Some evolutionary and ecological implications of colour variation in the sea urchin Heliocidaris erythrogramma

by

Jane Growns B.Sc. Jt. Hons. (U.C.N.W., Bangor)

submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

University of Tasmania Hobart

December 1989 I hereby declare that this thesis contains no material which has been accepted for the award of any degree or diploma in any university and that, to the best of my knowledge and belief, the thesis contains no copy or paraphrase of material previously published or written by another person, except where due reference is made in the text.

Jane Growns TABLE OF CONTENTS Page

ABSTRACT

ACKNOWLEDGEMENTS I I

CHAPTER 1 GENERAL INTRODUCTION 1

CHAPTER 2 THE POLYMORPHISM, ITS PIGMENTS AND POSSIBLE GENETIC BASIS 7 2.1 INTRODUCTION 7 2. 2 DESCRIPTION OFTHE POLYMORPHISM 9 2.3 METHOOS 14 2.3.1 Combinations of pigmentation 14 2.3.2 Identification of pigments from the calcareous parts 1 6 2.3.3 Identification of echinochrome A and histology 1 7 2.3.4 Diet preferences among morphs 1 8 2.3.5 Colour change experiments 1 8 2.4 RESULTS 19 2.4.1 ·Variation in test and spine colours 19 2.4.2 Distribution of spinochromes among phenotypes 22 2.4.3 Identification of naphthoquinone pigments 2 5 2.4.4 Composition of the dermal pigment granules 3 0 2.4.5 Diet preferences among morphs 3 0 2.4.6 Colour change 3 0 2.5 DISCUSSION 33

CHAPTER 3 TEMPORAL STABILITY OF MORPH FREQUENCIES 3 7 3 .1 INTRODUCTION 3 7 3.2 METHODS 39 3.3 RESULTS 42· TABLE OF CONTENTS (continued) Page 3.3.1 Stability of morph proportions during the study 4 2 3.3.2 Variation in dermis colour proportions between size classes 4 9 3.4 DISCUSSION 6 1

CHAPTER 4 GEOGRAPHICAL VARIATION IN MORPH FREQUENCIES AND ENVIRONMENTAL ASSOCIATIONS 64 4.1 INTRODUCTION 64 4. 2 MATERIALS AND METHODS 68 4.2.1 Collection of data 68 4.2.2 Analysis of data 74 4.4 RESULTS AND DISCUSSION 82 4.4.1 Small scale geographic variation in proportions of morphs 8 2 4.4.2 Distribution of morphs over entire study ar ea 9 0 4.4.3 Spatial patterns in dermis colour . proportions 9 9 4.4.4 Association between dermis and spine colour 99 4.4.5 Relationships between dermis colour and environmental data 102 4.4.6 Evidence for processes affecting population differentiation 1 07 4.4.7 Water currents within and between the geographical regions 1 1 7 4.5 GENERAL DISCUSSION 1 1 9

CHAPTER 5 VARIATION BETWEEN MORPHS IN MORPHOLOGY, MICROHABITAT, REPRODUCTION AND TUBE FEET STRENGTH 1 24 5. 1 INTRODUCTION 1 2 4 5.2 METHODS 1 26 5.2.1 Morphometr ies and meristics 126 TABLE OF CO NTENTS (continued) Page 5.2.2 Microhabitat and behavioural variation I 29 5.2.3 Reproductive cycle and investment I 31 5.3.4 Tube feet strength experiment 1 31 5.3 RESULTS 132 5.3.1 Morphometries and meristics 132 5.3.2 Microhabitat and behavioural variation I 34 5.3.3 Reproductive cycle and investment I 52 5.3. 4 Tube feet strength experiment 152 5. 5 DISCUSSION 158

CHAPTER 6 GENERAL DISCUSSION I 61

REFERENCES 169

APPENDICES 179 Appendix I Laborato ry breeding trials. 179 Appendix 2a Dermis colour data for all sites I 89 Appendix 2b Morph data for all sites I 90 Appendix 3 Environmental data for all sites 1 91 Appendix 4a Means, standard errors and sample sizes for morphometric and meristic variables, for red and white dermis urchins and pooled data from Tinderbox 192 Appendix 4b Means, standard errors and sample sizes for morphometric and meristic variables, for red and white dermis urchins and pooled data from Ling Reef. I 9 3 Appendix 4c Means, standard errors and sample sizes for morphometric and meristic variables, for Fortescue Bay urchins. 1 94 Appendix 4d Means, standard errors and sample sizes for morphometric and meristic variables, for red and white dermis urchins and pooled data from Cowrie Pt. 195 ABSTRACT

An investigation into the evolutionary and ecological implications of variation in the external colou ration of the sea urchin Hefiocidaris erythrogramma was made. Two different pigment systems create a complex polymorphism; red granules of echlnochrome A in the dermis occur in varying densities, and purple and green naphthoquinone pigments are found in the calcareous test and spines. Many morphs may occur within one population, but the proportions of morphs vary markedly between sites. Evidence from the observed variability and chemistry of the pigments strongly indicates that the variation has a genetic basis. Breeding studies which would have resolved this question were unsuccessful, but did show that all crosses between morphs developed and metamorphosed successfully. Repeated sampling of 15 sites showed that morph proportions were stable at most sites over the 35 months of the study. Geographic variation in the proportions of morphs was determined from samples from 49 sites. Environmental variables were recorded and the exposure of each site to wave action was estimated using algal communities to develop an Algal Exposure Index (A.E.I.). Stepwise linear regression analysis indicated that the A.E.I. and amount of algal cover were the only environmental factors noted that were useful predictor? dermis colour proportions. Five hypotheses were developed (two selective and three stochastic) of processes which might be affecting morph proportions in the study area; these were tested using Mantel's non-parametric test. The results suggest that four geographical regions each ·have different patterns of morph distribution which are controlled by unique combinations of selection (related to exposure) and gene flow. These results are generally supported by what is known of water currents in each region, as most gene flow in H. erythrogramma will occur due to movement of pelagic larvae. Morphological data showed slight differences between urchins of different dermis colour at one site, but no differences between urchins with different coloured spines. There were significant differences between urchins at different sites. Surveys of urchin microhabitats indicated that (I) urchins of the same dermis colour tend to occur next to each other, (2) white dermis urchins tend to occur under rocks more often than red dermis urchins, and (3) urchins which are hidden under rocks tend to 'cover' with pieces of shell, algae or pebbles to a lesser extent than urchins which occur on the upper surfaces of rocks. A laboratory experiment indicated that, although the podia (tube feet) of red and white dermis urchins were initially of comparable strength, red dermis urchins tended to tire more quickly. No differences between morphs were found in the time of maturation of gonads or the size of gonads relative to body weight. ACKNOWLEDGEMENTS

I gratefully acknowledge all the people thq.t helped me in the preparation of this thes is. Peter Whyte, who suggested the project in the first place. Mike Bennett, Paul Cramp, Mike Driessen, Andrew Fleming, lvor Growns, Premek Hamr, Lee Hamr, Rowan Hug hes, Paul Humphries, Jean Jackson, John Kalish, Klobs, Ron Mawbey, Peter Mooney, Sarah Munks, Mark Nelson, Dominic O'Brien, Steve Reid, Sean Riley, David Ritz, Craig Sanders on, Andrew West, Peter Whyte, Gus Yearsley and especially Richard Holmes, who acted as diving partners and field assistants and without whom this project would not have been possible. Andrew Constable, for col lecting the data from Port Phillip Bay for nie and for comments on part of the thesis. Richard Holmes, Wayne Kelly, Ron Mawbey, and Barry Rumbold, for technical assistance both in the field and in the laboratory. The Tasmanian Department of Sea Fisheries, for the use of their aquarium facilities. Dr. Adrian Blackman, for the use of facilities in his laboratory and helping me understand the chemistry. Charlie Dragar, without whom I might not have survived the chemistry. Glen McPherson, Paul Humphries, Bob Black, Mike Johnson, Alastair Richardson, and Kit Williams, for statistical and computing he I p. Craig Sanderson, for help in algal taxonomy and developing the AEI. Adrian Bradley, for help with the histology. Bob Black, Paul Humphries, Mike Johnson, and my supervisor, David Ritz, for careful and constructive criticism of the manuscript. I would like to thank the Department of Zoology of the Univers ity of Western Australia and particularly Bob Black and Mike Johnson for hosting me during the writing of the thesis. All my friends who supported and encouraged me, especially Sarah and Jean, Prem and Lee , and Humphries l suppose. Special thanks must go to lvor for making it all worthwhile.

II 1

CHAPTER 1 GENERAL INTRODUCTION

It is now generally accepted that most visible and some non-visible polymorphic variation is subject to natural selection. Genetic polymorphism, when two or more discrete morphs of a species occur together (i.e. excluding seasonal variation and isolated populations which differ from the rest of the species), was defined by Ford (1945). Ford argued that genetic polymorphisms could only arise and be maintained by the action of selection. Previously the extensive shell colour and banding polymorphism of the landsnail Cepaea nemoralis had been used as a classic example of non-adaptive vari at ion (Mayr, 1942). However, further studies revealed not only that different morphs occurred in higher proportions in habitats where they were cryptic (Cain and Sheppard, 1950), but that visual selection by thrushes against the less cryptic morphs was indeed occurring (Sheppard, 1951 ). A large number of subsequent studies have shown that the situation is far more complex than was at first thought and at least six selective processes are now known to affect the C. nemoralis polymorphism, several of which rnay be acting on one population (reviewed in Jones et at. , 1977 and Goodhart, 1987). Random (stochastic) processes are also known to affect visual polymorphisms with founder effects and bottlenecks being demonstrated for colonies of the butterflies Melitaea aurinia and Mania/a jurtina (Ford , 1975) and C. nemoralis (Goodhart, 1962). The relative effects of genetic drift versus natural selection in affecting variability in natural populations has been the subject of much discussion, however, it is now generally agreed that genetic drift will only be important when effective population sizes are very small or if populations are subject to periodic extinctions and recolonisations (Goodhart, 1987). Most of the early work on genetic polymorphisms was carried out on lepidopterans, such as Panaxia dominula (Ford, 1975) and Biston betularia (Kettlewell, 1955, 1958), and the landsnail Cepaea spp. (Cain and Sheppard, 1 950, I 954 ). Ford developed the mark-recapture technique which allowed direct estimates of selection pressure acting on the lepidopterans to be made, although the agent of selection was unknown (Ford and Sheppard, 1969). The industrial melanism in B. betularia is an example of how the visual effects of a polymorphism may be selected for and showed that strong selection could rapidly change populations (Kettlewell, 1973). Cepaea nemoralis again showed the importance of the visual differences among morphs, as well as physiological and thermal differences, but it soon became clear that selection could not explain all of the observed patterns 2

(Jones et a/., 1977). In each of these species the effective population size is small because of the limited mobility of the adults and larvae and variability of recruitment. Thus random processes may periodically override the effects of any selection which would normally control the polymorphism . Adults of many species of marine invertebrates may often be sessile or of limited mobility but often have planktonic larvae which may travel great distances because of ocean currents. Thus the effective population size of these species is greatly increased and the likelihood of random processes overriding selection is much less (Janson, 1987). Recently there has been much interest in polymorphic marine snails, particu larly littori nes. The shells of these snails vary morphologically among habitats but the polymorphic variation is restricted to colour and in some cases sculpture. Several studies have found evidence tor selection in maintaining the colour polymorphisms. Selection for crypsis was suggested by the correlation of shell and substratum colour for Littorina rudis and L. nigrolineata (Heller, 1975). A combination of selection for crypsis and apostatic selection was indicated for L. rudis and L. arcana (Atkinson and Warwick, 1983), Littorina sp. (Hughes and Mather, 1986), and Littoraria filosa (Reid, 1987). A balance between crypsis and the density of snails in a habitat was

proposed tor L. mariae by hypothesizing that the visual predator, a blenny r preys preferentially on conspicuous morphs and secondarily in microhabitats where high densities of snails occur (Reimchen, 1979). Cook (1986) found that dark morphs of Littoraria pallescens move to more protected microhabitats on the lower surfaces of leaves when temperatures are high, probably because their internal temperature rises faster and higher than that o t paler morphs. This also gives them a cryptic advantage as the lower surfaces of leaves are darker, whereas paler morphs are cryptic on the upper surfaces. Most of the above species, however, do not have planktonic larval stages (Mileikovsky, 1975) and therefore have small effective population sizes. Selection can therefore affect the proportions of morphs in localised areas but random processes, such as founder effects, may cause differences between two populations to arise, even if the same selective forces are operating on both. Littoraria filosa is an exception and does have a planktonic larva although the extent of its dispersal is unknown (Reid, 1987). Reid contends that strong visual and apostatic selection are the mechanisms by which the polymorphisms must be maintained in the face of extensive gene flow. Colour variation both within and among populations appears also to be widespread among echinoids (Table 1.1 ) . In all cases where test colour variation is described, however, it is not certain whether this 3

Table 1.1 Known colour variation in echinoids. Where spines were multicoloured, the proximal colour is given first followed by the distal colour. Where different morp'hs of the same species were described by different authors, the location is given.

Species and source Test Spines

Psammechinus miliaris brown ish-green violet tips Lindahl and paler grey-g reen violet tips may Runnstr o m ( 1929) extend almost to base

Strongylocentrotus dark purple pale purple droebachiensis green Vasseur ( 1952)

S. droebachiensis dermis is purple to white, slightly violet Jensen (1974) dark violet or o I ive-g reen almost black

S. pallidus red brown Vasseur (1952) whitish-g reen green-brown bluish-purple

S. pal!idus . pale green olive green Jensen (1974) greenish-brown olive brown reddish-pur pie amethyst-violet

S. intermedius dark violet olive green Jensen (1974) pale reddish-brown

Lytechinus variegatus green (solid or green variegatus mottled) green and purple Serafy (1973) white green and red white and purple

L. v. at/anticus purple purple Serafy (1973) green green red red green and purple green and red 4

Table 1.1 {continued) Known colour variation in echinoids.

Species and source Test

L. v. carolinus red red Serafy (1973) white

Paracentrotus /ividus purple-blue purple-blue Gamble (1967) red-brown red-brown ye llow-bro wn yellow-brown

Echinometra lucunter black or bluish black black or bluish black McPherson (1969) bright red apical area bright red apical area Florida dull red dull red

E. lucunter dark purple, brown or Tsuchiya and Nishihara green-brown with (1984) Japan white tip and base. dark all dark or green­ brown, less distinct white base.

E. mathaei pink pink Lawrence (1983) black black Gulfs of Aqaba and Suez

E. mathaei dark red pale pink through to J. Prince (pers. comm.) dark purple-brown Western Australia green 5 refers to the colour of the overlying dermis, which is likely to affect the appearance of living specimens, or to the colour of the underlying calcareous test. Colour variation within pop�lations has been described for Strongy locentrotus droebachien sis and S. pallidus (Vasseur, 1952 ; Jensen, 1974) , Echin ometra lucunter (McPherson, 1969), E mathaei (Tsuchiya and Nishihara, 1984; J. Prince, personal commun·ication), three subspecies of Lytechinus variegatus: at/ant icus, caro/inus and variegatus (Serafy, 1973; Pawson and Mi!!er, 1982 ), and Paracentrotus lividus (Gamble, 1967) . However, in all but one of these examples (Tsuchiya and Nishihara, 1984) it is not known whether morphs occur in different microhabitats or differ behaviorally. Geographical variation in colour was reported for S. droebachiensis and S. pallidus (Vasseur, 1952 ; Jensen, 1974) and obviously occurs in E. mathaei because of the different morphs found in Western Australia (J. Prince, personal communication), Japan (Tsuchiya and Nishihara, 1984) and the Gulfs of Suez and Aqaba (Lawrence, 1983) . Almost all echinoids have planktonic larvae (Moore, 1966) and for the majority of species the dispersal phase may last several weeks or even months. The ecological implications of the colour variation are unknown except for limited work on Psammechinus miliaris and on Echinometra mathaei in Japan. These studies showed that two morphs of P. miliaris occur in different habitats and that there are morphological and· behavioural differences between them (Lindahl and Runnstrom, 1929). Tsuchiya and Nishihara (198 4, 1985) found that two morphs of E. mathaei occur together but prefer different microhabitats and show different levels of agonistic behaviour both within and between morphs. Tsuchiya et al., (1 987) observed an occasion when exceptionally high wat er temperatures caused mass mortality of the urchins and it is possible that there was differential mortality between the morphs because of their microhabitat separation. There have been no studies which have addressed the evolutionary processes through which such variation might have originated or be mai ntained in the face of extensive gene flow. Heliocidaris erythrogramma is a regular echinoid which forms a major component of the rocky subtidal and seagrass communities around Tasmania and south-eastern Australia. It reaches a maximum test diameter of 125 mm in southern Tasmania where it occurs in densities of up to 15ur chins m-2. It shows extensive colour variation of the calcareous test and spines, and the dermis, which overlies the test, varies from red to white and gives the colour of the main body of 6 the live animal. The latter is of particular interest because such variation has not been described for any other echinoid, although Jensen (1974) says that the dermis· of Strongylocentrotus droebachiensis varies from purple to dark violet. H. erythrogramma is unusual among echinoids in having lecithotrophic larvae which settle after about a week in laboratory conditions. Thus larval dispersal will be restricted in comparison to other urchins but is still likely to be extensive. This study provides a detailed description of the colour variation of H. erythrogramma both within and among populations and identifies the pigments involved. The evidence for the genetic basis of the variation is assessed, but breeding studies which would have unequivocally answered the question were unsuccessful. The stability of proportions of morphs within populations over the time period of the study was determined to validate the use of samples collected over almost three years in a study of geographical variation. The major portion of the work involved a survey of the distribution patterns of proportions of morphs in south eastern Australia and correlations of the patterns with e nviro nmental variables. These data were used to assess models of both selective and stoch'astic processes which might be affecting the variation. Morphological, microhabitat and physical variation among morphs was investigated in orde.r to develop specific hypotheses about the selective forces which might be acting on the urchins. The evolutionary processes which may be maintaining the variation are discussed and related to other studies of visual polymorphisms. 7

CHAPTER 2 THE POLYMORPHISM, ITS PIGMENTS AND POSSIBLE GENETIC BASIS

2.1 INTRODUCTION

lntra�specific variation in the colours of echinoids, particularly shallow water forms, is well known (Serafy, 1973) and is often used for taxonomic studies (Clark, 1923, 1938; Mortensen, 1943 ; Jensen, 1974) and field identification (Hagstrom and Lanning, 1961; McPherson, 1969). However, most descriptions in the literature are confusing or incomplete. The colours of the spines, and sometimes the tests, are described but the extent of variation in intensity of pigmentation is usually unclear (McPherson, 1969; Serafy, 1973; Lawrence, 1983; Lewis and Storey, 1984; Tsuchiya and Nishihara, 1985). The numbers of urchins observed may be small and thus the proportions of different spine colours in natural populations are unknown (Clark, 1923; Mortensen, I 943; Jensen, 197 4 ). Occasionally the colour of tube feet 1974) but the dermis is usually ignored or may be confused with test colour (Vasseur, 1952; Serafy, 1973; McPherson, 1969; Lewis and Storey, 1984; Tsuchiya and Nishihara, 1985). This may be because species were described from preserved or dried specimens in many of the older works (Mortensen, 1943; Clark, 1938), in which case the dermal pigmentation would have been lost. The dermis plays a major role . in the polymorphism of living H eli ocida ris erythrogramma and the lack of descriptions of variation in dermal pigmentation for other species indicates that this is unusual among echinoids. The calcareous tests and spines of echinoids are usually coloured in various shades of purple, red, green and brown, although occasionally white specimens do occur. These colours are caused· by the presence of the calcium salts of naphthoquinone pigments in the test and spines (Thomson, 1957; Goodwin, 1969). These pigments have been given the trivial name of spinochromes, with different pigments being given suffixes A, B, C etc. in the order in which they were first isolated. By the mid-1 960's it was thought that there were only five spinochromes (A-E), however it was later found that two Echinothrix species contained at least 30 pigments, either naphth oq uinones or closely rei ated compounds (Moore et a/., 1966). Many of these have now been found in other species, however, they occur in small quantities (Kol'tsova et a/") 1977) and consequently are probably of minor importance to the colour of the urchins. Naphthoquinone pigments are unique to echinoderms in the Animal Kingdom, 8 although they are widespread among plants. The origin of the pigments has not been fully determined but they are known to be synthesized de novo by the urchins (Salaque et a/., 1967). The soft parts of echinoids, such as the dermis, podia, pedicillariae, gonads, gut wall, and coelomic fluid, also contain various types of pigments. Of these only those which are external to the test affect the appearance of the urchin. The dermis covers the test, podia and pedicillariae and consequently the same pigments occur in all three areas. Red-brown and black dermal pigment granules have been described for the diadematoid urchins Diadema spp. (Millott, 1964) and Cent rostephanus longispinus (Gras and Weber, 1977). Millott (1 964) found that the granules contained a mixture of echinochrome A, a melanin, chromolipid and an unknown iron-containing pigment. Echinochrome A, a naphthoquinone very similar to the spinochromes, occurs in the dermis and coelomic fluid of many species and is common in developing et a/. , 1983). de novo larvae (Bay It is syn.thesized by the embryo from tyrosine and acetate (Asashima, 1971 a and b) and is thought to be involved in lipid peroxidation (Kol'tsova et a/., 1981 ). It has also been reported in small quantities from the spines and tests of some species but it is unclear if this is due to contamination by dermal tissue (Thomson, 1957). It is not known whether the production and distribution of these pigments are genetically or environmentally determined in H. erythrogramma. Keeping young Diadem a antil/arum under different light regimes has been shown to affect their pigmentation (Kristensen, 1964); those kept in the dark did not develop the completely black dermis of those kept in ambient light and 40% became white. However, variation in test and spine colour of geographically distant populations of Lytechinus variegatus has been shown to be genetically determined (Pawson and Miller, 1982). Differences in diet were suggested as the reason for colour differences between Echinometra /ucunter at different sites (Lewis and Storey, 1984) but many morphs of H. erythrogramma may be found at one site, so the different morphs would have to be highly selective in their feeding if pigmentation was determined by diet. In this chapter the variation in dermal and calcareous pigmentation which occurs in natural populations of H. eryth rogramma is described and the pigments involved are identified. The possibilities of colour change due to different light regimes and of selective feeding by the different morphs are addressed, and the hypothesis that the polymorphism has a genetic basis, rather than being caused by environmental influences, is discussed. 9

2.2 DESCRIPTION OF THE POLYMORPHISM

There are two different pigment systems involved in the colour polymorphism of Heliocidaris eryth rogramma; one in the dermis and one in the calcareous test and spines.

Vari;ation in QQIQurof dermis Red granules occur in the dermis, tube feet and pedicillariae; these may occur in very high densities, giving the body of the urchins a dark red colour (R), or in very low densities when the live urchin's test appears white (W). These granules start to appear 18-24 hours after fertilisation (personal observations) and are easily visible on newly metamorphosed urchins (Plate 2.1 ) . Occasionally there are urchins which are an intermediate pink (p); these are rare in southern and eastern Tasmania ( <1% of the population) but more common on the north coast of

Tasmania and in Port Phillip Bay l Victoria (see Chapters 3 and 4). Podia and pedicillariae were the same colour as the dermis overlying the test on every urchin observed duri ng the study.

Variation in testan d spine colour In the calcareous test and spines there are several pigments which give purple (P), green (G) and brown (B) colours, although occasionally white (W) is seen (no pigmentation). The brown pigmentation is visible on relatively few urchins and as it is always associated with green pigment, it has not been included in the analyses presented later. The test pigmentation is nearly always obscured by the dermal pigmentation, so only the spine colours can be seen in live animals. The spines may be all one colour or they may have several pigment bands, in which case the colour at the base of the spine is denoted by the first letter and colours further up the spine by subsequent letters (e.g. PBG). For PG spines, the purple is always at the base of the spine, then there may or may not be an area which is brown and the tip is green. Pigmentation of the test also varies but this can only be scored for dead animals which have had the spines removed. Test colour varied from white or pale green to dark green, purple or both. When both purple and green were visible the colours appeared either in different areas of the test or the whole test might be a greeny-purple. 10

Plate 2.1 Juvenile of Heliocidaris erythrogramma showing red pigment granules. 11

Notation for the variation The large number of possible morphs described above is unwieldy and would require very large sample sizes to allow any statistical analyses to be made. Therefore morphs were combined in three different ways: by dermis colour, spine colour and overall morph (Plate 2.2; Table 2. 1 ).

Table 2.1 Notatio n fo r overall morphs. Spine colour p G PG w

R RP * RPG RW Dermis colour w WP WG WPG ww

(p pP pG pPG pW) - rare morphs

* Many RPG urchins were examined under a dissecting microscope to determine whether a purple ring was present at the base of the spine. In almost all the urchins examined a purple ring was visible. RG urchins probably occur in very low frequencies, but as it was not possible to determine in the field whether the purple ring was present; all such urchins were scored as RPG. All of the above morphs could be easily distinguished except on the north coast of Tasmania where dermis pigmentation was almost continuous (Plate 2.3). Red dermis urchins could be distinguished, but white urchins were rare and pink dermis urchins varied considerably.

Addedco mplications The polymorphism in the live animals is more complicated than described so far: PG spines may have a discrete ri ng of purple at the base or the purple may extend varying distances up the spine (Plate 2.4 ). If any extension of the purple was noticed a 1+' was added to the colour code for the spine (i.e. PG+). The other major variation is in the intensity of the pigmentation, which varies from very dark to barely visible for each pigment. This was scored by the darkest colours being denoted by upper case letters and anything noticeably paler by lower case. For example, an urchin with a white test colour and pale purple spines was scored as Wp. There was some difficulty in distinguishing between WW and Wg urchins and this may be an unnecessary distinction. Thin-layer chromatography on WW urchins did show very small amounts of pigment to be Plate 2.2 Six of the rnost common colour morphs of

Heliocidaris erythrogramma in Tasmania. 12 13

Plate 2.3 Urchins from the north coast of Tasmania showing continuous variation of dermal pigmentation

Plate 2.4 Urch in from the east coast of Tasmania showing PG+ spines. 14

present, thus true WW urchins may not occur. Urchins were only scored as WW if no purple or green pigmentation could be seen by eye.

2.3 METHODS

2.3.1 Combinations of pigmentation

To determine how dermis and spine colours were associated and how test colours vary between individuals within each overall morph, animals from four sites were examined in detail (Figure 2.1 ). These sites were chosen so that two (Tinderbox and Ling Reef) had highly variable populations whereas the other two had a restricted number of morphs present (Fortescue Bay has almost all white dermis urchins and Marion Bay nearly all red dermis urchins). All the urchins in an area were taken using SCUBA equipment from Tinderbox, Ling Reef, Fortescue Bay and Marion Bay, until about 15 individuals of the most common overall morphs present had been collected, (83, 78, 30 and 45 urchins respectively) and scored for their phenotype using the scheme outlined above. Test pigmentation is only visible when spines and dermis have been removed so they were soaked in 0.5% sodium hydroxide solution for 24 hours and dried at 60°C overnight. Spines and remaining muscle tissue were separated from the tests using a hard brush. A detailed examination of test and spine colouration was made for each urchin. The proportions of different morphs at these sites will be discussed in Chapter 4; in this chapter the variation within each morph (RP, RPG, WP, WG, pP etc.) will be considered. The degree of association between dermis and spine colour was determined using G-tests for independence for the data from Ling Reef and Tinderbox only, as the other two sites were nearly monomorphic for dermis colour. Dermis colour was categorised as 'white' or 'red', with pink dermis lumped with white dermis urchins, and spines were categorised as 'purple' or 'not purple'. Other associations are possible as four spine types had been identified, but the numbers of urchins in at least one category would have been too small for the test to be performed. For example, white spines are rare for both red and white dermis urchins and green spines were almost never observed on red dermis urchins. Echi noids have large primary spines and smaller secondary spines (Fell and Pawson, 1966); the secondary spines of H. erythrogramma are very small, 5 mm in length or less, whereas the primary spines may be up to 40 mm in length. The colour of the secondary spines is often different to that of the larger splne� 15

VICTORIA

··' N

•' .

Bass Strait

TASMANIA

Ling Reef

100km

Figure 2.1 Map of study area. 16

Secondary spines are visible on close inspection but because they are so much smaller than the primary spines they have a negligible effect on the ove rail appearance of the urchin. The colour of secondary spines was noted on two occasions for Tinderbox and Ling Reef and one occasion for Fortescue Bay, using urchins which were dissected for another part of the study (see Chapter 5).

2.3.2 Identification of pigments from the calcareous parts

Thin-layer chromatography (TLC) was used to determine the number of pigments involved and how they were distributed among the different phenotypes. Preparative chromatography was used to obtain sufficient pigment for nuclear magnetic resonance and mass spectroscopic analyses. These were used to identify the pigments by comparison with results in the literature. Tests and primary spines were prepared as described above (2.3.1) and tests were broken into small pieces.

Thin-layerQh romatography Spines and tests were analysed separately and for each individual. Pigments were extracted and analysed using the method described by Chang and Moore (1971 ). Approximately 10 g of spines or the entire test were gradually dissolved in concentrated hydrochloric acid ( 2.5 m I per g of material). The red or purple solution was then filtered through acid-washed celite to remove any tissue debris. Sodium bicarbonate was added in small amounts until the naphthoquinone pigments were converted to their sodium salts and the solution turned black. Lipids which might interfere with the chromatography were removed by extraction with ether. The solution was then acidified with the minimum amount of concentrated hydrochloric acid until it regained its initial colour. The pigments were repeatedly extracted with ether which was then dried for several hours using anhydrous sodium sulphate. The ether was evaporated off using a vacuum condenser and the pigments were redissolved in a small amount of ether for transfer to the chromatographic plate.

The plates (Merck silica gel 60 F- 254) had been dipped in 0.5M hydrochloric acid and air dried. The characteristics of the plates meant that reproducible Rf values were not obtained. Therefore pigments from PG spines were run on each plate to allow comparisons to be made between plates. Pigments from each individual were run on three separate plates to determine whether consistent results were obtained. Chromatograms were run using 5% methanol I dichloromethane as the solvent. 17

Preparativech romatography In order to isolate enough pigment for identification, 1 kg of PG+ spines were treated as above and separated using preparative chromatography. The 25 x 100 em plates were made up with silica gel in dilute hydrochloric acid and air dried overnight. The plates were run in 5% methanol I dichloromethane for 2 hours and allowed to air dry. The three major pigment bands were scraped off the plates and eluted from the silica gel using ethyl acetate. The pigments were purified by recrystallisation from hot methanol, methanol and chloroform, or hot toluene.

Anal�sisof purifiedpigments Nuclear magnetic resonance spectroscopy (NMR} is a technique whereby a purified sample, dissolved in a suitable solvent, is placed in a strong magnetic field and irradiated with radio frequency radiation. It is .used to detect certain nuclei, most commonly 1 H or 13C nuclei. Each of the 1 H (or 13C) atoms is uniquely affected by the other atoms surrounding it and thus gives a unique signal which is measured as o, a proportion of a standard frequency of radio waves. Comparison with spectra produced by known compounds allows the structure of the compound to be determined. The NMR spectra obtained from 1 H nuclei may be integrated to obtain the relative numbers of protons represented by each peak. Integration in a similar fashion is not possible for 13C spectra. Although the 1 H spectra of spinochromes (naphthoquinones found in the calcareous parts of echinoids and a few other echinoderms} are well understood, I know of no published 13C-NMR spectra. Pigments were dissolved in deuteriochloroform or dimethyl sulphoxide (DMSO-d6} for nuclear magnetic resonance spectroscopy. Both 1 H and 13C spectra were determined. Mass spectroscopy was also used for confirmation of the pigment structures.

2.3.3 Identification of echinochrome A and histology

To determine whether echinochrome A was present in the dermal granules of H. erythrogramma a crude extract of the dermal pigment was prepared and analysed using light spectroscopy. Histological techniques were used to detect the presence of a melanin and iron�containing pigments. Echinocrome A was extracted using the method of Bay et at. (1 983). A freshly killed red dermis urchin was placed in a beaker containing acidified ethanol (1 part 25% HCI and 3 parts 95% ethanol} and shaken for 10 seconds. The resulting bright orange solution was filtered and diluted before being 18

analysed using a Shimadzu UV- 1 20-02 spectrophotometer. A reading was taken every 10 nm from 250 to 500 nm. A sample of pure echinochrome A, (obtained from Professor Thomson, University of Aberdeen) was dissolved in acidified ethanol and its spectrum determined in the same way. Histology was performed on tube feet and peristomial membranes, both of which are covered by dermis, from white and red dermis urchins. These tissues were used because the dermis over the test is a single layer of cells which is difficult to separate. Tissue was fixed in 10% formalin for 24 hours and then transferred to 70% alcohol. Mallory's triple staining technique was used to observe the distribution of pigment granules. Schmorl's ferricyanide method (Humason, 1 967) was used to determine the presence of melanin, together with the suggested control to determ ine whether ferrous iron is present. Lillie's ferric ferricyanide technique (Humason, 1967) was also used to detect the presence of melanin as this technique is considered to be highly specific for melanins (Lillie, 1957).

2.3.4 Diet preferences among morphs

To determine whether the morphs fed on different macroalgae, urchins were sampled at Tinderbox on 25 i 88. The morphs were identified underwater (only those which were unambiguous were used) and if a piece of alga was present between the teeth of the urchin it was removed and placed in a plastic bag labelled for that morph. Algae were identified later in the laboratory. Although about 150 urchins were observed by two divers, only 50 were feeding at the time.

