Molecular and morphological phylogenetics of the faviidae (scleractinia) in singapore

Note on coauthorship Chapter 2 Slow mitochondrial COI sequence evolution at the base of the metazoan tree and its implications for DNA barcoding has been published Appendix II, while Ch

PHYLOGENY OF THE FAVIIDAE (SCLERACTINIA) IN SINGAPORE BASED ON MOLECULAR AND MORPHOLOGICAL DATA

HUANG DANWEI

(B.Sc.(Hons.), NUS)

A THESIS SUBMITTED FOR THE DEGREE OF

MASTER OF SCIENCE

DEPARTMENT OF BIOLOGICAL SCIENCES

NATIONAL UNIVERSITY OF SINGAPORE

2008

Acknowledgements

First and foremost, I thank my supervisors Dr Peter Alan Todd and Professor Chou Loke Ming for their tireless supervision and guidance, the stimulating discussions, as well as the invaluable advice and motivation for me to delve into marine science The last three years have been productive and fulfilling because of the opportunity to carry out my research in the Marine Biology Laboratory I have also been very fortunate to receive the gracious advice of A/P Rudolf Meier, who gave me permission to pursue molecular work in the Evolutionary Biology Laboratory My heartfelt gratitude goes

to him for patiently coaching me in phylogenetics and scientific writing

Both laboratories mentioned above have been an important part of my life as a

graduate student, and I appreciate the people who have made it such To Abby Ng, Angie Seow, Farhan Ali, Gaurav Vaidya, Gwynne Lim, Hwang Wei Song, Karenne Tun, Lin Juanhui, Nalini Puniamoorthy Tay Ywee Chieh and Zhang Guanyang, for rendering assistance, constant reviews and encouragement I would like to especially mention the help of Kathy Su, Sujatha Narayanan Kutty and Zeehan Jaafar, who gave guidance on various aspects of taxonomy, phylogenetics and laboratory work

This project would not have been possible without the expert advice of Professor Nancy Knowlton (Scripps Institution of Oceanography) on coral systematics, and Dr Hironobu Fukami (Seto Marine Biological Laboratory) on DNA extraction and PCR

My participation in the Coral Molecular Biology Techniques Workshop conducted by the Hawaii Institute of Marine Biology helped to jumpstart my molecular work, and I appreciate Lady McNeice’s generosity that made it financially possible I thank Drs Andrew Baird (James Cook University), Wilfredo Licuanan (De La Salle University),

Emre Turak and Lyndon DeVantier (Australian Institute of Marine Science) for their assistance with specimen identification and ideas on coral systematics I am also indebted to Professor Gregory Rouse (Scripps Institution of Oceanography), my PhD advisor, who gave me time to complete this work as I began my doctoral candidature

Several staff members of the Department of Biological Sciences have been of

tremendous help I thank the R.V Mudskipper crew of Salam, Rahmat and Ishak for

skillful handling of the department boat, and I am also grateful to Latiff for offering help in many ways Kelvin Lim kindly facilitated my access to the coral collection at the Raffles Museum of Biodiversity Research, and I acknowledge his assistance I would like to dedicate this work to the late Yeo Keng Loo who had tirelessly curated the invertebrate collection and managed the coral specimens that I used as reference

Last but not least, I am grateful to my family for supporting me through my

endeavours

Note on coauthorship

Chapter 2 (Slow mitochondrial COI sequence evolution at the base of the metazoan tree and its implications for DNA barcoding) has been published (Appendix II), while Chapter 3 (Phylogenetic relationships in the Faviidae based on molecular and

morphological markers) is in review In both instances, I am the first author, while A/P Rudolf Meier, Dr Peter A Todd and Professor Chou Loke Ming are coauthors Although I received substantial advice and guidance from RM, PAT and CLM as advisors, the data and thesis are my own work

The first examining of volcanic rocks, must to a geologist be a memorable epoch, and little less so to the naturalist is the first burst of admiration at seeing corals growing on their native rock

Charles Darwin

1.4 Objectives of the present study 17

CHAPTER 2: SLOW MITOCHONDRIAL COI SEQUENCE EVOLUTION AT THE BASE OF THE METAZOAN TREE AND ITS IMPLICATIONS FOR

CHAPTER 3: PHYLOGENETIC RELATIONSHIPS IN THE FAVIIDAE

BASED ON MOLECULAR AND MORPHOLOGICAL MARKERS

technique for non-bilaterians, including the Scleractinia (hard corals), has not been empirically assessed Here, I present a comprehensive analysis of intra- and

interspecific COI variabilities in Porifera (sponges) and Cnidaria (corals, jellyfish and hydrozoans) using a dataset of 685 sequences from 283 species Variation within and among species was found to be much lower in Porifera and Anthozoa (containing Scleractinia) compared to the Medusozoa (Hydrozoa and jellyfish, i.e Scyphozoa), which has divergences similar to typical metazoans Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely

to be plesiomorphic for the Metazoa Higher rates of evolution could have originated independently in Medusozoa and Bilateria, or acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa, and hence the Scleractinia, unsuitable for DNA barcoding Caution is advised for Porifera and Hydrozoa because of relatively low identification success rates It has been suggested previously that the barcoding limitation generally exist for Cnidaria, but here, I

confirm that it is restricted to the Anthozoa and caution against the use of COI for species delimitation in this taxon

Due to the likely futility of coral DNA barcoding using COI, reconstructing

evolutionary relationships within the Faviidae remains as difficult as before Relying solely on conventional taxonomic traits used in this family, I collected 84 colonies

from 42 ingroup species as well as two outgroup specimens (Acanthastrea echinata and Scapophyllia cylindrica) These were sampled for two mitochondrial genes (COI

and a mitochondrial noncoding region adjacent to COI) GenBank sequences were

also extracted for four Caribbean faviid species and an Acropora outgroup A

morphological dataset for monocentric species was generated with 12 descriptive and eight morphometric characters Maximum parsimony analysis was carried out

separately on the molecular and morphological data using both dynamic (optimisation alignment) and static (ClustalX) homologies Both data types were also combined for total analysis I found that phylogenies based on both data types are incongruent and did not recover traditional taxonomic classification Of the eight genera with more than one species examined using molecular data, only two are monophyletic

Furthermore, the outgroup Scapophyllia cylindrica is deeply nested within Faviidae, while the Indo-Pacific Montastrea spp are distinct from the Atlantic M annularis

complex Data show clearly that conventional taxonomy has masked morphological convergence and reticulate evolution within this family Results also support the hypothesis that some species within genera spanning both the Indo-Pacific and

Caribbean are less closely related to one another than to taxa from other families

List of Tables

Table 1.1 List of publications since Romano & Palumbi (1996) that use DNA

sequence data to build phylogenies among scleractinian species Number of species analysed, molecular markers employed and the citation for each work are included 12S and 16S are mitochondrial rRNA genes; 5.8S and 28S are nuclear rDNA genes; ITS = nuclear internal transcribed spacer regions, including 5.8S (ITS1-5.8S-ITS2); cytB = cytochrome b (mitochondrial); COI = cytochrome oxidase c subunit 1

