Molecular Delineation of Species in the Coral Holobiont

C H A P T E R

O N E

Molecular Delineation of Species in the Coral Holobiont Michael Stat*,†,‡,1, Andrew C. Baker§, David G. Bourne}, Adrienne M. S. Correajj,#, Zac Forsman*, Megan J. Huggett*, Xavier Pochon*,**, Derek Skillings*, Robert J. Toonen*, Madeleine J. H. van Oppen} and Ruth D. Gates*

Contents 1. Introduction 1.1. Coral reefs 1.2. Climate change and other impacts to coral reefs 1.3. The need for coherent species delineation in coral reef research 2. The Species Debate 2.1. The species debate in context 2.2. Species concepts 3. Systematics 3.1. Traditional taxonomy using phenetics 3.2. The utility of a molecular approach in delineating eukaryotic species 3.3. Species delineation using an integrative approach 4. The Coral Holobiont 4.1. Coral: The animal host 4.2. Symbiodinium: The dinoflagellate symbionts of reef corals 4.3. The prokaryotic symbionts: Bacteria and Archaea 5. Concluding Remarks

2 2 3 4 5 5 6 9 9 9 12 14 14 25 38 45

* Hawaii Institute of Marine Biology, School of Ocean and Earth Science and Technology, University of Hawaii, Kaneohe, HI, USA The UWA Oceans Institute and Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley, WA, Australia { Australian Institute of Marine Science, The University of Western Australia, Crawley, WA, Australia } Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, USA jj Department of Microbiology, Oregon State University, Corvallis, OR, USA } Australian Institute of Marine Science, Cape Ferguson, Townsville, Qld, Australia # Department of Biological Sciences, Florida International University, North Miami, FL, USA ** The Cawthron Institute, Aquaculture and Biotechnology, Nelson, New Zealand 1 Corresponding author.: Email: [email protected] {

Advances in Marine Biology, Volume 63 ISSN 0065-2881, http://dx.doi.org/10.1016/B978-0-12-394282-1.00001-6

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2012 Elsevier Ltd All rights reserved.

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Acknowledgements References

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Abstract The coral holobiont is a complex assemblage of organisms spanning a diverse taxonomic range including a cnidarian host, as well as various dinoflagellate, prokaryotic and acellular symbionts. With the accumulating information on the molecular diversity of these groups, binomial species classification and a reassessment of species boundaries for the partners in the coral holobiont is a logical extension of this work and will help enhance the capacity for comparative research among studies. To aid in this endeavour, we review the current literature on species diversity for the three best studied partners of the coral holobiont (coral, Symbiodinium, prokaryotes) and provide suggestions for future work on systematics within these taxa. We advocate for an integrative approach to the delineation of species using both molecular genetics in combination with phenetic characters. We also suggest that an a priori set of criteria be developed for each taxonomic group as no one species concept or accompanying set of guidelines is appropriate for delineating all members of the coral holobiont. Key Words: coral; bacteria; Symbiodinium; dinoflagellate; species; symbiosis

“No one definition has satisfied all naturalists; yet every naturalist knows vaguely what he means when he speaks of a species.” Charles Darwin 1859

1. Introduction 1.1. Coral reefs Tropical coral reef and rainforest ecosystems represent the most biologically diverse environments on earth (Reaka-Kudla, 1997). Coral reefs cover around 255,000 km2 of the earth’s surface and are predominantly restricted to tropical and subtropical waters between 30 N and 30 S (Johannes et al., 1983; Spalding and Grenfell, 1997). The architectural framework of corals (Fig. 1.1) creates habitat complexity that provides space for thousands of species of marine organisms. In addition to their biological and ecological significance, coral reefs have enormous economic and societal value through tourism, a supply of fish and other marine fauna as a source of protein for human consumption, and provide coastal protection from storm and wave damage (Pendleton, 1995; Spalding et al., 2001). Corals are able to grow and thrive in the characteristically nutrient-poor water environments of tropical and subtropical regions due to their ability to form mutually beneficial symbioses with unicellular photosynthetic

Molecular Delineation of Species in the Coral Holobiont

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Figure 1.1 Underwater image of the coral reef ecosystem on the fore-reef in Moorea, French Polynesia, taken in 2006. The calcium carbonate structures of corals provide habitat complexity for a wide range of marine organisms.

dinoflagellates belonging to the genus Symbiodinium (Freudenthal, 1962; Muscatine and Porter, 1977). The Symbiodinium reside in the gastrodermal tissue of the coral and translocates fixed organic carbon and other nutrients to their host in return for inorganic waste metabolites from host respiration and protection from grazing (Muscatine, 1967; Yellowlees et al., 2008). This exchange of nutrients allows both partners to flourish and helps the coral secrete calcium carbonate that forms the skeletal structure of the coral colony and contributes to the formation of the reef (Goreau and Goreau, 1959). While this interaction has received much attention, corals also associate with a wide range of other organisms including prokaryotes. The coral animal host and its taxonomically diverse portfolio of symbionts are referred to as the coral ‘holobiont’ (Rohwer et al., 2002). Coral-associated prokaryotes, like their neighbouring dinoflagellates, are also beneficial to the animal host; cyanobacteria provide nutrition through nitrogen fixation (Lesser et al., 2004), and a consortium of bacteria resides in the mucous layer of corals acting as a first line of defence against pathogens by occupying space and producing antimicrobial compounds (Schnit-Orland and Kushmaro, 2009).

1.2. Climate change and other impacts to coral reefs Climate change and other local anthropogenic impacts are having a significant negative effect on the world’s coral reefs (Hoegh-Guldberg, 1999; Hoegh-Guldberg et al., 2007). Over the past 25 years, an estimated 30% of coral reefs around the world have been severely damaged through loss of

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coral cover and declining habitat quality, and this decline is expected to continue in the coming decades (Hughes et al., 2003; Carpenter et al., 2008; Halpern et al., 2008). Coral bleaching not only refers to the breakdown in the symbiosis between the coral host and its Symbiodinium but can also occur through the loss of chlorophyll pigment from algal cells (Hoegh-Guldberg and Smith, 1989). It can lead to coral mortality and has increased in frequency and magnitude as a result of rising sea surface temperatures (Glynn, 1993; Hoegh-Guldberg, 1999; Baker et al., 2008). Coral symbioses, while highly successful in tropical waters, are very sensitive to changes in ocean temperature and live close to their upper thermal tolerance limits. A prolonged temperature increase of as little as 1  C above the normal average maximum for a region leads to stress and potentially to bleaching (Jokiel and Coles, 1990). Thermal stress and declining habitat quality have also been linked to the increased incidence of coral disease (Porter et al., 2001; Bruno et al., 2007; Brandt and McManus, 2009). Over 29 coral diseases have been described so far, each having varying effects on coral mortality and fecundity (Rosenberg and Loya, 2004). An understanding of disease-causing agents and their transmission strategies is of primary importance in determining the aetiology of coral disease. Thermal sensitivity and disease in corals are believed to be augmented by ocean acidification (Orr et al., 2005; Anthony et al., 2008). The dissolution of atmospheric carbon dioxide into seawater leads to changes in ocean chemistry and a lowering of seawater pH and has the potential to inhibit the calcification process of corals and other marine biota. A recent study highlights that a reduction in coral diversity, recruitment, and abundance, as well as shifts in species composition, is likely to occur over large geographic scales as a result of ocean acidification (Fabricius et al., 2011). A suite of local anthropogenic impacts including pollution, coastal development, and overfishing all act synergistically, further contributing to localised degradation of coral reefs (Hoegh-Guldberg, 1999). It is therefore not surprising that the collective barrage of these insults to corals results in the reduction of reef health and a decline in coral cover that has dire consequences for the future of coral reef ecosystems.

1.3. The need for coherent species delineation in coral reef research There has never been a more timely need for focused research on the biology of corals to help understand and address the multiple threats facing coral reef ecosystems. A consistent outcome of current research investigating the effects of bleaching, disease, and ocean acidification points to variability in the response of corals (e.g. Brown et al., 2000; Loya et al., 2001; Borger and Sascha, 2005; Page and Willis, 2006; Anthony et al., 2008). The cause of this variation is no doubt complex, but one important

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factor contributing to the variability is the organismal composition of the holobiont. Different species of coral show different levels of susceptibility to bleaching, disease, and ocean acidification. Within a host species, the genetic identity of the dinoflagellate symbionts and their tolerance to thermal stress play an important role in determining whether a coral bleaches (Rowan, 2004). With the breadth of current research investigating the variability in response of corals to stressors, it is of paramount importance that comparative analyses and integration of results from studies conducted in different geographic locations and from different research laboratories can be performed. To this end, the process by which the partners in the holobiont are described, and the species names attributed to the coral hosts, as well as their dinoflagellate and prokaryotic symbionts, needs to be consistent. In addition, for species in the coral holobiont to be listed as endangered, data supporting the delineation of a proposed species (or subspecies, or population) are required (Green, 2005; Fallon, 2007) and necessary under the US Endangered Species Act (ESA), the Canadian Species at Risk Act (SARA), and the Australian Environment Protection and Biodiversity Conservation Act. This chapter reviews the species and systematics debate and summarises the diversity of approaches, along with their benefits and limitations, used in describing species for the three major partners of the coral holobiont: the coral host, dinoflagellate symbionts, and prokaryotic symbionts. It is our intention that this review will provide a platform to promote the delineation of species within the coral holobiont and facilitate much needed integrative and comparative research among coral biologists worldwide to further our understanding of coral reef ecosystems.

