still have lasting effects on the life histories of single colonies.
Because regeneration requires energy, damage to a colony may force the re-allocation of resources from functions such as growth and reproduction, to repair and regeneration, thus potentially compromising defensive capabilities. These energy drains subsequent to damage can also have lasting effects on resource stores for future growth and reproduction. In effect, the energetically costly process of regeneration may compete with another costly process, reproduction. Wahle (1983) found that reproductive gorgonians regenerated at a slightly slower rate following damage than non-reproductive colonies and lesion infliction in reef-building corals may decrease growth rates subsequent to damage (Meesters et al. 1997).
Other potentially lasting effects may result from the way damage alters colony structure. Colony damage most obviously decreases colony area, by removing component modules within the colony. Since colony size is closely linked, in many species, to the likelihood
113 of survival, growth rate, and disease (e.g. Hughes and Jackson 1980; Hughes and Connell 1987; Davis 1988; Babcock 1991), this reduction in colony size can often have deleterious effects on subsequent colony life history. Damage also, by extension, decreases the feeding capacity of a colony through removing feeding modules. The loss of resources gained through feeding, coupled with the extra energetic demands of injury regeneration can lead, in many species, to cumulative reductions in fitness, especially when coupled with other stressors, such as loss of symbiotic zooxanthellae in coral bleaching (Meesters and Bak 1993; Baird and Marshall 2002; Fine et al. 2002), or previous fragmentation (Smith and Hughes 1999; Cumming 2002).
Effects of damage are, however, also mediated by the age structure of the component modules within a colony, and the subsequent module maturity and capacity. For example, the removal of a few old, mainly structural modules may not compromise colony fitness to a significant degree, whereas removing healthy, reproductive modules or young developing modules may represent a larger loss of resources and thus slow regeneration (e.g. Wahle 1983; Bone and Keough 2005). Similarly, in modular plants, the type of tissue that is removed has important consequences for the effects of damage (e.g. Honkanen and Haukioja 1994; Senn and Haukioja 1994; Honkanen et al. 1994; Ruohomäki et al. 1997). Due in part to these altered patterns of resource transfer within colonies, damage can have varying consequences for reproductive output and quality. For example, damage or growth obstruction in Membranipora membranacea may induce rapid onset of reproductive maturity (Harvell and Helling 1993), while damage in the arborescent bryozoan Bugula neritina can delay reproduction and lower eventual reproductive output (Bone and Keough 2005). Further, Marshall and Keough (2004) found that damage in Bugula neritina also resulted in a decrease in the size of larvae produced at reproduction, which potentially leads to important carry-over effects, since larval size in this species is closely linked to adult colony size and fitness (Marshall et al. 2003).
The efficiency and rapidity of regeneration is also linked to the size, shape, position and type of injury. These effects are especially important in reef-forming coral species, where
114 living tissue is removed from the underlying skeleton and the responsibility for regeneration lies with modules surrounding the damage. In these taxa, the rate of regeneration may be affected by the type of injury (Hall 1997) the shape of the injury (Oren et al. 1997a) and the numbers of healthy polyps at the perimeter of the injury (Meesters et al. 1997) as well as the injury location. Thus, effects of partial mortality can vary depending on the location of damage, and to ascertain the likely effects of damage to a modular invertebrate, we need to know more about how colony structure is related to module function.
The interaction between modules within a bryozoan colony, and the properties of those modules, determine the ability of the colony as a whole to grow, regenerate, and allocate energy to reproduction and defence. Properties of modules that may be important in determining to what extent they are able to perform functions important to the overall health of the colony include their role within the colony (feeding, reproduction, structural support or defence as dictated by their nature as autozooid, kenozooid or avicularium), their maturity, their condition (i.e. healthy, degenerative, damaged), their history, their position within the colony, and their ability to feed and transport nutrients to other modules. We have seen in preceding chapters that, while modules in a typical unilaminar encrusting bryozoan, such as Watersipora subtorquata, may be quite uniform in size and shape, the ways in which these modules interact to determine a colony’s capacity to grow and reproduce are also highly dependent on a number of the aforementioned properties of individual modules. First, the ability to and tendency for modules to share resources appears to be linked to module age; younger modules may have a higher capacity for nutrient sharing than older modules, for example. Second, modules near the edges of colonies appear to direct their resources towards further growth of this edge in a number of species, including W. subtorquata (Hart 2001; Chapter 2, this volume). In this chapter, I describe two experiments investigating regeneration patterns in W. subtorquata. The first experiment assesses the effects of repeated damage to the growing edges of colonies, on colony growth, zooid survival and overall reproduction, while the second experiment examines the relative importance of different colony regions by measuring changes in
115 colony area following the removal of these regions. Further, I examine the capacity of colonies to regenerate damage both with and against the predominant flow of resources, by measuring regeneration both towards and away from the growing edge.
1. Repeated removal of the growing edge
Reproduction in Watersipora subtorquata is a clearly localised process, and once colonies have matured, at around four weeks (this chapter), a ring of developing embryos is usually evident at around in the middle zone between the ancestrula and the growing edge of the colony (E. K. Bone, personal observation; Hart 2001). More centrally, the older zooids have typically senesced, while at the edge, new growth is occurring. The position of these reproductive zooids, in undamaged colonies, is very consistent, and appears to be a result of zooid maturation. Alternatively, since reproduction occurs around four zooid rows behind the growing edge, this edge may effectively act as a buffer zone for the developing embryos, giving protection from predators and any damage that may occur at the edges of colonies. I sought to test whether the position of reproductive zooids is a programmed process in W. subtorquata, through the repeated removal of zooids at the growing edge.
If maturity and reproductive onset is a programmed process in W. subtorquata, we should see no difference in the position of reproductive zooids within colonies following damage. Reproductive zooids will remain at an equivalent position relative to the ancestrula of the colony following the removal of the edge, and therefore be closer to the edge in damaged colonies than in undamaged colonies. In addition, the expansion of the dead area in the centre of the colony should remain consistent between undamaged and damaged colonies. However, the edge region of a colony, being the site of growth, may be an important resource sink, and damage may have deleterious effects additional to the removal of this buffer zone. In this case, we might expect negative consequences from this damage to include a reduction in growth at colony edges, a reduction in colony-wide reproduction, or changes in the pattern of reproductive onset across a colony. The extra demands placed on remaining zooids may also result in resource depletion within the
116 colony, and possibly in higher levels of zooid death. Such cumulative effects of damage and stress have been in seen in corals (bleaching effects: e.g. Baird and Marshall 2002; Cumming 2002), ascidians (greater effects of predation: Davis 1988) and other bryozoan species (temperature effects: Menon 1972).
Removal of the edge may also alter the patterns of resource translocation patterns within colonies. Without the large resource sink that the growing edge constitutes, nutrients may be relocated elsewhere to offset the effects of this damage, resulting in, for example, the development of defensive spines, or induce early reproduction (Harvell and Grosberg 1988; Harvell 1991, 1992). This then provides an indirect test of the flexibility of the funicular system (Chapter 3). If zooids within the dead central zone are able to regenerate, or if reproduction within this zone exceeds that of undamaged colonies, if may be indicative of a redirection of nutrients back towards this colony centre. Further, these patterns would suggest that these colonies retain the capacity to alter the direction of communication pores within the zooid walls, or are able to form new connections that run opposite to the prevailing flow of nutrients. I explored these possibilities by examining patterns of colony growth and reproduction following repeated damage to the growing edge.
Methods
I collected a number of mature Watersipora subtorquata colonies from Workshops pier in Williamstown, Victoria, and transported them back to the flow-through seawater system at the University of Melbourne. Colonies were kept in a light-tight box for 48 h, after which, on January 15, 2004, they were light-shocked to induce the release of larvae. I constructed artificial substrata from four 240 mm × 240 mm transparent PVC sheets, of 0.38-mm thickness. I roughened the surface of the sheets with sandpaper to facilitate settlement of larvae, and placed them in individual shallow trays of seawater. Upon release, I retrieved each larva with the aid of a plastic pipette, and transferred it to one of the trays. These trays were covered with black garbage bags to prevent any light entering, and thus any modification of settlement behaviour. Once larvae had settled and
117 metamorphosed, after a period of around 48 h, I divided each settlement plate into four quadrants and mapped the position of each settler within these divisions. The four settlement plates were then bolted to the backing plate and deployed at Workshops Pier in Williamstown at a depth of approximately 2 m below the low water mark.
The plates were retrieved after two weeks and brought back to the laboratory, whereupon newly-settled competing organisms and epifauna were removed from the colonies using a pair of fine forceps and a fine paintbrush. Any colonies that appeared to be crowding each other were also removed. This process ensured uninterrupted growth of a selected number of colonies. After a period of four weeks, on February 17, the plates were retrieved from the pier and brought back to the lab, whereupon the health of all mapped colonies on the plates was assessed. I removed any colonies that appeared to have irregular growth or excessive epifauna, and any colonies that were crowded and growing less than a centimetre apart were removed. This process resulted in a total of 113 colonies being chosen for experimentation. Within each quadrant of each plate, colonies were evenly and randomly divided into control and treatment colonies. At this point I measured colony size as the total number of zooids, and noted the number of dead zooids, after which I removed the edge zooids of treatment colonies using a scalpel. I only removed partially developed zooids in this process, and as such the procedure did not alter the number of feeding zooids within the colony. After the damage procedure was completed, the plates were returned to the pier. I repeated this process each week for three more weeks following the initial damage treatment. At each census time, and for each colony, I measured the total number of zooids, the number of dead zooids in the central region, and from the second week onwards, the number of live and dead zooids at the colony edge. I also noted in which week colonies became reproductively active, and counted the number of embryos produced by each colony.
Initially, there were 58 control colonies and 55 colonies in the damage treatment. However, in the final two weeks of the experiment, three control colonies died, a pair of control colonies fused, one control colony fused with one damaged colony, and a triplet formed with three control colonies fusing. At the final census of four weeks, this left a
118 total of 49 control colonies, and 54 colonies in the damage treatment. Missing colonies were excluded from all analyses with the exception of calculations of average rates of growth.
Analysis
I analysed the effects of treatment on most variables using one-way analysis of variance. I analysed final size and the average growth rate of colonies in this fashion, as well as the size and average growth of the central zone of dead zooids. In addition, I analysed the proportion of live zooids over the entire colony, and at the colony edge. The reproductive potential of colonies is often tightly linked to the number of modules within a colony, i.e. colony size. I analysed the number of embryos per zooid at the final census in control and treatment colonies using analysis of variance.
Results
While colonies in both treatments were initially the same size, control colonies increased in size more rapidly than colonies in the damage treatment (Fig. 1) and at the final census
were significantly larger than damaged colonies (F1,102 = 3.934, P<0.0001). Growth of control colonies was therefore higher, and was relatively steady, while damaged colonies did not appear to grow at all in the first week, and showed slow growth for the remainder of the experiment (Fig. 1). The average growth over time was slightly higher in control colonies than in colonies damaged at the edge although this difference was not significant
(F1,102 = 0.627, P = 0.430). When growth was measured as the increase in zooid number proportionate to the previous number of zooids, the average proportionate growth of control colonies was significantly higher than that of colonies with their edge removed
(F1,102 = 72.766, P<0.0001).
The number of dead zooids in the central region of colonies was very similar after experimental manipulation, but in control colonies this number increased rapidly over time, and by the final census was significantly higher than in damaged colonies (Fig. 2,
119 F1,102 = 9.850, P = 0.002). As a proportion of the total zooid numbers in colonies, the numbers of dead central zooids were higher at each census in the damaged colonies, but
not significantly higher at the final census (F1,101 = 2.883, P = 0.093). Over the four weeks of the experiment, there was a change in the ratio of live zooids to dead zooids across the entire colony, and at the final census, control colonies had ratios of live to dead zooids that were significantly higher than the ratios of live to dead zooids in damaged
colonies (F1,101 = 15.654, P<0.001). At the colony edge, differences in the treatments were again strongly evident. The proportion of zooids at the edge that were alive was relatively even between treatments in the second and third weeks, but by the end of the experiment, the proportion of live zooids at the colony edge, while still high in control colonies, had dropped significantly in damaged colonies (Fig. 3). These differences were significant at the final census, with control colonies again having a higher proportion of
live zooids at the colony edge (F1,101 = 42.022, P<0.001).
Colonies became reproductive in the third week, and the final measurement at four weeks was the only recorded measure of reproductive effort. After this time, the colonies began to overgrow each other and fuse in high numbers, so measurements were ceased. At the final census of four weeks, control colonies had significantly higher numbers of embryos than damaged colonies (Fig. 4a, F1,99 = 12.41, P = 0.0006). Embryo production did not appear to change markedly as a function of the ratio of live to dead zooids (Fig. 4b), although some damaged colonies were able to produce relatively more embryos at very small ratios of live to dead zooids. Embryo production per zooid was higher in control colonies (Fig. 4), but one-way analysis of variance showed that this difference was not significant (F1,109 = 0.819, P = 0.367). However, the number of live edge zooids was a reasonable predictor of embryo production in control colonies (Pearson’s correlation coefficient r2 = 0.652), but in damaged colonies, this was not so (Pearson’s correlation coefficient r2 = 0.323) (Fig. 4b).
I also observed that embryo production in damaged colonies appeared to be more random in location, deviating from the expected ring or doughnut patterns apparent in undamaged colonies. In many cases, there were high numbers of dead zooids within colonies,
120 coupled with significant tissue necrosis in damaged colonies, especially at the colony edge. Despite this, embryo production was still achieved in 49 of 54 damaged colonies.
2. Recovery from damage in three patterns in Watersipora subtorquata: an examination of the flexibility of recovery
As we saw in the previous section, damage to the growing edges of Watersipora subtorquata colonies can be detrimental to eventual colony fitness, reflected in the relative proportions of live functioning zooids, and in the eventual reproductive output of colonies. I saw an increase in the number of dead zooids in the internal regions of colonies, and, more strikingly, an increase in the number of dead or dying zooids at the colony edge, in colonies whose edge was sequentially removed over time. The edges of colonies thus appear to be important resource sinks, and their removal compromises colony fitness in a number of ways. Despite the negative impacts of edge removal, there was some evidence that repeatedly removing this resource sink may trigger a re- allocation of resources away from this area. In this experiment, I aimed to further explore the importance of the growing edge as a resource sink, and to investigate the flexibility in internal resource transfer.
I divided colonies of Watersipora subtorquata into three size classes after a period of high settlement onto artificial substrata deployed at St. Kilda pier, Victoria, and manipulated the position of damage within these colonies in three different patterns that removed zooids of varying levels of maturity. I predicted the damage treatments to have mixed effects on colonies according to their size class; on the one hand, small colonies are susceptible to further tissue loss or mortality through predation and less able to recover than large colonies in many modular invertebrates (e.g. Davis 1988; Cumming 2002; reviewed in Hughes 2005). However, analyses of feeding patterns in Chapter 2 and of the funicular system in Chapter 3 also suggest that young colonies may also be more flexible in their recovery responses, and may thus show higher levels of regeneration against the predominant flow of nutrient transfer (i.e. backwards). Adding to the complexity, while large colonies have potentially better regeneration capabilities, due to
121 higher numbers of feeding zooids that may contribute to the process, with increasing maturity, the central zones of colonies begin to senesce. This means that any damage to the central region is likely to not be regenerated efficiently, while growth can continue at the colony edge.
Methods
I deployed an array of artificial substrata, made from 4 sheets of 240 mm × 240 mm 0.38- mm thick clear PVC sheeting, attached to a single 600 mm × 600 mm grey PVC backing plate with stainless steel bolts, from St. Kilda pier, Victoria, in late January, 2003. The array was hung at a depth of approximately 1 m below the low water mark, and the surface of the sheeting was roughened by sanding to facilitate larval settlement. The plates were regularly checked for settlement of Watersipora subtorquata recruits, and cleared of any other competing organisms. After a period of high settlement, on February 6, I chose a number of colonies of varying sizes for experimentation, and divided these into three groups on the basis of their size. After the experimental manipulation of removing colony tissue in three different patterns (described below), small colonies were an average size of 191.898 ± 10.28 (s.e.) mm2, medium colonies were an average size of 368.921 ± 28.812 (s.e.) mm2, and large colonies were an average size of 653.107 ± 35.045 (s.e.) mm2. At this stage, I measured the sizes of colonies by carefully placing a thin transparency over the colonies and tracing the outlines of each colony using a permanent marker. I scanned the resulting images and measured sizes using SigmaScan 2.0.
