Coral Reefs (2007) 26:475486

DOI 10.1007/s00338-007-0206-1
1 3
REVIEW
Genomic and microarray approaches to coral reef conservation
biology
S. Fort K. S. Kassahn L. C. Grasso
D. C. Hayward A. Iguchi E. E. Ball D. J. Miller
Received: 20 November 2006 / Accepted: 5 February 2007 / Published online: 28 February 2007
Springer-Verlag 2007
Abstract New technologies based on DNA micro-
arrays and comparative genomics hold great promise
for providing the background biological information
necessary for eVective coral reef conservation and
management. Microarray analysis has been used in a
wide range of applications across the biological sci-
ences, most frequently to examine simultaneous
changes in the expression of large numbers of genes in
response to experimental manipulation or environ-
mental variation. Other applications of microarray
methods include the assessment of divergence in gene
sequences between species and the identiWcation of
fast-evolving genes. Arrays are presently available for
only a limited range of species, but with appropriate
controls they can be used for related species, thus
avoiding the considerable costs associated with devel-
opment of a system de novo. Arrays are in use or prep-
aration to study stress responses, early development,
and symbiosis in Acropora and Montastraea. Ongoing
projects on several corals are making available large
numbers of expressed gene sequences, enabling the
identiWcation of candidate genes for studies on gamete
speciWcity, allorecognition and symbiont interactions.
Over the next few years, microarray and comparative
genomic approaches are likely to assume increasingly
important and widespread use to study many aspects
of the biology of coral reef organisms. Application of
these genomic approaches to enhance our understand-
ing of genetic and physiological correlates during
stress, environmental disturbance and disease bears
direct relevance to the conservation of coral reef eco-
systems.
Keywords Microarray Genomics EST WGS
Acropora Fast evolving genes
Introduction
We are entering an era when genomic, or systems
biology, approaches can be applied to study the biol-
ogy of coral reef organisms. Genomic approaches diVer
signiWcantly in scale from other types of molecular
Communicated by Biology Editor M. van Oppen.
S. Fort and K.S. Kassahn contributed equally.
S. Fort
Mathematical Science Institute,
Australian National University,
Canberra, ACT 0200, Australia
K. S. Kassahn
School of Tropical Biology,
James Cook University, Townsville,
QLD 4811, Australia
L. C. Grasso D. C. Hayward E. E. Ball
ARC Centre for the Molecular Genetics of Development,
Research School of Biological Sciences,
Australian National University, PO Box 475,
Canberra, ACT 2601, Australia
A. Iguchi D. J. Miller (&)
ARC Centre of Excellence in Coral Reef
Biology and Comparative Genomics Centre,
James Cook University,
Townsville, QLD 4811, Australia
e-mail: david.miller@jcu.edu.au
Present Address:
K. S. Kassahn
Institute for Molecular Biosciences,
University of Queensland,
Brisbane, QLD 4072, Australia
476 Coral Reefs (2007) 26:475486
1 3
analyses; these are large-scale methods that allow the
investigation of a substantial fraction of, or sometimes
the entire, transcriptome or genome. The scale of sys-
tems biology, dealing as it does in thousands or tens of
thousands of genes, introduces requirements for auto-
mation, the deployment of robotics and computational
analyses of large datasets.
One outstanding advantage of genome-scale
approaches is that these free us from some of the limi-
tations imposed by candidate gene investigations; one
no longer needs to propose simple hypotheses to test.
EVectively, hypotheses can be broadened from some-
thing like Are genes encoding homologs of metazoan
heat shock proteins induced in corals during a coral
bleaching event? to What changes in gene expres-
sion occur during a coral bleaching event?. This shift
to less constrained hypotheses and the exploratory
nature of genomic approaches are likely to be particu-
larly important in the case of corals, given the complex
nature of their genomes and the presence of a substan-
tial number of non-metazoan genes (Technau et al.
2005).
Sequencing a whole genome is an expensive under-
taking (e.g., the human genome project as a whole cost
an estimated $US3 billion) and inevitably this leads to
questions about the value or value for money of such
undertakings. What can we learn from having available
to us one or more genomes from our favourite organ-
isms? Or perhaps the question should be, What can
we learn from a genome above and beyond that which
could come from more targeted approaches? In
many cases, the more directed approach of sequencing
large numbers of ESTs (expressed sequence tags; com-
plete or incomplete sequences of cDNA clones repre-
senting expressed genes) is a cost-eVective alternative
(a poor mans genome project; Rudd 2003). It has
often been argued that ESTs provide what you need to
knowinformation about the ability of an organism
to make speciWc proteins, but one major advantage of
genome sequences is that they allow non-coding DNA
and regulatory sequence to be investigated. Moreover,
evolutionary inferences based on limited numbers of
loci can be compromised by population history and
linkage eVects, whereas whole genome data permit the
identiWcation of those areas of the genome that are or
have been functionally important. Whole genome
sequence data have also become enormously valuable
resources for evolutionary biologists as well as those
interested in the biology of particular lineages. Com-
parisons between closely related taxa at the whole
genome level are a potentially powerful means of
addressing the molecular mechanisms underlying the
evolution of novel characteristics. For example, com-
parative genomic studies of primates permit investiga-
tions of the molecular changes associated with the
expansion of the cerebral cortex in man (Popesco et al.