2. The Species Debate 2.1. The species debate in context The debate over how to define species has persisted for centuries. Despite the vast amount of literature devoted to the subject, it can seem at times that little headway has been made. This lack of resolution has been variously attributed to an incomplete knowledge of the natural world, the multifarious nature of biological organisms, imprecision in human language, and doubt about the existence of an entity that encompasses a species (Hendry et al., 2000; Hey, 2001, 2006). One of the primary causes of dispute is in part due to the many different roles species play in biology (Mallet, 2007). Species are the basic taxonomic unit used as an index of biodiversity, are the largest putative group affected directly by evolutionary forces, and represent the taxonomic unit in ecology and biodiversity studies—the unit within which variation can be largely ignored when trying to

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understand community interactions and abiotic interactions. Biodiversity and conservation hotspots are chosen based on the number of species they contain, or the rate at which the area is thought to generate new species, while extinction rates are made in reference to number of species (Agapow et al., 2004). As a result, much of the legislation and politics surrounding conservation are centred on species preservation (Agapow et al., 2004). Therefore, in our opinion, the most important task in taxonomy is to delineate species, that is, to recognise when different specimens are members of the same cohesive lineage (Dayrat, 2005).

2.2. Species concepts Mayden (1997) lists 22 species concepts, but his review omits at least some of the suggestions put forth in the literature. A list of the most commonly used species concepts can be found in Table 1.1. These are divided by general themes that are based, at least in part, on differing conceptions of the defining properties of species. These themes include reproductive isolation, shared mate recognition or fertilisation systems, morphological distinctiveness, phylogenetic relationship and monophyly, fixed character differences, ecological similarity or distinctiveness, and exclusive coalescence of alleles. Differences in opinion on the important properties of species not only lead to different concepts but often lead to vastly different numbers of species as the perceived boundaries between species shift. In addition, defining characteristics of a species concept may not be applicable to all groups of organisms (e.g. shared mate recognition in asexually reproducing organisms). Alternatives to a single universal definition for species include using different definitions depending on the group to be studied, excluding some parts of life from having species, or denying that species exist at all. Recently, more integrated and/or hierarchical approaches have been offered. Mayden (1997, 1999, 2002), de Queiroz (1998, 1999), and Naomi (2011) propose a similar approach whereby a core or primary species concept is used to unify all organisms, such as a modified version of Simpson’s (1961) evolutionary species concept in the case of Mayden. In addition, secondary criteria modifying the primary concept are further applied and would include other species concepts applicable to the organism in question, such as those based on mate recognition for sexually reproducing species. Another integrative approach by Pigliucci (2003) and Hull (1965) based on Wittgenstein’s (1953) explanation of family resemblance concepts proposes that the word ‘species’ is also a family resemblance concept: that is, there is no definition that will singularly pick out everything that we mean by the word ‘species’. Instead there are overlapping criteria or properties that link all of the different uses of the word ‘species’. This sense of species reflects the idea that there is no single process responsible for the splitting, or unifying, of groups of organisms into lineages. It also explains

Table 1.1 Summary of major species concepts (modified from de Queiroz 2007) Species concept

Definition

References

Biological

Groups of actually or potentially interbreeding natural populations resulting in viable and fertile offspring which are reproductively isolated from other such groups Intrinsic reproductive isolation (absence of interbreeding between heterospecific organisms based on intrinsic properties, as opposed to extrinsic geographic barriers) Shared specific mate recognition or fertilisation system (mechanisms by which conspecific organisms, or their gametes, recognise one another for mating and fertilisation) Same niche or adaptive zone (all components of the environment with which conspecific organisms interact); a lineage which occupies an adaptive zone minimally different from that of any other lineage in its range and which evolved separately from all lineages outside its range A lineage evolving separately from others and with its own unique evolutionary role and tendencies Phenotypic cohesion (genetic or demographic exchangeability); the most inclusive population of

Wright (1940), Mayr (1942), and Dobzhansky (1950)

Isolation

Recognition

Ecological

Evolutionary Cohesion

Mayr (1942) and Dobzhansky (1970)

Paterson (1985), Masters et al. (1987), and Lambert and Spencer (1995)

Van Valen (1976) and Andersson (1990)

Simpson (1951), Wiley (1978), and Mayden (1997) Templeton (1989) (continued)

Table 1.1

(continued)

Species concept

Definition

References

individuals having the potential for genetic and/or demographic exchangeability Phylogenetic Hennigian Monophyletic

Ancestor becomes extinct when lineage splits

Hennig (1966), Ridley (1989), and Meier and Willmann (2000) Rosen (1979), Donoghue (1985), and Mischler (1985)

Monophyly (consisting of an ancestor and all of its descendents, commonly inferred from possession of shared derived character states) Genealogical Exclusive coalescence of alleles (all alleles of a given gene Baum and Shaw (1995) and Avise and Ball (1990) are descended from a common ancestral allele not shared with those of other species) Diagnosable Diagnosability (qualitative, fixed difference) Nelson and Platnick (1981), Cracraft (1983), and Nixon and Wheeler (1990) Phenetic Form a phenetic cluster (quantitative difference) Michener (1970), Sokal and Crovello (1970), and Sneath and Sokal (1973) Genotypic Form a genotypic cluster (deficits of genetic intermediates, Mallet (1995) cluster e.g. heterozygotes) Morphological The smallest groups that are consistently and persistently Cronquist (1978) distinct and distinguishable by ordinary means Population level Metapopulation-level evolutionary lineages de Queiroz (2007) lineages Hierarchy Operational and theoretical species concepts hierarchically Mayden (1997) related to each other in primary/secondary relationships Integrated Operational species concepts are used as delineating Pigliucci (2003) and Naomi (2011) criteria; species is a cluster concept

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how almost every person has no problem using and understanding the word ‘species’ and yet is not able to define exactly what it means.

3. Systematics 3.1. Traditional taxonomy using phenetics Binomial classification used in systematics was introduced by Carolus Linnaeus in the eighteenth century and included only two levels in the taxonomic hierarchy—genus and species. This nomenclature system has persisted through time with additional taxonomic ranks included in the hierarchy at various stages following its introduction (Raven et al., 1971). Classification of organisms using the binomial system has predominantly been achieved using phenetic characters, mostly morphology for eukaryotes and biochemistry for prokaryotes (Winston, 1999; Packer et al., 2009). Phenetic characters are scored and taxa are grouped into species based on the overall similarity of observed features without reference to how these features have evolved. Identification of the earth’s biodiversity using morphology is a fundamental skill required of any naturalist, without which biology would not be possible. Although delineating species using phenetics has been the primary approach in taxonomy, and is the methodology of choice in certain situations (e.g. when comparing fossil records with living taxa), there are notable limitations to its resolving power and efficacy (Wiens, 2004). Firstly, an intact specimen is usually required for identification, and in many situations, there are only fragmentary remains of the organism available, or, as is the case for the majority of prokaryotes (and some eukaryotes), the complete absence of a type culture (Pace, 1997). Secondly, in the case of most animals, identification is based solely on the adult form (e.g. Ehrlich and Ehrlich, 1967), and juveniles can exhibit very different phenotypes (often so different that it has been identified as a different species in many instances) that add a level of complexity to identification and delineation. Thirdly, morphological variation can be minimal or absent among many species (e.g. Symbiodinium see Section 4.2). Fourthly, dimorphic species where traits can be variable adds confusion, such as colour polymorphism in birds (Galeotti et al., 2003). And fifthly, subjectivity can play a major role in delineating species among specialists within a particular field (e.g. corals see Section 4.1).

3.2. The utility of a molecular approach in delineating eukaryotic species Molecular genetic information to aid in the delineation of species is extremely powerful and can overcome many of the limitations described above. In contrast to phenetic systematics, similarity based on gene

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sequence or genome composition coupled with phylogenetic reconstruction can also infer the evolutionary relatedness of taxa in addition to its application as a tool to delineate species. Further, DNA can also be used to assess phylogeographic patterns within species (Avise et al., 1987; Avise, 2000). Importantly, defining species boundaries in a phylogenetic context is only achieved through assessing multiple populations from closely related species over broad geographic scales to determine the extent of intra- versus interspecific variation. In the identification of eukaryotic species through the monophyletic grouping of taxa using phylogenetic species concepts (Table 1.1), phylogenetic reconstructions should be based on multiple gene targets to avoid any discordance that may arise between the species tree and a particular gene tree (Goodman et al., 1979; Pamilo and Nei, 1988; Doyle, 1992; Maddison, 1997; Nichols, 2001). Ideal gene candidates should also be single copy and target multiple organelles. This is exemplified by the fact that species supported by data from multiple genes (both nuclear and mitochondrial in the case of animals) have a higher chance of being listed as endangered by the United States Fish and Wildlife Service and the National Marine Fisheries Service, as compared to candidates based on single genes (Fallon, 2007). In the case where monophyletic groupings do not occur, there are various reasons that can account for the resulting poly- or paraphyly that can be difficult to determine, confusing the identification of species and their relationships (reviewed in Funk and Omland, 2003). These include introgressive hybridisation, where phylogenetically divergent alleles cross species boundaries, and incomplete lineage sorting following recent speciation events (Fig. 1.2). Misidentification of multi-copy genes with paralogous copies as single copy, misidentified specimens, species boundaries (intraand interspecific variation), inadequate sampling of both individuals within a species and closely related taxa, cryptic species, and inadequate phylogenetic information from utilising too small a gene fragment or slow-evolving gene that does not capture enough synapomorphies to differentiate species all contribute to false identification of polyphyly and inaccurate conclusions related to the evolution and relationships of taxa (Fig. 1.2). Therefore, even though polyphyly can occur representing a true picture on the evolution and organisation of genes within species, care must be taken to rule out all other possibilities. The utility of a molecular approach to delineating species can also result from the discovery of a gene ‘barcode’ that can be applied once the evolutionary relationship of taxa has been defined, and the boundaries set for inter- and intra-genomic variation. DNA barcoding to identify species can be simpler than identification based on traditional phenetic approaches. Barcoding is based on the idea that genetic variation is greater between species than within species. The utility of barcoding is to identify species