2.3.5 Colour change experiments

An experiment was performed to determine whether urchin pigmentation was var!able under different light regimes. Urchins were collected from Ti nderbox using SCUBA on 2 iv 86 and kept in the laboratory in dark, aerated bins overnight. The test diameters of the urchins ranged from 35 to 49 mm; these were the smallest urchins which could be found in sufficient numbers for the experiment. Small urchins were used as any ability to vary pigmentation would probably be greatest in young urchins (Millott, 1952). The urchins were scored for dermis and spine colours and their test diameters measured to enable later identification. Two experimental bins were set up; (1) the light box was white and two fluorescent tubes were placed 30 em above the water surface and illuminated it continuously, and (2) the dark bin which was black and had a tight fitting black lid 19 through which a small hole was drilled to allow for an air hose. A control bin was white, without a lid and was illuminated at moderate light intensity by the tungsten bulb of the room, which was on a 12 hours on /12 hours off cycle. Urchins were distributed between the bins as follows: Light Bin Dark Bin Control Bin 4 RPG 5 RPG 5 RPG 1 RP 1 RP 1 WP 2Wg 1 Wg 3WG 2WG 3WG 1 Wp 1 Wp 1 Wp The bins were placed side by side in a constant temperature room at 1ooc, which is comparable to sea temperatures in winter in southern Tasmania. Each bin was supplied with kelp, Ecklonia radiata, once a week and remaining kelp removed. Urchins had been observed feeding on E. radiata in the field. The water in the boxes was continually aerated and replaced once a week. Urchins were kept under these conditions for 6 months, until 18 ix 86 when they were again scored for dermis and spine colouration. This experiment was repeated from 25 i 88 to 29 iv 88 with water temperatUres maintained at ambient sea water temperature (14-170C) using a flow through aquarium system. On this occasion 9 bins were used with 3 assigned to each of the light regimes described above. Each bin contained one urchin of the following r'norphs; RP, RPG, WG, WP, WW, The test diameters of the urchins ranged from 50 to 70 mm.

2.4 RESULTS

2.4.1 Variation in test and spine colours

· A significant excess of RP urchins relative to WP urchins was found among No Tinderbox urchins (G = 8.50, df = 1, p < 0.01; Table 2.2a). association of dermis and spine colour was found for Ling Reef urchins (Table 2.2b). Test pigmentation is related to dermis colour (Table 2.3); urchins with red dermis always have a darkly pigmented test, which may be purple, green or a mixture of both whereas urchins with white dermis have pale green or white tests (a pale purple test was never observed throughout the study). The test colour is not related to primary spine colour, e.g. a RP urchin may have a green test. Urchins with pink dermis have test pigmentation similar to that of white urchins. 20

Table 2.2 Results of G-test for independence to determine character asso ciat ion between dermis and spine colour. Dermis colours = red� white (pink lumped with white) Spine colours = purple, not pl}rple (i.e. green, purple green and white)

(a) Tinderbox

Spine co lour pu rple not purple Total

De rmis red 1 8 20 38 colour white 8 37 45

Total 26 57 83

G = 8.50 df = 1 p < 0.01

:b) Ling Reef

Spine colour purple not pu rple Total

Dermis red 1 5 1 9 34 co lour white 1 7 27 44

Total 32 46 78

G = 0.24 df = 1 p > 0.05 21

Table 2.3 Variation in test colour in relation to overall morph. Overall morph - first letter refers to dermis colour (R :::: dark red, W = white, p = pink); remaining letters refer to primary spine colour (P = purple, G :::: gre en, W = white). Spines of different intensities of pigmentation have been lum ped. Test colours were determined from clean, dried tests. They were scored as for spine colours but upper case letters refer to dark colours and lower case letters to pale. Number of urchins and percentages within each overall morph are given.

Overall Test Ling Reef Tinderbox Marion Bay Fortescu.e Bay morph col our n o/o n % n o/o

RP p 7 46.7 11 61.1 7 43.8 G 7 46.7 2 11 .1 8 50.0 R3 1 6.6 5 27.8 1 6.2

Rffi p 4 21.1 6 30.0 5 21 .7 G 8 42.1 5 25.0 16 69.6 R3 7 36.8 9 45.0 2 8.7

p 1 50.0 G 1 50.0

WP w 1 5.9 3 37.5 1 100.0 g 16 94.1 5 62.5

w 2 20.0 4 26.7 4 100.0 g 8 80.0 11 73 .3

w 3 21.4 8 38.1 1 25.0 21 80.8 g 11 78.6 13 61.9 3 75.0 5 19.2

g 1 1 00.0

pP w 1 100.0

g 1 100.0

pPG g 1 100.0

Number of urchins 78 83 45 30 22

The proportions of test colours among red dermis urchins differ; the proportion of dark purple tests is higher among the RP urchins than among RPG urchins (Ling Reef, 46.7 and 21.1 %; Tinderbox, 61.1 and 30.0%; Marion Bay, 43.8 and 21.7%). Thus red dermis urchins with purple-green spines are more likely to have green pigment in their tests than are those with only purple spines. Among white dermis urchins, pale green tests are more common than white tests, except at . . Fortescue Bay (pale green tests: Ling Reef, 85.4%; Tinderbox, 66.7%; Marion Bay, 75.0%; Fortescue Bay, 16. 7%). The proportions of test colours within each overall morph vary between sites. For example, a G-test for independence among WPG urchins showed that the proportion of pale green tests at Fortescue Bay (19.2%) was significantly lower

< than at both Ling Reef (78.6%) and Ti nderbox (61 .9%) (G = 16.50, df = 2, p 0.001 ). However the numbers of urchins observed were too small to determine whether there were consistent differences between sites. Geographical variation in morph distributions are discussed in Chapter 4. All the red dermis urchins had dark secondary spines and most white dermis urchins had pale secondary spines (Ling Reef, 90.1 %; Tinderbox, 97.9%; Fortescue Bay, 86.7%; Table 2.4). Occasionally white dermis urchins had dark secondary spines, however it was observed that these urchins always had very dark primary spines, i.e. an urchin with pale primary spines never had dark secondary spines. All observed WW urchins had white secondary spines. Dark and pale green spines were the .most common types of secondary spine, even for urchins with purple primary spines (Ling Reef, 89.6%; Tinderbox, 77.8%; Fortescue Bay, 81 .6%). Urchins with pink dermis had secondary spine pigmentation similar to that of white dermis urchins.

2.4.2 Distribution of spinochromes among phenotypes

· The results from the TLC showed that each of the individuals of a particular morph had identical, pigments present. There are at least five naphthoquinone pigments present in most morphs but that relative amou nts of pigment vary among morphs (Tables 2.5a and b). Pigments from bands 2, 3 and 5 probably have the greatest effect on the phenotype (i.e. they occur in the highest concentrations). Band 2 occurs in all morphs and is the dominant pigment in purple tests and spines. Dark green tests and dark purple-green spines have both band 2 and 5 pigments in high concentrations, but Band 5 is predominant in pale green parts. Brown spines have similar pig ments to dark purple-green spines but they have a higher concentration of band 3 pig ment. White tests and spines contain at least 23

Table 2.4 Variation in secondary spine colour in relation to overa ll morph.

Overall morph - first letter refers to dermis colour (R ==- dark red,

W = white, p :; pink); remaining letters refer to primary spine colour (P = purple, G :; green, W :; white). Primary spines of different intensities of pigmentation have been lumped. Secondary spine colours were scored in the .same way but upper case letters refer to dark colours and lower case letters to pale. Number of urchins and percentages within each overall morph are given.

Overall Secondary Ling Reef Tinderbox Fortescue Bay morph spines n % n % n %

RP p 2 4.8 8 25.0 G 35 83.3 24 75.0 FG 5 '1'1.9

RFG p 2 4.2 G 6'1 98.4 46 95.8 FG 1 '1.6

G 1 100.0

G 3 '14.3 2 100.0 g 18 85.7 13 68.4 w 6 31.6

G 1 33.3 g 20 87.0 8 57.1 1 33.3 w 3 13.0 6 42.9 1 33.3

Pg 1 3.2 G 4 '12.9 2 4.0 3 7.5 g 24 77.4 42 84.0 30 75.0 w 2 6.5 6 12.0 7 17.5

w 6 100.0 12 100.0

pP g 3 75.0 3 100.0 w 1 25.0

g 7 100.0

pPG g 5 100.0 1 100.0 3 75.0 w '1 25.0

Number of urchins scored 20'1 1 80 49 24

Table 2.5 Distribution of spinochrome pigments. Results of thin-layer chromatography. Relative amounts of pigments are shown : +++ = major band� ++ = obvious band�

+ = faint band. The bands occurred in order along the chromatograrT with band 1 travelling furthest from the original pigment spot. Colours of bands are those seen on the chromatograma.

(a) Spines.

Spine Colour w Band Colour p FG Pg B p g

1 Pink + + + + + +

+ 2 Purple +++ +++ +++ +++ ++ +

+ 3 Brown + ++ + +++ + +

4 Pink + + + + +

5 Orange ++ +++ ++ ++ + ++ Number of urchins 5 4 2 4 4 4

{b) Tests.

· Test colour Band Colour p PIG G g w

1 Pink + + + +

2 Purple +++ +++ +++ ++ +

3 Brown + + + +

4 Pink + + + +

5 Orange ++ +++ ++ +++ Number of urchins 1 1 1 1 1 25 two pigments, but in very low concentrations. The other pigments were not observed in white tests·and spines but this may be due to the extremely low level of pigment present making these bands undetectable by eye.. This would be very hard to verify because many of the rare WW urchins would need to be analysed. These results indicate that the band 2 pigment produces the purp le colour in tests and spines, band 5 pigment produces the green colour and band 3 produces the brown colour. Bands 1 and 2 are accessory pigments which occur in low concentrations and their effect on colouration is unknown. All the pigments are probably found in all the morphs.

2.4.3 Identification of naphthoquinone pigments

The 1 H-NMR spectrum of band 2 (Table 2.6a) indicates that the pigment is probably spi nochrome A (2-acetyl-3,5,6,8-tetrahydroxy-1 ,4-nap hthoquinone) (Moore and Scheuer, 1966; Chang et a/., 1964). The 13C-NMR spectrum was consistent with this, showing peaks for 12 C atoms (Figure 2.2). The results from mass spectroscopy confirm this identification (Fig. 2.4a), indicating a molecular mass (M) of 264 and a spectrum similar to that found by Becher eta/. (1966). It was not possible to purify sufficient pigment from band 3 for 13C NMR and the results from 1 H and mass spectrum were difficult to interpret. The 1 H-N M R spectrum (Table 2.6b) suggested that the struct ure was 2-acetyl-1 ,4,7- tetrahydroxy-1 ,4-naphthoquinQ ne (M=264) or its isomer, which have not previously been found in echinoids. However the mass spectrum (Fig. 2.4b) indicated a molecular ion of mass 280 and the spectrum was almost identical to that of spinochrome C (2-acetyl-3,5,6,7,8-pentahydroxy-1 A-naphthoquinone) as determined by Miyauchi and Okajima (1970). All previous workers had determined the 1 H-NMR spectrum of spinochrome C in DMSO-d6 and found only one peak at 8=2.59 (Chang et a/., 1964; Miyauchi and Okajima, 1970). The different solvent used makes these results not comparable to those of the present study and thus these anomalous results cannot be explained. However, it seems most likely that band 3 is spinochrome C. The 1 H and 13C-NM R spectra for band 5 (Table 2.6c and Figure 2.3) suggested that it was probably spi nochrome B (2 ,3,5, 7 -tetrahydroxy-1 ,4- naphthoquinone) (Moore and Scheuer, 1966; Gough and Sutherland, 1964). This was confirmed by the mass spectrum (Figure 2.4) which indicated a molecular mass of 222. 26

Table 2.6 Results from H�NMR spectroscopy. The chemical shift ( o) indicates where peaks occurred in the spectrum. The integral indicates how many protons are implied by the peak.

(a) Band 2 in deuteriochloroform. o Integral Substituent 16.15 0.91 aromatic -OH (hydroxy group) 14.33 1.00 aromatic -OH 12.07 1.09 aromatic -OH 11.75 6.89 0.18 aromatic -H (proton) 6.65 0.94 3.50 0.60 quinoidal -OH 2.87 3.40 quinoidal -COCH (acetyl group) 2.80

(b) Band 3 in deuteriochloroform.

Integral Substituent 15.53 1.04 quinoidal -OH 13.82 1.07 aromatic -OH 12.17 1.07 aromatic -OH 6.98 1.24 1 quinoidal and 6.58 1.03 1 aromatic -OH 2.85 2.86 gu inoidal -COCH

(c) Band 5 in dimethyl sulphoxide. o Integral Substituent 11.99 1 .05 -OH 11.23 11.00 3.10 3-0H 10.27 9.86 6.99 1.04 aromatic -H 6.52 1.00 aromatic -H

N.B. A second smaller peak (shown by brackets) sometimes accompanied the main signal; this is probably due to the presence of isomers. 1t�Jr: 1 , . ,, ,, � , � ,

dimethyl sulphoxide .....,.

tv -....]

acetone? t I

I I v!IIM""'l\,_'�'><�t;y,.W"fN'"w'\Y'.,\../ \v�W-.v ....

��t.vw'�'·""��\'1''''l''''l' '''l''''l'·''l'''' l''''l'fflui�v'�Jy'''l''''l''''l'''' �l''''l''' 'l''''l''''l''''l''''l''''l' 200 150 100 50 0 Figure 2.2 13C�NMR spectrum for band 2 in dimethyl sulphoxide.

··---- . ,.,...... �-····�·.. ------·--·-·- ...-··- ··-···' , � ,, � , ,,, I

I [\.) I (}) ,,

·��

� � 180 160 8 140 120 Figure 2.3 13C-NMR spectrum for band 5 in dimethyl sulphoxide. 29

{a) Band 2.

i ?S

{b) Band 3.

{c) Band 5.

1 3'7&

! t I' (ij c ·0>en

-0 · >. ' ...... ·w c:: 2 l 8 c::

Atomic mass of fragment

Figure 2.4 Mass spectra of naphthoquinones from H. erythrogramma. 30

2.4.4 Composition of the dermal pigment granules

The spectrum of the orange pigment extracted in acidified ethanol was almost ' identical to that of the pure echinochrome A (Figure 2.5) and to published data on echinochrome A (Ball and Cooper, 1949; Service and Wardlaw, 1984). Histology showed that pigment granules of varying sizes were present in the epithelium covering the podia and peristomial membrane of both red and white urchins. Schmorl's ferricyanide technique stained the granules dark blue, but when the tissue was treated with ferricyanide only the granules were stained red. Thus the positive result indicated the presence of iron and not melanin (Humason, 1 967). Lillie's ferric�ferricyanide technique gave a negative result with all tissues stained pale green indicating that melanin was not present. The dermal granules are therefore composed mainly of echinochrome A with iron or an iron�containing pigment also present.

2.4.5 Diet preferences among morphs

Heliocidaris erythrogramma feeds on a wide variety of macroalgae. The 50 urchins observed were feeding on 15 different species, 4 species of brown algae (Phaeophyta, 2 species of green algae (Chlorophyta) and 9 species of red algae (Rhodophyta) (Table 2.7). All the morphs were feeding on all three phyla, except for WP urchins which were only observed feeding on green and red algae. Each morph was feeding on a range of algae, from 2 to six species. The large number of food species made comparisons between morphs difficult but there was no evidence of specialisation in the diet of different morphs.

2.4.6 Colour change

For the experiment ru n in 1986 at winter temperatures, four urchins died during the experiment; one WG and one RPG from the control box; one Wg and one WG from the dark box. Of the remaining urchins, there was no change in the dermis or spine colours of any of the urchins in both the control box and the dark box. In the light box there was no change in any of the red dermis urchins but four of the five originally white dermis urchins now had slightly pink dermis. For the experiment run in 1 988 in summer temperature conditions, there was no mortality. This was probably because of the improved water quality of the flow through system. There was no change in the dermis or spine colours of any of the urchins. 31

(a) Pure echinochrome A

1.2

0.8

0.4

f 0.0 4---r------r----..--.-----r---r-----., 200 300 400 500

(b) Dermal pigment of H. erythrogramma

2.0

u c: ro ..0 L 0 {I") l.O ..0 -<(

0.0 +---r---.---,.--.----r----.----. 200 300 400 500 Wavelenth (nm)

Figure 2.5 Comparison of absorption spectra of echinochrome A from different sources. 32

Table 2.7 Diet of colourmorphs at Tinderbox, surveyed on 25 Jan 88. Moq2h Phylum SQecies RP RFG W3 WPG WP WN Phaeophyta Ecklonia radiata 2 2 2 1 Sargassum sp. 1 Ca rpog lossum confluens 1 Unknown sp. 1 4 Chlorophyta Caulerpa trifaria 6 1 2 2 Ulva sp. 1 1 1 Rhodophyta Jeannerettia Iabata 2 3 1 Lophurella periclada 4 Lenormandia sp. 1 Polysiphonia sp. 1 Phacelocarpus alatus 2 3 Ca l lophyllis coccinea 1 1 Porphyra sp. 1 Wrangelia sp. 1 Unknown SQ. 1 Number of urchins 9 14 4 1 0 6 7 !·· . [: 33

2.5 DISCUSSION

The colours of the dermis, test, primary and seco ndary spines of H. erythrogramma all vary between individuals and among overall morphs, creating a complex polymorphism. The colour of one part of an urchin is not dependent on the others, but some combinations of pigmentation are more common than others. An example of this was provided by the excess of RP urchins found at Tinderbox. The intensities of colour are also often interrelated, thus a red dermis always overlays a dark test and a white dermis always overlays a pale or white test. The relationship with spine pigmentation is slightly more complex; most red dermis urchins also have dark coloured spines but white dermis urchins may have pale or dark spines. The intensity of colour of secondary spines may be similar to that of the test (and therefore of the dermis) or of the larger spines. Thin-layer chromatography indicated that there were three major naphthoquinone pigments in the calcareous parts of the urchins, with at least two other minor pigments present in most morphs. All pigments were present in most of the morphs but different combinations give the colours seen in live urchins. It therefore seems probable that all morphs can produce each of the pigments but in varyi ng proportions. Spinochromes A, B and C were identified as the major pigments in H. erythrogramma and these are widespread among other echinoids. The combination of these three spinocttromes is found in many species (Anderson et a/., 1969). The pigments of most species have not been identified, but it seems unlikely that the spinochromes can be used to determine taxonomic affinities (Anderson et a/., 1969 ). Lederer ( 1952 ) states that the calcium salts of spinochromes A, B and C give violet, green and brown-pink colours, respectively, which agrees with the results from TLC on the distribution of pigments among morphs of H. erythrogramma. The variation in dermal pigmentation of H. erythrogramma is the most unusual aspect of the polymorphism. The dermis may be dark red or white, with few intermediates except on the north coast of Tasmania. Variation in dermal pig mentation of other species is usually not described and therefore probably does not occur or is minimal. For example, Jensen (1974) states that the test of Strongylocentrotus droebachiensis varies from purple to almost black because of a very dark violet pigment in the dermis. The few exceptions, such as the diadematoid urchins Diade m a spp. (Millott, 1952) and Centros tephanus longispinus (Weber and Dambach, 1972), are examples of physiological colour 34 change rather that permanent differences in dermis colour. Young Oiadema have black, white, grey or striped spines (Kristensen, 1964) and both Diadema and Centrostephanus exhibit reversible colour change, especially in young urchins, from white to black by extension or contraction of dermal chromatophores (Millott, 1975). The major pigment is a melanin although echinochrome, chromo lipid and an iron-containing pigment are also present (Millott, 1964). As the urchins age, chromatophores in the dermis break down and melanin is deposited in the skin so that, under normal light conditions, the urchins are completely black after a few months (Kristensen, 1964). Heliocidaris erythrogramma showed no ability to change the pigmentation of their tests and spines under different light regimes over 3 to 6 months. There was a slight increase in dermal pigmentation in white dermis urchins kept in continuous high light intensity for 6 months but the resulting pale pink colour did not approach the deep red of the red dermis urchins. Even this slight change was not observed when the experiment was repeated. The ability of H. erythrogramma to change colour is therefore minimal. Echinochrome A is the major pigment in the dermis of H. erythrogramma and is also known to occur in the dermis of other species of echinoids (Led�rer, 1952; Millott, 1964). No physiological function for echinochrome A in adult urchins has been proven although it may have antibacterial and algi stat properties (Kittredge, 1972; Service and Wardlaw, 1984). It has been suggested that echinochrome A may be involved in respiration (MacMunn, 1885) and photoreception (Millott and Yoshida, 1957) but both of these theories are now considered unlikely (Goodwin, 1969; Millott, 1975). Heliocidaris erythrogramma is similar to Diadema spp. in having an iron-containing pigment present in the dermis, but no melanin was detected. As white dermis urchins have only a small fraction of the echinochrome A present in the dermis of red urchins, it seems unlikely that it performs a I. physiological function in H. erythrogramma unless one of two situations exists: (1) the level of echinochrome A in the white dermis urchins is sufficient' for its physiological function and the extra pigment in red dermis urchins is excess, which may be genetically determined or due to environmental factors, or (2) white and red dermis urchins occur in different habitats, or have different metabolisms, which would require different levels of echinochrome A. Red and white dermis urchins may be found next to each other in natural populations although they may occur in slightly different microhabitats (see Chapter 5). Also, there is evidence of physiological differences between red and white dermis urchins (see Chapter 5), but these differences are slight, so the latter possibility seems unlikely. 35

The definition of 'polymorphism' assumes that the variation is genetically controlled (Ford, 1 945) but until breeding experiments have been performed this cannot be proven for the colour variation in H. erythrogramma. Breeding experiments were attempted during this study but failed to rear juve niles to the point where adult pigmentation could be determined (see Appendix 1 ). However, results from this chapter indicate that environmental control is unlikely, although the possibility of some modification to dermal pigmentation cannot be ruled out. Each urchin usually has differently coloured test, primary and secondary spines due to the presence of naphthoquinone pigments. It is very unlikely that any environmental factor, or combination of factors, could cause this complicated distribution of pigments. The field survey indicated that all morphs eat a wide range of algae and no evidence was found of any dietary prefere nces between morphs. Other possible environmental influences include differences in temperature or light regimes during larval development but these possibilities cannot be eliminated until it is possible to breed the urchins in the laboratory. Urchins are pentamerous with five ambulacral and five interambulacral areas alternating. During this study two urchins were found on separate occasions at Tinderbox which had obvious zones of different pigmentation corresponding to multiples of one ambulacrum and one interambulacrum. Thus one urchin was 3/5 RPG and 2/5 Wg and the other was 1/5 RP and 4/5 Wp. Such a pattern is unlikely to be produced by environmentally induced processes. Vari ation in phenotypic characters of Lytechinus variegatus from Bermuda and Florida has been shown to be inherited (Pawson and Miller, 1982) by rearing test crosses through to two year old urchins. The characters included spine length relative to test diameter and skeletal morphology, but also pigmentation. Bermudan L. variegatus have purple tests and spines whereas those from elsewhere have red to green tests and spines (Serafy, 1973). Juveniles of the two different types were distinguishable mainly by their pigmentation at 2-3 days after meta!'llorphosis. This difference then disappeared but by 17 weeks the two groups of juveniles resembled the adults from each area. Pawson and Miller (1982) concluded that the colour variation in L. variegatus was not re lated to environmental conditions and that the differe nee in Bermudan populations was maintained because of genetic isolation. The available evidence on colour variation in H. erythrogramma also does not suggest environmental control. The hypothesis that the variation in H. eryt hrogramma is genetically determined therefore seems reasonable until future breeding studies can answer the question. Assuming that the colour variation is genetically based, the associations 36

between pigmentation of different parts of the urchins suggest that there may be linkage between at least some of the genes. In the case of dermis and primary spine colour there is a disequilibrium in favour of RP urchins at Tinderbox. In some other instances the disequlibrium is almost complete. For example, the intensity of pigmentation; all red dermis urchins observed had darkly pigmented tests and most had dark primary and secondary spines whereas all the white urchins had pale or white tests. Linkage of genes can occur through random processes such as population bottlenecks or mutations such as chromosome inversions, but in the absence of selection the population will return to equilibri um at a rate depending on the degree of linkage. If similar patterns of disequlibrium were found in many populations of urchins and as the possibility of a historical accident affecting all the populations seemed unlikely, this would be further support for the action of selection in maintaining, if not developing, the disequlibrium and perh aps linkage.

! ' 37

CHAPTER 3 TEMPORAL STABILITY OF MORPH FREQUENCIES

3.1 INTRODUCTION

Studies of temporal variation of genetically based traits have been widely used to detect the action of natural selection (Cain, 1983; Manly, 1985; Endler, 1986). If selection is not acting on a trait then the trait would be expected to vary randomly over time (Endler, 1986). Thus both long-term stability or regular directional change (either long-term or seasonal) may provide evidence for selection. For quantitative traits such as size, shape, fecundity the variation in mean and variance among age groups or generations is used (Berry and Crothers, 1968; O'Donald, 1971 ; Beatson, 1976), but for disconti nuous traits the proportions of different morphs before and after selection are compared (Van Val en, 1965). Data of two types may be collected; longitudinal data for which populations are sampled repeatedly over time, and cross-sectional data, for which there is only one sampling occasion and comparisons are made between age classes (Arnold and Wade, 1984 ). Longitudinal data have been used in studies of visual polymorphisms in the landsnails Cepaea nemoralis and C. hortensis (Clarke and Murray, 1962; Walda, 1969; Williamson et al. , 1977; Murray and Clarke, 1978; Wall et al. , 1980); and the lepidopterans Mania/a jurtina (Dowdeswell, 1961) and Panaxia dominula (Ford and Sheppard, 1969) to show that selection was involved in maintaining or changing morph proportions over time intervals varying from two to fifty years. Adults of M. jurtina and P. dominula live for less than a year, thus samples from successive years represent different generations. Cepaea spp. live for several years, so samples represent average frequencies for several generations, making the detection of selection more difficult. This is because changes in morph frequencies for the whole population will occur more slowly. I ,. . Cross-sectional data have rarely been used in studies of visual I po!ymorphisms but this approach has been used to demonstrate selection on quantitative characters such as the size of dog-whelk shells (Berry and Crothers, 1968). Age classes were determined somewhat arbitrarily, using size and sculpture of the shell, and the variance of the ratio of shell length to cube root of shell weight fo r different age classes was compared. Variance was found to be reduced in older animals and this reduction was greatest on shores with the highest exposure to wave action. Thus stabilising selection was shown to be occurring and the intensity of the selection varied among habitats. 38

Although in some of the long-term studies on Cepaea spp. juvenile and adult snails could be distinguished on the basis of the presence of a lip on the shell, the morph proportions between the two groups were not compared (Clarke and · Murray, ; 962; Murray and Clarke, 1 978; Wall et at. , 1 980). However, Williamson et at. (; 977) found no differences in morp h proportions among size classes varying from 6-8 to 1 6-1 8 mm and adults of C. nemoralis. Walda ( 1 969) found small differences (4-5%) in the proportions of yellow unbanded and yellow banded morphs between adults and juveniles of C. nemoratis at forty sites. Walda indicated that selection was probably responsible but did not suggest any specific mechanisms. The maximum age to which H. erythrogramma may live is unknown but is probably over ten years (Ebert, 1982; A.Constable, personal communication) and recruitment is patchy, with urchins of test diameter less than 35 mm occurring in very low frequencies at most sites (personal observations). For this reason, and because the length of the present study was restricted to three years, analysis of longitudinal data would only be able to detect strong selection. The longevity of H. erythrogramma allows the use of cross-sectional data to determine whether morph proportions vary between age classes. Echinoids h.ave very variable growth rates, depending mainly on the availability of food, but also on factors such as the amount of spine regrowth needed because of damage (Ebert, 1 968; Dix, 1971 ; Andrew and Choat, 1985). Aging of H. e1ythrogramma by counting growth rings in the genital plates and pyramids from the lantern has shown that urchins of the same size may be different ages at different si�es (A. Constable, personal communication). Therefore, although it may be possible to identify different age classes within a site, urchins cannot be compared among sites. Size frequency distributions can be used to identify different age classes, although the largest urchins may be of varying ages as older cohorts tend to merge. If selection were acting on the visual polymorphism of adult H. erythrogramma then the proportions of morphs might vary among age classes. This approach would not, of course, detect selection acti ng at other times in the life cycle, such as differential reproductive success or recruitment. This chapter describes the temporal stability of morp h proportions of H. erythrogramma over three years, at sites around Tasmania and in Port Phillip Bay, Victoria. Size frequency distributions from some Tasmanian sites were used to determine whether there was evidence of changing proportions of dermis colours between size classes. The main reason for this study on temporal variation was to determine whether morph proportions of H. erythrogramma were stable at a given site during the study. This was neccessary because the 39

collection of data on geographical variation occurred over almost three years (see Chapter 4). If morph proportions were stable then it would be valid to compare sites which had been sampled at different times, in order to detect geographical _ patterns. However, if morph proportions were not stable this would suggest that strong selection was acting differentially on the morphs.

3.1 METHODS

Three sites in Tasmania (Tinderbox Cliffs, Ling Reef, and Fortescue Bay) were sampled repeatedly from 1985 to 1988; at Tinderbox Cliffs and Ling Reef samples were initially taken approximately every three months, but later this was increased to six months. Several other sites were sampled two or three times (Figure 3.1) and the sites in Port Phillip Bay were sampled by A. Constable (Department of Zoology, University of Melbourne) from two to four times between 1985 and 1987. Permanent transects were placed at two depths at Tinderbox and Ling Reef to ensure that the same areas were sampled each time. The restricted distribution of urchins at Fortescue Bay made this precaution unnecessary. A series of small orange buoys was suspended 1 m above the substratum by rope attached to a piece of angle iron which was wedged in cracks or under rocks. Each transect comprised 10 buoys placed at approximately 3 m intervals along a depth contour (Figure 3.2). Care was taken to place the transects within areas which were homogeneous for substratum and algal communities, so that urchins collected from any part of a transect would have come from the same habitat type. Transects remained in place for over two years and were not lost during storms, although they had to be resecured occasionally. The rope and buoys were cleaned of epiphytic growth each dive so that they remained easily visible. Transects were placed at 3 m and 7 m depths off the Tinderbox Cliffs on 2 May 1986 and at 3 m and 13 m at Ling Reef on 15 April 1986 . . Urchins were collected using SCUBA by starting at one end of a transect and working along it to a distance of 1-2 m on each side. The area was thor�ughly searched to ensure that there was no bias towards the more obvious morphs. A sample size of 100 (the same as was used for the study of geographic variation, see Chapter 4), was aimed for, as this was the largest number of urchins which could be collected at most sites without requiring excessive diver time. Less than half the transect had to be searched to find 100 urchins in each case. The urchins were later returned to the water but not to the transect. Urchins were found to repopulate the denuded areas within 3 months (when the next sample was usually taken), but as a precaution the sample was taken from opposite ends of 4V Point Point

VICTORIA

N

•

•

Rocky Cape

TASMANIA

100km

Figure 3.1 Map of study sites. shoreline

Transect lines were placed along contour at the required depth.

!t

�� . �� a. �

Figure 3.2 Diagram to show structure of permanent transects. 42 the transect on successive dives, to allow a minimum of 6 months for the area to recover. However, I acknowledge the slight possibility that this may have led to different subsets of the population being sampled. Sites without transects were sampled by searching a patch carefully. The colours of the dermis and spines were scored according to the description in Chapter 2. This was done on shore as it was often difficult to accurately identify spine colours underwater because of turbidity causing low light levels. On some of the first dives green and purple-green spines were not distinguished. Data for the Port Phillip Bay sites were collected by underwater observations and therefore only dermis colour data have been used. At some sites test diameter was measured to the nearest 1 mm using Vernier calipers. At sites where it was impossible to search the area completely (for example because of very large boulders which could not be turned) diameters were not measured because the resulting size frequency would not have been complete. G-tests for independence were used to determine whether there was significant temporal change in morph frequencies at any of the sites. The G-tests were calculated using BIOI,TAT which applies the Yates correction where neccessary. The morphs were divided into dermis colour only and spine colour only, as well as overall morph. The Kolmogorov-Smirnov two-sample test was used to determine whether the size frequency distributions of red and white dermis urchins differed at each site. This test is sensitive to differences in the location, dispersion and skewness of distributions but gives no indication of which of the parameters are important (Sakal and Rohlf, 1981 ). Where the overall size frequency (i.e. red and white dermis urchins together) showed two or more reasonably distinct peaks, the dermis colour data were lumped into size classes and G-tests for independence were used to determine whether the proportions of red and white dermis urchins varied significantly.

3.3 RESULTS

3.3.1 Stability of morph proportions during the study Dermis colour Both of the Ti nderbox transects showed evidence of change over time (3 m transect; G = 17.41 , df = 6, p < 0.01 : 7 m transect; G = 14.73, df = 5, p < 0.05) but there were no clear seasonal or long term trends (Figure 3.3a and b). The main change at the 3 m transect was a sharp decrease in the proportion of red dermis urchins in January 1988, but this was increased again in the final sample. At the 43 (a) Tinderbox 3 m transect. 106 136 101 200 99 98 93 100 -

- G = 17.41 60 - df = 6

- 0.01 p < 20-

I . I

(b) Tinderbox 7 m transect.

117 98 195 107 1 03 106 100:-

- G = 14.73

df = 5 so- 0.05 p < -

20-

,I I

(c) Ling Reef 3 m transect.

111 108 101 1 03 113 1 0 1

100 - · G = 2.54 - df = 5

60- p > 0.05 -

20 -

I I

(d) Ling Reef 13 m transect.