2006).
Although it has been predicted that the cost of
sequencing individual human genomes may tumble to
a few thousand dollars in the near future (e.g., Mardis
2006), at present the expense involved in sequencing
entire genomes and printing microarrays of whole tran-
scriptomes puts these technologies beyond the budget
of single investigators or even moderate sized teams of
investigators; and despite extensive lobbying, there is
no immediate prospect of a complete coral genome
sequence. However, whole genome sequence data for
related animals (see below) can provide powerful com-
parative data. For example, comparison of existing
coral EST data with the complete genome sequence for
the sea anemone Nematostella vectensis should permit
insights into the molecular bases of coral-speciWc bio-
logical processes, such as the deposition of the arago-
nite skeleton. EST collections can also be used to
provide estimates of divergence rates and for the iden-
tiWcation of fast-evolving genes, as outlined below.
Since their development in the mid-1990s (Schena
et al. 1995) microarrays, which potentially allow moni-
toring the expression of thousands of genes at once,
have found a broad range of applications, including
functional genomic studies in ecology and evolution
(Feder and Mitchell-Olds 2003). In functional genomic
studies, assessment of the transcriptome allows infer-
ences about how the genetic make-up of an organism
translates into biological function. One major focus of
microarray-based research has been the study of envi-
ronmental stress responses (Gracey et al. 2001, 2004;
Williams et al. 2003; Podrabsky and Somero 2004;
Krasnov et al. 2005). In addition to studying responses
to environmental factors or the passage of time, micro-
arrays can also be used for comparative genomic stud-
ies and the identiWcation of fast evolving genes (Fig. 1).
Available resourcesgenome sequence data and EST
collections
It is often said that we live in the post-genomic era,
but the reality is that whole genome sequences are only
available for a limited number of animal species, the
selection being strongly biased towards vertebrates.
Nevertheless, the sequence databases are still enjoying
massive expansion, and comparison of coral genes with
their homologs in a diversity of well studied species can
provide insights into structure/function relationships
that would not be available by comparison with other,
Coral Reefs (2007) 26:475486 477
1 3
less studied species. One purpose of this review is to
summarise the available resources that are of direct
relevance to those of us with interests in corals and
other reef organisms.
Whilst no coral genome has been completely
sequenced, pilot sequencing was carried out in 2005
on three speciesAcropora millepora (25,152 whole
genome shotgun reads), Acropora palmata (11,024
whole genome shotgun reads) and Porites lobata
(13,824 whole genome shotgun reads). Our best guess
as to the size of the A. millepora genome is 200 Mb, but
we stress that this is a very rough estimate based on
limited data. These pilot data have demonstrated the
feasibility of sequencing an entire coral genome and
are in the trace archive at NCBI (http://www.ncbi.
nlm.nih.gov/Traces/). There are three substantial sets
of coral ESTs presently available in Genbank. The spe-
cies from which these ESTs were made and the num-
bers of ESTs as of mid-October 2006 are: A. millepora
(10,247), A. palmata (4,017) and Montastraea faveolata
(2,156) (see Schwarz et al. 2006, for background on
the latter two collections). One important diVerence
between these datasets is that the A. millepora ESTs
speciWcally exclude the dinoXagellate symbionts,
whereas for the latter two species no attempts were
made to resolve the two partners (although it appears
that symbiont representation is very low). The EST
collections for A. palmata and M. faveolata are likely
to be signiWcantly expanded in the near future, with
Department of Energy support via the Joint Genome
Institute (see http://www.jgi.doe.gov/sequencing/why/
CSP2007/coralalgal.html). The A. millepora ESTs that
are presently publicly available represent three devel-
opmental stages, the pre-gastrulation prawn chip
(3,285 ESTs), the post-gastrulation motile planula
(3,754 ESTs), and the immediate post-settlement
crown stage (3,208 ESTs) (see Ball et al. 2002; for
descriptions of these stages). These ESTs yielded 6,021
predicted genes and 5,063 predicted peptides (Technau
et al. 2005, Table S1). On the basis of these ESTs and a
collection of 16,598 ESTs from the sea anemone
Nematostella it was estimated that the gene number for
Acropora would be 2025,000 (Technau et al. 2005).
Two additional sets of A. millepora ESTs, from
bleached adult colonies (4,838 ESTs), and eggs (1,088
ESTs), have been sequenced but are not yet public.
The Marine Genomics Project (http://www.marinege-
nomics.org/) is home for two additional sets of coral
ESTs from Montastraea annularis (5,066 ESTs) and
Porites porites (247 ESTs). These coral resources are
complemented by EST projects for Symbiodinium
spp., the dinoXagellate symbionts of corals. Strains
from A. palmata and M. faveolata are likely to be
extensively represented in the near future (http://www.
jgi.doe.gov/sequencing/why/CSP2007/coralalgal.html),
and 5,161 ESTs from the clade C3 Symbiodinium (from
Acropora aspera) have been sequenced (W Leggat, D
Yellowlees personal communication) but are not yet
public.
