THE UNIVERSITY LIBRARY

PROTECTION OF AUTHOR ’S COPYRIGHT

This copy has been supplied by the Library of the University of Otago on the understanding that the following conditions will be observed:

1. To comply with s56 of the Copyright Act 1994 [NZ], this thesis copy must only be used for the purposes of research or private study.

2. The author's permission must be obtained before any material in the thesis is reproduced, unless such reproduction falls within the fair dealing guidelines of the Copyright Act 1994. Due acknowledgement must be made to the author in any citation.

3. No further copies may be made without the permission of the Librarian of the University of Otago.

August 2010 Genetics of the snake star Astrobrachion constrictum ( Ophiuroidea: Asteroschematidae) in Fiordland, New Zealand

Debbie Steel

~-

'.>

A thesis submitted for the degree of Master of Science i' in Marine Science at the University of Otago, Dunedin, ~ New Zealand

"-..

>-

April 1999 Abstract

This thesis was initiated to address two of the questions posed by Stewart's 1995

study on the biology of Astrobrachion constrictum; Is gene flow between fiords

restricted in this ophiuroid species and are the colour morphs as different genetically

as they are visually? Specimens of A. constrictum were collected from four sites

within Doubtful Sound and one site in each of Nancy Sound, Preservation Inlet and

7 Chalky Inlet. The genetics of these populations of the snake star were assessed using

mitochondrial DNA and allozyme analysis. These results were analysed using G-test and AMOVA. Both G-test and AMOV A anlaysis showed that there was no

significant genetic differentiation between populations within the same fiords (G[21J =

11.97; P > 0.9) (Fsc = -0.037, P > 0.05) or among fiords (G[21J = 23.32; P > 0.2) (FsT = 0.013, P > 0.05). This result was unexpected as the Fiordland environment appears to

present several barriers to dispersal, and genetic differentiation of Fiordland

populations has been demonstrated in other echinoderm species. Genetic differences

between the five colour morphs of A. constrictum were also assessed using

mitochondrial DNA and allozyme analysis. G-test and AMOVA analysis of these

:~ results also showed no significant differences between colour morphs (G[lSJ = 13.88; P

> 0.9) (FsT = -0.089, P > 0.05) indicating that they are conspecific. While studying the population genetics of the snake star two individuals were discovered which

> appeared to be heteroplasmic. Through the use of PCR cloning, SSCP and sequencing, the presence of heteroplasmy within these two individuals was demonstrated. This is

the first reported case of heteroplasmy in an ophiuroid species and probably arose - through paternal leakage. Cytochrome Oxidase I sequence obtained from the population analysis was also used in conjunction with sequences from Genbank to

;,- assess the phylogeny of echinoderms. Several studies have addressed echinoderm

phylogeny over the past century but have failed to clarify the relationship between the echinozoa ( echinoids and holothuroids ), ophiuroids and asteroids. A relatively

fast evolving gene was used in this study in an attempt to clarify this relationship. Of the nineteen trees generated with PAUP (Swofford, 1993), only one gave a topology

11 indicating monophyly for all five classes. This was trimmed and compared with three previous hypotheses using the Kishino-Hasegawa test. Results from this showed no significant difference between hypotheses. This is probably due the large amount of noise introduced into the study through the use of a rapidly evolving gene.

7

'l

:,

,~

I>-...,

}-

ill Acknowledgements

There are just a few people who I would like to thank for all their effort in helping me

with this wee project of mine.

First, my supervisors, Dr Brian Stewart, Dr Graham Wallis and Dr Steve Trewick,

7 thanks for all your patient guidance and words of wisdom over the years. ,,

Thanks to those who helped with the collection of all my wonderful stars, and made

trips into Fiordland an interesting and fun time, Paul Meredith, Les Raj and Brian. A

special thanks to Pete Stratford who did most of the collecting while I sorted myself

out underwater! Thanks also to Jo and Nicki for help in fetching my samples from the

deep south.

Big thanks to people who helped me in the lab, Karen I would have been lost without

you, Marty you made the lab meetings almost fun to attend and PAUP easy .... .ish to

understand!! Steve, without you pushing me all the way I doubt I ever would've ,. finished.

Finally thanks to the Warrender St crew and Mikey who housed me during my write

up, Susan that massage was fantastic, and a big thanks to Harne. ;,_

'--< Cheers!!!

;,-

IV Table of Contents

List of Tables Page No ...... • .• ...... • ...... •...... • . .. · · · · · · ·· · Vil

List of Figures ...... v111

List of Abbreviations ...... IX

Chapter One: Introduction...... 1

Methodology ...... 5

Chapter Two: Population genetics of the sea snake star Astrobrachion constrictum in Fiordland New Zealand...... 7 Introduction ...... 7 Materials and Methods ...... 11

Sample collection and storage ...... 11 DNA Analysis ...... 13 Allozyme Electrophoresis ...... 16 Results...... 19 " DNA Analysis ...... 1 9 Allozyme Analysis ...... 2 4

;> Discussion ...... 25

~ c, Chapter Three: Evidence of Germ-line Heteroplasmy in the Mitochondrial DNA of the brittle star Astrobrachion ~>----< constrictum (Ophiuroidea: Asteroschematidae ) ...... 28 Introduction ...... : ...... 28 Materials and Methods ...... 3 0 Results...... 32 Discussion...... 35

V Chapter Four: Echinoderm Phylogeny ...... 37 Introduction ...... 37 Materials and Methods ...... 39 Results...... 42 Discussion...... 56

Chapter Five: General Discussion...... 58 References ...... 61 AppendixA: Collection Information...... 76 Appendix B: Buffer and Stain Recipes...... 77 Appendix C: Enzymes Assessed ...... 79

\>

c, ... t,

Vl List of Tables

,) Page No.

Table 2.1: Summary of haplotype data. 19

Table 2.2: PGI allele frequency results for fiords. 24

') Table 4.1: Species of echinoderm used in this analysis. 40

,), Table 4.2: Summary of relationship between data and weighting

schemes used and which analyses were conducted. 41

'I Table 4.3: Distance information for the reduced taxa list. 43

" Table 4.4: Results from Kishino-Hasegawa tests. 53 Table 4.5: Results from permutation tests and analysis of random trees. 54

;,

~,,,. •·.

;·';,- .,

vii List of Figures

Page No. Figure 1.1: Striped colour morph of Astrobrachion constrictum. 4 Figure 2.1: Estuarine-like circulation pattern within Fiordland. 9 Figure 2.2: Map of the Fiordland area, South Island, New Zealand. 12 '! Figure 2.3: Region of ophiuroid mitochondrial genome showing ,_, location of amplified segments. 13 Figure 2.4: SSCP gel showing haplotypes A, B, D, E and F. 20 'f ., Figure 2.5: Sequence and amino acid data for the eight COI haplotypes. 21 ., Figure 2.6: Minimal spacing network constructed from sequence data. 22 Figure 3 .1: SSCP gel showing heteroplasmic individual and haplotypes. 33

'.• Figure 3.2: Sequence and amino acid data for the three haplotypes present in the two heteroplasmic individuals. 34 Figure 4.1: Echinoderm phylogenies proposed by previous authors. 38 Figure 4.2: Base by base transitions and transversions calculated from the tree in Fig. 4.4. 42

',, Figure 4.3: Percentage of steps by codon from the tree shown in Fig 4.4. 44 Figure 4.4: MP tree from analysis of the full taxa list data set with non-synonymous weighting. 45 Figure 4.5: Bootstrap analysis of the parsimony tree shown in Fig 4.4. 46 Figure 4.6: An example of the most commonly produced topology ':- from parsimony analysis. 47 Figure 4.7: Tree trimmed from the tree shown in Fig. 4.4. 48

(' Figure 4.8: NJ tree produced from analysis of the full taxa list, unweighted. 49 '} Figure 4.9: Bootstrap of the tree shown in Fig. 4.8. 50 ..::-',"-<( Figure 4.10: NJ tree produced from analysis of the full taxa list j>....t weighted for non-synonymous changes. 51 Figure 4.11: ML analysis ofreduced taxa list weighted for -, non-synonymous changes. 52 Figure 4.12: DNA sequence variation for the region of COI assessed for different levels of taxonomy. 55

Vlll List of Abbreviations

mtDNA mitochondrial DNA

COI Cytochrome Oxidase subunit I con Cytochrome Oxidase subunit II -" SSCP Single Stranded Conformational Polymorphisms

·j' RFLP Restriction Fragment Length Polymorphism

'/ TBE Tris Borate EDTA r

-1 TEMED Tetramethy1-ethy lene-diamine AMOVA Analysis of Molecular Variance

CAEA Citric acid-aminopropyl morpholine

HK Hexokinase

PGI Phosphoglucose isomerase

PEP Peptidase

SOD Superoxide dismutase cs Continental Shelf PE Perkin Elmer NJ Neighbour Joining

MP Maximum Parsimony ·.i ML Maximum Likelihood

BS Bootstrap

\• CI Consistency Index •. ,. RI Retention Index ;,.,.., "~ Ti/Tv Transition/Transversion ..

·;:.·;r

.J

IX Chapter One

Chapter One: Introduction

The phylum Echinodermata is one of the best characterised and most distinctive phylum of the Animal Kingdom (Hyman, 1955). Species of this marine phylum are recognised by the _\

"! presence of pronounced radial symmetry, the notable absence of either head or posterior

7 end and the presence of a water vascular system (Hyman, 1955; Nichols, 1962; Lawrence, 1987). Traditionally, five living classes of Echinodermata are recognised; Crinoidea (sea r lilies and feather stars); Holothuroidea (sea cucumbers); Echinoidea (sea urchins and sand '( dollars); Asteroidea (sea stars) and Ophiuroidea {brittle stars and basket stars) (Hyman, 1955; Nichols, 1962; Lawrence, 1987). A sixth class, Concentricycloidea, has also been proposed (Baker et al., 1986). The phylogenetic relationship of these classes has been . I studied over the past few years but is still not entirely clear today (Smith et al., 1993; Wada and Satoh, 1994).

Ophiuroidea is derived from the Latin words ophis (snake) and ura (tail) in reference to the snake-like appearance of the star's arms (Hyman, 1955). They are diverse and widespread '.> with some 250 described genera and 2000 species (Smith and Paterson, 1995). There are two orders within the class Ophiuroidea; Euryalida and Ophiurida (Hyman, 1955; Smith

·; and Paterson, 1995). Of these, euryalids are thought to be the most primitive and least

> studied order (Baker, 1980). They mainly exist in deep waters and are therefore predominantly known only from dredged material (Hyman, 1955). There are four families I'

• j within the Euryalida; Asteronychidae, Asteroschematidae, Gorgonocephalidae and ,_, Euryalidae (Baker, 1980; Smith and Paterson, 1995). Astrobrachion constrictum 1s a "'~ member of the family Asteroschematidae (Baker, 1980). "'

• r Astrobrachion constrictum is found off the south-east coast of Australia from Tasmania to New South Wales, around Lord Howe Island and around New Zealand, from Fiordland to North Cape (Baker, 1980). As with other euryalids, it is normally a deep water species, existing at depths between 50 and 180 m (Baker, 1980) which makes collection of specimens difficult. Difficulty in collection has been suggested as one of the main reasons

1 Chapter One

why euryalids are one of the least studied orders of ophiuroids (Baker, 1980). This is perhaps also true for A. constrictum as until a recent study by Stewart (1995) little was known about this species. As well as assessing several aspects of the brittle star's biology, Stewart's study posed several questions. This study was initiated to answer two of those \ questions: To what degree, if any, are fiord populations of the brittle star genetically isolated? and are the colour morphs of the brittle star as different genetically as they appear !,

"/ visually?

'!

Miller (1997) showed genetic differentiation among populations of the black coral -; Antipathes jiordensis within a fiord. This study also found slight significant differentiation . I among fiords and between fiord populations and a population on Stewart Island. These >' results are supported by the Mladenov et al. (1997) study of the sea urchin, Evechinus chloroticus which also found slight genetic differentiation between the Doubtful Sound population and other populations around the New Zealand coastline. Although this study did not make a comparison among fiord populations recent studies on Evechinus chloroticus (Wing and Skold, in prep) and the starfish Coscinasterias muricata (Skold et al., in prep) have showed slight genetic differences among populations in different fiords .. This study utilises allozyme electrophoresis and mitochondrial DNA (mtDNA) analysis to assess the degree of genetic differentiation between populations of the brittle star,

'.> Astrobrachion constrictum, within Fiordland, New Zealand. Results from this study will add to our knowledge on the dynamics of gene flow within Fiordland. It was hypothesised > that, due to the expected short larval period of the brittle star and the isolating nature of Fiordland's geography and hydrology, gene flow among fiords would be limited. " r;

-H It was also possible to address the question of genetic differentiation between colour "~ morphs by the above methods. Both the external and the internal anatomy of the colour morphs of Astrobrachion constrictum are identical and they appear to occupy the same . ' habitat (Stewart, 1995), indicating that they are variants of the same species. Several other studies of marine invertebrates have shown that near identical organisms are so significantly different on a molecular level as to warrant being identified as separate species (Grassle and Grassle, 1979; Hedgecock, 1979; Kwast et al., 1990). It was hypothesised that different

2 Chapter One

colour morphs of A. constrictum are conspecific, and should not show any correlation with haplotype frequencies.

While analysing the mtDNA of sampled Astrobrachion constrictum two individuals were discovered which appeared to be heteroplasmic. Mitochondrial heteroplasmy, either due

~- to tandem repeats or biparental inheritance, has been observed in several different taxa ·, including mussels (Zouros et al., 1994), rabbits (Biju-Duval eta!., 1991), spruce budworms

"/ (Sperling and Hickey, 1994), elephant seals (Hoelzel et al., 1993) and several different 1' r' species of fish (Jorstad et al., 1994; Miracle and Campton, 1995). However, it has only

( been observed once before in echinoderms (De Giorgi, 1988). Therefore, the possibility of ,,. the presence of heteroplasmy within this species of echinoderm was assessed. A novel approach, using PCR cloning, SSCP and sequencing was used to determine the presence of

, I heteroplasmy.

Finally, echinoderm phylogeny has been studied extensively throughout the past century by a variety of techniques. The latest of these studies (Littlewood et al., 1997) suggested that the failure of previous genetical studies to resolve phylogeny is a result of the rapid radiation of echinoderms. As these studies used relatively slowly evolving genes it was thought that a more rapidly evolving gene such as Cytochrome Oxidase I (COI) may be " able to better resolve phylogeny. Chapter four of this thesis used COI sequence obtained

> from the population study in conjunction with sequences from GenBank to analyse the

phylogeny of echinoderms. ,,

·; .,,.., These studies are presented in this thesis as three separate chapters. The population study ... and the heteroplasmy analysis ( chapters two and three) have been prepared for submission to journals for publication.

3 ·(p10Jimis

Jg:J.gd JO Asgµno::, O:J.Ol[d) ·utnJ;JJ.J.JSUO;J UOJl,f:JV.J.qo.J.JSV JO l[WOUI mo10::, pgdpis : rI ;}.lll~!.tl

_,

I ,

y Chapter One

Methodology

Before the development of isozyme electrophoresis, morphological and behavioural

mutants were the only means of analysing genetic variation (Avise, 1994). In 1966, isozyme electrophoresis was shown to be a simple and relatively fast way of gathering - " Mendelian data (Avise, 1994). Since then, this method has been used worldwide for the analysis of populations, species boundaries and phylogenetics (Richardson et al., 1986; '! Avise, 1994). Isozyme electrophoresis has been used extensively in population studies of

/ benthic marine invertebrates, including many species of echinoderms. These studies have analysed a range of species with varying modes of reproduction and scale of geographical -1 distribution (Ward and Andrew, 1995; Edmunds et al., 1996; Nishida and Lucas, 1988; Hensley et al., 1995; Hess et al., 1988). Recent developments in DNA analysis techniques

- I have permitted a shift away from isozyme electrophoresis to direct analysis of DNA " (Avise, 1994). These methods, however, have not changed the fact that 1sozyme electrophoresis is a simple, relatively cheap and fast method of determining variation in

natural populations (May, 1992; Murphy et al., 1990).

Since the development of the Polymerase Chain Reaction (PCR) procedure, the number of evoiutionary studies involving DNA analysis have increased dramaticaily. A large number of these studies have used mtDNA to assess population structure, gene flow, phylogenetic

> relationships, and biogeography (Moritz et al., 1987). Several population studies have incorporated both mtDNA and protein analysis. Some of these have shown similar results ;c from both types of analysis (Gardner and Skibinski, 1991; Edmunds et al., 1996), while

.,, others have shown remarkably different results (Burton and Lee, 1994; Foltz et al., 1996) . l • Several factors may be responsible for these differing results; smaller effective population size due to maternal mode of inheritance and haploid nature of the mitochondrial genome; physical independence from the nuclear genome; weak selection acting on mtDNA proteins and relatively rapid rate of evolution due to inefficiency of repair (Barton and Jones, 1983; Wilson et al., 1985; Harrison, 1989). Therefore, it is often useful to assess both allozyme and mtDNA variation when carrying out a population study.