On February 7, I split the colonies from each size class into groups, and manipulated the colonies in three different ways, as shown in Figure 5. The first treatment, edge removal, removed four zooid rows from the colony edge on one side of the colony. Treatment 2 removed four zooid rows of tissue from a position of four zooid rows behind the growing edge on one side of the colony, while treatment 3 involved removing the central part of colonies on one colony side at a distance of eight zooid rows back from the colony edge, or in the case of smaller colonies, four zooid rows from the edge. I manipulated colonies
122 in this way in order to investigate the relative capacities of colonies at different sizes to regenerate lost tissue both with and against the predominant direction of growth. All colonies were monitored since settlement, and cleared of any competing organisms, so it could be assumed that no colony had suffered any other damage that would alter the age structure of the component modules. As a result, small colonies were younger than larger colonies, and the relative maturity of central parts of colonies increased with colony size.
Over a period of 17 days, I measured the sizes of colonies across the three treatments and three size classes at three intervals; 5 days (t1), 10 days (t2) and 17 days (t3). I analysed size and growth of colonies over time using repeated measures analysis of variance, with time as a repeated factor, and treatment and size class as fixed factors. While I initially included plate as a blocking factor, there was no effect of plate, and I excluded this factor from the final analysis. This was a test of the growth capacities of colonies following damage. I predicted that colonies with more of their growing region removed (treatment 1) would perform worse than those where the central region was removed (treatment 3), as this would normally contribute little in the way of nutrient transport to the edge regions (Chapter 2). I also predicted that small colonies would have a proportionately lower capacity to re-grow following damage than large colonies.
After 17 days, I removed a sample of colonies from each size class at 23, 28 and 33 days and returned them to the lab for specific analysis of the regeneration patterns within colonies in treatments 2 and 3. After taking digital images of the colonies, I measured total colony size using SigmaScan 2.0, and noted the number of zooids that had regenerated forwards (towards the growing edge) and backwards (towards the colony centre). I predicted that, since the bulk of nutrient transfer is likely to be towards the growing colony edge, backward regeneration should be lower than regeneration in a forward direction. In small colonies, however, zooids may be immature and more capable of making new inter-zooid connections to enable backwards regeneration. Large colonies, due to increasing maturity of zooids and the onset of zooid senescence in the central part of colonies, may not show regeneration in either direction, as these zooids may be dead and incapable of regeneration. Since the sequential removal of colonies reduced the
123 sample size in each combination of treatment and size class at each removal period, I pooled colonies of different size classes and used actual colony size, measured as millimetres squared, rather than size class, in the analysis. The effect of treatment on the amount of forward and backwards regeneration was analysed using analysis of covariance, with colony size in mm2 as a covariate (Quinn and Keough 2002). In both these analyses, colonies in treatment 1 were excluded, as regeneration was only possible at the colony edges, and I was interested in the relative strengths of regeneration both forward of and behind the ancestrula.
Results
In the first few days after the initial damage, most colonies suffered a decrease in colony size (Fig. 6a–c), with large colonies in all treatments showing the sharpest rate of decrease (Fig. 6c), and medium colonies in treatments 1 and 3 the next fastest in terms of colony size reduction (Fig. 6b). Small colonies, in contrast, showed no substantial reduction in colony size in the first time period (Fig. 6a). After the initial decrease in the first five days, all colonies appeared to grow at a reasonably steady rate for the remainder of the experimental period (Fig. 6), and there were no significant effects of any factor on growth across treatments over time (Table 2).
Regeneration in either direction did not appear to be affected by the damage treatment. Fig. 7 shows examples of regeneration in the forwards and backwards direction, in colonies between 10 and 17 days after damage. The amount of regeneration in the direction of the edge (forwards) was not predicted by either the treatment, or colony area (Fig. 8, Table 3). Similarly, the amount of regeneration away from the predominant direction of growth (backwards) was not predicted by treatment. However, there was a significant effect of colony size on backwards regeneration, with larger colonies tending to regenerate more tissue away from the edge, regardless of treatment (Fig. 9, Table 4).
Discussion
124 Edge damage
Repeatedly damaging the edges of W. subtorquata colonies had significant impacts on their subsequent growth, their reproductive rates, and their ability to add new modules at the colony edge. Colonies whose edge had been removed at weekly intervals were smaller than undamaged colonies, had lower growth rates and higher proportions of dead zooids, both across the whole colony, and more importantly, at the colony margins. Since growth of colonies is by the addition of new modules, I would expect this deterioration of the colony edge to have profound negative impacts on the ability of colonies to re-grow and eventually reach a size where colony reproduction is comparable to that of undamaged colonies. While one damage event may produce a delay in colony recovery (e.g. Bone and Keough 2005), it seems here that repeated damage is having a cumulative negative effect on colony fitness. Damage in modular invertebrates can cause a re- allocation of resources towards the damaged area, with the site of injuries becoming new resource sinks (e.g. Harvell and Helling 1993; Oren et al. 1997b; Oren et al. 1998). It follows that repeated damage might sequentially create and remove those resource sinks, effectively compounding the effects of damage.
In many cases, necrosis of colony tissue at colony margins was an indicator of the deterioration of that edge, and the lack of investment in new growth at the perimeter. With the edge coming under repeated damage, we may expect the colony to re-allocate resources to other regions to compensate for this damage. These responses have been noted in a number of species, with Harvell and Helling (1993) providing a particularly neat example. Membranipora membranacea colonies that were damaged on one side grew proportionately faster on the other side to compensate. In a similar vein, colonies where growth was obstructed on one side reproduced earlier than those whose growth was uninterrupted, suggesting that the cost of persisting to allocate nutrients to growth outweighed the benefits of continued growth to reach a larger size, where embryo production is maximised. This response does, however, rely on a rapid natural growth rate due to low levels of investment in zooid development. Therefore, it is most likely to occur in species with simple, monomorphic zooids with little skeletal ornamentation, of
125 which M. membranacea is an example.
In the current experiment, some evidence for the re-allocation of nutrients away from the edge was found, with the aforementioned necrosis of the edge, and in the patterns of zooid death in colony centres. In the centres of colonies, the number of dead zooids increased at a greater rate in control colonies than in damaged colonies, and these control colonies showed a higher average growth rate when measured as a proportion of the total number of zooids within a colony. This suggests that damaged colonies may be able to direct nutrients back into the central areas, causing slower deterioration, or even regeneration, of the central zone. It also follows that zooid ontogeny, while appearing to be under endogenous control, can be altered by the schedule of resource allocation within colonies. Regeneration of injuries prior to resumption of normal growth has been seen in a number of modular species (e.g. Tardent 1963; Bak 1983; Wahle 1983; Meesters et al. 1994). Similarly, while embryo production was reduced in colonies that were repeatedly damaged, damaged colonies also appeared to be able to reproduce even when a high proportion of zooids within the colony were dead. This suggests that the additional drain of resources by removal of the edge may cause a shift in resource allocation towards reproduction, despite the small sizes of colonies (e.g. Kojis and Quinn 1984). I removed the growing edge completely every week, effectively removing the entire resource sink; more subtle effects on growth, reproduction may be apparent if the colony remained intact, but energy re-allocation was enforced in an alternative manner. Obstruction of growth on one side of the colony, for example, can cause a shift in energy allocation towards the opposite side (Harvell and Helling 1993). However, the effectiveness of this method may be limited in W. subtorquata, as the flexible growth patterns of this species often allow colonies to overgrow adjacent objects.
Regeneration patterns
In the week immediately following treatment, most colonies shrank, losing tissue additional to that removed in the damage treatment. Initial reductions in size were greatest in large colonies, suggesting that they had lower powers of recovery than smaller
126 colonies; this was contrary to predictions that small colonies would be least likely to be able to recover rapidly from damage, due to fewer component modules. However, following this initial decline in colony size, the growth trajectories of all colonies were similar. The positions in which colonies were able to regenerate differed according to treatment, with colonies in the first treatment, where part of the edge was removed, arguably best equipped to recover from damage. Colonies that had been damaged both in an arc behind the edge, and with a wedge of tissue removed towards the centre of the colony, had intact edges, but were also able to, in some cases, form new tissue into the gaps created by the damage process.
The number of zooids developed due to regeneration forward of the inflicted damage appeared to be reasonably linked to the sizes of individual colonies; larger colonies tended to have a higher level of forward regeneration than medium and small colonies. Forward regeneration was also commoner in the second treatment rather than the third treatment, showing that younger zooids near the edge were more able to invest resources in regeneration than those in the older colony centre. In contrast, backwards regeneration was achieved in colonies in both the second and third treatments, and was highly dependent on individual colony size. In encrusting bryozoans, the predominant flow of nutrients between zooids appears to be in the direction of the growing edge (e.g. Best and Thorpe 1985; Miles et al. 1995). However, the incidence of regeneration away from the edge in W. subtorquata indicates that this process may be flexible and responsive to changes in the structure and internal organisation of the colony. Thus, the capacity for regeneration, and particularly for development of new zooids against the predominant direction of growth, is retained across a range of colony ages.
In summary, damage to the growing regions of colonies appears to have a profound negative impact on pre-reproductive W. subtorquata colonies, reducing both growth and reproductive output significantly, and thus imparting severe negative effects on colony fitness. In larger colonies, however, the potential to regenerate lost tissue is enhanced, and some redirection of resources away from the colony edge is seen through the production of new zooids in the colony centre.
127
250
200
Colony size 150 (number of zooids) 100
Control 50 Damaged
0 123 4
Time (weeks)
Figure 1. Colony size over time in control (circles) and damaged (triangles) colonies of Watersipora subtorquata, over the 4-week experiment. Data points are mean values, and error bars represent ± one standard error.
128
40
35
30 Number of dead zooids 25 in colony centre 20
15 Control
10 Damaged
5
0 1 2 3 4 Time (weeks)
Figure 2. Increase in the number of dead zooids in the central region of control (circles) and damaged (triangles) Watersipora subtorquata colonies over four weeks. Data points are mean values, ± one standard error.
129
1 e g 0.9 ed y
0.8
0.7
0.6 Control Damaged
ortion live zooids at colon at ortion live zooids 0.5 p Pro 0.4 234
Time (weeks)
Figure 3. The proportion of live zooids at the colony edge over time in control (circles) and damaged (triangles) Watersipora subtorquata colonies over four weeks. Data points are mean values, ± one standard error.
130 a)
0.14
Number of 0.12 embryos per zooid at the 0.1 final census 0.08 Control 0.06 Damaged
0.04
0.02
0
b) 80
70
60
50 Number of embryos at 40 final census 30
20 Treatment 10 Control Damaged 0 0 2 4 6 8 10 12
Ratio of live to dead zooids at the final census
Figure 4. Embryo production in control and damaged Watersipora subtorquata colonies. (a) the number of embryos at the final census of control and damaged colonies. Bars show mean values, ± one standard error, and (b) the number of embryos in control (circles) and damaged (crosses) colonies as a function of the proportion of live zooids to dead zooids at the final census.
131
Treatment 1.
Damage to colony edge
Treatment 2.
Damage behind colony edge
Treatment 3
Damage to colony centre
Figure 5. Treatments in the Watersipora subtorquata regeneration experiment. Shaded areas represent colony portions removed at the first census period. In larger colonies, treatment 1 removed the first 4 zooid rows of the colony, treatment 2 removed rows 4–8, and treatment 3 removed the central region behind the first 8 rows. In smaller colonies, portions were scaled to individual colony size, so that an equivalent proportion of the colony was removed.
132 c) Large a) Small b) Medium colonies colonies colonies 1000 1000 1000
800 800 800
Colony 600 size 600 600 (mm2) 400 400 400
200 200 200
0 0 0 0 5 10 17 0 5 10 17 0 5 10 17
Days after damage Treatments 1 – Damage to colony edge 2 – Damage behind colony edge 3 – Damage to colony centre
Figure 6. Sizes of Watersipora subtorquata colonies over seventeen days following damage in three different treatments. Small (a), medium (b), and large(c) colonies are shown for each of three damage treatments. Data points shown are mean values, ± one standard error.
a)
b)
Figure 7. Examples of (a) forward regeneration and (b) backward regeneration in damaged colonies of Watersipora subtorquata. Areas of regeneration are circled in each figure. The colony in (a) was damaged according to treatment 2, whereas the colony in (b) was damaged according to treatment 3 (see Fig. 6 for descriptions). Note the differences in colour between the bright orange of new growth (both at regeneration sites, and around the perimeter of colonies) and the grey to black of degenerated, dead zooids.
134
80
70 a)
60
Forward 50 regeneration (zooids) 40
30
20 Treatment 10 2 0 3 SML Colony size class
80 b) 70
Forward 60 regeneration (zooids) 50 40
30
20 Treatment 10 2 0 3 0 500 1000 1500 2000 Colony size (mm2)
Figure 8. Regeneration forwards towards the edge in damaged colonies of Watersipora subtorquata. (a) the number of zooids regenerated in a forwards direction according to the colony size class for both treatments, and (b) the number of zooids regenerated in a forwards direction according to the actual size of the colony, in both treatments.
135
70 a) 60
50 Backward regeneration (zooids) 40
30 Treatment 2 20 3
10
0 S M L colony size class
70 b)
60
Backwards 50 regeneration (zooids) 40
30 Treatment
20 2 3 10
0 0 500 1000 1500 2000 Colony area (mm2)
Figure 9. Regeneration away from the growing edge (backwards regeneration) in damaged Watersipora subtorquata colonies. (a) the number of zooids regenerated in a backwards direction according to colony size class; (b) the number of zooids regenerated in a backwards direction according to actual colony size, in both damage treatments that removed internal zooids.
136
Table 1. Analysis of covariance on embryo production in Watersipora subtorquata. Treatment is a fixed factor, and colony size is a covariate. Squared multiple R = 0.357. Significant effects are highlighted in bold.
Source of variation df MS F P
Treatment 1 101.088 1.156 0.285
Colony size 1 2761.781 31.592 <0.001
Residual 93 87.417
137
Table 2. Repeated measures analysis of variance for colony growth over time, relative to colony size, in three groups of damaged colonies of Watersipora subtorquata. Time is a repeated factor, treatment and size are fixed factors, and plate is a fixed blocking factor. Significant effects are highlighted in bold.
Source of variation df MS F P Between subjects Size 2 50592.677 5.584 0.007 Treatment 1 131630.092 14.529 <0.001 Size*treatment 2 6679.529 0.737 0.485 Residual 39 9059.919 Within subjects Time 3 73884.668 29.380 <0.001 Time*size 6 18124.232 7.207 <0.001 Time*treatment 3 2960.753 1.177 0.321 Time*size*treatment 6 295.689 0.118 0.994 Residual 117 2514.787 0.118 0.994
138
Table 3. Analysis of covariance for the amount of regeneration in a forwards direction in damaged colonies of Watersipora subtorquata. Treatment is a fixed factor, and colony size in mm2 is a covariate. Squared multiple R: = 0.101. Significant effects are highlighted in bold.
Source of variation df MS F P Treatment 1 573.759 1.697 0.203 Colony size 1 472.438 1.397 0.247 Residual 28 338.169
139
Table 4. Analysis of covariance for the amount of backwards regeneration in damaged colonies of Watersipora subtorquata. Treatment is a fixed factor, and colony size in mm2 is a covariate. Squared multiple R = 0.282. Significant effects are highlighted in bold.
Source of variation df MS F P Treatment 1 158.010 0.510 0.482 Colony size 1 2831.345 9.132 0.006 Residual 28 310.048
140 Chapter 5
Colony growth and damage recovery in the presence of conspecific colonies
Introduction
Space is a vital resource on marine hard substrata, and the limitation of this space promotes high levels of competition between organisms within a sessile assemblage. Modular and colonial forms often dominate in these environments due to their theoretically indeterminate growth rates and their often flexible growth trajectories and morphologies (e.g. Jackson 1977; 1979b; 1985). Encrusting and runner-like growth forms are particularly able to adapt to changes in the amount, type and shape of space available, by growing into that space. Intense competition for space in sessile marine assemblages means that the likelihood of contact between individual colonies is extremely high. In encounters between colonies of different species, competitive and defensive abilities tend to be the primary determinants of the outcomes. Possible interactions include the overgrowth of one colony by another, the cessation of growth by one or both species at the inter-colony border (essentially a stand-off between the two colonies), or the retreat of one colony and the advance of the superior competitor. These competitive abilities tend to operate as hierarchies; for example, species A dominates B, which dominates C, and so on, and are highly influential in shaping assemblage structure in marine communities. Such strict hierarchies have been observed in coral communities (e.g. Connell 1976) and intertidal assemblages (e.g. Dayton 1971) and appear to be the most likely determinant of competitive outcomes in marine assemblages on hard substrata in both shallow (e.g. Quinn 1982) and subtidal environments (e.g. Osman 1977).