Extensive genomic and EST datasets are available
for two other cnidarians, and these are enormously
useful comparative resources for coral biology. The sea
anemone, Nematostella vectensis, was selected as the
Wrst representative cnidarian for whole genome
sequencing (WGS); the genome has been sequenced to
>7.8-fold coverage and assembly version 1 is available
via the JGI Nematostella pages (http://genome.jgi-psf.
org/Nemve1/Nemve1.home.html). Various sequence
comparison algorithms such as BLAST, the Basic
Local Alignment Search Tool (Altschul et al. 1997),
Fig. 1 Examples of
applications of microarray
methods for studies in ecology
and evolution. The image
shown is of a zebraWsh oligo-
nucleotide array hybridised
with Xuorescently labelled
cDNAs from the coral reef
Wsh Pomacentrus moluccensis
478 Coral Reefs (2007) 26:475486
1 3
which allows comparison of a given nucleotide or pep-
tide sequence to appropriate databases, can be used to
search both the genome, and large predicted protein
and EST collections for Nematostella via the Stellabase
site (www.stellabase.org). Somewhat more distantly
related to corals, but also a useful comparator for many
purposes, Hydra magnipapillata was the second cnidar-
ian targeted for WGS; its genome is much larger than
that of Nematostella, but has presently been deter-
mined to approximately 6-fold (R. Steele personal
communication). The J. Craig Venter Institute is con-
ducting the genome sequencing, and hosts the latest
data summaries on its website (https://research.venter-
institute.org). More than 150,000 ESTs have been
determined for this species (H. Bode personal commu-
nication), and are searchable via Hydrabase (www.
hydrabase.org).
Although there are currently no whole genome
sequencing projects underway for coral reef Wshes,
other teleosts have been completely sequenced, e.g.,
the zebraWsh Danio rerio, the puVerWshes Takifugu
rubripes and Tetraodon nigroviridis, the medaka Oryz-
ias latipes and the stickleback Gasterosteus aculeatus.
These genome sequence data can be accessed via the
Ensembl Genome Browser at http://www.ensembl.org/
index.html. More detailed information regarding indi-
vidual Wsh genomes can be obtained from the respec-
tive research communities, for example, the ZebraWsh
Information Network at http://zWn.org/. There are also
signiWcant sequencing eVorts for Atlantic salmon
Salmo salar, rainbow trout Oncorhynchis mykiss, and
some cichlid species, e.g., Oreochromis niloticus. These
teleost sequences and ESTs can be accessed via data-
bases at NCBI (http://www.ncbi.nlm.nih.gov/) and pro-
vide useful resources for comparative genomic studies.
Researchers interested in coral reef Wsh biology thus
have a wealth of genomic resources available for com-
parative studies.
Microarray basics
Next we turn to a technique that allows the visualisa-
tion of the simultaneous expression of large numbers
of genes, the microarray. There are a number of excel-
lent sources of information on microarrays. Among the
most comprehensive are Bowtell and Sambrook (2003)
and Kimmel and Oliver (2006a, b). Here we provide a
brief description of the principles underlying micro-
array analysis and some considerations for planning a
microarray experiment.
For non-commercial marine organisms, at least for
the near future, most microarrays will consist of an
array of spots of complementary DNA, made on an
RNA template originating from the expressed genes,
on a glass slide. The identity of the cDNAs spotted
onto the array may be known or unknown and they
may be redundant to varying degrees. Therefore the
Wrst step in the development of a new cDNA micro-
array is the isolation and cloning of a cDNA library,
which can then be PCR ampliWed and spotted onto
microarray slides.
The spots on the array can be random expressed
genes from a speciWc developmental stage, some exper-
imental treatment or a particular condition, such as a
disease state. Alternatively, a speciWc set of clones
known or suspected to be related in some way, e.g., in a
regulatory pathway, may be spotted onto the micro-
array. In most microarray experiments, the species
being investigated is the species from which the cDNA
was derived. However, other species, especially if
closely related, can also be analysed in heterologous
microarray experiments, as discussed below. In order
to carry out a microarray experiment mRNAs, or their
complementary DNAs, from two or more samples are
each labelled with two diVerent Xuorophores and are
then competitively hybridised to the same microarray
slide (Fig. 2). In contrast to older methods for analy-
sing changes in the level of transcript abundance, such
as northern blots, microarrays are a high throughput
technology allowing the analysis of thousands of genes
simultaneously, with the ultimate possibility of being
able to survey every transcript encoded by an organ-
isms genome. The output of a microarray experiment
is an ordered list of diVerentially expressed genes and
interest is usually focused on those that are most sig-
niWcantly up or down regulated.