5 Chapter One

The mitochondrial genome of most species consists of twenty two tRNAs, thirteen protein coding genes, two rRNAs and a non-coding region, each with a differing evolutionary rate (Moritz et al., 1987). The most quickly evolving of these is the non-coding control region (Moritz et al., 1987) and because of this it is often used in population genetic studies.

,::,\ Several of the protein coding genes are also frequently used in population genetic analysis, including COL For example, Burton and Lee (1994) used COI analysis in conjunction with allozyme and nuclear analysis to show a phylogeographic break in populations of the copepod Tigriopus californicus, and Sperling and Hickey (1994) used COI to assess

7' intraspecific variation over a large geographic distribution for various species of budworm. ., A recent analysis of ten conserved insect COI primers by Lunt et al. (1996) showed that different regions of the insect COI gene evolve at different rates. It was found that the 5' "" end of COI was faster evolving than the middle or 3'end, although this will probably vary 7 amongtaxa.

..> Several screening techniques have been developed to increase the efficiency of DNA analysis across large sample sizes (Lessa and Applebaum, 1993). These techniques include, heteroduplex analysis, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis and single-stranded conformational polymorphism (SSCP) (Lessa and ~> Appiebaum, 1993). SSCP is a simple and relatively inexpensive method for discovering > sequence variation at a chosen locus (Lessa and Applebaum, 1993). This technique has

predominantly been used in human genetic studies (Strautnieks et al., 1992; Newcombe et

~"-..,. al., 1997; Tanaka et al., 1997) but recently the utility of this technique for population '> ·., studies of other species has been demonstrated (Ostellari et al., 1996; Li and Hedgecock,

i .. 1998).

,f· 1'·'

:,.

......

6 Chapter Two

Chapter Two: Population Genetics of Astrobrachion constrictum (Ophiuroidea: Asteroschematidae) within . ~ Fiordland, New Zealand.

Introduction ·,

7 Long, deep coastal fiords have the potential for harbouring discrete marine populations, particularly for species with limited dispersal ability. There have been studies assessing / genetic variability between Scandinavian fjords and open ocean populations but few " assessing differentiation of populations among fjords. Herring (Clupea harengus L.) in a ·., 'I- Norwegian fjord were shown to be genetically different from populations in the Atlantic

·, ··" outside the fjord (Jorstad and Pedersen, 1986, cited in (Jorstad et al., 1994)) but contained some allozyme alleles identical to the more common alleles present in Pacific populations >> of the herring species Clupea pallasi (Jorstad et al., 1994). Isozyme studies of Zoarces (eelpout) have shown a cline between inner and outer fjord populations for morphological structures and certain alleles (Christiansen et al., 1984; Christiansen et al., 1988;

[,. Frydenberg et al., 1973).

't --~ Mladenov et al. (1997) showed slight genetic differentiation between the Doubtful Sound

/ population of the sea urchin Evechinus chloroticus and other populations around the New

:, Zealand coastline but did not compare populations from different fiords. This result has ;-,1.. been supported by recent unpublished allozyme data for the starfish Coscinasterias muricata (Skold et al., in prep) and E. chloroticus (Wing and Skold, in prep) which both

~,t~ showed slight genetic differentiation among fiords. Miller (1997) showed significant t-;.;\ genetic differentiation among populations of the black coral Antipathes fiordensis within fiords, and also slight differentiation a.rnong fiords and between fiord populations and a

·.? population on Stewart Island. ;,

;,·}' Several studies on the population genetics of benthic marine invertebrates, including echinoderms, have sought to clarify the relationship between larval type, gene flow and genetic variation within species. Most have confirmed a relationship between larval type

7 Chapter Two

and the differences in genetic structure of separate populations. Populations of species with long living planktonic larvae tend to be genetically homogeneous (Selander et al., 1970; Benzie and Stoddart, 1992; McMillan et al., 1992; Stickle et al., 1992), whereas species

. ~ with benthic developing larvae or short-lived planktonic larvae are genetically more diverse (Gaines et al., 1974; Day and Bayne, 1988; McMillan et al., 1992; Stickle et al., 1992).

Some studies, however, have shown the reverse to be true (Palumbi, 1995; Hess et al., ~'

:;,- 1988). Several factors could be responsible for this pattern, including differential selection ·, , pressure on adults and larvae (Ayre, 1990; Burton and Feldman, 1982; Koehn et al., 1980), ·, larval behaviour (Burton and Feldman, 1982; Helmuth et al., 1994), geological history (Voight, 1988) and strength and direction of current flow (Benzie and Stoddart, 1992). '.( Therefore, while in most studies a direct correlation between the length of larval period and ;':;, the genetic variation of populations can be made, in other studies the situation is more

,'" complex.

: > The south-west coast of New Zealand comprises fourteen glacial fiords extending from Preservation Inlet in the south to Milford Sound approximately 200 km to the north (Augustinus, 1992). Although information on early glacial activity in this area is lacking due to intense erosion during subsequent stages (McKellar and Soons, 1982), speleotherm

~ ."', deposits in the Aurora-Te Ana-Anau caves suggest five glacial events in the last 180,000 years (Williams, 1982). The last of these events is thought to have been between 10,000 •' and 18,000 years before present (Pillans et al., 1992; Pickrill et al., 1992). Associated with

·;·,) the end of this final glacial period was a rise in sea level of between 110 and 120 m (Pillans et al., 199'.f), stabilising at today's level around 6500 years before present (Pickrill et al., :) 1992). ~-..

"t~);,i.,

;r- Due to the mountainous nature of Fiordiand and its exposure to prevailing westerly

'? weather systems, the region has a very high amount of rainfall (Stanton and Pickard, 1982).

;r The resulting freshwater runoff forms a low salinity surface layer in the fiords (Stanton and Pickard, 1981), creating an estuarine-like circulation system in the upper layers of the fiords (Fig 2.1 ). The surface layer flows seaward entraining water of higher salinity from beneath, driving this lower layer towards the head of the fiord (Gamer, 1964; Stanton and

8 Chapter Two

Pickard, 1981; Stanton, 1978; Stanton, 1986). Sea water below sill depth lacks direct communication with the open ocean which can result in extended periods of isolation. Replacement of this water can only occur when coastal water at or above sill depth is '"~ denser than the deep water of the fiords (Stanton, 1986; Stanton and Pickard, 1981). There is still some debate as to how often deep water renewal occurs. <-.. \

77

Low salinity• layer • • • ,''

Deep Water

1>

:>-

> Figure 2.1: Estuarine-like circulation pattern within Fiordland. The surface layer flow ·; seaward entraining water from beneath, causing this layer to flow towards the head of the •;, 1 fiord. ' :1 ,,~ High levels of humic acids from soil runoff are contained within the low salinity surface "'"' layer (Corson, 1999). Their presence lowers light penetration to deeper water within the ~·

.,' fiord (Grange et al., 1981). Other 'deep' water conditions such as low current and wave

;r action are also promoted within the fiords due to their sheltered nature (Grange et al., ·. I 1981). These factors combine to allow species that normally occupy deep offshore

habitats, such as the snake star Astrobrachion constrictum, to exist within the fiords

(Grange et al., 1981).

9 Chapter Two

The snake star lives exclusively on the antipatharian black coral, Antipathes fiordensis

(Stewart and Mladenov, 1997) in a mutualistic relationship (Vasques, 1997). It displays

•\ five colour morphotypes (Baker, 1980; Grange et al., 1981), dark red, dark red with stripes, dark red with spots, yellow, and pale with red stripes although graduations in pattern are '' evident in some individuals (Stewart and Mladenov, 1997). Although Stewart's (1995) ·y study was unable to determine conclusively the type of larval development employed by this animal he suggested that development was lecithotrophic (Stewart, 1995; Stewart and ,·,

7 Mladenov, 1993). Females may produce 5000-6000 large (- 415 µm diameter) yolky eggs

which is within the range described by Strathmann and Rumrill (1987) for ophiuroid ·r species with lecithotrophic larval development. Recent observations of early larval ~· ;..• development support this conclusion (Dr Brian Stewart, pers. com., 1998). Lecithotrophic larvae often develop in the pelagic zone but remain there only for a short period of time, typically less than a week (Strathmann and Rumrill, 1987).

Due to the probable lecithotrophic larval phase of Astrobrachion constrictum, the dispersal potential of this species is presumed to be relatively low. The fiord environment may also provide barriers for long range dispersal; estuarine circulation in the upper water layers ,·i...,

> could trap larvae within the fiords, limiting mixing between fiords; the narrow and shallow > entrances to the fiords may limit larval movement into the fiords from populations on the

·J continental shelf. These factors should result in genetic differentiation among fiord , ..i populations of this species. Conversely, the fiords are relatively young, having been •' :1 flooded with water no more than 10,000 years ago (Pillans et al., 1992). This is not n~ sufficient time for the evolution of novel haplotypes but lineage sorting may have occurred . .. 'k

This study was undertaken to assess the level of genetic structure of populations of .! Astrobrachion constrictum within and among fiords, on a scale ranging from a few hundred

' { metres to several fiords apart. It was also possible to examine the degree of genetic differentiation between colour morphs.

10 Chapter Two

Materials and Methods

Sample Collection and Storage Specimens of Astrobrachion constrictum were collected by SCUBA from four sites

~~ (Espinosa Point, Tricky Cove, Oz and Crowded House) in Doubtful Sound, and one in each of Nancy Sound, Chalky Inlet and Preservation Inlet (Fig. 2.2). Several collecting trips were made between Oct, 1996 and Sept, 1998 (Appendix A). Due to the patchy distribution of this species and the rarity of some colour morphs (Stewart and Mladenov, >), 1997) sample numbers vary considerably between sites and between morphs. At all sites except Crowded House and Oz, animals were collected from several different host coral colonies within the dive site. Where possible, animals were taken in groups from the ends of coral branches to minimise damage to the coral colonies. All animals were kept m seawater for transport to the laboratory alive, where they were immediately stored at

-80°C. One hundred and seventeen animals were collected in total.

:;--

CT

;,-1..

;y

,, t~.-'1

' '"'

7

11 Chapter Two

165 180 i:t I NEW ZEALAND I 167 168 Milford Sound

10 40 '" I.

\

:-; 45

165 180

·, \c,_1 Doubtful Sound

461 ~/ 46 Chalky Inlet ? ~

Preservation Inlet {;)fl v--,

.. ,.

4 '*' 166 167 168

1 Figure 2.2: Map of the Fiordland area, South Island, New Zealand. Dots mark collection T

'j locations. Doubtful Sound sites are; 1. Espinosa Point, 2. Tricky Cove, 3. Oz, 4. Crowded House. (Adapted from Stewart 1995, pg. 14)

12 Chapter Two

DNA Analysis

DNA extraction Approximately 50 mg of gonad tissue was dissected from the upper-arm of each specimen

and macerated with 50 µl ofmilliQ water. Samples were placed in a boiling water bath for ;L

·"- approximately 10 mins then centrifuged at 13,000 rpm for 2 mins. The supernatant was \ removed to a clean tube and DNA precipitated with 1120th volume of 5 M NaCl and 2x volume of cold 100% ethanol. The tubes were centrifuged at 13,000 rpm for 10 mins,

-c- )' ethanol removed and the dried DNA dissolved in 30 µl of milliQ water. This DNA

solution was used directly for i>CR.

:,' l A 473 bp fragment of the COI mitochondrial gene was amplified using Cl-J-1718 and Cl­ N-2191 primers (Simon et al., 1994) and a 511 bp fragment of the large ribosomal subunit

(16S) was amplified using insect primers LR-J-12887 and LR-N-13398 (Simon et al., 1994). A 2.5 kb fragment of the genome between COI and Cytochrome Oxidase II (COII) .. > was also amplified with some success using the primers C2-N-3661 and Cl-N-2183

(Simon et al., 1994). (Fig 2.3).

~

IG I 16S IL [ == = ] cm IR E4~ corr I ~ ~ :>·~ (12887) (13398) (1718) (2191) ~;r- ~ •I (2183) (3661)

''Ill-,~

..!.,,b]-t Figure 2.3: Region of the ophiuroid mitochondrial genome showing location of amplified segments. Numbers represent base pair location on Drosophila yakuba genome (Simon et '.) al., 1994).

{

13 Chapter Two

PCR

PCR reactions were carried out using 3.5 mM MgCb, 200 µM each dNTP, 0.25 µM each

primer, 0.025 units of Qiagen© Taq polymerase, lOx Qiagen© buffer in a total volume of 10 l µl (1 µl of template) for SSCP and 25 µl (2 µl of template) for sequencing and Restriction ·"" fragment length polymorphism (RFLP) analysis. The following thermocycling protocol ( ,, was used for amplification: initial denaturation at 94 °C for 1 minute, 40 cycles of "f

denaturation at 94°C for 15 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min,

followed by a further extension at 72°C for 3 mins.

Sequence variation of the COI fragment was surveyed across individuals using single

·>1 stranded conformational polymorphism (SSCP) and representatives of the haplotypes

.~ ~ were sequenced. Representatives of COI haplotypes were also surveyed for 16S using

:, SSCP. The 2.5 kb fragment was used for RFLP analysis.

~ - ~ SSCP

For SSCP, 10 µl PCR reactions were carried out under the same conditions as described

above with an additional 0.05 µl of 33P labelled dATP per reaction. Following PCR, an ·,

> p, equal volume of formamide buffer (95% formamide, 20 mM EDTA pH 8, 0.05% Bromophenol Blue, 0.05% Tylene cyanol) was added to each reaction and denatured at 94°C for 5 mins. Reactions were placed on wet ice and loaded immediately. Four -;;.:i microlitres of sample was applied to a 6% bis-acrylamide (37.5:1 ratio, BioRad premix) gel ' , ,_,.. with 5% glycerol in 0.5x TBE, with 0.25% ammonium persulphate and 1% tetramethyl­

"'. ethylene-diainine (TEMED). Electrophoresis was carried out at 4°C for 200 watt-hours ,, .,,' running at approximately 13 W. Completed gels were iifted from the plates with blotting \>- ·~ paper and dried using a Bio-Rad 583 Gel Dryer. Kodak BioMax™ film was exposed to the

~ dried gels for two to three days. Haplotypes were scored by comparison of renatured ; 7

n, single stranded DNA migration patterns and this information was used to select individuals for sequencing.

14 Chapter Two

Sequencing

One to five representatives of each haplotype were sequenced. Twenty five microlitre

PCR products and a 1 kb Plus DNA ladder (Gibco BRL) were electrophoresed in 2% l agarose gels stained with ethidium bromide. Products of expected length were excised and ;t

'\.,, purified using Prep-A-Gene® (BioRad). Purified PCR product was quantified by

\" electrophoresis with 1 kb Plus DNA ladder (Gibco BRL) on a 2% agarose gel stained with

·7 ethidium bromide. Cycle sequencing was carried out with BigDye™ chemistry according to

.... , '! manufacturer's recommendations (PE Applied Biosystems). The product was precipitated

~ using ethanol/sodium acetate (PE Applied Biosystems) and the sequence read on an ABI377 (PE Applied Biosystems).

>/

~t ) Sequences were aligned by eye and nucleotide differences noted using SeqEd vl.03 (PE,

Applied Biosystems). From this a network showing the most parsimonious relationship " between haplotypes was drawn. Haplotype frequency and sequence information were

>~ analysed using G-tests and AMOVA (Amova 155, Excoffier et al., 1992).

RFLP ,, A 2.5 kb segment of the mitochondrial genome from the 3' end of COI to the 5' end of >- .. COII was successfully amplified and used for RFLP analysis. Twenty five microlitre reactions for representatives of SSCP haplotypes 1, 2, 3, 4, and 5 were obtained by the

above method. This product was then cut with Mael, Tsp509I, Alul, Rsal, Ndell, Cfol, '>'). Dral, Hinfl., Msp and Taql according to Hillis et al. (1996). Restriction digests were run on

:::r- an 0.8 mm thick 8% bis-acrylamide (19:1 ratio, BioRad premix) gel with 5% glycerol in 2x ..._. TBE. A molecular weight marker (1kb ladder, Gibco BRL) was also run to size restriction ., 4-- .. fragments. Electrophoresis was carried out at 70 V for 16 hr. Differences in the resulting banding patterns were noted .

• 1

·;:,- ·;,

15 Chapter Two

Allozyme Electrophoresis Tissue Preparation A portion of gonad and connective tissue was sliced from an arm of each animal and placed in an Eppendorf tube on ice with an equal volume of homogenisation buffer (0.02 M tris HCl, 0.1 % mercaptoethanol, 0.1 % bromophenol blue). The tissue was then homogenised "'.~

\ .). by maceration with a glass rod for approximately 10 seconds to break open cells and

' release proteins. Each sample was centrifuged quickly and returned to -80°C for storage.

·;

" Electrophoresis Two methods of electrophoresis were used to screen 30 enzymes, starch-gel electrophoresis and cellulose-acetate electrophoresis. 'I

·, '/ 1. Starch-gel electrophoresis >. Horizontal starch-gel electrophoresis was carried out at 4 °C using 10% hydrolysed starch.

'·-.\ Two continuous systems and two discontinuous systems were used: tris-citric EDTA, pH 7 [TE] (Ayala et al., 1973), tris-EDTA maleate (Selander et al., 1971), tris-citrate (Poulik, 1957) and Lithium Hydroxide (Selander et al., 1971). Gels were prepared the day before

they were used. They were wrapped in cling wrap and stored at 4 °C to prevent moisture

>- c.., loss overnight.