Buss and Jackson (1979; Jackson 1979a), however, described the interruption of these competitive hierarchies in cryptic coral reef communities, and the formation instead of intransitive competitive relationships. In these interactions, for example, species A may out-compete species B, which out-competes species C, but species C in turn out- competes A. Such competitive networks or loops may thus promote the co-existence of a
141 number of competitively dominant species, and so maintain species diversity in communities. A strict competitive hierarchy, on the other hand, would promote the outright dominance of a few superior competitors, and lower diversity in the absence of other modifying factors such as predation or disturbance. In addition, interactions between sessile organisms may involve other factors, with Day (1977) showing that competitive outcomes may depend on the relative sizes of colonies and Jackson (1979b) showing that the angle of encounter may be important in bryozoan interactions. The size of patches of free space may also be crucial to development of assemblages, interacting with the capacity for vegetative growth to influence competitive outcomes on hard substrata (Kay and Keough 1981).
Encounters between colonies on marine hard substrata are not limited to those between different species; intraspecific interactions are very common, and may be facilitated by a number of mechanisms operating before, during and after settlement and assemblage development. Prior to settlement, larvae may aggregate due to kin-recognition systems, and in some species, have been shown to settle preferentially near conspecific colonies (e.g. Keough 1984; Grosberg and Quinn 1986; Craig 1995). Following aggregative settlement, colonies may experience increased intraspecific competition leading to higher rates of mortality. Conversely, colonies within aggregations may actually survive better than isolated colonies (e.g. Keough 1984, 1986). The combination of aggregative settlement and increased survivorship may increase the likelihood of monoculture formation, and may be maintained through superior competitive dominance of that species, or the dispersal abilities of larvae and possible subsequent aggregative settlement.
Encounters between conspecific modular colonies may have a number of different outcomes. Overgrowth of one colony by another may still occur, and Ivker (1972) and McFadden (1986) have described overgrowth interactions to be governed by differences in genotypes between colonies, where the dominant genotype overgrows the inferior genotype. Differences in size may instead result in aggressive interactions such as the development of offensive stolons in large colonies of the anascan bryozoan
142 Membranipora membranacea (Harvell and Padilla 1990). In monocultures of species where overgrowth is not apparent, and abutting colonies instead cease growth along their contact margins, the effects of such high-density living may be more subtle. A particularly prominent example of a colonial invertebrate which is often found in monocultures is the encrusting bryozoan Membranipora membranacea, an epibiont on blades of the giant kelp Macrocystis pyrifera in stands off the west coast of the USA. In these monocultures, higher population density reduces maximum colony size, but lowers the size and age at first reproduction (Harvell et al. 1990). High density conditions may also lead to interference competition, for example between feeding lophophores of adjacent bryozoan colonies, decreasing feeding efficiency across the monoculture (Okamura 1985). Intraspecific competition can also reduce growth rates and reproduction in branching corals, and also cause abnormal growth trajectories (Rinkevich and Loya 1985). Since effects of high population density are often very important to population demographics, and given the variation in their effects across modular taxa, continuing discussion of the consequences of monoculture living is constructive in understanding population dynamics in modular organisms.
In encounters between colony pairs, we may see the cessation of growth at the inter- colony margin, or fusion to form a single colony. The mechanisms promoting and maintaining fusion in marine invertebrates have been widely studied, with the histocompatibility and kin recognition systems of colonial tunicates being particularly well understood. Kin recognition in the colonial ascidian Botryllus schlosseri operates to both increase aggregative settlement of larvae, and the formation of chimeras by the fusion of compatible individuals (Grosberg and Quinn 1986). In Botryllus, the capacity for fusion or rejection is dictated by a single highly polymorphic locus with co- dominantly expressed alleles. If colonies share one or more alleles at this locus, they may fuse. A second allorecognition mechanism is employed following this initial fusion, leading to the resorption or morphological elimination. Thus, a successfully fused individual is not composed of competing genotypes, but is a fully integrated unit with a single dominant genotype (Buss 1982; Rinkevich and Weissman 1992). Fusion is therefore more to be initiated and maintained colonies with a high level of genetic
143 relatedness, a pattern found in many other marine invertebrates (e.g. Craig 1995).
Grosberg and Quinn (1986) identified four main ways in which chimera formation is of benefit to the colonies involved; genetic variability, developmental synergism, mate location and size-specific ecological processes. Fusion between two or more related individuals allows a colony to increase in size rapidly, and, since many demographic processes in sessile marine invertebrates are closely related to size (e.g. Jackson 1985; Hughes and Connell 1987; Yoshioka 1994), fusion may enhance survivorship, competitive abilities and capacity for further growth, while reproductive capacity may be enhanced through reducing the age at first reproduction (Harvell el al. 1990). Fusion may therefore be adaptive in small colonies.
Small encrusting bryozoan colonies appear to be particularly vulnerable to factors limiting growth, and the threat of overgrowth by competing neighbouring organisms, fouling and predation is high (e.g. Craig 1995). This may be due to the immaturity of the component modules, and thus potentially underdeveloped defence system, or may be a result of the inability of colonies to allocate any resources to the maintenance of the colony's position on the substratum and thus to defence of the colony. In Watersipora subtorquata, for example, growth in small colonies appears fairly high relative to the numbers of feeding zooids. Colony size, as we have seen, is such an important parameter that growth of a colony in the early stages appears to take precedence over any sort of defence mechanism. It follows that, when a colony is small, it must allocate a large proportion of its resources to the formation of new zooids to reach a size sufficient to be able to resist sources of mortality such as overgrowth and predation (e.g. Yoshioka 1994). For instance, the ratio of W. subtorquata colony area composed of feeding zooids (those that are capable of contributing resources to the further growth of the colony) to non- feeding area (developing zooids or extra-zooidal space; still developing and reliant on feeding zooids) approaches 1 in some small colonies comprising 1–3 zooids (Chapter 2). It follows that growth in small colonies constitutes a large drain on their resources, and as such they are especially vulnerable to damaging processes until they reach a size and a feeding to non-feeding ratio where they have the capacity to divert superfluous resources
144 to defence. Fusion in small colonies thus may be an adaptive response to the crowding of colonies on a substratum.
Despite the numerous theoretical advantages to fusion and chimera formation, few studies have confirmed fusion as beneficial, although Foster and colleagues (2002) found increased mobility in larger, fused slime mould slugs, but Maldonado (1998) found no evidence of improved survival in chimeric sponges, and Rinkevich and Weissman (1992) found no improvement in survivorship, growth rates or reproductive onset in Botryllus chimeras. If fusion in modular invertebrates is adaptive, and kin recognition systems sustain aggregations of colonies and promote contact between colonies, we might expect growth trajectories of colonies to be positively influenced by the proximity of a compatible conspecific colony, and negatively influenced by the presence of an incompatible conspecific colony.
The three experiments I describe in this chapter offered an opportunity to conduct some preliminary investigations into the relationship between the likelihood of fusion and changes in colony form in an encrusting bryozoan, and to examine the potential of colonies to recover from damage whilst in the crowded conditions of a monoculture. In the first experiment, changes in growth patterns are examined in closely paired Watersipora subtorquata colonies from different areas within the Santa Cruz Marina in Santa Cruz, California. The second experiment looks at similar patterns of growth in paired W. subtorquata colonies of different sizes from Williamstown, Victoria. These two experiments aimed to investigate the effects of presumed colony relatedness and colony size respectively on growth patterns and fusion initiation. In the third experiment, I examined a plate on which Conopeum seurati colonies had naturally settled to form a monoculture, and evaluated the ability of colonies to recover from damage caused by barnacle settlement according to colony size. Colony size is often an important indicator of survival, growth, recovery and reproductive potential in modular organisms, and I investigated whether this discrepancy is retained in crowded conditions.
1. Fusion in Watersipora subtorquata colonies at two locations
145
A. Colony fusion at Santa Cruz, California: effects of apparent relatedness
Aggregative settlement of Botryllus larvae is regulated by the same kin recognition system as that which regulates fusion in conspecific individuals (Grosberg and Quinn 1986), while larvae of the bryozoan Bugula neritina settle in aggregations when in the presence of sibling larvae (Keough 1984). If these processes are instrumental in determining the likelihood of aggregative settlement, they may also modify growth patterns of colonies after settlement to increase or decrease the likelihood of contact and eventual fusion. In this experiment, I manipulated colonies to pair them with colonies derived from either the same maternal colony, from different colonies in the same location, or from different locations, and examined their growth patterns. While I did not definitively test for genetic relatedness, I assumed that colonies from different locations were less likely to be related than colonies from the same location (under the same floating pontoon) or from the same maternal colony.
Methods
I conducted this study within the Santa Cruz Marina, Santa Cruz, California. Santa Cruz is located at the northern end of Monterey Bay. Fig. 1 shows the location of the marina relative to the shore, and the positions of the two collection locations. I completed all laboratory work within the Long Marine Laboratory, University of California, Santa Cruz.
Collection of parental colonies. Mature colonies of Watersipora subtorquata were located growing on the undersides of floating pontoons at the Santa Cruz Marina. The colonies were large and healthy, and were three-dimensional bilaminate and foliose in colony form. Several colonies were collected at each of two locations in the marina; one close to where the mouth of the marina fed out into Monterey Bay (henceforth referred to as the ‘near’ location), and one at the very far end of the marina towards the land (‘far’). Fig. 1 shows the location of the two collection locations. The large colonies
146 were transported separately in large snap-lock bags filled with local seawater, within insulated containers, back to the laboratory.
Water flow into the marina from the Bay was high at the ‘near’ end of the marina, whilst at the ‘far’ end the water was fairly static and movement was predominantly from small changes in the tidal flux and from passing boats. It was therefore anticipated that water transfer, and therefore larval supply, between the two locations in the marina, would be minimal. In addition, the arrangement of pontoons within the marina, in two rows with a common channel for vessel thoroughfare between the rows, would appear to be a limiting factor in the supply of larvae between locations. The lecithotrophic larvae of W. subtorquata are active swimmers, and to some extent can ‘choose’ a suitable settlement site. While it is theoretically possible that a larva could, upon release, swim from, for example, the ‘far’ site, between the pontoons into the main channel branch, up this branch, and into the smaller branch where the ‘near’ site is located (see Fig. 1), it seems much more feasible that, even notwithstanding the possibility of aggregative settlement (e.g. Keough 1984), they would settle at an earlier time, given the number of suitable settlement sites between these two locations in the form of pontoons,. With these issues in mind, I predicted that the two locations would have minimal mixing of larvae over time. Therefore, I hypothesised that the genetic relatedness of larvae from colonies between locations was likely to be lower on average than those larvae obtained from colonies within each location, which in turn was lower than those obtained from the same maternal colony.
Collection of larvae. In the laboratory, I placed the colonies from each location in separate covered plastic tubs within a flow-through seawater table, to block out any light to the colonies. They were kept in these covered tubs for approximately 48 hours. After this amount of time, they were removed from the seawater table and placed in separate plastic tubs (‘near’ and ‘far’) filled with seawater from the flow-through system underneath a bank of halogen lights to induce larval release. An additional two large colonies from each location were kept in separate individual tubs. Larvae that were released from all tubs were retrieved with separate pipettes and released into four
147 additional shallow containers filled with seawater; two for each general location, and two for each colony within each location. These containers also held a number of artificial substrata, which were 100 mm × 100 mm sheets of 0.375-mm thick clear PVC plastic.
Inducing larvae to settle to these novel substrata proved tricky, however. W. subtorquata larvae are initially photo-positive, and swim towards the surface. Due to this tendency, settlement onto the artificial substrata, which were by necessity on the bottom of the containers, was fairly low, with most larvae settling onto the sides of the containers near the surface. To attempt to rectify this problem, the containers were covered with black plastic garbage bags to block out light. Another tactic was to float the inverted substrata on the surface of the water within the tub, so that larvae which swam towards the surface would encounter the plastic and settle. Neither technique proved particularly successful.
Although many larvae were released from maternal colonies, only a small number from each location were settled onto the required substratum. The larvae were allowed to settle within the tubs for a period of 24 h, after which the settlement sheets were attached to larger backing plates using stainless steel bolts and returned to the field. I marked the position of each settler on the settlement plate using a graphite pencil. Each plate was returned to its location of origin; settlement plates containing settlers released by ‘near’ colonies were replaced at the near location, while those containing settlers released by ‘far’ colonies were replaced at the far location. The backing plates were hung by ropes at a depth of 1.5 m from the low water mark at each of the locations.
Colony pairing. All colonies were left out in the field and checked periodically for growth over four weeks. Although growth of the colonies was lower than expected, due to time constraints (i.e. my imminent departure from California), they were removed from the field on October 5 and rearranged into pairs. All colonies had been out in the field for the same amount of time, to account for possible effects of age. In addition, pairs were formed between colonies of similar size, where possible, to exclude any effects of size. Combinations of colony pairs were:
148 1. Same maternal colony 2. Maternal colonies from same collection locations 3. Maternal colonies from different collection locations
A total of 15 colony pairs were formed: 6 in combination 1, 6 in combination 2, and 5 in combination 3. I hypothesised that colony pairs in combination 1 would have a higher likelihood of fusion than those in combination 2, due to a greater likelihood of relatedness, and that those in combination 3, from opposite sides of the marina, would have the lowest likelihood of fusion. Therefore, I predicted that colony pairs in combination 1 would be more likely to exhibit growth patterns consistent with fusion attempts, while colonies in paired combinations 2 and 3 would show growth patterns that did not differ from normal, circular growth.
Each colony was isolated by carefully cutting the PVC sheet around the colony. Colonies thus isolated on small pieces of sheet were arranged in pairs on glass slides, with the distal edges of each colony (those forward of the ancestrula) placed together as close as possible (less than 2 mm) so that time to encounter was minimised, and fastened to the slides using silicone sealant. The slides were placed in a plastic slide box with the walls cut out, and retained within the flow-through seawater system for observation. The slides were kept clean by brushing off accumulated silt and algae every day with a fine paintbrush. I checked the colony pairs three times, at 8, 13 and 15 days after pairing. On these days, the colonies were analysed under a dissecting microscope, where the total number of zooids, number of dead zooids, amount and direction of new growth and fusion status were recorded. On the 2nd and 3rd census dates, additional information was able to be gathered through taking digital photographs of each colony pair using a Motic digital camera mounted on the dissecting microscope. The subsequent digital images were analysed using Image J software. Due to a calibration error, size is recorded and analysed as the standardised number of pixels, rather than mm2. Most analyses were performed on data obtained at 13 and 15 days after pairing, with the exception of zooid number over time, when data from 8 days after pairing are also used.
149 Owing to time constraints, colonies were not analysed for skeletal evidence of fusion in this experiment. However, it was noted whether the presence of a close neighbour influenced the growth patterns of colonies within pairs. In this analysis, the degree of elongation of the colony was recorded as a simple length-width ratio. The maximum length of the colony was measured from the base of the ancestrula to the edge closest to contact with the paired colony, while the width measurement was made at the widest point perpendicular to the length measurement. The resulting length-width ratios of colonies were compared across pairing combinations, with the prediction that colonies that were potentially closely related would have higher length-width ratios than those that were not, due to a predicted tendency for these colonies to actively seek fusion with the paired neighbour. A length:width ratio greater than 1 would indicate a tendency away from circularity and towards a more ellipsoid shape, while a length:width ratio of approximately 1 would indicate a more typical circular pattern of growth. In colonies that are not related, I predicted that the action on contact would be active rejection and pre-contact growth patterns would therefore be directed away from the paired neighbour. I therefore recorded the predominant growth pattern of colonies as:
F: growth predominantly forward of the ancestrula, towards paired neighbour Interpreted as actively seeking contact P: normal growth around colony perimeter Interpreted as a neutral reaction to the paired neighbour B: growth predominantly backward of the ancestrula This type of growth is uncommon in small colonies, but if noted, would indicate an active avoidance of contact L: growth predominantly at either side of the ancestrula of the colony, excluding direct forward growth. This type of growth I interpreted as active avoidance of contact
I predicted that colonies in pair types 1 and 2 would be most likely to show forward growth, due to a high likelihood of relatedness, while those in pair type 3 would be the most likely to show growth patterns consistent with avoidance or neutrality. I analysed differences in colony size, growth, proportion of dead zooids, and length to width ratios between combinations of pairs using one-way analysis of variance, using Systat 10.2.