106 109 102 105 100 104 L 100- :::J 0 - G = 3.72 0 df = 5 u 60- 1./) E - p > 0.05 L Q) 20- '0 � I I 1986 1987 1988 Year Figure 3.3 Temporal variation in dermis colours at n permanent transects. 1m Pink 0 White G-tests for independence were used to determine whether • Red there was temporal variation 1n morph frequencies. 44

7 m transect the proportions of red urchins decreased in 1 987 but again this trend was reversed in 1988. There was no significant change in dermis colour proportions over time at either of the Ling Reef transects ( 3 m; G = 2.54, df = 5, P >

0.05: 13 m; G = 3.72, df = 5, p > 0.05: Fig. 3.3c and d). At Tinderbox the proportions of red urchins were consistently higher at the deeper transect, by 10.4 to 38.4%. At Ling Reef the difference between transects was less marked � ut the shallow transect had higher proportions of red urchins in every sample except one (mean difference between transects was 8.6%). The numbers of red urchins at Fortescue Bay were so low that a test could not be performed, but Figure 3.4a shows that there was little variation at this site. None of the other sites in Tasmania showed temporal variation in dermis colours

(Tinderbox Beach; G = 0.28, df = 2, p > 0.05: Betsy Island, 6 m: G = 0.47, df = 1, p>0.05: Betsy Island, 13m; G == 0.84, df = 1, p > 0.05: Rocky Cape; G = 2.1 1, df = 1, p > 0.05: Figure 3.4b-e). In Port Phillip Bay the overall picture was again one of stability (Figure 3.5). The numbers of white and pink urchins at Point Lillias and Point Henry were so low, even when pooled, that no tests could be made, but Figure 3.5a and b shows that the proportions were stable over time. Point Franklin (G = 0.1 4, df = 2, ·p >

0.05) and Tablerock Point (G = 0.20, df = 2, p > 0.05) showed no significant change over time. At Point Cook, Barren 1 showed no evidence of temporal change (G = 4. 76, df = 6, p > 0.05) but Barren 2 showed an increase in the proportion of pink urchins at the expense of the white urchins (G = 20.00, df = 6, p < 0.01 ). Both of the sites with heavy algal cover, Kelp 1 and 2, showed significant increases in the proportions of red urchins with time and a peak of pink urchins in

August 1 986 (Kelp 1, G = 35.31 , df = 4, p < 0.01 ; Kelp 2, G = 31 .98, df = 4, p < 0.01 ).

Spi ne colour

· At the permanent transects only the Ling Reef 3 m transect showed significant temporal variation (Tinderbox 3 m, G == 21 .04, df = 15, p > 0.05; Tinderbox 7 m, G

= 1 3.40, df = 12, p > 0.05; Ling Reef 3 m, G = 17.81, df == 8, p < 0.05; Ling Reef 13 m, G = 7.38, df = 8, p > 0.05). For both of the Ling Reef transects white spines were not included in the analysis because of their low frequencies. The proportion of purple spines at the 3 m transect decreased from 32.4 to 1 8.6% over 1 986-7 but increased again to 31.7% in 1 988 (Figure 3.6c). For Fortescue Bay purple and white spines were not included in the analysis as they only comprised 2.3 and 0.5% of the samples respectively. When only 45 (a) Fortescue Bay. 78 61 4 0 31 69 71 4 9 88 1 00 -

- Invalid 60- test -

20-

I (b) Tinderbox Beach. 49 256 192 100- G = 0.28 - df = 2 so- p > 0.05 -

I I I

(c) Betsy Island$ 6 m. 1 0 1 100 100

G = 0.47 df = 1 60 p > 0.05

20

(d) Betsy Island, 13 m. 55 94 1 00 G = 0.84

60 df = ' p > 0.05

20

(e) Rocky Cape. L ::l 0 144 1 02 -0 1 00 c...> G = ·(/) 2.1 1 E so df = 1 L 0.05 Cll p > -o � 20 1985 1986 1987 Year n Figure 3.4 Tempo ral variation in derm is colours at 1m Pink Tasmanian sites without permanent transects. 0 White G-tests for independence were used to determine whether • Red there was temporal variation in morph frequencies. 46

(a) Point Franklin. 95 185 100 ::·:•: �-;.:

G = 0.14

df = 2 60 p > 0.05

20

(b) Point Henry.

155 1 0 1 133 115 100 �

Invalid 60 test

20

I ,...-,

(c) Point Lillias. 106 155 197 1 00

Invalid so- test

20-

I I

(d) Tablerock Point.

105 107 5 100 I 0 0 G = 0.20 (..) 60 = 2 ·�Cl.l df E p > 0.05 � 20 -o �-,�-.�!� 1985 1986 1 987 Year n Figure 3.5 1m Pink Temporal variation in derm is colours at 0 White sites in Port Phillip Bay. • Red G-tests for independence were used to determine whether there was temporal variation in morph frequencies. 47

(e) Point Cook, Barren 1. 174 153 154 107 1 00 - at: � � �r::?a - G = 4.76

df = 6 60- p > 0.05 -

20- Lr---1 I I

(f) Point Cook, Barren 2. 1 09 1 51 173 1 06 100- = F �1'$. � - G = 20.00

60- df = 6 0.01 - p <

20 - -i----T I I

(g) Point Cook, Kelp 1. 155 155 1 15 100 - F � � - G = 35.31

60- df = 4

- p < 0.01

20-

I I

(h) Point Cook, Kelp 2. 207 146 107 5100- :;z F"' 0 �-

- = 0 G 31 .98 (..) ·CI) 60- df = 4

E - p < 0.01 L a.> 20- -o � � � -1--r I I .,..._, Key 1985 1986 1987 I Year n Figure 3.5 (continued) Temporal variation in dermis 1m Pink D White colours at sites in Port Phillip Bay. • Red G-tests for independence were used to determine whether there was temporal variation In morph frequencies. 48

(a) Tinderbox 3 m transect.

G=21.04

df = 15 0.05 p >

(b) Tinderbox 7 m transect.

98 195 1 00- 7

- G = 13.40 df = 12 so- 0.05 - p >

I I

(c) Ling Reef 3 m transect. 113 1 0 1 1 00- G = 17.81 - df = 8 60- p 0.05 - <

20-

I

(d) Ling Reef 13 m transect.

109 105 1 00 104 L 100- ::J * 0 ·� .-- - G 7.38 0 = () 60- df = 8 Q) c: - 0.05 p > 0.. (I) 20- � I

1986 I 1987 I 1988 Year n Figure 3.6 Temporal variation in spine colours at D White sites with permanent transects. fm Purple-green 123 Green G-tests for independence were used to determine whether 8 Purple __ _ there was temporal variation in morph frequencies. .__ 49

graen C�nd purple-green spines were used there was weak evidence of temporal ViJriiJtion (G ::: 11.82, df ::: 5, p ::: 0.04). Purple-green spines varied from 95.0 to /' SA% oi the samples but there were no seasonal or long term trends . to the v�riation (Figure 3.7). For some of the early samples purple-green spines were not diliorentia ted from green spines, so Betsy Island and Rocky Cape cannot be compared for spine colour. At Tinderbox Beach there was no significant temporal

> variation in spine colours (G = 9.87, df = 6, p 0.05).

OvAral l moroh At the permanent transects only the Tinderbox 3 m transect showed significant

temporal variation in morph proportions (Tinderbox 3 m, G = 52.27, df = 25, p <

> 0.01 ; Tinderbox 7 m, G = 23.53, df = 20, p 0.05; Ling Reef 3 m, G = 28.60, df =

> > 20, p 0.05; Ling Reef 13 m, G = 30.69, df = 20, p 0.05). The major variation occurred in the January 1988 sample when there was a marked increase in WPG urchins and a decrease in RPG urchins (Figure 3.8a). This reflects the change that was observed for the dermis colours. There was some evidence of change at

Fortescue Bay (G = 12.05, df = 5, p = 0.04) which reflects the variation found for spine colours (Figure 3.9). At Tinderbox Beach there was no evidence of temp.oral

> variation (G = 16.1 0, df = 1 0, p 0.05). The overall picture from these results is one of stability of morph proportions during the study. At some sites there was evidence of temporal variation but the only consistent trends which were observed were for dermis colours at Point Cook Barren 2, Kelp 1 and Kelp 2, in Port Phillip Bay. The dermis and spine colours shov1ed different patterns of variation and their combination to give overall morph did not increase the amount of temporal variation observed.

3.3.2 Variation in dermis colour proportions between size classes

At Tinderbox, only the sample taken from the 3 m transect in January 1988 showed significant variation in the size frequency distributions of red and white dermis urchins (D = 0.343, p < 0.05); the median test diameter of the white urchins was 80 mm whereas that of the red dermis urchins was 7 4 mm (Table 3.1 ; Figure 3.1 0). The overall size frequency at the 3 m transect sampled in May 1987 showed two peaks at test diameters of about 66.5 and 86.5 mm. Although these peaks may not represent single year classes, they will represent different age groups. The first peak appeared to have a higher proportion of red dermis urchins than the second, but a G-test for independence showed that the diffe renee

> between the size classes was not significant (G = 2.06, df = 1, p 0.05). 5 0

n 0 White 1il Purple-green � Green • Purple

(a) Fortescue Bay. N.B. Only green and purple-green spines were used for the G-test.

7 1 49 8 8 . 78 61 4 0 100

G = 11.82

60 df = 5 p = 0.04

20

(b) Tinderbox Beach.

49 256 192 L 100 :::J 0

0 G = u 9.87

60 = cv df 6 c p > 0.. 0.05 (I) � 20

1986 1987 1988 Year

Figure 3.7 Temporal variation in spine colours at Tasmanian sites without permanent transects.

G-tests for independence were used to determine whether there was temporal variation in morph frequencies. 51

(a) Tinderbox 3 m transect. 106 99 98 93 100

G = 52.27

60 df = 25 0.01 p <

20

(b) Tinderbox 7 m transect. 98 195 107 103 106 100

G = 23.53

df = 20 w- 60 , ?, .- p > 0.05

20

.

(c) Ling Reef 3 m transect. 1 08 1 0 1 103 11 3 1 01 100

G = 28.60

60 df = 20 p > 0.05

20

(d) Ling Reef 13 m transect. 109 102 105 100 104 .s::: 1 00 D. G = 30.69 ,_ 0 E df = 20 p > 0.05 - 60 ·co ,_ Q) > 0 n 20 � OWN E3 WP 1987 1988 Year rzJ Wffi Figure 3.8 Temporal variation in overall morphs at lffi] 'AG permanent transects. � Rffi G�tests for independence were used to determine whether • RP there was temporal variation in morph frequencies. 52

n OWN 6J WP [2J WPG I]V\G �RPG • RP

(a) Fortescue Bay.

100

G = 12.05

60 df = 5

p = 0.04

20

(b) Tinderbox Beach.

4 9 256 192 1GO ..c 0. L 0 o G = 16.1 E 6o df = 10 co L p > 0.05 Q.l � 20 �

1986 1 987 1 988 Year

Figure 3.9 Temporal variation in overall morphs at Tasmanian sites without permanent transects. G-tests for independence were used to determine whether there was temporal variation in morph frequencies. 53

Table 3.1 Results of Kolmogorov-Smirnov two-sample tests for differences between size freq uency distributions of red and white dermis urchins.

sa mple size )ite and date D red white p

Tinderbox 3 m � 29 v 87 0.146 44 54 ns Tinderbox 3 m - 7 i 88 0.343 25 68 < 0.05 Tinderbox 7 m - 29 v 87 0.097 57 46 ns

Tinderbox 7 m - 7 i 88 -0.043 66 35 ns Tinderbox Beach - 30 iv 87 0.000 27 22 ns Tinderbox Beach - 22 v 87 -0.027 151 105 ns Tinderbox Beach - 5 x 87 -0.032 108 84 ns Ling Reef 3 m - 4 iii 87 -0.250 67 36 ns Ling Reef 3 m-10 vi 87 -0.059 71 42 ns Ling Reef 3 m - 12 i 88 -0.091 68 33 ns

Ling Reef 13 m - 4 ii 87 -0.065 57 48 ns Ling Reef 13 m - 10 vi 87 -0.194 65 35 ns

Ling Reef 13 m - 12 i 88 0.000 36 59 ns Ling Reef Barren - 10 vi 87 -0 .312 78 26 < 0.05 Ling Reef Slope - 19 vi 87 -0.074 55 46 ns Satellite Island - 11 v 87 -0.256 86 15 ns Roaring Beach - 9 vii 87 -0.023 72 61 ns Betsy Island 6 m - 24 vii 87 0.487 19 19 < 0.01 Betsy Island 13 m - 24 vii 87 0.318 21 77 < 0.05 Stewarts Bay A - 4 xi i 86 0.392 22 83 < 0.01 Stewarts Bay B - 4 xii 86· -0.026 62 39 ns (a) 3 m transect - 29 v 87.

G = 2.06 n = 98 df = 1

p > 0.05 4

(b) 3 m transect - 7 i 88. 8 n = 93

4

(c) 7 m transect - 29 v 87. 8

4

(d) 7 m transect - 7 i 88.

8 � u Key c ::} • Red 0 L 4 D White L-1.-

10 30 50 70 90 110 Test diameter (mm )

Figure 3. 10 Size frequency distributions of red and white derm is urchins at Tinderbox transects.

Insets show overa ll size frequencies. Arrows show where distributions were divided into size cl asses. G-tests for independence were used to determ ine whether proportions differed between size cl asses. 55

Three samples were taken from a seagrass habitat at Ti nderbox (Tinderbox Beach) between April and October 1987. This was one of only two sites found during the entire study where urchins less than 35 mm test diameter could be found in reasonably large numbers. The substrate was coarse ·sand with patches of large oyster and scallop shells where adult urchins occurred. The smaller urchins were found beneath shells but only in patches where adults occurred. For the first sample only small urchins were collected, and the median test diameter tor the whole sample was 18.5 mm (Figure 3.11 a). The two later samples showed three peaks (Figure 3.11b and c) each of which showed increases of their median values of 4-5 mm over the 5 month period. Kolmogorov-Smirnov tests showed no significant differences in distributions of red and white dermis urchins for any of the three samples (Table 3.1 ). When the numbers of each dermis colour were pooled tor each size class for the three samples, a G-test for independence showed no significant variation in morph proportions between size classes. Neither of the permanent transects at Ling Reef, each sampled three times over 10 months, showed significant variation between the size frequency distributions of red and white dermis urchins and only one clear peak was observed for each transect (Figure 3.12; Table 3.1 ). Two other areas at Ling Reef were sampled once in 1988; a flat rock area which was devoid of algal cover (Ling Reef Barren) and a boulder slope, also with very low algal cover (Ling Reef Slope) (Figure 3.13a and b). Kolmogorov-Smirnov tests showed that the size frequency distributions of red and white dermis urchins varied significantly at Ling Reef ' Barren (D = 0.312, p < 0.05) but not at Ling Reef Slope (Table 3.1 ). The median test diameters of red and white dermis urchins at Ling Reef Barren were similar (68.5 and 66.5 mm respectively), so the difference was probably due to the greater negative skew of the white urchins' distribution. Roaring Beach 13 m was the second site where small urchins occurred; the overall size frequency distribution showed two obvious peaks with median values of 2? and 70 mm (Figure 3.13d). A G-test for independence showed that there was a significantly higher proportion of red dermis urchins in the larger size class

(G = 7.07, df = 1, p < 0.01). Of the remaining sites which were sampled once only, three showed different size frequency distributions between red and white dermis urchins: Betsy Island

6 m; D = 0.487, p < 0.01 : Betsy Island 13 m; D = 0.318, p < 0.05: Stewarts Bay A; D

= 0.392, p < 0.01 (Figures 3.13e, t and g; Table 3.1 ). Three peaks were visible in the overall size frequency distribution for Betsy Island 6 m (median values of 38, 64 and 83 mm test diameter) and a G-test for independence indicated that there were significantly different proportions of red and white dermis urchins between 56 (a) 30 iv 87. N.B. Large urchins were not collected for this sample.

- Data were pooled from the three n = 49 samp les for each of the size classes. 8 G = 2.22 df = 2

4- p > 0.05

-

I I I I I

(b) 22 v 87.

12 n = 256

8 llnli�lllllllulllllliiJill,, o1,

4 � - � I� I I I Ifi _I I I

(c) 5 x 87.

12 - n = 205

! JJj��� ili 1llli

Kev ! ----- 4 - • Red

- 0 White I � �� � I I I I 10 30 50 70 90 110 Test diameter Cm m)

Figure 3. 1 1 Size frequency distribution of red and white derm is urchins at Tinderbox Beach.

Insets show overall size frequencies. Arrows show where distr1but1ons were divided into size classes. G-tests for independence were used to determ ine whether proportions differed between size classes 57

Ca) 3m transect - 4 iii 87.

n • 103 - I lllilI.lilll,i, I

-

-

I JlUl l ] I I I I

(b) 3 m tra nsect - 10 vi 87.

-

I) • 113

-

- ""' L

-

-

-

�I �� �I I I I I

(c) 3 m transect - 12 1 88. 12

I) .. 101

>.. u c a

4 � • Red o White

10 30 50 70 110 Test diameter

Figure 3. 12 Size frequency distributions of red and White derm is urchins at Ling Reef transects.

Insets show overa ll size frequencies. 58

(d) t 3 m transect - 4 i i i 8 7

n ... 104 I ,I,I,III,IIIIIII� IJII!uI ,

(e) 13 m transect - 10 vi 87.

n = 100

(f) 13 m transect - 1 2 i 88.

Ke.¥. • Red D White

10 so 70 90 110 Test diameter (mm)

Figure 3. 12 (continued) Size frequency distributions of red and white dermis utchins at Ling Reef transects. Insets show overa 11 size frequencies. 59

(a) Ling Reef Barren - I o vi 87.

n = 104 12 - Ill� 8 -

4 -

h h n11 1 I I I I

(b) Ling Reef Slope - 19 vi 87.

8

4

(c) Satellite Island A- 1 1 v 87.

8

4

(d) Roaring Beach 13 m - 9 vii 87.

G = 7.07 12 n = 133 df = I >. u p < 0.0 I c Q) 8 :::> 0" Q) Key L LL 4 • Red 0 White

10 30 50 70 90 II0 Test diameter (mm)

Figure 3. 13 Size frequency distributions of red and w hite dermis urchins.

Insets show overa ll size frequenc ies. Arrows show where distributions were divided into size classes. G-tests for independence were used to determ ine whether proportions differed between size classes. 60

(e) Betsy lslandJ 6 m-24 vii 87.

n = I 01 G = 1 4.48 - df = 2

'111111I ll ll1 p < 0.0 1 4 "'" I � � II � � � � � � � I I I I I

(f) Betsy Island 13 m-24 vii 87. 8 n = 98

4

(g) Stewarts Bay A-4 xii 86 8 G = 0.03 n = I 07 df = 1

p ) 0.05 4

(h) Stewarts Bay B-4 xii 86. Key 8 • Red n = 102 >­ D Whi te u c Cl.> ::J 4 0"' Cl.> L LL

lO 30 50 70 90 110 Test diameter (mm) Figure 3. 1 3 (continued) Size frequ ency distributions of red and white dermis urchins.

Insets show overa ll size frequencies. Arrows show where distributions were divided into size classes. G-tests for independence were used to determ ine whether proportions differed between size classes. 61

the different size classes (G = 14.48, df = 2, p < 0.01 ). The middle size group contained only one red dermis urchin out of forty whereas the other two groups consisted of 33.3 and 27.9 % red dermis urchins respectively. For Betsy Island 13 m the median test diameter for the red urchins was 77 mm, but 87 mm for the white urchins. The overall size frequency for Stewarts Bay A was divided into two size classes but a G-test for independence indicated that there was no significant differe nee in the proportions of red and white dermis urchins between the two

> groups (G = 0.004, df = 1, p 0.05).

3.4 DISCUSSION

Overall the data indicated that proportions of morphs were stable at a given site over the three year time period of the study. Thus it is valid to use data collected over this time to study geographical patterns of variation (see Chapter 4). Although the repeated sampling of sites allowed the stability of morph proportions over the time period of the study to be determined, the sampling frequency and sample sizes were only sufficient to detect the action of strong selection. The sampling regime used was similar to that for the study of geographic variation and was not designed specifically to detect selection acting over time. Changes in morph proportions of 1 to 1 0% of the population would indicate that weak but biologically significant selection was occurring, but the sample size of 1 00 urchins was too low to allow such changes to be detected. For example, at Tinderbox the average proportion of red dermis urchins sampled was 0.44. The binomial 95% confidence limits for this value are 0.34 and 0.54 (Pearson and Hartley, 1954) indicat ing that only changes of more than 20% of the population could be detected. Also, the sampling intervals were short compared to the generation time of H. erythrogramma, so it was not possible to detect changes in morph proportions between generations. However, the variation which was observed at Tinderbox and Point Cook does suggest that changes in morph frequencies may occur over time periods of 3 to 6 months. As such changes are unlikely to have been caused by differential mortality or recruitment this suggests that morphs may be migrati ng between habitats. Migration of morphs to preferred habitats has been shown in several types of organisms; land snails (Johnson, 1981 ) , limpets (Giese I, 1970 ), and freshwater isopods (Christensen, 1977). Johnson (1981) found that frequencies of banded and effectively unbanded morphs of the land snail Th eba pisana varied between habitats in Western Australia. The degree of association of morph frequencies with habitat varied seasonally and among years at a boundary between habitats. 62

These variations were shown to be due to differential migration of morphs from the more sheltered habitat in winter and to the more sheltered habitat for summer aestivation. Johnson (1 981 ) concluded that differential habitat choice between morphs increased the probability of mai ntaining a stable polymorphism in a heterogeneous envi ronment. Behavioural differences have been found among morphs of the limpet Acmaea digitalis in relation to their preferred habitat (Giesel, 1970) . All limpets settled on the rock face and then light coloured and striped limpets migrated to colonies of the white goose-neck barnacle, Pollicip es polymerus, whereas darker limpets remained on the bare rock. Giesel (1 970) suggested that selection by visual predation was responsible for the evolution of these beh a vi aural differences as the morphs were more cryptic in their preferred habitats. Limpets with intermediate pigmentation were shown to suffer higher selection intensities than either of the extre me morphs. Giese I ( 1 970) concluded that the polymorphism could only be stable if such behavioural differences existed, as selection would be directional if the paler morph did not migrate to the Pollicipes colonies. The behaviour of different morphs of H. eryth rogramma is unknown but'the potential exists for differential migration between habitats because of the variability of their environment. The subtidal habitat of H: erythrogramma shows variation in factors such as the morphology and type of substratum, algal communities and depth. Marked seasonal changes in the amount of algal cover occur and sand may be redistributed during winter storms to cover previously rocky areas. A change in the amount of algae present would change the need for camouflage by the urchins; white dermis urchins are far more easily visible against bare rock than red or pink urchins. The normal amount of movement of adult H. erythrogramma is uncertain but Connolly (1986) found that it was minimal unless urchins were disturbed, when they could move tens of metres before becoming sedentary again. Significant migration is therefore possible and if morphs showed behavioural differe nces in their choice of microhabitat, then changes in the proportions of morphs in an area could change over the short time scales observed in this st udy. Five out of thirteen sites showed different patterns of size frequency distribution between red and white dermis urchins. Of these only two sites showed differences in morph proportions between size classes. The differences at the other three sites were either between the median test diameters or the skew of the distributions of the red and white dermis urchins. These differences might 63 either indicate that the morphs had different growth rates at these sites, or that cohorts from different years were merging to form one peak in the distribution and that the morphs had had differential recruitment successes in different years. However, the sample sizes were not large enough to determine whether biologically significant variation in morph proportions occurs over the life time of a cohort. Juvenile urchins were only found in large numbers at two sites, one of which (Roaring Beach 13 m) showed a significant decrease in the proportion of white dermis urchins among the larger urchins and at the other site (Tinderbox Beach) there were no differences between size classes. There was almost no algal cover at Roaring Beach 13 m and the substratum was mainly flat rock which suggests that the more visible white urchins may be subject to heavier visual predation than red urchins at this site. The availability of large shells at Tinderbox Beach allows juveniles to shelter beneath them and small urchins are often completely invisible. The effects of visual predation are therefore likely to be reduced at this site, so that the differential crypsis of morphs is not selected for. The results from this study vary markedly from those of Walda (1969), who found consistent changes in morph proportions between adult and juvenile Cepaea nemoralis at the majority of sites sampled. Fifty-two sites on a dam next to the river Rhine had sufficient numbers of juveniles to allow the comparison to be made. Forty sites had a higher percentage of yellow unbanded she lis in the juveniles and thirty-nine had a lower percentage of yellow banded shells in the juveniles. Although the differences were small (usually 4-5%) the trend was clear and Walda suggested that selection was probably responsible. This hypothesis is strengthened by the large numbe r of sites which showed this trend. Walda (1969) also showed that a very steep cline in morph proportions along part of the dam was stable over twelve years. He decided that the results could only be explained by the presence of a governing mechanism, such as heterozygote advantage or frequency-dependent selection. The stability of a cline is stronger evide nee of selection than stability at one or two locations as such a pattern is far less likely to persist (Endler, 1986). The existence of geographic patterns in morph frequencies of H. erythrogramma is addressed in Chapter 4, but the present study was too short to determine whether any such patterns were stable over time. To determine whether morph proportions do vary duri ng the lifetime of a cohort much larger sample sizes would be required and the study should be extended for several years. Also, attempts should be made to find additional sites where juvenile urchins can be sampled in reasonable numbers. 64

CHAPTER 4 GEOGRAPHICAL VARIATION IN MORPH FREQUENCIES AND ENVIRONMENTAL ASSOCIATIONS

4.1 INTRODUCTION

Observations on the patterns of abundance and variability of organisms over their geographical range have played a m ajar role in the development of evolutionary theory (Gould and Johnston, 1972). The variation may be continuous, such as quantitative or morphometric traits, or discontinuous, such as genetic or visual polymorphisms, (Endler, 1986). Initially much of this variation was dismissed as being due to random variation (Dobzhansky, 1941 ; Huxley, 1942), but gradually it became clear that although variation may arise through random processes, it will only become widespread and be maintained by natural selection or by the patterns of gene flow within a particular species. Theories about both selective and stochastic evolutionary processes have been developed through studies of geographical variation. In some cases the presence of a non-random geographical pattern in itself is taken as evidence for selection (Manly, 1985); however, more commonly the associations between the geographical distribution of an organism and environmental variables are studied. Specific patterns, such as clines or patches, may be predicted to test models of stochastic processes. The evidence for the evolutionary models which may be provided by these methods is �nly circumstantial but it often leads to the formation of new hypotheses which may then be tested experimentally. There have been many studies of geographical variation in the visual polymorphisms of terrestrial and marine organisms. Probably the most well known example is that of industrial melanism in moths, where it was first noticed in the late 19th century that the occurrence of previously unusual melanic forms was increasing in industrial areas in Britain, whereas rural areas continued to be .dominated by paler forms (Ford, 1975). The correlation of high proportions of melanic forms of Biston betularia with industrial areas, where air pollution had eliminated most of the lichens �hich grew on tree trunks, was detailed by Kettlewell (1958, 1973). He also showed experimentally that the melanic forms were at a strong disadvantage on lichen covered tree trunks, as was the lighter form on dark tree trunks where lichen was not present, because of selective predation by birds (Kettlewell, 1955, 1956). Thus recognition of geographical patterns in the distribution of the various morphs led to a detailed study and a greater understanding of the evolutionary forces controlling the polymorphism. 65

Cepaea nemoratis has the most studied of all visual polymorphisms, with variation occurring in shell colour and the number and position of bands on the shell (Jones et at., 1977). Many selective and stochastic evolutionary forces have now been shown to act upon this polymorphism. The low mobility of C. nemoralis, and hence restricted gene flow, has meant that the types and proportions of morphs present in a population may vary considerably over a few hundred metres. Small scale geographical variation in C. nemoralis has been shown to be influenced by visual selection by predators; brown, pink and effectively unbanded snails occur in high proportions in woodlands where they are cryptic against the carpet of dead leaves whereas yellow and banded shells predominate in short grass (Cain and Sheppard, 1950). Climatic selection has been linked to both small and large scale geographic patterns. Yellow shells reflect sunshine better than the darker morphs and thus are less subject to heat stress, whereas in cold climates pink and brown snails warm more quickly and thus can become active earlier in the day than yellow-shelled snails. The former has been shown to lead to higher mortality rates in pink and banded snails than in yellow snails on sand­ dunes (Richardson, 197 4) and the latter is thought to explain the lower proportions of yellow shells in the colder parts of Europe (Jones et at. , 1977). Stochastic processes are also involved in producing the geographic patterns observed in C. nemoratis. For example, populat ions occurring near boundaries between woodland and grassland habitats may have high proportions of visually inappropriate morphs because· of gene flow from adjacent populations (Cain and Sheppard, 1954). Founder effects are also thought to be important for many populations as C. nemoratis is opportunistic and may colonise transient habitats, leading to frequent local extinctions and recolonisations. The morphs present in areas recolonised after flooding in East Anglia, and isolated populations in northern Scotland were also probably due to the founder effect (Goodhart, 1962, � 973; Jones et at. , 1977). In North America the populations are often dependent on their origin rather than selective forces (Brussard, 1975; Richards and Murray, 1975). There have also been many studies on colour polymorphisms in marine gastropods. In many of these, small scale geographic variation in morph frequencies have been found and these have been linked to predation pressure either thro ugh apostatic selection or because of cry psis in different habitats (Heller, 1975; Hoagland, 1977; Reimchen, 1979; Atkinson and Warwick, 1983; Hughes and Mather, 1986; Reid, 1987). In at least one study physiological differences between morphs have been considered to be the selective factor 66 involved (Etter, 1988). Stochastic processes are thought to be involved only in as much as many of these species do not have pelag ic larval stages and thus there is restricted gene flow between nearby populations. Many echinoid species are polymorphic for colour, and the proportions of morphs may vary over small and large scale distances (Lindahl and Runnstrom, 1929; Vasseur, 1 952; Gamble, 1967; Pawson and Miller, 1982; Lawrence, 1 983; Lewis and Storey, 1984; Tsuchiya and Nishihara, 1984). Although some of these authors have suggested possible reasons for the patterns of variation observed, there have been no studies specifically addressing the problem. Colour has been shown to be genetically controlled in Lytechinus variegatus (Pawson and Miller, 1982) and this is probably also the case for H. erythrogramma (see Chapter 2). Unlike some of the colour polymorphic marine gastropods, almost all echinoids have planktonic larval stages and so the patterns of geographic variation must be maintained in the face of considerable gene flow. The most common approach in studies of geographical variation has been to produce distribution maps of the occurrence of specific genes or morphs which are used to detect spatial patterns. Correlations between proportions of morphs and environme ntal factors are then looked for as evidence of the action of nat,u ral selection. Stochastic processes may be inferred but usually are not tested for because there are no satisfactory methods of describing spatial differentiation quantitatively (Endler, 1977). Sokal and co-workers have developed methods of determining whether a spatial pattern is non-random or fits a particular type of distribution (Gabriel and Sokal, 1969; Royaltey et a/. , 1975; Sokal, 1979) but these methods have yet to be extensively applied to studies of polymorphic variation. Sokal (1 978) described four ways in which population differentiation of genetically controlled traits might occur: (1 ) a character may be differentiated in response to an environmental gradient producing a cline; (2) the environment is �ade up of patches with heterog�neous populations between patch types, but homogeneous populations within; (3) isolation of populations with increasing distance leads to a cline; (4) historical accidents may have occurred, such as the founder effect causing random patches, two founder effects followed by diffusion of genes between the two populations causing a cline, or bottleneck phenomena. The first two models imply selection and the last two, stochastic or historical effects. Sokal and Oden (1978b) pointed out that it is unlikely that only one of these mechanisms will be responsible for an observed geographical pattern; C. nemoralis provides a good example of the many different evolutionary processes which can affect one species. Jones et a/. (1977) suggest that unique 67 combin��\lons oi �'Jolutiomny processes may control each population of Cepaea. The problt:m in rn:::ny studies is to distinguish between selective and swciK! Stic rneciKwisrns. This is partly because of the lack of statistical methods �1v�1il�tblu to t0st tho signiiicance of geographical patterns as evidence for specific mac!0ls oi population differentiation. This problem has now been partially ovcrcomo with the development of Mantel's non-parametric test (Sokal, 1 979; �·.!Jnly, 1985}. Mantel's test was first used to detect space and time clustering of dise:.1ses (Mantel, 1967) and has since been developed by Sokal and co-workers to determine the significance of geographical patterns of morphological and en:::yme variation. It is now being more widely used in many areas of biology: evolutionary biology (Jones et a/. , 1980; Douglas and Endler, 1982; Francis et a/., 1 986; Able and F elley, 1986; and see D ilion, 1984); ani mal behaviour (Schnell et a/. , 1985}; biogeography (Page, 1987}; and plant ecology (Burgman, 1987; Harvey eta/. , 1988}. The advantage of Mantel's test is that several competing hypotheses may be tested against the same data set if each of the hypotheses predicts different patterns of variation. It may therefore be used to distinguish between selective and stochastic processes by developing specific models of population differentiation for each process (Douglas and Endler, 1982; Dillon, 1984; Francis et a/. , 1 986). Each model is tested by comparing pairs of matrices based on morphological or genetic, envi ronmental and geographical distances (although Dillon uses Kendall's tau (Kc) rather than Mantel's statistic). Environmental and geographic matrices are compared to determine whether selective and stochastic models are independent. Douglas and Endler (1982} concluded that selection was the major force in determining the spot pattern s on male guppies, whereas Dillon (1984} and Francis et a/. (1986} found that both selective and stochastic processes were important, but for different suites of morphological or enzyme characteristics. Burg man ( 1987} also used Mantel's test to distinguish between various ecological models of plant distribution. He found that specific predictions could be proposed for each hypothesis and tested to distinguish between competing models using this method. · This chapter describes small and medium scale geographic variation in the frequencies of colour morphs of Heliocidaris erythrogramma in south eastern Australia. Associations with environmental variables are examined and both selective and stochastic models of population differentiation are tested using Mantel's non-parametric test. 68

4.2 MATERIALS AND METHODS

4.2.1 Collection of data

In order to determine the variation in proportions of morphs around Tas mania samples were collected from the south, east and north coasts (Figure 4. i ). The west coast was not sampled because it is a very exposed coast and few, if any, H. erythrogramma occur there (Dix, 1977). Sites in Port Phillip Bay, Victoria (Figure 4.2) were sampled by A. Constable, Zoology Department, University of Melbourne. Small scale geographic variation was determined by sampling five sites at Tinderbox (sites i 3 -1 7), four sites at Ling Reef (7 - 1 0) and two at Satellite Island (5 - 6) and Stewart's Bay (24 - 25). Ling Reef was mapped using five 1 00 m transects of leaded ro pe placed perpendicular to the shoreline. Depth, substratum and the main algal species present were recorded every 1 0 m along each transect. The map of the seabed at Tinderbox was adapted from Sanderson and Thomas (1 987).