Microarrays oVer the possibility of identifying genes
not previously known to be involved in a process, e.g.,
the response to a potential stressor such as heat. Previ-
ously unknown interactions between genes or gene
products may also become apparent. Genes which
share an expression pattern are frequently co-regu-
lated, with the implication that they are operating in
the same molecular pathway, leading to a co-ordinated
response. This has been shown to be the case for genes
involved in several developmental and metabolic path-
ways such as cell cycle genes (Cho et al. 1998; Iyer et al.
1999) and yeast mitochondrial genes (Eisen et al.
1998). There are various software packages available
for taking the extremely large amount of data gener-
ated by a microarray experiment, and integrating it
into a more easily interpretable representation. For
example Cluster 3.0 (Eisen et al. 1998) allows hierar-
chical clustering of gene expression proWles and is
available as free open source software.
Coral Reefs (2007) 26:475486 479
1 3
The design of a microarray experiment should be
carefully considered in order to do the most informa-
tive experiment possible with the (sometimes limited)
amount of biological material available. This involves
determining how many biological replicates and tech-
nical replicates are needed, which samples should be
labeled with which Xuorophore and which samples
should be directly compared on the same slide (Chur-
chill 2002; Yang and Speed 2002; Bowtell and Sam-
brook 2003). This is particularly pertinent when doing
microarrays with material from small animals, embry-
onic stages or larvae, as material from which to extract
RNA can be in short supply and sometimes yields
insuYcient RNA to do an experiment without ampliW-
cation. One solution to this problem is the use of com-
mercially available RNA ampliWcation kits. These kits
synthesise large numbers of transcripts in a linear,
rather than an exponential ampliWcation, and ideally
the transcripts will be represented in the same relative
amounts as they were in the original sample. This may
not always be the case with PCR (exponentially)
ampliWed samples. The potential problem that some
transcripts may be preferentially transcribed, skewing
their relative abundance, remains, but comparisons
between microarray experiments done with ampliWed
and unampliWed mRNA suggest that ampliWed mRNA
can give accurate results (Stoyanova et al. 2004; Zhu
et al. 2006). Adult hard coral material poses less of a
problem in terms of quantities available than embry-
onic material, but the relatively small amount of living
tissue per sample means that even large-scale extrac-
tions can sometimes yield little RNA. The situation is
further complicated by the presence of dinoXagellate
symbionts (zooxanthellae) in at least some life stages
of most species. As more sequence data for both corals
and symbionts become available this will be less of a
consideration, but at present knowing the source of the
genes that you are probing is of critical importance for
interpretation of results.
It is particularly important to involve a statistician at
the planning and experimental design stage of micro-
array experiments and to understand possible sources
of variability. Microarray experiments are expensive
and typically substantial time is spent performing and
analysing the experiment, so it is very important to
design well. Sometimes the statistically best experi-
ment may be either practically or Wnancially impossi-
ble, and then it becomes a matter of doing the best one
can with the available time and resources. The topic of
experimental design is too broad to be covered here,
but excellent discussions can be found in Yang and
Speed (2002, 2003).
The biological material used for microarray analysis
also warrants some consideration. The most reliable
results will be obtained using fresh tissue samples
that have been transferred into liquid nitrogen immedi-
ately upon collection. Where this is impossible RNA-
later and similar products may be suitable. RNAlater
has been used to successfully preserve total RNA from
A. millepora embryos, however, problems may arise in
using RNAlater to preserve adult coral tissue due to
the relatively impermeable nature of the coral skele-
ton, although this has not been systematically tested.
Direct comparisons of yields from diVerent RNA pres-
ervation methods do not appear to be available. Once
RNA is extracted from the sample it is used as a tem-
plate for reverse transcription into cDNA. During the
reverse transcription reaction the sample is Xuores-
cently labelled with either red or green Xuorescent dye.
Fluorescently labelled cDNA samples are then com-
petitively hybridised to microarray slides. For each
gene represented on the microarray, the relative tran-
script abundance in both samples is inferred from the
red and green signal intensities of hybridised tran-
scripts. The brightness of all of the spots is measured by
scanning the slides with lasers. A number of diVerent
types of scanners and software packages are available
Fig. 2 Diagram showing the steps involved in a microarray
experiment in which gene expression in two samples is being com-
pared
Preparation of Test Samples for Comparison
Sample1 Sample 2
(RNA extraction)
RNA
cDNA
(incorporation
of aminoallyl-dUTP)
Sample 1cDNA
labeled with Cy3
Sample 2 cDNA
labeled with Cy5
(dye coupling) combine samples
Hybridise to array
Scanning
Analysis
RNA
cDNA
(reverse transcripiton)
(incorporation
of aminoallyl-dUTP)
480 Coral Reefs (2007) 26:475486
1 3
for converting the raw signal intensity data into relative
transcript abundance. The statistical analysis of extracted
cDNA microarray data includes three main steps; nor-
malisation, transformation and testing for diVerential
expression. Various statistical techniques for analysing
microarrays are represented by an abundance of soft-
ware packages available for purchase or for free on the
web (www.bioconductor.org). A number of reviews and
books have been published on the various packages
available for microarray analysis (Kimmel and Oliver
2006b; Gentleman et al. 2005). However, the statistical
analysis of microarray data is an active area of
research. Hence it is prudent to employ the help of a
statistician (Allison et al. 2006).