150
Results
All colonies showed fairly low rates of growth across the experiment, with six pairs in contact with each other at the final census, and only two of those six showing apparent morphological fusion. At the final census of 15 days after pairing, there was no significant difference in the sizes of colonies measured in pixels, between the three different paired combinations (Fig. 2a, F2,27 = 0.779, P = 0.469). Between 13 and 15 days following pairing, however, the change in area appeared to be very different amongst combinations, with colonies in paired combinations 1 and 2 having much higher rates of growth than those in paired combination 3 (Fig 2b), an effect that was statistically
significant (F2,27 = 4.784, P = 0.017).
Net increases in number of zooids across the experiment were also low in all colonies, with only colonies in pair combinations 1 and 3 showing a net increase in the number of functional zooids within the colony between days 8 and 15 after pairing, while those in pair combination 2 showed a net decrease in the number of functioning zooids (Fig. 3a). At the final census, there was a significant difference in the total number of functioning zooids between colonies across paired combinations (F2,25 = 4.105, P= 0.029). Colonies in combination 2, derived from maternal colonies from the same location, had the highest increase in zooid number from day 8 to 15 after pairing, however this result was not
significant (F2,27 = 3.209, P = 0.056).
The proportion of dead zooids within each colony at the final census of 15 days after pairing did not appear to differ between the three combinations, with colonies in combination 2 showing a marginally lower proportion of dead zooids than those in
combinations 1 and 3 (Fig. 3b). This difference, however, was not significant (F2,25 = 0.665, P = 0.523). The ratio of colony length to colony width did not appear to differ between the two census dates (Fig. 4a) and at the final census of 15 days, there was no
difference in length to width ratios according to the paired combination (Fig. 4b, F2,27 = 1.302, P = 0.288).
151
Predominant growth directions differed in colonies across the three paired combinations. Examples of forwards, lateral and perimeter growth as described in the Methods section are shown in Fig. 5. In paired combination 3; colonies derived from maternal colonies from different locations, 10 of 12 colonies showed predominantly forward growth, while few colonies in paired combinations 1 and 2 grew in a mostly forwards direction (Fig. 6). Only colonies in paired combinations 1 and 3 showed normal, perimeter growth, while all combinations had low numbers of colonies that showed predominantly lateral growth. All incidences of contact and likely fusion occurred in colony pairs where at least one colony grew in a predominantly forward direction.
B. Colony pairing and growth at Williamstown, Victoria: effect of colony size
Where growth is limited by crowding, fusion represents a rapid increase in colony area. Such a size increase holds numerous benefits, including increases in feeding capacity and the potential for sexual reproduction. Small colonies appear to have the most to gain from fusing with a conspecific, since crowding may prevent these colonies from independent growth to reach this potential themselves. Thus, we may expect that small colonies are more likely to fuse with conspecific colonies in a paired situation than large colonies. On the other hand, aggressive interactions have been recorded between paired bryozoan colonies of different sizes, including overgrowth (Craig 1995) and stolon production in Membranipora membranacea (Harvell and Padilla 1990). Here I test the prediction that small colonies of Watersipora subtorquata are more likely to initiate fusion with conspecifics than large colonies, and the converse scenario that large colonies are more prone to aggressive actions, by pairing colonies of different sizes in three combinations.
Methods
I deployed artificial substrata composed of four 240 mm × 240 mm squares of 0.38- mm thick clear PVC plastic sheeting bolted to a 600 mm × 600 mm 6-mm thick PVC backing sheet, at approximately 1.5 m below the low water mark at Workshops Pier,
152 Williamstown, Victoria. A number of Watersipora subtorquata colonies were allowed to settle on this substratum during the course of a normal spawning season. Following settlement, competitors and other fouling organisms were removed from the plates at regular intervals. This process ensured that growth of new colonies was not impeded. In early April, I selected a number of colonies for experimentation on the basis of their regularity of growth and apparent health, and grouped these into small and large colonies, keeping the sizes of colonies within these two groups as uniform as possible. Following selection, each colony was removed by carefully cutting the PVC sheet around the colony. Colonies thus isolated on small pieces of sheet were arranged in pairs on glass slides, with the distal edges of each colony (those forward of the ancestrula) placed together as close as possible (less than 2 mm) so that time to encounter was minimised, and fastened to the slides using silicone sealant. I organised colonies into 27 pairs in three combinations: small/small (8 pairs), small/large (10 pairs), and large/large (9 pairs). Once colonies were paired, I took digital photographs using an Olympus C-5050 digital camera mounted on an Olympus SZ-40 binocular dissector microscope. I fastened the slides to two modified settlement plates of 240 mm × 240 mm 6-mm thick grey PVC plastic, bolted these to a 600-mm-square backing plate, and returned the colonies to the field at Workshops pier. After a period of 8 days in the field, I brought the colonies back into the lab to measure the amount of growth, direction of growth and incidence of fusion between colony pairs. Unfortunately, due to a memory card error, many of these data were lost, and by the next census date of 18 days, zooid death within colonies was high, and fusion data was lost. Therefore, I present simple descriptions of the fate of colony pairs.
Results
Table 1 shows the outcomes of 27 pairings of colonies of Watersipora subtorquata in each of three combinations: small/small, small/large and large/large, after eight days in the field. Of 27 pairs, 12 did not achieve contact due to zooid death, colony loss or growth away from the paired colony. In nine pairs, one (usually large) colony overgrew the paired colony, while of six pairs that contacted each other, two grew against each
153 other, and four pairs showed possible fusion. Small colonies were no more likely to fuse than large colonies, but large colonies were more likely to overgrow small colonies and other large colonies. Whether this is due to explicitly aggressive actions by the large colonies, or through zooid death in small colonies, and thus a decline in their defensive capacities, is unclear.
2. Damage recovery and colony size in a monoculture of Conopeum seurati
Studies of encrusting bryozoans in monocultures have shown that these crowded conditions can lower maximum colony size and induce earlier reproduction (Harvell et al. 1990), or reduce feeding capacity in small colonies (Okamura 1985). These conditions may also enhance survival in arborescent bryozoans (Buss 1981; Keough 1986), possibly through the risk of predation being dissipated across a number of colonies or through reducing the likelihood of encounters leading to overgrowth by a superior competitor. These responses suggest that there may be a re-allocation in the energetic resources of colonies in a crowded situation away from growth, which would be unfeasible and therefore a waste of energy, to reproduction. In this instance, the common size-related demographic processes are interrupted, and changes in life history variables become more density-dependent.
In this experiment, I was interested in whether high density living also affects the ability of colonies to allocate resources to damage repair. I examined damage recovery in an existing monoculture of colonies of the encrusting anascan Conopeum seurati. All colonies were in contact with each other, with no incidences of overgrowth or abutting growth evident. Damage was incurred by the recruitment of a number of barnacles whilst the colonies were submerged at Workshops pier, Williamstown, and the subsequent interruption of colony growth at points of barnacle settlement. Removing adult barnacles created an empty space in the monoculture. Measuring the amount of regeneration of lesions created in this way provides a test for the likelihood of colony recovery according to size under crowded conditions. If colonies are redirecting energy stores away from growth processes, or are fused physiologically, we may expect the sharing of resources
154 throughout the monoculture to weaken the expected positive effects of colony size on damage recovery. To this end, I examined a number of borders between colonies to ascertain whether the appearance of morphological fusion was reinforced with physiological fusion at the level of the funicular system
Methods
Damage recovery
I allowed natural settlement onto a 240 mm × 240 mm square of 0.38-mm-thick clear PVC sheeting, attached to a 600-mm-square PVC backing plate as part of an array deployed at Workshops pier, Williamstown, Victoria. I allowed a monoculture of Conopeum seurati colonies to develop, with all colonies in contact with each other. After this had occurred, a large recruitment event of barnacles fouled parts of this monoculture. After a period of time, on January 18, 2005, I removed these barnacles, effectively creating holes in the C. seurati sheeting upon removal, and outlined the areas of damage on the sheeting using a graphite pencil. I carefully laid a clear transparency over the sheeting, tracing around the margins of each colony and marking the location of the ancestrula. I analysed these tracings using Image J software, and measured a number of colony variables, including area and perimeter, and counted the number of neighbouring colonies.
I returned the sheet of colonies to the field for a period of 10 days, after which I brought it back into the lab and cut the sheet into quarters. I used a 1 cm × 1 cm grid overlay to divide the four quarters into blocks. I randomly selected 50 1 cm × 1 cm replicate blocks based on their column and row, of which 42 were suitable for analysis, and photographed these blocks using an Olympus C-5050 digital camera mounted on an Olympus SZ-40 dissector microscope. I analysed the resulting images using Image J software, and for each block measured the number of damaged parts, the area and perimeter of damage, and the amount of regeneration. Regenerated areas were also calculated as proportionate to the initial area of damage. These functions were compared to colony area, the
155 perimeter of the damaged areas, the number of damaged parts and the number of neighbours for each colony using multiple linear regression analysis, performed with Systat 10.2. Measures of colony perimeter were not included due to the risk of collinearity. Similarly, the total area of damage per block was also excluded as a predictor variable, due to its close relationship with the perimeter of damage.
Border analysis
I selected inter-colony borders for scanning electron microscopy (SEM) based on the number of blocks within colonies that were selected for analysis (see Table 2). A total of ten borders between colonies were selected (allocontact borders), while three borders formed when a single colony split, then regained contact with itself (isocontact borders) were also examined for comparison. After preparation of colonies using sodium hypochlorite, fixation of specimens and initial examination, most proved unsuitable for analysis, so final examination of borders was limited to three allocontact borders and 1 isocontact border. I examined each border for the presence of communication pore plates.
Results
Damage recovery
The amount of damaged area that regenerated over time did not appear to change according to colony size (Fig. 8a) or perimeter (Fig 8b). Similarly, I found only weak effects of colony area and perimeter on the total area that was regenerated per block (Fig. 9a, b), and these appeared to be primarily driven by a few outlying values. In contrast, as the area and perimeter of damaged areas increased, the percentage regeneration and total regeneration of those areas decreased (Fig. 10a, b). Smaller injuries were therefore more likely to be regenerated than larger injuries. Table 3 shows results of a multiple regression analysis on both the total area regenerated per block following damage, and on the average proportion of the damaged area per block that was regenerated, according to colony size, damage perimeter, the number of damaged parts and the number of
156 neighbours. In both cases, only damage perimeter was a significant predictor of the amount of regeneration that was achieved in each replicate block.
Border analysis
All four borders examined showed evidence of physiological fusion, as indicated by the presence of communication pore plates within the inter-colony and intra-colonial borders. However, the types of communication pore plates found within these borders differed between that formed within a single colony (isocontact border) and those formed between distinct colonies (allocontact borders). Isocontact borders generally showed communication pore plates consistent with those found within whole, undamaged colonies (see Chapter 3 for descriptions), while allocontact borders contained pore plates consistent in morphology with the communication plates described in Membranipora membranacea by Shapiro (1992), but different to those described within Thalamoporella californica by Chaney (1983). Fusion pore plates in this study consisted of a double-sided dome (a sphere) of calcium carbonate within the lateral walls of contact borders between colonies. These spheres were perforated with a number of communication pores, indicating physiological fusion between the two colonies. Fig. 11 shows an example of an allocontact border with a number of these fusion pore plates within the lateral wall at the point of contact between two colonies.
General Discussion
Growth patterns in presence of conspecific colonies
At Santa Cruz, W. subtorquata colonies showed few differences in total growth rates across paired combinations, and no real increase in colony growth towards the conspecific colony with increasing apparent relatedness. There were, however, differences in the predominant growth patterns by colonies in different paired combinations. Interestingly, the highest occurrence of forward growth, which I predicted to be indicative of compatibility, was in colonies from opposite sides of the marina. I
157 expected colonies from larvae originating from the same maternal colony to have both the highest length-width ratios, and the highest incidence of forward growth; this result is clearly counter to that prediction. A number of factors may be contributing to this apparently anomalous result. The maternal colonies from which I obtained larvae were very large, and growing in three-dimensional foliose configurations. While they appeared to be cohesive colonies, they were extremely complex in structure, and it is possible, although unlikely, that they may have been chimaeras of a number of smaller colonies. As such, larvae obtained from these colonies may not have been siblings as presumed, reducing the likelihood of compatibility. More likely explanations are that an absence of compatibility between colonies from different locations has lowered the colonies’ capacity to recognise the presence of conspecific colonies, or that larval dispersal within the marina was higher than anticipated. Another possible explanation stems from the experimental procedure. I cut the PVC substratum fairly close to the forward edges of settled colonies to enable close pairing, and in some cases, there appeared to be a degeneration of zooids near this edge a few days after experimentation. This reaction may be due to handling trauma or due to the silicone sealant being toxic and causing zooid death, and may explain higher than predicted levels of lateral growth, and low overall rates of growth. Repeating the experiment, but increasing sample sizes, the distance between populations, and the experimental period, would go some way to identifying the mechanisms underlying changes in growth patterns in the presence of conspecific colonies.
At Williamstown, although data were extremely limited, there seemed to be no apparent effect of the paired combination on the likelihood of fusion. Large colonies, however, overgrew conspecific colonies more often, while small colonies tended to die, be removed from the substratum, or suffer partial mortality. These patterns suggest that competitive interactions between colonies of different sizes may be overriding any advantage small colonies may gain through fusion. Similar antagonistic interactions have been noted in Membranipora membranacea (Harvell and Padilla 1990), and suggest that, while the benefits of fusion may be great for small colonies, the benefits for large colonies are few, and their superior competitive abilities reduce the likelihood of this
158 interaction.
Regeneration in a monoculture
After damage through the removal of epifaunal barnacles, colonies within a monoculture of Conopeum seurati regenerated rapidly, and after ten days, most lesions had been totally grown over. The degree of recovery of lesions was not related to a number of factors shown to be influential in damage regeneration in isolated colonies. Increases in colony size and perimeter had no effect on the amount of regeneration, contrary to studies on lone colonies, which suggest that larger colonies may have a greater capacity for regeneration (Chapter 4). Similarly, there were no effects of the number of damaged parts within a colony, or the number of adjacent colonies, on the amount of regeneration. The area and perimeter of damage appeared to be a limiting factor in the regeneration of injuries; smaller lesions with a shorter perimeter were more likely to be fully regenerated after ten days.
Examination of monocultures of Membranipora membranacea by Harvell et al. (1990) suggests that the high density conditions may induce changes in the resource allocation schedules within colonies, limiting growth and inducing reproduction at a younger age and smaller size than isolated colonies. These changes may also be operating in Conopeum seurati. Obstruction of growth by the presence of conspecific colonies may cause a shift in resource allocation away from growth and towards reproduction or damage repair. While I did not measure reproduction in colonies within the monoculture, the high rate of regeneration amongst lesions regardless of colony size appears to be indicative of such a shift.
Another possible explanation for the high regeneration of lesions in C. seurati is that these colonies within the monoculture are fused, and are subsequently able to share resources and direct them to damage repair. In effect, they may act as one large ‘supercolony’; size effects on regeneration efficiency would thus be reduced or eliminated. I examined four borders within the monoculture for evidence of physiological
159 fusion between colonies, and in each case found that fusion had occurred, from the presence of communication pore plates. Although this is only a small sample, it does point to the possibility of widespread resource sharing across the population. However, while the presence of fusion pore plates indicates some physiological fusion between the distinct colonies, and thus the capacity for resource sharing, little is still known about the actual rates of resource transfer between individual zooids, both within colonies and between colonies. Radiolabelling studies on single colonies have suggested that metabolites are transported predominantly towards the growing edge (Best and Thorpe 1985; Miles et al. 1995), at a rate of approximately one zooid per hour (Best and Thorpe 1985), and Craig (1995) showed movement of metabolites between fused colonies. In Chapter 3 (this volume), examination of pore plates between zooids within C. seurati colonies appears to validate this finding, with high numbers of directional connections near colony edges, and the appearance of communication pore plates between fused C. seurati colonies in this chapter strongly suggests the capacity for metabolite movement between colonies.