-·- ' To load samples into the gel, a slit was cut parallel to a side of the gel, approximately 3 cm '";>-'•/

? -; from one edge. Filter paper wicks (Whatman No.3) were dipped into thawed samples and excess liquid removed by gently wiping the wicks on the side of the Eppendorf tube. One ...... , ...... side of the slit was pulled back and soaked wicks were inserted, taking care to keep

'f different samples from coming into contact from one another.

Gels were aligned with the loading slit nearest the cathode electrode tank. Each was ,_ r

-,. ;, covered with cling wrap to prevent moisture loss and an ice tray placed on top to maintain a temperature near 4 °C. All gels w~re run at 45 mA and 4 °C until the buffer dye neared the

anode wick.

16 Chapter Two

2. Cellulose acetate electrophoresis

Cellulose acetate electrophoresis was also carried out at 4 °C using Titan III© cellulose

acetate plates. Again several different buffer systems were used to screen a variety of

different enzymes. These were, citric acid-aminopropyl morpholine (CAEA) (pH 7.2)

- "' (Hebert and Beaton, 1989), tris-glycine (pH 8.5), 0.13 M tris-EDTA borate (pH8.9), 0.02 -' ,, M phosphate (pH7) (Richardson et al., 1986) and tris-EDTA maleate (Daly et al., 1981). ', The cellulose acetate plates were soaked in the appropriate buffer for 20 mins prior to use.

/' ·1 A Super Z Application Kit© (Helena Laboratories) was used to apply the samples to the

plates. Eight micro litres of thawed sample was loaded into the individual wells of a chilled

-·) loading plate. The samples were then blotted onto a soaked cellulose acetate plate three or

~· ...,, four times to give a high concentration of sample on the plate. During the screening of

enzymes, samples were loaded into the middle of the plate so that both enzymes that c, migrated towards the cathode and those that migrated towards the anode would be

:<, detected. The plates were placed face down in a tank and run at 150 V until the buffer dye

neared the end of the gel. The time required for this varied for the different buffer systems.

;-- Staining

>- C>- Both the starch and the cellulose acetate gels were stained in a similar fashion. The starch gels were cut into three or five slices depending on the original thickness of the gel and each

_,.,,_, slice was placed on a perspex plate. The cellulose acetate plates were removed from the

c, ~ tank and placed directly onto a perspex plate for staining. Stain reaction mixes were

'.)-- applied to the gels as an agar overlay. After the agar began to solidify the gels were ..... incubated at 37°C. All the buffer and stain recipes used are listed in Appendix B. \!-"-4<---<,

> y

-;--

,, r

-,,._ v.,.,

17 Chapter Two

Analysis ofResults The most common band was designated '100' and the other band was scored proportionately to this. The fast band was later referred to as 'a' and the slow band 'b'. Those samples initially difficult to interpret were rerun and on each gel a marker of known

( genotype was run. Allelic frequencies (q) for populations and colour morphs were

·.l ., calculated by the following formula: ; q = Het+2Hom 2N

'? where Het is the number of heterozygotes, Hom is the number of homozygotes for that .• ·1 allele and N is the total number of individuals sampled at that site. Ninety five percent

" confidence intervals (CI) for the allelic frequencies were also calculated. This was done . t using the following formula:

-) ",Y

_'.,.., 95%CI = ±l.96~(q(l~ q))

:', where q is the allelic frequency and N is the number of animals assessed at that locus

,. " (Griffiths et al., 1993).

)-

, ·,

> '.>

> ,.

';-·-,

·., 1

-'_'r- ... ,,

~ ;...,

/ 7

j ' . ,•

·,· v

18 Chapter Two

Results

DNA Analysis

Using SSCP, 114 individuals were scored for COI and nineteen individuals representing all .1.

.\ COI haplotypes were scored for 16S. Eight COI haplotypes (Table 2.1, Fig. 2.4) and a ' ,J ' single 16S haplotype were recognised from the SSCP gels. Four individuals of COI

haplotype A, two of haplotypes D and E, and one of haplotypes B, C, F, G and H were sequenced (Figure 2.5). ·, Table 2.1: a) Summary of haplotype data for the four sites within Doubtful Sound,

Crowded House (C.H.), Tricky Cove (T.C.) Espinosa Point (Esp.) and Oz, and the sites ·-1

'f ·f within Nancy Sound, Chalky Inlet and Preservation Inlet. b) Summary of haplotype information for colour morphotypes.

:_ a)

Doubtful Sound Nancy C. Inlet P. Inlet : ~- '"" Hap. C.H. T.C. Esp. Oz A 14 13 23 3 17 5 9 B 0 0 0 0 0 0 1 C 0 1 1 0 0 0 0 D 2 3 3 1 0 1 5 E 1 0 2 0 0 0 0 F 1 0 0 0 1 0 0 G 1 1 0 0 1 1 0 .,. H 2 0 1 0 0 0 0 <""~-,

~ -f. b)

"'·• Hap. Red Yellow Striped Spotted Light :,. .Ji"' A 63 8 7 3 1 B 1 0 0 0 0 .... 7 C 1 0 1 0 0 ) D 14 1 0 0 0

~ E 3 0 0 0 0 F 2 0 0 0 0

·~- \r G 3 0 0 1 0 H 3 0 0 0 0

19 Chapter Two

\._

,( ..

7

.,

~ .,

;>- ,,

~ 'T

!>.. Figure 2.4: Photo of SSCP gel showing from left to right, four individuals ofhaplotype A,

•. one of haplotype B, one of haplotype D, a blank lane, an individual of haplotype E, a

(h\.. group of haplotype D, a group of haplotype A with two lanes of haplotype F in the middle, two more of haplotype D, a blank lane, two ofhaplotype A, a blank lane, two of 'I._ haplotype F, a blank lane and the heteroplasmic individual CR6. -r

t> ~

~

~

~-y

"' - ...... ,.

7 .- ~r ·~.,

20 Chapter Two

70 A GGAAATTGAT TAGTTCCCTT AATGATAGGA GCGCCAGACA TGGCATTCCC TCGAATGAAT AACATGAGAT B C .. A ...... D E F G , L H aa G N W L V P L M I G A P D M A F P R M N N M S _I 140 A TCTGATTAAT TCCCCCTTCA TTTATACTTC TTATAGCCTC CGCTGGAAAT GAGAGAGGAG TAGGTACTGG \ B C .( c< D .. c ...... E F G ...... c. H ...... aa F W L I p p s F I L L I A S A G N E S G V G T G 210 A TTGAACCGTT TACCCCCCCT TATCAGGCCC AGTAGCCCAT GCAGGAGGTT GTGTAGACCT AGCAATATTT B C D E F ...... T. G H aa W T I y p p L S G P VAH AGG CVDL AI F 'l 280 A TCCCTTCATC TAGCCGGTGC CTCCTCAATT ATGGCATCAA TAAAATTTAT AACAACCATA ATAAAAATGC ,_;;( ·7 B ..... c ...... C D ...... c. E F " G H aa S L H L A G A S S I M A S I N F I T T I I N M 350 A GAGCACCAGG AATTACAATG GACCGAACCC CTTTATTTGT ATGATCAATT CTCCTCACAA CCTTTCTACT B ...... C ...... T. D ,\..... E . ... c ..... F G H aa RAPG ITM DRT PLFV WS I LLT TFLL 420 :,r ') A ACTCCTTTCC CTTCCAGTAC TAGCTGGAGC AATAACAATG TTATTAACAG ATCGAAATAT AAAAACAACT B C D ,, E F G ~, H aa LLS LPV LAGA ITM LLT DRNI N T T ' ( 473 A TTCTTCGACC CAACTGGAGG AGGTGACCCC ATACTATTCC AGCATCTATT TTG B C D .. " E ·,,, ,~, F G H ...... T. .... 7t aa F F D PTGG GDP I L F Q H L F

'7' Figure 2.5: Sequence and amino acid data for the eight COi haplotypes.

--..-. ;r

21 Chapter Two

Of the 4 73 bp sequenced, there were nine variable sites, all being the third position and none being phylogenetically informative. The transition:transversion ratio was 7:2. Haplotypes B, C, D, E, F, G, and H appear to be derived from the most common 'l haplotype, A (Fig 2.6).

( )

'-,

-"

- .} Figure 2.6: Minimal spacing network constructed from sequence data. Letters represent haplotypes (Fig 2.5, Table 2.1) and the line length is proportional to the number of

-; substitutions (no mark on line indicates one substitution, mark on line indicates two).

~ ·r

;'>-

" 6 Results from the G-test analysis showed no significant difference in haplotype frequency ''1< among the four fiords (G[2lJ = 23.32; P > 0.2) or among the four populations in Doubtful :7

Sound (G[2IJ = 11.97; P > 0.9). There was also no significant difference among colour

morphs (red, striped, spotted, yellow and light) (G[28i = 13.88; P > 0.9). Due to the large number of haplotypes at some locations with zero frequency, G-test analysis was also -,, ;, done after grouping haplotypes with low frequency i.e. A, D and other. Results from this were also not significant. Further analysis incorporating information on nucleotide

22 Chapter Two

differences between haplotypes was conducted using AMOVA (Excoffier et al., 1992). This analysis also showed no significant difference among the fiords (FsT = 0.013, P > 0.05) or among populations within Doubtful Sound (Fsc = -0.037, P > 0.05). These

,L results indicate that nucleotide variation between populations or fiords is not significantly ;,

\ greater than variation within a population. There was also no significant difference ,L _. between colour morphs (FsT = -0.089, P > 0.05).

•7 •.,. As only a few individuals were able to be amplified for the region used in RFLP analysis,

..._..,., "! results are limited. Of the ten restriction enzymes used to cut the 2.5 kb segment of DNA '·' only one (Mael) was variable over the individuals tested. Three of the enzymes, Cfol, ,, :z Hinfl, and Msp had no cut sites within this region, all other enzymes gave the same cut sites over all the individuals tested. Although amplification of this region was only -,')( )'

:, partially successful, individual representatives of several SSCP COI haplotypes were

·}, " amplified and cut. The low level of variation with several enzymes across haplotypes 1s

consistent with results from SSCP and sequence analysis. &, ',

r~

:.~ :;,..,

'Y

.,. ')

<>;,

~ :,, ~·

...-<·) -.,, .

"'" ~~

~ ·;,.

".r

?"":.."-

23 Chapter Two

Allozyme Analysis

A total of thirty systems were assessed with starch and cellulose acetate electrophoresis

across ten different buffer systems (Appendix C). Of these, only five systems, hexokinase

-{ (HK), phosphoglucose isomerase (PGI), superoxide dismutase (SOD) and two peptidases

.\ (PEP-1, PEP-2), gave scorable activity. All of these systems except PGI were

_( ·' homozygous. Allele frequency information for PGI is shown in Table 2.2a. No individuals

homozygous for allele b were discovered in any population sampled. All 95% confidence

. ,~ \l intervals overlap, indicating no difference in the allelic frequency of the sampled

-7 populations.

-..r > Allele frequency results for colour morphs are shown in Table 2.2b. Again all 95% -·,

.._j,_ ·-; confidence intervals overlap indicating lack of genetic differentiation between colour morphs.

'!,, .")..

Table 2.2: a) PGI allele frequency results for fiords. b) PGI allele frequency results for

colour morphs. Frequency (Confidence Interval).

a) ;:-;--,, Fiord Doubtful Nancy P. Inlet C. Inlet

;_"'..\ Site T. C. C.H. Esp.

N 13 18 27 19 10 3

-r ~ a 0.92 (0.15) 0.97 (0.08) 0.94 (0.09) 0.97 (0.08) 0.85 (0.22) 1

b 0.08 (0.15) 0.03 (0.08) .0.06 (0.09) 0.03 (0.08) 0.15 (0.22) 0 -:'- )

·:_,..

-, -~ b)

~ "1' It Colour Dark Red Striped Spotted Yellow Light ,;. ? N 75 7 7 1 0 '7.., a 0.94 (0.05) 0.93 (0.19) 1 1 0 .:..,~ b 0.06 (0.05) 0.07 (0.19) 0 0 0 ;,-

Results from allozyme analysis are also consistent with SSCP and sequence results.

24 Chapter Two

Discussion

Analysis of the genetic structure of seven fiord populations of Astrobrachion constrictum . , showed no significant differentiation among population samples within the same fiord or ,'- .. among fiords. Both allozyme and mitochondrial RFLP data support the finding from the l . J principal SSCP/sequence analysis of mitochondrial COL As has been shown for other

benthic marine invertebrates, there was a lack of heterozygotes found during allozyme

",' ';I analysis (Hensley et al., 1995; Ayala et al., 1973; Kwast et al., 1990). Several explanations

for this effect in other species have been offered (Zouros and Foltz, 1984; Koehn et al.,

',Y 1976; Gaffney, 1990) but due to a lack of allozyme data in this study conclusions about

--{ ':J low levels of heterozygosity cannot be made . .. ,

' I ·' The lack of differentiation among fiord populations could be due to a number of factors.

~\... _\. First, larvae from the continental shelf (CS) population of Astrobrachion constrictum could

be settling in the fiords providing a continual source of genetic material to all fiords. This

scenario would require the CS population to be genetically similar from north to south.

The prevailing current flow for this region is strong and towards the south-west (Stanton, ~:.., 1976). Regardless of this flow, Mladenov et al. (1997) showed some differentiation

between the Doubtful Sound population of sea urchin and populations on the coast while

Miller (1997) showed similar results for black coral. These findings suggest that there is

·r ' some limitation to gene flow between Doubtful Sound and other populations for these

species. Evechinus chloroticus has a long planktotrophic larval phase and thus has a high ' ·, dispersive potential (Walker, 1984; Dix, 1969); it also inhabits shallow waters (Dix, 1970).

.. -~ These two factors should make it easier for this echinoid species to colonise the fiords from

·;,,. the coast than for A. constrictum. Therefore, it is unlikely that the lack of genetic ;r differentiation between fiords is due to ongoing colonisation from the CS population. 7

·:,-' However, without sampling the CS population this possibility cannot be discounted. A. constrictum lives beyond SCUBA range on the continental shelf and the only method ,•;r · available for collection is dredging. Unfortunately this method is not viable as the brittle star inhabits the protected black coral species, Antipathes fiordensis.

25 Chapter Two

Another possibility is the relatively new development of the fiords. Evidence suggests the last glacial period ended between 10,000 and 18,000 years before present (Pillans et al., 1992; Pickrill et al., 1992). Associated with the end of this final glacial period was a rise in

'( sea level of between 110 and 120 m (Pillans et al., 1992), stabilising at today's level around 6500 years before present (Pickrill et al., 1992). The timing of the flooding of the fiords

( ,\ would be dependent on the height of the entrance sill. Fiords such as Preservation Inlet with high sills would have been flooded much later than fiords with low sills. Therefore ·r -1 Fiordland as it is today has probably only been in existence for 7,000 to 10,000 years. This is too short a period of time for significant molecular change to occur in situ without :r strong selection pressure. However if effective population size were restricted, differential

.., )' sorting of haplotypes from an original heterogeneous population would be possible. This

,._ -r appears to have happened between coastal and Doubtfu~ Sound populations of the sea urchin Evechinus chloroticus (Mladenov et al., 1997) and the black coral Antipathes

fiordensis (Miller, 1997), between fiord populations of E. chloroticus (Wing and Skold, in "':"" prep), the starfish Coscinasterias muricata (Skold et al., in prep) and A. fiordensis, but not ::-.,:'- with fiord populations of Astrobrachion constrictum.

~~'-

'~-', ' A third possibility is migration by juveniles, either among fiords or from the continental sheif into the fiords. Recruitment of juvenile Astrobrachion constrictum onto coral colonies is thought to occur soon after their first year (Stewart and Mladenov, 1997). The smallest 1' ~} recorded individual found either on coral colonies or the surrounding substrate had a disc ,, diameter of 2.5 mm. At present, it is unknown what happens to juvenile stars before recruitment (Stewart and Mladenov, 1997).

,-,4

s:," Finally, limitations to sampling and to the region of the mitochondrial genome able to be :> assessed, may have had an effect on the results. Sample sizes were influenced by patchy / distribution of the snake stars and by a limit to the number of collecting trips able to be ',· made to Nancy Sound, Preservation Inlet and Chalky Inlet. Although COI has shown

,. ;.,..- differentiation between populations in other studies (Burton and Lee, 1994; Sperling and Hickey, 1994), it is more conserved than the non-coding control region of the mitochondrial

26 Chapter Two

genome. Analysis of the control region may have highlighted more subtle differences to genetic structure among the sampled populations. However several attempts at amplification of this region were unsuccessful. Another method sensitive enough to reveal

small differences in genetic structure is the analysis of microsatellites. Any further fiord genetic studies of Astrobrachion constrictum should utilise either the control region or

I_-! microsatellite analysis.

··rf This study also assessed the possibility of genetic differentiation between the five colour .• 1

.._.,, morphs of Astrobrachion constrictum. Results of both allozyme and mtDNA data analysis

:r showed no significant difference between the genetic structure of any of the colour morphs. Colour morphs of A. constrictum occupy the same habitat and have identical external and internal anatomy (Stewart, 1995). Therefore, this result is not unexpected.

