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

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),

ii


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.

iii


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

iv


Table of Contents

Acknowledgements

ii

Table of Contents

v

Summary

vii

List of Tables

ix

List of Figures

xi

CHAPTER 1: GENERAL INTRODUCTION
1.1

The Scleractinia

1

1.2

Coral taxonomy, barcoding and phylogenetics

5

1.3

Faviidae in Singapore

15

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
DNA BARCODING
2.1

Introduction

18

2.2

Materials and Methods

20

2.3

Results

22

2.4

Discussion

26

v


CHAPTER 3: PHYLOGENETIC RELATIONSHIPS IN THE FAVIIDAE
BASED ON MOLECULAR AND MORPHOLOGICAL MARKERS
3.1

Introduction

32

3.2

Materials and Methods

35

3.3

Results

47

3.4

Discussion

60

GENERAL CONCLUSIONS

68

References

70

Appendix I

104

Appendix II

128

Appendix III

137

vi


Summary
The Faviidae constitutes one of the most important families of hermatypic corals on
Indo-Pacific reefs. Several species in this group are taxonomically difficult and little
is known about their phylogenetic relationships at the species level. DNA barcoding
holds enormous potential for species identification and subsequent resolution of

evolutionary relationships among faviid corals. However, the efficacy of this
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.

vii


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.

viii


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).

ix


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).

x

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

xi

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).

xii


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).

1


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; RodriguezLanetty et al. 2006). This is known to affect the response of the holobiont (host +

2


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

3


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

4


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).

5


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

6


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

7


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 (WijsmanBest 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

8


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
& Fiedler 2007).

In the Scleractinia, low COI divergence has been detected. Hebert et al. (2003b), in

determining Cnidaria’s suitability for barcoding, noted that interspecific COI
variability is unusually low. Of all pairwise congeneric interspecific distances in the

9


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

10


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

11


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.

12


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
Scleractinia

Acroporidae

Acropora

No. of species Markers

Reference

34

16S

Romano & Palumbi (1996)

15

28S

Veron et al. (1996)

34

16S

Romano & Palumbi (1997)

22

28S

Chen et al. (2000)

68

16S; 28S

Romano & Cairns (2000)

28

12S

Chen et al. (2002)

40

28S

Cuif et al. (2003)

70

cytB; COI;
tub

Fukami et al. (2004b)

84

16S

Le Goff-Vitry (2004)

9

mt-genome

Medina et al. (2006)

15

cytB; ATP6

Fukami et al. (2000)

29

cytB; h2ab

Wallace et al. (2007)

6

MC2

Hatta et al. (1999)

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)

13