(mitochondrial);
tub =
-tubulin (nuclear); mt-genome = complete mitochondrial genome sequence; ATP6 = ATPase 6 (mitochondrial); h2ab = partial histone 2A and 2B (nuclear); MC2 = mini-collagen intron 2 (nuclear); Pax-C = DNA 46/47 Pax-C intron (nuclear); MIR = mtDNA intergenic/control region; MNC = mtDNA

noncoding region (adjacent to COI) (pp 13–14)

Table 2.1 COI distances, pairwise Mann-Whitney U-statistics, significance tests and

the rank of each taxon (largest to smallest distances) among Porifera, Anthozoa, Hydrozoa and Scyphozoa for intraspecific (a) and closest congeneric interspecific distances (b) Distance values denote means and standard errors (p 25)

Table 2.2 Frequencies of sequences (percentages in parentheses) with accuracy of

species attribution using three threshold values: 3.0%, 10
mean intraspecific distance (10
) and point of minimum overlap (MO) An accurate identification of a sequence

is classified as ‘correct’; sequence with best matches to the correct barcode and at least an incorrect one is ambiguous; erroneous species attribution is ‘incorrect’; and

‘unmatched’ sequence does not have any match closer than the threshold (p 26)

Table 3.1 List of 86 specimens from 44 species sampled in this study (asterisk

denotes taxon designated as outgroup) Successful PCR amplifications of two genes, including the presence of an intron embedded within COI, are shown Analyses carried out for each taxon are also indicated ALL represents 91-taxon molecular analysis; ALN is reconstruction with all taxa for which the gene fragment MNC can

be aligned; MOR is the morphological analysis of monocentric taxa (pp 37–41)

Table 3.2 List of genera with long segments of T repeats that cannot be fully

amplified with MNC1f and MNC1r Internal primer sequences designed for these taxa and melting temperatures employed for PCR are shown (p 41)

Table 3.3 List and synopses of morphological characters, including descriptive and

morphometric parameters, used to analyse the monocentric species Character states and corresponding codes are indicated (pp 43–45)

List of Figures

Figure 1.1 Illustration of generalised Scleractinia corallites, indicating common

morphological features used for taxonomic descriptions The live tissues of a polyp are also shown (reproduced with permission from Veron 2000) (p 6)

Figure 2.1 Bar chart showing proportion of pairwise comparisons of the COI gene at

each range of sequence divergence Intraspecific and closest congeneric interspecific matches of the following taxa are represented: (a) phylum Porifera; (b) class

Anthozoa; (c) class Hydrozoa; and (d) Scyphozoa (p 24)

Figure 2.2 Two most parsimonious evolutionary scenarios for slow mtDNA

evolution in Anthozoa and Porifera From a slow ancestral mtDNA, (A) fast evolution originated in the Medusozoa and Bilateria independently, or (B) fast mtDNA evolved

in the Cnidaria + Bilateria clade but was lost in Anthozoa Black bars labelled ‘fast’ and ‘slow’ respectively denote acceleration and deceleration of mitochondrial

sequence evolution (p 28)

Figure 3.1 Alignments of DNA sequences of five types of COI group I intron found

in Faviidae Taxa labelled with intron type in parenthesis are GenBank sequences,

while those without are from the present study Echinopora gemmacea and E

lamellosa possess the type 5 intron that is new to science (p 49)

Figure 3.2 Maximum parsimony tree based on combined COI and MNC sequence

data using dynamic homology Numbers indicate bootstrap support values; only values >50 are reported Taxa with crosshatched circles contain type 1 group 1 intron

in COI, filled circles denote taxa with type 4 intron, while open circles are taxa with type 5 intron (pp 52–53)

Figure 3.3 Maximum parsimony tree based on combined sequence data from aligned

COI and MNC Clade II is paraphyletic and hence labelled with inverted commas Numbers indicate bootstrap support values; only values >50 are shown Taxa with filled circles contain type 4 group 1 COI intron, while open circles denote taxa with type 5 intron (p 54)

Figure 3.4 Maximum parsimony tree based on morphological data comprising 18

characters Numbers indicate Bremer support values; only positive values are shown (p 57)

Figure 3.5 Maximum parsimony trees based on molecular data (COI and MNC) on

the left and morphological data on the right Numbers on molecular tree indicate bootstrap supports (>50 only) while those on morphology tree are Bremer support values (positive values only) Relationships among morphological clades A, B1 and B2 are coded with different colours; other taxa are black (p 58)

Figure 3.6 Maximum parsimony tree based on combined morphological and

molecular (COI and MNC) data Numbers above branches are bootstrap support values (>50 only) and those below are Bremer supports (positive values only) for the same node (p 59)

CHAPTER 1: GENERAL INTRODUCTION

1.1 The Scleractinia

Corals of the order Scleractinia (Cnidaria; Anthozoa; Hexacorallia) constitute the largest taxon of the Anthozoa, which is a class with an exclusively polyp adult life stage (Wells & Hill 1956; Bridge et al 1995; Ruppert et al 2004) The order is also distinguished by a calcareous exoskeleton made up of radial septa between the

mesenteries (developed in multiples of six) as well as a complex array of surrounding and supporting structures (Wells 1956) The taxon includes over 700 recorded

zooxanthellate species, i.e those containing symbiotic dinoflagellates, and these form the basis of coral reefs along the tropical and subtropical coasts of the Indo-Pacific and Atlantic Azooxanthellate members, on the other hand, tend to have wider ranges

of depth and latitude (Wells 1956; Veron 1995)

Corals are generally colonial, although some solitary species exist—the most

prominent being members of the family Fungiidae, or mushroom corals For colonial species, the coelenteron or body cavity of each polyp is connected to adjacent clonal individuals through which water and nutrients are transported (Veron 2000) Living polyps secrete crystalline fibres of aragonitic calcium carbonate calcification centres

at the basal disc of the polyp These centres and associated fibres, known as

sclerodermites, form the basis of the skeleton that support the soft tissues, allowing for expansive growth in the colony for as long as the constituent polyps survive and

reproduce (Wells 1956) Porites, for instance, can grow up to several metres in height

with more than 100,000 polyps (Ruppert et al 2004)

As in other cnidarians (traditionally known as coelenterates), the body wall of corals consists of three cell layers: the external ectodermis, middle mesoglea and the

gastrodermis (or endodermis) that surrounds the coelenteron (Wells 1956; see also Willmer 1990; Hayward et al 2004) With well-developed nervous, muscular and reproductive systems, corals are able to sense and respond to mechanical, chemical and light stimuli (Veron 2000) Most species utilise their tentacles, lined with stinging cells or cnidocytes, to feed on animals ranging from zooplankton to small fish Some have reduced tentacles and feed exclusively on suspended nutrients with the aid of mucous (Ruppert et al 2004)