-·,

,\ :~,

·1 ~j

~·~~.,-

,'

,-,)I

,f'-

,-·,

9-',7

27 Chapter Three

Chapter Three: Evidence of Germ-line Heteroplasmy in the Mitochondrial DNA of the brittle star Astrobrachion constrictum (Ophiuroidea: Asteroschematidae ) . . ,. ,\

( -l Introduction '(

.,. ., Mitochondrial DNA (mtDNA) has become a powerful tool for assessing relationships

among individuals, populations and species of animals (Avise, 1994; Moritz et. al., 1987). :/

.\. 7 As the number of studies using this genome increases, knowledge of the genetics of the genome itself is also increasing. Two of the more surprising discoveries have been the j.. ,'! extent of heteroplasmy in animal populations (Lunt et. al., 1998) and cases of biparental

inheritance of the genome (Gyllensten et. al., 1991; Hoeh et. al., 1991). Heteroplasmy is ,\.. "· the occurrence of more than one type of mtDNA in the same organism and can arise either from mutation of the genome within the individual, heteroplasmy of the original oocyte, or from biparental inheritance. Most published examples ofheteroplasmy involve variation in

,:--· ... the number of repeats within the control region of the genome (Lunt et. al., 1998). Although the control region is non-coding, it probably contains sequences that initiate

;:- :.. replication and transcription (Clayton, 1982). In echinoids and vertebrates, the

displacement loop (d-loop) structure evidences replication (Matsumoto et. al., 1974). The 1i

-<•·--;, length of repeats found in this region can vary from small microsatellite-like repeats

J--,. (Wenink et. al., 1994) to very large repeats of 1100 bp (Wallis, 1987). Length heteroplasmy is generally -explained by slipped strand mispairing during replication i" "" (Densmore et. al., 1985), and high frequencies oflength heteroplasmic individuals can occur --:-.-"ii.

;, in some species (Lunt et. al., 1998). In a few cases observed heteroplasmy has been attributed to biparental inheritance (Magoulas and Zouros, 1993; Kondo et. al., 1990).

';- )"'T Paternal leakage is still thought to be rare in animals, with mussels of the genus Mytilus

being a prominent exception (Zouros et. al., 1994; Wenne and Skibinski, 1995).

28 Chapter Three

De Giorgi (1988) reported heteroplasmy in the eggs of the sea urchin Arbacia lixula using RFLP analysis. However, in the absence of additional data, it is difficult to rule out partial digestion for the single restriction enzyme (BamHI) site that was seen to vary both within and among females. To date, no cases of heteroplasmy have been reported in ophiuroids. While conducting a population genetic study of the brittle star Astrobrachion constrictum ' \

' I in Fiordland, New Zealand, two of 117 sampled individuals were found to be '( heteroplasmic. The first was identified from an SSCP gel where the banding pattern of the ·;, ';' individual appeared to be a combination of two previously recognised cytochrome oxidase I ') (COI) haplotypes. The second was identified through a sequence which appeared to be a

'J combination of two previously sequenced COI haplotypes. Through a combination of

,( SSCP, cloning and sequencing, heteroplasmy is confirmed in this echinoderm species.

>

~-~"'

-~ l

.~

'.>'

;,-

/-

,?

29 Chapter Three

Materials and Methods

Sample Collection Specimens of Astrobrachion constrictum were collected by SCUBA from seven sites in -~ \ Fiordland, southern New Zealand: four sites within Doubtful Sound (Espinosa Point, Tricky Cove, Crowded House and Oz) and one site each from Nancy Sound, Preservation '--·l Inlet and Chalky Inlet,. All animals were kept in seawater for transport to the laboratory .-,, ,.,, alive, where they were stored at -80°C. Total genomic DNA was extracted from

- r approximately 50 mg of gonad tissue by boiling for 10 mins in 50 µl of water. Extracted :;- ·:r DNA was precipitated in cold 100% ethanol, washed once with 70% ethanol, and resuspended in 30 µl of milliQ water.

,,,...__¥( PCR > A 473 bp fragment ofCOI was amplified using Cl-J-1718 and Cl-N-2191 primers (Simon ,_' ~ ' ~

et. al., 1994). Twenty five microlitre PCR reactions contained 3.5 mM MgC12, 200 µM each dNTP, 0.25 µM each primer, 0.25 units of Qiagen© Taq polymerase, lOx Qiagen©

buffer and 2 µl of template DNA. The following thermocycling protocol was used for the

amplification: initial denaturation at 94 °C for 1 min, 40 cycles of denaturation at 94 °C for

15 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min, followed by a further

;r> extension at 72°C for 3 mins. PCR products and a 1 kb molecular weight marker (Gibco

_,,," BRL) were electrophoresed in 2% agarose gels stained with ethidium bromide. Gel plugs containing DNA of expected length were excised and purified using Prep-A-Gene® )· ·r (BioRad).

/

'y-S:.'* I Cloning f}' Purified PCR product was cloned using the Blunt Ended PCR Cloning Kit-pMOS blue ! J

7 (Amersham Pharmacia Biotech). Transformed cells were plated on LB agar (Sambrook et. ,- ,' al., 1989), treated with X-gal and IPTG for blue/white screening and grown overnight at 37°C. White colonies were picked and cultured overnight in LB broth (Sambrook et. al.,

-c.-.,.... 1989). Plasmids were purified from these cultures as follows. Approximately 1.5 ml of culture was tipped into an Eppendorftube and centrifuged at 13,000 rpm for 30 s. All but

30 Chapter Three

50-100 µl of supernatant was removed and the pellet was resuspended in the remainder.

300 µl of TENS buffer (lx TE, 0.5% SDS, 0.1 M NaOH) was added and the mixture

vortexed briefly. Quickly following this, 150 µl of 3 M NaOAc (pH 4.8) was added, then vortexed and centrifuged for 2 mins at 13,000 rpm. The supernatant was removed and placed in a new tube, ethanol precipitated and the pellet resuspended in 30 µl of milliQ '' I water with 1 µl of added RNase (Hillis et. al., 1996). The purified plasmid DNA was screened using SSCP. _..T ';y

) ; SSCP Ten microlitre PCR reactions were carried out under the same conditions as described above with the addition of 0.05 µl of 33P labelled dATP per reaction. Following the PCR cycle an equal volume of formamide buffer (95% formamide, 20 mM EDTA pH 8, 0.05% )

,, bromophenol blue, 0.05% xylene cyanol) was added to each reaction and denatured at 94°C J for 5 mins. Reactions were placed on wet ice and loaded immediately. Four microlitres of each sample were applied to a 6% bis-acrylamide (37.5:1 ratio, BioRad premix) gel containing 5% glycerol in 0.5x TBE. Electrophoresis was carried out at 4°C for 200 Whr running at approximately 13 W. Completed gels were lifted from the plates with blotting

paper and dried using a Bio-Rad 583 Gel Dryer. Kodak BioMax™ film was exposed to the dried gels for two to three days. Haplotypes were scored by comparison of renatured ;,. > single stranded DNA migration patterns and this information was used to select cloned DNA for sequencing. -·, ·t

~ i

~ "r Sequencing Minipreped DNA was quantified by electrophoresis with 1 kb Plus DNA ladder (Gibco } ~)t BRL) on a 2% agarose gel stained with ethidium bromide. Cycle sequencing was carried ;r. out with BigDye™ chemistry according to manufacturer's recommendations (PE Applied Biosystems). The product was precipitated using ethanol/sodium acetate (PE Applied

;;:,,-·/-- Biosystems) and sequenced on an ABI377. Sequences were aligned by eye using SeqEd vl.03 (PE Applied Biosystems) and compared with previous sequence obtained from the population study (Chapter Two).

31 Chapter Three

Results

One hundred and seventeen individuals were screened for 473 bp at the 5' end of COi using

SSCP and sequencing (Chapter Two). Of these, 113 showed clear, single SSCP products, and three failed to amplify. Eight haplotypes (A to H) were recognised from SSCP and

,. I sequence. SSCP of one individual, ERl 1, showed a combination of two haplotypes, A and

D. From the intensity of the bands on SSCP autoradiographs these haplotypes appeared / to have amplified approximately equally. Another individual, CR6, showed a single SSCP

' -, product, but sequence results indicated a combination of haplotypes A and G.

--,.

c\...-';>,- To determine whether these individuals were heteroplasmic, COi PCR products for both

individuals and for a control individual of haplotype A (NR7) were cloned. Seventeen

ERl 1 colonies, twenty CR6 colonies and five control colonies were picked and cultured

,'__....,)- overnight. Plasmid preparations were made from all overnight cultures and subjected to

SSCP analysis. On each gel, representatives of haplotypes A, D and G were run as -, ~\._ positive controls. Eleven clones from ERl 1 produced a banding pattern identical to haplotype A, while a single clone produced a banding pattern identical to haplotype D (Fig

·;- ,,_ 3.1). All clones from CR6 produced identical banding patterns to haplotype A as did

clones from the control, NR7. As colonies were chosen at random for SSCP screening, the

proportion of different SSCP haplotypes from the sampled colonies is probably an ~). 1 approximation of the haplotype frequencies within the individuals. This suggest that there ~r is a higher proportion of haplotype A in both of the heteroplasmic individuals.

~ /.ls

)--,

-;r.,,.... ,

·--,,-

32 t~ Chapter Three

\

\.. ... (

.T r:-~,

- '{

,,_ 1<'

,._ ~T"

.!> I). Figure 3.1: Photos of SSCP gels. The top photo shows the heteroplasmic individual,

-.:, ERl 1 (fourth from the right) before cloning, with individuals of haplotype A to the immediate left and D to the immediate right. The bottom photo shows the haplotypes of " clones obtained from ERl 1 aligned with individuals ofhaplotype A and D. From left to ,c /) . right, one individual of halpotype A, three clones of haplotype A, heteroplasmic ERl 1

.. ·- (four bands), one clone of haplotype D and two individuals of haplotype D . ..

-·, One clone representative of each haplotype observed in ERl 1 and two clones

~ representative of the haplotype observed in CR6 were sequenced and compared to . "'

<,> reference sequence from a population study (Chapter Two). Sequences from ERl 1 clones .. .:.. were identical to haplotypes A and D as expected. Of the two clones sequenced for CR6, • ;, , one gave sequence identical to haplotype A but the other gave a novel sequence haplotype (I). Of the 480 bp cloned and sequenced from the 5' end of COI, haplotypes present in ~

rr ERl 1 differed by two bases, while haplotypes present in CR6 differed by only one (Fig ? 3.2). All three base substitutions are synonymous, third codon position transitions.

- v'

33 Chapter Three

70 A GGAAATTGAT TAGTTCCCTT AATGATAGGA GCGCCAGACA TGGCATTCCC TCGAATGAAT AACATGAGAT D G -1 ~ aa GNW LVPL MIG APD MAFP RMN NMS 140 A TCTGATTAAT TCCCCCTTCA TTTATACTTC TTATAGCCTC CGCTGGAAAT GAGAGAGGAG TAGGTACTGG D ...... c ...... G ...... j ...... c . aa F w L I p p s F I L LIAS AGN ESG VGTG '._.,, 210 A TTGAACCGTT TACCCCCCCT TATCAGGCCC AGTAGCCCAT GCAGGAGGTT GTGTAGACCT AGCAATATTT D G J aa W T I Y P P L S G P V A H A G G C V D L A I F 280 A TCCCTTCATC TAGCCGGTGC CTCCTCAATT ATGGCATCAA TAAAATTTAT AACAACCATA ATAAAAATGC ~ D ...... c ...... G ...... aa s L H L A G A S S I M A S I N F I T T I I N M 350 A GAGCACCAGG AATTACAATG GACCGAACCC CTTTATTTGT ATGATCAATT CTCCTCACAA CCTTTCTACT D

'> G aa RAPG ITM DRT PLFV WS I LLT TFLL 420 A ACTCCTTTCC CTTCCAGTAC TAGCTGGAGC AATAACAATG TTATTAACAG ATCGAAATAT AAAAACAACT D G aa L L s L p V L A G A I T M L L T D R N I N T T 473 A TTCTTCGACC CAACTGGAGG AGGTGACCCC ATACTATTCC AGCATCTATT TTG D G aa F F D P T G G G D P I L F Q H L F

Figure 3.2: Sequence and amino acid data for the three haplotypes present in the two

~"-., l \ heteroplasmic individuals.

'J,

•,N

"'°"

? '.;

''?-' ;-

/ - ,·

34 Chapter Three

Discussion

The majority of reported heteroplasmy is due to differences in repeat number within the

.[ ) non-coding control region of mtDNA. This length variation is easily identified through RFLP analysis (Solignac et. al., 1983), or more recently through PCR. This study

'· .( demonstrates the presence of heteroplasmy for base substitution variation in the COi

·.J coding region of the mitochondrial genome in two individuals of the brittle star Astrobrachion constrictum. As gonad material only was examined, observations are specifically germline heteroplasmy. Sampling of other tissues could reveal higher levels of

'\ organismal heteroplasmy. The haplotypes present in each heteroplasmic individual are also found in homoplasmic individuals in the Fiordland population of A. constrictum ··\ (Chapter Two). Sequence information for these haplotypes shows that they differ by two base pairs in one individual and by a single base in the other. Base differences on this scale can be detected conveniently with methods such as SSCP that can separate short segments of DNA as a result of conformational differences resulting from minimal sequence variation ~·, (Lessa and Applebaum, 1993). Cloning provides a relatively quick and easy way of separating different mitochondrial PCR products for SSCP screening. Direct sequencing of :, PCR product alone would not usually be sufficient to demonstrate heteroplasmy of this type as results may often be misinterpreted as unreliable sequence. Indeed, until closer inspection of SSCP gels and fresh DNA extractions were made, sequence from CR6 was -.> ·;,.· assumed to be contaminated. This could also be the case with other studies and therefore ~-- ;)- this type of heteroplasmy could be more common than reported.

..,. The haplotypes present in ERl 1 are two of the more divergent pairs of haplotypes found

< .... within the Fiordland population of Astrobrachion constrictum (Chapter Two). It is not :.,~ likely that one of these variants arose de nova within this animal, since this scenario would require two mutations very early in development. The two more likely hypotheses are .( "f

·"1'·.;--- that heteroplasmy has persisted through several germ-line replications, or that there has been paternal leakage. For the first to occur, heteroplasmy would have to have persisted ,· long enough in the same lineage to evolve two independent mutations. From studies on Drosophila (Solignac et. al., 1983) and crickets (Rand and Harrison, 1986) it has been suggested that fixation is complete within a few hundred generations. This is a short time

35 Chapter Three

for two mutations to have evolved before sorting out into homoplasmic lineages, but without exact knowledge of the sorting out rates and mutation rates within COi of echinoderms this possibility cannot be discounted. c

> >· The second explanation, paternal leakage, seems more plausible. Experiments designed to detect low levels of paternal leakage through repeated back-crossing have shown partial

J 7 paternal mitochondrial inheritance in Drosophila (Kondo et. al., 1990) and mice --J (Gyllensten et. al., 1991). These studies suggested that the observed heteroplasmy may be a result of reduced compatibility between egg and sperm due to the use of hybrid strains. However, heteroplasmy attributed to paternal leakage has been observed in natural \ ~ populations of anchovy, Engraulis encrasicolus (Magoulas and Zouros, 1993), and

·'( mussels of the genus Mytilus (Zouros et. al., 1994; Wenne and Skibinski, 1995). Although the heteroplasmy observed in anchovy was a not a result of recent hybridisation, the authors suggested that historical separation events may have made it possible for heteroplasmy to occur (Magoulas and Zouros, 1993). The extent and type of biparental mitochondrial inheritance in mussels appears to be unique to this genus. Magoulas and Zouros (1993) suggested that a lack of mitochondrial diversity within a population may give a false impression of the extent of paternal inheritance due to inheritance of the same molecule from both parents. This could be the case for Astrobrachion constrictum as the recent study of the Fiordland population showed low levels of mitochondrial diversity

; '~)-' (Chapter Two) therefore heteroplasmy could be greater than currently observed in this species. /

-, Low levels of heteroplasmy resulting from a small number of base substitutions are

..~"'{ difficult to detect without methods designed to differentiate DNA molecules differing by :,-,, only one base pair. This study uses PCR and SSCP to identify heteroplasmy, and cloning

';> and SSCP to isolate and identify the haplotypes. This approach, if employed widely, 7 j could reveal even higher levels of heteroplasmy in natural populations than are currently T -I- believed to exist.

36 Chapter Four

Chapter Four: Echinoderm Phylogeny

'. 1 Introduction

Since the early 20th century, several approaches have been taken to determine the 7 {

' ·;I relationship between five extant classes of echinoderm. Initial studies were limited to the

-~ / use of adult morphology before techniques in palaeontology and embryology were "/ improved in the late 20th century (Smith, 1984). More recent studies have taken advantage of molecular genetics, using nuclear and mitochondrial DNA to classify echinoderms (Wada ... t ::... and Satoh, 1994; Smith et al., 1993). There have also been studies using a combination of methods (Smith, 1984; Littlewood et al., 1997).