The growing edge of an intact colony may function as a strong resource sink, but damage creates an additional sink requiring the redirection of resources to regeneration. In scleractinian corals, metabolites appear to be directed towards the site of regeneration of artificially inflicted lesions (Oren et al. 1997b) suggesting that, in these taxa at least, directional movement of resources away from growth and towards damage repair is possible. In encrusting bryozoans, the mode of resource transfer is more complicated, but examination of the funicular system (Chapter 3, this volume) and of patterns of regeneration following damage (Chapter 4, this volume) suggest that it is a reasonably flexible system, with the potential to alter the direction of nutrient flow after disturbance. Thus, the damaged C. seurati colonies seen in this study appear to be rapidly redirecting nutrients towards damage repair, and the cessation of growth at the colony edges in crowded conditions may increase the store of resources in order to facilitate this response.
160
Collection site 1 – ‘NEAR’ Collection site 2 – ‘FAR’
Figure 1. Map of Santa Cruz marina, showing the collection sites for Watersipora subtorquata colonies used in the pairing experiment. Colonies from larvae originating from both collection sites were paired in three combinations: 1) larvae originating from the same supercolony (from either collection site) 2) larvae originating from same collection site (different colonies). 3) larvae originating from different collection sites. The approximate distance between the two collection sites is 200 m. Map is courtesy of http://www.santacruzharbor.org.
161 250000 a)
200000 Area (pixels)
150000
100000
50000 13 15 Days after pairing
100000
b)
50000
Change Paired in colony combination area (pixels) 0 1 2 3
-50000
Figure 2. Size of colonies, measured as the number of pixels, at each of two census periods in paired colonies of Watersipora subtorquata (a) and the change in colony area between these two periods (b), according to the paired combination. Combination 1: settlers from same maternal colony, combination 2: settlers from same location, combination 3: settlers from different locations. Size is measured as the total number
162 a)
2
Change in 1 number of zooids 0 from day 8 to day -1 15 after pairing -2
-3
-4
0.8 b)
0.7
0.6 Proportion of dead 0.5 zooids Paired within 0.4 combination colony at 1 15 days 0.3 2 3 0.2
0.1
0.0
Figure 3. Net change in the total number of zooids per Watersipora subtorquata colony in each of three paired combinations between days 8 and 15 after pairing (a) and the number of dead zooids per colony in the three combinations at day 15 after pairing (b). Data shown are mean values, ± one standard error.
163 2.0 a)
Ratio of 1.5 colony length to width
1.0
0.5 13 15 Days after pairing b)
1.6 1.4
Length to 1.2 width ratio 1 at final Paired census 0.8 combination 0.6 1 2 0.4 3 0.2 0
Figure 4. The length to width ratio per colony in paired colonies of Watersipora subtorquata at 13 and 15 days after pairing (a) and at the final census of 15 days (b) according to the combination of pairing. Combination 1 represents a pair of colonies from the same maternal colony, combination 2 pairs colonies from the same location, and combination 3 pairs colonies obtained as larvae from different marina locations. Bars are mean values, ± one standard error.
164 a) Forward growth
b) Lateral growth
c) Perimeter growth
Figure 5. Examples of directional growth in Watersipora subtorquata following pairing, showing (a) forward growth, (b) lateral growth, and (c) perimeter growth, with perimeter growth being the closest to growth of colonies under normal, uncrowded conditions. Note the lengthening of the colony at the frontal edge in forward growth.
165
12
10 Number of colonies 8 exhibiting growth 6 type
4
2
0 123 Paired combination Type of growth Forward growth Lateral growth Perimeter growth
Figure 6. Types of growth exhibited by individual colonies of Watersipora subtorquata within each of three pair combinations at the final census. Refer to figure 4 for examples of each growth type.
166
A
B
Figure 7. Examples of actions 8 days after pairing of Watersipora subtorquata colonies at Williamstown. (A) growth of colonies against each other on contact (abutment) (B) overgrowth; the colony on the right is growing over the colony on the left
167
a)
100.0
Average percentage 80.0 regeneration of damaged areas 60.0
40.0
20.0
0.0 0 10203040 Colony area (cm2)
b)
100.0 Average percentage regeneration 80.0 of damaged areas 60.0
40.0
20.0
0.0 0 5 10 15 20 Colony perimeter (cm)
Figure 8. Average proportion of regeneration occurring in damaged areas per block according to (a) the area of the damaged colony in cm2 and (b) the perimeter of the damaged colony in cm, in damaged colonies of Conopeum seurati.
168
a) 1 0.9 0.8 Total area regenerated 0.7 per block 0.6 (mm2) 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 30 35 40 Colony area (cm)
1 b) 0.9
0.8
0.7
Total area 0.6 regenerated per block 0.5 2 (mm ) 0.4
0.3
0.2
0.1
0 0510 15 20 Colony perimeter (cm)
Figure 9. Total area of damage regenerated per block according to (a) colony area in cm2 and (b) colony perimeter in cm, in damaged colonies of Conopeum seurati.
169 a)
100
Average 80 percentage regeneration per block 60
40
20
0 0 0.2 0.4 0.6 0.8 1 Total area of damage per block (mm2)
b)
100
Average 80 percentage regeneration per block 60
40
20
0 0 0.5 1 1.5 2 Total perimeter of damage per block (mm)
Figure 10. The percentage regeneration of damaged areas according to (a) total area of damaged areas per block, and (b) total perimeter of damaged areas per block, in damaged colonies of Conopeum seurati.
170
A position of inter-colony border
200µm
B 100µm
Figure 11. Examples of communication pore plates within a contact border between two Conopeum seurati colonies. (A) the contact border between two distinct colonies, showing the varying morphology of pore plates at the site of contact (marked with the solid yellow line) and those in other regions of the abutting colonies. (B) close-up view of two of the allocontact pore plates found within the contact border. Again, the solid yellow line indicates the position of the contact border.
171
Table 1. Actions at 8 days following Watersipora subtorquata colony pairing in three combinations at Workshops pier, Williamstown. Possible actions recorded are: no contact, contact (possible fusion), contact (abutment) and overgrowth of one colony by the other.
Pair Combination Notes Action 1 S/L Small colony mostly dead; some live zooids near No contact contact border, 1 embryo. Large colony fine. No fusion 2 S/L Small colony removed. Large colony contains dead No contact zooids near edge of substrate 3 S/L Large colony broken in half, resulting in increased No contact distance between colonies 4 S/S Both colonies have high numbers of dead zooids No contact 5 L/L Contact, some evidence of fusion, border 10 zooids Contact; possible wide. Some zooid death; regrowth is towards paired fusion colony 6 L/L Contact, but no fusion apparent – abutting growth. Contact; colonies abutting 7 S/S One colony removed No contact 8 S/L Large colony overgrowing small colony Overgrowth 9 S/L Contact, possible fusion – contact border 7 zooids wide Contact; possible fusion 10 L/L Contact, possible fusion – contact border 2 zooids wide Contact; possible fusion 11 S/S Both colonies missing No contact 12 S/L Contact, possible fusion – contact border 9 zooids wide Contact; possible fusion 13 S/L Contact, colonies growing against each other Contact; abutting growth 14 L/L Some tissue loss in one colony, no contact No contact 15 S/S Colonies gone 16 S/S Colonies gone 17 L/L Fusion likely to occur; both colonies healthy. No No contact contact yet 18 L/L Overgrowth of one colony by the other Overgrowth 19 S/L Large colony overgrowing small colony Overgrowth 20 S/S One colony died, was overgrown by other colony Overgrowth 21 S/L Large colony overgrowing small colony Overgrowth 22 S/L Zooid death in both colonies; fusion unlikely No contact 23 L/L Overgrowth of one colony by the other Overgrowth 24 L/L Overgrowth of one colony by the other Overgrowth 25 L/L Overgrowth of one colony by the other Overgrowth 26 S/S One colony died No contact 27 S/S One colony died No contact
172
Table 2. The number of blocks selected randomly within different colonies across the monoculture of Conopeum seurati. Highlighted rows indicate the colonies whose borders were selected for scanning electron microscopy (SEM) analysis.
Colony Number of number blocks selected 1 5 2 1 3 3 4 1 5 2 6 4 7 1 9 2 11 3 12 1 13 1 15 2 17 2 18 1 19 2 21 1 23 1 24 1 26 1 27 2 31 1 34 1 44 1 49 1 52 1
173 Table 3. Multiple least-squares linear regression analysis of the a) total area of regeneration, and b) average proportion of area regenerated, in damaged parts of Conopeum seurati colonies according to colony area, damage perimeter, the number of damaged parts and the number of neighbours. Squared multiple R = 0.333. Significant effects are highlighted in bold.
a) Total area regenerated
Standard Coefficient Tolerance t P error Constant -0.023 0.072 -0.325 0.747
Colony area <0.001 <0.001 0.944 0.627 0.534
Perimeter of damaged 0.240 0.073 0.568 3.293 0.002 area Number of damaged -0.005 0.021 0.533 -0.237 0.814 parts per colony Number of -0.005 0.011 0.955 -0.439 0.663 neighbours per b) Average proportion area regenerated
Constant 0.766 0.126 <0.001
Colony area <0.001 <0.001 0.944 6.087 0.545
Perimeter of damaged -0.439 0.127 0.568 0.610 0.001 area Number of damaged 0.048 0.037 0.533 -3.456 0.205 parts per colony Number of 0.014 0.019 0.955 1.2910 0.456 neighbours per colony
174 Chapter 6
Damage recovery under field conditions in Parasmittina delicatula
Introduction
Although growth in modular organisms, due to the addition of new, reasonably independent modules and module regeneration, can theoretically be indeterminate (e.g. Jackson 1985; Lidgard and Jackson 1989), their patterns of growth and module maturation can have a significant impact on the way colonies recover from physical damage. In encrusting unilaminar bryozoans, growth usually occurs at the colony perimeter; if growth is continuous, these peripheral zooids will, in the absence of damage, be the youngest, whilst those in the centre of the colony will be older. These age gradients may have significant effects on the life history of the colony, not considered under the indeterminate growth model. For example, Palumbi and Jackson (1983) noted more waste accumulation, less feeding activity and slower lesion regeneration in proximal sections of the encrusting bryozoan Steginoporella sp., and reported this as a clear sign that ageing and senescence was occurring in those older zooids. In the fast- growing Watersipora subtorquata this ageing process gives rise to a centralised dead zone of senesced zooids after around 4–5 weeks of uninterrupted growth, and a ring of reproductively active zooids distal to this dead centre is also evident upon maturation (Hart 2001; E. Bone, personal observation). Growth at the colony edges also means that this edge is a valuable resource sink in bryozoans. New zooids, formed by budding at the colony edges or branch tips, require resources and nutrients to continue their development into independently feeding zooids, and this investment must come from feeding zooids further back in the colony. Source-sink control of resource allocation has been demonstrated for a number of modular plants (e.g. Senn and Haukioja 1994; Kaiteniemi and Honkanen 1996; Charpentier et al. 1998), and is also important in lesion recovery in corals (Oren et al. 1997b, Oren et al. 1998).
In unilaminar encrusting bryozoan colonies, where growth is at the colony edge, that
175 edge can also become a valuable resource sink, and the ages of component modules can have a profound effect on the likelihood and magnitude of recovery in damaged bryozoans. In the common bryozoan Membranipora membranacea, radiolabelling studies showed a preferential shift in resources towards this growing edge (Best and Thorpe 1985; Miles et al. 1995). Prior to reproduction, young colonies may be expected to allocate a significant portion of energy into further growth, and damage to this growing region represents a significant loss of energy through the reduction of this resource sink. Bone and Keough (2005) showed that damage to the growing tips of branches in pre- reproductive colonies of the arborescent bryozoan Bugula neritina had a significant negative effect on their subsequent reproductive output, and on the recovery of the damaged areas. In the encrusting species Mucropetraliella ellerii, edge fragments had better rates of re–growth than those formed from central parts of colonies (Klemke 1993), and Hart (2001) showed that the regeneration abilities of fragments of the encrusting bryozoan Watersipora subtorquata were increased when those fragments incorporated edge regions. In Chapter 4 of this volume, repeated damage to the growing edge significantly reduces both recovery rates and embryo production in this species, and the level of investment in the edge was reflected in higher numbers of interzooid connections towards the edge (Chapter 3). However, I completed most of this work in W. subtorquata, a species with high rates of growth, simple zooids, and fairly regular colony construction, but it is likely that species with different patterns of construction and demography are investing resources in the growth of the colony edge in different ways. I also used colonies settled on artificial substrata that were cleared of competitors, but competition and substratum condition are factors that may greatly influence colony growth and recovery in ways that have yet to be examined in this volume.
Here I describe experiments examining the responses to artificial physical damage in the encrusting bryozoan Parasmittina delicatula (Busk, 1884), conducted under field conditions at two different sites in Victoria, Australia. These two sites are superficially similar, being located near the openings of large embayments and subject to high levels of water flow. Two size classes of colonies were used, and these were subjected to single instances of damage, which removed varying amounts of the growing edge. If the amount
176 of growing edge in this species is a significant resource sink, we should expect similar patterns of recovery as seen for previously studied species. That is, the amount of growing edge removed should dictate the level of recovery and eventual reproduction; essentially, the greater the damage to the edge of the colony, the slower the re-growth and recovery (see Chapter 4). However, P. delicatula colonies have very different patterns of intra-colonial connections to those found in W. subtorquata (Chapter 3), and are much more heavily calcified. As a result, they may exhibit different types of responses to damage at varying regions across the colony as a result, while competition from neighbouring organisms is likely to have a strong effect on colony growth patterns. I compare the effect of size on colony survival, and discuss the influence of damage in fragments originating from colonies of similar size. Finally, I consider any differences in these experimental findings between sites, and discuss these differences with respect to assemblage structure.
Methods
Species description
Parasmittina delicatula (Busk, 1884); synonyms Mucronella avicularis, Parasmittina decorata, Smittina unispinosa (Bock 2005) is an encrusting cheilostome bryozoan in the family Smittinidae, and is widespread across the Pacific basin. It has a unilaminar growth form, with growth by the asexual addition of new modules occurring at the colony perimeter. P. delicatula is a brooding bryozoan species, and larvae develop in calcified ovicells associated with maternal zooids until maturity. These larvae are visible within the ovicell housing, and appear as creamy white spheres on the surface of the colony. P. delicatula appears to be a fairly long-lived species, and reproduction tends to occur only once colonies have reached large sizes, when they produce up to thousands of embryos (M. J. Keough, personal communication).
Site descriptions
177 Fragmentation experiments on existing colonies of Parasmittina delicatula were run concurrently at two sites in Victoria; Flinders pier and Queenscliff pier (Fig. 1). Flinders pier is located near the entrance to Westernport, a large embayment, and is exposed to the waters of Bass Strait. Queenscliff pier is located on the Bellarine Peninsula, near of the entrance of another large embayment, Port Phillip Bay. The two sites, although located in different areas, appear to be subjected to similar flow regimes.
Experimental set-up
The experiment was devised by Prof. Mick Keough and Dr. Craig Styan, and experimental treatments and underwater photography were carried out by members of the lab in 1999. The treatments were devised to examine how the presence or absence of a growing edge affects fragment performance, and to compare the performance of fragments with that of intact colonies of similar size. Two size classes of colonies were also used to compare the effects of size on the recovery capacities of colonies. Colonies were divided into 6 treatments (see Fig. 2). The same treatments were used at both sites, with the exception that all six treatments were represented at Flinders pier, while only treatments one to five were set up at Queenscliff. Small colonies were defined as those with a diameter of 10–15 mm, while large colonies were those with a diameter of 30mm or more.
Divers on SCUBA located colonies of suitable size, and removed colony parts, according to the assigned treatment, by scraping. The location of each colony was marked using cattle tags fastened by nails to the pier piling. At intervals of one, two, three, four and eight months, colonies were photographed in situ by a Nikonos V underwater camera, fitted with a 1:3 extension tube, using slide film. I projected the slides onto graph paper, and traced the outlines of colonies. I then scanned these images and analysed the resultant digital images using SigmaScan 2.0.
Sampling and analysis
178 Flinders
A total of 56 established Parasmittina delicatula colonies were photographed over a period of eight months at Flinders Pier, Victoria. These colonies were divided into six treatments; treatments 1–6 in Fig. 2.