The gastrodermis of zooxanthellate species harbour symbiotic dinoflagellate protists

of the genus Symbiodinium (Wells 1956; Veron 2000) These organisms, commonly

known as zooxanthellae, are ubiquitous members of the coral reef (Taylor 1974; Trench 1993; Rowan 1998) They are the source of up to 50% of the host’s nutrient intake, provided in the form of nitrogen and carbon, while obtaining metabolic

products such as PO4, NH3 and CO2 from the coral host (Ruppert et al 2004) A remarkable aspect of these symbionts, known only recently through molecular

approaches, is their genetic diversity While the complexity of the group’s taxonomy

has been suspected (see Baker 2003), several molecular clades of Symbiodinium have

been identified and the discovery of novel types continues to rise (Rowan & Powers 1991; Rowan & Knowlton 1995; LaJeunesse 2001, 2002, 2005; Karako-Lampert 2004) Interestingly, the specificity of the symbiont for host species, and vice versa,

vary among Symbiodinium clades and coral types (Baker 2003; Knowlton & Rohwer

2003; Ulstrup & van Oppen 2003; Little et al 2004; Garren et al 2006; Lanetty et al 2006) This is known to affect the response of the holobiont (host +

Rodriguez-symbiont) under different environmental stresses, including coral bleaching

(expulsion of zooxanthellae) and subsequent recovery of the symbiosis (Buddemeier

& Fautin 1993; Rowan et al 1997; Toller et al 2001a,b; Baker 2003; LaJeunesse et

al 2003; Baker et al 2004; Warner et al 2006; Visram & Douglas 2007)

Scleractinians can reproduce both asexually and sexually The production of clones can occur by intratentacular budding (division of two or more polyps within one tentacular ring), extratentacular budding (development of new polyp outside the tentacular ring) or transverse division (formation of daughter polyps by splitting of parent into two along the oral-aboral axis) (Wells 1956) These processes are

responsible for colony formation and generation of new individuals or colonies They are also significant diagnostic features in coral taxonomy, though not easily

distinguished from one another (A H Baird, pers comm.; pers obs.) More than one mechanism may be present in a single species and sometimes within an individual colony (Veron et al 1977; Veron 2000) New daughter colonies can also develop from fragments produced by external disturbances such as wave action, and this process is important in species with low rates of larval recruitment (Highsmith 1982; Wallace 1985; Hughes 1992; Okubo et al 2007)

Most scleractinian species are hermaphroditic, and there are fewer gonochoric

species, i.e those with individuals having separate sexes (Harrison & Wallace 1991; Carlon 1999) Gamete and larval dispersal also vary among species Some undergo internal fertilisation and brood their larvae within the parent while, in the majority of species, gametes are broadcasted into the water column where they fertilise and develop (Babcock & Heyward 1986; Szmant 1986) One of the most exciting

discoveries in coral biology is the synchronous multispecific release of gametes at various geographic locations (Harrison et al 1984; Babcock et al 1986, 1994;

Hayashibara et al 1993; van Veghel 1993; Guest et al 2005a; Nozawa et al 2006) More than 30 species in sympatry may spawn within two hours of each other, as recorded in the Great Barrier Reef (Willis et al 1985; Babcock et al 1986) Buoyant gametes are subsequently mixed by currents at the water surface, increasing the likelihood for introgressive hybridisation (Willis et al 2006) This effect is balanced

by instances where reproductive isolation is achieved For instance, although

spawning times of different species can overlap, the modal release of gametes may be temporally separated (Knowlton et al 1997; Fukami et al 2003) For species without isolated spawning periods, hybridisation trials have shown that successful fertilisation

or larval development is absent (Knowlton 1997; Fukami et al 2004a; Levitan et al 2004) Nevertheless, perfect reproductive barriers are very rare (Mallet 2005)

Hybridisation is believed to be common in the Scleractinia, especially at the periphery

of species’ ranges, where hybrids may be able to exploit niches not suitable for their parents (Miller & Ayre 2004; Willis et al 2006) This process is possibly a primary driver of coral diversification and reticulate evolutionary pathways (Veron 1995; Vollmer & Palumbi 2002; Seehausen 2004)

In Singapore, the distribution of coral assemblages has been studied extensively during the last two decades (e.g Chou 1988; Leng & Lim 1990; Leng et al 1990a,b; Chua and Chou 1991; Lane 1991; Goh & Chou 1992, 1993; Goh et al 1994; Ang 2007) Using the line intercept transect method (Dartnall & Jones 1986; English et al 1994), 197 species were recorded on the reefs south of the mainland A recent update

by Huang et al (manuscript accepted; see Appendix I) on the zooxanthellate species

alone augmented this figure to 258 with 30 new records, although only 165 species have been encountered in the last three years This is comparable to the reefs of neighbouring countries if habitat area is taken into account—the area of Singapore’s reefs (~10 km2) is only 0.25% of Malaysia’s (4,006 km2; 348 species) and 0.02% of Indonesia’s (50,875 km2; 443 species) reef expanses (Spalding et al 2001; Burke et

al 2002; Goh 2007) As only about half of species with distribution ranges

encompassing Singapore have been found, the actual alpha diversity is likely to be

greater (Huang et al manuscript accepted; Appendix I)

1.2 Coral taxonomy, barcoding and phylogenetics

Species are the basic units of biodiversity and their precise definitions are vital to our understanding of the natural environment and evolution (Claridge et al 1997)

Taxonomy of the Scleractinia has primarily been based on morphological features of the coral skeleton and, to a lesser extent, the living polyp tissue (Lang 1984; but see Potts et al 1993; Todd et al 2001a,b, 2004c; Tambutté et al 2007) While such

characteristics can be very complex (sensu Wells 1956), identification has

conventionally relied on easily observable traits (Veron & Pichon 1976, 1980, 1982; Veron et al 1977; Wallace 1999; Veron 2002) These include the corallite wall, septa, costae, coenosteum, paliform lobes, columellae, colour, tissue expansion, asexual budding mode and various morphometric dimensions (Wells, 1956; Lang 1984; Wallace 1999; Veron 2000) (Figure 1.1)

Figure 1.1 Illustration of generalised Scleractinia corallites, indicating common

morphological features used for taxonomic descriptions The live tissues of a polyp are also shown (reproduced with permission from Veron 2000)

There have been inconsistencies in the use of these characters for species descriptions before and after 1970 (Zlatarski 2007) Prior to the work of Veron et al in the 1970s, each species was defined based on multiple character states that are not easily

distinguishable (e.g Wells 1956) Today, species are differentiated based on the same characters, but with decreased number of states For instance, the mode of asexual reproduction is used to differentiate several taxa Two states are now commonly used, i.e intra- and extratentacular budding (e.g Veron et al 1977; Wijsman-Best 1977a), while previously, intratentacular budding may be ‘monostomadeal’, ‘distomadeal’,