Smith (1984) gave a good summary of the evolution of ideas about echinoderm classification up to 1984. Since then, the number of studies assessing the relationships

]'" :, both within and among classes of echinoderm appears to have increased. These studies have generated several different hypotheses about the relationships of five extant :'F ,"_,._ echinoderm classes (Fig 4.1 ).

1 . Paul and Smith collectively published several of these studies in 1988. One of these combined 18S sequence data to suggest the phylogeny shown in Fig 4.la (Raff et al., 1988). > Jacobs et al. (1988), used rearrangements in the mitochondrial genome between echinoids

' ., and other animals as well as partial alignments of the mitochondrial genome between .:x ., echinoids and asteroids to assess phylogeny of echinoderms. This work was continued by ;.-..-'*1( Smith et al. (1993) who added the mitochondrial genomes of two other classes,

,Y .... Ophiuroidea and Holothuroidea. This study grouped together ophiuroids and asteroids, and holothuroids and echinoids (Fig 4.1 b), primarily on the basis of an inversion of a large /' af fragment of the genome, but gave no indication of further relationships within the two groups. It has since been discovered that ophiuroids do not share this inversion event with

1 ,' asteroids but have a unique rearrangement. Wada and Satoh (1994) added to the 18S study by Field et al. (1988) with an increase in sequence data. Analysis of these data agrees with

37 i': Chapter Four

the phylogeny proposed by Raff et al. (1988). The most recent study by Littlewood et al. (1997) used a combination of morphological characters and molecular data for a large number of species from the five extant classes of echinoderm. Analysis of these data gave two

.t ' competing phylogenetic solutions (Fig 4.la and 4.lc).

Cri Oph Ast Holo Ech Cri Ast Oph Holo Ech A A A ~ ~

[j '/

.• I V a) c) . ·; \

~..- :~ t·-'./ Oph Ast Holo Ech

' (

LT,:.,' / b)

''. ·... Figure 4.1: Echinoderm phylogenies proposed by previous authors.

,.. • ,~ ~> l-,.·;. These three proposed phylogenies (Fig 4.1) differ in the relationship of asteroids and ophiuroids to the echinozoans (holothuroids and echinoids). Littlewood et al. (1997) I:, suggested that these three groups separated around the same time, a theory supported by fossil ) ' evidence (Smith, 1984). Until now molecular studies have used relatively slow evolving

)- "} genes such as 18S (Field et al., 1988; Wada and Satoh, 1994; Littlewood et al., 1997) and 28S ! (Littlewood et al., 1997). The failure of these genes to resolve the relationship among four of ~

•'k< A faster evolving gene may be able to better resolve the relationship among ophiuroids, v-• asteroids and echinozoans by providing more signal for the area of dispute. The danger of using a fast gene is incorporating a greater amount of noise with the signal. This study uses v' recently obtained ophiuroid cytochrome oxidase I (COi) sequence (Chapter Two; R. Sponer, ,~ unpublished sequence) with COi sequences from Genbank to analyse the relationships among / s r ·.,~ five extant classes of echinoderm. fi 38 \ 11 Chapter Four

Materials and Method

COI sequences were obtained from Genbank for species representing the echinoderm \ classes; Asteroidea, Echinoidea, Holothuroidea and Crinoidea. The Ophiuroidea were ,~ .\..

' ( represented by partial COI sequences recently obtained for Astrobrachion constrictum (Chapter Two) (submitted to Genbank) and Amphipholis squamata (R. Sponer,

1.- '7/ unpublished data). A full list oftaxa is presented in Table 4.1. A weta (Insecta) sequence was used as an outgroup (S. Trewick, unpublished data). DNA sequences (up to 520bp) were imported into SeqEd (PE Applied Biosystems), aligned by eye and translated to amino acids using the echinoderm codon table (Himeno et al., 1987).

' ( Analysis of both amino acid and DNA sequence data was conducted with PAUP

~ " (Swofford, 1993) using a variety of analyses. Maximum parsimony (MP) and Neighbor Joining (NJ) were performed using both the full list oftaxa and the reduced list (Table 4.1). Maximum Likelihood (ML) analysis was undertaken with the reduced list due to the length of time taken to complete this analysis. Two weighting schemes were employed: increased ~ weighting of non-synonymous changes and reduced weighting of transitions in relation to transversions. Heuristic search methods were employed for all analyses and data sets were bootstrapped with 100 replicates. ML analysis was performed with transition/

\ ·, transversion ratio being estimated first via ML. Permutation and random tree tests were performed for both amino acid and DNA full list data sets and for all the weighting schemes applied to the DNA data. The types of analyses performed on each data set with different

I ]~ weighting schemes are summarised in Table 4.2.

.;'j(

n,

', 7

39 1.,J Chapter Four

Table 4.1: Species of echinoderm used in this analysis;*= those used in reduced analysis. Phylum Echinodermata Class Echinoidea ® Class Ophiuroidea '¥­ Order Arbacoida Order Euryalida -1 le Family Arbaciidae Family Astroschematidae .. ' Saa. Ar.bacia dufresnei* 3. Astrobrachion constrictum* ( Sab. Arbacia incisa Order Ophiurida i. Sac. Arbacia lixula Family Amphiuridae

~>' ;/ Order Echinoida 4. Amphipholis squamata* ---r Family Paracentrotidae Class Holothuroidea I}' 3. Paracentrotus lividus* Order Dendrochirota

._ .. { Family Strongy locentrotidae Family Cucumariidae ,: l. Strongylocentrotus purpuratus* Sa. Cucumaria vegae* Class Asteroidea t} Sb. Cucumaria pseudocurata .... 7, Order Forcipulatida Sc. Cucumaria piperata Family Asteriidae Sd. Cucumaria pallida

\ :,. 3. Pis aster ochraceus* Se. Cucumaria miniata Sf. Cucumaria lubrica .::, '::. 2. Leptasterias polaris* Order Valvatida Sg. Cucumaria curata Family Asterinidae 4a. Pseudocnus californicus* 4a. Asterina pectinifera* 4b. Pseudocnus astigmatus ·,. 4b. Asterina miniata Family Phyllophoridae 4c. Asterina gibbosa 3. Peritarnera lissoplaca* 4d. Asterina pseudexigua pacifica Family Psolidae la. Patiriella exigua* 2. Psolus chitonoides* Family Sclerodactylidae -, ·, 1b. Patiriella paravivipara le. Patiriella pseudoexigua l. Eupentacta quinquesemita* ld. Patiriella regularis Order Aspidochirota -. \ le. Patiriella gunnii Family Synallactidae lf. Patiriella brevispina 6. Pseudostichopus mollis* >- :.-. lg. Patiriella vivipara Family Stichopodidae ,., ~,ca 7a.Parastichopus parumensis* .,.,.. lh. Patiriella ca/car Class Crinoidea ~ 7b. Parastichopus californicus ·~ Order Comatulida T Family Antedonidae .,,. - l. Florometra seratissima* --/

..... ,.~

40 Chapter Four

Table 4.2: Summary of the relationship between the data sets and weighting schemes used and which analyses were conducted; BS = bootstrapped.

- l Weighting MP NJ ML PTP MP NJ ML PTP -- \

'( Scheme random random

- _, Full data list Reduced data list -,r-; Unweighted vl',BS vl',BS '1 vl',BS vl',BS - - ~-1 - Nsyn/syn vl',BS vl',BS - '1 vl',BS vl',BS '1 - --:, Tv/Ti vl',BS vi', BS - '1 vi', BS vi', BS - -

...... ,L y Amino acids - vl',BS - - '1 '1 - -

-.-(

The MP tree obtained from analysis of DNA sequence data weighted for non-synonymous changes was used to generate a hypothesis for the relationship of five extant classes using representatives of each class. This was done using MacClade (Maddison and Maddison, 1992) by trimming the appropriate branches to give the shortest possible tree. This tree was compared to the tree topologies shown in Fig 4.1 using the Kishino-Hasegawa test to see if this phylogenetic hypothesis differed significantly from those previously proposed. Permutation and random tree tests were also performed on the data set from the trimmed tree. DNA sequence variation along the 520 bp of COI assessed in this study was I -, compared for different levels of taxonomy; within a family, within a class and among five

:,..') classes . .. '

.,,...., ;,., -"I,,.

,...... ,,

./ -r

~ -,

/

41 Chapter Four

Results DNA analysis

A total of 520 bp from the 5' end of COI was aligned for 37 species of echinoderm '- ... -~ representing the five extant classes and one outgroup species. Of the 520 bp, 233 sites are ,.I, parsimony informative. The transition transversion ratio is approximately 1: 1 (Fig 4.2)

j.

To ,- " A C G T

•'/'

r A .,

.... lo; . "' .~ From c

I>, G • ~

J,..

T • C>

.t,..

> e Transitions 9 Transversions I> t ., Figure 4.2: Base by base transitions and transversions calculated from the tree in Fig. 4.4

"' Distance information for uncorrected and Tamura-Nei corrected data is shown in Table 4.3. -,. . "'

~ '' ... r .,.., .... r

'I""

42 Chapter Four

Table 4.3: Distance information for the reduced taxa list, a) shows uncorrected distances,

b) shows Tamura-Nei corrected distances.

a)

Cri Oph3 0.29 - Oph4 0.27 0.23 - Astla 0.28 0.25 0.25 - Ast2 0.23 0.26 0.24 0.20 - Ast 3 0.25 0.25 0.27 0.20 0.18 - Ast4 0.26 0.26 0.26 0.19 0.20 0.20 - Holo 1 0.26 0.20 0.24 0.22 0.21 0.22 0.22 - Holo 2 0.27 0.21 0.26 0.21 0.21 0.22 0.22 0.17 -

7 IHolo 3 0.26 0.22 0.27 0.24 0.22 0.24 0.23 0.14 0.18 - .,, Holo 4a 0.30 0.22 0.24 0.18 0.22 0.24 0.21 0.19 0.15 0.18 - Holo 5a 0.27 0.23 0.26 0.22 0.22 0.22 0.22 0.17 0.17 0.19 0.16 - 'l 1Holo 6 0.24 0.25 0.26 0.24 0.23 0.24 0.23 0.19 0.19 0.21 0.18 0.20 - Holo 7a 0.26 0.24 0.26 0.23 0.22 0.24 0.22 0.18 0.19 0.23 0.20 0.21 0.21- Ech 1 0.28 0.27 0.29 0.22 0.21 0.22 0.23 0.24 0.26 0.25 0.25 0.24 0.27 0.24 - Ech3 0.28 0.25 0.26 0.21 0.20 0.21 0.19 0.18 0.20 0.19 0.20 0.18 0.22 0.21 0.19 - Ech5aa 0.23 0.26 0.28 0.23 0.21 0.23 0.24 0.23 0.22 0.24 0.23 0.22 0.25 0.25 0.26 0.21 - Weta 0.27 0.30 0.30 0.30 0.27 0.28 0.30 0.27 0.30 0.28 0.30 0.29 0.30 0.29 0.30 0.27 0.30 -

b)

Cri Oph3 0.37 - Oph4 0.35 0.28 - Astla 0.37 0.30 0.31 Ast 2 0.28 0.32 0.29 0.24 - Ast 3 0.31 0.31 0.33 0.25 0.21 Ast 4 0.33 0.33 0.31 0.23 0.23 0.25 - ·, Holo 1 0.33 0.24 0.29 0.27 0.25 0.26 0.26 - Holo 2 0.35 0.25 0.32 0.25 0.25 0.26 0.25 0.19 - Holo 3 0.33 0.26 0.34 0.29 0.27 0.29 0.27 0.16 0.21 ·( Holo 4a 0.41 0.26 0.30 0.21 0.26 0.30 0.25 0.22 0.17 0.20 - ;,.. Holo 5a 0.34 0.28 0.32 0.26 0.26 0.27 0.26 0.20 0.19 0.22 0.18 -

:, Holo 6 0.30 0.30 0.32 0.29 0.28 0.29 0.28 0.22 0.23 0.25 0.21 0.24 - Holo 7a 0.33 0.29 0.32 0.28 0.26 0.29 0.27 0.21 0.22 0.28 0.24 0.25 0.24 - ~ ..... Ech 1 0.37 0.33 0.38 0.27 0.26 0.27 0.27 0.29 0.32 0.31 0.31 0.29 0.34 0.29 - Ech3 0.35 0.31 0.32 0.25 0.23 0.25 0.22 0.21 0.24 0.22 0.24 0.21 0.27 0.25 0.22 - Ech5aa 0.29 0.32 0.36 0.28 0.25 0.27 0.29 0.28 0.27 0.29 0.28 0.26 0.31 0.32 0.32 0.25 - Weta 0.34 0.38 0.39 0.39 0.34 0.35 0.39 0.35 0.39 0.36 0.40 0.37 0.39 0.38 0.39 0.34 0.39 -

43 Chapter Four

The distances among and between classes of echinoderm are as great as those between echinoderm species and the outgroup .

.._ ·- 100 i 0, 90 ,{ =. 80 .....Glo ;I. I ....en 70 - !" = 60 Glo 1:1'1 50 -·r( «:I I ..... 40 r I =Glo ~ 30 ,- I,,. I Glo 20 Cl;. '.,- I 1 0

·V'. 0 1 2 3 >( I Codon position

- J,

1>-, Figure 4.3: Percentage of steps by codon from the tree shown in Figure 4.4. >-

J:,.

The percentage of steps by codon (Fig. 4.3) shows that 85% of substitutions come from - D- third position, 13% come from first position and 2% come from second position. ,I>-

~

1>- Parsimony Analysis of DNA data \~.. Parsimony analysis with non-synonymous weighting 10: 1 gave one MP tree for the full data set (Fig 4.4) and 2 MP trees for the reduced data set. Analysis with >,;

t transition/transversion (Ti/Tv) weighting gave one MP tree for both the full data set and

I)\ the reduced data set. Analysis with unweighted data gave two MP trees for both the full ilt,, and the reduced data sets. Bootstrap analysis of all the MP trees collapsed the majority of .. branches (Fig 4.5) giving weak support for the relationships shown by the MP trees. Only

' 'I'"' one tree (Fig 4.4) showed monophyly of all five classes of echinoderm. This was produced .-.,- from analysis of the full data set weighted for non-synonymous changes. Therefore only ' this MP tree could be reduced to five discrete classes for KH analysis (Fig 4.7). An V example of the topology produced from MP analysis of the Ti/Tv weighted data set and the non-weighted data set for full and reduced taxa lists is shown in Fig 4.6.

44 Chapter Four

.------F. seratissima~ A. constrictum y ....______A. squamatcl\ .______P. mollist{J

P. chitonoides (:J P. californicus (y' { 5 P. astigmatus /1 C. lubrica fY

·" C. curata/.) C. piperata (:/ C. vegae () C. pseudocurata :

C.pallida ~ C. miniata (Y P. parumensis /,) P. californicus(J) f? E. quinquesemita (} ...._ ___ P. lissoplaca 1 P. exigua t, P. paravivipara .__ ___ P. vivipara P. pseudoexigua r:;,. A.pacifica P. brevispina ,"-. P. calcar ~

·' A. pectinifera 'Q'" A. miniata '----- P. ochraceus :·.1. .------P. regularis A. gibbosa <> t ../ L.polaris ~ S. purpuratus @ ...._ __ P. lividus (&

~>..

~ .) A. incisa (fjJ .__ A. lixula Weta 50 changes

Figure 4.4: MP tree from analysis of the full taxa list data set with non-synonymous ·, weighting. (Tree length = 3150, CI= 0.393, RI= 0.590)

45 Chapter Four

Weta F seratissima V

JOO I A. constrictum y I A. squamata J

\fo I P. exigua -{J. I ( 73 I P. paravivipara l ·'- I P. vivipara '¢ .l It\'.) I P. pseudoexigua I r ;, A.pacifica

/001 P. regularis '\>' I A. gibbosa ·r ', ..,, P.gunnii P. brevispina '1 P. ca/car .lJ. L. polaris

' ( P. ochraceus

q71 A. pectinifera '£i, I A. miniata

"IOI E. quinquesemit~ P. lissoplaca ,, P. chitonoides /;) P. californicus (.;/

/CIC) I P. astigmatus /,) I ,.. n I C lubrica 6/ "5'1 ~71 I C curata I) I Cpiperata ;,, :J.,, 11+- C vegae 6J C pseudocurata I C pallida tffJ ':: C miniata P. mollis tfJ ·-·t 'J 1001 P. parumensis i' I P. californicus(J)

') S purpuratustJ>.. •>< P. lividus '2Y ""-~ ,ool A. dufresnei A. incisa @ Cl I ~I I A. lixula f >

r 'J Figure 4.5: Bootstrap analysis of the parsimony tree shown in figure 4.4.