Colony survival and causes of mortality. I noted the number of months each of the 56 colonies persisted on the pier pilings, and defined mortality as the complete removal of the colony from the substratum, the complete overgrowth of the colony, or the death of all component zooids. I assumed complete zooid death had taken place when all zooids took on a distinct brown (indicating the formation of degenerative brown bodies) or green (indicating algal overgrowth) colour. I also noted differences in survival as the proportion of colonies in each treatment surviving to a given time, while I categorised the primary causes of death as overgrowth, removal or total zooid death. I used analysis of variance to analyse differences in the survival of colonies according to treatment. All analyses were done using Systat 11. I used post-hoc power analysis to gauge the power of the main ANOVA test to identify a 50% difference in survival of colonies in different treatments, using G Power software.
I conducted planned comparisons between treatments originating from large colonies where the edge was intact (treatments 1 and 2) and those where the edge was removed (treatments 3 and 4). If these comparisons were non-significant, I pooled treatments 1 and 2 and compared them to the pooled treatments 3 and 4. I compared treatments 5 and 6 to examine changes in survival between undamaged and damaged small colonies, while a comparison between treatments 1 and 5 examined differences in survival between large and small undamaged colonies. Finally, I compared treatments 4 and 6 to identify any differences in survival rates between central fragments originating from large and small colonies.
Size, growth and fragmentation I used colonies in treatments originating from large colonies (treatments 1 through 4) in this part of the experiment. Because there was a
179 very high rate of mortality among colonies in all six treatments, the numbers of colonies in the analysis were further reduced to only include colonies that survived a minimum of 3 months. This gave a final total number of 21 colonies; 6 in treatment 1, 5 in treatment 2, 6 in treatment 3 and 4 in treatment 4. I recorded the sizes of colonies, measured as total live colony area, and their growth, measured as the change in total live colony area, at each sampling time. Since growth is highly dependent on the number of live modules that can contribute resources to growth, I also expressed growth values proportional to colony size at each sampling time. I noted embryo production and any incidences of partial or total overgrowth, fragmentation or fusion of colonies. Those parts of a colony that were overgrown or obscured by other animals were not counted towards total colony size, as I assumed that these were not viable areas of the colony. At Flinders pier, no colony fragmented or split into smaller colonies. I analysed the size of colonies after four months, their average growth rate to four months, and the average net growth after four months according to treatment, using analysis of variance. The total numbers of colonies available for analyses of survival, size and growth rates are listed in Table 1. I used post- hoc power analysis to gauge the power of the main ANOVA test to identify a 70% difference in the average net growth of colonies over four months using G Power software. This effect size was based on the differences in the net growth of large control colonies and central fragments given an ideal rate of module addition of four zooid rows per month, and using the initial sizes of colonies in these two treatments at Flinders pier.
I used planned comparisons to identify any fine-scale differences in the average rates of growth, scaled to colony size, after four months, in colonies that had their edge removed compared to those that retained their edge. As in the previous section, treatments 1 and 2 were compared first, then treatments 3 and 4. If these separate comparisons were non- significant, treatments 1 and 2 were pooled and compared to the pooled treatments 3 and 4. In addition, I compared the undamaged controls, treatment 1, to colonies where only the central portion remained, treatment 4.
Queenscliff
180 A total of 47 established Parasmittina delicatula colonies were photographed over a period of eight months at Queenscliff Pier. These colonies were divided into five treatments; 1–5 in Fig. 2. Owing to insufficient numbers of small colonies found on pilings at Queenscliff pier, treatment 6 was not included.
Colony survival and causes of mortality. For all 47 individuals, I noted the number of months they persisted on the pier pilings. I defined mortality as the complete removal of the colony from the substratum, the complete overgrowth of the colony, or the death of all component zooids (as above). I noted differences in survival as the proportion of colonies in each treatment surviving to a given number of months, and differences in the causes of eventual colony mortality. I analysed differences in the number of months survived according to treatment using analysis of variance. I used post-hoc power analysis (G Power software) to gauge the power of the main ANOVA test to identify a 50% difference in survival of colonies in different treatments.
As for the Flinders colonies, I conducted planned comparisons between specific treatments, first comparing treatments 1 and 2, then treatments 3 and 4. If these individual comparisons were non-significant, I pooled treatments 1 and 2 and compared them to the pooled treatments 3 and 4. I also compared treatments 1 and 5 to examine differences in survival between large and small undamaged colonies.
Size, growth and fragmentation. For this part of the experiment, I only considered treatments originating from large colonies (treatments 1 through 4). There was a very high rate of mortality among colonies in all six treatments, so I only used colonies that survived a minimum of three months so as to provide meaningful analyses of changes in colony size and growth over time. This resulted in a final total of 24 colonies; 7 in treatment 1, 6 in treatment 2, 5 in treatment 3 and 6 in treatment 4. I noted the following variables for each time period: colony size, measured as total live colony area; colony growth, measured as the change in total live colony area; colony growth relative to colony size, number of embryos, and any incidence of partial or total overgrowth, fragmentation or fusion of colonies. I discounted any parts of a colony that were
181 overgrown or obscured by other animals were not counted towards total colony size, assuming that they were not viable areas of the colony. During the course of the experiment, five colonies at the Queenscliff site fragmented into smaller colonies. One colony from treatment 1 split into two separate fragments after one month, and another colony from treatment 1 split into two fragments after one month, while a third fragment was formed at two months. One colony from treatment 2 split into two fragments after two months, one colony from treatment 5 split into two fragments after one month, while another split into two fragments after four months. I included these fragments in the analysis as additional replicates of the treatment of the original colony. I analysed differences in the size of colonies after four months, their average growth rate to four months, and the average net growth after four months according to treatment, using analysis of variance. I used post-hoc power analysis to gauge the power of the main ANOVA test to identify a 50% difference in the average net growth of colonies over four months using G Power software, based on the differences between the net growth of large control colonies and that of central fragments, given a rate of module addition of four zooid rows a month, and using the initial sizes of colonies in these treatments at Queenscliff pier.
Again, I used planned comparisons to compare the average net growth to four months across specific treatments. I first compared treatments 1 and 2, then treatments 3 and 4. If these individual comparisons were non-significant, I pooled treatments 1 and 2 and compared them to the pooled treatments 3 and 4. I also compared treatment 1 (undamaged control) to treatment 4 (central fragment).
The total numbers of colonies available for analysis in each section are listed according to treatment in Table 1.
Results
Flinders
182 Colony survival and causes of mortality. The survival of colonies varied across treatments. Small colonies with the edge removed began to die quickly, with only 40% remaining after one month, and only 20% remaining at the end of the eight-month period (Fig. 3). Small control colonies had a less dramatic decline, but after eight months, no colony in this treatment was alive. Large fragments with the edge removed had the slowest rate of decline, and at the end of eight months, around 50% of colonies were still alive, while large control colonies also performed well. Treatments originating from large colonies appeared to survive for longer than small colonies or fragments originating from small colonies (Fig. 3). However, there was a large amount of variation both within and between treatments and there were no significant differences between treatments in the
average number of months that colonies persisted on pilings (one-way ANOVA; F5,50= 1.132, P = 0.356). Planned comparisons also showed no significant effects (Table 2). The number of colonies that succumbed to the three main causes of death was also fairly even (Fig. 4). In each treatment, there was at least one incidence of colony overgrowth, removal or death. Overgrowth was not a particularly common cause of death. When overgrowth did occur, it was more likely to be by algae or clumps of hydroids than other large sessile colonial animals such as bryozoans, sponges and ascidians. The mortality of colonies resulted in a decrease in sample sizes for many aspects of analysis across the experiment. As a result, only colonies that survived more than three months were used in analyses of size and growth. In addition, many colonies were absent at the final census period of eight months, further reducing the sample sizes to levels considered too low for meaningful statistical analysis. Therefore, I analysed total growth rates across the experiment after four months, and calculated average growth rates across four census periods to four months.
Size, growth and fragmentation. Only colonies that survived more than three months were used in this analysis. However, colonies continued to die after this time, and only 12 colonies survived until the final census. Treatment 3, the large fragments with the edge removed, was the only treatment in which a colony did not die after three months, with treatments 1, 2, and 4 having one, three, and two colonies remaining respectively. Owing to this high mortality rate, the sizes and growth rates of colonies were analysed
183 after four months, rather than after the final census at eight months. After four months, 19 of the original 21 colonies were surviving; two in treatment 1, four in treatment 2, six in treatment 3 and five in treatment 4. The sizes of colonies in treatments 1 to 4 appeared to follow similar trends over time, although colonies in treatment 1, the large control, were much larger at every census except for the last measure at eight months (Fig. 5a). There appeared to be a dramatic decline in colony size across all treatments at four months, after which colonies in all treatments appeared to recover to a comparable degree, but there were no significant differences between treatments in the sizes of colonies after four months (Table 3). Initially, large colonies in treatment 1 grew at a faster rate than colonies in all other treatments, but declined in size towards the end of the experiment (Fig. 5a), and the average net growth rates of large control colonies was lower than the rates of both large fragments with their edge removed, and central fragments (Fig. 5b). No treatment recorded a net positive average growth rate over time when growth was scaled to colony size, with large fragments with the edge retained having the lowest net growth rate of colonies in all treatments (Fig. 5b). Analysis of the average growth to four months showed no significant differences between treatments (Table 3). Similarly, when these average growth rates were scaled to the original size of the colony, there were also no significant differences between treatments after four months, and planned comparisons between colonies that retained their edge and those whose edge was removed showed no significant differences in net growth (Table 3). At Flinders, no colony fragmented into smaller colonies, and no colony reproduced during the experiment.
Queenscliff
Colony survival and causes of mortality. The survival rates of colonies in the different treatments varied over time, with large fragments with the edge removed showing a rapid decrease in the proportion of colonies surviving over time. After eight months, only around 20% of colonies in this treatment remained (Fig. 6). Centre fragments were the next worst performing colonies, with around 40% of colonies surviving to eight months. The remaining three treatments performed relatively well, and
184 after eight months, around 60% of large colonies, large fragments with the edge removed, and small control colonies were still alive (Fig. 6). The average number of months that colonies survived on the pier pilings was not, however, significantly different between
treatments (one-way ANOVA: F4,42 = 0.926, P = 0.458), while planned comparisons also yielded no significant results (Table 4).
All five treatments had colonies that were killed as a result of complete overgrowth or removal. The only colonies to die were one small control and one large fragment with the edge intact. Complete overgrowth, typically by didemnid ascidians, was the commonest cause of death, with 17 colonies succumbing to overgrowth (Fig. 7). Overgrowth was more prevalent in small control colonies than in any other treatment, although this difference was not significant. Of 30 fatal and non-fatal overgrowth events across all treatments, ten (33.33%) were by didemnid ascidians, four (13.33%) were by polyclinid ascidians, one (3.33%) was by the ascidian Botrylloides leachii and one (3.33%) was by an unidentified polyclinid ascidian. In total, ascidians were responsible for over half (16 events: 53.33%) of all overgrowths. An unidentified yellow sponge (7 events, 23.33%), hydroids (4 events: 13.33%) and the sponge Dendrilla rosea (1 event: 3.33%) accounted for the remainder of overgrowth interactions. A further seven colonies were completely removed from the substratum.
Size, growth and fragmentation. Only colonies that survived three months or more were used in this analysis; however, as at Flinders pier, there was a large amount of mortality after this time. Of 29 colonies used in the analysis, 23 survived until four months; eight in treatment 1, eight in treatment 2; four in treatment 3 and four in treatment 4. At the final census of eight months, however, only 16 colonies were surviving, decreasing sample sizes to an unacceptable level. Analyses of size, growth and average growth were therefore restricted to the four-month time period. Over the eight months of the experiment, the size of colonies in all five treatments showed similar trends over time, except for the final census at eight months, when central fragments showed a rapid increase in size and were much larger than colonies in other treatments (Fig. 8). However, there was a large amount of variation within treatment, and the sizes of
185 colonies after four months were not significantly different according to treatment (Table 5) Colonies in treatment 4 (central fragments) appeared markedly larger than those in all other treatments (Fig.8), and large fragments with the edge removed were the poorest performers in terms of total colony size. Centre fragments also appeared to have the highest growth rates of all treatments, while large controls had the lowest (Fig. 8). However, average growth rates across four months treatments did not differ significantly (Table 5). Similarly, when growth was scaled to colony size, there were no significant differences across treatment after four months, and there were no significant results amongst the planned comparisons (Table 5). Although no colony at Flinders pier reproduced, 12 colonies at Queenscliff were able to produce embryos. Due to such small sample sizes, comparisons of embryo production across treatments were not made, and reproductive effort across all treatments was analysed with reference to the incidence of overgrowth and colony size.
Effects of overgrowth. Overgrowth, aside from being the most prevalent cause of death at Queenscliff, also caused lasting changes to the life histories of colonies. Fifteen of 29 colonies in treatments 1–4 were overgrown at least once. As the number of partial overgrowth events increased, the average net growth rates of colonies across all treatments declined significantly (Fig. 9, average net growth = 0.196 × number of
overgrowth events – 0.192, F1,44 = 11.277, P = 0.002), and colonies that were never overgrown had significantly lower average growth rates than those that had been
overgrown once or more over the course of the experiment (one-way ANOVA: F1,36 = 9.231, P = 0.004). Within treatments 1–4, this effect was retained, with colonies that had never been overgrown having significantly higher average growth rates to four months than those that had been overgrown at least once (paired t-test; t = 3.754, P = 0.0017). While these results are not unexpected, since overgrowth occludes part of the colony, a more important result was the reduction in embryo production with increasing number of overgrowth events, although this decline was not significant (number of embryos =
669.809 × number of overgrowth events – 311.864, F1,44 = 3.637, P = 0.063). Only 13.33% colonies that had been overgrown once or more during the experiment (2 of 15 colonies) were able to produce embryos. These overgrown colonies produced 114.5 ±
186 37.47 embryos. In contrast, 71.43% of colonies that had never been overgrown (10 of 14 colonies) were able to produce embryos, at a mean of 3157.2 ± 2294.79.
Discussion
Damage to different parts of Parasmittina delicatula colonies did not have a noticeable impact on the recovery and growth of colonies found at either site. At Flinders, small colonies tended to survive for fewer months than large colonies or fragments derived from large colonies, although this difference was not strong, and planned comparisons between colonies that retained their growing edge and those that had their edge removed showed no difference in survival. Despite a substantial increase in the size of large control colonies up to four months, there were no differences in the sizes of colonies after four months across treatments or in the average growth rates of colonies after this time. Colonies at Flinders pier showed extremely low overall rates of growth, there were no incidences of fragmentation or fusion of colonies, and no colony was reproductively active over the course of the experiment. At Queenscliff, in contrast, small undamaged colonies showed fairly high rates of survival, while the lowest longevity was shown by both large and small colonies in treatments that completely removed the growing edge of the colony.
In treatments derived from large colonies, there were no differences in the final sizes of colonies, although central fragments appeared larger at the final census and had higher levels of growth towards the end of the experiment. Although these patterns were not statistically significant, they were nevertheless contrary to our initial predictions, which were that this treatment would be the worst performed, due to the importance of the growing edge as a resource sink. Central fragments were formed by removing the entire edge region of colonies, effectively clearing a space around the central part of the colony. The relatively high growth rates and survivorship shown by fragments created in this way suggests that, in contrast to W. subtorquata (examined earlier in this thesis), not only does the central region of a P. delicatula colony retain a fair capacity for module addition, but that the presence of neighbouring organisms may limit a colony’s ability to
187 grow.
In unilaminar, encrusting species of bryozoan, it is presumed that the bulk of nutrients are directed towards the growing edge of the colony. Examination of the connective pores within the funicular system suggests that this is the case (Chapter 3, this volume), and radiolabelling studies provide direct evidence of such directional transfer (Best and Thorpe 1985; Miles et al. 1995). Losing that growing edge would then remove all of these nutrient stores, such that recovery and further growth may be compromised. Responses of this nature have been seen in other cheilostome bryozoan species, for instance the arborescent Bugula neritina (Bone and Keough 2005), and a number of encrusting species including Mucropetraliella ellerii (Klemke 1993), Watersipora subtorquata (Hart 2001; Chapter 4, this volume) and Membranipora membranacea (Harvell and Helling 1993). In B. neritina, removing tissue from the growing periphery of the colony had a profound negative effect on its subsequent reproductive effort, while the encrusting species exhibited a range of responses indicating a shift in resource allocation, including lower growth rates, induced reproduction, or lower total reproductive effort.