‘tristomadeal’, etc (Wells 1956) These temporal differences of term usage could

have contributed to the unstable nature of coral taxonomy; changes in nomenclature

are common and can be drastic, e.g Favites spp (Veron et al 1977)

An advanced taxonomic tool that has enhanced the reliability of species descriptions

is skeletal analysis at the microstructural level, usually based on traits related to biomineralisation patterns (Chevalier & Beauvais 1987; Yamashiro 1989; Roniewicz

& Morycowa 1993; Cuif et al 1997, 2003; Perrin 2003) This approach is adapted from the field of coral palaeontology and has helped to strengthen classifications at the family level (Veron et al 1996) Likely mistakes in familial placement of some

taxa such as Cladocora and Eusmilia were detected through their microstructure, and

confirmed by 28S rRNA sequence data (Cuif et al 2003) However, this

‘microstructural revolution’ has apparently jolted confidence only in the taxonomy of

geologically older genera (e.g Cretaceous–Recent Cladocora and Diploastrea)

(Stolarski & Roniewicz 2001) Most Recent genera remain well-supported within their present families In fact, externally observable traits appear to distinguish

families more reliably compared to internal microskeletal characters (Veron et al 1996)

At the species level, not only have taxonomic descriptions been carried out differently since the 1970s, Veron’s (2000) treatise on the Scleractinia has revolutionised how specimen identification is being carried out today Although it has often been

dismissed for lack of taxonomic details no different from general coral and reef guides (e.g Ditlev 1980; Gosliner et al 1996; Allen & Steene 2003; Edward et al

2004), Corals of the World (Veron 2000) has attempted to consolidate the taxonomy

of Scleractinia under a single framework It has allowed coral researchers not

concerned about complex taxonomic issues (sensu Wells 1956) to identify

morphospecies easily for their work (Márquez et al 2002; see also Dupré 1999) There is also a strong emphasis on the appearance of the coral in its natural

environment, with a large number of specimen images from various geographic regions and habitats presented Hence, features used for the purpose of species

recognition can include those that are easily observable

Unfortunately, due to the great amount of variability within each taxonomic unit, there is substantial overlap in morphological characters between species (Wijsman-Best 1974a; Randall 1976; Brakel 1977; Best et al 1984; Budd 1993; Veron 2000)

As a result, various workers are likely to interpret species limits differently (Veron 2001; Zlatarski 2007) Indeed, experts in certain taxonomic groups often have distinct

opinions For instance, Veron (2000) recognises 170 species of Acropora, while

Wallace (1999), a specialist in this speciose genera, lists only 113 species globally Classification of corals has a long history dating back to the time of C Linnaeus in the 18th century, but new information on the lineage’s ecology and evolutionary history, coupled with conflict among taxonomists, e.g Hoeksema (1989) vs Veron (2000), Wallace (1999) vs Veron (2000), may result in the continual instability of this taxon (Knowlton & Jackson 1994)

It is currently acknowledged that, in addition to morphological characterisation of species, genetic boundaries have to be considered in order to validate the taxonomy of scleractinian corals (Wallace and Willis, 1984; Willis 1990; Knowlton and Budd, 2001) In a broader perspective, Hebert et al (2003a,b) suggested that animal

specimens should be diagnosed to species based on a DNA barcode, a ~650 bp long

sequence of the mitochondrial cytochrome oxidase c subunit 1 (COI) gene They envisioned that this technique would increase the efficiency and objectivity of species identification However, the accuracy of DNA barcoding depends on the presence of a comprehensive COI sequence database against which specimen DNA can be

evaluated (Meyer & Paulay 2005; Ekrem et al 2007) In most taxa this information is lacking, either due to logistical constraints or general deficiency in biodiversity data (Wilson 2003; Meyer & Paulay 2005; Cameron et al 2006; Meier et al 2006)

Several studies have claimed that current barcoding success rates warrant sufficient merit for the method to be extensively employed (Hebert et al 2004; Hajibabaei et al

2006, 2007; Pfenninger et al 2006; Clare et al 2007; Gómez et al 2007; Kerr et al 2007; Min & Hickey 2007) Others are more cautious, but remain optimistic that several of the limitations can be minimised For instance, increased gene sampling and the use of appropriate taxon-specific thresholds can reduce erroneous species attributions (Dasmahapatra & Mallet 2006; Lefébure et al 2006; Seifert et al 2007) The accumulation of data in the barcode library is also likely to increase identification success (Webb et al 2006; Ekrem et al 2007; Waugh 2007) Conversely, many researchers have called for the integration of other data types when diagnosing

species, e.g morphology and ecology (Tautz et al 2003; Meyer & Paulay 2005; Dasmahapatra & Mallet 2006; Meier et al 2006; Vogler & Monaghan 2007; Wiemer

phylum, 94.1% are less than 2%, while most other animal taxa in a broad survey of major metazoan groups has much higher interspecific variability (Hebert et al

2003b) This is believed to hinder the barcoding of corals since intra- and interspecific distances may not be separable (Medina et al 1999; Hellberg 2006) However, the extent to which this can affect DNA barcoding is unknown Species boundaries and identification schemes have been proposed for scleractinian corals based on the COI (Medina et al 1999) Other mitochondrial regions have also been used for this

purpose, e.g cytochrome b and ATPase 6 (Fukami et al 2000) The reliability of results from such techniques must be questioned unless identification success is evaluated Outside the Scleractinia, it is also unclear when in evolutionary history slow sequence evolution originated Some data are present for other anthozoans (e.g France & Hoover 2002; McFadden et al 2004), but these have not been consolidated Furthermore, low COI variability is not found in all cnidarians as the sister taxon Medusozoa seems to have typical metazoan COI evolution rates (e.g Dawson & Jacobs 2001; Govindarajan et al 2006) Porifera, a more ancient phylum comprising the sponges, possesses limited speeds akin to the Anthozoa (e.g Watkins &

Beckenbach 1999; Lavrov et al 2005) Evidently, a better understanding of the

evolutionary processes determining mitochondrial sequence variation is desirable before use of the COI barcode becomes a standard practice among coral scientists

The increasing ease and falling cost of DNA sequencing has not only generated interest in the barcoding of the Scleractinia—reconstructions of its evolutionary history using sequence data have also called to question the phylogenetic significance

of traditional taxonomy In the seminal work by Romano & Palumbi (1996, 1997), analysis of the mitochondrial 16S ribosomal RNA (rRNA) from 34 scleractinian

species grouped the Scleractinia into two well-supported major taxa known as the

‘robust’ and ‘complex’ clades, with a mean divergence of 29.4% between them They are also named ‘short’ and ‘long’ clades respectively based on the length of the 16S sequences—a range of 406–565 bp in PCR amplified length This split was estimated

to have occurred 300 million years ago (mya), before the appearance of the calcium carbonate skeleton in fossil records 240 mya Hence, the scleractinian skeleton may have, in fact, evolved more than once from a soft-bodied, anemone-like ancestor (Romano & Palumbi 1996, 1997)