,..' 'r

~

46 Chapter Four

,------F. seratissimaV A. constrictum ¥ '------A. squamata

- L E. quinquesemita ,() '---- P. lissoplaca (JI' P. chitonoides J '.). IJ P. californicus I ( P. astigmatus (J ) 7 C. lubrica {:/ C. curata

~·r ·~~ C. piperata A C. pallida {:/ C. miniata tJ C. vegae C. pseudocurata /,) . ( ----- P. mollis f:7

I \ P. parumensis r{jl P. californicus(l) S. purpuratus ® '----- P. lividus _, P. pseudoexigua A. pacifica t}

P. gunnii ~

} P. brevispina

A. pectinifera ¢ J :,., A. miniata

I I pp ~::,Mpara ft !~ :f

- ·_; ~ P. vzvzpara P. regularis i\ ·,:, .____ -, A. gibbosa "

;"' n A. incisa f/9 ,__ __ A. lixula __'f L------Weta l :,,- - JO changes 1;, Figure 4.6: An example of the most commonly produced topology from parsimony -y J analysis. This tree is the first tree of four produced from unweighted analysis of the full ,' taxon list. (Tree length= 1829, CI= 0.273, RI= 0.493)

47 Chapter Four

Cri Holo Oph Ast Ech

1 '

)

Figure 4.7: Tree trimmed from the tree shown in Figure 4.4. Branches were trimmed to -, produce the shortest possible tree while retaining the overall topology.

' NJ analysis for DNA data All trees produced from NJ analysis showed paraphyly of asteroids and echinoids and monophyly for the remaining three classes, similar to that shown in Fig 4.6 (Fig. 4.8 and

:, 4.10). Again bootstrap analysis of these trees failed to support many of the clades (Fig.

;, 4.9)

-, -,

,,I

,;..,,

~ ""

f

48 Chapter Four

.------F. seratissima ~ .------11 A. constrictum I A. squamata ':f- E. quinquesemitah1 .______P. lissoplaca f:/

P. chitonoides J .~ tJl .----- P. californicus P. astigmatu~ - -; C. lubrica

- I .._ C. curata

_.,, •/ C. piperata j9 -, C. vegae C.pseudocurat~ C. pallida {:/

7 ·) ------P. mollis P. parumensis tJ P. californicus(J)

P. paravivipara J:;, ·. P. vivipara > P. regularis '------1 A. gibbosa {::r P. pseudoexigua ~ A. pacifica 0 ~ P. gunnii '.,. ---- P. brevispina 'I;- .______P. calcar ~ A .. p.ectinifera {J.. --- A. mmzata ., S. purpuratus,d:>i ------P. lividus IZ.Y ') L. polaris ' c} I} i P. ochraceus ..-.:;"" A. dufresnei :-- .. "y,,: A. incisa ®

Weta ' - IOchanges ' Figure 4.8: NJ tree produced from analysis of the full taxa list, unweighted. (Tree length= T r 1852, CI= 0.269, RI= 0.484)

49 Chapter Four

F seratissima "1.f

(.( I A. canstrictum \.!.._ I A. squamata~ P. exigua /Of> I ~P.paraviv ao P. vivipl z /00 P. pseudoe; a .\ A.pacifica -Q

- J /0() 1 P. regul, I A. gibbo

fl I P.gunnii • I P. brevispina -- ( P. calcar L. polaris ~ P. ochrace

·/ I qJ I A. pectinifera I t:r A. miniata E. quinqu !c-. mi~ 6'f I ·~ '"' P. lissoplac P. chitonoides ~ :\., P. californicus ~ ;.r 1001 l tigmatus 100 I I ( brica (/ I C ·ata C. vegae 51 100 () C.pseud ata ',. C. piper ,'/ 1l:. C. pallida I C. minia1 (/ .. :( P. mollis tf) .. , /001 P.parume, P. californicus S.purpi, us P. lividus © 1;.-. A.1 esnei Cfq r--i: \--< A. i, a © lixula Weta r ·.------50 changes

Figure 4.9: Bootstrap analysis of the NJ tree shown in Figure 4.8. -y

T

50 Chapter Four

F. seratissima ~ A. constrictum * ------A. squamata E. quinquesemita Ii) ---- P. lissoplaca ti P. chitonoides /j ~ .\ P. californicus -~ P. astigmatus 7 C. lubrica tJ --- C. curata - C. piperata fJ C. vegae t:/ C. pseudocurata C. pallida /J • •/ C. miniata ; "( '.\ '------P. mollis P. parumensis P. californicus(J) fJ '.. P. exigua {l P. paravivipara ~ '~ '------P. vivipara ,t1, > .______. P. regularis ,9 A. gibbosa P. pseudoexigua . ,·. A. pacifica I} ...---- P. gunnii 'r P. brevispina ~ ---- P. calcar

- ·:::., A. pectinifera [;,

S. purpuratus ~ @ .,. '--- P. lividus .---- L. polaris r)- L------P. ochraceus -Jr• A. dufresnei

~ -.~ A. incisa (Jg

Weta ' 50changes ; Figure 4.10: NJ tree produced from analysis of the full taxa list weighted for non­ 1 7 r synonymous changes. (Tree length= 3283, CI= 0.377, RI= 0.562).

51 Chapter Four

ML analysis for DNA data ML analysis of the reduced data list produced a tree which also showed paraphyly of asteroids and echinoids similar to that shown in Fig 4.6 (Fig 4.11 ). This tree also failed to show the accepted notion that crinoids diverged from other echinoderms very early on.

,------F seratissima ~ -\ .- L---- P. exigua .l;). ,---

'-- A. pectinifera J;}-

~ ...,_, .----- A. constrictum y 'j( - -( '------A. squamata

I !) >

r-- E. quinquesemita ,!} .-- ( ::,_ P. lissoplaca J} .\ ...

.______P. chitonoidesj} ;~ ~ .----- P. californicus tfJ .--- C. vegae tffl ....

" .------P. mollis .-- tP .__

'------P. parumensis tfJ)

-, .------S. purpuratus ® .--- "'I ....._ L--- P. lividus ® eir-- ._____ A. dufresnei @ , "~ '----- P. ochraceus r:} t ·;- 1------L. polaris Q ' ' 50 changes Weta i Figure 4.11: ML analysis of reduced taxa list weighted for non-synonymous changes. (Ti/Tv estimated via ML= 3.372875).

52 Chapter Four

Amino acid analysis Of the 173 amino acid characters, 28 were found to be parsimony informative. MP analysis of the full list of taxa was aborted after it found 1525 trees of equal length (tree length 120). Analysis of the reduced taxa list found 11 equally short trees, the strict

./ ~l consensus of which failed to show monophyly of asteroids and echinoids. NJ analysis of

\ both the full and reduced data list also showed paraphyly for these two classes.

7

., In the majority of trees for all the above analyses, the relationship between holothuroids,

7 ophiuroids and crinoids was consistent. However the relationship between asteroids and

V echinoids was not stable as shown by the trees in Fig 4.6, Fig 4.8, Fig. 4.10 and Fig 4.11.

. ·(

..., ·-~ KH testing of the trimmed tree (Fig 4.7) showed that the phylogenetic tree suggested by this study is not significantly different from any of those previously proposed (Table 4.4). s - Table 4.4: Results from Kishino-Hasegawa tests.

> Tree Length t value P value

Trimmed tree 1075 y Test tree a) (Fig 4.1) 1056 0.9172 0.3595 Test tree b) (Fig. 4.1) 1070 0.2780 0.7812 Test tree c) (Fig. 4.1) 1056 0.9172 0.3595 ~ ·,

·\

:L

PTP and random tree tests were performed to assess the level of signal in the data for the ~... ~',... full taxa list and for the taxa used in the trimmed tree. Results from tests on the full taxa list indicate that there is sufficient signal within the data (Table 4.5). However, results r from analysis of the taxa list from the trimmed tree indicate that there is insufficient signal -, at a deep phylogenetic level (Table 4.5).

53 Chapter Four

Table 4.5: Results from permutation tests and analysis ofrandom trees;* indicates a significant value.

Data (number oftaxa used in analysis) P value from PTP gl value from 1000

I .\ test random trees

-L, Unweighted (35) 0.01 * -0.506437* 7 Ti/Tv weighted (35) 0.01 * -0.238898* y Non-synonymous 10:1 weighted (35) 0.01 * -0.416960* --· ~7 Trimmed data (6) 0.22 -0.228028

-~r The variability of DNA sequence along the region of the COi gene assessed in this study

-( was analysed for several levels of taxonomy; within a family, within a class and between :, >- the five classes (Fig. 4.12). The height of the peaks varies between graphs due to the ,,.' number of taxa used in each analysis. The pattern of variation along the sequence is similar -- ,"- ·, for all four graphs with highs and lows of variation occurring in approximately the same places.

r ;,

" .,.

,:·.....

-~ \!'< ,,

~ ·./ f

54 Chapter Four

Full taxon list

60 50 40 1~ . l 30

I, 20 10 V \ 0 -+11JJII WI n1111 n1 n1 Ui Ill 01 u1 DI WI 11101111 Ui Ill DI DI "I Ell DI DI n1111 "'I DI "I n1 n1 Hi Ill Ill n1 DI Hi "I RI n1 "I "'I n1 n1 Ill Hi n1 n1 "I 01 "I "I Ill DI "I "'I "I 111

~ ~ M O ~ v - 00 ~ N ~ ~ M O ~ v - 00 ~ 0 M ~ ~ - ~ ~ N ~ ~ 0 M ~ 00 - ~ ~ N I .J, v v - - - - N N N M M M M v v v v ~

l

"( Class Holothuroidea

,- •;r 60 50 40 y 30 -1

201 l ,_ ~ ~1~ 111•1m1B 1n1D 1 1l 1l 111l 1l101l 1l 1l 1l 1l 1l 1l 1B 1l 1l 1l 1~ 11111n111n 1l 1l 1B1l1l1l1 l1l11111D1D1 II D11111 •1 n1l1 l1D101111111 ~ ~ M O ~ v - 00 ~ N ~ ~ M O ~ v - 00 ~ 0 '( M ~ ~ - v ~ ~ N ~ ~ 0 M ~ 00 - v ~ ~ N - - - - N N N M M M M v v v v ~ f " -~- Class Asteroidea - 60 50 40 30 20 10 ,·__,._ 0 l~~~~~~~~~~~~~~D1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

m ~ M O ~ V - ro ~ N m ~ M O ~ V - ro ~ 0 ;... ; M ~ m - V ~ m N ~ ~ 0 M ~ ro - V ~ m N N N N M M M M v v V V ~

Family Cucumariidae (Holothuroidea)

',C 60 50

~ 40 i ~'- 30 20 ;'- 10 I I "I 111 "I I '"I 111 n1 "I I "I "l"I "I 111 I n1 n1 "I Ill I "I "I "I "I "I 111 "I "I n1 I "I 1n1n1 "I "I o1 "I n1 "I n1 "I I n1n1n1 "l"I I "I u1a1 n1 "I '"I "I ~ ,r, 0 Pi ~ ~ M O ~ v - 00 ~ N ~ ~ M O ~ v - 00 ~ 0 M ~ ~ - v ~ ~ N ~ ~ 0 M ~ 00 - v ~ ~ N - - - - N N N M M M M v v v v ~

i'

;- Nucleotide position along region of DNA :~. ;.~

f Figure 4.12: DNA sequence variation for the region of COI assessed for different levels of taxonomy

55 Chapter Four

Discussion

The COI gene was chosen for this phylogenetic analysis because it is a relatively fast

( :( evolving gene. It was thought that this gene would be able to provide more signal for the I -

\t .. \ region of echinoderm taxonomy currently in dispute, that is, the relationship of asteroids \ and ophiuroids to the echinozoa. Unfortunately as well as providing more signal a large

quantity of noise was also introduced to the analysis. Information from distance data, the ratio of transitions to transversions and the number of substitutions by base, suggests that this gene is near saturation for this level of phylogenetic analysis. -~ - The variability of DNA sequence along the COI gene was assessed for several levels of 11 taxonomy. As COI is a coding gene the DNA sequence is somewhat constrained by the i final protein structure. This means that some regions of DNA will be able to vary more 1: than others depending on the final structure of the protein. Results from the comparison of --· ~>- DNA variation at different taxonomic levels showed that regions of relatively high variation within a family were also regions of high variation within the phylum indicating that :,. saturation of these regions has occurred at an early taxonomic level. In· an attempt to reduce the amount of noise introduced through saturation, the DNA data were weighted i ,>- with two different weighting schemes and the amino acid sequence was also used for analysis with PAUP (Swofford, 1993). Only MP analysis of the full DNA data set weighted for non-synonymous changes produced a tree topology showing monophyly for r- ..,, all five classes of echinoderm. The general topology of this tree was compared to

_, previously proposed phylogenies but was not significantly different. All other analyses ':~-- including those done on the amino acid data produced trees showing monophyly of

~ \,4 ophiuroids, crinoids and holothuroids but paraphyiy of asteroids and echinoids. A r possible explanation for the lack of a tree showing monophyly from amino acid data

T analysis is that saturation has occurred at the amino acid level also. A study by Lunt et al.

.'-1 .; (1996) showed that the 5' end of insect COI contains relatively variable regions when T compared to the 3' end. This may also be the case with echinoderm COL However, the number of substitutions able to occur in these regions will still be constrained by the final

56 Chapter Four

structure of COL Therefore, in most cases, any amino acid substitutes will have to be replaced with an amino acid of similar biochemical properties. This limits the amount of

variation able to occur. ! L

I • \ 1. ,, . Phylogenetic analysis of echinoderm COI failed to adequately resolve the relationship ,' ·-\. between the five extant classes. As mentioned, this is probably due to the amount of noise

introduced by the use of a fast evolving gene. Results from PTP tests and the analysis of 1000 random trees of the full data set suggest that there is sufficient signal within the data. I- However when these analyses were conducted on the taxa from the trimmed tree used in

I > KH analysis, there was no significant signal. This suggests that at a deep phylogenetic I. level the degree of noise has completely obscured any signal. Therefore COI appears to be

evolving too fast for this type of phylogenetic analysis.

Several approaches have been taken to determine the relationship between the five extant -"-· classes of echinoderm. These include analysis of fossil records, larval morphology (reviewed in Smith, 1984), mitochondrial gene arrangement (Smith et al., 1993) and molecular analysis of 18S, 28S and COI (Wada and Satoh, 1994; Littlewood et al., 1997).

~ .... So far none of these analyses have been able to adequately resolve the relationship between II- :, ophiuroids, asteroids and echinozoa. Further analysis of mitochondrial gene arrangements

,, may be able to resolve this. However it is possible that these groups radiated in such a short period of time that no analysis will be able to determine their relationship

" conclusively.

-'

\ t~

;r

57 Chapter Five

Chapter Five: General Discussion

,,,\,11 .I,

' \ This thesis has analysed several aspects of the genetics of the brittlestar ) I .\ I -ic Astrobrachion constrictum; population genetics in Fiordland, differentiation of colour

;, morphs, presence of heteroplasmy and the phylogeny of echinoderms. These studies

are independent ideas and as such will be summarised separately in this chapter.

Despite the assumed short larval period of Astrobrachion constrictum and the

apparent isolating nature of Fiordland's geography and hydrology, there is no - ·;

., ) significant genetic differentiation among the sampled fiords. It is argued that the homogeneity is not likely to be due to continual larval input from the continental shelf ,, population of snake stars as some differentiation between fiord and coastal

;~ ::,, populations of another echinoderm species with a long larval period has been shown

(Mladenov et al., 1997). Assuming that there is minimal input of larvae from the

continental shelf population it also seems unlikely that there is sufficient larval flow

between fiords to maintain genetically homogeneous populations. As has been shown

with other benthic marine invertebrates (Helmuth et al., 1994) migration or passive

dispersal of juveniles and adults can have a significant impact on the genetic structure

.> I• of separate populations. As it is currently unkown what happens to A. constrictum

./',: )c before recruitment at one year old (Stewart and Mladenov, 1997) migration of I juveniles could have an influence on the genetic structure of different fiord

\ J 1' populations.

c-

Physical features such as geological history and, in the marine environment, current

direction and strength, can have an influence on the degree of difference in genetic ' r

~ structure between geographically separate populations (Voight, 1988; Benzie and

Stoddart, 1992). The fiord environment as it is known today has only been in

58 Chapter Five

existence since the last ice age, 6,000 to 10,000 years ago (Pillans et al., 1992; Pickrill

et al., 1992). This is a relatively short period of time for genetic differentiation to

have occurred without strong selection pressure. Therefore there may not have been ~

.A sufficient time for differences in population genetic structure to occur to a significant ) I level able to be detected by COI analysis.

;;"'- __j,.

The five colour morphotypes of Astrobrachion constrictum are not significantly

" different genetically therefore the morphs are, as expected, not cryptic species. The presence of such striking colour differences between individuals of the snake star is

curious. Sublethal predation, most probably by fish, appears to be common in A.

., constrictum (Stewart, 1995). However, occurrences of lethal predation have not been

J > documented. For predation to be the selection pressure behind the presence of colour I ~'..

'- morphs it would either have to have a negative effect upon individual fitness or be ,,. lethal. The effect of sublethal predation on the fitness of A. constrictum has not been

::,. studied.

' The presence of heteroplasmy from either tandem repeats or base substitutions, has

not been reported for any other echinoderm species. It is unlikeiy that the

heteroplasmy in Astrobrachion constrictum arose by mutation de nova or that the :, mutations have accumulated and been maintained through several generations.

\.. ~ However, without more information on mutation and sorting out rates of the ,> ) echinoderm COI gene this possibility cannot be completely discounted. The most probable explanation for the presence of two previously recognised COI haplotypes

' i.,. differing by up to 2 bp in one animai is paternai input. Leakage of fhe paternal

~ mitochondrial genome has been shown for other invertebrate species (Kondo et al.,

.~ 1990; Wenne and Skibinski, 1995) .