The rapid re-growth of central fragments indicates that the central zooids of P. delicatula colonies may not be as prone to mortality as those of other encrusting bryozoans, and are capable of rapid regeneration after damage. This re-growth appears to be facilitated by the formation of giant buds, where the lateral walls are formed first and radiate outwards from the colony perimeter (Fig. 10). Transverse walls are added after this first stage is complete. Giant bud formation has been observed in a number of bryozoan species, including other smittinids such as Parasmittina trispinosa (Silén 1982), as well as Membranipora membranacea (Lutaud 1961, in Silén 1982) and has been associated with rapid colony growth (Ryland 1976; Lidgard 1984). Another important function of these giant buds may be to increase the colony’s ability to compete with neighbouring organisms, and their formation has been documented in the presence of competitors (Nandakumar and Tanaka 1994). The development of these extensive lateral walls may allow P. delicatula to continue to successfully occupy space on the substratum and protect the remainder of the original colony while it is being repaired, essentially acting
188 as a buffer zone for the colony. Although frontal budding is present in P. delicatula colonies, no instance of frontal budding was noted in any colony in this experiment. This is an interesting finding, as frontal budding may have been a valid response to crowding at the colony perimeter, allowing the colony to increase in size even in the presence of competing organisms at Queenscliff pier. Queenscliff pier is, however, subject to high swells, and high levels of sand scouring on the pier surfaces (E. K. Bone, personal observation). The deleterious effects of sand scour could have precluded frontal budding in these colonies, although further careful investigation is required.
Another explanation may be that P. delicatula is not as vulnerable after zooid removal from the colony edges as other previously studied taxa. Species that show rapid growth and high levels of investment in that growth and subsequent reproduction must necessarily divert large energy stores towards the growing edge. For example, repeated damage to the colony edges in Watersipora subtorquata induces a reduction in the total growth of colonies, and a subsequent reduction in reproductive output (Chapter 4, this volume). Damage in the cosmopolitan encrusting cheilostome Membranipora membranacea results in a re-allocation of resources to towards early reproduction, while obstructing colony growth on one side leads to compensatory growth on the opposite colony side (Harvell and Helling 1993). Across the experiment, P. delicatula colonies exhibited very low rates of overall growth even in undamaged colonies, and at Flinders pier, where competition was low compared to Queenscliff pier, colonies in all treatments recorded a zero net average growth across 8 months. In addition, where colonies did reproduce at Queenscliff, embryos were spread across the surface of colonies, including the central parts of colonies, suggesting that zooid age and senescence does not limit reproduction in the colony centre. P. delicatula may therefore transfer fewer resources to the growing edges of colonies than presumed for other species. For example, in the cosmopolitan bryozoan Watersipora subtorquata, growth at the colony margins is rapid, reproduction is quite localised, and zooid senescence in the central parts of colonies begins to occur after 4–5 weeks (Hart 2001). In Membranipora membranacea, the most studied species of extant cheilostome bryozoan, populations are subject to seasonal fluctuations and growth is rapid, due to its reliance on ephemeral algal substrata
189 (Bernstein and Jung 1979; Harvell et al. 1990). Predation by nudibranchs also calls for rapid development of defensive structures (Harvell 1984), while on the south-west coast of the U.S., predation by fishes limits the lifespan of colonies to around 6 weeks (Yoshioka 1982). The life history strategy of P. delicatula, in contrast, seems predisposed towards slower growth, the maintenance of colony form over time, late reproduction, and very high eventual reproductive output, promoting lower rates of resource translocation to growth at colony edges. These inherent characteristics may in part explain the relative equality of growth rates across treatments, although further detailed research is required.
Competition from other invertebrates proved to be a significant factor in the survival, growth and ultimate reproduction of P. delicatula colonies at Queenscliff pier. Visual inspection of the assemblages surrounding target colonies showed abundant large sessile invertebrates – commonly colonial ascidians – at Queenscliff pier and much crowding of colonies. In contrast, at Flinders pier, competition appeared to be greatly reduced, and large sessile modular invertebrates were few. Competition is a major influence on the development and maintenance of sessile assemblages on hard substrata (e.g. Russ 1982; Buss 1990), and the competitive hierarchies of sessile assemblages have been well studied (e.g. Dayton 1971; Buss and Jackson 1979). Russ (1982) demonstrated that, in Port Phillip Bay, ascidians and sponges were effective in overgrowing bryozoan colonies, while bryozoans in turn win out over taxa such as tubiculous polychaetes and hydroids. In addition, Kay and Keough (1981) showed that the amount of free space, and the size of these clear patches, could also be crucial to development of assemblages, interacting with the capacity for vegetative growth of individuals to influence competitive outcomes on hard substrata. Lower apparent abundances of sponges and colonial ascidians at Flinders than at Queenscliff may have reduced the importance of overgrowth as a modifier of P. delicatula life histories at that site. In contrast, at Queenscliff, overgrowth was a major cause of mortality for colonies, outnumbering total zooid death and colony removal as causes of mortality. Moreover, incidences of partial overgrowth, where a colony’s live area is reduced by the encroachment of another sessile organism, also had significant impacts on the subsequent recovery and eventual reproductive output of colonies. Incidences of overgrowth reduced the total growth of colonies in a highly competitive
190 environment, and this in turn reduced the number of colonies that could reproduce. No colony that was fatally overgrowth by a competitor managed to produce embryos at any stage of the experiment, and as the number of overgrowth events increased per colony, the average growth rates and reproductive output of colonies decreased. P. delicatula appears to reproduce only when colonies have reached quite a large size and are relatively old (M. J. Keough, personal communication), so any variable that reduces growth, or limits the size that a colony can reach will have an adverse impact on the reproductive output of that colony.
Although colonies at both sites exhibited low total growth and fairly high rates of mortality, there were fewer incidences of overgrowth at Flinders, compared to Queenscliff pier, where overgrowth was a key mediating factor in colony recovery. This may be a result of Flinders pier supporting lower numbers of the major overgrowing species found at Queenscliff pier, such as didemnid ascidians, but this reduced rate of competition by other large modular species at Flinders did not lead to increases in growth rates or reproduction. In effect, while competition was reduced at Flinders pier, so too was the success of P. delicatula colonies, as reflected in the relatively low growth rates and absence of reproduction over the eight month period. This could indicate that the conditions for growth of colonial invertebrates were less favourable at Flinders pier than at Queenscliff pier, or that the colonies themselves were less viable at Flinders. Alternatively, high levels of competition at Queenscliff pier may put more pressure on colonies, forcing them to increase their level of investment in the maintenance of the colony and its defences in order to avoid overgrowth or invasion by competitors. Increased investment in maintenance may in turn result in the early induction of reproduction of colonies at Queenscliff, while colonies at Flinders were not subject to such pressure, and may have been yet to reproduce by the end of the experiment. These possibilities require further investigation, but the differences between colony responses at the two sites nevertheless highlight the importance of considering repeat experiments at multiple sites, while the unexpected responses to damage seen in these colonies emphasises the need to be careful about extrapolating patterns from well-studied species to other taxa.
191 Melbourne
Port Phillip Bay
Geelong
Q Westernport Bay
F
Figure 1. Location of study sites Queenscliff (Q) and Flinders (F) piers, relative to Melbourne and Port Phillip Bay, Victoria, Australia
192 1. Large control (≥ 30mm diameter) An undamaged large colony of 30 mm diameter or greater
2. Large fragment, edge intact A fragment taken from a large colony, with the growing edge left intact
3. Large fragment, edge removed A fragment taken from a large colony, but with the growing edge removed
4. Central fragment A fragment created from the centre part of a large colony, with all the growing edge removed
Small control (10–15mm diameter) An undamaged colony of diameter 10-15mm
6. Small fragment, edge removed (Flinders only) A small colony with the growing edge removed
Figure 2. Treatments used in the Parasmittina unispinosa fragmentation experiments at Queenscliff and Flinders piers. Treatments 1–4 all involved large colonies of greater than 30mm diameter; treatment 1 was a control, and treatments 2–4 were selectively damaged. The growth rates and reproduction of treatments 1–4 are compared. Treatments 5 and 6 involved small colonies less than 15mm in diameter, but only treatment 5 was implemented at Queenscliff due to insufficient numbers. For these reasons, treatments 5 and 6 were only used in comparisons of survival. All colonies were pre-reproductive at the time of damage.
193
1.0
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0 1 2 3 4 8 Months
treatment
Large control
Large fragment, edge intact
Large fragment, edge removed
Centre fragment Small control
Small colony, edge removed
Figure 3. Survival of Parasmittina unispinosa colonies in all treatments at Flinders pier over the eight months of the experiment.
194 20
15
10
5 Number of colonies
0 Zooid Overgrowth Removal death from substrate
Cause of death
Figure 4. Causes of death for all colonies (treatments combined) at Flinders pier. Total zooid death was noted as the presence of brown bodies within zooids across the whole colony, while total overgrowth and total removal were noted as the presence of another organisms, or empty space, over the area where the colony was present at the previous census.
195 a) 7000
6000 Treatment Large colony 5000 Large colony, Colony edge removed size 4000 Large colony, (mm2) edge intact Large central 3000 fragment
2000
1000
0 0 1 2 348
0.5 Months b)
0.0 Average net growth of colonies (mm2 -0.5 per month) treatment Large colony Large colony, -1.0 edge removed Large colony, edge intact Large central -1.5 fragment
Figure 5. Colony size over time (a) and the average level of colony growth per unit area (b), across treatments 1-4 at Flinders pier. All treatments originated from large colonies. Data shown are mean values, ± one standard error.
196
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Proportion colonies surviving 0.6
0.4
0.2
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0 1 2 3 4 8
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Large control Large fragment, edge intact Large fragment, edge removed Centre fragment Small control
Figure 6. Survival of Parasmittina unispinosa colonies in all treatments over the eight months of the experiment at Queenscliff pier.
197
20
15
10
Number of colonies 5
0 Zooid Overgrowth Removal death from substrate
Figure 7. Causes of death in colonies at Queenscliff pier across all treatments. Treatments have been combined. Total zooid death was noted as the presence of brown bodies within zooids across the whole colony, while total overgrowth and total removal were noted as the presence of another organisms, or empty space, over the area where the colony was present at the previous census.
198 12000 a)
10000
Colony 8000 size 2 (mm ) 6000
4000
2000
0 0 1 2 3 4 8
Months Treatment
Large colony Large colony, edge removed Large colony, edge intact Large central fragment 1.5 b)
1.0
Average 0.5 net growth of 0.0 colonies Treatment (mm2 Large colony per -0.5 month) Large colony, edge removed Large colony, edge intact -1.0 Large central fragment
-1.5
-2.0
Figure 8. Size over time (a) and the average growth rates of colonies across the experiment, scaled to colony size (b), of colonies in treatments 1–4 at Queenscliff pier. Data shown are mean values, ± one standard error. 199
0.003
0.002
Average 0.001 colony growth per unit area 0 (mm2/ month)
-0.001
-0.002 0 1 2 345
Number of overgrowth events per colony
Figure 9. The average net growth rate of colonies according to the number of overgrowth events that affected each colony in Parasmittina delicatula colonies at Queensliff pier.
200
Figure 10. Re-growth in Parasmittina delicatula, showing the formation of giant buds at the colony perimeter. The colony is completely surrounded by an unidentified species of sponge, whose perimeter abuts that of the P. delicatula colony. The formation of giant buds is one of the responses elicited by certain bryozoan species in response to competition.
201 Table 1. Number of colonies per treatment used in each test in analyses of variance at both Flinders and Queenscliff piers.
Total Analysis Treatment sample size 1 2 3 4 5 6 Flinders – survival 11 9 9 8 14 5 56 Flinders – size/growth 3 5 6 5 – – 19 Queenscliff – survival 8 8 8 7 16 – 47 Queenscliff – size/growth 10 8 5 6 – – 29
202 Table 2. Analysis of variance comparing the number of months that colonies survived at Flinders pier, according to treatment. Additional planned comparisons were conducted between: large colonies that had an intact edge (pooled treatments 1 and 2) and those that had their edge removed (pooled treatments 3 and 4), between undamaged large (treatment 1) and small (treatment 5) colonies, and between undamaged small (treatment 5) and damaged small (treatment 6) colonies. The power of the ANOVA test to detect a 50% difference in survival was 0.790.
Source of variation df MS F P Treatment 5 10.670 1.129 0.357 Planned comparisons (t1 + t2)/2 = (t3 +t 4)/2 1 12.697 1.344 0.252 t1 = t5 1 10.286 1.089 0.302 t5 = t6 1 0.019 0.002 0.965 Residual 50 9.447
203 Table 3. Differences in colony size (a), average growth (b), and average net growth after four months (c) between colonies in treatments 1-4 at Flinders pier. All traits were analysed using one-way analysis of variance, with treatment as a fixed factor. Planned comparisons were conducted between: colonies that had an intact edge (pooled treatments 1 and 2) and those that had their edge removed (pooled treatments 3 and 4), and between undamaged colonies (treatment 1) and central fragments (treatment 4). The power of the ANOVA test to detect a 70% difference in average net growth to four months was 0.602.
Source of variation df MS F P a) Colony size after four months Treatment 3 3769569.719 2.698 0.083 Residual 15 1397333.862 b) Average colony growth per month to four months Treatment 3 19685.185 0.238 0.868 Residual 15 82593.047 c) Average net colony growth to four months, scaled to colony size Treatment 3 0.126 0.454 0.718 Planned comparisons (t1+t2)/2 = (t3+t4)/2 1 0.327 1.180 0.295 t1 =t 4 1 0.334 1.208 0.289 Residual 15 0.277
204 Table 4. Analysis of variance comparing the number of months that colonies survived at Queenscliff pier, according to treatment. Additional planned comparisons were conducted between: large colonies that had an intact edge (pooled treatments 1 and 2) and those that had their edge removed (pooled treatments 3 and 4), and between undamaged large (treatment 1) and undamaged small (treatment 5) colonies. The power of the ANOVA test to detect a 50% difference in survival was 0.740.
Source of variation df MS F P Treatment 5 10.670 1.129 0.357 Planned comparisons (t1 + t2)/2 = (t3 + t4)/2 1 14.985 1.461 0.234 t1 = t5 1 0.188 0.018 0.893 Residual 50 9.447
205 Table 5. Differences in colony size (a), average growth (b), and average net growth over four months (c) between colonies in treatments 1-4 at Queenscliff pier. All traits were analysed using one-way analysis of variance, with treatment as a fixed factor. Planned comparisons were included to compare the average net growth of colonies after four months in two combinations; between large colonies that retained an intact edge (pooled treatments 1 and 2) and those that had their edge removed (pooled treatments 3 and 4). A further comparison was made between large control colonies (treatment 1) and central fragments (treatment 4). The power of the ANOVA test to detect a 50% difference in average net growth to four months was 0.536.
Source of variation df MS F P a) Colony size after four months Treatment 3 2417853.851 1.035 0.404 Residual 16 2336446.751 b) Average colony growth per month to four months Treatment 3 139981.262 1.216 0.324 Residual 25 115090.466 c) Average net colony growth to four months, scaled to colony size Treatment 3 0.021 0.508 0.681 Planned comparisons (t1 + t2)/2 = (t3 + t4)/2 1 0.316 2.774 0.110 t1= t4 1 0.460 3.999 0.057 Residual 25 0.042
206 Chapter 7
General Discussion
In unilaminar encrusting bryozoans, the shape and growth form of a colony can be indicative of the degree of colonial integration, but this may be modified through disruptions to the colony’s ontogeny, and may also be variable between species. Factors that influence the way a colony grows and its ability to recover from damage include the colony’s size and the maturity of the component zooids, and the strength of morphological and physiological connections between these components. Generally, the level of colonial integration in bryozoans has been inferred from the numbers and types of specialised zooids, or polymorphs, within a colony. However, this information is limited, and does not take into account features of the internal nutrient transport system, or any discrepancies in zooid function across colonies that may arise from the ontogenetic stages of zooids or their position within a developing colony. Moreover, in relatively simple colonies with generally monomorphic zooids and consequent low levels of polymorphism, changes in both the characteristics of individual zooids and in the pattern of zooids across colony regions may influence the ways that colony are able to utilise resources in order to grow and reproduce.