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Molecular Delineation of Species in the Coral Holobiont

A

B

A1

A1

A1 D? B2 B2

A1

C3

B2

Incomplete lineage sorting

Hybridization (introgression)

C

D Gene duplication (xy)

A1

x

B2

x

A1

y

B2

y

A1 A1

Speciation

B1 B1

C2

Species limits Intraspecific variation mistaken for interspecific variation

Paralogy

E

F A1

A1 A1

A1 C2

A3

Cryptic species

A2 A2

C3

Inadequate phylogenetic information

Figure 1.2 Processes and interpretations resulting in paraphyly. Capital letters refer to nominal species, while subscript numbers refer to ‘true’ species. (A) Introgression between two different species will result in a hybrid and disrupt the monophyletic groupings of species; (B) the line diagram within the species phylogeny shown for incomplete lineage sorting would result in an inaccurate phylogeny (A1, (A1,B2)) if that particular gene was utilised; (C) paralogy results when a gene is assumed to be orthologous but has undergone a duplication event (gene xy) prior to a speciation event, and different orthologs used in the phylogenetic reconstruction; (D) defined species limits may be inaccurate and what was initially identified as representing different species (e.g. morphological variation or geographic variation), correspond to intraspecific variation; (E) cryptic species may be initially overlooked in delimiting species; and (F) inadequate phylogenetic information from the application of a gene or fragment of a gene that does not contain adequate numbers of synapomorphies to differentiate species.

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based on a gene sequence that have already been characterised, and, to exclude novel organisms for further phylogenetic and phenetic investigation (Rubinoff and Holland, 2005; Hajibabaei et al., 2007). Barcoding can also aid in the discovery of cryptic species that were not resolved when using phenetic approaches. In some cases, back-checking samples that differ based on a barcode also identify morphological variation that was initially overlooked but is concordant with the molecular data (e.g. Smith et al., 2008). For barcoding, it is now generally accepted that there is no single gene that can be used across all organisms (Packer et al., 2009). The most applied gene and global standard barcode is a 650-bp fragment of the 50 end of the mitochondrial (mt) gene cytochrome c oxidase 1 (CO1, cox1). However, a single mtDNA gene can be problematic and inadequate for some taxa. Primarily, the rate of mitochondrial evolution can be too slow, limiting its resolving power in distinguishing species as is the case in the Anthozoa and plants (Shearer et al., 2002; Kress et al., 2005; Hellberg, 2006; Shearer and Coffroth, 2008). Therefore, different gene barcodes will be needed to delineate species for different taxa. One alternative gene that has been investigated and applied in plants and fungi is the nuclear ribosomal gene (rDNA) internal transcribed spacer (ITS) region (Fig. 1.3; Kress et al., 2005; Kelly et al., 2011). However, due to the highly repetitive nature of this gene and resulting intra-genomic variation, coupled with the high variability among taxa in the amount of genetic divergence defining intra- and interspecific variation, similarly to the cox1 gene, its utility is not universal. It is important to note that the international standard for species identification using barcoding requires that the sequence be catalogued with an image of the organism and associated geographic information (CBOL, http://www. barcodeoflife.org/). In other words, even in barcoding, a gene sequence is not enough to define a species; associated attribute information is also required.

3.3. Species delineation using an integrative approach It is clear that the species concept that a researcher adopts will have significant effects on how that researcher delineates species. It is therefore of paramount importance that the species concept is made clear in any research study involving species delineation, description, or classification. The operational criteria that will be used to delineate species must be set forth in an a priori manner at the beginning of a study to prevent purely post hoc speculation about what are the important criteria in delineating species in that group. As new candidate criteria are discovered, they can be worked into future studies and applied reflectively to previously published studies, but it is important that the framework within which research is done is as transparent as possible so that decisions on what criteria count and which do not for species delineation can be applied consistently and accurately.

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Eukaryotic cell

rDNA array

rDNA IGS

5¢ ETS

18S

5.8S

28S

3¢ ETS

ITS1 ITS2

Figure 1.3 The internal transcribed spacer regions and rDNA in eukaryotes. ITS regions 1 and 2 are coloured red and functional rRNA elements involved in protein structure are shown in blue. IGS: intergenic spacer region, ETS: external transcribed spacer region.

Even though there are clear advantages in applying a molecular approach to the delineation of species, the breadth of inaccurate conclusions that can arise and the lack of universal genes that can be used across taxa clearly provide caution for its sole application. Further, how is molecular data interpreted when there is a disagreement between gene phylogenies? A combinatorial approach using molecular data coupled with phenetic characteristics can provide a more integrative and accurate representation of species for both eukaryotes and prokaryotes (e.g. Stackebrandt et al., 2002; Carew et al., 2005; DeSalle et al., 2005; Smith et al., 2008). An ideal study in the delineation of a species would include phylogenetic information from multiple genes along with closely related congeners, across a large geographic range with multiple individuals from each location encompassing all sources of biological variation including morphological variants. Phenetic characteristics that support molecular information can then be used in the delineation of species. We favour this approach and suggest a combination of molecular information with phenetic classification be used in the delineation of species for the three major partners of the coral holobiont.

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4. The Coral Holobiont 4.1. Coral: The animal host 4.1.1. Early examples of taxonomic confusion Scleractinian corals are among the most taxonomically challenging and problematic group of organisms. Skeletal morphology has been the primary character for systematic and taxonomic studies over the past few centuries, yet morphology can vary wildly and is likely to be under selection pressure. Reef building corals have been plagued with a convoluted history of complete taxonomic confusion; from the beginning they were incorrectly classified as plants until the mid-seventeen hundreds (Edmunds and Gates, 2003; Humann and DeLoach, 2006). The genus Porites is an excellent example of extreme taxonomic confusion and is considered a prime example of ‘the coral species problem’ (Brakel, 1977). There are approximately 500 named Porites species (D. Potts, personal communication), about an order of magnitude higher than currently considered valid (Cairns, 1999; Veron, 2000). The origin of the genus was problematic, when Link (1807) originally described two species: ‘Porites polymorphus’ with a variety of forms and ‘Porites damicornis’ which was later moved to the genus Pocillopora. Lamarck (1816) described 16 Porites species, but this ‘catch-all’ group contained species that were later moved to other genera such as Alveopora, Stylophora, and Montipora. Bernard (1905) completely abandoned the Linnean binomial classification system for Porites, instead grouping by geographic region followed by a number for each form with the rationale that the “assumed genetic value of resemblance is not justifiable in dealing with the more plastic forms of life. . . They seem to be more easily moulded to the environment, so that genetically related forms, dispersed into slightly different conditions, quickly become different, and genetically different forms, cast into the same environment, quickly become alike.” Phenotypic plasticity, phenotypic polymorphism, recent divergence, and hybridisation between species are the most commonly invoked explanations for the overlap between intra- and interspecific variability that is commonly referred to as the ‘species problem’. 4.1.2. Hybridisation and reticulate evolution Veron (1995) proposed the hypothesis of ‘reticulate evolution by sea surface vicariance’ to explain species-level variation in Scleractinia. Veron argued that synchronous mass spawning events provide many opportunities for gametes from recently diverged species to come in contact, which will result in hybridisation if pre-zygotic barriers are absent. Veron further argued that episodic changes in sea level and ocean currents are likely to drive patterns of divergence and hybridisation between species within

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complexes. As with plants, these species complexes are referred to as a ‘syngameon’ or ‘meta-species’, which is defined as the highest taxon that never hybridises (Veron, 1995). Veron (2000) hypothesised that modern distribution patterns arose following episodic glaciation during the Pleistocene, while most extant recognisable species and biogeographic provinces arose in the Miocene (24–5.2 mya). Pleistocene glaciation cycles occurred at roughly 80,000– 110,000 year intervals, with roughly seven major glaciation events having occurred in the past 650,000 years (Barnola et al., 2003). These cycles resulted in changes in sea level and sea surface circulation patterns, providing ample opportunities for geographic isolation and the potential for allopatric speciation events. Veron’s (1995) hypothesis has stimulated interest in the evolution of reef building coral, but it has been difficult to adequately test, in part because mitochondrial markers evolve particularly slowly in corals, with temporal resolution to detect differences only after several million years of reproductive isolation (Shearer et al., 2002; Hellberg, 2006; Shearer and Coffroth, 2008). Single- or multi-copy nuclear genes have frequently shown high levels of genetic variation within a given individual, which has made it challenging to distinguish between the similar signatures of hybridisation and incomplete lineage sorting. Furthermore, depending on the criteria or species concept used, it is hard to differentiate a ‘syngameon’ with ‘seven species’, from a ‘good species’ (sensu the Biological Species Concept; Table 1.1) with ‘seven subspecies’, ‘seven races’, or ‘seven morphs’. Regardless of the technical or conceptual challenges, Veron’s (1995) hypothesis has had a profound influence on all studies of coral evolution and biodiversity ever since. 4.1.3. Early molecular studies and surprises Early genetic studies on corals used allozyme electrophoresis and made the first contributions to understanding of clonal (e.g. Stoddart, 1983; Willis and Ayre, 1985), population (Stoddart, 1984a,b), and potential ‘species level’ (Ayre et al., 1991; Knowlton et al., 1992; Van Veghel and Bak, 1993) genetic variation in coral. Molecular studies using DNA began in the late 1990’s and immediately yielded surprising patterns of incongruence with existing taxonomy and existing hypotheses about evolutionary relationships. In only the past few decades, these studies have fundamentally revolutionised the current understanding of the evolution of reef building corals. Early molecular studies began with a ‘top-down’ approach, examining broad taxonomic relationships among families (e.g. Chen et al., 1995; Romano and Palumbi, 1996; Veron et al., 1996). These studies found a surprising lack of correspondence between traditional taxonomy and evolution by descent as inferred from DNA. Romano and Palumbi (1996) discovered major incongruence between scleractinian suborders based on gross morphology and two strongly supported and deeply divergent genetic