The two-clade phylogeny, later supported with larger taxon-sampling by two other genes—nuclear 28S rRNA (Romano & Cairns 2000) and mitochondrial 12S rRNA (Chen et al 2002)—and the entire mitochondrial genome (Medina et al 2006), does not correspond to morphological hypotheses about relationships among families and suborders Each major clade consists of taxa from different suborders defined by Veron (1995) Of the seven extant suborders, only three are monophyletic, while four morphological families (Caryophylliidae, Faviidae, Oculinidae and Poritidae) are paraphyletic (Romano & Cairns 2000) More recently, Fukami et al (2004b)

examined evolutionary relationships among Indo-Pacific and Atlantic corals using the mitochondrial COI, cytochrome b (cytB) as well as two exons from the nuclear
-tubulin (
tub) gene Atlantic corals conventionally placed in Faviidae and Mussidae form a well-defined clade distinct from Indo-Pacific congeners The Atlantic lineage probably became isolated more than 34 mya before the Tethyan connection between the tropical Indo-west Pacific and Atlantic closed, and has since been undergoing morphological convergence under similar ecological conditions as Indo-Pacific corals Several microskeletal characters used by Wells (1964) seem to distinguish the

Atlantic species effectively; but until Fukami et al (2004b), these were disregarded Contrary to Veron et al (1996), phylogenies based entirely on externally observable features of the coral skeleton may be unreliable

Since Romano & Palumbi (1996), phylogenetic reconstructions have been carried out for several scleractinian taxa using primarily DNA sequence data A list of such work published between 1996 and 2007 is presented in Table 1.1 All of the studies

uncovered discrepancies between phylogenies obtained from molecular analyses and those derived from morphological data or traditional classification Disagreements are due to incongruence in tree topology (e.g Romano & Palumbi 1996; Fukami et al 2004b) or paraphyletic species resulting from reticulate evolution (e.g Odorico & Miller 1997; van Oppen et al 2001; see also Willis et al 2006) Surprisingly, only one taxonomic revision has been formally proposed to date Based on cytB, nuclear histone 2a and 2b, as well as a morphological analysis, Wallace et al (2007) elevated

the subgenus Isopora to the genus level, having demonstrated significant distinction between Acropora (Isopora) and A (Acropora) They also changed the placement of Acropora togianensis to the new subgenus Given the burgeoning amount of evidence

illustrating inadequacies of conventional Scleractinia classification, more effort should go into taxonomic revisions to reflect accurate coral phylogeny (see Wheeler 2004; Padial & De la Riva 2007) Additionally, apart from work carried out at the suborder or family level, there is limited species- and genus-level information on taxa other than the Acroporidae Focus should thus be diverted to non-acroporid lineages

Table 1.1 List of publications since Romano & Palumbi (1996) that use DNA

sequence data to build phylogenies among scleractinian species Number of species analysed, molecular markers employed and the citation for each work are included 12S and 16S are mitochondrial rRNA genes; 5.8S and 28S are nuclear rDNA genes; ITS = nuclear internal transcribed spacer regions, including 5.8S (ITS1-5.8S-ITS2); cytB = cytochrome b (mitochondrial); COI = cytochrome oxidase c subunit 1

(mitochondrial);
tub =
-tubulin (nuclear); mt-genome = complete mitochondrial genome sequence; ATP6 = ATPase 6 (mitochondrial); h2ab = partial histone 2A and 2B (nuclear); MC2 = mini-collagen intron 2 (nuclear); Pax-C = DNA 46/47 Pax-C intron (nuclear); MIR = mtDNA intergenic/control region; MNC = mtDNA

noncoding region (adjacent to COI)

Taxon No of species Markers Reference

Scleractinia 34 16S Romano & Palumbi (1996)

29 cytB; h2ab Wallace et al (2007)

5 ITS Odorico & Miller (1997)

3 Pax-C; ITS van Oppen et al (2000)

25 Pax-C; MIR van Oppen et al (2001)

5 ITS van Oppen et al (2002a)

3 Pax-C; MIR Márquez et al (2002)

Taxon No of species Markers Reference

4 28S Wolstenholme et al (2003)

5 ITS Diekmann et al (2003)

3 ITS; MNC Fukami et al (2004a)

Coral reefs are being degraded at an alarming rate (Knowlton 2001; Hughes et al

2003; Pandolfi et al 2003, 2005; Bellwood et al 2004; Wilkinson 2004; Bruno &

Selig 2007) Studies on the phylogenetic history of their member organisms may offer

prognoses for the future of reefs (van Oppen & Gates 2006) They provide for a

thorough understanding of speciation and biogeographic events that shape the

distribution of reef corals, enabling researchers to make predictions about responses

and the evolution of reefs in light of oceanographic modifications resulting from

climate change (Pandolfi 1992; Palumbi 1997; Barber et al 2006) There has also

been a call for conservation efforts directed at the maintenance of evolutionary

processes that generate diverse assemblages of taxonomically complex organisms

such as scleractinian corals (Ennos et al 2005) This approach, however, will only

work with detailed knowledge about their phylogeny

1.3 Faviidae in Singapore

Corals in the family Faviidae are an especially problematic group in terms of species identification and characterisation, yet it is one of the most prominent hermatypic coral taxa with high abundance and the most numerous genera in Indo-Pacific reefs (Wijsman-Best 1974b, 1976, 1977b, 1980; Veron et al 1977) For example, the

genera Favia, Favites and Goniastrea are classified according to morphological traits

such as presence of paliform lobes and sharing of corallite walls, most of which are arbitrary and may have no phylogenetic basis (Veron et al 1977) This situation is exacerbated by morphological plasticity shown to occur in this family (e.g Todd et al 2001b, 2004a,b) For instance, corallite expansion and exsertion were enhanced in

Favia speciosa and Diploastrea heliopora for specimens transplanted to shallower

depths, possibly increasing the effective surface area for light capturing and/or shading as a response to increased irradiance (Todd et al 2004b) The family is thus

self-an importself-ant model group to examine the problems with coral taxonomy

The coastal environment in Singapore is heavily impacted, especially by sediments from land reclamation and dredging (Hilton & Manning 1995; Chou 1996, 2006) As

a result, corals tend to exhibit morphologies that are atypical with respect to those from less disturbed reefs and identification based on conventional taxonomy can be

problematic (E Turak & L DeVantier pers comm.; Huang et al manuscript

accepted; Appendix I) Unfortunately, detailed taxonomic work has only been

conducted on the family Fungiidae and genus Acropora in 1987 and 1992

respectively Twelve free-living fungiid species out of 40–50 recorded in the world

have been found in Singapore waters (Koh 1987) and 18 Acropora spp were recorded

(Leow 1992) For the Faviidae, surveys conducted from 1985 to 1991 revealed that

this taxon was dominant in Cyrene Reefs, Pulau Hantu (West) and the reef flat of Pulau Salu (Chua & Chou 1991; Chou & Wong 1985) A short review of this family was published in 1992 but it is largely descriptive (Low & Chou 1992) Guest et al (2002, 2005b) recently found that several members of the taxon participate in the synchronous spawning up to six days after full moon in March and/or April So far, the only study specifically targeted at Singapore faviids is Ang (2007), who found that diversity of the taxon is greater for reefs further away from the mainland, likely due to a declining gradient of sedimentation However, no clarification of species limits in these marginal habitats was attempted