'··;,-

To discount the possibility of the reported heteroplasmy arising from mutation within an individual or germline, analysis of mutation and sorting out rates of echinoderm

59 Chapter Five

COI are necessary. It should also be noted that as low levels of heteroplasmy

resulting from small numbers of base substitutions are hard to detect, methods such as

those used in this thesis could perhaps reveal higher levels of heteroplasmy than

currently believed to exist.

I , ~.i. I '·;,, .\ Analysis of echinoderm COI failed to produce a significantly different phylogeny than

--r those previously proposed. This is probably due to the large amount of noise

'l introduced into the study by analysing a relatively fast evolving gene. Several

,~ '.r attempts to reduce the amount of noise obscuring the signal were unsuccessful. I , ·, As well as answering the questions initially posed, this thesis has also highlighted f-~) several areas of further study. To more fully understand the mechanisms controlling 1~ gene flow among fiord populations of the snake star, studies on the genetic structure

.:-.... of populations from other areas around the New Zealand coast, such as the continental shelf off the Fiordland coast, would be beneficial. Studies identifying the mode of larval development and location ofjuveniles before recruitment would also be i : useful in understanding the mechanisms behind gene flow in this species. Any further studies assessing the population genetics of A. constrictum should utilise more

~ sensitive genetic techniques, such as microsatellite analysis, to more accurately resolve i ;, genetic differences. ,',' i X i, Although COI has been shown to be too rapidly evolving to resolve echinoderm phylogeny the idea of using a faster evolving gene than those currently used warrants further attention. Such genes could include, 12S, 16S and ATPase. Further analysis ' .... " of rearrangements in the echinoderm mitochondrial genome could also provide the ·:; :, answer to the phylogeny of this phylum.

'

60 :ji~i! References

References

Augustinus, P. C., 1992. Outlet glacier trough size-drainage area relationships, Fiordland, j New Zealand. Geomorphology 4: 347-361. i .J

·> ' Avise, J.C., 1994. Molecular markers, natural history and evolution. New York: Chapman '/ and Hall. ·r

\ t~·---

~ Ayala, F. J., Hedgecock, D., Zumwalt, G. S. and Valentine, J. W., 1973. Genetic variation ., in Tridacna maxima, an ecological analog of some unsuccessful evolutionary lineages . ? Evolution 27: 177-191. ;;

>- -) t '... Ayre, D. J., 1990. Population subdivision in Australian temperate marine invertebrates: &. Larval connections versus historical factors. Australian Journal ofEcology 15: 403-411. " Baker, A., 1980. Euryalinid Ophiuroidea (Echinodermata) from Australia, New Zealand and the south-west Pacific Ocean. New Zealand Journal ofZoology 7: 11-83. >

'-( Baker, A. N., Rowe, F. W. E. and Clark, H. S., 1986. A new class of Echinodermata from

'.r- ;),. New Zealand. Nature 321 (26): 862-864.

> Barton, N. and Jones, J. S., 1983. Mitochondrial DNA: new clues about evolution. Nature 306: 317-318. '

;, Benzie, J. A. H. and Stoddart, J. A., 1992. Genetic structure of crown-of-thorns starfish .7-

- J'i_,;, (Acanthaster planci) in Australia. Marine Biology 112: 631-639.

;,- Biju-Duval, C., Ennafaa, H., Dennebouy, N., Monnerot, M., Mignotte, F., Soriguer, R. C., :, Gaaied, A. E., Hili, A. E. and Mounolou, J.-C., 1991. Mitochondrial DNA evolution in Lagomorphs: origin of systematic heteroplasmy and organization of diversity in European -, rabbits. Journal ofMolecular Evolution 33: 92-102.

61 References

Burton, R. and Feldman, M., 1982. Population genetics of coastal and estuarine invertebrates: does larval behaviour influence population structure? In: Estuarine Comparisons, (Kennedy, V. S., ed.) Academic Press; 537-550.

·)-- Burton, R. S. and Lee, B-N., 1994. Nuclear and mitochondrial gene genealogies and

--- -t allozyme polymorphism across a major phylogeographic break in the copepod Tigriopus

californicus. Proceedings ofthe National Academy ofScience, USA 91: 5197-5201.

"(

~ Christiansen, F. B., Frydenberg, 0. and Simonsen, V., 1984. Genetics of Zoarces

~{ populations XII. Variations at the polymorphic loci Pgml, Pgmll, Hbl and Estill in fjords.

'7 Hereditas 101: 37-48. ;,

( "J

,,~ Christiansen, F. B., Nielsen, V. H. and Simonsen, V., 1988. Genetics of Zoarces t populations. XV. Genetic and morphological variation in Mariager Fjord. Hereditas 109:

"). 99-112.

~y- Clayton, D. A., 1982. Replication of animal mitochondrial DNA. Cell 28: 693-705.

'r

Corson, J., in prep. Burnie substances in southern New Zealand aquatic environments.

~ (M.Sc. thesis ),University of Otago, Dunedin.

j-

7 Day, A. J. and Bayne, B. L., 1988. Allozyme variation in populations of the dog-whelk ~-

)- Nucella lapillus. (Prosobranchia: Muricacea) from the south-west peninsula of England. ,- Marine Biology 99: 93-100. l !,--d

Daly, J. C., Wilkinson, P. and Shaw, D. D., 1981. Reproductive isolation in relation to ' tr allozymic and chromosomal differentiation in the grasshopper Caledia captiva. Evolution

,- 35 (6): 1164-1179.

·;

62 References

De Giorgi, C., 1988. Mitochondrial polymorphism in the eggs of the sea urchin Arbacia lixula. Cell Biology International Reports 12 (5): 407-412.

Densmore, L. D., Wright, J. W. and Brown, W. M., 1985. Length variation and .,, heteroplasmy are frequent in mitochondrial DNA from parthenogenetic and bisexual lizards ~- (Genus Cnemidophorus). Genetics 110: 689-707.

Dix, T. G., 1969. Larval life span of the echinoid Evechinus chloroticus (Val.). New --r -· Zealand Journal ofMarine and Freshwater Research 3: 13-16. I ,

'./" Dix, T. G., 1970. Biology of Evechinus chloroticus (Echinoidea: Echinometridae) from r ? ! different localities: 3. Reproduction. 1; ·1 New Zealand Journal of Marine and Freshwater \' j.__ t Research 4 (4): 385-405.

I ,:,.. ! Edmunds, S., Moberg, P. E. and Burton, R. S., 1996. Allozyme and mitochondrial DNA evidence of population subdivision in the purple sea urchin Strongylocentrotus purpuratus.

Marine Biology 126: 443-450.

)..

I , Excoffier, L., Smouse, P. E. and Quattro, J. M., 1992. Analysis of molecular variance - ;,- inferred from metric distance among DNA haplotypes: Application to human

mitochondrial DNA restriction data. Genetics 131: 4 79-491.

'l

-~~ Field, K. G., Olsen, G.J., Lane, D. J., Giovannoni, S. J., Ghiselin, M. T., Raff, E. C., Pace,

T ...... N. R. and Raff, R. A., 1988. Molecular phylogeny of the animal kingdon. Science 239: 748- ,._.. 753. ,'-

?"

'r Foltz, D. W., Stickle, W. B., Campagnaro, E. L. and Himel, A. E., 1996. Mitochondrial

I T DNA polymorphisms reveal additional genetic heterogeneity within the Leptasterias ' hexactis (Echinodermata: Asteroidea) species complex. Marine Biology 125: 569-578.

63 References

Frydenberg, 0., Gyldenholm, A. 0., Hjorth, J. P. and Simonsen, V., 1973. Genetics of Zoarces populations. III. Geographic variations in the esterase polymorphism Est!II. Hereditas 73: 233-238.

) -), Gaffney, P. M., Scott, T. M., Koehn, R. K. and Diehl, W. J., 1990. Interrelationships of

,l heterozygosity, growth rate and heterozygote deficiencies in the Coot Clam, Mulinia 7 lateralis. Genetics 124: 687-699.

,., Gaines, M. S., Caldwell, J. and Vivas, A. M., 1974. Genetic variation in the mangrove ., periwinkle Littorina angulifera. Marine Biology 27: 327-332 . ,7

) :> Gardner, J. P. A. and Skibinski, D. 0. F., 1991. Mitochondrial DNA and allozyme 1 \' J .,_ covariation in a hybrid mussel population. Journal of Experimental Marine Biology and I ~ ' Ecology 149: 45-54. I .._,,

,' Garner, D. M., 1964. The hydrology of Milford Sound. In: Studies of a New Zealand fiord. New Zealand Oceanographic Institute Memoir, (Skerman, T. M., ed.) 25-33.

}

>- - Grange, K. R., Singleton, R. J., Richardson, J. R., Hill, P. J. and Main, W. deL., 1981. .· Shallow rock-wall biological associations of some southern fiords of New Zealand New -l Zealand Journal ofZoology 8: 209-227.

'1 ,

:" Grassle, J. F. and Grassle, J. P., 1979. Life histories and genetic variation in manne ,- invertebrates. In: Marine Organisms: Genetics, Ecology and Evolution, (Battaglia, B.and "'-' Beardmore, J. A., eds.). New York: PlenTu.11 Press; 347-364. c, i ;r

1 Griffiths, A. J. F., Miller, J. H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W. M., 1993. f 7 An introduction to genetic analysis, 5th Edition. New York: WH Freeman Company.

7'

64 References

Gyllensten, U., Wharton, D., Josefsson, A. and Wilson, A. C., 1991. Paternal inheritance

of mitochondrial DNA in mice. Nature 352: 255-257.

Harrison, R. G., 1989. Animal mitochondrial DNA as a genetic marker in population and

evolutionary biology. Trends in Ecology and Evolution 4: 6-11. J f

l

Hebert, P. D. N. and Beaton, M. J., 1989. Methodologies for allozyme analysis using

cellulose acetate electrophoresis; A practical handbook. Helena Laboratories.

j

I " Hedgecock, D ., 1979. Biochemical genetic variation and evidence of speciation m J Chthamalus barnacles of the tropical eastern Pacific Ocean. Marine Biology 54: 297-214.

! ?

Helmuth, B., Veit, R. R. and Holberton, R., 1994. Long-distance dispersal of the

subantartic brooding bivalve (Gaimardia trapesina) by kelp-rafting. Marine Biology 120: :,._ 421-426. J;,

~~ Hensley, R. T., Beardmore, J. A. and Tyler, P.A., 1995. Genetic variance in Ophiomusium

£ lymani (Ophiuroidea: Echinodermata) from lower bathyal depths in the Rockall Trough )c (northeast Atlantic). Marine Biology 121: 469-475.

Hess, H., Bingham, B., Cohen, S., Grosberg, R. K., Jefferson, W. and Walters, L., 1988.

<\ The scale of genetic differentiation in Leptosynapta clarki (Hetling), an infaunal brooding ;cc holothuroid. Journal ofExperimental Marine Biology and Ecology 122: 187-194.

''N TT Hillis, D. M., Moritz, C. and Mable, B. .I',._,, 1996. A1olecular systematics. Sunderland,

Massachusetts: Sinauer Ass. Inc. 7

7 Himeno, H., Masaki, H., Kawai, T., Ohta, T., Kumagai, I., Miura, K.-I. and Watanabe, K.,

1987. Unusual genetic codes and a novel gene structure for tRNA (Ser, AGY) in starfish mitochondrial DNA. Gene 56: 219-230.

65 References

Hoeh, W. R., Blakley, K. H. and Brown, W. M., 1991. Heteroplasmy suggests limited biparental inheritence of Mytilus mitochondrial DNA. Science 251: 1488-1490.

'( Hoelzel, A. R., Hancock, J. M. and Dover, G. A., 1993. Generation of VNTRs and

..J heteroplasmy by sequence turnover in the mitochondrial control region of two elephant

J seal species. Journal ofMolecular Evolution 37: 190-197.

--y

7 Hyman, L. H., 1955. The Invertebrates: Echinodermata. New York: McGraw-Hill Book

"r Company.

y

·< Jacobs, H. T., Balfe, P., Cohen, B. L., Farquharson, A. and Comito, L., 1988. Phylogentic implications of genome rearrangement and sequence evolution in echinoderm mitochondrial DNA. In: Echinoderm phylogeny and evolutionary biology, (Paul, C. R. C. and Smith, A. ,._" B., eds.). New York: Oxford University Press: 121-137. ·"'

(; Jorstad, K. E. and Pedersen, S. A., 1986. Discrimination of herring populations in a

:-;. northern Norwegian fjord: genetics and biological aspects. Council Meet. Cons. int. Explor.

Mer: 1986/M:63. I ·~

Jorstad, K. E., Dahle, G. and Paulsen, 0. I., 1994. Genetic comparison between Pacific

Ii I herring (Clupea pallasi) and a Norwegian fjord stock of Atlantic herring (Clupea harengus).

I ~ Canadian Journal ofFisheries and Aquatic Science 51 (Suppl. 1): 233-239.

k Koehn, R. K., Milkman, R. and Mitton, J. B., 1976. Population genetics of marine

clt,,-4 pelecypods. IV. Selection, migration and genetic differentiation in the blue mussel Mytilus edulis. Evolution 30: 2-32. T '

), 7 Koehn, R. K., Newell, R. I. E. and Immermann, F., 1980. Maintenance of an aminopeptidase allele frequency dine by natural selection. Proceedings of the National Academy ofSciences, USA 77 (9): 5385-5389.

66 References

Kondo, R., Satta, Y., Matsuura, E. T., Ishiwa, H., Takahata, N. and Chigusa, S. I., 1990.

Incomplete maternal transmission of mitochondrial DNA in Drosophila. Genetics 126:

657-663 . . (

I ·I Kwast, K. E., Foltz, D. W. and Stickle, W. B., 1990. Population genetics and systematics

.\ of the Leptasterias hexactis (Echinodermata: Asteroidea) species complex. Marine Biology

7 105: 477-489. ' '7 , Lawrence, J., 1987. A functional biology ofechinoderms. London: Croom Helm.

'y

Lessa, E. P. and Applebaum, G., 1993. Screening techniques for detecting allelic variation in "cf DNA sequences. Molecular Ecology 2: 119-129. I 1.~ I Li, G. and Hedgecock, D., 1998. Genetic heterogeneity, detected by PCR-SSCP, among ... samples of larval Pacific oysters (Crassostrea gigas) supports the hypothesis of large ,:,_ variance in reproductive success. Canadian Journal of Fisheries and Aquatic Science 55:

,'> 1025-1033.

i Littlewood, D. T. J., Smith, A. B., Clough, K. A. and Emson, R. H., 1997. The

interrelationship of the echinoderm classes: morphological and molecular evidence.

I t Biological Journal ofthe Linnean Society 61: 409-438. ,,

' ';,. Lunt, D. H., Zhang, D.-X., Szymura, J. M. and Hewitt, G. M., 1996. The insect I :- ·~. cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic ,,_. studies. Insect Molecular Biology 5 (3): 153-165. "

;r

1 Lunt, D. H., Whipple, L. E. and Hyman, B. C., 1998. Mitochondrial DNA variable number I ,, tandem repeats (VNTRs): Utility and problems in molecular ecology. Molecular Ecology 7: .- 1441-1455.

67 References

McKellar, I. C. and Soons, J. M., 1982. Fiordland. In: Landforms of New Zealand, 2nd

Edition (Soons, J.M. and Selby, M. J., eds.) New Zealand: Longman Paul: 505-515.

( McMillan, 0. W., Raff, R. A. and Palumbi, S. R., 1992. Population genetic consequences

-i of developmental evolution in sea urchins (Genus Heliocidaris). Evolution 46 (5): 1299- ) ,l 1312.

7

Maddison, W. P. and Maddison, D. R. 1992. MacClade: analysis of phylogeny and

" character evolution, version 3. 0. Sinauer Associates, Sunderland, Massechusetts. '>

J Magoulas, A. and Zouros, E., 1993. Restriction-site heteroplasmy in anchovy (Engraulis ':I encrasicolus) indicates incidental biparental inheritence of mitochondrial DNA. Molecular i .l. Biology and Evolution 10 (2): 319-325 .

'., Matsumoto, L., Kasamatsu, H., Piko, L. and Vinograd, J., 1974. Mitochondrial DNA '.; replication in sea urchin oocytes. The Journal of Cell Biology 63: 146-159.

:>-

May, B., 1992. Starch gel electrophoresis of allozymes. In: Molecular genetic analysis of ) populations, a practical approach, (Hoelzel, A. R., ed.) Oxford: Oxford University Press: 1-27. ·~

" Miller, K.J., 1996. Piecing together the reproductive habits of New Zealand's endemic

.c- black corals. Water and Atmosphere 4: 18-19 . ..,

~ Miller, K. J., 1997. Genetic structure of black coral populations in New Zealand's fiords. ·' Marine Ecology Progress Series 161: 123-132. 7

? Miracle, A. L. and Campton, D. E. 1995. Tandem repeat sequence variation and length

)' heteroplasmy in the mitochondrial DNA d-loop of the threatened Gulf of Mexico sturgeon, Acipenser oxyrhynchus desotoi. Journal ofHeredity 86: 22-27.