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clades based on mitochondrial 16S genes. Corals in the ‘robust’ clade tend to have a more solid skeleton, while the interface between tissues and skeleton is more perforated in the ‘complex’ clade. Additional studies confirmed these results and yielded additional major discrepancies between genetic and morphological groupings (e.g. Chen et al., 1995, 2002; Veron et al., 1996; Romano and Palumbi, 1997; Romano and Cairns, 2000; Cuif et al., 2003; Fukami et al., 2004a; Le Goff-Vitry et al., 2004; Huang et al., 2009). In 2004, Fukami and colleagues (Fukami et al., 2004b) presented strong evidence that many traditionally recognised families and genera are not monophyletic: instead, it appears that there are remarkable examples of convergent evolution that have occurred independently in the Atlantic and Pacific Oceans. Huang et al. (2009) further confirmed the results of Fukami et al. (2004a) and reviewed the literature to reveal that a lack of monophyly is pervasive across many morphological groups, with numerous examples across the family, genus, and species level. 4.1.4. Species-level studies in corals The following is a summary of examples of some of the major species-level studies on scleractinian corals. Many aspects of the more widely studied taxa (particularly Acropora and Montastraea) have been previously reviewed elsewhere (e.g. Frank and Mokady, 2002; van Oppen and Gates, 2006; Willis et al., 2006; Fukami, 2008). Here we integrate more recent studies and focus specifically on the molecular delineation of species. Acropora is arguably the most widely studied coral; from the start, Acropora have played a central role in a debate over the significance of hybridisation on the evolution of reef building corals. Odorico and Miller (1997) found several ITS variants within single individuals that were highly divergent (up to 29%) within individual Acropora colonies. The authors maintained that the sequences were unlikely to be pseudogenes based on secondary structure, and because these species are known to hybridise, the overall pattern was more consistent with reticulate evolution. Hatta et al. (1999) crossed morphologically defined Acropora species in breeding experiments and found heterozygosity in the lab and the field consistent with reticulate evolution, although extreme phenotypic polymorphism cannot be ruled out. van Oppen et al. (2001) examined 28 Acropora species (including some examined by Hatta et al., 1999) with the putative mitochondrial control region, and a Pax-C intron. The major genetic clusters corresponded well to groups with little difference between spawning times, but there were several disagreements between mitochondrial and nuclear genes, a pattern which is consistent not only with reticulate evolution but also with extreme phenotypic polymorphism, or incomplete lineage sorting. Interestingly, the van Oppen et al. (2001) and Hatta et al. (1999) studies were not all together congruent with each other, which may be due to different geographic, taxonomic, or genetic sampling (i.e. sampling genes with different

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evolutionary histories). van Oppen et al. (2000) and Vollmer and Palumbi (2002, 2004) demonstrated that Acropora prolifera results from hybridisation between Acropora palmata and Acropora cervicornis. Based on the high frequency of heterozygous hybrids sampled, Vollmer and Palumbi (2002) disputed the reticulate hypothesis in corals and suggested that A. prolifera is an F1 hybrid that is effectively a sterile ‘immortal mule’, whereas van Oppen et al. (2000) and Miller and van Oppen (2003) argue that backcrossing with at least one of the parental species is highly likely. The frequency and evolutionary consequences of hybridisation and backcrossing have been a subject of ongoing vigorous debate, which illustrates that there are often several competing interpretations for conflicting genetic data. Based on examples with Acropora, Vollmer and Palumbi (2004) warned against the use of the ITS region for systematic inference and recommended caution in interpreting either hybridisation or reticulation based on data from the multi-copy gene due to potential problems with intra-genomic variation (divergent copies within a single individual), parology (lack of homology among copies), and alignment ambiguity due to insertion and deletion events (see Alverez and Wendel, 2003; Coleman, 2009 for contrasting reviews of the utility of the ITS region). In a more broad taxonomic survey, however, Chen et al. (2004) and Wei et al. (2006) found that high ITS intragenomic variation is an exception limited to Isopora and Acropora and not the general rule across most coral species: ITS intra-genomic variation was relatively low in most taxa examined and generally concordant when compared with other markers. Ma´rquez et al. (2002) further examined the high intra-genomic variation of ribosomal genes in Acropora. The authors examined RT-PCR and structural analysis of 5.8S rDNA and suggested that the high intra-genomic variation of ribosomal genes in Acropora may be in part due to non-functional pseudogenes that may have originated from ancient hybridisation events. LaJeunesse and Pinzo´n (2007) took this concept further and argued that some ribosomal ITS copies in Acropora are more meaningful than others. LaJeunesse and Pinzo´n (2007) phylogenetically compared Acropora ITS sequences obtained from cloning as opposed to isolating the brightest ‘dominant’ bands from denaturing gradient gel electrophoresis (DGGE). They argued that ITS sequences obtained from dominant DGGE bands tend to be more congruent with other markers and that these dominant bands were more ‘evolutionarily relevant’ than cloned sequences which tend to yield higher variation with increased sampling effort. The debate over the interpretation of molecular data and the evolutionary significance of hybridisation have important conservation implications; for example, the Caribbean Acropora are listed for federal protection under the US ESA, and Richards et al. (2008) argued that many rare Acropora are probable ‘hybrids’ based on allele/haplotype sharing and incongruence between the putative mitochondrial control region and Pax-C. Richards et al. (2008) argued that the propensity for hybridisation may actually reduce

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vulnerability to extinction by increasing genetic variation and thus adaptive potential. Richards et al. (2008) argued that incomplete lineage sorting was unlikely due to an estimate of low effective population sizes of the rare corals based on the estimated global census size. This study assumed that hybridisation between morphospecies was very rare; however, if hybridisation is more common than assumed, the effective population sizes would be much larger. Furthermore, Richards et al. (2008) defined species based solely on morphological criteria, and did not mention the possibility of extreme phenotypic polymorphism, and is an example of how species concepts can change underlying assumptions; for example, reproductive or morphological criteria for delineating species can have very different conservation implications. The Montastraea annularis species complex is probably the most thoroughly studied system for species-level evolution in reef building corals (previously reviewed by van Oppen and Gates, 2006; Fukami et al., 2008). Weil and Knowlton (1994) argued that M. annularis consists of a species complex (M. annularis, Montastraea faveolata, Montastraea franksi) based on morphology, allozymes, behaviour, and isotopic differences. Knowlton et al. (1997) reported evidence for reproductive isolation, while in contrast Szmant et al. (1997) reported no evidence of reproductive isolation. Lopez and Knowlton (1997) found genetic differences using AFLP, but Medina et al. (1999) showed that members of the M. annularis species complex did not show differences using the ITS region or cox1 and therefore argued that the complex represents a ‘single evolutionary entity’. Fukami et al. (2004b), on the other hand, argued that cox1 and ITS may lack sufficient resolution and found statistically significant genetic differences (AFLP, mitochondrial noncoding region, ITS) and morphological differences between M. faveolata, M. annularis, and M. franksi in Panama, but not in the Bahamas. The authors suggested that a possible explanation for the conflicting results in previous studies is that there is geographic variation to the permeability to species boundaries, or a ‘hybridisation gradient’. The sequence differences, however, were subtle (non-overlapping genotypes in the mitochondrial non-coding region, and statistically different allele frequencies of ITS) and not based on reciprocal monophyly. Levitan et al. (2004) provided reproductive evidence that M. franksi may be reproductively isolated temporally and M. faveolata may be isolated by reproductive incompatibility; however, these isolation mechanisms may be geographically and temporally variable. In an attempt to resolve the issue of slow mitochondrial evolution, Fukami and Knowlton (2005) sequenced the entire mitochondrial genomes of the M. annularis species complex. Only 25 variable positions were found, and they did not resolve the species complex; intriguingly, 16 positions differed between two M. franksi individuals. The authors’ explanation is that either M. franksi contains a cryptic lineage or it retains two ancestral mitochondrial lineages.