In the recent update of Singapore’s zooxanthellate Scleractinia, Huang et al

(manuscript accepted; Appendix I) extracted all Faviidae specimens from the coral

reference collection at Raffles Museum of Biodiversity Research (RMBR) and

reidentified them based on updated taxonomic descriptions and nomenclature in Veron (2000, 2002) Of 127 species from 24 genera described globally, 60 species from 14 genera have been recorded in Singapore With knowledge of inconsistencies

in Faviidae systematics emerging, e.g Leptastrea (Romano & Palumbi 1996, 1997;

Romano & Cairns 2000) plus Atlantic Faviidae and Mussidae (Fukami et al 2004b), these data may not reflect actual species diversity locally and globally It is striking that the first report of paraphyly in the family appeared 35 years ago in a coral study employing numerical taxonomy (Powers & Rohlf 1972) But until recently, this problem has been methodically ignored (e.g Veron et al 1977; Veron 2000) It is therefore pertinent to reconstruct a species-level phylogeny of this taxon as a step towards resolving these long-standing issues

1.4 Objectives of the present study

The purpose of the present study is to first examine mitochondrial COI sequence evolution and DNA barcoding efficacy at the base of the metazoan tree, and then reconstruct the first broad-based species-level phylogeny of the Faviidae using

molecular and morphological data The following hypotheses are tested:

(1) COI sequence evolution in non-bilaterian taxa is slow relative to the Bilateria; (2) Identification success using DNA barcodes varies among lineages in Cnidaria and Porifera;

(3) Phylogeny of the Faviidae is incompatible with conventional taxonomy of this group; and

(4) Phylogenetic recontructions based on mitochondrial sequence data and

morphological characters are incongruent

The results of this study will provide crucial information on the evolutionary history

of the Faviidae and fuel the impetus for taxonomic revisions of the Scleractinia Findings will also be useful for future forecasts of reef evolution in the region and may contribute to the designation of conservation priorities in Singapore

CHAPTER 2: SLOW MITOCHONDRIAL COI SEQUENCE EVOLUTION AT THE BASE OF THE METAZOAN TREE AND ITS IMPLICATIONS FOR DNA BARCODING

2.1 Introduction

Most biologists assume that metazoan mitochondrial DNA evolve up to 10 times faster than nuclear DNA (e.g Dawid 1972; Brown et al 1979, 1982; Brown 1983; Avise et al 1987) Recent evidence, however, has suggested that this feature of

mitochondrial sequences has evolved within the Metazoa and is different among various animal groups In particular, Anthozoa and Porifera have unusually slow-evolving mtDNA (e.g Watkins & Beckenbach 1999; France & Hoover 2002;

McFadden et al 2004, 2006; Smith et al 2004; van Oppen et al 2004; Lavrov et al 2005; Tseng et al 2005; Calderón et al 2006) Two hypotheses have been proposed

by Shearer et al (2002) to explain the rate variation in ‘basal’ Metazoa Firstly,

sequence evolution was slow in the metazoan ancestor and later gathered speed in the Bilateria The second hypothesis was fast-evolving mitochondrial DNA in the

metazoan ancestor and a secondary slow down in Anthozoa Here, we use COI as a model to present a consolidated analysis of our own and GenBank data pertaining to this question We furthermore add outgroup information and summarise a large

amount of new evidence to test the hypotheses by Shearer et al (2002) Lastly, we discuss the implications of our results for DNA barcoding

Unfortunately, insufficient data are available for the choanoflagellates, the putative sister group to Metazoa However, recent analysis of the next closest outgroup of

Metazoa, Fungi, revealed slow COI evolution given that many species of Penicillium

shared the same COI sequence (Seifert et al 2007) Similarly, recent studies on the Porifera suggested slow-evolving mtDNA To date, three analyses of sponge COI gene have been performed Schröder et al (2003) found that COI sequences of four species from the family Lubomirskiidae were identical, and only 1–2% different from

a confamilial species Duran et al (2004) demonstrated few differences in the COI

gene of western Mediterranean and Atlantic Crambe crambe populations despite them

being separated by up to 3,000 km Wörheide (2006) also showed similar results for

the Indo-Pacific desmosponge Astrosclera willeyana over a range of 20,000 km For

Cnidaria, Hebert et al (2003b) noted that interspecific COI variability was also

unusually low More than 90% of congeneric interspecific divergences were <2%, while only 1.9% of species pairs in other metazoan groups had such distances

However, their conclusion was based on only 17 species pairs of Cnidaria, while an average of 950 pairs were included for the other major animal taxa Furthermore, all

three Cnidaria genera (10 spp.) in the survey (Corallium, Narella and Urticina)

belonged to the Anthozoa Thus, the status of the remaining Cnidaria remained

uncertain Subsequently, Hellberg (2006) confirmed low sequence divergence for Anthozoa, while results of Govindarajan et al (2005, 2006) implied that Hydrozoa has more conventional patterns of interspecific COI distances Research on the other major cnidarian class, the Scyphozoa, also suggested a fast-evolving COI gene For example, interspecific divergences varied from 13% to 24% in the cosmopolitan

genus Aurelia (Dawson & Jacobs 2001), 11.8–15.3% between two putative species of Cyanea (Dawson 2005), and 10.9–23.4% in Cassiopea (Holland et al 2004)

The evolution of divergence rates in COI is not only interesting from an evolutionary point of view, but also important for the prospects of DNA barcoding (see Hebert et al

2003a) The success of this species identification tool is often thought to depend on the presence of a barcoding ‘gap’; i.e., the separation between intra- and interspecific variation of COI (Meyer & Paulay 2005) Here, we test the data for the presence of a barcoding gap and assess identification success using three threshold values for species delimitation—3% as initially proposed by barcoding advocates (Hebert et al 2003a), 10
mean intraspecific variability suggested by Hebert et al (2004), and a taxon-specific value that minimises overlap (or maximises separation) between intra- and interspecific distances (Lefébure et al 2006) In order to test the hypotheses by Shearer et al (2002) with regard to COI evolution rates at the base of the Metazoa tree, as well as to assess DNA barcoding identification success, we summarise