Morph data .A quick visual survey of each site was made to determine the area of greatest urchin abundance, from wh ich the sample would be taken. All divers kept within sight of each other to ensure that they were collecting from the same habitat. Urchins were collected by searching the area thoroughly to ensure that the more cryptic morphs were not under-represented. Where possible a sample of 1 00 urchins was taken, but in areas of low urchin density this had to be reduced to 50- 60. The urchins were taken to the shore and scored for colour and intensity of pigmentation according to the scheme outlined in Chapter 2. Most sites were sampled only once, but two transects at Ti nderbox (sites i 3 and 14) and Ling Reef (7 and 8) and one area at Fortescue Bay (26) were sampled repeatedly to determine the temporal variation in proportions of morphs (see Chapter 3) . For these sites the total numbers of urchins collected throughout the study were used in analyses presented in this chapter.

Environ mental data Site descriptions included information on the depth at which urchins were collected, substratum, algal species present and amount of algae present, density of the urchin population, and the exposure of the site to wi nd driven waves. Depth was measured using a Technisub, oil-filled depth gauge. The presence or Figure 4.1 Site locations around Tasmania

Key 1 Pelican Island 2 2 Betsy Island 13 m 2 Blubber Heads 2 3 Dart Island 3 Roaring Beach 6 m 2 4 Stewarts Bay A 4 Roaring Beach 13 m 2 5 Stewarts Bay B 5 Satellite Island A 2 6 Fortescue Bay 6 Satellite Island B 2 7 Marion Bay 7 Ling Reef 3 m transect 2 8 Reidle Bay 8 Ling Reef 13 m transect 2 9 Stapleton Point 9 Ling Reef Barren 3 0 Painted Cliffs 1 0 Ling Reef Slope 3 1 Shelly Beach 11 Gordon 3 2 Co�es Bay 1 2 Coningham 3 3 Bicheno 1 3 Tinderbox 3 m transect 3 4 Skeleton Bay 1 4 Tinderbox 7 m transect 3 5 Stumpys Rocks 1 5 Tinderbox Slope 3 6 South Crappies Point 1 6 Tinderbox Barren 3 7 Low Head 1 7 Tinderbox Beach 3 8 Greens Beach 1 8 Blackmans Bay 3 9 Rocky Cape 1 9 Alum Cliffs 4 0 Cowrie Point 2 0 Dennes Point 41 Trousers Point 21 Betsy Island 6 m 69

N

3 6 {) 35

50km 70

Melbourne •

N

10km

Key 42 Point Franklin 43 Point Henry 44 Point Lillias 45 Tablerock Point 46 Point Cook - Barren 1 47 Point Cook - Barren 2 48 Point Cook - Kelp 1 49 Point Cook - Kelp 2

Figure 4.2 Site locations in Port Phillip Bay, Victoria. 71

absence, and size of boulders was noted. If the substratum was flat rock, the presence and extent of crevices were also recorded. The rock type was noted and later verified using the map ��Geology of Tasmania" (Lands Department, Hobart). The substratum ty pe was classified as follows: 1 Sand 2 Flat rock 3 Flat rock with some crevices 4 Medium boulders with crevices 5 Large boulders with extensive crevices Algal cover was estimated visually and macroalgae were collected for later identification. The density of the urchin population and percentage algal cover were measured using a 0.25 m2 quadrat (20 replicates per site). As it was only possible to use the quadrat in calm weather and when diving partners were available, these data were not collected at all sites. The quadrat data were used to roughly calibrate the visual assessment of algal cover: 1 Very low <1 0% 2 Low 10-40% 3 Moderate 40-80% 4 Heavy >80% The directions open to wind driven waves were noted using a compass and a rough assessment of the exposure of the site was made, for example by noting whether bull kelp (Ourvillea potato rum) was present in the intertidal zone indicating a very exposed site, o'r noting the presence or absence of fine sediment on algal fronds which helped to identify very sheltered sites.

Exposure indices To determine whether exposure to wave action was correlated with proportions of morphs an exposure index was developed. Exposure indices may be divided into two types: biological and physical. Biological indices are based on communities of plants and animals which occur on shores of different exposures (Ballantine, 1961 ; Lewis, 1964), whereas physical indices attempt to model the strength of wave action using measurable parameters such as wind strength and direction and length of fetch (Moore, 1935; Baardseth, 1970). A physical index was preferred to a biological scale in the hope that it would provide a more objective measure and might give greater resolution. The factors affecting the strength of wave action hitting a coastline are many and most physical indices oversimplify the situation and hence are not reliable for fine scale resolution. However, Thomas (1 986) developed an index using wind 72

energy and direction, total fetch and the distance of water shallower than 6 m next to the coastline. This index was calculated for each of the sites around Tasmania but failed to provide values consistent with what was already known of the exposure of Tasmanian coastlines (Bennett and Pope, 1960; Davies, 1978). It gave values for the north coast (adjacent to the shallow Bass Strait) of the same magnitude as those for the east coast which is exposed to oceanic swells. The physical exposure index (P.E.I.) was modified to allow adjustment for the depth over the total fetch, and to increase the distance which was assumed to be the maximum fetch over which waves increase wave energy. Biological exposure indices have been developed for Tasmanian coastlines based on intertidal communities (Ben nett and Pope, 1960) and on subtidal macroalgae (Edgar, 1984). The latter approach was adopted for this study. Edgar's (1984) index appears to be based mainly on data from the D'Entrecasteaux Channel in southern Tasmania (Sanderson, personal communication) so an index was developed, with the help of C. Sanderson (Plant Science Department, University of Tasmania), which could be used for all Tasmanian coastlines. The index is based on the biological communities described by Bennett and Pope (1960), Edgar (1984), and Sanderson and Thomas ( 1987), modified by our own observations. The dominant large brown algae were used as indicators of the different communities which occur with differing exposure (Figure 4.3). To apply this index, the vertical distribution of the algae at each site must be observed, although it was usually only necessary to survey the algae down to a depth of 7-1 0 m. Although the algal communities present at different depths were used in applying the index, the resulting value for the exposure of a site does not contain a depth component, e.g. two sites in the same area, at 3 and 10 m respectively, would be given the same A.E.I. even though the deeper site would receive less force from surface wave action. The application of this index was usually straight forward except for Bass Strait sit�s which have somewhat different algal communities to the rest of Tasmania. This is probably due to many factors including different nutrient levels in the water and a water temperature, on average, 2°C warmer than elsewhere in Tasmania (Sanderson, personal communication). Category 1 can include communities of seagrass (which grow on sand) or Cystophora/Sargassum (which grow on rocks) because this difference reflects the substratum rather than a difference in exposure to wave action. Algal Exposure Index

6 5 4 3 2 I

5

10

Depth -...) (m) Vl J D= Durvillea 15 y p = Phyllospora E L = Lessonia E = Ecklonia M= Macrocystis 20 � / C= Cystophora

s = Sargassum A= Aerocarpi a 25 .J/ G= Seagrass

Figure 4.3 Exp lana1ion of Algal Exposure Index. 74

4.2.2 Analysis of data

G-tests for independence were used to determine whether the small scale geographic variation between sites was significant for each of the four areas. Character association within populations was determined by calculation of the index D' =AD - BC (Jones, et a/., 1980), where the letters refer to the proportions of individuals that were (A) red dermis, purple spined, (B) white dermis, purple spined, (C) red dermis, not purple spined and (D) white dermis, not purple spined. The significance of the association was determined by G-tests for independence, although for some sites the test was not appropriate because of the small numbers of individuals in one or more categories. Algal and physical exposure indices were considered to be independent variables, hence the I eve I of agreement between the two was determined by correlation. To determine whether either index could be used to predict dermis colour proportions, linear regressions of arcsine percentages of red dermis urchins against A.E.I. and P .E.I. were calculated. This transformation is used when small (<30) or large (>70) percentages occur in a data set, in order to improve the normality of the data (Zar, 1974). The Kolmogorov-Smirnov test did not indicate that the normality of the data had improved, but frequency plots showed that the data were less negatively skewed. The relationships between transformed perce ntages of red dermis urchins present at each site and the environmental variables were analysed using stepwise multiple regression (Sakal and Rohlf, 1981 ). As the densities of urchins were only known for 27 out of the 49 sites, these data were regressed separately. Simple linear regressions of transformed proportions of dermis colour against A.E.I. were calculated for each of the four geographical regions; southern, eastern, and northern Tasmania and Port Phillip Bay. Analysis of covariance was used to determine whether the slopes of the regressions varied significantly. Tukey's test was used to determine which of the pairs of regressions differed significantly. Mantel's test (Mantel, 1967; Sakal, 1979; Manly, 1985) was used both to determine whether the geographic distribution of morph frequencies was non­ random (Sakal, 1979) and to test specific models of population differentiation. This test compares two similarity (or distance) matrices (site x site) which may represent gene frequencies, morph frequencies, morphological similarity, environmental data or various types of geographic distances. Mantel's test determines the extent of a linear relationship between the two variables. Consequently plots of each pair of matrices should be produced and any non- 75

linear relationships should be adjusted by a suitable transformation (Smouse et a/.,1986).

The matrices are usually designed so that the null hypothesis assumes a non­ significant negative relationship between them (Douglas and Endler, 1982). A test statistic (Z), its standard normal variate (G) and Pearson's coefficient of correlation (r) between the elements of the two matrices are calculated. The sig nificance level of G may be determined by comparing it with the standard normal distribution but this assumes that G is normally distributed and this may not be the case (Manly, 1985). An alternative approach is to use a randomization test (Edgington, 1987). A large number of random permutations of one of the matrices against the other matrix are produced and G is calculated for each permutation. The probability value is the proportio n of these G values which is greater than or equal to the experimental G. For example, if the test G is greater than 950 of 1000 randomly produced G's, then the result is significant at the 5o/o level. Manly (1985; pp. 176-

182) gives a detailed description of the mathematics of Mantel's test and calculations were perfo rmed using the computer program provided.

The Matrices DERMIS

Differences in the proportions of dermis colours between pairs of all 49 sites were determined using the equation from Manly (1985): k dmor = 0.5 2: ·I (p; - qi) I i="1

where dMoR = distance measure k = number of morphs

Pi = proportion of i morphs at site p q = proportion of i morphs at site q

Identical sites = 0 Different sites ::; 1 SPINE

Differences in proportions of spine colours were determined as for DERMIS, but data were only available for 38 sites, i.e. excluding Coningham (site 12),

Bicheno (33), Trousers Point ( 41 ) and all the Victorian sites (42-49). EXPOSURE IN DE X

Differences in exposure between each pair of sites, p and q, were calculated

as; d AEI - AEi EXP = P q where AEI = algal exposure index. Identical sites = 0 Different sites ::;; 5 76

OVERALL ENVIRONMENT Distance measures based on all the environmental data collected (AEI, depth, substratum, algal cover) were produced by B IOLT AT cluster analysis, using Gower's coefficient which is designed for use with mixed continuous and multistate data. Identical sites = 0 Different sites � 1 BINARY PATCH Patch types were defined on the basis of type and amount of algae present, substratum type and depth: 1 Macrocystis forest, large boulders, urchins at < 6.5m 2 Macrocystis forest, large boulders, > 7 m 3 Low algal cover, flat rock substratum, > 7 m 4 Low algal cover, sand substratum, > 7 m 5 Low algal cover, flat rock substratum, < 7 m 6 Heavy algal cover, boulders, < 7 m 7 Low algal cover, boulders, < 7 m 8 Seagrass bed, sand substratum, < 7 m A binary connectivity matrix was produced. Sites of same patch type = 0 Sites of different patch type = 1 SHORTEST SEA DISTANCE The shortest sea distance (SSD) between each pair of sites was measured in kilometres around the coast, from 1 :50,000 maps. Very close sites = 0 Distant sites � 869 BINARY CONTIGUITY A modified Gabriel network was produced to identify geographically contiguous sites. Gabriel and Sakal (1969) defined two sites (A and B) as contiguous if no other site fell within the circle of which AB was the diameter (Figure 4.4). In this study SSD (instead of straight line distance) was used to check that two sites were not considered contiguous if there was a third site which was closer to A or B (in terms of SSD), than they were to each other. This was done because straight line distances would not accurately describe the probable patterns of gene flow for marine species with pelagic larvae. In practice the only differe nee this made was that Dart Island (23) was considered contiguous with Betsy Island (2"1 and 22) but not with Stewart's Bay (24 and 25). No contiguity was allowed between sites in different geographical regions, as previously defined. A binary connectivity matrix was produced. Contiguous sites = 0 Non-contiguous sites = 1 77

Figure 4.4 Definition of contiguity for Gabriel networks. Pairs of sites A with B and B with C are contiguous, but A is not contiguous with C.

CONTIGUOUS DISTANCE Values for contiguous sites {i.e. 1's) fro m BINARY CONTIGUITY were replaced with the equivalent kilometric distance from SHORTEST SEA DISTANCE. Non-contiguous sites {designated by O's) would have an infinite distance between them; in practice they were given a value slightly larger than the greatest SSD between any two sites. Contiguous sites = 0 - 89 Non-contiguous sites = 870 BINARY AREAS The four geographical regions {i.e. sites 1-23, 24-35, 36-41 and 42-49) were assumed to be genetically isolated from each other but gene flow was assumed to occur between all sites within a region. A binary connectivity matrix was produced: Connected sites = 0 Unconnected sites = 1 DISTANCE WITHIN AREAS Values for connected sites from BINARY AREAS were replaced with SSD's from SHORTEST SEA DISTANCE. Unconnected sites were given a value slightly larger than the greatest distance between sites. Connected sites = 0 - 269 Unconnected sites = 870 Each of the environmental and geographical distance matrices were produced for data from all 49 sites for comparisons with DERMIS, and for data from only the 38 sites for which spine colour data were avai lable, for comparisons with SPINE. A 38-site matrix of DERMIS was also produced to determine how strongly correlated dermis and spine colours were. 78

The models Each of the four mechanisms of population differentiation suggested by Sakal (1978) and described in the Introduction was used to develop a specific hypothesis for the H. erythrogramma polymorphism: Model 1 Proportions of morphs are related to an environmental gradient which may or may not take the forrn of a geographical eli ne. Sakal's mode I assumes that environmental differentiation increases with the distance between sites. However, this is not necessarily true; the exposure of a site to wave action may depend on whether it is on an open coastline or within a sheltered bay. In this case the organisms at the site might be responding to an environmental gradient, but show patchy variation in space. Two possibilities were tested; the exposure of a site to wave action may be of paramount importance or, alternatively, several environmental factors could be involved. Model 2 Proportions of morphs are dependent on the types of habitat (defined as patches), which are not re lated to geographical position, and there is no correlation between proportions of morphs of different patch types even if ·the environmental factors used to define the patches are similar. Model 3 Proportions of morphs vary because isolation by distance has allowed divergence between distant sites to occur, givi ng a geog raphical cline over the entire study area. Model 4 Each of the four geographical regions previously defined was subject to a founder effect. Thus proportions of morphs of the sites within each region should be similar and not necessarily related to those of adjoining regions. Model 5

· This model extends Model 4 to predict isolation by distance may have occurred within each region after the initial proportions of morphs were established by founder effects. Thus a geographical cline would be observed within each area.

The tests Each of the five models suggests a specific and unique pattern of morph distribution. Limitations of the models and alternative processes by which such patterns might have been produced are discussed later. The results of 79 comparisons of morph matrices (DERMIS and SPINE) with each of the environ mental and geographic distance matrices may be predicted for each. model

(Table 4.1 ) . It can be seen that it should be possible to distinguish between selective (Models 1-2) and stochastic (Models 3-5) processes. However, the three environmental matrices are all derived from similar data, as are the five geographic distance matrices. Thus correlations of matrices within the two groups are to be expected and it is only possible to determine which of the models fits the data best, without excluding the possibility of other models being valid. For example, if both EXPOSURE INDEX and OVERALL ENVIRONME NT gave positive results, the one with the highest correlation coefficient would be considered to have the greatest predictive power. Where two models might predict similar patterns (e.g. Models 2 and 4 predict patches and Models 1 and 5 predict clines) the two models may be distinguished if the patches (or clines) do not correspond. For example, both OVERALL ENVIRONMENT and SHORTEST SEA DISTANCE might give a positive result when compared with DERMIS because sites which are geographically far apart tend to have different environments. If OVERALL EN VI RON MENT and SHORTEST SEA DISTA NCE were significantly correlated with each other it would then be difficult to determine which of the two processes was occurring. However, if they were not correlated, this would be evidence that both environmental factors and isolation-by-distance were important. Therefore when two models gave significant results, the relevant pairs of environmental and geographic distance matrices were compared. Comparisons between DERMIS and BINARY CONTIGUITY and CONTIGUOUS DISTANCE were made to determine whether the distribution patterns were non-random. A significant positive relationship would indicate that sites were positively autocorrelated, i.e. the proportions of morphs at one site were dependent on those of contiguous sites.

_ In case the large matrices were obscuring different patterns present in each geographical region, similar matrices were produced for each region and tested in the same way as before. BINARY AREAS and DISTANCE WITHIN AREAS were not applicable because this approach makes the assumption that the four regions are different. These regional matrices were given suffixes as shown; southern Tasmania - S, eastern Tasmania - E, northern Tasmania - N, Port Phillip Bay, Victoria - V. For most comparisons 1 000 random permutations were computed in order to determine the significance level of the test. Where this gave a borderline significance (i.e. close to p==0.05) 50,000 simulations were used (Jackson and Table 4. 1 Predictions of results of Mantel's tests comparing morph matrices (DERMIS or SPINE) with each of the environmental and geographic distance matrices.

++ + = highly significant positive result, = significant positive result, ns = non-significant or negative result.

Model 1 - Morph distribution related to environmental gradient.

Model 2 - Morph distribution related to environmental 'patch' type.

Model 3 - Morph distribution due to isolation by distance.

Model 4 - Morph distribution due to a founder affect in each region.

Model 5 - Morph distribution modified by isolation by distance within each region. Comparisons with BINARY CONTIGUITY and CONTIGUOUS DISTANCE were to determine whether the geographical patterns were random or showed I ow-order autoco rrel atio n.

Model 1 Model 2 Model 3 Model 4 Model 5 ++ + EXPOSURE INDEX ns ns ns OJ Environmental OVERALL EN VI RONM ENT ++ + ns ns ns 0 matrices BINARY PATCH + ++ ns ns ns SHORTEST SEA DISTANCE ns ns ++ + + Geographic BINARY AREAS ns ns + ++ + distance DISTANCE WITHIN AREAS ns ns + + ++ matrices BINARY CONTIGUITY ns ns + + + CONTIGUOUS DISTANCE ns ns + + + 81

Somers, 1989). To reduce the chance of Type 1 errors occurring, a family . significance level was then determined using the Bonferroni technique (Douglas

and Endler, 1982). The probability value was calculated as p = 0.05 In, where n was the number of comparisons made. Where both selective and stochastic models gave significant results, the relative importance of the different models was determined by calculating partial correlation coefficients and the coefficient of multiple determination (Smouse et a/., 1986). The environmental (Y;) and geographic distance (Yj) matrices were regressed against the morph matri.x (Yk) and each other, and new matrices (Di.j, Di.k and Dj.k) of the residual deviations from these regressions were produced. When one of the residual matrices (e.g. Di.k) is compared with another (e.g. Dj.k) the correlation coefficient is the partial correlation, rij.k of Yi and Yj , given Yk (Smouse et a/., 1986). The advantage of this approach is that the significance level of the partial correlation coefficients may be determined by comparison with a null distribution produced by randomly perm utating one of the residual deviations matrices and comparing it with the other, as before. The spatial patterns of dermis colour proportions were further clarified by plotting the percentages of red dermis urchins against shortest sea distance. As , each region (apart from Port Phillip Bay) approximated a straight line, the distance from a site at one extremity of the region to each of the other sites was used (i.e. site 1 in southern Tasmania, site 24 for eastern Tasmania and site 41 for northern Tasmania). For Port Phillip Bay Point Franklin (site 42) was taken as the initial site. This type of plot is used to determine whether spatial patterns form continuous or stepped clines or patches (Endler, 1977). In order to determine whether pelagic larvae of H. erythrogramma were likely to be transported from northern Tasmania to the east coast or across the Bass Strait to Port Phillip Bay, current patterns were investigated using a computer simulation developed by C. Fandry, C.S .I. R.O. Division of Oceanography. The mqdel assumes that particles are always at or near the surface and their movement is simulated under different wind conditions which commonly occur in summer (i.e. when H. erythrogramma breeds). Winds of 25 knots from four directions were considered; northerly, north-westerly, westerly and south-westerly. Particles (representing urchin larvae) were released from three points; South Crappies Point (site 36), Cowrie Point (40) and Trousers Point ( 41 ). The positions of the larvae at 24 hour intervals were recorded for 20 days, which is comparable with the length of larval life found in laboratory trials (Appendix 1 ). 82

4.4 RESULTS AND DISCUSSION

4.4.1 Small scale geographic variation in proportions of morphs

Dermis colour The dermis colour proportions at five sites at Ti nderbox show highly significant variation between sites (Figure 4.5; Table 4.2a). There is a slight trend of decreasing proportions of white dermis urchins from west to east except for the 3 m transect (site 13), which has a much higher proportion of white dermis urchins than any other site. This site is the only one which is affected by the oceanic swells from Storm Bay (Sanderson and Thomas, 1987); the 7 m transect will not be so affected because of its greater depth and all the other sites are protected by Bruny Island. The other feature which distinguishes site 13 from all the others is its heavy algal cover; there is almost no algae at sites 14 and 15, site 16 had no algae present when sampled although it has later become overgrown by the Ecklonia zone, and the seagrass bed of site 17 is very sparse. The four sites at Ling Reef again show highly significant variation in dermis colour proportions (Figure 4.6; Table 4.3a). The three most shallow sites show a trend of increasing proportions of white dermis urchins with decreasing depth but this is reversed at the deepest site (8), which has almost the same proportions of white dermis urchins as the shallowest site (9). All four sites have low algal cover but site 8 is the only one with a sand substratum. There will be an increasing level of exposure to wave action with decreasing depth. Two sites on opposite sides of Satellite Island (5 and 6) and two sites at Stewarts Bay (24 and 25) also show significant variation in dermis colour proportions (Figure 4. ?a and b; Tables 4.4a and 4.5a). Satellite Island A (24) is shallow, has almost no algae present and is very sheltered in a small bay on the north-east of the island. Only about 1.5 km around the coast of the island is site B (25j, which is deeper, has heavy algal cover and is exposed to oceanic swells entering the southern end of the D'Entrecasteaux Channel. Satellite Island B has more than three times the proportion of white dermis urchins as A Stewarts Bay A and B are 400 m apart, on opposite sides of the mouth of a small bay on the Tasman Peninsula; A has double the proportion of white dermis urchins as B. Both have heavy algal cover and similar boulder substrata and depth but A is part of a Macrocys tis forest whereas the main algae at B were Ecklonia and Sargassum spp. indicating that it is less exposed. The shape of the bay is such that A will be subject to wave action from southerly oceanic swells, although they 83

� (site number)

Substrate Dermis colours 13 3 m transect � boulders • R 14 7 m transect D w 15 Slope tlat rock [ill p O 16 Barren - sand tZJ 17 Beach N (a) Dermis colour proportions and substratum. I

Q)� 0 .c. U) E 0 .::: 40

(.)Q) c ro ..... (/)

0 200 400 Distance along shore (m) Algal Communities Key R Acrocarpia - . [j.·:·:·: Eck ron Ja Carpoglossum seagrass L::.JJeanne retti a m j;rj �Cys tophora v-:::1 . mrrn c ule pa low algal U z onana � : � moniliformis Willl t r 1fana D cover {b) Algal communit;es.

Figure 4.5 Variation in dermis colour proportions with habitat at Tinderbox. 84

Table 4.2 Results of G-tests for independence on morph proportions at Tinderbox sites.

(a) Dermis colours Any p urchins were lumped with w�s because they occurred at very low frequencies.

Site Dermis (13) 3 m (14) ?m ( 15) ( 1 6) ( 1 7) colour transect transect Slo e Barren Beach Total

R 367 460 55 62 285 1229

w 466 256 39 32 212 1005

Tot a l 833 716 94 94 497 2234

G = 71 .47 DF =4 p < 0.001

(b) Spine colours

Site Spine (13) 3 m (14) ?m ( 1 5) ( 1 6) ( 1 7) colour transect transect Slo e Barren Beach Total

p 112 1·55 1 9 24 106 416

FG 508 370 57 58 306 1299

G 52 46 15 1 0 74 197

w 25 33 3 2 1 1 74

Total 697 604 94 94 497 1986

G = 55.14 OF= 12 p < 0.001 � Alga l Communities Key

Substrate F0J Acrocarpia - 2m Li:J 2m Jeanneretti a �boulders � Cystophora � mo niliformis Otlat rock low algal D cover liJ�s and

Dermis colours

• A 8 0 w Ol Ul E1 p 10

(site number) 12 7 3 m transect 8 13 m transect 9 Barren 10 Slope N� � 25 m (a) Dermis co lour proportions and substratum. (b) Algal communities.

Figure 4.6 Variation in dermis colour proportions with habitat at Ling Reef.

-It""..;:-�-�·- 86

Table 4.3 Results of G�tests for independence on morph proportions at Ling Reef.

{a) Dermis colours Any p urchins were lumped with W's because they occurred at very low frequencies.

Site Dermis (7) 3 m (8) 13 m (9) ( 1 0) colour transect transect Barren Slo e Total

R 422 361 55 78 916

w 215 265 46 26 552

Total 637 626 1 01 104 1468

0.001 G = 20.09 OF = 3 p <

{b) Spine colo urs

Site Spine (7) 3 m (8) 13 m ( 9) ( 1 0) colour transect transect Barren Slo e Total

p 140 130 2 7 1 3 310

ffi 293 318 63 69 743

G 81 63 1 2 1 7 173

w 1 2 9 2 2 25

Total 526 520 104 1 01 1 251

G=14.10 OF = 9 p > 0.05 87

Storm Bay

0. Satellite Island A

.

.

. /.

5 km

Dermis colours • A N ow {a) Satellite Island. 13 p

(25)

Tasman Peninsula

5 km

(b) Stewart's Bay.

Figure 4.7 Small scale variation in dermis colour proportions. 88

Table 4.4a Results of G-test for independence on dermis colour proportions at Satellite Island. Any p urchins were lumped with W's because they occurred at very low frequencies.

Site Dermis ( 5) (6) colour Site A Site 8 Total

R 86 51 137

w 1 5 55 70

Total 1 01 106 207

G = 33.22 DF = 1 p < 0.001

Table 4.4b Results of G-test for independence on spine colour proportions at Satellite Island.

Site Spine ( 5) (6) colour Site A Site 8 Total

p 20 23 43

FG 73" 66 139

G 7 1 0 17

w 1 7 8

Total 1 01 106 207

G = 6.02 OF = 3 p > 0.05 89

Table 4.5a Results of Gwtest for independence on dermis colour proportions at Stewarts Bay. Any p urchins were lumped with W's because they occurred at very low frequencies.

Site Dermis {24) {25) colour Site A Site B Total

R 21 63 84

w 86 39 1 23

Total 1 07 102 209

== G 63.24 DF = 1 p < 0.001

Table 4.5b Res ults of G-test for independence on spine colour proportions at Stewarts Bay.

Site Spine {24) {25) colour Site A Site B Total

p 9 1 1 20

FG 74 . 80 1 54

G 23 9 32

w 1 2 3

Total 1 07 102 209

G = 7.00 OF = 3 p > 0.05 90

will be decreased in intensity as they passed through Port Arth ur, whereas B is more protected.

The variations in dermis colour proportions therefore seem to be related to exposure, algal cover and the type of substratum. The proportions of white dermis

urchins increase with increasing exposure and algal cover for each area, except at

the deepest site at Ling Reef which is the only site with a sand substratum. The high proportion of white dermis urchins at this site may be due to visual predation

pressures; white dermis urchins are less obvious on sand than red dermis urchins. The urchins cover themselves extensively with pieces of broken shell and drift algae at this site which may also indicate the need for camouflage from visual

predators. The correlation with exposure may be due to different abilities of the morphs to maintain their position during storms or it may be that exposure is itself

correlated with other aspects of the habitat which affect urchin colour. The

correlation of increased proportio ns of white urchins with heavy algal cover at

rocky sites again suggests that visual predation may be important; the algae will help hide the white urchins. Pink dermis urchins occurred mainly at the shallowest

sites, suggesting that higher light intensities might cause increased dermal pigmentation.

Spine colour Proportions of spine colours varied sig nificantly at Tinderbox (Table 4.2b); site 13 had a higher proportion of purple-green spines than the other sites.

Proporti ons of spine colour did riot vary significantly between sites at Ling Reef, Satellite Island and Stewarts Bay (Tables 4.3b, 4.4b and 4.5b). Proportions of

spine colours therefore showed less variation than dermis colour and were little

affected by habitat variation. The only observed pattern was an increased

proportion of purple-green spines at the most exposed site at Tinderbox. In summary, small scale geographic variation in proportions of morphs occurs

mo9:inly for dermis colours, with spine colours showing little significant vari ation. Dermis colour showed consistent correlations with exposure to wave action, algal

cover and type of substratum at four areas in south eastern Tasmania.

4.4.2 Distribution of morphs over entire study area

De rmis colour

Several reg ional differences can be seen in the distribution of dermis colours (Figure 4.8; raw data given in Appendix 2a). In southern Tasmania (sites 1-?2) the populations were quite variable, whereas on the east coast, the exposed sites (26, 91

Key to dermis colours • Red D White lm Pink

N

50 km

Figure 4.8 Variation in dermis co lour proportions around Tas mania. 92

28, 33, 34, 35) have urchin populations which were almost 100% white and the sheltered sites (27, 29 and 31) were almost 100% red. On the north coast the proportions are ve ry consistent and the proportions of pi nk dermis urchins were

much higher than anywhere else in Tasmania (up to 20% of the population). In southern Tasmania the proportion of white dermis urchins gradually decreased

northwards in the D'Entrecasteaux Channel until it increased again at Tinderbox

(Figure 4.9), where the effects of oceanic swells from Storm Bay can be felt.

There are large variati ons in proporti ons of morphs in Port Phillip Bay (Figure

4.1 0) with the two very sheltered seag rass bed sites having almost 100% red dermis urchins and Point Franklin having the highest proportion of white dermis urchins. At Point Cook the barren sites had higher proportions of red dermis urchi ns than those with heavy algal cover. The proportions of pink dermis urchins

(0.7 · 15.6%) were higher than those found in southern and eastern Tasmania but not as high as found on the north coast of Tasmania.

Spine colours The patterns of distri bution of spine colours around Tasmania are not as clear

as for dermis colours, but similar groups of sites can be seen (Figure 4.1 1; .raw data given in Appendix 2b). The exposed east coast sites (26, 28, 34 and 35) had

very few purple spined urchins, but a very high proportion of purple-green spines. In the D'Entrecasteaux Channel and Derwent Estuary the proportions were fairly

stable with about 25% of the urchins having purple spines. On the north coast the

proporti on of purple spined urchins was about a third. The group of sites in the

southeast which had almost 100% red dermis urchins (23, 27, 29 and 30) have the highest proportion of purple spines: about 45%. The proportion of purple­

green spines in southern Tasmania decreased northwards in the D'Entrecasteaux Channel, whereas the more northern sites, exposed to swells from Storm Bay, had higher proportions again (Figure 4.1 2).