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Fukami et al. (2004a) demonstrated that many traditionally recognised families and genera (including Faviidae which includes the M. annularis complex) are not monophyletic according to cox1, cytb, and b-tubulin. Some Atlantic Mussids look morphologically similar to Favids, in other words; there are extraordinary examples of morphological homoplasy (convergent or parallel evolution) that obscured deep phylogenetic divergence between Atlantic and Pacific groups. The families Faviidae, Mussidae, Pectiniidae, Mullinidae, Trachyphylliidae, Meandrinidae, and Ocullinidae were not monophyletic and are now referred to as the ‘Big Messidae’. Huang et al. (2009) further confirmed these findings in an examination of 41 faviid species with mitochondrial cox1 and a non-coding region, finding that paraphyly is pervasive at the family and genus level. Huang et al. (2009) also included a tree based on morphological data, which was quite discordant with the genetic data. In a review of most of the published DNA sequencebased literature until that time, Huang et al. (2009) showed that paraphyly is pervasive at the species, genus, and family levels in scleractinian corals. Pocillopora is also a relatively well-studied group, and a fascinating body of conflicting studies is now accumulating. Flot and Tillier (2006) examined five Pocillopora species in Hawaii with ITS2 and found slight congruence with two traditionally recognised species. Flot et al. (2008) added to this dataset and found general agreement between mtDNA (putative control region, ORF) and five widely recognised morphological species in Hawaii. The ITS2 only delineated two species while single copy nuclear genes for ATP synthase b subunit, calmodulin, and elongation factor-1a had deeply divergent alleles and resolved no groups. The general conclusion of the study was that although mtDNA is slow to accumulate mutations, it may have a faster coalescent time and more accurately reflect the species tree, while the other genes show incomplete lineage sorting (Flot et al., 2008). Combosch et al. (2008), on the other hand, examined some of the same taxa in a study comparing the Eastern Pacific using only the ITS region. The ITS region resolved two groups and an intermediate, which the authors interpreted as evidence of rare hybridisation between Pocillopora in the Central Pacific and the Eastern Pacific with occasional dispersal and hybridisation across the Eastern Pacific Barrier. Pinzo´n and LaJeunesse (2010) examined corals in the Eastern Pacific, with a strikingly different result. They examined microsatellites, mtDNA (ORF), and the ITS2 of coral and zooxanthellae (using the dominant band of DGGE), and all methods resolved three clear and distinct genetic groups; however, these groups did not remotely correspond to morphologically recognised species. The interpretation favoured by the authors is that colony morphology is unreliable in this group, that is, rampant and extreme phenotypic polymorphism of colonylevel morphology. The authors argue that the reason for conflict with the Combosch et al. (2008) study is because Combosch and colleagues only examined one marker and relied on direct sequencing as opposed to the

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‘dominant band’ DGGE approach. Flot et al. (2008) by contrast found general agreement between mtDNA and morphology but focused on Hawaii and did not sample intermediate or difficult to identify specimens. Flot et al. (2010) did, however, find similar results to Pinzo´n and LaJeunesse (2010) in a study on Pocillopora from the same geographic region (although focused only on Clipperton atoll). Flot and colleagues examined mtDNA (ORF þ CR), ITS, and the ATPsb genes and found support for two cryptic groups that had little correspondence to any recognisable taxonomy based on gross colony morphology. The authors argued for extreme phenotypic polymorphism of colony-level morphology as the most likely explanation. Flot et al. (2010) based this conclusion on a novel technique that they refer to as ‘haplowebs’, which is a graphical method of representing the criteria of mutual allelic exclusivity. The authors argued that the advantage of this technique is that mutual allelic exclusivity will delineate species more rapidly than reciprocal monophyly. The haplowebs method consists of drawing a network (or tree) for a given gene with lines interconnecting between terminal branches that represent alleles found within a single individual. The thickness of these lines is in proportion to the frequency of occurrence. These networks are then compared for congruence between markers, and circles are drawn based on shared bipartitions of groups that do not share the same alleles. On the opposite end of the enormous geographic range of Pocillopora, Souter (2010) found similar evidence for a lack of correspondence between genes and morphology on the coast of Africa and Mauritius. The study found two cryptic species of Pocillopora damicornis according to mitochondrial genes (ORF þ CR) and microsatellites, with only weak and partial support from ITS2. At the population level, Combosch and Vollmer (2011) found fine-scale genetic structure over tens of metres in P. damicornis in the Eastern Pacific but largely dismissed the possibility of cryptic species as suggested by Pinzo´n and LaJeunesse (2010) and Souter (2010). The genus Pocillopora therefore remains an interesting example of conflicting studies and an unresolved debate over the nature of species level variation. Hunter et al. (1997) were among the first to suggest that the ITS region had utility for both coral and zooxanthellae, after amplifying both from Porites and observing adequate levels of polymorphism. Using Porites and Siderastrea, Forsman et al. (2006) explicitly examined two of the most widely cited problems with ITS: intra-genomic variation and multiple alignment ambiguity due to alignment gaps from insertion and deletion events (Alverez and Wendel, 2003; Vollmer and Palumbi 2004). Intra-genomic and intra-species variation in Porites was low (0.05–0.1%, respectively), and ITS groups from nearly all alternative multiple alignments were generally consistent with morphology in the Atlantic and Eastern Pacific Porites. Forsman et al. (2009) added mitochondrial genes (COI, putative control region) which were concordant with the ITS groups. With the addition of

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more Central Pacific taxa, however, an interesting and surprising pattern emerged; three separate clades contained both branching and mounding species (genetically similar yet morphologically distinct), while conversely mounding species with similar appearance and nominal identification were found in three highly divergent clades (genetically different yet morphologically similar). While colony morphology was wildly variable, corallitelevel characters appeared to be broadly consistent with discrete differences between the genetic groups. The authors concluded that colony morphology may be extremely phenotypically polymorphic within some species (Forsman et al., 2009). Phenotypic plasticity is a more specific case of phenotypic polymorphism that is easier to unequivocally demonstrate. Phenotypic plasticity has been observed in branching Porites (Muko et al., 2000) and has been shown to be pervasive in scleractinian coral in response to a wide variety of biotic and abiotic factors (reviewed by Todd, 2008). Increasingly, integrated genetic and morphological approaches are becoming the new standard; for example, Stefani et al. (2008) used an integrated approach including an examination of type material, on 11 nominal branching species of Psammocora. The study found that morphology (corallite and colony-level traits) and genetics (b tubulin) retained only two species and revised a complex history of their synonymies. In a later study, Benzoni et al. (2010) examined 12 nominal species of the genus Psammocora, with an integrated approach using genes (cox1 and ITS) and morphological characters. The results were consistent with previous studies, highly congruent among molecular markers, with high concordance to morphology, yielding five clear groups. This study is notable in that it is one of the very few that includes type material and makes very thorough recommendations for taxonomic revision with a detailed description of the history of taxonomic confusion, which the authors refer to as the ‘name game’ in the genus. The molecular delineation of coral species can have critical conservation implications, for example, Acropora in the Caribbean are listed for federal protection under the US ESA; yet the status of A. prolifera is vague because it is considered an F1 hybrid of A. palmata and A. cervicornis. Several studies have raised doubts of the species status of very rare corals, which has refocused conservation priorities. Siderastrea glynni, for example, in the Eastern Pacific was considered the rarest coral known until it was discovered that it shared identical ITS sequences with Siderastrea siderea in the Atlantic (Forsman et al., 2005). Given the range of previously estimated rates of evolution of the ITS region, and the closure of the Isthmus approximately 3.5 mya, Forsman et al. (2005) argued that transport through the Panama canal was more likely than the alternative hypothesis (remnant 3.5-mya old population) for the origin of these five colonies. Montipora dilatata is also one of the rarest known corals, with only a handful discovered in Hawaii despite extensive surveys; however, it is difficult to positively identify, and putative

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hybrids have been postulated since Montipora turgescens and Montipora capitata can have some similarities (Forsman et al., 2010). According to five mitochondrial genes (control region; cox1, cytB, atp6, 16S) and the ITS region, all seven Hawaiian Montipora sorted into four groups (I: Montipora patula/ Montipora verrilli, II: Montipora cf. incrassata, III: M. capitata, IV: M. dilatata/ Montipora flabellata/M. cf. turgescens). M. patula, M. dilatata, and M. flabellata were petitioned to be listed under the US ESA (Sakashita and Wolf, 2009), yet they were genetically indistinguishable from more common and geographically widely distributed species. Insipient speciation cannot be ruled out because the markers may only detect differences between species isolated in the past 0.5–1 mya; however, if speciation has recently occurred, then it is an example of speciation in sympatry with no clear geographic barriers (Forsman et al., 2010). As genetic distance between taxa decreases (incipient speciation), it becomes more difficult to distinguish or even define hybridisation between groups that are not sufficiently divergent or reproductively isolated. Forsman et al. (2010) also examined a single copy nuclear marker (ATPsb) and a principal component analysis of microskeletal features. The single copy nuclear marker had high allelic variation and failed to resolve most groups, but since mitochondria, ITS and micro-morphology agree, the authors argue that ATPsb has not yet sorted among lineages and that extreme colony-level polymorphism is the most likely explanation. van Oppen et al. (2004) came to different conclusions when studying Montipora from Indonesia and the Great Barrier Reef using the putative mitochondrial control region and the nuclear single copy marker Pax-C intron; the markers were generally concordant, except for several key groups with similar spawning times. This was interpreted as evidence of hybridisation, although phenotypic polymorphism and incomplete lineage sorting could not be ruled out. 4.1.5. Future directions for studying coral species diversity There are several consistent patterns that emerge from an overview of species-level studies on reef building coral. Firstly, a surprising majority of studies on reef building coral have found genetic structure that does not map well onto morphologically defined species. Studies on the same organism, sometimes in the same geographic region, can have very different interpretations of the molecular patterns (e.g. Lopez and Knowlton, 1997; Odorico and Miller, 1997; Medina et al., 1999; van Oppen et al., 2001; Vollmer and Palumbi, 2002, 2004; Fukami et al., 2004b; Combosch et al., 2008; Flot et al., 2008; Pinzo´n and LaJeunesse, 2010). It is clear that there is a lack of consensus on the genetic and morphological scale defining species as opposed to population level genetics for reef building corals. Secondly, most studies that have integrated several molecular markers or combined molecular and morphological approaches have shown conflict between