GenBank data and examine intra- and interspecific variabilities for Porifera and Cnidaria, the latter of which is analysed separately for Anthozoa, Hydrozoa and Scyphozoa We also sequenced COI for 28 species of Cnidaria to assemble a dataset totalling 685 sequences from 283 species, the largest thus far to examine COI

evolution rates in ‘basal’ metazoans

2.2 Materials and Methods

All poriferan and cnidarian mtDNA COI sequences available in GenBank, including those from mitochondrial genome sequences, were downloaded (as of April 2006) In addition, 66 scleractinian (class Anthozoa) samples representing 28 species were collected from Singapore to expand upon the taxon coverage in GenBank From these samples, genomic DNA was extracted from tissue digested in twice their volume of CHAOS solution (4M guanidine thiocyanate, 0.1% N-lauroyl sarcosin sodium, 10mM Tris pH 8, 0.1M 2-mercaptoethanol) (Sargent et al 1986) for at least three days at room temperature before DNA extraction using the phenol-chloroform method with a

phenol extraction buffer (100 mM TrisCl pH8, 10 mM EDTA, 0.1% SDS) (Fukami et

al 2004a) COI was amplified with Scleractinia-specific primers MCOIF(5'–TCT ACA AAT CAT AAA GAC ATA GG–3’) and MCOIR (5'–GAG AAA TTA TAC CAA AAC CAG G–3’) using the following protocol: 95°C for 2 min, 35 cycles of 94°C for 45 s, 55°C for 45 s and 72°C for 1.5 min, ending with 72°C for 5 min

(Fukami et al 2004b) PCR products were purified with SureClean (BIOLINE,

London, U.K.) following the manufacturer’s protocol and cycle sequencing reaction was carried out using the BigDye Terminator kit (Perkin Elmer, Massachusetts,

U.S.A.) Sequences were obtained from the ABI 3100 capillary genetic analyser

Sequences were placed in four datasets—Porifera, Anthozoa, Hydrozoa and

Scyphozoa—using TaxonDNA (Meier et al 2006) They were aligned using

AlignmentHelper 1.2 (McClellan & Woolley 2004), which translates the nucleotide sequences into amino acid sequences, aligns them using ClustalW 1.81(Thompson et

al 1994, and translates the results back to DNA data Alignments were then optimised manually Sequences not identified to species and those with >30% sequence

divergence for all pairwise comparisons were excluded as they may represent

specimens that have been incorrectly identified (see Meier et al 2006) In all, 122 poriferan sequences from 75 species, 317 anthozoan sequences from 153 species, 112 hydrozoan sequences from 44 species, and 134 scyphozoan sequences from 11

species were analysed (total 685 sequences; 283 species) Uncorrected and Kimura two-parameter (K2P; method based on different treatments of transitions and

transversions; Kimura 1980) pairwise distances for the sequences within each dataset were calculated in TaxonDNA (Meier et al 2006) Data were then separated into three categories: (i) intraspecific; (ii) congeneric (i.e., same genus) interspecific; and

(iii) closest congeneric interspecific match (i.e., smallest distance between species in the same genus)

Due to vast disparity in numbers of pairwise combinations among various groups, the data did not conform to assumptions required for parametric analysis of variance (ANOVA) Hence, nonparametric Kruskal-Wallis ANOVA (for multiple independent

groups) and Mann-Whitney U test (for two independent groups) were used to test for

significant differences in pairwise distances among the four taxa All statistical

analyses were performed using the software STATISTICA 6.0 (StatSoft) To assess overlap between the intra- and interspecific variabilities of COI, relative abundances

of sequences at each range of intraspecific and closest matched congeneric

interspecific divergences were plotted as histograms Identification success for each taxon was also examined using the ‘best close match’ strategy: assigning to a query sequence the best-matching barcode from all the other sequences, and designating it the name of the barcode if the distance was smaller than a particular reference value (Meier et al 2006) The above was tested using three thresholds, 3%, 10
mean intraspecific distance, and a taxon-specific value that minimised intra- and

interspecific distance overlap, measured using the mean of two parameters: (i)

proportion of intraspecific pairwise divergence values greater than the threshold; and (ii) proportion of interspecific pairwise distances smaller than the threshold

2.3 Results

Intraspecific COI variation was distinct among taxa: Kruskal-Wallis ANOVA

revealed highly significant differences in intraspecific distances among the four

datasets (H=307.09, d.f.=3, p<0.001) The divergence range of Porifera and Anthozoa

was 0–6%, while the Hydrozoa and Scyphozoa (together the Medusozoa) scored intraspecific distances of between 2% and ~20% (Figure 2.1) Sequences with

divergences of 0–2% accounted for the majority of variation in all taxa, but this was much greater in Porifera and Anthozoa than medusozoans (Porifera: 94.20%;

Anthozoa: 97.35%; Hydrozoa: 59.74%; Scyphozoa: 56.92%) Medusozoa was

significantly more divergent than Porifera and Anthozoa, as shown by the

Mann-Whitney U test; Anthozoa scored the lowest intraspecific distances (Table 2.1)

Closest congeneric interspecific COI evolution rates were more variable than

intraspecific divergences in all taxa, with large ranges of about 20%, overlapping with their respective intraspecific distances A majority of the interspecific distances in the Porifera and Anthozoa were
6% or invariant (Porifera: 82.54%; Anthozoa: 98.06%), resulting in substantial overlap between intra- and closest interspecific variation The Medusozoa generally had greater interspecies distances, i.e., 93.62% of hydrozoan sequences and 82.09% of scyphozoan sequences registered >6% and >12% mean pairwise divergence respectively Significant differences existed among taxa

(Kruskal-Wallis H=296.55, d.f.=3, p<0.001) By rank, anthozoan closest congeneric

interspecific divergences were the lowest, followed by Porifera, Hydrozoa and the Scyphozoa; distances differed from one another significantly (pairwise Mann-

Whitney U test; Table 2.1) Mean uncorrected interspecific distances were 4.21% (±

S.E 0.54%), 1.60% (± S.E 0.16%), 12.20% (± S.E 0.21%) and 15.41% (± S.E 0.34%) respectively for Porifera, Anthozoa, Hydrozoa and Scyphozoa

Figure 2.1 Bar chart showing proportion of pairwise comparisons of the COI gene at

each range of sequence divergence Intraspecific and closest congeneric interspecific matches of the following taxa are represented: (a) phylum Porifera; (b) class

Anthozoa; (c) class Hydrozoa; and (d) Scyphozoa

Table 2.1 COI distances, pairwise Mann-Whitney U-statistics, significance tests and

the rank of each taxon (largest to smallest distances) among Porifera, Anthozoa,

Hydrozoa and Scyphozoa for intraspecific (a) and closest congeneric interspecific

distances (b) Distance values denote means and standard errors

Taxon Uncorrected

distance (%)

K2P distance (%)