Qverall morpb The distribution of overall morphs around Tasmania summarises the variation

in dermis and spine colours (Figure 4.13). The exposed east coast sites (26, 28, 34 and 35) are almost completely dominated by WG and WPG urchins, whereas

the very sheltered sites in the southeast (23, 27, 29 and 30) are almost completely dominated by RP and RPG urchins. A pattern which was not shown by the distribution maps was that of PG+ spines (purple ri ng at the base of the spine

extends up the spine). Sites which are dominated by WPG urchins also have very

high proportions of PG+ spines, so that at these exposed sites the dominant morph 93

Key to dermis co lours • Red 0 White lD Pink

N

Storm Bay

20 km

Figure 4.9 Variation in dermis colour proportions in southern Tasmania. 94

N Key to dermis colours • Red D White � t.::a Pink

15 km

Figure 4.10 Variation in dermis colour proportions in Port Phillip Bay. 95

Key to spi ne colours • Purple . · · " . ()Q ,• f2:i Purple-green G

. IEl . Green . 0 White of?be�.,(.) H q

50 km

Figure 4.11 Variation in spine colour proportions around Tasmania. 96

Key to spi ne colours N • Purple � Purple-green f.illl Green 0 White

Storm Bay

20 km

Figure 4.1 2 Variation in spine colour proportions in southern Tasmania. 97

Key to overall morph s

e · • RP

� RG ; I!) � !• ••••- •• WG ... ·.·.·. · D ,. e :ae B WPG (}) . WP . D . ww 'D9o

N

50 km

Figure 4.13 Variation in overall morph proportions around Tasmania. 98

is a distinctive WPG+. The sites in the Derwent Estuary were the most highly polymorphic with all morphs present, although RW's are very rare. On the north coast all morphs were present but RPG urchins formed almost half of the population and RW urchins were found at all sites, although they were only a small proportion of the population (<6%). Patterns of variation over the entire study area therefore occurred for both dermis colour and spine colour although they were clearer for dermis colour. The patterns were similar for both; the distribution of overall morphs, where dermis and spine colour data were combined, showed the same patterns. Four geographical regions were identified where diffe rent amounts of variability of proportions of morphs occurred: southern Tasmania had large numbers of morphs present at each site, the sites in eastern Tasmania showed more extreme variation with sites tending to be do min a ted by a few morphs, northern Tasmania showed little variation between sites and Port Phillip Bay was similar to eastern Tasmania. Most morphs were present in each of these regions but northern Tasmania had an exceptionally high proportion of pink dermis urchins at all sites. Northern Tasmania was also unusual for several ot her reasons. There was little variation in proportions of morphs between sites and the proportions of red urchins, for sites of the same exposure, were higher than elsewhere. Also, there were higher frequencies of morphs which are rare in the other regions; pink dermis and RW urchins (i.e. red dermis and white spines) were found at every site and were far more common than elsewhere. Data on the intensity of the spine co lours were not included in the s'tatistical analyses but dark red urchins with pale spines (Rp, Rg etc.) were common (mean of the 6 sites = 15.3%), whereas they almost never occurred elsewhere, except Marion Bay ( 4.4% ), Stapleton Point (6.7%) and Dart Island (6.7% ). Intermediate, p, dermis colours are more co mmon than in the other regions, but they were also more difficult to identify. Elsewhere pink dermis urchins were obvious because they were unusual and quite distinct from red or white dermis urchins, but on the north coast the dermis colour seemed to vary continuously (see Chapter 3), with true W's being very rare. These observations show that different patterns of morph distributions occur in each region. Also, there seems to be a general correlation of increasing proportions of white dermis and purple-green spined urchins at sites which are known to be very exposed to wave action, suggesting that selection may be occurring. A detailed analysis of correlations with exposure and other environ mental variables was thereto re needed and alternative stochastic explanations for the observed patterns need to be assessed. 99

4.4.3 Spatial patterns in dermis colour proport ions

The patterns of dermis colour variation observed in the previous section were clarified by the plots of percentage of red dermis urchins against shortest sea distance. These show that each region had a different type of distribution (Figure 4. 14). Southern Tasmania shows a cline of increasing proportions of red dermis urchins northwards in the D'Entrecasteaux Channel; exposure also decreases northwards as oceanic swells lose their energy because of refraction in the narrow channel. However, sites in the Derwent Estuary which are again affected by oceanic swells entering Storm Bay, show the opposite trend. Dart Island (site 23) does not follow this pattern with the highest proportion of red dermis urchins for the region. This was not unexpected as this site is isolated from the rest of the sites in a very sheltered area near the Tasman Peninsula and perhaps should not be included in this geographical region (Figure 4.1 ). Eastern Tasmania shows little patterning with adjacent sites differing widely in dermis colour proportions except for the three highly exposed sites on the nort hern part of the coast (sites 33-35). Northern Tasmania shows little variation over distances between sites as great as those for eastern Tasmania, and Port Phillip Bay shows a steep cline. It must be remembered, however, that the sites in Port Phillip Bay do not occur in a straight line and hence the variation does not form a true cline. If gene flow was occurring between adjacent sites, this would tend to reduce the difference in dermis colour proportions between the sites. These results would therefore suggest that gene flow between sites was greatest in northern Tasmania, moderate in southern Tasmania and Port Phillip Bay and minimal in eastern Tasmania.

4.4.4 Association between dermis and spine colour

·Six sites were not included in the analysis, three because spine colour data were not available and three because the population was monomorphic for dermis colour. Thirty of the remaining thirty-nine sites showed excesses of red dermis, purple spined (RP) and white dermis, not purple spined (WG, WPG, WW) urchins (Figure 4.1 5). The association was significant for ten sites, all of which had relatively large, positive values for D' (Table 4.6). These results show that the disequilibrium between dermis and spine colour described for Tinderbox in Chapter 2 occurs at many sites and this is evidence that it has developed because of selection. The chance of disequilibrium in the same direction occurring at many X ::::;; >- 0> L ....., ro>- ::::;; ro (f)ro c a:; �co u c ID ro w � "-E �u<-.c c 0 w (!) ....., 3:(!) 0 L U QJ c: (J) o ro .. site 23 c:: 100 � .C:: 1 u L 80 :::J t (I) � E 60 L 1---' (J) 0 D 0 D 40 Q) L sites 33 3 4 35 � 20 / ' '

50 100 0 100 200 300 0 100 200 300 0 50

Distance (km )

Ca) Southern Tasm ania (b) Eastern Tasmania (c) Northern Tasmania (d) Port Phillip Bay

Figure 4. 14 Variation in % red derm is urchins with distance for each geographical region.

Shortest sea distances between sites in each region were measured from sites 1, 24, 41 and 42 respectively. Table 4.6 Disequilibrium between dermis and spine colour.

D' = AD - BC, where letters represent proportions of individuals that were (A) red dermis, purple spined, (B) white dermis, purple spined, (C) red dermis, not purple spined and (D) white dermis, not purple spined.

Site D' G df 12 Site D' G df 12 Pelican Island 0.003 0.029 1 > 0.05 Stewarts Bay B 0.041 7.643 1 < 0.01 Blubber Heads 0.020 0.730 1 > 0.05 Fortescue Bay -0.001 1 .609 1 > 0.05 Roaring Beach 6 m 0.029 1 .540 1 > 0.05 Marion Bay -0.001 0.187 1 > 0.05 Roaring Beach 13 m 0.001 0.085 1 > 0.05 Reidle Bay -0.010 1.824 1 > 0.05 Satellite Island A 0.000 0.048 1 > 0.05 Stapleton Point 0.004 0. 01 1 1 > 0.05 Satellite Island B -0.010 0.071 1 > 0.05 Painted Cliffs 0.061 8.491 1 < 0.01 Ling Reef 3 m 0.035 15.1 81 1 < 0.01 Shelly Beach 0.012 0.060 1 > 0.05 Ling Reef 13 m 0.011 1 .1 61 1 > 0.05 Coles Bay 0.020 0.521 1 > 0.05 Ling Reef Slope 0.029 2.176 1 > 0.05 Sth. Crappies Point -0.006 0.007 1 > 0.05 .....&. 0 Ling Reef Barren 0.036 3.105 1 > 0.05 Low Head -0.002 0.023 1 > 0.05 .....&. Gordon 0.049 4.989 1 < 0.05 Greens Beach -0.002 0.019 1 > 0.05 Tinderbox 3 m 0.030 15.403 1 < 0.01 Rocky Cape 0.021 0.595 1 > 0.05 Tinderbox 7 m 0.029 11.1 89 1 < 0.01 Cowrie Point -0.006 0.002 1 > 0.05 Tinderbox Slope 0.009 0.040 1 > 0.05 Point Franklin 0.029 11.308 1 < 0.01 Tinderbox Barren 0.034 1 .860 1 > 0.05 Point Henry -0.003 1 .512 1 > 0.05 Tinderbox Beach 0.021 4.714 1 < 0.05 Point Lilias 0.002 1.423 1 > 0.05 Blackman's Bay 0.049 4.047 1 < 0.05 Tablerock Point -0.01 8 1.306 1 > 0.05 Alum Cliffs 0.025 0.658 1 > 0.05 Point Cook - B1 0.004 0.277 1 > 0.05 Dennes Point 0.026 0.371 1 > 0.05 Point Cook - B2 -0.012 2.1 01 1 > 0.05 Betsy Island 6 m 0.039 6.327 1 < 0.05 Point Cook - K1 0.028 3.539 1 > 0.05 > Betsy Island 13 m 0.001 0.067 1 > 0.05 Point Cook - K2 -0.014 2.036 1 0.05 Stewarts Bav A 0.021 1.972 1 > 0.05 102

sites interspersed with sites where no disequilibrium was found is unlikely to have arisen by chance.

-

- x = 0.018

.

.

.

I I I I I I I I I -0.04 -0.02 0 0.02 0.04 0.06 0.08 Disequilibrium (D')

Figure 4.15 Disequilibrium between dermis and spine colour for 39 sites.

4.4.5 Relationships between dermis colour and environmental data

The Algal and Physical Exposure Indices were quite strongly correlated, with a coefficient of 0.786 (n=49). However, the P .E.I. did not take into account diffraction of waves entering bays and this led to some obviously anomalous re sults; Pelican Island (site 1) and especially Fortescue Bay (26) and Reidle Bay (28) are known to be very exposed sites (personal observations), but occur within small bays. The P .E.I. underestimated the sectors from which wind driven waves affect these sites because diffraction causes waves which would not directly hit the sites to have some effect The P.E.I. was thus unreliable, so the A.E.I. was considered to be the better indicator of exposure of a site to wave action. The values provided by the A.E.I. were consistent with previous studies of exposure regimes around Tasmania (Bennett and Pope, 1960; Davies, 1978). The west coast is maximally exposed and this is probably the main reason that H. erythrogramma is not found here (Dix, 1977). The east coast is only slightly less exposed and is subject to heavy oceanic swells because of the long fetch and narrow continental shelf here. There is a sharp decrease in exposure on the north coast because the Bass Strait is relatively shallow (approximately 200 m) and the fetch is restricted to less than 375 km in all directions because of the islands at the western and eastern ends of the Strait The south coast is exposed to oceanic swells but the effect of these is reduced as they enter Storm Bay and the D'Entrecasteaux Channel is the most sheltered area, because of Bruny Island. 103

Port Phillip Bay is a large bay with a very restricted connection to the Bass Strait, thus none of the sites will be very exposed because the maximum fetch is about 50 km and the average depth of the Bay is less than 20 m. The A.E.I. had a stronger relationship with the transformed percentages of red dermis urchins than the P.E.I., with coefficients of determination of 0.586 and 0.360 respectively (Figures 4.16 a and b). The four geographical regions identified previously ( 4.4.2) showed slightly different relationships with A.E .I. although the proportion of red urchins decreased with increasing A.E .I. for each region except northern Tasmania (Figure 4. 1 6b). Regressions for each region were found to be highly significant, except for northern Tasmania (Table 4.7). The slopes of the three significant area regressions were significantly different (ANCOVA, F = 7.906, p

= 38.72, regression OF = 2, residual OF = 36) and the slope of -3.722 was significantly different from zero (t = -2.00, p < 0.05). The density of urchins was not 10 4

(a) Physical Exposure Index

100 2 y = 64. 1253 - 7.8707x r "'0.36 tf) c .c:. 80 u • (._ :::J • I • (site number) 0::: • • 60 • • � • c • I • (j) u 40 • (._ <(

• • I ( 1 ) (42) 20

• • ( 26) (28)

2 3 4 5 6 Physical Exposure Index

(b) Algal Exposure Index

regression for pooled data 100 2 y = 85.76 - 12.89x r ;::; 0.59 • tf) c Key 80 • South Tasmania .c:. � u 0 0 L East Tasmania ::J • + North Tasmania a: � + 60 0 � t <> Port Ph illip Bay � I c I I I tf) • • u 40 • (._ <(

<> 0 I 20

0 0

2 3 4 5 6 A 1 ga 1 Exposure Index

Figure 4. 16 Regressions of exposure indices on % R derm is for all sites 105

Table 4.7 Results of linear regressions of Algal Exposure Index (Y) vs arcsine 0/o red test urchins (X). ns = not significant

intercept slope a b r d f F p

All sites 85.76 -12.89 0.59 48 83.50 <0.001

South Tasmania 72.43 -7.94 0.55 22 25.64 <0.001

East Tasmania 92.18 -1 8.14 0.87 11 66.14 <0.001

North Tasmania 55.82 1.52 0.11 5 0.48 ns

Port Phillip Bay 109.10 -26.09 0 .46 7 77.88 <0.001

Table 4.8 Results of Tukey's test with unequal sample sizes on A.E.I. vs arcsine 0/o red test urchins regressions. Critical values: q(o.os, 37, 3) = 3.455; q(o.oo1 . 37, 3) = 5.579 ns = not significant

s Compari on SE q [2, South Tasmania vs Port Phillip Bay 6.63 2.52 ns

South Tasmania vs East Tasmania 1.91 5.35 <0.01

East Tasmania vs Port Phillip Bay 6.63 0.98 ns 106

Table 4.9 Results of stepwise linear regression of arcsine 0/o R u rchi n s on environ men tal variables. F to enter = 4.0 F to remove = 3.996

Regression equation: Y = 93.84 • 12.33x1 - 3.72x2 Where y = arcsine % R dermis urchins x1 = Algal Exposure Index x2 =Algal cover

R when OF Variable variable added Reg. I Res. F p

Algal Exposure Index 0.586 1 I 47 69.04 <0.001

Algal cover 0. 61 1 2 I 46 38.72 <0.05

Variables not Partial F

m equation Correlation (r) to enter

Depth -0.131 0.78

Shelter -0.105 0.50 107 significantly related to transformed percentages of red dermis urchins. The exposure of a site to wave action, as estimated by the A.E.I, was therefore strongly correlated with dermis colour proportions over the entire study area and within each of the geographical regions except northern Tasmania. Adjoining regions also showed different relationships with exposure; the decrease in the proportions of red dermis urchins with exposure was greater in eastern than southern Tasmania and there was no relationship for northern Tasmania whereas eastern Tasmania and Port Phillip Bay gave significant regressions. The identification of exposure and algal cover as the best predictors of dermis colour proportions supports the selective hypotheses put forward to explain the patterns of both small and medium scale geographic variation observed. However, the possibility that stochastic processes might have produced similar patterns has not been considered and the relative merits of selective and stochastic hypotheses must now be addressed.

4.4.6 Evidence for processes affecting population differentiation

None of the environmental or geographic distance matrices showed non­ linear relationships with DERMIS. Consequently there was no need to transform any of the data before performing Mantel's tests. Five of the eight 49-site environment and distance matrices were significantly correlated with DERMIS (Table 4.1 0). Thus the interre lationships between the OVERALL ENVIRONMENT and the geographical distance matrices had to be investigated. The significant correlations of BINARY CONTIGUITY and CONTIGUOUS DISTANCE with DERMIS (r=0.1 8 in each case) indicated that the geographical pattern is non-random (Royaltey et al. , 1975) and that there is significant positive autocorrelation between sites (Sokal and Oden, 1978a; Sokal, 1979). _OVERALL ENVIRONMENT gave a correlation coefficient of 0.35 when compared with DERMIS but was not correlated with any of the geographical distance matrices, thus Model 1 was clearly supported. All the distance matrices were highly significantly corre Iated with each other so it was not clear whether Models 3 and/or 4 were supported. The correlation coefficients for BINARY AREAS and DISTA NCE WITHIN AREAS vs DERMIS (0.33 and 0.34 respectively) were almost double those of the other three comparisons, indicating that Model 4 gave the best fit to the data. Thus the four regions showed different patterns of morph distribution and isolation by distance might be occurring within each area. These results suggest that both environmental and stochastic processes are . Table 4.1 o Results of Mantel's tests for 49 site�matrices. Correlation coefficients (r) are shown with family significance levels of test statistic (G) determined by the Bonferroni technique (p = 0.05 I number of tests) . * indicates p<0.0025

DISTANCE OVERALL Bl NARY WITHIN BINARY DERMIS ENVIRONMENT AREAS AREAS CONTIGUITY

EXPOSURE INDEX 0.04

OVERALL ENVIRONMENT 0.35*

* BINARY PATCH 0.14 0.47 _,. 0 OJ SHORTEST SEA DISTANCE 0.15 -0.05

BINARY AREAS 0.33* 0.01

DISTANCE WITHIN AREAS 0.34* 0.01 1.00*

BINARY CONTIGUITY 0.18* 0.05 0.45* 0.56*

CONTIGUOUS DISTANCE 0.18* 0.05 0.45* 0.47* 1 .00* 109 involved in controlling the distribution of dermis colours. However, the power of the tests for stochastic processes is limited because patterns predicted by models 3 to 5 might be due to selective forces which were not considered by the selection models (i and 2). That is, a cline which does not correspond to variation in the environ mental variables measured in this study may correspond to some other environmental variable. Also, other types of selection may be acting which cannot be addressed by this study, such as selection acting through the genetic environment because of coadaptation, linkage or epistasis. SPINE and DERMIS-38 were significantly related to each other (r=0.75), and showed almost identical patterns of correlation with the environmental and geographic distance matrices (Table 4. i i). The major difference to the results from the 49-site matrices was that both SPINE and DERMIS-38 were significantly related to EXPOSURE INDEX (r=0.28 and 0.42 respectively) but SPINE was not related to OVERALL ENVIRONMENT. Of the geographical distance matrices BINARY AREAS and DISTANCE WITHIN AREAS again gave the best correlations but by a smaller margin (comparisons with SPINE, r=0.36 and 0.38; comparisons with DERMIS, r=0.29 and 0.30 respectively). The lack of a significant relationship between DERMIS and EXPOSURE INDEX for the 49-site matrices was unexpected after the strong relationship regression analyses had shown. Also, the major difference between the 49- and 38-site matrices was the exclusion of one region, Port Phillip Bay. It therefore seemed likely that the geographical regions had different patterns which were being obscured in the large matrices. A similar analysis on matrices produced for each of the regions was performed to see if this was the case. This also allowed the importance of Model 3 to be assessed, as Model 4 is automatically excluded· from the analysis. For southern Tasmanian sites, EXPOSURE INDEX-S was significantly correlated with DERMIS-S (r=0.55), whereas OVERALL ENVIRONMENT-S was not _(Table 4. i 2). All of the geographical distance matrices were significantly correlated with DERMIS, with SHORTEST SEA DISTA NCE-S giving the highest corre lation coefficient of 0.39. EXPOSURE INDEX and SHORTEST SEA Dl STANCE were not significantly related, therefore both Models 1 and 3 are supported. The partial correlation coefficients indicate that both matrices were important factors in predicting dermis colour proportions; EXPOSURE INDEX gave an r2 of 0.30 and with the addition of SHORTEST SEA DISTANCE, the coefficient of multiple determination was increased to 0.60 (Table 4. i 3). These results show that both selection due to exposure to wave action and isolation by distance may be affecting dermis colour proportions in this region. SPINE-S was significantly 110

Table 4.11 Results of Mantels's tests for 38�site matrices Correlation coefficients (r) are shown with family significance levels of test statistic (G) determined by the Bonferroni technique

(p = 0.05 I number of tests). * indicates p<0.0029

SPINE DERMIS-38

EXPOSURE INDEX-38 0.28* 0.42*

OVERALL ENVIRONMENT-38 0.12 0.22*

BINARY PATCH-38 0.01 0.06

SHORTEST SEA DISTANCE-38 0.26 0.24

Bl NARY AR EAS-38 0.36* 0.29*

DISTANCE WITHIN AREAS-38 0.38* 0.30*

BINARY CONTIGUITY-38 0.21 * 0.21 *

CONTIGUOUS DISTANCE-38 0.22* 0.21 *

DERMIS-38 0.75* Table 4.12 Results of Mantel's tests for data from Southern Tasmania (23 sites). Correlation coefficients (r) are shown with fa mily significance levels of test statistic (G) determined by the Bonferroni technique (p = 0.05 I number of tests). * indicates p<0.0026 - comparison not tested

SHORTEST BINARY EXPOSURE SEA CONTIGUITY DERMIS-S SPINE-S INDEX-S DISTANCE -S

...... SPINE-S 0 .59* ......

EXPOSURE INDEX-S 0 .55* 0.27

OVERALL ENVIRONMENT-S 0.21 0.10

BINARY PATCH-S 0.03 0.01

SHORTEST SEA DISTANCE-S 0.39* 0.25 0.20

BINARY CONTIGUITY-S 0.20* 0.06 0 .11 0.57*

CONTIGUOUS DISTANCE-S 0.20* 0.07 0.12 0 .57* 1 .0 0 * 112

Table 4. 13 Partial correlation coefficients from Mantel's tests for geographical regions. AB.C represents the correlation of matrix A with B, keeping C constant. Probabilities are based on 1000 simulations. A= DERMIS * P<0.05 B = EXPOSURE ** p<0.01 C = SHORTEST SEA DISTANCE *** p<0.001 D = OVERALL ENVIRONMENT

Matrix Partial correlation Coefficient of multiple R egion comparison coefficient (r) determination (R }

Southern AB.C 0.529* ** Tasmania AC.B 0.352 *** 0.603

Port AC.D 0.062 *** PhilliQ Ba;t AD.C 0.517 113 related to DERMIS-S (r=0.59) but not to any of the environmental or geographical distance matrices. For eastern Tasmanian sites, EXPOSURE INDEX-E gave a better correlation than OVERALL ENVIRONMENT-E when compared with both DERMIS-E (r=0.78 and 0.63 respectively) and SPINE-E (r=0.69 and 0.60 respectively), indicating that Model 1 was supported for both types of colour variation (Table 4.1 4). D ERMIS-E and SPINE-E were highly correlated (r=0.87) but neither gave significant relationships with any of the geographical distance matrices. No significant correlations were found for northern Tasmania (Table 4.1 5). For Port Phillip Bay sites, OVERALL ENVIRONMENT-V and SHORTEST SEA DISTANCE-V both had significant relationships with DERMIS-V (Table 4. 16). However, the high correlation between OVERALL ENVIRONMENT and SHORTEST SEA DISTANCE (r = 0.75) means that one of the relationships is probably spurious. The partial correlation coefficients show that OVERALL ENVIRONMENT (r=0.51 ) gave the only significant relationship (Table 4.1 3). This indicates that isolation by distance may be occurring in Port Phillip Bay, however, this is unlikely because of the current patterns in the bay (see 4.4.7). In summary, these results provide evidence that the patterns over the et}tire study area were affected by selection and that isolation by distance may also be involved; models of these processes can be distinguished by Mantel's test because the relevant environmental and geographic distance matrices were shown not to be interrelated for the 49-site matrices and for southern Tasmania. Specifically, overall proportions· of morphs were correlated with OV�RALL ENVIRONMENT, which includes data on A.E.I., algal cover, depth and substratum, suggesting that selection was involved. However, within eastern and southern Tasmania at least, exposure to wave action was shown to be the overriding environmental variable. The founder effect is unlikely to be responsible for the different patterns of morph variation found in the different regions because most mo�phs may be found in each region and also the large effective population size due to movements of pelagic larvae means that populations of H. erythrogramma are not likely to be subjected to mass extinctions followed by recolonisation. Also, it is not supported by the other results presented in this chapter and it seems more likely that the significant relationships for DERMIS with BINARY AREAS and DISTANCE WITHIN AREAS were caused by the different patterns of morph distribution within each region resulting from the different environmental regimes. The low order, positive autocorrelation which was found suggests that gene flow does occur between contiguous sites, at least in southern Tasmania. Analyses for each region separately showed proportions of morphs in each 114

Table 4.14 Results of Mantel's tests for data from Eastern Tasmania (12 sites). Correlation coefficients (r) are shown with family significance levels of test statistic (G) determined by the Bonferroni technique

(p = 0.05 I number of tests). * indicates p<0.0036 - companson not tested

EXPOSURE DERMIS-E SPINE-E INDEX-E

SPINE-E 0.87*

EXPOSURE INDEX-E 0.78* 0.69*

OVERALL ENVIRONMENT-E 0.63* 0.60* 0.71 *

BINARY PATCH-E 0. 11 0.01

SHORTEST SEA DISTANCE-E 0.10 0.13

BINARY CONTIGUITY-E 0.15 0.24

CONTIGUOUS DISTANCE-E 0.15 0.25

Table 4.1 5 Results of Mantel's tests for data from Northern Tasmania (6 sites). Correlation coefficients (r} are shown with family significance levels of test statistic (G) determined by the Bonferro ni technique

(p = 0.05 I number of tests) . * indicates p<0.0045 - comparison not tested

DERMIS-N SPINE-N

SPINE-N 0.56

EXPOSURE INDEX-N 0.58 0.52

OVERALL ENVI RONM ENT-N 0.37 0.64

SHORTEST SEA DISTANCE-N 0.55 -0.13

BINARY CONTIGUITY-N 0.34 0.21

CONTIGUOUS DISTANCE-N 0.35 0.21 115

Table 4.16 Resu Its of Mantel's tests tor data from Port Port Phillip Bay (8 sites). Correlation coefficients (r) are shown with family significance levels of test statistic (G) determined by the Bonferroni technique

(p = 0.05 I number of tests).

* indicates p

� comparison not tested N .B. There were no spine data available for th is site.

OVERALL DERMIS-V ENVIRONMENT-V

EXPOSURE INDEX-V 0.05

OVERALL ENVIRONMENT-V 0.50*

BINARY PATCH-V -0.17

SHORTEST SEA DISTANCE-V 0.67* 0. 70 *

BINARY CONTIGUITY-V 0.04

CONTIGUOUS DISTANCE-V 0.07 116

region were probably controlled by different combinations of processes. Both exposure and isolation by distance were indicated for southern Tasmania, although, as previously discussed, the clinal variation which did not correlate with variation in exposure may have been caused by other selective processes. Exposure to wave action was found to be the only useful predictor of morph proportions in eastern Tasmania. The results for Port Phillip Bay are difficult to interpret and further information is needed for this region. Only for northern Tasmania were none of the models supported. This may have been because there was little variation in morp h frequencies or environme ntal data for the six sites. However, this uniformity itself suggests that either gene flow is so extensive that little differentiation between populations can develop, or that the environment is homogeneous so all populations are controlled by the same selective pressures. In the latter case, only moderate gene flow would be necessary to ensure that genetic drift did not lead to differentiation of populations. Dermis and spine colours were strongly correlated overall and for southern and eastern Tasmania. However, spine colour was not related to the environmental or geographic distance matrices, except for eastern Tasmania where they were correlated with EXPOSURE INDEX and for the 49-site matrices where they showed the same results as dermis colours but with lower correlation coefficients. The similar geographic patterns may be a result of linkage between dermis and spine colour, such as that described previously (4.4.4). The results from the Mantel's. tests again predict the same patterns of gene flow within the three Tasmanian regions as before, with least gene flow between sites in eastern Tasmania, mod.erate gene flow in southern Tasmania and maximal gene flow along the north coast. The results suggest that the amount of gene flow in Port Phillip Bay is probably restricted if isolation by distance is occurring. Adult H. erythrogramma are not known to migrate over large distances and probably only move significantly after a disturbance (Connolly, 1986), although at Point Cook, urchins may be washed into some areas after storms (A. Constable, personal communication). Therefore, for most sites, the only significant gene flow will be due to movement of the pelagic larvae. The ova of H. erythrogramma float in mats at the surface in calm water (personal observations, Fortescue Bay, Dec. 1985), because of their high fat content. The lecithotrophic larvae are ciliated but swim weakly; they remain at the surface under laboratory conditions but in the sea their position would probably be determined by currents. They metamorphose and attach themselves to the walls of the aquarium after 5-7 days in the laboratory 117

(Appendix 1 ), although Williams and Anderson (1975), in Sydney, found that they took 4-5 days to settle. The distribution of settling larvae and patterns of gene flow will therefore be determined by the local current patterns.

4.4.7 Water currents within and between the geographical regions

There have been several studies on current patterns within these regions, but little is known of water movements between them. The main currents affecting the sites in southern Tasmania flow north in the D'Entrecasteaux Channe I, on the western side of Bruny Island, and south on the eastern side of the Derwent Estuary (Cooper et al., 1982), but the flow in the D'Entrecasteaux Channel is reversed on the ebb tide. Cooper et a/.(1982) do not give current speeds but surface water movement may be in the order of metres per second, particularly in the restricted D'Entrecasteaux Channel (personal observations). Gene flow is therefore likely between close sites 1-22, and possibly also with site 23. However, the complex coastline with frequent bays may restrict the exchange of surface water for some sites. There is little published information on east coast currents but it is known that the warm East Australian Current may extend to the far south of Tasmania in summer (Harris, et al. , 1988). Subantarctic waters flowing north meet the warmer water but the position of the confluence varies from year to year. The currents off the east coast may therefore flow north or south at different places and this will vary between years. However, there are many enclosed bays, particularly on the southern half of the east coast, and thus gene flow may be restricted to and from these sites. This is consistent with the finding that isolation by distance was restricted for the east coast. It is not possible to predict whether southern and eastern Tasmania are isolated from each other from the available data. The computer simulations of surface particle movements in the Bass Strait i ndic_ated that if there are westerly components to the prevailing winds, particles from the north coast of Tasmania may be carried to the east coast (Figure 4.1 7 and unpublished data). Particles released from South Crappies Point (site 36) or Trousers Point (site 41 ), on the eastern end of the northern coastline, took about six days to reach the east coast. Particles released from Cowrie Point (site 40), on the western end of the north coast, never reached the east coast within 20 days. Under no wind conditions did any of the particles cross the Bass Strait to reach Port Phillip Bay. The simulations make it clear that surface particles could move between all sites on the north coast within 20 days and gene flow is therefore likely to be extensive, particularly as there are few semi-enclosed bays. However, _j � - � Victoria !\'

I ,, 1-----.J \, � I _) \ Bass Strait � CJ .� CJ

{a } Northerly winds. {b} Westerly winds.

� i'--" co

� � \ CJ CJ L f\•

{c} No rt h-w ester I y winds. South-w esterI y winds.

Figure 4.17 Examples ot simulated surtace particl e movements in the Bass Strait under ditter ent wind conditions. From a computer simuration deveroped by C. F andr,t. C ircres represent i nitiar position of particre. Ticks re present position of particre at 24 hour in terv ar s. 119

all the Tasmanian sites wi ll be isolated from those in Port Phillip Bay. The currents in Port Phillip Bay have been studied in some detail (Environmental Study of Port Phillip Bay, 1973); net water movement over one tidal cycle (two ebb and two flood tides) varies from 0.1 to 2.8 km in a clockwise direction, but may be much greater under short-term wind effects. As the bay has a maximum width of 40 km, substantial gene flow probably occurs between all sites. These results confirm the predictions made by this study of the amount of gene flow within the three Tasmanian regions. The occurrence of gene flow between regions is less clear although it seems likely that it does occur between the north and east coasts. The results for Port Phillip Bay are difficult to explain if gene flow is as extensive as suggested by the Environmental Study. The large differences in proportions of morphs between different sites are surprising given that variation in exposure to wave action (the most important environmental factor identified in southern and eastern Tasmania) is restricted because of the enclosed na.ture of the bay. Unfortunately, the sites which were very close together (Point Henry and Point Lillias and the four sites at Point Cook) had similar environments and identical A.E.I.'s making it difficult to distinguish between the effects of environmental differentiation and gene flow.

4.5 GENERAL DISCUSSION

The overall picture to emerge from this study is that four geographical regions have been identified within each of which a different regime of selective factors and gene flow exists. These regions may or may not be isolated from one another in terms of gene flow. Thus although the same selective factors are probably affecting all populations of H. erythrogramma, patterns of morph distributions are different for each region. The present study differs from other studies because mod�ls of both stochastic and selective processes which could affect proportions of morphs were supported. This was possible because the use of Mantel's test enabled the effects of differe nt models of population differentiation to be distinguished. The ability of Mantel's test to show that Model 1 (selection due to exposure) and Model 3 (isolation by distance) were both independently useful predictors of dermis colours overall and in southern Tasmania was crucial. In several other studies Mantel's test was used to investigate the evolutionary processes affecting population differentiation. Douglas and Endler (1982) found that only selection by visual predation was important in maintaining a complicated polymorphism involving the number, size and distribution of coloured spots in 120

male guppies. Suites of visual and non-visual predators varied between sites and where the former predominated the need to be inconspicuous had to be balanced against the ability of males to attract females (Endler, 1978). Mantel's tests indicated that neither isolation by distance nor gene flow were correlated with spot patterns. Dillon (1984) showed that both environmental and distance measures correlated with population divergence (measured by morphology and allozyme frequencies) for a freshwater snail, but the environmental and distance matrices were also strongly interrelated. No correlation coefficients were given so it was impossible to determine the relative importance of selective and stochastic processes, although that was one of the stated aims of the paper. However, an interesting result from the study was that the environme ntal data were only correlated with morphological divergence, whereas the distance matrices were correlated with both morphological and allozyme variation. Francis et at. (1986), working on the threespined stickleback, produced four morphological distance matrices from the factors identified by factor analysis. They used Mantel's test to compare each of these with three geographical distance and ten envi ron mental matrices. They found that factors 1 and 2 were best explained by stochastic processes, but factors 3 and 4 were correlated with various selective pressures. This and Dillon's (1984) study therefore indicate that different aspects of population differentiation may respond independently to different evolutionary forces. In contrast, the present study of H. erythrogramma used only one measure of population differentiation, proportions of colour morphs, but found that both selective and stochastic forces may affect this trait. Correlations of proportions of morphs with environmental variables and geographical patterning provide only circumstantial evidence in support of different models of population differentiation. More definite evidence could be provided if new hypotheses were developed from the results of this study, about the _specific mechanisms affecting the polymorphism, which could be tested experimentally. Two potential areas for further study have emerged: quantification of gene flow between sites and particularly between regions, and identification of the selective forces which led to the correlations of proportions of morphs with exposure and algal cover. The former could be approached directly (release and recapture of markers to determine direction and speed of water movements) or indirectly (electrophoretic techniques). The second question is likely to be less tractable and would best be answered by extensive manipulations of nat ural populations. Three hypotheses about the selective forces involved wi ll now be put forward.