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nuclear and mitochondrial genes or between genes and morphology. The most controversial studies have tended to make sweeping generalisations or to not give due consideration to multiple alternative hypotheses that can explain the data. Extreme phenotypic polymorphism, hybridisation, incomplete lineage sorting, misidentification, or even occasional sample mislabelling can all result in very similar data (the latter, however, can be easily ruled out by proper technique and repeat confirmation). Although molecular studies have great potential for resolving patterns of evolution and biodiversity below the genus level, at present, there is little or no consensus on which molecular markers are the most effective in stony corals. Although there is a plethora of markers used, there is generally wide agreement that studies require at minimum both mitochondrial and nuclear markers to attempt species level resolution. The ITS region is the most widely used nuclear marker for many basal eukaryotic groups with slowly evolving or unusual mitochondria (e.g. plants, fungi, corals), but due to its multi-copy nature and complex evolutionary history, it is also among the most controversial (Alverez and Wendel, 2003; Vollmer and Palumbi, 2004). Concerted evolution is assumed to homogenise the multiple copies through recombinant mechanisms (unequal crossover and gene conversion); therefore, proponents have argued that it is an ideal marker for determining what has the potential to interbreed (Coleman, 2009). Opponents have urged caution, because highly divergent and probably ancient copies coexist within a single genome (Alverez and Wendel, 2003; Vollmer and Palumbi, 2004). Since the ITS region is non-coding, it is subject to insertions and deletion events, which can result in multiple alternative sequence alignments, which means there are multiple alternative methods of analysis. The same set of sequences can yield more than one interpretation of the results; however, this may not be a problem if alternative alignments are compared or if taxonomic sampling is sufficient (Forsman et al., 2006). Protein-coding mitochondrial genes often yield little or no polymorphism between coral species (Shearer et al., 2002; Hellberg, 2006; Shearer and Coffroth, 2008); therefore, non-coding mitochondrial markers (putative control region, intergenic regions) usually provide the only possibility of any adequate species-level polymorphism for resolution of recent evolutionary time (ca < 5 mya). Due to the numerous rearrangements of the mitochondrial genome throughout Scleractinia (Medina et al., 2006), each taxon is idiosyncratic with a marker that can only be applied to that particular group and often cannot be aligned outside the genus or family. Thus, non-coding regions are very difficult to calibrate with the geologic or fossil record to estimate rates of evolution or dates of divergence. Although mitochondria genes evolve more slowly and may have less polymorphism, they are likely to have a faster rate of coalescence due to smaller effective population size resulting from their haploid nature, and maternal mode of inheritance (Rand, 2001). Mitochondrial genes might

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be expected to more accurately reflect the species tree because of this faster coalescent time; however, there are numerous examples of asymmetric introgression of mitochondrial genes between hybridising species, and organelles are often preferentially introgressed into invasive species (see Currat et al., 2008 for a review); therefore, additional evidence is needed to corroborate whether mitochondrial groups correspond to reproductively isolated species. Future studies may take advantage of large amplicon sequencing, or whole mitochondrial genome sequencing with next generation tools, which have great potential to overcome the lack of polymorphism and resolution due to slow mitochondrial evolution. As an example, Morin et al. (2010) used whole mitochondrial genome sequencing to examine 160 killer whales and were able to resolve groups that were suspected based on behaviour and morphological differences but could not be resolved in previous mitochondrial studies due to slow evolution. Along with increased capability for taxonomic and gene sampling, computational methods are emerging that can distinguish between hybridisation and incomplete lineage sorting (e.g. Holland et al., 2008), but these methods require data from more gene regions than are currently routinely sampled in corals with current technology. Next generation tools are providing unprecedented levels of data, and recent and ongoing temporal processes can be revealed; for example, Emerson et al. (2010) used next generation tools (RAD tags) to resolve postglacial phylogeography in mosquitoes (22–19 ka bp). Increasingly, integrated approaches and new methods are raising the bar as the new standard with multiple nuclear and mitochondrial markers, in addition to morphological data. Evidence from multiple studies is just beginning to mount, and a coherent picture of the scale of species-level variation is starting to form for some well-studied groups. Due to the enormous geographic range of many Pacific taxa, standardisation of methods will allow studies from different regions to be compared (e.g. comparable DNA sequence data, accessible voucher photographs both microscopic and in situ). If molecular studies include morphological data, then it will become clear which traits are under strong selection, and which are more evolutionarily neutral and may be tied to the well preserved scleractinian fossil record, providing a ‘Rosetta stone’ for interpreting the fossil record (Budd et al., 2010). Even if methods can be standardised in an ideal dataset, there will still be significant challenges that remain when interpreting conflicts between distinct types of data (mitochondrial, nuclear, and morphological). These challenges will only be overcome when data from many studies can be compiled and compared. Increased taxonomic, geographic, and genetic sampling will require larger-scale collaborative efforts across the globe. These efforts are critically important to understand the past, present, and uncertain future of coral reefs in the face of the present biodiversity crisis.

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4.2. Symbiodinium: The dinoflagellate symbionts of reef corals 4.2.1. Taxonomy of Symbiodinium The genus Symbiodinium is classified within the family Symbiodiniaceae, order Suessiales, and class Dinophyceae (Freudenthal, 1962; Fensome et al., 1993; Steidinger and Tangen, 1997; Taylor, 2004). Members of the genus are unicellular photosynthetic cocci that can be found in symbiosis with a wide range of invertebrate hosts and protists or free-living in the ocean environment (reviewed in Stat et al., 2006; Fig. 1.4). Phylogenetically, the genus Symbiodinium groups within the Gymnodiniales–Peridiniales– Prorocentrales dinoflagellate complex (Wisecaver and Hackett, 2011) and has long been described as closely related to members of the predominantly free-living genus Gymnodinium (Taylor, 1974; Blank and Trench, 1986; McNally et al., 1994). More recently, the genus Symbiodinium was shown to form a monophyletic group sister to the new dinoflagellate genus Pelagodinium (Siano et al., 2010), a symbiotic dinoflagellate found in the planktonic foraminifer Orbulina universa (Shaked and de Vargas, 2006).

Figure 1.4 Scanning electron microscope image of Symbiodinium cells in culture isolated from the anemone Aiptasia pulchella. Cells are approximately 10 mm in diameter. Photo courtesy of Scott R. Santos, Department Biological Sciences, Auburn University, USA.

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4.2.2. A history of Symbiodinium species and diversity The taxonomy of Symbiodinium and description of species in the genus are hampered by an abundant and confusing literature generated in the past 130 years. The nomenclature of algal endosymbionts harboured by animal hosts dates to 1881 when Brandt (1881/1882) first employed the term ‘zooxanthellae’ to describe the yellow-brown endosymbiotic algae of animal cells. Brandt (1881/1882) created the genus Zooxanthella to classify the yellow cells of radiolarians, certain hydrozoans, and actinians. At the same time, Geddes (1882a,b) suggested that the yellow cells should be called according to their habitat and introduced the genus Philozoon and several species names (Philozoon medusarum for the symbionts of medusae, Philozoon actiniarum for the symbionts of sea anemones, Philozoon siphonophorum for the symbionts of siphonophores, Philozoon radiolarum for the symbionts of radiolarians). Ten years later, Klebs (1884) created the family Zooxanthellidae and considered it as ancestor of both dinoflagellates and chrysomonads. Pascher (1911) regarded all the organisms previously believed to be Zooxanthella nutricula as cryptomonads and renamed the entire group of zooxanthellae as Chrysidella nutricola (C. nutricula; ICBN Art. 73 and IRZN Art. 32). Hovasse (1924), however, introduced the name Endodinium chattoni (E. chattoni; ICBN Art. 73) for the endosymbiotic dinoflagellate of the jellyfish ‘By-the-wind-Sailor’ Velella velella. A significant advance was reached with the pioneering work of Kawaguti (1944), and following investigations from McLaughlin and Zahl (1959), whose successful culture of symbiotic algae from cnidarian hosts and observations of Gymnodinium-like features of motile cells proved unequivocally that these algae are dinoflagellates. Following the work of Kawaguti (1944), Freudenthal (1962) used light microscopy to provide the first description of the taxonomy, life cycle, and morphology of the symbiotic dinoflagellates isolated from the scyphozoan Cassiopeia xamachana and introduced a new genus and species Symbiodinium microadriaticum. Initially, despite early cautions to the contrary (e.g. McLaughlin and Zahl, 1966), S. microadriaticum (Freudenthal) was reported as a unique and pandemic species comprising all dinoflagellate symbionts associated with marine invertebrates (Freudenthal, 1962; Taylor, 1973, 1974). This paradigm lasted for more than 20 years. Beginning in the mid-1970s and culminating in the 1990s, however, evidence drawn independently from a variety of approaches (Table 1.2) suggested that these dinoflagellates were, in fact, characterised by a high degree of taxonomic diversity. The characters used are largely phenetic and include ultra-structure morphology and chromosome number (Blank and Trench, 1985a; Trench and Blank, 1987; Blank and Huss, 1989), cell size (Schoenberg and Trench, 1980a; Domotor and D’lia, 1986), chloroplast number, size and arrangement (Blank and Trench, 1985b), isoenzyme profiles (Schoenberg and Trench, 1980b; Blank and Trench, 1985b), fatty acids and sterol composition (Blank and Trench, 1985b), photoadaptive