68 References

Mladenov, P. V., Allibone, R. M. and Wallis, G. P., 1997. Genetic differentiation in the

New Zealand sea urchin Evechinus chloroticus (Echinodermata: Echinoidea). New Zealand

Journal ofMarine and Freshwater Research 31: 261-269. a(

I .. j Moritz, C., Dowling, T. E. and Brown, W. M., 1987. Evolution of animal mitochondrial

,\ DNA: Relevance for population biology and systematics. Annual Review of Ecology and

··~7 Systematics 18: 269-292.

,, Murphy, R. W., Sites, J. W., Buth, D. G. and Haufler, C. H., 1990. Proteins 1: Isozyme

Electrophoresis. In: Molecular Systematics, (Hillis, D. M. and Moritz, C., eds.). -< Sunderland, Massechusetts, U.S.A.: Sinauer Associates, Inc.: Chapter 4. -;I

( (}. Newcombe, J., Dyer, S., Blackwell, L., Cartwright, K., Palmer, W. H. and McFadden, J. ,,­ 1997. PCR-single-stranded confirmational polymorphism analysis for non-culture-based i' I 'o, subtyping of meningococcal strains in clinical specimens. Journal of Clinical Microbiology ,S 35 (7): 1809-1812. I> I

~ Nichols, D., 1962. Echinoderms. London: Hutchinson & Co. LTD. >

>

If'' Nishida, M. and Lucas, J. S., 1988. Genetic differences between geographic populations of 1, the Crown-of-thorns starfish throughout the Pacific region. Marine Biology 98: 359-368. "" ' ,. Ostellari, L., Bargelloni, L., Penzo, E., Patamello, P. and Patarnello, T., 1996. Optimization

of single-strand conformation polymorphism and sequence analysis of the mitochondrial

:>- control region in Pagellus bogaraveo (Sparidae, Teleostei): Rationalized tools in fish " .. population biology. Animal Genetics 27: 423-427 .

' Palumbi, S. R., 1995. Using genetics as an indirect estimator of larval dispersal. In: Ecology ofMarine Invertebrate Larvae, (McEdward, L., ed.) Boca Raton: CRC: 369-388.

69 References

Paul, C. R. C. and Smith, A. B., 1988. Echinoderm phylogeny and evolutionary biology.

New York: Oxford University Press.

,,( Pickrill, R. A., Fenner, J. M. and McGlone, M. S., 1992. Late quaternary evolution of a I 1·) fjord environment in Preservation Inlet, New Zealand. Quarternary Research 38: 331-346. I 1· ; 1·7 Pillans, B., Pullar, W. A., Selby, M. J. and Soons, J.M., 1992. The age and development of I, the New Zealand landscape. In: Landforms ofNew Zealand, 2nd Edition (Soons, J. M. and t

? Selby, M. J., eds.) New Zealand, Longman Paul: 31-62.

Poulik, M. D., 1957. Starch gel electrophoresis in a discontinuous system of buffers.

7 Nature 180: 1477-1478. C)-

l. ,- Rand, D. M. and Harrison, R. G., 1986. Mitochondrial DNA transmission genetics in "· crickets. Genetics 114: 955-970. >

Raff, R. A., Field, K. G., Ghiselin, M. T., Lane, D. J., Olsen, G. J., Pace, N. R., Parks, A. I'>- L., Parr, B. A. and Raff, E. C., 1988. Molecular analysis of distant phylogenetic > relationships in echinoderms. In: Echinoderm phylogeny and evolutionary biology, (Paul, C. ·' R. C. and Smith, A. B., eds). New York: Oxford University Press: 29-41.

Richardson, B. J., Baverstock, P. R. and Adams, M., 1986. Allozyme Electrophoresis: A "' ,. handbook for animal systematics and population studies. Sydney: Academic Press. ,...

-, Sambrook, J., Fritsch, E. F. and Maniatis, T., 1989. Molecular cloning: A laboratory 1 >-.. I ; manual. U.S.A.: Cold Spring Harbour Lab Press. j I • : Selander, R. K., Yang, S. Y., Lewontin, R. C. and Johnson, W. E., 1970. Genetic variation ' in the horseshoe crab (Limulus polyphemus), a phylogentic relic. Evolution 24: 402-414.

70 References

Selander, R. K., Smith, M. H., Yang, Y. S., Johnson, W. B. and Gentry, J. B., 1971. Biochemical polymorphism and systematics in the genus Peromyscus I: variation in the old-field mouse (Peromyscus polionotus). University of Texas Publication 7103. Studies in .• £ _,, Genetics 6: 49-90.

,\ Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. and Flook, P., 1994. Evolution, -, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annual Entomological Society ofAmerica 87 ., (6): 651-701.

.- ? Skold, M., Wing, S. R. and Mladenov, P. V., in prep. Genetic subdivision of the sea star ;, Coscinasterias muricata in the fiords of New Zealand .

...... "

~ c,, Smith, A., 1984. Classification of the Echinodermata. Paleontology 27 (3): 431-459. ',

;,- Smith, M., Arndt, A., Gorski, S. and Fajber, E., 1993. The phylogeny of echinoderm t,- > classes based on mitochondrial gene arrangements. Journal of Molecular Evolution 36: 545-

- '"' 554. :,.I

Smith, A. B. and Paterson, G. L. J., 1995. Ophiuroid phylogeny and higher taxonomy: '}- '

'\ morphological, molecular and palaeontological perspectives. Zoological Journal of the Linnean Society 114: 213-243.

Solignac, M., Monnerot, M. and Mounolou, J.-C., 1983. Mitochondrial DNA

C>-.. heteroplasmy in Drosophila mauritiana. Proceedings of the National Academy of Sciences,

USA 80: 6942-6946. -;,- H

"1" ' Sperling, F. A.H. and Hickey, D. A. 1994. Mitochondrial DNA sequence variation in the -., spruce budworm species complex (Choristoneura: Lepidoptera). Molecular Biology and Evolution 11(4): 656-665.

71 References

Stanton, B. R., 1976. Circulation and hydrology off the west coast of the South Island, New Zealand. New Zealand Journal ofMarine and Freshwater Research 10 (3): 445-467 .

•• I_ ~ Stanton, B. R., 1978. Hydrology of Caswell and Nancy Sounds. In: Fiord Studies: Caswell

,J and Nancy Sounds, New Zealand New Zealand Oceanographic Institute Memoir, (Glasby, G. P., ed.): 73-82 . ..,.

Stanton, B. R., 1986. Winter oceanographic observations in some New Zealand fiords. New -r Zealand Journal ofMarine and Freshwater Research 20: 299-314.

-.; .,

Stanton, B. R. and Pickard, G. L., 1981. Physical oceanography of the New Zealand '?

> Fjords. New Zealand Oceanographic Institute Memoir 88: 37pp.

'->-:,.

1·- ~ Stanton, B. R. and Pickard, G. L., 1982. Physical oceanography of the New Zealand -~ Fjords. In: Fjord Oceanography, (Freeland, H. J., Farmer, D. M. and. Levings, C. D, eds.). ). New York: Plenum Press: 329-332. v- :> r !> Stewart, B., 1995. The biology of the euryalid snake star Astrobrachion constrictum ::, (Echinodermata: Ophiuroidea) (Unpublished PhD thesis). University of Otago, Dunedin. I\ :,

-.--l ->

' Stewart, B. and Mladenov, P., 1993. Aspects of reproduction and anatomy of the

~ euryalinid snake star Astrobrachion constrictum. In: Echinoderms through time: t'

>--.,, Proceedings ofthe eighth international echinoderm coriference Dijon, France. (Balkema, A.

- -t' ~;;I A. ed.): 491-497. ,,_

" Stewart, B. G. and Mladenov P. V., 1997. Population structure, growth and recruitment of v--">

-, ,' the euryalinid brittle-star Astrobrachion constrictum (Echinodermata: Ophiuroidea) in ' Doubtful Sound, Fiordland, New Zealand. Marine Biology 127: 687-697.

72 References

Stickle, W. B., Foltz, D. W., Katoh, M. and Nguyen, H. L., 1992. Genetic structure and

mode of reproduction in five species of sea stars (Echinodermata: Asteroidea) from the

Alaskan coast. Canadian Journal ofZoology 70: 1723-1728. -I, >

,.J ,j .. Strathmann, M. F. and Rumrill, S.S., 1987. Phylum Echinodermata, Class Ophiuroidea. In: Reproduction and development of marine invertebrates of the northern Pacific coast. Data

--r· "'I and methods for the study of eggs, embryos, and larvae, (Strathmann, M. F., ed.) Seattle:

University of Washington Press: 556-573. I y

IV ,.i Strautnieks, S., Rutland, P., Winter, R. M., Baraitser, M. and Malcom, S. 1992. Pelizaeus­ I,

Merzbacher disease: detection of mutations Thr181 -Pro and Leu223-Pro in the proteolipid 7 protein gene, and prenatal diagnosis. American Journal ofHuman Genetics 51: 871-878. >

4-- '.'.\

1 Swofford, D. L., 1993. PAUP: Phylogenetic analysis using parsimony version 4.64d. )_ .,. Computer program distributed by the Illionis Natural History Survey, Champaign, Illinois .

I~> Tanaka, Y., Nakashima, H., Otsuka, T., Nemoto, Y, Niiro, H., Yamaoka, K., Ogami, E.-I., Ir ~ I Arinobu, Y., Tachida, H., Imamura, T. and Niho, Y. 1997. Detection of polymorphisms :,. within the human ILIO receptor cDNA gene sequence by RT-PCR RFLP. {> Immunogenetics

~ ·, 46: 439-441.

'

\, Vasques, J., 1997. Sedimentation and housecleaning: the interactions of Astrobrachion I ,. I constrictum and its host Antipathes fiordensis. (Unpublished MARI 480 project). ~

. . I University of Otago, Dunedin. ,,__ I " Voight, J. R., 1988. Trans-Panamanian geminate octopods (Mollusca: Octopoda). 't';

-, 'i Malacologia 29 (1): 289-194.

I , I •

73 References

Wada, H. and Satoh, N., 1994. Phylogenetic relationships among extant classes of

echinoderms, as inferred from sequences of 18S rDNA, coincide with relationships deduced

from the fossil record. Journal ofMolecular Evolution 38: 41-49. -<

") I., Walker, M. M., 1984. Larval life span, larval settlement, and early growth of Evechinus

,l chloroticus (Valenciennes). New Zealand Journal of Marine and Freshwater Research 18:

-, -; 393-397.

~ .,, Wallis, G. P., 1987. Mitochondrial DNA insertion polymorphism and germ line y -1 heteroplasmy in the Triturus cristatus complex. Heredity 58: 229-238. r ''" Ward, R. D. and Andrew, J., 1995. Population genetics of the northern Pacific seastar '.> ,, Asterias amurensis (Echinodermata: Asteriidae): allozyme differentiation among Japanese,

Russian, and recently inroduced Tasmanian populations. Marine Biology 124: 99-109.

I J. "'' Wenink, P. W., Baker, A. J. and Tilanus, M. G. J., 1994. Mitochondrial control-region

V" :> sequences in two shorebird species, the turnstone and the dunlin, and their utility m

J- '.> population genetic studies. Molecular Biology and Evolution 11 (1 ): 22-31. >

! > Wenne, R. and Skibinski, D. 0. F., 1995. Mitochondrial DNA heteroplasmy in European

.... ")- populations of the mussel Mytilus trossulus. Marine Biology 122: 619-624 . ,,.

~

.. ,r:,.. Williams, P. W., 1982. Speleothem dates, quaternary terraces and uplift rates in New

:, -, Zealand. Nature 298 (15): 257-298.

>;.,---·

" Wilson, A. C., Cann, R. L., Carr, S. M., George, M., Gyllensten, U. B., Helm-Bychowski, ~;,'

,., :.,- K. M., Higuchi, R. G., Palumbi, S. R., Prager, E. M., Sage, R. D. and Stoneking, M., 1985.

"," Mitochondrial DNA and two perspectives on evolutionary genetics. Biological Journal of

the Linnean Society 26: 375-400.

74 References

Wing, S. R. and Skold, M., in prep.. Genetic subdivision of the sea urchin Evechinus chloroticus in the fiords of New Zealand.

·< ' Zouros, E. and Foltz, D. W., 1984. Possible explanations of heterozygote deficiency in

I,, bivalve molluscs. Malacologia 25 (2): 583-591.

~

-,. y Zouros, E., Ball, A. 0., Saavedra, C. and Freeman, K. R., 1994. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proceedings of the National 't Academy ofSciences, USA 91: 7463-7467.

·"' •I

/

'7

"- -- "

\.

,\.

\

> I).. t I:,.

?

I>

-- ~t

v

t>

-, ,,

\ ~'7

......

c,.

'l'- ·/ -,,

~ .,,

75 Appendices

Appendix A

( .., Collection information for specimen of Astrobrachion constrictum.

~

" Name of Site Map Ref. Date Depth No . . :' Crowded House, Doubtful Sound 45°25.0'S/167°07.3 'E 30/10/96 20m 9

Crowded House, Doubtful Sound 29/6/98 20m 11

Tricky Cove, Doubtful Sound 45°21.0'S/167°03.3 'E 26/5/97 20m 7 ' .·(

•} Tricky Cove, Doubtful Sound 29/6/98 20m 11

~ Espinosa Point, Doubtful Sound 45°19.0'S/167°00.0'E 31/10/96 15-25 m 31 " .., Oz, Doubtful Sound 45°22.8' S/167°06.0 'E 5/8/98 15-20 m 4

&. Nancy Sound 45°07.0'S/167°03.7'E 27/5/97 10-25 m 19

The Narrows, Preservation Inlet 46°03.78'S/166°44.2'E 26/11/96 21 m 5 "' The Narrows, Preservation Inlet 31/7/97 15-22 m 10

~ ,'). Chalky Inlet 45°57.81 'S/166°45.13'E 22/11/96 20-23 m 7

I:,

I>

" .,,

,. r- ,,.., .... r r

76 Appendices

AppendixB

,, Homogenization Buffer: 0.02 M Tris HCl 0.1 % mercaptoethanol

~ 0.1 % bromophenol blue 7'

'/ Running Buffers

Discontinuous Tris Citrate .,' Electrode (pH 8.2): 300 mM Borate Gel (pH 8.7): 76 mM Tris 5mMcitrate

~

..__ 0.025 M Tris Glycine (pH 8.5) "' 0.25 M Tris 2M Glycine * Tris EDTA Maleate (pH 7.4) t, lOOmMTris

1 mMMgC12 1 rnM N azEDTA

~

1 0.13 M Tris EDTA Borate (pH 8.9) 130 mMTris

2.2 mM Na2EDTA 6mMNaOH " 71.3 mM Boric acid "r

CAEA (pH 7.2) } 0.05 M Citric Acid r " 57.6 g N-(3-aminopropyl morpholine) 'r

0.02 M Phosphate (pH 7.0) y 11.6 mM Na2HP04

8.4 mM NaH2P04

77 Appendices

Stains Staining Buffers

PGI Tris HCl (pH 8) 3mg fructose-6-phosphate 0.09 M Tris ~ • l 2mg NAD 0.062MHC1 2mg MIT ,. trace PMS Tris HCl (pH 7) 0.09 M Tris '/' 2 µl/2 units G6PD 0.087 M HcCl 10 ml Tris HCl pH 8 8ml Agar Phosphate

0.0061 M NazHP04 S' PEP 0.0039 M NaH P0 5mg di/tri peptide 2 4 2mg L-amino acid oxidase

"' 2mg peroxidase ,;. 2 mg o-dianosidine "' 10 ml Phosphate Buffer 8ml Agar ,, HK

- t,- 100 mg D-glucose "' 5mg ATP 2mg NAD

<> 5mg MgC12

1 2mg MIT

{> 10 ml Tris HCl 2 µ1/2 units G6PD

~ 8ml Agar

.,_, SOD trace PMS "' trace MIT I ~r'

'r'

T

78 Appendices

Appendix C

·t ., Enzymes assessed in allozyme analysis. " = unscorable activity, * = scorable activity

A. (all 100), " = no activity, * ! = scorable activity with differences. TG = Tris Glycine,

7' TC8 = Tris Citric pH 8, TC7 = Tris Citric pH 7, TEM = Tris EDTA Maleate.

Cellulose Acetate Starch En es TG CAEA Phos. Mal. Bor. Poulic TC8 TEM Lith. TC7 IE.C. No. . IDH .1.1.42 I\ I\ .,. LAP LDH I\ I\ I\ 1.1.1.27 MDH - - I\ I\ I\ - I\ - - 1.1.1.37 ME I\ I\ I\ I\ I\ I\ I\ l.1.1.40 )- MPI - - I\ I\ I\ I\ I\ 5.3.1.8 NP - I\ ~ PEP.pp * * I\ I\ 13.4.11/13 i' PEP.II * * * * * PEP.lg * PEP.tg - I * ,. PEP.gs I\ PEP.pl ,l PEP.lgg .,,.. PEP.It * PEP.gt * PGI - *! *! I\ t.3.1.9 PGM - - - - I I\ I\ I\ .7.5.l 3PGD I\ I\ .... 6PGD I\ I\ 11.1.144 SOD I\ *MPI 1.15.1.1 .,. XDH I I\ 1.2.1.37 ·,

79