Once statistical support for diVerential expression of
a subset of genes has been achieved, any further analy-
sis depends on how much information is available for
these. Where the identity of the cDNA spots repre-
sented on the microarray is known, hierarchical clus-
tering of gene responses (mentioned above) can
identify the types of genes that are regulated in
response to a certain treatment. The more that is
known about the cDNA spots on the array, the more
accurate and complete will be the testing of hypotheses
and drawing of conclusions concerning possible inter-
relationships between the genes represented on the
microarray.
Model organisms and beyondstrategies for applying
microarray technology to non-model species
Developing a microarray for a new species is usually
time-consuming and expensive. It may be possible to
avoid this process by using microarrays developed for a
closely related species in heterologous array experi-
ments. Human cDNA microarrays, for example, have
been used to study bovine, pig and salmon gene regula-
tion (Medhora et al. 2002; Tsoi et al. 2003; Adjaye
et al. 2004). Oligonucleotide microarrays can also
be successfully applied in heterologous microarray
experiments (Ji et al. 2004; Kassahn et al. in press).
Oligonucleotide microarrays may consist of either
short (25 bp, e.g. AVymetrix GeneChip

arrays) or
long (5065 bp) oligomers deposited on the micro-
array. The success of such experiments depends on the
ability of the heterologous DNA species to bind to the
microarray and the level of sequence similarity
between the two taxa. In general, microarray analysis is
robust to some level of sequence divergence and genes
with less than 25% sequence divergence have been
shown to produce signiWcant cross-hybridisation on
long (50mer) oligonucleotide microarrays (Kane et al.
2000). With increasing sequence divergence, the ability
to accurately measure gene regulation decreases due to
poor cross-hybridisation (Renn et al. 2004). However,
the level of sequence divergence diVers across genes
with some genes diverging more rapidly than others
(Makalowski et al. 1996). Therefore, heterologous
microarrays may be more successfully employed to
measure gene expression responses at conserved gene
loci, while genes that are rapidly diverging may fail to
cross-hybridise. Furthermore, an important distinction
has to be made between the use of heterologous micro-
array experiments to measure changes in gene expres-
sion within a species, e.g., comparison of two diVerent
treatment types within the same species, and the use
of a single microarray to compare expression levels
between two species. In the latter case, true biological
diVerences in expression level between two species
may be confounded with poor cross-hybridisation due
to sequence divergence between the two species (Gilad
et al. 2005). In contrast, when using heterologous
microarrays for within-species comparisons, the poten-
tial eVects of sequence divergence on cross-hybridisa-
tion are accounted for as they aVect both treatment
types similarly.
Inter-species genomic hybridisation experiments can
be used to identify which genes are suYciently con-
served to signiWcantly cross-hybridise in heterologous
microarray experiments (Kassahn et al. in press). For
this purpose, genomic DNA is extracted from the spe-
cies of interest and the species for which the micro-
array was designed and both gDNA samples are
competitively hybridised to the microarray. Genes
which share signiWcant sequence similarity between the
two species will produce equivalent signal intensities,
while genes with signiWcant sequence divergence will
show reduced signal intensities in the heterologous
species. In general, cross-hybridisation on microarrays
is positively correlated with sequence similarity (Wu
et al. 2001; HinchliVe et al. 2003; Brunelle et al. 2004).
Hence, relative signal intensities can be used to infer
the level of sequence similarity for each gene on the
microarray and several approaches for analysis are
available (Kim et al. 2002; Le Quere et al. 2006; Kas-
sahn et al. in press). Using this technique it is possible
to eliminate from consideration those genes that may
not cross-hybridise in heterologous experiments.
DiVerences in gene copy number can also potentially
confound the results of such experiments (Pollack et al.
1999; HinchliVe et al. 2003), but with increasing phylo-
genetic distance such diVerences will play a minor role
(Brunelle et al. 2004). Researchers using cDNA micro-
arrays may also need to account for diVerences arising
from the diVerent structure of genomic DNA and
Coral Reefs (2007) 26:475486 481
1 3
cDNA. Because genomic DNA includes introns and
non-coding regions, spots on the microarray with
sequences spanning an exon-intron boundary in the
genomic DNA sample may not bind genomic DNA,
but would successfully bind cDNA during gene expres-
sion analysis. In these instances, Xuorescently labelled
cDNA derived from the species of interest and the spe-
cies for which the microarray was designed may be
competitively hybridised to the cDNA microarray and
provide information about the potential for cross-
hybridisation (Renn et al. 2004). However, true biolog-
ical diVerences in gene expression, gene copy number
diVerences, and gene dosage eVects can confound the
results of competitive cDNA hybridisation experi-
ments across species. When using cDNA microarrays,
the best strategy for selecting those genes that can be
successfully measured across species would thus consist
of a combination of comparative genomic DNA and
cDNA hybridisations.