Table 1.2 A brief summary for evidence in support of Symbiodinium diversity in invertebrate symbiosis (modified from Baker Ph.D. dissertation) Evidence

Behavioural

References

Motility pattern Infectivity

Morphological Ultrastructure Morphometric

Biochemical

Physiological

Genetic

3D-reconstruction PCP complexes Fatty acids, sterols, and terpenes MAAs Growth rates Photosynthetic response Thermal response Isozymes DNA base composition DNA hybridisation RFLPs Sequence comparison

Fitt et al. (1981) and Fitt and Trench (1983) Trench (1971), Kinzie (1974), Kinzie and Chee (1979), Schoenberg (1976), Schoenberg and Trench (1980b), Trench et al. (1981), and Colley and Trench (1983) Leutenegger (1977) Schoenberg and Trench (1980a), Domotor and D’lia (1986), and Sandeman (1996) Blank and Trench (1985a) and Blank (1986a,b, 1987) Haxo et al. (1976), Chang and Trench (1982, 1984), and Govind et al. (1990) Withers et al. (1979, 1982) and Kokke et al. (1984) Banaszak et al. (2000) Fitt and Trench (1983) and Chang et al. (1983) Trench and Fisher (1983) and Iglesias-Prieto and Trench (1994) Iglesias-Prieto et al. (1992) and Warner et al. (1996) Schoenberg and Trench (1980c) and Schoenberg (1976) Blank et al. (1988) Blank and Huss (1989) Rowan and Powers (1991a,b), Rowan and Knowlton (1995), Baker and Rowan (1997), Loh et al. (1998), and Darius (1998) Rowan and Powers (1991a,b), Rowan and Powers (1992), McNally et al. (1994), Wilcox (1997), and Darius (1998)

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physiology (Blank and Trench, 1985a; Iglesias-Prieto and Trench, 1994), and host infectivity (Trench, 1971; Kinzie and Chee, 1979). Beginning in the 1990s, advances in molecular biology and the use of phylogenetics on symbiotic dinoflagellates left little doubt that the genus Symbiodinium is much more diverse than originally thought. Rowan and Powers (1991a,b) revealed that the amount of genetic diversity in Symbiodinium is equal to that seen between some free-living dinoflagellates belonging to different orders (Rowan and Powers, 1992; Rowan, 1998). There has since been a plethora of studies investigating the molecular diversity of Symbiodinium focusing on biogeography, host specificity, and environmental partitioning (see Section 4.2.3). There are currently 16 Symbiodinium species names reported in the literature (Table 1.3), but only 6 (S. microadriaticum, Symbiodinium pilosum, Symbiodinium kawagutii, Symbiodinium goreauii, Symbiodinium natans, Symbiodinium linucheae) have been formally described (Freudenthal, 1962; Trench and Blank, 1987; Trench and Thinh, 1995; Trench, 2000; Hansen and Daugbjerg, 2009). Life cycle and morphological features were used in the description of S. microadriaticum by Freudenthal (1962). Trench and Blank (1987) further described S. pilosum, S. kawagutii, and S. goreauii based on size of the coccoid and motile stages, number and volume of chromosomes, number of chloroplasts and pyrenoid stalks, thylakoid arrangement, and also isoelectrical characteristics of peridinin–chlorophyll a–protein complexes (PCP). In addition, Trench and Thinh (1995) described the dinoflagellate symbionts of the jellyfish Linuche unguiculata as Gymnodinium linucheae. However, further large subunit (LSU) analysis of this species revealed high genetic similarity (>98%) with S. microadriaticum (Wilcox, 1998) and later referred to as S. linucheae by LaJeunesse (2001). More recently, a free-living strain named S. natans isolated from the water column of Tenerife in the northeast Atlantic Ocean was described using morphological analysis of plate tabulation and ultra-structural characteristics, as well as genetic analysis of the nuclear-encoded LSU rDNA (Hansen and Daugbjerg, 2009). However, the remaining Symbiodinium species names that are used in the literature are either based on unpublished data or lack an adequate description to justify their classification as a binomial species. These nomina nuda or ‘naked names’ should be considered invalid at this time until evidence in support for their delineation is published. For example, six species names (S. bermudense, S. cariborum, S. corculorum, S. meandrina, S. pulchrorum, and S. californium) were introduced in Banaszak et al. (1993). Five of the names in their Table 1 are referred to as ‘Trench (unpublished)’, and evidence in support for these species delineations have never been subsequently published. More recently, S. californium, S. muscatineii, S. trenchii, S. glynni, and S. fitti have all been added to the literature based on a single gene sequence or DGGE ITS2 fingerprint (LaJeunesse and Trench, 2000; LaJeunesse, 2001; LaJeunesse et al., 2009, 2010; Pinzo´n et al., 2011). There is no formal

Table 1.3 List of Symbiodinium species names and associated attributes Species

Description

Reference Holotype Synonyms

Symbiodinium microadriaticum Formal

[1]

Symbiodinium goreaui

Formal

[2, 3]

Figs. 13– Gymnodinium 18 [1] microadriaticum [13], Zooxanthella microadriaticum [14] Fig. 3 [2] None

Symbiodinium kawagutii

Formal

[2, 3]

Fig. 4 [2] None

Symbiodinium pilosum

Formal

[2, 3]

Symbiodinium natans Gymnodinium (Symbiodinium) linucheae Symbiodinium bermudense Symbiodinium californium Symbiodinium cariborum

Formal Formal

[4] [5]

Nomen nudum Nomen nudum Nomen nudum

[6] [6, 8] [6]

Symbiodinium corculorum

Nomen nudum

[6]

Symbiodinium meandrinae

Nomen nudum

[6]

Symbiodinium pulchrorum Nomen nudum Symbiodinium microadriaticum Nomen nudum subsp. condylactis

[6] [7]

Fig. 5 [2] S. meandrinae; S. corculorum [15] Fig. 3 [4] None Figs. 12, None 13 [5] None S. pulchrorum [15] None None None S. microadriaticum subsp. condylactis [6, 15] None S. pilosum, S. meandrinae [15] None S. pilosum; S.corculorum [15] None S. bermudense [15] None S. cariborum [6, 15]

Cultures

Dominant ITS2 sequence

Trench: #61; CCMP: #421

A1 [15]

Trench: #113; CCMP: #2466 Trench: #135; CCMP: #2468 Trench: #185

C1 [15] F1 [15]

None Trench: #368

Clade A [4] A4 [15]

Trench: #13 Trench: #383 Trench: #80

B1 [8, 15] E1 [8, 15] A1.1 [7, 15]

Trench: #350

A2 [15]

Trench: #130

A2 [15]

Trench: #8 Trench: #80

B1 [8, 15] A1.1 [7, 15]

A2 [8, 15]

(continued)

Table 1.3 (continued) Species

Description

Reference Holotype Synonyms

Cultures

Symbiodinium muscatineii Symbiodinium trenchii Symbiodinium glynni Symbiodinium fitti

Nomen nudum Nomen nudum Nomen nudum Nomen nudum

[8] [9, 10] [11] [12]

None CCMP: #2556 MAC: #A001 Trench: #77, #220, #292

None None None None

None S. trenchiii [9] None None

Dominant ITS2 sequence

B4 [15] D1a [9, 10] D1 [11] A3 [12, 15]

[1] Freudenthal (1962), [2] Trench and Blank (1987), [3] Trench (2000), [4] Hansen and Daugbjerg (2009), [5] Trench and Thinh (1995), [6] Banaszak et al. (1993), [7] Blank and Huss (1989), [8] LaJeunesse and Trench (2000), [9] LaJeunesse et al. (2005), [10] LaJeunesse et al. (2009), [11] LaJeunesse et al. (2010a), [12] Pinzo´n et al. (2011), [13] Taylor (1971), [14] Loeblich and Sherley (1979), [15] LaJeunesse (2001).