'• 121

(1) Exposure may be directly affecting the polymorphism. One of the major causes of urchin mortality is probably dislodgement and damage during storms. If urchins are responding directly to exposure to wave action, this suggests that white dermis urchins are able to attach themselves to the substratum more firmly, either because of stronger tube feet or because they wedge themselves in with their. spines more firmly. Thus higher proportions of white dermis urchins would survive at exposed sites. Many other studies of variation in marine invertebrates have identified exposure as an important environmental influence. For example, the shells of Littorina saxatilis, in Sweden, are smaller, thin-walled and have larger apertures at exposed sites than sheltered sites (Janson, 1983). The larger aperture is thought to allow a greater area for attachment by the muscular foot, whereas the thinner shell may be due to reduced predation pressure from crabs which are more common at sheltered sites. Shell sculpture in Littorina picta is highly variable with smooth shells predominating in exposed habitats, whereas most individuals from sheltered sites have raised spiral ribs bearing many regularly­ spaced tubercles (Struhsaker, 1968). Larvae from smooth and sculptured populations were found to differ in shell morphology, size, growth and mortality rate. The smooth-shelled adults were found to be less resistant to high salinity and desiccation whereas sculptured individuals were more susceptible to dislodgement by wave action and could not withstand long periods of submersion. In each of these cases the advantage of the morphological variation is clear but experiments are needed to determine whether the colour morphs of H. eryth rogra mma show similar adaptations. Several studies of echinoids have shown that morphology and proportions of colour morphs vary with exposure but the selective pressures involved have not been identified. In Barbados, the echinoid Echinometra lucunter have thicker tests and are smaller, flatter and narrower in exposed sites (Lewis and Storey, 1984). The exposed and sheltered populations also varied in the proportions of test and spine co lours. The difference in food sources was suggested as the reason for this, however, this is unlikely as it is now known that echinoids synthesise the pigments de novo (Salaque et at. , 1967; Asashima, 1971 a) . Individuals of E mathaei on Reunion Island in the southern Indian Ocean were found to be small and pink on the exposed side of a headland, and large and black on the leeward side (Lawrence, 1980). Identical colour differences were found between populations in the Gulfs of Aquaba and Suez in the Red Sea but the exposure of the sites was not described (Lawrence, 1983). 122

(2) The urchins are not responding directly to exposure, but to some other factor which is correlated with exposure. The exposure of a site affects many features of the habitat such as turbidity, amount of sedi ment, algal species and growth and this in turn may affect parameters such as the suites of predators present. Also, sheltered sites often have sand present whereas most sediment is removed at exposed sites leaving either boulders or flat rock substrata. This affects both the availability of physical refuges avai lable to urchins in which to hide from predators and the background against which they are seen. Selective pressures correlated with exposure have been shown to affect colour polymorph isms in several marine invertebrates. Shell colour of Littorina nigrolineata may be white, yellow or red-brown whereas L. rudis varies from white to yellow, red, brown, purple, grey or black. Heller (1975) found that different morphs of these species predominated on shores of differi ng exposure. He showed that crypsis against the different rock, barnacle and algal backgrounds found on shores of different exposure was the reason for this and suggested that selection by visual predators was responsible for maintaining the polymorphisms. Reimchen (1 979) found that brown-shelled L. mariae were common on exposed shores whereas yellow morphs predominated in sheltered areas. He showed that this was related to the availability of microhabitats on the brown alga Fucus serratus which varied with the morphology of the plants found in differe nt exposure regimes. He suggested that crypsis and density dependent predation were the selective forces involved. These two studies showed that although exposure was correlated with morphological or colour variation, it was crypsis against different backgrounds that was being selected for. The possibility that visual predation may be involved in maintaining the colour polymorphism of H. erythrogramma is plausible. The suites of predators probably do vary with the exposure of the site and white dermis urchins are far more obvious to the human eye and would probably need camouflage more than red dermis urchins. Exposed sites tend to have large boulders and extensive holes and crevices in which the urchins can hide. The correlation of increasing proportions of white dermis urchins with increasing algal cover is also consistent with this idea. Known predators of H. erythrogramma include Port Jackson sharks (Smith, 1942), large leatherjackets ( Meusch enia australis and M. freycineti; Last, 1983) and the Blue-throated wrasse (Pseudolabrus tetricus; personal observations). The three species of fish are visual predators but the Port Jackson shark may rely more heavily on chemoreception. (3) Another possibility is that the colours may not be important visually, but that the colour morphs are physiologically different. This has been shown to be the 123

case for the dogwhelk, Nucel/a lapillus, which is polymorphic for shell colour and darkly pigmented morphs predominate at exposed sites (Etter, 1988). Brown morphs heated up and desiccated more quickly than white morphs and suffered greater mortality in sunny microhabitats on sheltered shores. It has been suggested that echinochrome A (the dermal pigment) may be a respiratory pigment (MacMunn, 1885) although this was never proven. It is possible, therefore, that the respiratory abilities of red and white dermis urchins differ. The red urchins, with very high concentrations of echinochrome A in their dermis', predominate in very shallow, sheltered sites where water temperatu res may exceed 2ooc in summer (in Tasmania) and it is possible that they would be oxygen stressed. Urchins kept in aquaria always died if the water temperature rose to 2ooc (personal observations). High water temperatures have been shown to cause mortality in the echinoid Echinometra mathaei although whether this was due to oxygen stress is not known. In southern Japan, two colour morphs of E. mathaei occur in different habitats on coral reef flats, but it has not been shown whether this is due to different settlement patterns of larvae or habitat preferences of the adults

(Tsuchiya and Nishihara, 1984). · Urchins with purple, brown or green-brown spines with a white ring at the base and white tips (Type A) occur in calm water environments. Urchins with completely dark green-brown spines (Type B) occur in slightly deeper habitats which are more exposed to wave action. ln the summer of 1986 mass mortalities of E. mathaei occurred because of unusually high water temperatures (Tsuchiya et a/., 1987). It is possible that differential mortality may have occurred between the morphs occurring in different microhabitats as water temperatures were probably lower where the reef was affected by waves but this was not investigated. The study of small scale geographic variation of H. erythrogramma found significant differences in proportions of morphs between sites, which could be related to aspects of the habitat. This suggests that morphs may differ behaviourally in their choice of microhabitat, and this was also suggested by the results from Chapter 3. Differences in the behaviour and microhabitats of colour morphs would therefore probably be another fruitful area for further study. 124

CHAPTER 5 VARIATION BETWEEN MORPHS IN MORPHOLOGY, MICROHABITAT, REPRODUCTION AND TUBE FEET STRENGTH

5.1 INTRODUCTION

Most studies of colour polymorphisms have emphasised the importance of the visual differences among morphs and many invoke selection for crypsis as the major factor controlling the variation. This has been demonstrated directly by some studies (Clarke and Murray, 1962; Reid, 1987) but in many cases there may be other factors involved, such as physiological variation between colour morphs which is thought to affect survival rates of Cepaea nemoralis (Richardson, 197 4; Jones et at. 1977), a freshwater snail (Heller, 1979), a snake (Forsman and As, 1987) and a marine snail (Etter, 1988). Thermal stress was the selective agent in these examples, with more heavily pigmented morphs heating up more quickly. Although thermal stress is thought to have caused mortality in tropical urchins (Tsuchiya et at. , 1987), it is unlikely to do so in Tasmanian sea urchins which do not occur intertidally and where water temperatures rarely reach 18° C (personal observations). Morphological variation has also been shown to affect survival in different habitats; Struhsaker (1968) showed that genetically based differences in shell sculpture of a marine snail were associated with the exposure of the habitat to wave action. It is clear t�at studies on physiological, morphological and ecological variation among morphs are necessary to fully understand the selection pressures acti ng on polymorphisms. Morphological variation has been correlated with the degree of exposure to wave action for intertidal snails; shells of a whelk, Oicathais sp. (Phillips et at. , 1973), and a periwinkle, Littorina saxatitis (Janson, 1 983), from exposed sites are smaller and have a larger aperture (and therefore a larger su rface area to attach by). Individuals of the sea urchin Echinometra /ucunter from high wave-energy habitats have thicker tests and are smaller, flatter and narrower than those from low wave-energy sites (Lewis and Storey, 1984). In each case the variation is such that it would decrease the chances of individuals from exposed habitats being swept away by waves. If similar variation were found between morphs of H. erythrogramma this would help to explain the correlation of morph proportions with the Algal Exposure Index. Urchins attach themselves to the substratum by both suction by their tube feet (podia) and by wedging themselves into cracks using their spines. Thus another 125

possible mechanism to explain the correlation of white dermis urchins with exposed sites is that white dermis urchins might have stronger tube feet and be better able to maintain their position in rough water conditions than red urchins. This would be supported by the results of Sharp and Gray (1962) who used the differe nt strengths of the tube feet of Arbacia punctulata and Lytechinus variegatus to explain their distributions among sheltered or exposed habitats. The results from Chapter 4 showed that there were differences in morph proportions betwe en sites and that the variation was related to certain environmental factors. The major environmental influence was found to be the exposure of a site to wave action and although morph proportions were not significantly correlated with the type of substratum, the A.E.I. was correlated with substratum (r = 0.52). Sites which were dominated by red dermis urchins often had substrata which were either flat rock or sand, therefore the urchins were exposed to both wave action (although these sites were always very sheltered) and visual predators (personal observations). Urchins did not occur at sites where the substratum was flat rock and which were exposed to wave action. At sites where white dermis urchins predominated, the substratum was usually made up of large boulders underneath or between which the urchins were found wed,ged (personal observations). These observations suggest that red and white urchins may prefer different microhabitats on the basis of physical shelter. The 'covering response' is a well known behavioural pattern of sea urchins whereby they pick up pieces of drift algae, shells or small stones from the substratum or even dead urchin tests, and transfer them to their aboral surface where they are held by the tube feet (Millott, 1956; Dix, 1970). Several possible functions for this behaviour have been suggested, such as protection from high light intensities in tropical sea urchins (Millott, 1956; Sharp and Gray, 1962) or as camouflage from visual predators for temperate species (Dix, 1970). The turbidity of Tasmanian sea water and the relatively low light intensities compared to the t_ropics suggest that camouflage is the most likely explanation for 'covering' by 1-J. erythrogramma. The 'covering' response is well developed in adult H. erythrogramma (personal observations). and predictions may be made of the likely covering responses of different morphs, based on the results of the preceding chapters. First, in rocky areas white dermis urchins would be expected to cover to a greater extent than red dermis urchins if visual predation was involved, because they are more obvious against a rock background. However, red dermis urchins are more obvious than white dermis urchins against sand backgrounds and would therefore be expected to cover to a greater extent in such habitats. Second, in boulder 126

fields, white dermis urchins would be expected to seek shelter under rocks to a greater extent than red dermis urchins either seeking shade from sunlight or to hide themselves from visual predators. Finally, urchins which were under rocks would be less exposed to sunlight as well as less obvious to visual predators and would therefore cover themselves less than urchins on the upper surfaces of rocks. It is possible that the variation in H. erythrogramma does not constitute a true polymorphism because the morphs do not interbreed in natural populations. This could be accomplished if the morphs had developed different life history strategies so that they matured at different times of the year (Tauber and Tauber, 1977). Even if morphs interbreed freely, differential fitnesses of morphs in different habitats might l�ad to variation in reproductive investment between morphs. Assuming that interbreeding occurs between morphs, the position of the equilibri um between the proportions of morphs at a site would be affected if morphs differed in their reproductive investments. This might be detected in many ways, one of which would be to determine whether morphs had different weights of gonadal tissue during the reproductive season. This chapter describes a morphological study of H. erythrogramma which was designed to determine whether, at sites where many colour morphs occur, there are differences between morphs and if so, what the differences are. Several populations were used so that any differences between urchins from different sites could be identified. Two different approaches were used to determine whether microhabitat variation between urchins of different dermis colour occur. The timing of reproduction and gonadal investment of morphs and the strength of the tube feet of red and white dermis urchins were also investigated.

5.2 METHODS

5.2.1 Morphometries and meristics

The urchins from four sites were collected using SCUBA. Tinderbox and Ling Reef in southern Tasmania have many morphs present and 59 and 57 urchins were sampled, respectively. Fewer morphs occur at Fortescue Bay (mainly WPG and WG) on the south-east coast and Cowrie Point (mainly RP and RPG) on the north-west coast of Tasmania and 30 and 20 urchins were sampled respectively. Urchins from each site were collected from an area of roughly 1 00 m2 to ensure that urchins from the same breeding populations and habitats were sampled. The normal amount of movement of H. erythrogramma is uncertain but is thought to be 127

' t-..: n:;n:m�1t unr��is ciis:ucbt.:d'. .' i n th0y mo.y move several hundred metres (Connolly, l0J0). How:.1vcr, d·Jnuc!C!d 0JC3S of the permanent transects used in this study

·,·,-._.:r�: rJpopubt.;;cl ·•· ..it ilin tilrco months, presumably by immigration of nearby uc�hin�. Th0 sc:llc: of the 3rca from which urchins were collected is therefore we ll t,·:iti lin ti10 c:!p:!bilitius oi tho urcllins for movement. Live ','.'iJigiHs \'.rur�J moasurecl in the laboratory immediately after returning from n1u ti:.:tci, c:.:copt for Cowrie Point as it was not possible to keep the urchins alive ciwing tile journey b2ck to Hobart. When the urchins die the mouth opens and thu;' tend to lose fluid from the gut, thereby decreasing in weight. The urchins

','.'t:Ha scored fo r dermis and spine colour as outlined in Chapter 2 and 16 morphometric 2nd 3 meristic characters were recorded (Table 5.1 ). Twelve of those chamcters were used by Lessios (i 981 ) in his study of differentiation of throe species of sea urchins separated by the Isthmus of Panama. ;\ iter the live weight had been measured, urchins were killed by re moval of the l2ntern and internal organs. The test, spines and lantern were soaked in 5% sodium hydroxide solution for 24 hours and dried at 60° C overnight to give dry \':eights and also for the easy removal of spines from the test. Test dry weight was not me2sured for Cowrie Point urchins. Test thickness was measured to the nearest 0.01 mm using an optical micrometer on a low-power dissecting microscope whereas all other measurements were determined to the nearest 0.1 mm using Vernier calipers. The vertical edge of an ambulacral plate from the ambitus of each urchin was used as test thickness varies over each plate and at different parts of the test. The data were checked for homogeneity of variances and logarithmically transformed if necessary. For Tinderbox and Ling Reef individuals each morphological variable was then regressed against live weight and analysis of covariance (ANCOVA) was used to check whether significant differences in slopes and elevations occurred between dermis colours or between spine colours for each variable. Canonical variate analysis (CVA) was performed using the residuals of the regressions of variables against live weight, to remove the effect of size, on individuals from Tinderbox and Ling Reef. A similar analysis was performed between sites, pooling morphs, for all variables except live weight and test dry weight which had not been measured for Cowrie Point. Statistics were calculated using the statistical package SPSSX. 128

Table 5.1 Morphometric measurements and meristic counts made on urchins from Tinderbox, Ling Reef, Fortescue Bay and Cowrie Point.

* not measured for Cowrie Point urchins.

i Live weight* 2 Test and spines dried weight* 3 Lantern dried weight 4 Test diameter (average of 3 measurements) 5 Test height 6 Test thickness 7 Diameter of apical system 8 Diameter of peristome 9 Diameter of madreporite 1 0 Diameter of periproct 11 Maximum width of ambulacrum 1 2 Maximum width of interambulacrum i 3 Number of pore pairs per plate at ambitus 1 4 Maximum tooth length 1 5 Maximum tooth width 1 6 Number of plates in ambulacrum (average of 3 columns) i 7 Number of plates in interambulacrum (average of 3 columns) 1 8 Length of longest spine at ambitus 1 9 Maximum diameter of longe st spine 129

5.2.2 Microhabitat and behavioural variation

Choice of shelter and the covering response. Urchins were observed using SCUBA at five sites in southern Tasmania: Tinderbox; Ling Reef; Coningham; Alum Cliffs; and Betsy Island. Observations were made at 3 and 7 m at Tinderbox on four occasions, at 3 and 13 m at Li ng Reef on two occasions, and on one occasion at Coningham (3 m), Alum Cliffs (5 m) and Betsy Island (7 and 10 m). As observations were made underwater, only dermis colours were distinguished. An area was searched thoroughly and all urchins were scored for dermis colour, whether they were hidden under a rock or visible on the upper surface of a rock and whether they exhibited the covering response or not. Urchins were considered to be 'covered' if they were holding at least one piece of alga, shell or pebble on their aboral surface. Log linear models analysis, which is a method of analysing multidimensional contingency tables, was used to analyse the data. A series of increasingly complex models is developed; the first model assumes that all the variables are independent. A measure of the deviance of the observed from the expected frequencies is calculated and compared with the x2 distribution. If this model gives a reasonable fit to the data the analysis proceeds no further and the data are said to show random variation. If not, associations between variables are added, one at a time, until a reasonable fit to the data is obtained. The sampling distribution of ihe deviance approximates the x 2 distribution if; (a) the sample size exceeds 40; (b) the sample size is at least five times the number of cells (c) the proportion of expected frequencies with values < 1 does not exceed 10%; (d) no expected frequency is zero (an observed value of zero can have a non­ zero expected value (Fien berg, 1970). Three variables each with two categories were used; MOR, dermis colour or morph (R = red, W = white); HAB, microhabitat (H = hidden, V = easily visible);

COV, covering response (C = covered, U = uncovere d). Thus the maximum number of cells was 23 = 8. If all the variables are independent the model is represented by MOR + HAB + COV The more complex models are built up one step at a time. For example, if morph and habitat are related then the model becomes MOR + HAB + MOR.HAB +COV 130

However, this is usually represented as MOR * HAB + COV. Two other models have one dependent relationship; morph with covering

response (MOR * COV + HAB) and habitat with covering response (MOR + HAB * COV). More complex models would involve two or three dependent relationships

between variables (e.g. MOR * HAB + MOR * COV; MOR * HAB * COV). The models are compared both with the data and with the preceding model, i.e. differences between models which differ by the addition of one term only. The goodness of fit of the models is assessed as shown below;

Compari ng a model with the data

p > 0.1 the model is an adequate description of the data

0.1 > p > 0.01 weak evidence that the model is inadequate

p < 0.01 the model is not an adequate description of the data Comparing two models

p > 0.1 the extra term does not improve the fit of the model

0.1 > p > 0.01 weak evidence that the extra term is useful

p < 0.01 the extra term improves the fit of the model The degrees of freedom, when comparing a model with the data, are given by

• (n r), where n = total number of cells and r = number of independent restrictions imposed by the model. When comparing two models the degrees of freedom are given by the difference in the degrees of freedom associated with the two mode Is considered separately. The analyses were performed using the statistical package 'Genstat'. Only the sites at Tinderbox '(3 m), Coningham and Betsy Island (1 0 m) had boulders present and could be analysed using the log-linear models method because at the other sites the substratum was flat rock or sand so there was no choice of microhabitat. At these sites G-tests of independence were used to determine whether the morphs differed in their covering response. The G-tests were calculated using BIOI-TAT which applies the Yates correction where ne.cessary.

Nearest neighbour analys is On 12 vii 88 two divers observed urchins at Tinderbox (3 m) to determine whether those of a particular dermis colour (red or white) were randomly associated with urchins of both colours. The alternative hypothesis was that urchins were more likely to occur next to an individual of the same colour. If this was found to be the case then it would seem likely that urchins of the same dermis colour were choosing similar microhabitats, even if the nature of the microhabitat was not obvious to the observers. 131

Urchins were chosen haphazardly and their dermis colour and that of their nearest neighbour were recorded for 105 pairs of urchins. lf an urchin did not have a neighbour within 50 em it was not used in the analysis. A G-test for independence was calculated.

5.2.3 Reproductive cycle and investment

Urchins were retained from the repeated samples taken at Tinderbox (7 samples) and Ling Reef (5 samples) duri ng the study. The urchins were placed in aerated drums overnight before dissection. Measurements taken included live body weight and wet weight of the gonads, the dermis co lour and sex (where possible) of each urchin were recorded and if mature eggs or sperm were present the urchin was noted as 'ripe'. The proportions of ripe urchins of each morph in each sample were used to determine whether the morphs matured at different times. To determine whether reproductive investment varied between morphs, gonadosomatic ratios (gonad weight I body weight) were calculated, but were found to be stro ngly correlated with body weight for some samples. Therefore the data were log-transformed to improve the homogeneity of variances and regressions of log body weight against log gonad weight were calculated and compared between morphs for each sample by analysis of covariance (ANCOVA). Cochran's C test was used to determine whether the assumpti on of homogeneity of variance was met by the transformed data for each sampre. Statistics were calculated using the statistical package SPSSX.

5.3.4 Tube feet strength experiment

Twenty-eight urchins were collected from an area of about 100 m2 at Ti�derbox on 2 ii 88 and maintained in flow-through aquaria at ambient temperature for five days. The method of Sharp and Gray (1962) was modified as the urchins refused to attach themselves firmly to either a large rock or the underside of a piece of clear perspex. When placed on the gravel-covered bottom of an aquarium the urchins either remained where they were placed or moved slowly until they reached one of the sides. When they were placed with their oral surface against a glass side of the aquarium they attached themselves firmly and in some cases began to climb up the side. Urchins were chosen haphazardly and placed in a bag of nylon monofilament, placed against the side of the aquarium and allowed 5 minutes to attach 132 themselves. The bag did not impede the movement of the tube feet but allowed the attachment of a spring balance. After the urchin had attached itself, a force was applied through the spring balance which was gradually increased, over about 20 seconds, to the maximum pull of 3 kg. The force which was needed to remove the urchin was recorded but if it had not released its hold at 3 kg, the time for which it could resist this force was noted. The urchin was then placed on the gravel floor of the aquarium and allowed to rest for at least 5 minutes. The experiment was then repeated. The test diameter of each urchin was measured using Vernier calipers. Urchins which had resisted a 3 kg pull for varying lengths of time were scored as 3 kg for statistical tests, giving a conservative estimate as they would undoubtedly have resisted greater forces. Regressions of test diameter against Pull 1 and {Pull 1 - Pull 2) for each dermis colour were calculated to determine whether strength of tube feet varied with the size of the urchin. Unpaired t-tests were used to determine w�ether the mean force required to remove red and white dermis urchins differed. Paired t-tests were used to determine whether the forces required to remove urchins decreased for Pull 2 for each morph.

5.3 RESULTS

5.3.1 Morphometries and meristics

Means, standard errors and ·sample sizes for morphometric variables at each site are given in Appendix 4a-d. The data are given for both red and white dermis urchins and for the pooled sample, except for Fortescue Bay where only white dermis urchins occur. There were differences between red and white dermis urchins from Tinderbox in the maximum width of the ambulacrum {ANCOVA - for slopes, df1.55, F = 15.53, p � 0.001 ), the number of plates in the ambulacrum {ANCOVA - for slopes, df1,55, F = 8.16, p < 0.01) and the number of plates in the interambulacrum {ANCOVA - for slopes, df1,55, F = 8.03, p < 0.01 ); the maximum width of the ambulacrum increases . more rapidly with size for red dermis urchins whereas the number of plates in the ambulacrum and i nterambulacrum increase less rapidly with size for red urchins (Figure 5.1 ). No significant differences were found between dermis colours at Ling Reef for any of the variables. For the Tinderbox CVA classifying urchins by dermis colour (red or white) the canonical variate was significant {Wilks' A.=0.744, df = 4, p < 0.01) with red dermis urchins tending to have wider ambulacra {and correspondingly narrower 133

(a) 1.4 E ::J L u ro ::J 1.3 .0 E ro '-- 0 .c. 1.2 .... "0 �

X ro 1 . 1 E Q) 0

10

(b) E :::> '­ rou - ::J .0 E ro c:

en Q) +-> ro a. 0 c: 0) 0

( c£:::> J 5 '­ u ro :::> .0 1 4 E ro L Q) • ....c:

c: 13

(j) Q)

...... ro Key a. 12 0 • Red c: 0 White 0) 0 1 1 2 3 log 1 ive weight Figure 5. 1 Regressions for R and W dermis Tinderbox urchins of log live weight against (a) log max. width ambulacrum, Cb) log no. plates in ambulacrum, (c) no. plates in interambulacrum. 134 interambulacra) and heavier tests and spines (Table 5.2). Although 71 .1 9% of individuals were correctly classified, the Mahalanobis' distance indicated that the centroids of the two groups were not significantly different from each other (D = 1.153, p > 0.05). The CVA's using dermis colour at Ling Reef and spine colour at Tinderbox and Ling Reef showed no variation between morphs (Tables 5.3-5.4). When urchins were compared among all four sites (ignoring dermis and spine colours) the three canonical variates all explained a significant amount of the variation (1 st c.v., 57.61%; 2nd c.v., 33.04%; 3rd c.v., 9.35%; Table 5.5a). The centroids and individual points for the first two canonical variates are plotted in Figure 5.2 and the Mahalanobis' distances show that all pairwise comparisons are significantly different (Table 5.5b). The first canonical variate separated Cowrie Point from the other three sites and the second variate separated Tinderbox and Ling Reef, with Fortescue Bay intermediate. The third variate separated Fortescue Bay from the other two sites in southern Tasmania. The major components of the first variate were primarily lantern dry weight and secondarily length of the longest spine, with Cowrie Point having lower values for both. The major components of the second variate were test thickness and test height with Tinderbox having higher values for both. The major component of the third variate was test height with Fortescue Bay having relatively flatter tests than Tinderbox and Ling Reef. Overall 77.33% of individuals were classified into their correct sites with 90% of Cowrie Point urchins correctly classified (Table 5.5c). Most of the morphological v�riation therefore seemed to occur between sites rather than between morphs. To confirm this, urchins were classified into groups based on site and morph and reanalysed. The first three canonical variates explained a significant amount of the variation (1 st c.v., 51 .65%; 2nd c.v., 31 .67%; 3rd c.v., 11.20%; Table 5.6a). The Mahalanobis' distances again showed significant differences for all pairs of groups from different sites and no significant differences within sites (Table 5.6; Figure 5.3). This approach reduced the power of -the analysis with an average of only 56.67% of individuals being correctly classified into groups (Table 5.6c). However, it can be seen that very few of the Cowrie Point urchins were classified into any other site and the largest number of misclassifications occurred between Ling Reef and Fortescue Bay.

5.3.2 Microhabitat and behavioural variation

The study of the interrelationships between dermis colour, 'covering' response and position of the urchin showed that for two of the four samples taken at 135

Table 5.2 Resu Its of canonical variate analysis using dermis colour (red, white) for Tinderbox urchins.

(a) Percentage variation explained by the first canonical variate, canonical correlation, Wilks' lambda and the loadings (latent vectors) for the standardised morphometric variables.

0/o of variation explained 100.0 Canonical correlation 0.506 Wilks' lambda 0.744

p < 0.01

Variable Loading Max. width interambulacrum 0.5679 Test/spines dry weight 0.4457 Lantern dry weight 0.3569 Max. tooth width 0.3539 Max. width ambulacrum -0.3449 Diam. of madreporite 0.2650 Max. tooth length 0.2445 Test thickness 0.2183 No. plates in ambulacrum 0.2136 Max. diam. of longest spine 0.2008 Length of longest spine -0.1954 Test diameter -0.1897 Test height 0.1843 No. plates in interambulacrum 0.1777 Diam. of apical system 0.1415 Diam. of periproct -0.0426 Diam. pe ristome -0.0097

(b) Mahalanobis' distance and percentages of individuals classified into groups based on dermis colour.

= D 1.153 p > 0.05 GrouQ dermis colour Red White

Actual Red 79.3 20.7 dermis colour White 36.7 63.3 Mean percentaqe of correct classifications 71.19 136

Table 5.3 Results of canonical variate analysis using dermis colour (red, white) for Ling Reef urchins.

Canonical Variate 1 0/o of variation explained 100.0 Canonical correlation 0.356 Wilks' lambda 0.873 > 0.05

Table 5.4 Resu Its of canonical variate analysis using spine colour (green, purple-green, purple).

Canonical Variate I 2 Tinderbox - 60.00 40.00 0.585 0.508 0.488 0.742 > 0.05 > 0.05

Ling Reef - 0/o of variation explained 82.01 17.99 Canonical correlation 0.663 0.383 Wilks1 lambda 0.478 0.853 p > 0.05 > 0.05 137

Key 1 Tinderbox 2 Ling Reef 3 Cowrie Point 4 Fortescue Bay

+ centroids 4.0 1 1 1 1 1 1 1 1 1 1 11 (]) 2.0 1 1 - 1 11 3 3 3 3 1 11 + 1 4 3 3 1 1 11 11 1

-4.0

-4.0 -2.0 0.0 2.0 4.0 First canonical variate

Figure 5.2 Plot of values for the first and second canonical variates from the CVA of size corrected morphometric variables for urchins classified by site. 138

Table 5.5 Results of canonical variate analysis using site (Tinderbox, Ling Reef, Fortescue Bay, Cowrie Pt.)

(a) Percentage variation explained by the first canonical variate, canonical correlation, Wilks' lambda and the loadings (latent vectors) for the standardised morphometric variables.

Canonical Variate 1 2 3 % of variation explained 57.61 33.04 9.35 Cumulative % variation 57.60 90.65 100.00 Canonical correlation 0.808 0. 721 0.484 Wilks' lambda 0.128 0.368 0.766 p < 0.001 < 0.001 < 0.001

Variable Loading Lantern dry weight -0.957 -0.244 -0.41 8 Test height -0.027 0.610 0.612 Test thickness 0.473 0.665 0.167 Diam . of apical system 0.294 -0.146 0.237 Diam. of peristome -0.192 -0.198 0.016 Diam. of madreporite 0.402 -0.033 -0.162 Diam. of periproct 0.139 -0.128 0.569 Max. width of ambulacrum -0.236 0.256 -0.102 Max. tooth length 0.232 -0.090 0.508 Max. tooth width -0.016 -0.328 0.024 Length of longest spine 0.499 -0.018 0.452 Max. diam . of longest spine -0.095 0.488 -0.051 No. plates in interambulacru -0.293 0.240 -0.01 0

(b) Mahalanobis' distances for pairwise comparisons of sites. ** p < 0.01 *** p < 0.001

Tinderbox 0.00 Ling Reef 2.47** 0.00 Fortescue Bay 2.19** 1.762** 0.00

Cowrie Point 4.20** 3 .859*** 4.335** ; 0.00

Tinderbox Ling Reef Fortescue Cowrie Pt. 139

Table 5.5 Results of canonical variate analysis using site (Tinderbox, Ling Reef, Fortescue Bay, Cowrie Pt.)

(c) Percentages of individuals classified into categories based on sit e.

Groug based on site

Tinderbox Ling Reef Fortescue Cowrie Pt. Ba

Tinderbox 73.7 10.5 14.0 1.8

A ctu a l Ling Reef 8.9 73.3 17.8 0.0 site Fortescue Bay 0.0 17.9 82.1 0.0

Cowrie Point 5.0 5.0 0.0 90.0

Mean ge rcentage of correct classifications 77.33 Table 5.6 Results of canonical variate analysis using site (Tinderbox, Ling Reef, Fortescue Bay, Cowrie Point) and dermis colour (red, white).

(a) Percentage variation explained by the first canonical variate, canonical correlation Wilks' lambda and the loadings (latent vectors) for the standardised morphometric variables. Canonical Variate 1 2 3 4 5 6 % of variation explained 51 .65 31.67 11.20 3.02 1.74 0.72 Cumulative % variation 51 .65 83.32 94.52 97.54 99.28 100.0

Canonical correlation 0.807 0.731 0.537 0.314 0.243 0.159 -1. a 0.826 0.916 0.975 � Wilks' lambd 0.095 0.274 0.588 0 p < 0.001 < 0.001 < 0.01 > 0.05 > 0.05 > 0.05

Variable Loading Lantern dry weight -0.803 -0.263 -0.267 -0.055 -0.725 0.172 Test height -0.022 0.520 0.816 0.104 0.302 -0.269 Test th ickness 0.455 0.686 0.156 0.060 -0.432 0.225 Diam. of apical system 0.297 -0.1 08 0.048 0.337 -0.382 0.749 Diam. of peristome -0.172 -0.243 0.1 61 -0.228 0.203 -0.647 Diam. of madreporite 0.404 -0.029 -0.016 0.198 0.327 -0.605 Diam. of periproct 0.160 -0.186 0.632 -0.041 -0.014 -0.103 Max. width of ambulacrum -0.215 0.183 0.068 -0.246 0.708 0.203 Max. width of interambulacrur -0.051 0.030 -0.007 0.734 0.466 0.214 Max. tooth width 0.026 -0.366 0.064 0.028 0.293 0.213 Length of longest spine 0.543 -0.079 0.551 -0.155 -0.002 0.337 Max. diam. of longest spine -0.146 0.523 -0.066 0.277 -0.007 -0.355 No. plates in interambulacrum -0.296 0.216 0.021 0.1 11 0.357 0.253 Table 5.6 Results of canonical variate analysis using site (Tinderbox, Ling Reef, Fortescue Bay, Cowrie Point) and dermis colour (red, white, pink).

(b) Mahalanobis' distances for pairwise comparisons of sites. Only the first th ree canonical variates were used to calculate the Mah alanobis distances. TB - Tinderbox R - red ** p < 0.01 LR - Ling Reef W - white *** p < 0.001 FB - Fortescue Bay p - pink CP - Cowrie Point

...... � TB-R 0.00 ...... TB-W 0.69 0.00 LR-R 2.986*** 2.649*** 0.00 LR-W 2.386*** 2.141*** 0.69 0.00 FB-W 2.165*** 2.221*** 1.965 *** 1.579* 0.00 CP-R 4.035*** 4.155*** 3 .893*** 3.482*** 4.398 *** 0.00 CP-p 4.14 3 *** 4.488*** 4.473*** 3.954 *** 4.375*** 1.565 0.00

TB-R TB-W LR-R LR-W FB-W CP-R CP-p i42

Table 5.6 Results of canonical variate analysis using site (Tinderbox, Ling Reef, Fortescue Bay, Cowrie Point) and dermis colour (red, white, pink).