Because of their high throughput and ability to
screen thousands of gene transcripts at once, micro-
arrays are an attractive tool for environmental genomic
studies. Here, we have discussed strategies that can be
applied in situations where there are no commercial
microarrays available. In such cases, researchers can
either develop a custom cDNA microarray or use a
microarray developed for a close relative. We suggest
that inter-species hybridisation experiments using
genomic DNA and cDNA can be used to identify
which genes show loss of signal in heterologous micro-
array experiments. Such experiments can thus guide
the choice of which heterologous microarray is most
suitable for a particular application. Heterologous
microarray experiments are particularly suitable for
gene discovery studies where the aim is to identify a
small number of candidate genes with the most inter-
esting gene regulation. In this context, loss of signal for
some genes on the microarray would not compromise
the aims of the study. Once a selection of candidate
genes has been identiWed by means of heterologous
microarray experiments, diVerential expression can be
validated using an alternative method, such as a north-
ern or virtual northern blot or quantitative real-time
PCR.
The signiWcance of fast evolving genes
One of the major goals of evolutionary biology is to
understand the processes underlying the evolution of
species and populations, and these have direct implica-
tions for conservation genetics. Genes with high rates
of evolutionary change have been implicated in the
process of speciation and species diversiWcation
(Turner et al. 2005; Harr 2006). Furthermore, high
rates of evolutionary change may indicate strong direc-
tional selection and molecular adaptation, especially
where gene function can be directly related to the eco-
logical context of the species (Tautz and Schmid 1998;
Lecompte et al. 2001; Matzkin 2005; Fairhead and
Dujon 2006). The study of fast evolving genes may
therefore allow the identiWcation of unique adaptations
of the organism to its ecological niche, and pinpointing
sources of stress when the ecosystem is perturbed.
Fast evolving genes are typically found in systems
where equilibrium is never reached, such as the arms
race between host and pathogen. In a wide variety of
animals, genes in this category include those involved
in immunity, in reproduction (especially in sperm com-
petition and species recognition), and in various envi-
ronmental interactions. In addition, there are cases of
individual genes and classes of genes that show positive
selection in speciWc lineages (e.g., Castillo-Davis et al.
2004; Yu et al. in press), and this is often associated
with lineage-speciWc expansions of gene families. For
example, many members of the DUF1220 gene family
show hallmarks of positive selection in primates (Pope-
sco et al. 2006); these genes have not been functionally
characterised, but are predominantly expressed in
brain regions associated with higher cognitive func-
tions.
Amongst the numerous cases of rapidly evolving
reproductive proteins (see Swanson and Vacquier 2002
for a review), one of the best-characterised examples is
the co-evolutionary chase between abalone sperm
lysin and the vitelline envelope lysin receptor VERL.
External reproduction via broadcast gametes is com-
mon amongst marine animals, necessitating gamete-
speciWcity systems, and such systems appear to have
evolved independently in various animal lineages.
Abalone lysin and VERL appear to be a mollusc-spe-
ciWc solution to this problem. Lysin is a small (16 kD)
and highly basic protein that is a major constituent of
the abalone acrosomal vesicle. Lysin has the eVect of
solubilizing the vitelline envelope that is on the outside
of abalone eggs by non-enzymatic means, but its ability
to do so is species-speciWc; lysin eVectively creates a
hole in the vitelline envelope that allows the tip of the
sperm acrosomal process to fuse with the plasma mem-
brane of the egg (reviewed in Kresge et al. 2001). The
lysin protein is clearly under positive selection, and
shows hallmarks of this process throughout the entire
sequence, however VERL has a more complex evolu-
tionary history; it has a repeat structure that evolves by
concerted evolution in a neutral manner (Swanson
et al. 2001), whereas the N-terminal end of the molecule
482 Coral Reefs (2007) 26:475486
1 3
is under positive selection (Galindo et al. 2003). Other
animal groups appear to have solved the gamete-speci-
Wcity problem in similar ways but using unrelated pro-
teins; for example, the bindin protein and its receptor
fulWl analogous functions in the sea urchin (Gao et al.
1986). For this reason it is unlikely that these speciWc-
ity-conferring proteins have strict homologs in other
animals.
Many genes with roles in immunity and allorecogni-
tion are also known to be subject to positive selection,
the best-characterised system being the vertebrate
major histocompatibility complex (MHC). Genes
encoding other components of the immune repertoire,
such as antimicrobial peptides, are also under moder-
ate positive selection (Tennessen 2005). In populations
of estuarine Wsh that experience high levels of pollu-
tion, genes of the major histocompatibility complex are
under strong positive selection, signiWcantly stronger
than in populations living in less disturbed habitats
(Cohen 2002). Olfactory and gustatory receptors are
families of genes closely involved in the interaction
with the environment. In various species, they have
been found to evolve under positive selection (Thomas
et al. 2005; Shi and Zhang 2006). Adaptation to physi-
cal environmental factors, such as cold temperatures or
light can also leave their signature in the genome. For
instance, antifreeze proteins have been shown to
evolve by positive selection in Wsh and in beetles
(Swanson and Aquadro 2002), and the adaptive evolu-
tion observed in the opsin gene of cichlid species has
been hypothesised to be an adaptation to diVerent pho-
tic environments (Spady et al. 2005). Finally, positive
selection has been reported in genes acquiring new
functions, such as the sodium channel gene of electric
organs in electric Wshes (Zakon et al. 2006).