Porifera Anthozoa Hydrozoa Scyphozoa

(a) Intraspecific distances

Porifera 0.53 ± 0.09 0.57 ± 0.10 Rank=2 U=5429 U=502 U=605

Anthozoa 0.33 ± 0.05 0.34 ± 0.05 P<0.001 Rank=3 U=781 U=1140

Hydrozoa 3.82 ± 0.47 4.14 ± 0.53 P<0.001 P<0.001 Rank=1 U=4243

Scyphozoa 3.80 ± 0.39 4.23 ± 0.46 P<0.001 P<0.001 P=0.07 Rank=1

(b) Closest congeneric interspecific distances

Porifera 3.29 ± 0.52 3.29 ± 0.52 Rank=3 U=3125 U=961 U=511

Anthozoa 0.77 ± 0.13 0.82 ± 0.15 P<0.001 Rank=4 U=623 U=871

Hydrozoa 8.45 ± 0.34 9.16 ± 0.40 P<0.001 P<0.001 Rank=2 U=1255

Scyphozoa 12.94 ± 0.67 14.82 ± 0.77 P<0.001 P<0.001 P<0.001 Rank=1

The threshold values, based on 10
mean intraspecific distances, were 5.3%, 3.3%, 38.2% and 38.0% for Porifera, Anthozoa, Hydrozoa and Scyphozoa respectively To minimise overlap between intra- and interspecific divergences, distance thresholds employed are 1.2% for poriferans (overlap of 11.51%), 0.3% for anthozoans (overlap

of 24.48%), 5.8% for hydrozoans (overlap of 2.62%) and 11.8% for scyphozoans

(overlap of 14.91%) Proportions of sequences identified by DNA barcodes using

three different threshold values are shown in Table 2.2 Generally, frequencies of

sequences that were correctly identified or ambiguous do not change when different threshold values are used However, 10
mean intraspecific distance gave the highest frequency of inaccurate species attribution, and the least number without any match

minimum overlap, rates of accurate identification were not higher than other

thresholds, but many sequences labelled as incorrect otherwise were not named

Table 2.2 Frequencies of sequences (percentages in parentheses) with accuracy of

species attribution using three threshold values: 3.0%, 10
mean intraspecific distance

(10
) and point of minimum overlap (MO) An accurate identification of a sequence

is classified as ‘correct’; sequence with best matches to the correct barcode and at

least an incorrect one is ambiguous; erroneous species attribution is ‘incorrect’; and

‘unmatched’ sequence does not have any match closer than the threshold

Taxon Threshold Correct Ambiguous Incorrect Unmatched

3.0% 61 (50.0%) 6 (4.9%) 25 (20.5%) 30 (24.6%) 5.3% (10
) 61 (50.0%) 6 (4.9%) 26 (21.3%) 29 (23.8%) Porifera

1.2% (MO) 59 (48.4%) 5 (4.1%) 22 (18.0%) 36 (29.5%) 3.0% 97 (30.7%) 145 (45.9%) 65 (20.6%) 9 (2.8%) 3.3% (10
) 98 (31.0%) 145 (45.9%) 65 (20.6%) 8 (2.5%) Anthozoa

0.3% (MO) 94 (29.7%) 138 (43.7%) 43 (13.6%) 41 (13.0%) 3.0% 66 (58.9%) 0 (0.0%) 4 (3.6%) 42 (37.5%) 38.2% (10
) 68 (60.7%) 3 (2.7%) 41 (36.6%) 0 (0.0%) Hydrozoa

5.8% (MO) 68 (60.7%) 0 (0.0%) 6 (5.4%) 38 (33.9%) 3.0% 122 (91.7%) 1 (0.8%) 4 (3.0%) 6 (4.5%) 38.0% (10
) 124 (93.2%) 1 (0.8%) 8 (6.0%) 0 (0.0%) Scyphozoa

11.8% (MO) 123 (92.4%) 1 (0.8%) 4 (3.0%) 5 (3.8%)

2.4 Discussion

This study shows that intraspecific variability is much lower in Porifera and Anthozoa

compared to the Medusozoa, while interspecific distances are also low in Anthozoa

(uncorrected mean 1.60%) and high in Hydrozoa and Scyphozoa (12.20% and 15.41%

respectively) In Porifera the mean interspecific distance is 4.21%, very similar to the

5.6% registered for the outgroup Fungi (Seifert et al 2007) Both are considered low, given that Hebert et al (2003b) calculated a divergence mean of 11.3% for congeneric species pairs from 11 Bilateria phyla Distances in the Medusozoa are therefore comparable to those of bilaterians, while the Anthozoa, Porifera and Fungi exhibit low interspecific COI divergence It should be noted that Anthozoa and Porifera sequence evolution appears to be unusually slow for other mitochondrial genes as

well In Anthozoa, for instance, van Oppen et al (1999) observed that the cytochrome

b gene displays a maximum of 0.8% sequence divergence between Caribbean and

Pacific Acropora species Fukami et al (2000) surveyed eight species of Acropora

and found no differences for cytochrome b and only up to 0.46% in ATP6 genes In Porifera, Lavrov et al (2005) determined that the variability of mitochondrial SSU-rRNA genes relative to nuclear SSU-RNA genes is about 2.5 times lower in

poriferans than in mammals Conversely, the 16S rDNA in the hydrozoan Obelia geniculata and family Campanulariidae seem to mirror the COI gene in adhering to

evolutionary rates that are similar to those of typical triploblastic invertebrates

(Govindarajan et al 2005, 2006)

To explain speed variations, we use a metazoan tree as summarised from Kim et al (1999), Borchiellini et al (2001), Medina et al (2001) and Halanych (2004) (Figure 2.2) Note that poriferan paraphyly would not alter our interpretation Our results support two equally parsimonious reconstructions for the slow evolution of mtDNA, both with slow-evolving ancestral mitochondrial DNA We can thus reject the

hypotheses from Shearer et al (2002) of fast evolution at the base of the metazoan tree Higher rates could have originated twice—in the Medusozoa and Bilateria—possibly resulting from two independent losses of an mtDNA repair mechanism that

suppresses substitution rates (van Oppen et al 1999, 2002b; Shearer et al 2002; Hellberg 2006; Medina et al 2006) Alternatively, fast mtDNA could also have been acquired before the Cnidaria-Bilateria split, and subsequently lost in the Anthozoa Interestingly, medusozoans have unusual linear genomes, while Porifera and

Anthozoa possess typical circular mitochondrial DNA (Bridge et al 1992; Kingdon et al 2000) Proponents of slow mitochondrial sequence evolution in ‘basal’ metazoans have used genome organisation to lend support for our first reconstruction, suggesting that genome linearisation is related to mtDNA acceleration in Hydrozoa and Scyphozoa (Shearer et al 2002; van Oppen et al 2002b)

Pont-Figure 2.2 Two most parsimonious evolutionary scenarios for slow mtDNA

evolution in Anthozoa and Porifera From a slow ancestral mtDNA, (A) fast evolution originated in the Medusozoa and Bilateria independently, or (B) fast mtDNA evolved

in the Cnidaria + Bilateria clade but was lost in Anthozoa Black bars labelled ‘fast’ and ‘slow’ respectively denote acceleration and deceleration of mitochondrial

sequence evolution

Slow evolution of mitochondrial sequences at the Metazoa base is currently the best supported hypothesis, but additional data would be welcome In particular, more information on Ctenophora and Placozoa is needed Furthermore, we note that