A colony of a modular invertebrate is generally defined as an arrangement of individual modules (polyps, zooids, etc.) that are physically connected, have common ancestry through asexual reproduction from a single primary individual, and are also, by definition, genetically identical. The exceptions to this, chimaeras generated through fusion, are discussed in Chapter 5. The individuality of modules versus that of colonies can, however, differ widely, and is usually defined by the level of integration between modules within a colony. Indicators of high levels of integration can differ between taxa, but a common consensus is that colonies with a higher proportion of specialised modules or zooids are more likely to be well-integrated, whereas those that are composed of largely independently functioning modules should generally have lower levels of physiological interdependence (Boardman et al. 1973; reviewed in Hughes 2005). In
207 corals, levels of apparent integration may range from taxa which exhibit only skeletal connections, and thus low integration, to siphonophore colonies which comprise numerous zooids that are highly specialised for different functions within the colony (Boardman et al. 1973). While levels of polymorphism may thus be useful in order to define broad patterns of integration across taxa, fine-scale information regarding a colony’s capacity for growth and reproduction may only be accessible through a detailed analysis of systems that can enable nutrient transfer between colony regions. These analyses may in turn be strengthened by observation of responses to processes which disrupt a colony’s normal pattern of development. In this thesis, I aimed to determine the levels of colonial integration in a number of species of encrusting bryozoan, by combining examinations of overall colony form and individual zooid properties with analysis of the specific internal nutrient transport system.
Within this thesis, I did most experiments using the cosmopolitan species Watersipora subtorquata; its simple colony construction and regular arrangement of zooids means that it was an ideal candidate in which to explore issues of zooid integration and co-operation. It is also a common species in the study regions of both Port Phillip Bay and Monterey Bay, settles readily on a range of artificial substrata, and is easily manipulated in the laboratory. These factors made it an amenable study species, while the detailed knowledge on colony construction and integration I obtained from the W. subtorquata work allowed me to investigate the generality of these principles in a number of other species with contrasting life histories.
In colonies of the simple cosmopolitan unilaminar bryozoan Watersipora subtorquata, there appeared to be little variation in zooid measurements across very small colonies (Chapter 2). This is consistent with both Abbott’s (1973) findings on changes in zooid characteristics across colony development in Hippoporina and Silén’s (1977) identification of a ‘zone of astogenetic change’ close to the ancestrula. However, as colonies became larger, zooids generally became longer and more elongated across colony ontogeny, while their width increased slightly. These changes in zooid parameters developed even in the absence of significant levels of zooid polymorphism in W.
208 subtorquata, and were likely to be more pronounced with further colony development.
These data suggest that partitioning of roles among zooids may vary with increasing colony size or maturity, but confirmation comes with the finding that, as colonies grew, there was a relative decrease in the amount of growing area accounted for by the active feeding zooids. Since developing areas of colonies are not yet feeding, resources must be translocated towards these areas by fully-developed zooids that are capable of both feeding and nutrient delivery to other areas of the colony. Levels of resource acquisition by feeding zooids within a colony may therefore be highly indicative of the potential for that colony to develop new tissue, while the position of these feeding zooids can also influence feeding rates. Where resources must be directed towards the colony edge, it is likely that zooids behind this edge may be very important to the growth potential of the colony, and although feeding rates in this study did not reflect these trends, evidence from previous fragmentation work (Hart 2001) suggests this is the case. In addition, changes in the relative amounts of feeding and developing areas could incur a shift in the ways that colonies utilise resources as they increase in size. As a consequence, small colonies probably have higher resource demands than larger colonies, and this was supported by small W. subtorquata colonies having higher rates of feeding than larger colonies (Chapter 2). This imbalance in resource use and acquisition across colonies of different sizes may interact with the other effects of colony size, contributing to the lower likelihood of survival seen among many modular invertebrate species when colonies are small. High rates of mortality are common in juveniles of sessile marine invertebrates (e.g. Keough 1986; Gosselin and Quian 1997; Hunt and Scheibling 1997) and may be caused by various factors including sedimentation (e.g. Bak and Engel 1979) and predation (e.g. Osman and Whitlach 2004), but it is likely that such high investment in continued colony development, as seen in the W. subtorquata colonies in this study, can also serve to deplete a young colony’s energy reserves.
Transfer of nutrients, between zooids that are feeding and non-feeding zooids that may be developing or regenerating, is necessary for continued colony growth, and may be a valid indicator of the level of integration between component zooids. The funicular system has
209 been proposed by many researchers as the most likely facilitator of nutrient translocation between zooids within a bryozoan colony (e.g. Lutaud 1961, 1983; Banta 1969; Bobin 1971, 1977; Chaney 1983; Best and Thorpe 1995, 2002), and several studies utilising radio-labelled foodstuffs have noted the transport of material across colony regions (e.g. Best and Thorpe 1995, 2002; Miles et al. 1995), and Lutaud (1983) observed lipid movement between zooids within the funiculus of Membranipora membranacea. The general form of the system components have been described in numerous studies, including histological analyses of cells within the rosette complexes and electron microscopy of the communication pores, but this study is, to my knowledge, the first examination of patterns of communication pores over colony regions, with a view to assessing levels of colonial integration. Examining pore morphology using scanning electron microscopy first reinforced the assertions by Banta (1969) and others that the morphology of these structures is strongly suggestive of their function as active transporters of nutrients. Second, an examination of patterns of communication pores across colony regions in three species where polymorphism was low showed that, in general, the predominant direction of nutrient transfer is towards the growing edge (Chapter 3). These results strengthen the prediction that the growing edge is an important resource sink in bryozoan colonies, a feature observed in other modular invertebrates, particularly scleractinian corals (Oren et al. 1997b, 1998).
The trend for resource transfer to be directed towards the edge of the colony may be strengthened in small or young colonies, where we have seen that the relative ratio of feeding and developing areas incurs a high demand for resources (Chapter 2). In addition, these young colonies may also retain a level of flexibility in the delivery of resources between zooids, and I noted many colonies comprising three or fewer zooids possessed pore connections between the ancestrula and the secondarily developed zooid that were not able to be assigned to either ‘incoming’ or ‘outgoing’ pore morphologies (Chapter 3). This might suggest a two-way flow of nutrients between these early developed zooids. Alternatively, the morphology of porous connections may be unrelated to function in young developing colonies. The strength of directional nutrient movement can also be weakened where colonies have been damaged, component zooids are older, or
210 reproduction is evident, and resources instead are diverted from the process of growing towards the provision of resources for embryo production, polymorph functioning, or to the regeneration of senesced polypides.
Overall, my examination of the connections between zooids via the funicular system in Chapter 3 suggests that the zooids themselves may be able to control the fate of their acquired resources through actively transporting or retaining energetic stores gained through feeding by their functional polypide. Furthermore, the development of connective communication pores is the product of a process that requires dissolution of the zooid wall by both adjacent zooids, and is most likely initiated by the donor zooid. While this is likely a programmed process, the possibility that there may be discrepancies between the energetic stores and demands of adjacent zooids within a colony, and that these could influence the level of communication that may take place, is an intriguing possibility, and raises questions of just how well integrated and co-operative individual zooids within bryozoan colonies are, with possible implications for studies of the evolution of coloniality.
When the growth or development of a colony is disrupted, the way a colony responds is also indicative of the level of communication and physiological integration between zooids. Where growth is at the colony edge, this edge becomes a strong resource sink, and the removal of that edge can have large consequences for the fitness of a colony. For example, damage to the developing area of colonies, or obstruction of growth at the edge may compromise colony fitness in corals (e.g. Wahle 1983; Ruesink 1997), lower reproductive effort in the arborescent bryozoan Bugula neritina (Bone and Keough 2005) and change the kinds of larvae it produces (Marshall and Keough 2004), and induce rapid onset of reproduction in the encrusting bryozoan Membranipora membranacea (Harvell and Helling 1997), while several encrusting bryozoan species show slower recovery when the edge is removed through fragmentation (e.g. Klemke 1993; Hart 2001). In this study, the repeated removal of the colony edge in Watersipora subtorquata resulted in a concomitant decrease in the number of embryos produced by the colonies. Similarly, M. J. Keough and D. J. Marshall (unpublished observations) found that crowding can
211 severely compromise fecundity in W. subtorquata: crowded colonies produced fewer, smaller embryos than uncrowded colonies. These effects are likely to have strong carry- over effects on population demography. In the present study, rates of module addition were also lower in colonies that had been damaged, and these results suggest that the repeated removal of the key resource sink had a strong effect on colony function (Chapter 4). The removal of the colony edge may also disturb the pattern of resource transfer throughout colonies. In W. subtorquata, this was indicated through a less structured pattern of reproduction across colony regions, and through the degradation and necrosis of the colony edge following repeated damage. Removing modules from specific colony regions also suggests that the predominant direction of resource-transfer patterns may be flexible and responsive to zooid removal; the creation of new edges can create new resource sinks, triggering reversals of porous connections, or the formation of new connections to aid the movement of nutritious resources towards the edge created by this zooid removal. In the present study, W. subtorquata colonies, which had been damaged so as to create new internal edges, were able to effectively reverse the direction of growth to regenerate new zooids within the newly cleared area (Chapter 4).
Patterns of growth in encrusting bryozoans may be modified through local damage to the colony, but they may also be modified externally by the presence of obstructions, changes in environmental conditions, intra- and interspecific competition, and internal factors such as module age, damage history and, as we have seen, the capacity to transport nutrients and resources towards the growing area. Many species show aggregative settlement and this close proximity can increase the level of intraspecific competition between colonies. This can reduce colony size (e.g. Harvell et al. 1990) or cause interference competition (e.g. Okamura 1985), aggressive reactions (e.g. Ayre 1982) or abnormal growth (e.g. Rinkevich and Loya 1985). Close proximity can also, however, increase the rates of fusion between compatible colonies in some colonial invertebrate species. In the present study, the results of pairing in Watersipora subtorquata were unclear, with neither colony ancestry nor colony size proving decisive factors in the direction or degree of growth (Chapter 5). Nevertheless, both size and genetic relatedness are influential to rates of fusion in other marine invertebrates (e.g.
212 Stocker 1991; Feldgarden and Yund 1992; Shenk and Buss 2005) and further careful study is likely to yield better information on the effects of close proximity in adult W. subtorquata colonies and in other bryozoans.
When Conopeum seurati colonies in an established monoculture were damaged, there were no effects of colony size on the level of regeneration of injuries. We might expect larger colonies to be more able to recover from damage due to a higher number of modules, and consequent higher feeding capacity and energy stores, so this finding contradicts general predictions on the effects of colony size on regeneration in other sessile modular invertebrates. For example, W. subtorquata colonies appear more likely to be able to regenerate lost tissue against the predominant direction of growth when colonies are larger (Chapter 4), whereas larger fragments of modular colonies often recover faster and have higher survival rates than small fragments (e.g. Smith and Hughes 1999, but see Wahle 1983).
Scanning electron microscopy analysis revealed morphological evidence of fusion between C. seurati colonies in a monoculture at all locations where contact borders were able to be examined. In this case, fusion may therefore, by increasing colony size, enable more effective recovery from damage, and can be a beneficial response to a crowded situation. Depending on the complexity of the tissue recognition system, fusion in compatible colonies may also be less energetically expensive to initiate and maintain than avoidance tactics such as the re-direction of growth away from competitors, aggressive strategies such as overgrowth, or the development of structural defences. We saw in Chapter 2 that some colonies were more likely to transfer nutrients between zooids via the funicular system than to engage all capable zooids in active feeding, suggesting the former process is less energetically costly. A similar trade-off may be operating in fusion interactions. The initiation of fusion between two adjacent colonies requires recognition of the colonies’ compatibility, the amalgamation of both external walls, and the formation of connective pores. These processes may in turn be less energetically costly than aforesaid competitive responses, which would necessitate the development of several new zooids or kenozooids such as spines. For fusion to remain a viable and low-cost
213 response to the close proximity of conspecific colonies, however, requires an efficient mechanism to recognise levels of compatibility between colonies. Mechanisms of tissue histocompatibility have been widely studied in tunicates, particularly those in the genus Botryllus (e.g. reviewed in Brown and Ecklund 1994; Rinkevich 2002). Chaney (1983) followed paired colonies of the encrusting bryozoan Thalamoporella californica and found evidence of fusion only where colonies were siblings (from the same maternal colony) or where edges of a single colony met after growing around an algal stipe. Where non-sibling colonies contacted each other, fusion was not achieved, and colonies re- directed growth in a vertical fashion. These results suggest that a compatibility mechanism is operating in this species, and similar investigations in other encrusting bryozoans would yield valuable information on the mechanisms of fusion across this invertebrate group. A further consequence of the development of an efficient tissue compatibility recognition system in modular invertebrates is that, where both aggregative settlement and fusion is likely between compatible colonies, particular genotypes may become spatially dominant within an area.
Work to this point suggests that the interaction of a colony’s size, its growth form, and the age of the component modules interact to determine the strength of nutrient transfer towards the growing colony perimeter in that colony, and its likelihood of damage recovery depends of both these factors and on the characteristics of the damage incurred. I applied these findings and ideas in the analysis of an experimental investigation of the effects of module removal from different colony regions on the growth patterns and reproductive output of the encrusting bryozoan, Parasmittina delicatula. Conducted under field conditions, the experiment involved damaging colonies in two size classes at the colony edge, and forming fragments that included and excluded this edge tissue. While I did not quantify the internal nutrient transport system in P. delicatula within Chapter 3, investigating the effects of module removal on this species helped to clarify whether the importance of the colony edge is common to many encrusting species, or limited to those studied within earlier parts of this thesis. In addition, it provided an opportunity to test these assumptions in an environment where local competition is not controlled. I found that the relative importance of source-sink mechanisms in the
214 recovery of damaged P. delicatula colonies decreased where competition was high, but also where environmental conditions did not appear to favour the growth of large sessile colonial invertebrates. When competition was high, colony growth was also high, but was mediated by the effects of overgrowth. Reproductive output also decreased under this intense pressure from competitors. In contrast, where competition was low, local conditions appeared to restrict the level of recovery and growth of both damaged and undamaged colonies, so that any effect of reduced competition was minimal. These results highlight the importance of considering multiple species and multiple sites in an evaluation of colonial integration in modular invertebrates, and certainly with respect to field studies.
In encrusting cheilostome bryozoans, the functional separation of zooids and the lack of a common circulatory system imply that the movement of resources within a colony may be limited. Rapid regeneration of injuries and the apparent reversal of the predominant direction of growth after damage to inner colony regions in W. subtorquata suggest that this may not be the case, at least in this species. In addition, the prevailing theoretical view is that low levels of polymorphism result in consequent low levels of integration between modules, but rapid regeneration and numerous porous connections in the monomorphic species W. subtorquata, M. membranacea and C. seurati suggest that high levels of integration may also be achieved in these species. All three of these species are cosmopolitan weeds, and their high rates of growth may be facilitated by high levels of integration between zooids and strong directional transfer of nutrients between zooids. In contrast, the two other species examined in this study, M. ellerii and P. delicatula, are species with higher levels of polymorphism, but the strength of directional nutrient transfer appeared lower, reducing growth rates at the colony perimeter. Integration between zooids within colonies of these species may still be high, however, with colonies able to reproduce across all colony regions, and P. delicatula showing no differences in recovery after differing levels of damage to the colony edge. In this thesis, I have incorporated information on external zooid measurements, internal communication pores, and patterns of resource acquisition across colony regions. I have found strong evidence to suggest that the capacity for resource transfer, and thus inter-zooid physiological
215 integration, may be very high in the presumably well-separated zooids within several species of encrusting cheilostome Bryozoa, but that these patterns may change according to a colony’s damage history or age. This work confirms that simple measures of integration based on the level of polymorphism are inadequate across multiple species of encrusting cheilostome bryozoans, and that further work detailing actual rates of nutrient transfer under different conditions is required to confirm levels of integration across disparate bryozoan taxa.
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Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Bone, Elisa K
Title: Colonial integration and the maintenance of colony form in encrusting bryozoans
Date: 2006-11
Citation: Bone, E. K. (2006). Colonial integration and the maintenance of colony form in encrusting bryozoans, PhD thesis, Department of Zoology, University of Melbourne.
Publication Status: Unpublished
Persistent Link: http://hdl.handle.net/11343/39230
File Description: Colonial integration and the maintenance of colony form in encrusting bryozoans
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