Bioinformatic and microarray approaches
to the identiWcation of fast evolving genes
Most examples of genes under positive selection have
been identiWed by selecting candidate loci a priori, but
the availability of whole genome sequences and large
EST collections enables unbiased approaches which
are potentially much more informative. With the avail-
ability of a substantial number of large datasets for pri-
mates, this is a particularly active area, but similar
approaches are feasible for corals, Wsh and other reef
animals.
Adaptive evolution can be detected amongst either
paralogous or orthologous genes when there is a selec-
tion pressure (positive selection) for the diversiWcation
of the amino acid sequences of the proteins for which
they code. Most methods aimed at detecting positive
selection amongst paralogs or orthologs are based on
the comparison of the number of synonymous (non-
amino acid changing) and non-synonymous mutations.
When a sequence has an excess of non-synonymous
mutations, it may be under positive selection pressure.
A number of programs are available that enable the
detection of positive selection in datasets of orthologs
or paralogs; for reviews, see Yang and Bielawski
(2000), Suzuki and Gojobori (2003) and Nielsen
(2005). Several of these programs also allow the identi-
Wcation of individual sites under positive selection
(e.g., Suzuki et al. 2001; Bielawski and Yang 2004;
Massingham and Goldman 2005), which may provide
insights into the relationship between protein structure
and function. Positive selection can be detected at the
level of a whole gene family but methods also exist to
detect subfamilies that are under diVerent selection
pressures (Yang and Nielsen 2002; Zhang et al. 2005).
However, as a caveat, considerable debate has
occurred over the power and robustness of these tests
for positive selection (e.g., Nei 2005; Nunney and
Schuenzel, 2006), therefore great care should be taken
in the choice of test parameters and a combination of
tests is commonly used.
When data are available on gene polymorphism
within a species, population genetic techniques can be
used for the detection of positively selected mutations.
A large literature exists on these techniques, and an
excellent review of the subject can be found in Nielsen
(2005). Tests relying on population genetics data alone
can be highly sensitive to assumptions about demo-
graphic factors, but combining comparative data with
population genetic data can result in more robust tests,
such as the McDonald-Kreitman test (McDonald and
Kreitman 1991) or tests based on the ratio of non-syn-
onymous to synonymous mutations (see above).
In cases where direct bioinformatic approaches are
not possible, microarray-based methods provide an
alternative approach for the identiWcation of fast evolv-
ing genes. This approach may actually be more useful
for many marine organisms for which little sequence
data are available. This application of microarray tech-
nology follows the same principle as the inter-species
genomic DNA hybridisation experiments outlined
above. Two genomic DNA samples are competitively
hybridised to the microarray. Genes that have signiW-
cantly diverged between the two species and which
show accelerated rates of sequence evolution can be
identiWed on the basis of diVerences in hybridisation
signal between the two species (Kim et al. 2002; Le
Quere et al. 2006). Thus far, this application of micro-
array technology has mainly been used to study the
Coral Reefs (2007) 26:475486 483
1 3
comparative genomics of microbes (Murray et al.
2001). However, microarray approaches for the identi-
Wcation of fast evolving genes are feasible for any
organism and would be particularly useful for non-
model species and species for which bioinformatics
approaches are not applicable because of a lack of
genome sequence data.
Finally, it is worth noting that just as mutation of the
coding sequence can be of adaptive signiWcance, so can
changes in expression proWle. For example, northern
and southern populations of the killiWsh Fundulus
heteroclitus diVer in the expression level of lactate
dehydrogenase-B, resulting in important diVerences in
physiological function between these populations
(Powers and Schulte 1998). Microarrays have great
potential in detecting the evolution of gene expression
(Gilad et al. 2006).
Application to reef cnidarians
The application of systems biology approaches to study
the biology of reef organisms is still in its infancy, but
microarray methods have been applied to analyse
stress responses in corals. For example Edge et al.
(2005) used a small array containing 32 genes from
M. faveolata to examine the eVects of increased tem-
perature, salinity and UV light and found diVerential
gene expression patterns for each condition. Studies
are presently under way to document changes in gene
expression in A. millepora associated with normal
development (Grasso et al. unpublished) as well as in
response to heat (Seneca et al. unpublished) or sedi-
ment (Klueter et al. unpublished) stress using Acro-
pora millepora microarrays containing 13,000 or 17,000
ESTs. In addition, microarray studies are beginning on
M. faveolata and A. palmata in order to study changes
in gene expression associated with the initiation, main-
tenance and breakdown of the symbiosis of these cor-
als with their zooxanthellate symbionts (Schwarz et al.