(c) Percentages of individuals classified into groups. TB - Tinderbox R-red LR - Ling Reef W - white FB - Fortescue Bay p - pink CP - Cowrie Point

Group based on site and dermis colour

TB-R TB-W LR-R LR-W FB-W CP-R CP-p

TB-R 5i .7 34.5 0.0 0.0 i 0.3 3.4 0.0

TB-W 25.0 46.4 7.i 10.7 10.7 0.0 0.0

LR-R 0.0 5.3 57.9 26.3 i 0.5 0.0 0.0 Actual group LR-W 3.8 7.7 34.6 38.5 i5.4 0.0 0.0

FB�W 0.0 0.0 i 0.7 i 0.7 78.6 0.0 0.0

CP-R i 0.0 0.0 0.0 0.0 0.0 60.0 30.0

CP-p 0.0 0.0 0.0 0.0 0.0 20.0 80.0

Mean percentage of correct classifications 56.67 143

� 1 Tinderbox � red 2 Tinderbox - white 3 Ling Reef - red 4 Ling Reef � white 5 Cowrie Point - red 6 Cowrie Point - white 4.0 7 Fortescue Bay � white 1 2 2 + centroids 1 2 1 (]) 2 11 (ij 2.0 5 5 2 213 2 22 1 ·;;:: 2 2 1 2 6 5 6 6 1 2 + 2272 2 � 5 6 6 2 1222 3 2 ·c: �5 1 4 71 0 0.0 5 5 11 7 11 27 13 c 6

-4.0 -2.0 0.0 2.0 4.0 First canonical variate

Figure 5.3 Plot of values for the first and second canonical variates from the CVA of size corrected morphometric variables for urchins classified by site and dermis colour. 144

Tinderbox (3 m), the first model (all variables independent) was rejected (20 viii

85, deviance = 19.52, df = 4, p < 0.001 ; 2 v 86, deviance = 8.362, df = 4, p < 0.1 ; Tables 5.7 and 5.8). ·Although some of the models tested did not meet the requirement that less than 10% of the expected frequencies should be less than one, the same pattern of associations between variables occurred in both cases. The model which gave the best fit to the data included relationships between dermis colour and microhabitat and between microhabitat and cover. The observed frequencies showed that a higher proportion of red dermis urchins were visible on the upper surfaces of rocks than were white urchins (20 viii 85, n = 67,

96.7% red urchins and 70.3% white urchins were visible; 2 v 86, n = 136, 57.1% red and 41 .1% white urchins we re visible) and that visible urchins covered to a greater extent than hidden urchins (20 viii 85, n = 67, 63.6% visible and 1 00% hidden urchins were not covered; 2 v 86, n = 136, 68.2% visible and 82.9% hidden urchins were not covered). The other two samples showed no associations

> between variables (24 i 86, deviance = 3.359, df = 4, p 0.25; 1 0 xii 85, deviance > = 1.142, df = 4, p 0.75; Table 5.9). For Betsy Island (1 0 m) and Coning ham, sampled on 17 ix 85 and 23 iii 86, respectively, the independence model was also rejected (Betsy Island, deviance =

14.02, df = 4, p < 0.01 ; Coningham, deviance = 31 .27, df = 4, p < 0.001 ; Tables 5.1 0 and 5.1 1 ). In both cases the model which gave the best fit to the data included a relationship between microhabitat and cover. The observed frequencies showed that visible urchins covered to a greater extent than hidden urchins (Betsy Island, n = 125, 81 .1% visible and 97.2% hidden urchins were not covered; Coningham, n = 123, 37.0% visible and 84.0% hidden urchins were not covered). Fo r those sites where no physical shelter was available, there was no relationship between dermis co lour and coveri ng response (Table 5.12). In summary, there was no evidence that the covering response varied between urchins of different dermis colour. There was some evidence that white urchins were more likely to seek physical shelter where it was available and urchins (of either dermis colour) which were visible on the upper surfaces of rocks tended to cover more often than urchins under rocks.

Nearest neighbour analysis Urchins of a particular dermis colour are more likely to occur next to an urchin of the same colour than to one of a different colour (G = 7.35, df = 1, p < 0.01 ; Table 5.1 3). Therefore it seems likely that urchins of different dermis colour show different microhabitat preferences. 1 45

Table 5.7 Relationships between de rmis colour, covering response and microhabitat at Tinderbox (3 m) on 20 August 1985. Log- linear models analysis was used. See text for explanation. Models shown in brackets do not meet the req uirement that <1 0°/o of expected frequencies should be less than one.

(a) Observed frequencies.

Dermis Cover colour Microhabitat u c R v 20 9 H 1 0 w v 15 11 H 11 0 n = 67

(b) Comparisons between models. Beneath each model is shown; deviance (df) probability

MOR+HAB+COV 19.52 (4) P< 0 .001

MOR*HAB+COV MOR*COVt +HAB HAB*COV+MOR 9.18 (1) p < 0.005 0.00 (1 ) p > 0.975 9.58 (1) p < 0.005 t '- MOR*HAB+HAB*COV 0. 75 (1) p < 0.001

(c) Analysis of deviance table for comparisons with original data.

Model df Deviance !2 MOR+HAB+COV 4 19.52 < 0.001 MOR*HAB+COV 3 10.34 > 0.01 MOR*COV+HAB 3 19.52 < 0.001 HAB*COV+MOR 3 9.94 > 0.01 MOR*HAB+HAB*COV 2 0.75 > 0.5 146

Table 5.8 Relationships between dermis colour, covering response and microhabitat at Tinderbox (3 m) on 2 May 1986. Log-linear models analysis was used. See text for explanation. Models shown in brackets do not meet the requirement that <1 0% of expected frequencies should be less than one.

(a) Observed frequencies.

Dermis Cover colour Microhabitat u c R v 23 13 H 23 4 w v 22 8

H 35 8 n = 136

(b) Comparisons between models. Beneath each model is shown; deviance {df) probability.

MOR+HAB+COV 8.36 {4) p < 0.1 t MOR*HAB+COV MOR*COV+HAB HAB*COV+MOR

3.50 {1) p < 0.1 0 .47 { 1 ) p > 0. 25 4.01 {1) p < 0.05 f ...... MOR*HAB+HAB*COV , 4.01 {1) p < 0.05

(c) Analysis of deviance table for comparisons with original data.

Model df Deviance 12 MOR+ HAB+COV 4 8.36 < 0.1 MOR*HAB+COV 3 4.86 < 0.1 MOR*COV+HAB 3 7.89 < 0.05 HAB*COV+MOR 3 4.35 < 0.1

MOR*HAB+HAB*COV 2 0.85 > 0.25 147

Table 5.9 Lack of relationships between dermis colour, covering response and microhabitat at Tinderbox (3 m)and Betsy Island (7 m). Log-linear models analysis was used. See text for explanation.

Site Dermis Micro- Cover and date colour hab itat u c n Deviance d f p

Tinderbox R v 1 1 1 0 (3 m) H 2 1 10 Dec 1985 w v 45 26 H 9 5 109 1 .142 4 > 0.75

Tinderbox R v 1 2 8 (3 m) H 4 5 24 Jan 1986 w v 35 28 H 1 9 7 118 3.359 4 > 0.25

Betsy Island R v 1 7 3 (7 m) H 4 0 17 Sep 1985 w v 64 4 H 9 0 1 01 3.923 4 > 0.25 148

Table 5.10 Relationships between dermis colour, covering response and microhabitat at Betsy Island (1 0 m) on 17 September 1985. Log-linear models analysis was used. See text for explanation.

(a) Observed frequencies.

Dermis Cover colour Micro h abita t u c R v 20 3 H 22 0 w v 23 7 H 48 2 n = 125

(b) Comparisons between models. Beneath each model is shown; deviance (df) probability

MOR+HAB+COV 14.02 (4)+ p < 0.01

MOR*HAB+COV MOR*COV+HAB HAB*COV+MOR 2.17 (1) p > 0.1 0.73 (1) p > 0.25 9.43 (1) p < 0.01

(c) Analysis of deviance table for comparisons with original data.

Model df Deviance Q MOR+HAB+COV 4 14.02 < 0.01 MOR*HAB+COV 3 11.85 < 0.001 MOR*COV+HAB 3 13.29 < 0.001 HAB*COV+MOR 3 4.59 > 0.025 149

Table 5.1 1 Relationships between dermis colour, covering response and microhabitat at Coningham on 28 March 1986. Log-linear models analysis was used. See text for explanation.

(a) Observed frequencies.

Dermis Cover colour Microhabitat u c R v 21 33 H 27 7 w v 6 13

H 15 1 n = 123

(b) Comparisons between models. Beneath each model is shown; deviance (df) probability

MOR+HAB+COV 31.27 (4)� p < 0.001

MOR*HAB+COV MOR*COV+HAB HAB*COV+MOR

0. 52 ( 1 ) p > 0. 25 0.30 (1) p > 0.5 28.52 (1) p < 0.001

(c) Analysis of deviance table fo r comparisons with · original data.

Model df Deviance f2 MOR+HAB+COV 4 3'1 .27 < 0.001

MOR*HAB+COV 3 30.75 < 0.001 MOR*COV+HAB 3 30.97 < 0.001

HAB*COV +MOR 3 2.76 > 0.250 150

Table 5.12 Relationship between dermis colour and covering response at sites where no physical shelter was availab le. Observed frequencies of urchins are shown. G-tests for independence were calculated.

Dermis Cover Site and date colour u c n G df �

Tinderbox (7 m) R 26 19 20 Aug 1985 w 23 21 89 0.095 1 0.76

Tinderbox (7 m) R 23 19 10 Dec 1985 w 44 15 1 0 1 3.452 1 0.06

Tinderbox (7 m) R 8 61 24 Jan 1986 w 7 30 106 0.532 1 0.53

Tinderbox (7 m) R 15 66 2 May 1986 w 2 33 116 2.535 1 0.11

Ling Reef (3 m) R 44 27 15 Apr 1986 w 27 13 1 1 1 0.142 1 0.71

Ling Reef (3 m) R 74 0 8 Jul 1986 w 33 1 1 08 0.149 1 0.7

Ling Reef (12 m) R 3 61 15 Apr 1986 w 6 35 105 1.955 1 0.16

Ling Reef ( 12 m) R 1 1 49 8 Jul 1986 w 11 38 109 0.086 1 0.77

Alum Cliffs (3 m R 56 8 6 Sep 1985 w 38 5 107 0.028 1 0.86

Alum Cliffs (7 m R 26 7 6 Se� 1985 w 56 5 94 2.1 1 0.14 1 51

Table 5. 13 Results of nearest neighbour analysis. Urchins were observed at Tinderbox (3 m) on 12 July 1988. Dermis colours of urchins and their nearest neighbo urs were recorded for 105 pairs. Figures shown are the observed frequencies.

First urchin Red White

Red 24 24 Second urchin White 14 43

G = 7.354 df = 1 p < 0.01

UTAS 152

5.3.3 Reproductive cycle and gonadal investment

The proportions of red and white dermis urchins which were ripe in each sample followed the same pattern at both Tinderbox and Ling Reef (Figure 5.4). There were no consistent differences between the proportions of ripe urchins of red and white dermis colour at Ti nderbox. Although Ling Reef appeared to have higher proportions of ripe red dermis urchins than Tinderbox, for the three samples in which ripe urchins occurred, the differences were not significant when tested by G-tests for independence (15 iv 85, numbers of ripe urchins were too low to allow

> > a test to be made; 4 iii 87, G = 3.07, df = 1, p 0.05; 12 i 88, G = 2.79, df = 1, p 0.05; Table 5.14). The breeding season occurred in mid-summer at both sites. The proportions of urchins which were ripe during the breeding season at Ling Reef were always much lower than those at Tinderbox. There were no differe nces in gonad weights (as determined by ANCOVA's between log gonad weight versus log live body weight regressions) between red and white dermis urchins for males or females for six of the seven Tinderbox samples and all of the Ling Reef samples. For the Tinderbox sample taken on 7 i 88 there were significant differences between dermis colours for females but. not for males (ANCOVA - df1 , 46, F = 9.06, p < 0.01 ). The increase in gonad weight with size was greater for red urchins than for white urchins (Figure 5.5). However, this may have been due to the lack of large red urchins and small white urchins in the sample.

5.3.5 Tube feet strength experiment

There was no relationship between urchin size and the force required to remove them for Pull 1, Pull 2 or for the difference between Pulls 1 and 2, for either morph (Figure 5 .6). The mean force required to remove red dermis urchins on the first pull was slightly greater than for white urchins (mean force = 2.14 and 1.88 kg

> respectively) but the difference was not significant (t = 0.783, df = 26, p 0.05; Table 5.15). Eight urchins resisted the maximum force on Pull 1 for 15 to 90 seconds, of which six were red dermis urchins (Table 5.15). Thus red urchins showed a slightly greater resistance to the first pull than white urchins.

This trend was reversed for Pull 2 (mean force = 0.92 and 1 .19 kg) but the

> difference was again not significant (t = -1 .250, df = 25, p 0.05). The force required to remove the urchins for the second time was significantly lower for both morphs (red urchins, mean (Pull 1 - Pull 2) = 1.346 kg, t = 4.966, df = 12, p < 0.001 ; white urchins, mean (Pull 1 - Pull 2) = 0.689 kg , t = 2.614, df = 13, p < 0.05). The 153

(a) Tinderbox

9 18 19 43 45 35 46 12 24 29 47 54 45 53 100

80

60

40

20

(b) Ling Reef

16 65 56 47 49 23 89 52 45 52 100

80

Q) 60 Q. L � 40

• 20 0

Kev

n 1986 1987 +Red Year -o- Wl11 te

Figure 5.4 Variation in the proport ions of ripe urchins of different dermis colours. Numbers represent sample sizes - red urchins above, white below. 154

Table 5.14 Results of G-tests for independence on the proportions of ripe urchins between red and white dermis urchins at Ling Reef.

(a) Sample taken on 4 Mar 1987.

Dermis colour

Red White Total

Ripe 1 8 1 1 29

Unripe 29 34 63

Total 47 45 92

G = 3.074 df = 1 p > 0.05

(b) Sample taken on 12 Jan 1988.

Dermis colour

Red White Total

Ripe 1 2 9 2 1

Unripe 37 43 80

Total 49 52 1 0 1

G = 2.798 df = 1 p > 0.05 155

Red 2

log Q gonad • weight

0 Key • • Red •

c Wh ite

-1 14 1.6 1 .8 2.0 2.2 2.4 2 . 6 2.8 log live body weight

Figure 5.5 Regressions of Jog body weight against log gonad weight for red and white dermis urchins from Tinderbox (7 Jan 1 988). 156

(a) Pull 4

3 Kl D •• •• Force c D to • q, c • rem ove 2 D (kg) C D • D c • • c �

(b) Pull 2

3

Force a a • to 2 c remove • (kg) D • • • a • c •

(c) Pull I - Pul l 2 3 Key • • Red D • • • • c W h i te 2 D Change in c D force to rJ'!) • Cl • rem ove D • (kg) • 0 • c Cl D D c D • -1 40 50 60 70 80 90 100 Test diameter (mm )

Figure 5.6 Variation in tube feet strengths with test diameter between dermis colours. 157

Table 5.15 Results of tube feet strength experiment. Figures in brackets indicate the number of seconds the maximum force was maintained. * Urchin refused to attach firmly.

Dermis Test Force to remove (kg) colour diameter (m m) Pull I Pull 2 R 74 0.50 * w 75 1.50 0.75 R 74 1.00 0.70 w 1.70 1.45 R 78 3.00 (0) 0.95 w 71 1.00 I .30 w 70 2.70 0.80 R 80 3.00 (15) I .20 R 70 1.50 0.60 w 79 0.70 0.85 w 74 0.40 0.90 R 70 3. 0 0 (7 0) 0 . 6 0 w 91 2.20 2.20 R 73 3.00 (20) I .55 w 72 1.70 0.30 R 76 2.40 0.25 w 66 2.80 i .00 R 71 2.30 0.95 R 71 0.70 0.60 w 75 2.50 0.70 R 49 3.00 (30) 0.90 w 54 1.60 1.70 R 42 2.1 0 1.00 w 58 1.50 1.90 w 51 3.00 (60) 0.70 R 58 1.50 2.20 w 55 3.00 ( 40) 2 .I 0 R 50 3.00 (90) 0.50 158 decrease between Pull 1 and Pull 2 was significantly greater for red than white urchins (t = 1.736, df = 25, p < 0.05). These results indicate that the tube feet of rested red urchins are at least as strong as those of white urchins. However, white urchins seem to tire less quickly and would therefore be at an advantage at sites where rough water conditions occur for long periods.

5.5 DISCUSSION

The analysis of morphometric and meristic characters indicated that there were slight differences between red and white dermis urchins at Tinderbox, but this was not shown by Ling reef urchins. A more extensive study including more sites is needed to determine whether the differences found in ambulacral width, numbers of plates in the ambulacra and interambulacra are consistently different between red and white urchins. There were no differences found between urchins of different spine colours for Tinderbox or Ling Reef. The CVA also indicated that red urchins at Tinderbox have heavier tests and spines than white urchins. Ebert (1982) found that the longevity of seventeen species of sea urchins, including H. erythrogramma, was correlated with increasing relative size of the test and spines and with exposure to wave action. He suggested that the maximum level of water movement tolerated by a particular species was determined by the thickness, and hence strength, of the test, and that the minimum amount of water movement tolerable might be determined by the respiratory ability of the species; the thicker the test, the slower diffusion of oxygen into the body cavity. Following this line of reasoning, red dermis urchins at Tinderbox may have an advantage over white dermis urchins because their thicker tests allow them to better withstand wave action. This would support the hypothesis that the extra echinoch rome A in the dermis' of red urchins has a respiratory function, otherwise red urchins might be under oxygen stress on calm days. This would also agree with the finding that white dermis urchins from Tinderbox had stronger tube feet as they would be at greater risk than red dermis urchins if they were dislodged during a storm. Black et a/. (1984) found that the dry weight of the test increased with the dry weight of the lantern (when size was adjusted for) of Echinometra mathaei in Western Australia. This is also true for H. erythrogramma and the relationship is stronger for red urchins than for white at Tinderbox (partial correlations, adjusting for size by test diameter; red urchins, r = 0.78, white urchins, r = 0.67, all urchins, r 159

in = 0.72). Lantern dry weight was the third most important component of the CVA separating red and white urchins at Tinderbox. The same trend is found for Ling Reef urchins (partial correlations, adjusting for size by test diameter; red urchins, r no = 0.75, white urchins, r = 0.42, all urchins, r = 0.58) although the CVA found significant differences between dermis colours at Ling Reef. Both Ebert (1980), studying Diadema setosum and Strongylocentrotus purp uratus, and Black et at. (1982), studying E. mathaei, found that urchins with relatively larger lanterns were found in habitats where food was apparently scarce . Black et at. (1 984) also suggested that urchins with relatively large lanterns had poor food supply because they were found to have smaller gonads. The relatively larger lanterns of red dermis urchins at Tinderbox may therefore indicate that either (1) red urchins occur in a different microhabitat from white urchins, in which food is less available, or (2) red urchins are less able to utilise the available food than white urchins. Experiments on the feeding ecology of red and white morphs would be of interest in investigating these possibilities. Greater morphological variation was found between urchins from different sites, with Cowrie Point, on the north-west coast of Tasmania, being clearly separated from all other sites by the CVA. Cowrie Point urchins had smaller lanterns and shorter spines than those from the other sites. Thus it appears that more or better quality food is available at Cowrie Point than at the other three sites. Unfortunately test dry weights and gonad weights were not measured for Cowrie Point so the other trends sugg�sted by Black et at. (1 984) and Ebert (1 982) cannot be evaluated. The sites in southern Tasmania were separated mainly by test thickness and test height with Tinderbox urchins having the thickest tests and Fortescue Bay urchins having relatively flatter tests than either of the other two sites. As stated previously, thicker tests may increase the chance of survival for urchins (Ebert, 1982). The flatter tests from Fortescue Bay are consistent with its relatively high exposure to wave action; flatter urchins are less likely to be removed from their positions by waves because they will protrude less from the rock surface and because they will be able to attach by their oral tube feet over a relatively larger area. This is supported by the results of Lewis and Storey (1984) who found that Echinometra lucunter from high wave-energy habitats were flatter than those from low wave-energy sites. The nearest neighbour analysis indicated that red and white urchins may occur in different microhabitats but the nature of the difference is not clear. However, the microhabitat study indicated that white urchins may seek physical 160 shelter to a greater extent than red urchins. Thus it does seem that there may be behavioural differences between dermis colours, as suggested in Chapter 3. There was no indication of variation in the timing of reproduction between dermis colours but there were some indications that morphs may differ in their reproductive investmenri n terms of weight of gonad relative to body weight. Red urchins at Ling Reef showed consistently higher proportions of ripe individuals than white urchins. Ling Reef has low algal cover and therefore low food availability for urchins; if the observed trend was real (it was not statistically significant) this suggests that red urchins may be more efficient feeders or that they channel more of their resources into gonad development than white urchins. This is interesting as the morphometric analysis suggested that red urchins may be less efficient feeders than white urchins at Tinderbox. There was no evidence of differences in gonad weights between dermis colours except for the females of one sample from Tinderbox, and this result may have been due to the different sizes of the red and white urchins ·dissected. Although this does not exclude the possibility that the morphs differ in the number and/or the size of eggs produced, it does indicate that there are no differences in reproductive investment between dermis colours. The tube feet experiment again indicated slight differences between dermis colours. Red urchins initially resisted removal as strongly as white urchins but seemed to tire more quickly because they could not maintain their strength for the second pull. Sharp and Gray (1 962) found differences between the tube feet strength of Lytechinus variegatus and Arbacia punctulata; the latter could withstand a pull of 250 g for significantly longer than L. variegatus on both glass and rock surfaces. This supported their observations that L. variegatus was only found in sheltered habitats whereas A punctulata occurred in both sheltered and exposed areas. Thus the results from the present study indicate that white urchins may be at an advantage in areas exposed to wave action; this supports the correlation of high proportions of white urchins with high values for the A.E.I. found in Chapter 4. In summary, differences were found between red and white dermis urchins in morphology, microhabitat (and therefore probably in behaviour), and strength of tube feet. These differences wi ll probably be important in the maintenance of the colour polymorphism of H. erythrogramma because of differential mortality of morphs in different habitats. 161

CHAPTER 6 GENERAL DISCUSSION

In this study I have demonstrated the occurrence of a complex colour polymorphism in populations of H. erythrogramma around Tasmania and in southern Victoria. The polymorphism is unusual among echinoids, where colour variation is usually confi ned to the calcareous parts (Serafy, 1973; Jensen, 1974), because it involves variation in the colour of both the dermis and the calcareous test and spines (although test colour is not usually visible in the live animal). The most common morphs have either a dark red or, effectively, a white dermis. Those with red dermis usually have dark spines which can be purple or green with a purple ring at the base. Those with white dermis may have either. dark or pale spines, and these can be purple, green, green with a purple ring at the base, or white. The degree of polymorphism within a population varies markedly with site and, although some populations may be monomorphic for dermis colour, no populations which are monomorphic for spine colour occur. The proportions of morphs within a population show extensive geographic variation although most morphs are present in the majority of populations sampled. However, on the north coast of Tasmania there are high proportions of morphs which were rare in other areas. These unusual morphs include both urchins with intermediate pink dermis colours and urchins with combinations of dark red dermis and pale spines (either green, purple or brown). The variation in H. erythrogramma is unusual because significant variation m the proportions of morphs between neighbouring populations must be maintained in the face of considerable gene flow through pelagic larvae. Thus it seems clear that the colour polymorphism in H. erythrogramma can only be maintained by strong selection and that the selection pressure must vary between geographically close sites. This could occur if th e intensity of a selective force varied or if several types of selection occurred in different habitats, although a combination of the two is also possible. Most of the well known examp les of visual polymorphisms are species which have small effective population sizes because of the limited mobility of the adults and lack of larval dispersal, such as the landsnails Cepaea nemoralis (Jones et a/., 1977), Partula spp. 162

(Clarke and Murray, 1971) and Cerion spp. (Gould, 1969) and the lepidopterans Panaxia dominula (Ford and Sheppard, 1969), Mania/a jurtina (Dowdeswell, 1961) and Biston betularia (Kettlewell, 1955). In these organisms variations in proportions of morphs between geographically close populations are thought to be maintained by localised selection, random processes or a combination of the two. The selection occurs because morphs may be well adapted to their environment in one habitat but not in another, e.g. crypsis of morphs of C. nemoralis in woodlands and neighbouring grasslands (Cain and Sheppard, 1950), or different thermal properties of dark and light shell colour in C. nemoralis populations on sand dunes (Richardson, 1974). Random processes may affect populations because of their small size and their tendency to undergo periodic extinctions or bottlenecks (Ford, 1975; Jones et at., 1977). Organisms which have extensive gene flow because of either the high mobility of the adults or dispersing larvae or juveniles are usually thought to show little genetic variation between close populations (Janson, 1987). However, large scale clinal variation is common either because of climatic or other large scale environmental variation or because isolation by distance has allowed variation caused by genetic drift to be maintained (Endler, 1973, 1977; Ehrlich and White, 1980). This is the situation which would be expected to occur in echinoids and is supported by the results of Palumbi (1989) who found smaller genotypic differences between populations of Strongylocentrotus purpuratus and S. droebachiensis than are usual for terrestri al species with sma II e r effective popu I ati on sizes. However, H. erythrogramma does not show large scale clinal variation in proportions of morphs and although the amount of electropho retic variation is unknown, the colour variation at least shows substantial variation between geographically close populations. A group of marine snails, the littorines, provide an opportunity to test these hypotheses, as many species have variable shell morphology, sculpture and colour and there is a variety of larval types ranging from development within the parent to planktotrophic larvae which may be pelagic for several months. The majority of littorines have a planktonic larval stage (Rosewater, 1970; Mileikovsky, 1975) but almost all the colour polymorphic species are either viviparous or lay benthonic eggs which give rise to benthonic larvae (Heller, 1975; 163

Reimchen, 1979; Atkinson and Warwick, 1983; Hughes and Mather, 1986). Most studies on the colour polymorphisms have confirmed that local selection influences the morphs present in different hab itats; the most common selective pressures being visual and apostatic predation. Janson (1987) also found that allozyme and shell morphology variation differed between L. littorea (planktonic larvae, monomorphic for colour) and L. saxatilis (viviparous, colour polymorphic) which occur sympatrically in Sweden. Littorina littorea is homogeneous for both sets of characters, both with in and between populations whereas L. saxatilis shows marked variation in both sets of characters. These results support the hypothesis that restricted gene flow and therefore low effective population size is necessary for the maintenance of extensive genetic differentiation and visual polymorph isms. There is, however, one genus in which colour polymorphic species with a pelagic larval stage occur. Littoraria spp. live on mangrove trees in the tropics and some species are colour polymorphic while others are not, but all have a planktonic larval stage (Cook, 1986) . The polymorphic species tend to live in the heterogeneous habitats of the foliage whereas monomorphic species live on homogeneous substrata, such as bark (Cook, 1986). Both Cook (1986) and Reid ( 1987) found that visual selection was probably maintaining the polymorphisms and Reid also provided strong· evidence for the action of apostatic selection. However, the morphs of L. pallescens occurred in similar proportions in 39 samples along 80 km of the coast of Papua New Guinea and in samples from Kenya, in distinct contrast to the geographical variation found in H. erythrogramma. Cook (1986) suggested that such a situation could be maintained if similar selection pressures occurred in all areas with the same mangrove species. As H. erythrogra mma does not follow this pattern of differentiation, the selective pressures maintaining the polymorphism must vary among populations. The variation in some other echinoid species also does not follow the patterns which might be expected for well dispersed species. Different colour morphs occurring geographically close but in different habitats have been reported for th e echinoids Psammechinus miliaris (Lindahl and Runnstrom, 1929) and Echinometra mathaei (Lawrence, 1983). Lawrence (1 983) found small pink E. mathaei on 164

the exposed windward side and larg e black individuals on the sheltered leeward side of a small headland on Reunion Island in the South Indian Ocean, which parallels the correlation of red and white dermis individuals of H. erythrogramma with exposure. There were also differences between the two habitats in the algal growth and the burrowing habits of the urchins. Heliocidaris erythrogramma is found in burrows in limestone and sandstone areas (personal observations) but no attempt was made to correlate this behaviour with colour. They also occur in crevices where harder substrata occur and may be found on sand in seagrass beds in some areas. The former are often exposed sites where white dermis urchins predominate whereas the latter are invariably sheltered and are dominated by red dermis urchins, but no correlation between dermis colour proportions and substratum was found during this study. Lindahl and Runnstrom (1 929) described S- and Z-type morphs of Psammechinus miliaris on the west coast of Sweden which occur in deep areas of sandy shale (40 - 58 m) and shallow Zos tera beds respectively. The Z-type urchins have brownish-green tests with violet tips to the spines, whereas the S-type have lighter grey-green tests which sometimes tend to red or yellow, and their spine tips are also violet but this may extend almost to the base of the spine. The S-type urchins are usually smaller and have shorter aboral spines but longer and thinner lateral spines, th icker and flatter tests, and larger eggs. Lindahl and Runnstrom (1 929) concluded that the colour differences were due to differing light intensities in the two habitats because of the decreasing pigmentation with increasing depth and because of the distribution of pigment on the urchins. No correlation of colour with depth was found during the present study, but the observation that the aboral surface is more heavily pigmented than the oral surface is also true for H. erythrogramma. Two possible types of selection were identified by the present study. The first and the strongest was suggested by the association of dermis colour with the exposure of the habitat to wave action. In every area except northern Tasmania, where the uniformity of the enviro nment may have obscured the relationship, there was a significant decrease in the proportion of red dermis urchins with increasing exposure. Several hypotheses were put forward to explain this and it seems likely that white dermis urchins can withstand wave 165 action better than red dermis urchins. The results of the tube feet experiment suggested that white dermis urchins tire less quickly and are there fore less I ikely to be removed from the substratum and damaged during storms. The variation in the relative widths of the ambulacra (where the tube feet originate) and interambulacra found between red and white dermis urchins at Tinderbox may also affect the urchins' ability to cling to the substratum, although these differences were not found for Ling Reef urchins. The second type of selection, which probably only causes variation in the proportions of morphs on a local scale, was suggested by the increase in the proportion of white dermis urchins with the amount of algal cover. White dermis urchins are far more conspicuous against most substrata than red dermis urchins and are therefore more likely to be targeted by visual predators. Selection would therefore favour white dermis urchins which occurred where algal cover could help hide them. The microhabitat studies supported this hypothesis because they showed that there was some choice of microhab itat by the urchins and that white dermis urchins were more likely to be found hidden under rocks. Also, urchins which occur on the upper surfaces of rocks are more likely to 'cover' themselves with pieces of rock, shell and algae, sug gesting that these items are used as camouflage. However, there was no difference in the frequency of this behaviour between the morphs. The results of the study of temporal variation at Point Cook and Tinderbox also suggested that there may be behavioural differe nces between morphs in their migration between habitat types. Lindahl and Runnstrom (1929) found behavioural differences between the S- and Z-type urchins with S-type urchins moving more slowly and showing a greater tendency to 'cover' than Z-types. Also , the negative geotactic reaction of S-type urchins was much slower than that of Z­ types; in aquaria Z-type urchins usually migrated to the water surface in 30 minutes whereas S-types took 12 to 24 hours. Clearly further studies on the behaviour of the colour morphs of H. erythrogramma would be useful. Microhabitat separation of colour morphs has only been shown in one other study apart from the present one, and in that study behavioural differences between morphs was also demonstrated (Tsuchiya and Nishih ara, 1984, 1985). The two colour morphs of 166

Echinometra mathaei on Okinawan reef flats 1n Japan show overlapping distributions but type A urchins mainly occur in shallow areas and rock pools whereas type B urchins occur in slightly deeper water at the reef edge. Type A urchins occur in aggregations and may be found in crevices, burrows or in open areas whereas type B urchins occur singly in burrows. Type B urchins are also more agressive towards both other type B individuals and type A urchins when they intrude into their burrow. Type A urchins attempt to repel type B intruders but with a lower success rate and only rarely show agonistic behaviour against other type A urchins. Another indication that selection has influenced the polymorphism in H. erythrogramma is the association of dermis, test, and primary and secondary spine colours described in Chapter 2. A high intensity of test and spine colour is usually correlated with intense dermis pigmentation. Although white dermis urchins may have pale or dark spines the intensity of colour in the secondary spines always matched that of the primary spines. There are also associations among the actual colours, with disequilibrium being demonstrated for purple and non-purple spines with dermis colour. Significant disequilibrium was shown to occur in several sites and the same trend occurred over most of the geographical area studied (Chapter 4). Such patterns of variation suggest that the genes controlling pigmentation may be linked and this is evidence that th eir interaction is subject to selection (Jones et a/., 1977). Although it has been suggested that the influence of stochastic processes on the colour polymorphism is negligible because of the large effective population size of H. erythrogramma, isolation by distance was supported by the Mantel's test for southern Tasmania. This result may have been due to a correlation with another environmental variable which was not considered in this study, or it may be that the relatively short pelagic larval stage of H . erythrogramma (5-7 days in the laboratory, see Appendix 1) does not allow extensive dispersal, although this seems unlikely. A logical area for further study would be to use electrophoresis to determine the amount of genetic variation in H erythrogramma among populations and regions. This should provide data on the amount of gene flow which occurs within and between the four geographical regions. This approach would also allow the correlation of 167 morphological and allozyme differentiation to be determined. In several studies it has been found that patterns of morphological and allozyme variation do not coincide (Jones et al., 1980; Dillon,1984; Francis et al., 1986) suggesting that different aspects of differentiation may respond independently to different evolutionary forces. Although only minor differences in morphology between red and white dermis urchins at one site were found, there were significant differences among sites. The major differences were in lantern dry weight, spine length, test thickness and test height. The degree of morphological differentiation increased with increasing distance between sites with the urchins from Cowrie Point on the north coast being substantially different to those from each of the three other sites. It is not known whether this is due to divergence through isolation or because the environment is very different. It has been shown in several studies that morphological variation occurs among different populations of echinoids and this has been variously attributed to hybridization between species (Hagstrom and L o n n i n g, 1 9 61 , 1 9 6 4) , food a v a i I a b iIi ty ( B I a c k e t a/., 1 9 8 2) or to environmental influences such as exposure to wave action (Lewis and Storey, 1 984). Serafy ( 1973) found morphological differences between subspecies of Lytechinus variegatus which occur in different regions of the east coast from North to south America. However, he found that there was morphological overlap between the subspecies and suggested that test colour was the most reliable distinguishing character. There is also conclusive evidence for two species of echinoids that morphological variation within species has a genetic base. Individuals of Arbacia punctulata from Massachusetts and Florida were bred in identical laboratory conditions and pure-bred juveniles from the two populations had significantly different spine length : test diameter ratios (Marcus, 1980). However, crosses between the two populations produced juvenile urchins with intermediate values for the ratio. Pawson and Miller (1982) found differences betw een juveniles of L. variegatus from Bermuda and Florida but crosses between the two populations were not made. In addition to differences in the spine length : test diameter ratio, Pawson and Miller (1 982) also found differences in the pigmentation, activity and righting response 168 between the two types of juveniles. The success of these two studies in combination with results presented here suggests another major area for further work suggested by the present study, laboratory breeding of H. erythrogramma, would be worthwhile. This would allow the genetic basis of the polymorphism and the possible effects of different rearing conditions on colour to be determined. Also, the basis of the morphological variation between sites could be assessed. 169

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Appendix 1 Laboratory breeding trials

Aims The main aim was to determine whether dermal pigmentation in Heliocidaris erythrogramma has a simple genetic basis. The genetic basis of variation in spine colour was also to have been investigated but this is likely to be more complicated as three pigments are involved as well as multi-coloured spines. If both types of variation were found to have a simple genetic basis then the linkag e or assortative independence of the two systems would be determined. Thus a method had to be developed by which the urchins could be bred and reared in the I aboratory, in fairly large numbers, to the stage where adult pigmentation could be determined. Depending on the success of the crosses in demonstrating a simple genetic basis to the pigments present, further crosses could then be performed to investigate either the distribution of pigment on multicoloured spines or the variation in intensity of pigmentation. The influence of environmental factors such as light, temperature and food type and availability on juveniles of known genetic makeup could then be investigated.