2006).
The next few years are likely to see much broader
application of genomic and microarray approaches to
study coral reef biology, and these approaches should
considerably advance our understanding of how reefs
function at levels from the individual to the ecosystem.
Studies of the number of genes in individual gene fami-
lies, using a whole genome or large EST libraries, have
already revealed that cnidarian genomes encode a rich
assortment of genes (Kortschak et al. 2003; Technau
et al. 2005). The availability of the Nematostella and
Hydra whole genome sequences will enable the sys-
tematic investigation of gene family expansions and
reductions in these species. It is also anticipated that
lineage-speciWc genes will be discovered at many levels
within the Cnidaria, and that their identiWcation will
shed light on the genomic bases of the unique biology
of these animals. We call attention to the fact that,
while whole genome comparisons are necessary to be
conWdent about the extent of reduction of a gene fam-
ily, EST data may be suYcient to demonstrate signiW-
cant expansions. Similarly, selection pressures acting
on a group of paralogs can be estimated from EST data
alone. The study of orthologs may, however, require
whole genome sequences to reduce the possibility of
confusion arising from paralogous relationships.
The availability of EST collections for the conge-
neric coral species A. palmata and A. millepora oVers
the opportunity to compare these data and identify
individual genes under positive selection pressure. An
unbiased application of this approach holds great
promise for coral biology at every level, as it can
deliver candidate genes for roles not only in generic
processes such as gamete speciWcity and allorecogni-
tion, but also for coral-speciWc processes such as symbi-
ont recognition and determination of colony
morphology. Although signiWcant progress can be
made using existing EST resources, for comprehensive
analyses a full genome sequence for a coral is an urgent
requirement. In the face of ever-increasing anthropo-
genic pressures, systems biology approaches are likely
to play increasingly important roles in coral reef man-
agement and conservation.
Relevance to reef conservation
Coral bleaching is a stress response with many possible
causes including extremes of heat or cold, high irradi-
ance, prolonged low light levels or darkness, exposure
to heavy metals, herbicides, and pathogenic microbes
(reviewed by Douglas 2003). At present, in the absence
of an obvious cause, there is no way for marine conser-
vation authorities to determine the cause and take
remedial action. However, it seems quite likely that
diVerent molecules are upregulated in response to
diVerent stresses. Microarray studies are the most
eVective way of establishing this and identifying the
relevant molecules for each stress. Once this has been
accomplished it is quite likely that simple and cheap
diagnostic tests can be devised to allow marine manag-
ers to identify unknown stressors. Alterations in gene
expression are likely part of the early response to stress
and thus hold great promise as a sensitive tool to detect
organismal stress. Similarly for the case of diseases, it
may be possible to identify speciWc molecules that are
484 Coral Reefs (2007) 26:475486
1 3
upregulated in diVerent diseases and use this informa-
tion either in conjunction with or in place of culture
techniques to identify the causative organism.
Another way in which microarrays may be useful is
in aiding our understanding of the settlement and
metamorphosis of coral planulae, a critical process in
reef recolonisation. At present we know that extracts
of coralline algae (Heyward and Negri 1999; Morse
et al. 1988), a GLWamide neuropeptide (Iwao et al.
2002) and high concentrations of various ions such as
lithium (Mueller and Leitz 2002) are eVective in induc-
ing settlement. However, it might be possible to devise
even better and more eVective stimuli if we understood
the stimulus-response pathways involved in this pro-
cess at a biochemical level. By sampling larvae at vari-
ous times after induction with the above substances
it may be possible to work out the molecules involved
in the transduction-response pathway by looking for
upregulated genes.
Once this process is understood it should be possible
to devise more directed ways of manipulating the sys-
tem. This would open the way to artiWcially recolonis-
ing areas by moving larvae into areas devastated by
storms or anthropogenic disturbances and artiWcially
settling them. It is known that not all coral species
respond to the same settlement stimuli (Morse et al.
1988) thus facilitating selectively settling larvae in
areas where they will grow best.
Although direct applications of ESTs and micro-
arrays in conservation are still fairly limited, their
applicability will grow with time. To quote Feder and
Mitchell-Olds (2003) genomics has enabled EEFG
(evolutionary and ecological functional genomics),
rather than initiating or shifting its paradigm, and this
is essential for any Weld that attempts the challenging
task of integrating genes, function, ecology and evolu-
tion in its research programmes. Once genomics has
permeated these areas, on which conservation policies
are based, its use will almost certainly Xow on to man-
agers in the Weld.
Acknowledgments We gratefully acknowledge the contribu-
tions of various members of our laboratories and external collab-
orators, and the support of the Australian Research Council
(ARC) both directly to DJM and EEB (Grants A00105431,
DP0209460 and DP0344483) and via the Centre for the Molecular
Genetics of Development and the Centre of Excellence for Coral
Reef Studies.
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