Methods Ripe urchins were collected from natural populations during the breeding season from October to February each year (October 1985 - February 1988). Each urchin was injected through the peristomial membrane with 3-5 ml 0.5 M potassium chloride solution. Males were placed with the aboral side down on the top of a beaker full of seawater which allowed the sperm to settle at the bottom of the beaker. Females were placed oral side down at the bottom of a beaker of sea water and the eggs floated to the surface where they could be poured off. The eggs of each female were placed in a separate container and diluted with several litres of clean sea water. Several ml of concentrated sperm solution were pipetted into the egg suspension and the water was gently agitated to ensure that mixing occurred. Ten minutes were allowed for fertilisation to occur before more sea water was added to further dilute the suspension. Approximately 60 crosses were made during the three breeding seasons and these included most of the possible pairs of colour morphs. Two types of aquarium systems were available during the study: in the 1985- 86 and 1986-87 seasons single aquaria supplied with filtered sea water from a large storage tank at the Department of Zoology, University of Tasmania were used; for the I 987-88 season a flow through system at the Department of Sea 180

Fisheries laboratories at Crayfish Point, Taroona was available, supplied by direct intake from the Derwent Estuary. The latter had no reliable filtration system so that whenever a storm occurred large amounts of silt came through the system. ,In both systems the urchins developed best and had the fewest deformed larvae at 15- 180C, normal sea water temperatures for Tasmania in summer. As the larvae of H. etythrogramma are lecithotrophic there was no need to provide food until after metamorphosis.

Single aqu aria Each culture was placed in a white polyethylene box ( 15 I) which was gently aerated. Magnetic stirrers were used for some cultures in an attempt to increase the distribution of larvae in the water column as they tended to float at the surface. The water was replaced every few days by siphoning out water from the bottom and adding clean water. After most of the larvae had metamorphosed and settled, the remaining unhealthy larvae were removed and water replaced. Experiments were performed to determine whether the time taken to reach larval settlement could be reduced by placing portions of a culture in different boxes: one was initially sterile, one had a bacterial film because it had been used as an aquarium for several weeks previously and one had small rocks covered with coralline algae placed in it. When juveniles developed mouths, groups of twenty healthy individuals were placed in I I containers : one group was placed directly on small rocks which had recently been collected from natural urchin habitat; three groups were supplied with fresh Macro cys tis sp., Ecklonia radiata and U Iva sp. respectively, algae which had been seen to be eaten by adult urchins.

Flow�through system Green plastic rubbish bins which had been kept in an outdoor flow-through tank for several days were used so that each had a bacterial film present when the culture was added. When each cu lture had been fertilised the bin was half filled with unfiltered sea water and left until most of the larvae had metamorphosed and settled. The water in each bin was gently agitated each day. When most larvae had settled the flow-through system was connected; water entered through a tube, the mouth of which was placed at the base of the bin, and exited through a hole cut near the top of the bin. The flow rate was adjusted so that a slow but steady stream of water left the hole. For some cultures small rocks and pieces of macroalgae recently collected from natural urchin habitat were placed in the bin. In order to reduce the amount of silt which came through the system a settlement tank was set up and water was pumped in to the cultures from this tank through a 181 filter tower filled with cotton wool. However, the large amount of silt which occurred during rough weather regularly clogged the pump or simply passed straight through. The test diameters of the juvenile urchins were measured using an optical micrometer on a dissecting microscope.

Proposed Experimental Design The limited breeding season and amount of available aquarium space meant that a maximum of 20 crosses could be performed each year (assuming that juveniles would take several months to achieve scorable pigmentation). The crosses to be attempted in the first year are shown in Table 1; all the urchins would have come from Tinderbox in southern Tasmania. These crosses were chosen to allow the two pigment systems, dermal and spine, to be considered separately and because they could be made using the relatively common morphs at Tinderbox (RP, RPG, WW, WP, WG and WPG). The extreme rarity of RW and RG (if any) urchins made it impractical to rely on crosses involving these morphs. This design consists of 10 W x W, 6 R x R, and 4 W x R crosses which should have provided ample replication of results if dermal colour has a simple genetic basis. The greater number of possible crosses for spine colour meant that only 2 or 4 replicates were possible in each case but this should have been sufficient to allow the consistency of results to be indicated. If confirmation was necessary for any of the crosses, they would have been repeated in the second year's breeding season. If the results had shown that both dermal and spine pigmentation were probably each controlled by a si'ngle gene then these crosses would also allow the linkag e or independence of the two genes to be determined. The seco nd year's breeding season would have been used to further investigate any of the crosses which had given equivocal or confusing results during the first year. If enough aquarium space was available crosses would also have been performed to investigate either the distribution of pigment on multicoloured spines (if the results from the first year's crosses had suggested this would repay further study) or the variation in intensity of pigmentation on spines of the same colour. As the breeding was not successful it was only possible to try to repeat the crosses shown in Table 1 in the second and third years of the study. The time needed for H. erythrogramma to reach sexual maturity and the growth rates of newly metamorphosed juveniles are not known. However, observations on the gonads of small urchins from populations in southern Tasmania and on growth rates of Victorian H. eryth rogramma (A. Constable, personal communication) suggested that it would be possible to perform backcrosses during the third year of the study. Dissections of urchins from natural 182

Table 1. Proposed crosses to determine the genetic basis of dermis and spine pigmentation. (R = red W =white P =purple G =green)

Male Female Number of crosses dermis spine dermis spine

w w w w 2 w w w p 1 w p w w 1 w w w G 2 w G w w 2 w G w G 2 R p w w 1 w w R p 1 R p w G 1 w G R p 1 R p R p 2 R p R PG 2 R PG R PG 2 183 populations had indicated that urchins probably spawn every year. Thus it would be necessary to keep the adults from the first year's crosses alive for twq years either in the laboratory or, more probably, in separate labelled cages at the field site from which they originally came. This should have been possible as the inducement of spawning by injection of potassium chloride solution was not observed to cause any mortality and the urchins were known to feed on drift algae in the field. Crosses between what were thought to be homozygous recessive F1 progeny and parents from the first year's breeding would be necessary to confirm the genetic make up of the parents and thereby definitively demonstrate the simple genetic basis of the col our variation. Again the crosses performed would have been chosen to allow consideration of dermal and spine colour to be considered separately and together. The investigation into the influence of environmental factors would have concentrated on dermal variation as this was carrel ated with environmental variables in natural populations and showed the clearest geographical patterns. The most obvious factors to investigate in the laboratory are light intensity, temperature, and food type and availability. For each experiment the progeny from one cross would have been split into several experimental groups af\d a control group. For example, for the light intensity experiment, three aquaria would have been set up; the control would have been kept in ambient light, one aquarium would be kept in constant darkness and the third in constant bright light. The crosses chosen would initially have been R x R and W x W, preferably with known homozygotes as the parents. The R x R progeny in constant bright light and the W x W progeny in constant dark would also be acting as controls as R urchins could not become darker and W urchins could not become paler. If homozygous parents were used the progeny should be either all R or all W, if there was no environmental effect, so any deviation from this would indicate an en vi ron mental effect.

Results For all of the crosses the majority of the larvae developed and metamorphosed. Thus there is no evidence of barriers to reproduction between colour morphs, although it is possible that there would have been differential mortality between morphs or that some of the crosses might have produced sterile progeny. The larvae from all of the crosses developed as described by Williams and Anderson (1975), and developed red pigment granules after 1 8 - 24 hours. The concentration of pigment granules over the surface of the larvae was uneven with the densest area being at the tip nearest the developing echinoid rudiment, 184 but no differences between crosses were observed. After metamorphosis the aboral surface had the greatest density of pigment and the juveniles appeared red to the naked eye. Again no differences were observed between crosses but the amount of pigment decreased with time and sick or dead juveniles had very few pigment granules and appeared pale yellow. No differences were observed in the settlement time of larvae kept under different conditions; metamorphosis began after 2.5 days and was complete by 7 - I 0 days after fertilisation. After another week the mouth was open and teeth of the Aristotle's lantern could be seen so presumably the juveniles were ready to begin feeding. Si ngle aquaria The larvae swam weakly at the water surface and the effect of either aeration or magnetic stirrers was to force many of the larvae together in the corners of the bin where they failed to develop and soon degenerated into an orange scum which had to be removed regularly. Only those which had remained free in the water developed to metamorphose, at which point most attached themselves to the side of the bin near the water level. No growth was observed in any of the juveniles after metamorphosis was complete. None of the juveniles in the different food trials survived longer than those in the originally sterile boxes. Most cultwes died after about 2 months, presumably from starvation. Even though the tube feet and podia developed well the urchins were fairly sluggish and showed only a weak righting response. Flow-through system The larvae developed at the same rate as those in the single aquaria although they had no aeration and the water was only gently agitated once a day. When they settled they formed a tight band around the bin just under the water surface but when the flow-through system was switched on the band was covered by I 0 - 20 em of water. It was possible to induce more of the larvae that remained swimming at the water surface to settle by repeatedly raising the water level by a few em and leaving overnight. Thus several bands of newly settled juveniles were left on the sides of the bin. These bands gradually broke up as the larvae migrated slowly down over the next few weeks until the sides and bottom of the bin were fairly evenly covered with juveniles. The larvae were much more active than those reared in the single aquaria, so that it was often difficult to observe them under the microscope. They exhibited a strong righting response as soon as the tube feet were well developed (i.e. before mouth was open). The test diameter of newly settled urchins was approximately 0.5 mm. The juveniles showed no preference for rocks or macroalgae over the side of the bin and cultures with these additions induced no faster growth than other cultures. 185

Unfortunately, due to several storms, most of the cultures were swamped with silt and died. However, one culture (RP female with RPG male) which was fertilised on 2nd March 1987, survived until 13th May and individuals had grown to a maximum test diameter of 0.7 mm. The slow growth rate may be partly due to the low temperature as the culture· was at the ambient sea temperature which was much lower than newly settled urchins would normally have experienced in summer. On the 6th May about 100 of the juveniles were observed to have pale green pig mentation at the base of their lateral spines. This pig mentation had not increased by the time another covering of silt killed the culture.

Proposed analysis of results If dermal pigmentation has a simple genetic basis the simplest hypothesis is that it is controlled by one gene with simple dominance between two alleles for red (DR) and white(DW) colouration The expected proportions of phenotypes among the F 1 progeny would therefore be 1 : 0, 1 : 1 or 3 : 1 for different crosses. The observed proportions would have been compared with the relevant expected proportions using G-tests for goodness-of-fit. Which of the alleles was dominant would be shown by the results of the proposed crosses, e.g. if W x R crosses resulted in three times as many red progeny as white, then the DR allele would be dominant. Crosses of R x W with different sexes for each colour would determine

whe. ther the gene for dermal pigmentation was autosomal or sex-linked. � Spine pigmentation would be more complicated to determine as three colours (white, green and purple) are present and purple-green spines are also common. It is possible that this is controlled by one gene with three alleles for the three colours (SP, sG, SW) with complete dominance, e.g. P > G > W. Another gene might then inhibit the expression of the purple colouration in the tips of the spines for SPfSG urchins. Another possibility is that there may be incomplete dominance between the SP and SG alleles with the heterozygotes having purple-green spines. The results of the crosses with W spined parents would show whether P, G and W spines had a simple genetic basis using G-tests for goodness-of-fit, as before. The PG x PG crosses might help to distinguish between the two hypotheses described above, although more replicates of this cross would probably be necessary. If the latter was correct the expected ratio in the progeny would be 2 : 2 : 1 : 1, purple : green : purple-green : white. However, if the former was correct either the same 2 : 2 : 1 : 1 ratio or 3 : 1, purple : green progeny might occur, depending on the mode of action of the modifying gene and the genotypes of the parents for the modifying gene. 186

If the results suggested that both dermal and spine colour did have a simple genetic basis then crosses of double recessive homozygotes with urchins which were heterozygous at both loci should show whether the two genes are linked or independent. For example, if DR was dominant to ow and SP was dominant to both SG and SW then crosses of (DR/OW, SP/SW) x (OW/OW, SW/SW) should give progeny in the ratio 1 : 1 : 1 : 1 for the phenotypes RP : RW : WP : WW. This would again be tested using G-tests for independence.

Discussion There appear to be no barriers to fertilisation between colour morphs and all the crosses produced initially viable juveniles. The larvae do not require aeration if kept in a sufficiently large volume of water and appear to settle on the first surface they contact after the tube feet develop. The larvae are weak swimmers and were probably kept at the water surface because of the buoyancy of their yolk reserves. The newly settled juveniles migrated slowly downwards soon after settling but many remained close to the water surface. The juveniles were more active and developed better in a flow through system, although it is not known whether this was due to the flow of water over them or to the probably infE?rior water quality from the storage tank at the University of Tasmania. The juveniles were probably feeding on the film of algae and bacteria on the surface of the bins and showed no preference for rocks or macroalgae as a habitat or food source. No consistent differe nces in dermal pigmentation between crosses were observed. Pigmentation of the· calcareous parts began after approximately 2 months but this may have occurred sooner if ambient temperatures had been higher. The juveniles would probably have grown successfully if adequate filtration of the water in the flow-through system had been achieved. If the genetic basis for variation in dermis colour had been determined and followed the model of an autosomal gene with, for example, complete dominance of oR over ow. then it would have been possible to calculate the gene frequencies present in each of the populations sampled using Hardy-Weinberg theory. It would then be possible to determine whether each population was in Hardy­ Weinberg equilibrium. This is a weak test but could give direct evidence for the action of natural selection whereas correlations of morph frequencies with environmental data and geographical patterns of morph distributions can only provide circumstantial evidence. If the genetic basis of spine colour had been determined then it would also have been possible to determine whether there was linkage disequilibrium between the two types of colouration, as suggested i·n Chapter 2. 187

The genetic basis of colour polymorphisms for several other species have been determined. In gastropods there is usually one locus with several alleles for different colours, another determining the presence or absence of bands and sometimes others modifying the distribution of pigment (Murray, 1975). One of the simplest systems is that of Thais emarginata (Palmer, 1984, 1985). The banding of the outer shell is controlled by a single autosomal locus with two alleles for banded and unbanded shells with complete dominance of banding. This was easily determined although there was variation in the clarity with which the banding was expressed. Thus the occasional occurrence of pink dermis H. erythrogramma would not necessari ly obscure the genetic basis of the pigmentation. The colour of the outer shell of T. emarginata is also controlled by a single autosomal locus with the allele for orange usually completely dominant over the allele for black pigmentation. The two loci segregate independently and so do not form a 'super gene' as is usual among terrestrial pulmonates (Palmer, 1985). There are also other genes or alleles, however, which influence the intensity of pigmentation. The polymorphism of the land snail Cepaea nemoralis is well understood and has probably one of the most complicated genetic systems known for a polymorphism (Jones et a/. , 1977). There is one locus for shell colour with at least six alleles, one locus for banded or un banded shells, one locus which controls whether the bands will be punctate or not and at least six further loci controlling suppression of banding and pigmentation of other parts of the snails (Murray, 1975). Five of these loci including those for shell colour, banding and punctate bands, are tightly linked in a 'super gene', i.e. they are almost always inherited together. This allows natural selection to maintain the linkage disequilibrium often observed in natural populations. The shell of the oyster drill Urosalpinx cinerea may be purple, brown or white in juveniles although the distinctions may blur with age (Cole, 1975). This polymorphism was found to be controlled by a triallelic autosomal gene with complete dominance in the order purple > brown > white. This may be similar to the situation of the spine colours of H. erythrogramma. A different system of genetic control has been suggested for the marine periwinkle Littorina mariae (Reimchen, 1974 cited in Murray, 1975). It seems most likely that two loci are involved in the control of the two morphs, reticulata and citrina, and that reticulata occurs when the dominant alleles are present at both loci. Colour and spot pattern polymorphisms have also been well studied in fish such as the platyfish, Xiphophorus maculatus and the guppy Poecilia reticulata 188

(Kallman, 1975; Yamamoto, 1975). These two examples have many genes controlling the polymorphisms but each displays simple Mendelian inheritance with complete or incomplete dominance between the alleles. The genes differ from those previously described in that they are often sex linked in which case the polymorphism is not expressed In the females. Also, for X. maculatus, the expression of certain alleles and the relative proportions of alleles vary between populations in different river systems. This suggests that the polymorphic patterns have adaptive significance and evolved independently in the different river systems (Gordon and Gordon, 1950; Kallman, 1975). This type of analysis could have been applied to the results for H. erythrogramma if the breeding program had progressed far enough.

References

Cole, T .J. (1975) Inheritance of shell colour of the oyster drill Urosalpinx cinerea. Nature 257 : 794-795. Gordon, H. and Gordon, M. (1950) Colour patterns and gene frequencies in natural populations of a platyfish. Heredity 4 : 61-73. Jones, J.S., Leith, B.H. and Rawlings, P. (1977) Polymorphism in Cepaea: A problem with too many solutions? Ann. Rev. Ecol. Syst., 8 : 109- 143. Kallman, K.D. (1 975) The platyfish, Xiphophorus maculatus. In "Handbook of Genetics. Vol. 4". R.C. King, (ed.) pp. 81 -132. Plenum, New York. Murray, J. (1975) The genetics of the Mollusca. In "Handbook of Genetics. Vol. 4". R.C. King, (ed.) pp. 3-31 . Plenum, New York. Palmer, A.R. (1984) Species cohesiveness and genetic control of shell color and form in Thais emarginata (Prosobranchia, Muricacea): Preliminary results. Malacologia 25 : 477-491 . Palmer, A. R. (1 985) Genetic basis of shell variation in Thais emarginata (Prosobranchia, Muricacea). I. Banding in populations from Vancouver Island. Bioi. Bull., 169 : 638-651. Williams, D.H.C. and Anderson, D.T. (1 975) The reproductive system, embryonic development, larval development and metamorphosis of the sea urchin Heliocidaris erythrogramma (Val.) (Echinoidea: Ech inometridae ). Au st. J. Zool., 23 : 371-403. Yamamoto, T. (1975) The medaka, Oryzia latipes, and the guppy, Lebistes reticularis. In "Handbook of Genetics. Vol. 4". R.C. King, (ed.) pp. 133-1 49. Plenum, New York. 189

Appendix 2a Dermis colour data for all sites. See Chapter 2 for explanation of morph codes.

Site No. Site Name R w p n 1 Pelican Island 21 79 0 100 2 Blubber Heads 56 44 0 100 3 Roaring Beach 6m 56 49 0 105 4 Roaring Beach 15m 73 59 1 133 5 Satellite Island A 86 14 1 1 01 6 Satellite Island B 51 55 0 106 7 Ling Reef 3m 422 187 28 637 8 Ling Reef 13m 361 262 3 626 9 Ling Reef Slope 55 43 3 1 01 10 Ling Reef Barren 78 25 1 104 11 Gordon 58 44 1 103 12 Coning ham 88 35 0 123 13 Tinderbox 3m 367 462 4 833 14 Tinderbox 7m 460 255 1 71 6 15 Tinderbox Slope 55 31 8 94 16 Tinderbox Barren 62 30 2 94 17 Tinderbox Beach 285 203 9 497 18 Blackman's Bay 56 36 1 93 19 Alum Cliffs 38 50 0 88 20 Dennes Point 50 50 2 102 21 Betsy Island 6m 19 81 1 101 22 Betsy Island 13m 19 75 4 98 23 Dart Island 45 0 0 45 24 Stewart's Bay A 21 86 0 107 25 Stewart's Bay B 63 37 2 102 26 Fortescue Bay 6 477 5 488 27 Marion Bay 133 4 0 137 28 Reidle Bay 2 96 0 98 29 Stapleton Point 104 1 0 105 30 Painted Cliffs 68 19 0 87 31 Shelly Beach 56 4 0 60 32 Coles Bay 75 27 0 102 33 Bicheno 0 12 0 12 34 Skeleton Bay 0 52 0 52 35 Stumpy's Rocks 0 86 2 88 36 Sth. Crappies Point 80 16 19 115 37 Low Head 83 9 10 102 38 Green's Beach 74 4 16 94 39 Rocky Cape 74 7 21 102 40 Cowrie Point 75 7 20 102 41 Trousers Point 111 26 7 144 42 Point Franklin 62 208 20 290 43 Point Henry 493 4 7 504 44 Point Lilias 454 1 3 458 45 Tablerock Point 147 32 33 212 46 Point Cook -B 1 512 38 38 588 47 Point Cook -B2 451 44 33 528 48 Point Cook -K1 348 41 36 425 49 Point Cook -K2 381 59 25 465 190

Appendix 2b Morph data for all sites. See Chapter 2 for explanation of morph codes.

Site Site RP · RPG RW WG WPG WP ww n 1 Pelican Island 3 18 0 6 63 10 0 100 2 Blubber Heads 1 1 45 0 1 1 27 5 1 100 3 Roaring Beach 6m 15 41 1 1 1 26 7 4 105 4 Roaring Beach 15m 15 58 0 21 23 12 4 133 5 Satellite Island A 17 69 0 7 4 3 1 101 6 Satellite Island B 10 41 0 10 25 13 7 106 7 Ling Reef 3m 112 239 0 81 54 28 12 526 8 Ling Reef 13m 80 217 0 63 1 01 50 9 520 9 Ling Reef Slope 10 45 0 17 24 3 2 101 1 0 Ling Reef Barren 24 54 0 12 9 3 2 104 11 Gordon 18 40 0 12 26 5 2 103 1 2 Coningham Spine colour data not available. 1 3 Tinderbox 3m 64 214 1 47 220 35 23 604 14 Tinderbox 7m 11 6 262 6 46 108 39 27 604 15 Tinderbox Slope 12 43 0 15 14 7 3 94 1 6 Tinderbox Barren 19 43 0 10 15 5 2 94 17 Tinderbox Beach 71 213 1 74 93 35 10 497 18 Blackman's Bay 19 37 0 3 27 5 2 93 1 9 Alum Cliffs 13 25 0 12 26 12 0 88 20 Dennes Point 12 38 0 14 31 7 0 102 21 Betsy Island 6m 6 13 0 30 45 5 2 1 01 22 Betsy Island 13m 3 ; 6 ; 31 34 ; ; 2 98 23 Dart Island ; ; 32 2 0 0 0 0 45 24 Stewart's Bay A 4 17 0 23 57 5 1 107 25 Stewart's Bay B ; ; 51 ; 9 29 0 1 102 26 Fortescue Bay 0 2 0 46 329 9 2 388 27 Marion Bay 60 60 5 0 2 2 0 129 28 Reidle Bay l 1 0 24 70 1 1 98 29 Stapleton Point 47 55 2 ; 0 0 0 105 30 Painted Cliffs 28 37 3 0 18 ; 0 87 31 Shelly Beach 25 31 0 0 2 1 ; 60 32 Coles Bay 27 45 3 0 20 7 0 102 33 Bicheno Spine colour data not available. 34 Skeleton Bay 0 0 0 16 35 0 1 52 35 Stumpy's Rocks 0 0 0 12 75 ; 0 88 36 Sth. Crappies Point 32 45 3 13 5 15 2 115 37 Low Head 25 52 6 8 4 6 1 102 38 Green's Beach 27 46 1 7 5 7 ; 94 39 Rocky Cape 29 43 2 1 1 9 8 0 102 40 Cowrie Point 30 42 3 14 3 10 0 102 41 Trousers Point Spine colour data not available. 42 Point Franklin " 43 Point Henry " 44 Point Lilias II 45 Tablerock Point " 46 Point Cook -B 1 " 47 Point Cook -B2 " 48 Point Cook -K1 II 49 Point Cook -K2 " 1 91

Appendix 3 Environmental data for all sites. w data not collected Algal %Algal Urchin Site No. Site Name A.E.I. DeQt h Shelter Cover Cover Density: 6.80 1 Pelican Island 5 3.0 5 3 65.50 2 Blubber Heads 4 7.0 4 1 3 Roaring Beach 6r 4 6.0 3 3 4 Roaring Beach 1! 4 15.0 3 1 6.50 10.60 5 Satellite Island A 1 3.5 3 1 6 Satellite Island B 3 7.0 5 4 7 Ling Reef 3m 3 3.5 3 2 3.20 8 Ling Reef 13m 3 13.0 2 1 0.00 3.60 9 Ling Reef Slope 3 7.0 4 1 0.00 4.20 10 Ling Reef Barren 3 2.5 2 1 0.00 6.60 11 Gordon 1 4.0 3 3 97.60 5.10 12 Coning ham 2 3.0 4 3 13 Tinderbox 3m 3 3.5 4 4 92.50 3.20 14 Tinderbox 7m 3 7.0 3 1 34.80 13.00 15 Tinderbox Slope 2 3.5 2 1 8.50 12.80 16 Tinderbox Barren 2 3.0 2 1 17 Tinderbox Beach 1 3.5 1 3 51 .80 7.80 18 Blackman's Bay 3 4.0 4 4 19 Alum Cliffs 3 4.0 4 2 15.00 1.80 20 Dennes Point 4 6.5 4 4 88.50 2.80 21 Betsy Island 6m 5 6.5 4 2 14.50 4.80 22 Betsy Island 13m 5 13.0 3 2 19.80 2.80 23 Dart Island 1 2.0 4 2 24 Stewart's Bay A 3 4.0 5 4 25 Stewart's Bay B 2 3.0 4 4 26 Fortescue Bay 4 4.0 5 4 89.50 2.00 27 Marion Bay 1 2.0 1 3 28 Reidle Bay 5 8.0 5 4 29 Stapleton Point 1 4.0 2 1 30 Painted Cliffs 1 5.0 4 3 31 Shelly Beach 2 4.0 4 3 32 Coles Bay 2 3.0 4 3 77.30 6.00 33 Bicheno 6 10.0 5 3 34 Skeleton Bay 5 5.0 5 4 64.50 1.80 35 Stumpy's Rocks 4 3.0 3 3 36 Sth. Crappies Po 3 5.0 4 3 82.50 0.80 37 Low Head 4 3.0 4 3 78.50 0.80 38 Green's Beach 3 2.0 4 4 39 Rocky Cape 3 5.0 4 3 50.30 5.60 40 Cowrie Point 3 3.0 4 3 76.00 1.60 41 Trousers Point 2 6.5 4 3 42 Point Franklin 2 7.0 5 3 43 Point Henry 1 2.0 1 3 44 Point Lilias 1 1.0 2 2 45 Table rock Point 2 4.0 4 3 46 Point Cook WB 1 2 4.0 3 1 47 Point Cook WB2 2 4.0 3 1 48 Point Cook WK1 2 3.0 3 3 49 Point Cook WK2 2 3.0 3 3 Appendix 4a Means, standard errors and sample sizes for morphometric and meristic variables, for red and white dermis urchins and pooled data from Tinderbox.

Red urchins {n = 29) White urchins {n = 30} Total {n =59) Measurement X s.e. X s.e. X s.e. Live weight 146.04 15.948 202.36 18.276 174.68 12.607 Test/spines dry weight 18.97 2.216 23.28 2.133 21 .1 6 1.550 Lantern dry weight 1.67 0.151 2.12 0.156 1.89 0.112 Test diameter 68.86 2.462 77.91 2.363 73.46 1.792 Test height 33.88 1.570 38.70 1.474 36.33 1.113 Test thickness 1.25 0.064 1 .31 0.044 1.28 0.038 Diam. of apical system 13.29 0.41 4 14.63 0.423 13.97 0.306 Diam. of peristome 5.73 0.203 6.46 0.241 6.09 0.163 Diam. of madreporite 6.49 0.229 7.17 0.225 6.83 0.165 Diam. of periproct 19.22 0.479 21 .10 0.481 20.18 0.358 _.. c.o Max. width ambulacrum 16.45 0.520 18.33 0.363 17.41 0.336 1\) Max. width interambulacrum 25.59 1.038 28.53 1.078 27.08 0.767 No. pore pairs at ambitus 6.90 0.076 7.03 0.058 6.97 0.048 Max. tooth length 13.87 0.477 15.24 0.451 14.56 0.338 Max. tooth width 6.54 0.210 7.1 1 0.188 6.83 0.144 No. plates in ambulacrum 25.90 0.584 27.67 0.684 26.80 0.462 No. plates in interambulacrum 18.55 0.353 19.63 0.446 19.10 0.292 Length of longest spine 24.80 0.725 27.39 0.498 26.12 0.465 Max. diam. of lonqest spine 1.84 0.038 1.92 0.037 1.88 0.027 Appendix 4b Means, standard errors and sample sizes for morphometric and meristic variables, for red and white dermis urchins and pooled data from Ling Reef. ..

Red urchins (n = 21) White urchins (n = 26} Total (n = 47} Measurement X s.e. X s.e. X s.e. Live weight 113.37 14.316 95.27 11.137 103.36 8.881 Test/spines dry weight 13.19 1.616 11.47 1.285 12.24 1.01 0 Lantern dry weight 1.47 0.142 1.38 0.138 1.42 0.099 Test diameter 64.73 2.941 60.14 2.650 62.19 1.976 Test height 31 .18 1.560 28.82 1.482 29.88 1.078 Test thickness 0.86 0.048 0.88 0.036 0.87 0.029 Diam. of apical system 12.77 0.530 12.12 0.456 12.40 0.345 Diam. of peristome 5.67 0.250 5.28 0.21 1 5.45 0.162 Diam. of madreporite 6.02 0.274 5.72 0.246 5.85 0.182 Diam. of periproct 19.43 0.628 18.43 0.626 18.88 0.447 Max. width ambulacrum 15.29 0.578 14.23 0.558 14.70 0.406 ...... tO Max. width interambulacrum 23.95 1.245 22.00 1.055 22.87 0.81 0 w No. pore pairs at ambitus 6.91 0. 066 6.65 0.235 6.77 0.062 Max. tooth length 13.44 0.525 12.75 0.521 13.06 0.371 Max. tooth width 6.62 0.302 6.1 3 0.239 6.35 0.190 I i No. plates in ambulacrum 24.38 0.681 23.31 0.683 23.79 0.487 I { No. plates in interambulacrum 17.48 0.400 16.77 0.382 17.09 0.278 Length of longest spine 25.62 0.732 23.98 0.880 24.71 0.593 Max. diam. of lonqest spi ne 1.64 0.055 1.63 0.056 1.63 0.039 1 94

Appendix 4c Means, standard errors and sample sizes for morphometric and meristic variables, for Fortescue Bay urchins. N.B. Only white dermis urchins occur at Fortescue Bay.

30 (n = ) Measurement X s.e. Live weight 239.73 16.065 Test/spines dry weight 27.93 1.765 Lantern dry weight 2.47 0.139 Test diameter 86.70 2.372 Test height 39.88 1.272 Test thickness 1.20 0.030 Diam. of apical system 15.60 0.398 Diam. of peristome 6.85 0.191 Diam. of madreporite 7.94 0.237 Diam. of periproct 22.64 0.473 Max. width ambulacrum 19.57 0.449 Max. width interambulacrum 32.97 1.036 No. pore pairs at ambitus 6.83 0.069 Max. tooth length 15.95 0.376 Max. tooth width 7.69 0. 174 No. plates in ambulacrum 28.90 0.471 No. plates in interambulacrum 20.17 0.332 Length of longest spine 26.95 0.495 Max. diam. of longe st spi ne 1.94 0.040 Appendix 4d Means, standard errors and sample sizes for morphometric and meristic variables, for red and white dermis urchins and pooled data from Cowrie Pt.

Red urchins {n = 1 0} White urchins {n = 1 0} Total {n = 20} Measurement X s.e. X s.e. X s.e. Live weight Test/spines dry weight Lantern dry weight 2.76 0.107 2.47 0.196 2.62 0.1 14 Test diameter 77.05 2.313 73.58 2.466 75.32 1.693 Test height 42.45 1.442 38.04 1.758 40.25 1.21 7 Test thickness 1 .11 0.038 1.1 8 0.068 1 .14 0.039 Diam. of apical system 14.01 0.514 12.95 0.427 13.48 0.347 Diam. of peristome 6.48 0.253 5.82 0.21 1 6.15 0.177 Diam. of madreporite 7.05 0.335 6.38 0.231 6.72 0.21 3 Diam. of periproct 21 .58 0.572 20.28 0.519 20.93 0.404 Max. width ambulacrum 18.00 0.394 17.50 0.401 17.75 0.280 ..... (!) Max. width interambulacrum 28.90 1.100 27. 10 0.994 28.00 0.750 (.11 No. pore pairs at ambitus 7.00 0.000 6.90 0.100 6.95 0.050 Max. tooth length 16.34 0.263 15.60 0.51 4 15.97 0.294 Max. tooth width 7.75 0.175 7.32 0.256 7.54 0.158 No. plates in ambulacrum 29.80 0.593 28.90 0.674 29.35 0.449 No. plates in interambulacrum 20.60 0.427 20.10 0.277 20.35 0.254 Length of longest spine 22.72 0.665 21 .39 0.900 22.06 0.566 Max. diam. of lonqest spine 1.85 0.062 1.75 0.040 1.80 0.038