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description for these species, and the data in support for their delineation are loosely referred to across numerous different publications. For example, Symbiodinium trenchii was first introduced in LaJeunesse et al. (2005) and referred to as ‘unpublished results’ and as ‘sensu ITS2 D1a’. S. trenchii was later referred to by LaJeunesse et al. (2009), with reference as a provisional species based on the previous LaJeunesse et al. (2005) chapter. Finally, in LaJeunesse et al. (2010b), this species name was formerly introduced in the manuscript written as ‘designated S. trenchii hereafter (LaJeunesse et al., 2005)’—referencing back to the original 2005 manuscript that references S. trenchii as unpublished results. This circular reference across multiple manuscripts provides no description of S. trenchii and falls well short of the required and necessary information for the delineation of a species. This history of naming Symbiodinium species, where most is based on unpublished data, will clearly bring confusion to the field of coral-Symbiodinium research, and we argue against the inclusion of provisional names in the future that are likely to result in a similar scenario. 4.2.3. Molecular diversity of Symbiodinium There are a variety of molecular markers and techniques that have been applied to research investigating the genetic diversity of Symbiodinium, both conserved and variable. Originally, DNA/DNA hybridisation and allozymes were applied and used as phenetic characters in the description of some Symbiodinium species (Schoenberg and Trench, 1980c; Blank and Huss, 1989). Similar to sequence variation of 18S rDNA in Symbiodinium being comparable to other free-living dinoflagellates placed in different orders (Rowan and Powers, 1992; Stat et al., 2008a), DNA/DNA hybridisation showed a similar amount of variation, where DNA from Symbiodinium isolates differed as much as DNA from some other algae that belong to different classes. 18S rDNA sequences originally divided Symbiodinium into three phylogenetic groups referred to as clades A–C (Rowan and Powers, 1992), while today, the number of divergent lineages has expanded to nine and, following the same nomenclature, is now referred to as clades A–I (Pochon and Gates, 2010). While most studies on Symbiodinium diversity have employed the nuclear rDNA including the 18S, 28S, and ITS regions (reviewed in Baker, 2003; Coffroth and Santos, 2005; Stat et al., 2006), the ITS2 gene is by far the most utilised marker for studies investigating the biogeography, host specificity, and environmental partitioning of Symbiodinium variants within clades (e.g. LaJeunesse et al., 2003, 2004, 2009, 2010b; LaJeunesse, 2005; Pochon et al., 2007; Bongaerts et al., 2010; Silverstein et al., 2011; Stat et al., 2011). Additional gene markers include the chloroplast 23S rDNA, the chloroplast psbA, mitochondrial cox1, and cytochrome oxidase b (Santos et al., 2002a,b; Moore et al., 2003; Takishita et al., 2003a; Lewis and Coffroth, 2004; Takabayashi et al., 2004; Zhang et al., 2005; Sampayo

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et al., 2009; Stat et al., 2009; Pochon and Gates, 2010). Overall, these genes reveal similar evolutionary relationships among Symbiodinium clades; however, their utility in resolving fine-scale patterns of Symbiodinium diversity has yet to be explored. Other functionally important Symbiodinium genes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL; Rowan et al., 1996), a 33-kDa peridinin–chlorophyll a–binding protein (pcp; Reichman et al., 2003), and glyceraldehyde-3-phosphate dehydrogenase (gapdh; Takishita et al., 2003b), are complex multi-copy gene groups that show evidence of loci duplication, diverse isoforms, and potential lateral gene transfer, respectively, limiting their capacity to be used as a gene marker for diversity studies. More recently, actin sequence information and copy-number estimates using quantitative PCR (qPCR) have become available for Symbiodinium (Watanabe et al., 2006; Mieog et al., 2009). Other techniques that have been applied to Symbiodinium include allozyme analysis, RAPD, microsatellites, and DNA fingerprinting (Bythell et al., 1997; Goulet and Coffroth, 1997, 2003a,b; Baillie et al., 1998, 2000; Belda-Baillie et al., 1999; Santos et al., 2001, 2004; Santos and Coffroth, 2003; Magalon et al., 2004). 4.2.4. Current approaches to interpreting diversity and delineating species in Symbiodinium The most recent attempts at species delineation within Symbiodinium have attempted to correlate variation in the ITS2 with spatial, physiological, and/ or ecological attributes (Sampayo et al., 2009) or have used clustering approaches to identify groups of closely related ITS2 types (Correa and Baker, 2009). Some Symbiodinium ITS2 variants have been shown to vary in their thermal or light optima (e.g. Iglesias-Prieto et al., 2004; Frade et al., 2008), with host symbiont transmission strategy (Stat et al., 2008b), or are correlated with host disease resistance (Correa et al., 2009). Other Symbiodinium ITS2 variants appear to be restricted in terms of their host and/or geographic ranges, based on sampling to date (e.g. Baker, 2003; LaJeunesse, 2005). However, as the number of host individuals and reefs sampled increases worldwide, along with the sensitivity of molecular tools applied to detecting Symbiodinium diversity, some conclusions regarding specificity/ restricted ranges are being called into question (Baker and Romanski, 2007; Mieog et al., 2007; Correa and Baker, 2009; Correa et al., 2009; LaJeunesse et al., 2009; Stat et al., 2009; Silverstein et al., 2011). These observations have highlighted the utility of using the ITS2 for studies on Symbiodinium diversity and identified its potential as a species marker or barcode for systematics. Sampayo et al. (2009) performed 13 genetic analyses to characterise the Symbiodinium diversity within three coral species using ribosomal, mitochondrial, and chloroplast genes. The authors observed that phylogenetic reconstructions based on concatenated ITS1 and ITS2 sequences versus concatenated ITS1, ITS2, LSU rDNA, mtCob, and cp23S sequences

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produced congruent topologies, although the lack of branch support and polytomy in the phylogenies is not a conclusive result. The authors noted the presence of intra-genomic variation within sequenced Symbiodinium ITS1 and ITS2 types (e.g. one to three codominant repeats in a single DGGE band), particularly within cloned and sequenced ITS2 types (e.g. C42). Most cloned sequences from a given sample clustered together in maximum parsimony phylogenies, but some sequences showed little phylogenetic overlap. The authors assumed that variants other than the most commonly retrieved sequence did not have diagnostic value. Despite the lower statistical support for the concatenated ITS tree, and the fact that the Symbiodinium types examined represented <1% of the total documented Symbiodinium ITS2 diversity globally, Sampayo et al. (2009) concluded that Symbiodinium species could be assigned based on observed correlations among phylogenetically independent lineages and ecological and physiological attributes. No additional Symbiodinium ITS2 types have yet been tested using as many lines of evidence as Sampayo et al. (2009). It is therefore imprudent to extrapolate that all Symbiodinium types represent distinct species, particularly those that are minimally different (e.g. a single nucleotide or indel difference, that is, often not homogenised across the rDNA tandem repeat). Correa and Baker (2009) suggested an alternative cluster-based approach to interpretate Symbiodinium ITS2 variation. Sequences were iteratively grouped such that divergence was maximised between clusters. This approach identifies ITS2 types that are unlikely to belong to conspecifics based on their membership in different clusters. Although this method cannot designate which Symbiodinium ITS2 sequences represent conspecifics, it does identify the Symbiodinium ITS2 types that are in greatest need of rigorous testing to confirm that they are significantly different physiologically, ecologically, and/or in their geographic/host ranges. Stat et al. (2011) used cloning and sequencing of the Symbiodinium ITS2 to test how sequence diversity partitioned across spatial scales (region, site, and colony) and host genotypes in M. capitata. A high diversity of ITS2 sequences was recovered, with most variation partitioning among coral colonies. This study likely employs the most rigorous statistical support for differences in the distribution of Symbiodinium ITS2 sequences to date. The authors conclude, however, that it is not currently possible to determine the number of unique Symbiodinium types (or species) present within a given coral colony. This is because (1) the ITS2 is multi-copy and thus the relationship between ITS sequences and Symbiodinium cells is not 1:1, (2) no information is available regarding the potential function(s) of the nondominant sequences isolated, and (3) ITS2 variation within individual Symbiodinium genomes has never been assessed. Stat et al. (2011) supported the use of the ITS2 for studies of Symbiodinium diversity but cautioned against its use as a systematics barcode because of these limitations.

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4.2.5. Challenges in delineating Symbiodinium species Significant methodological challenges currently hinder our ability to partition Symbiodinium diversity using either a traditional approach based on morphology or a molecular approach. A primary difficulty in distinguishing Symbiodinium species arises from the lack of readily discernible morphological features, especially when observed in hospite as non-motile coccoid cells, and the infrequency of sexual recombination. Further complication arises due to the phenotypic plasticity of Symbiodinium. The morphology of Symbiodinium cells is affected by culture phase, nutrient exposure in hospite, the concentration of lipids and starch, and also irradiance, which can affect the cell size, and chloroplast size, and brings into question the validity of some of the original species designations based on such features (Rowan and Powers, 1991b; Muller-Parker et al., 1996; LaJeunesse, 2001). In addition, when Symbiodinium are harvested from a host and established in culture, the strain(s) that dominate the culture may not be those detected from host tissue using molecular markers. Since cultured strain(s) may represent cryptic or opportunistic symbiont strains in hospite, or surface contaminants, culturing techniques are limited in their capacity to identify the dominant and ecologically important symbiont(s) within a host individual (Trench, 1979, 1997; Goulet and Coffroth, 1997; Santos et al., 2001; Coffroth et al., 2006). Cultures are available for all but two of the formally described Symbiodinium species (Table 1.3). The culture of S. natans was lost after its description (see Hansen and Daugbjerg, 2009). S. muscatineii was isolated from the sea anemone Anthopleura elegantissima for DNA extraction only and was not cultured. The great majority of available cultures belong to the Bob Trench culture collection currently maintained by Todd LaJeunesse at Penn State University. Mary-Alice Coffroth at the University at Buffalo and Scott Santos at Auburn University also maintain several of these strains, as well as others. In order to conduct biochemical and physiological tests and investigate other phenetic characters of Symbiodinium, cultures are a necessary requirement, even though their biology is likely to differ when outside the host environment. However, similar to prokaryotes, most of the diversity remains unculturable with current techniques hindering such investigations (e.g. Krueger and Gates, 2012). Even though a holotype culture for each Symbiodinium strain destined for delineation may not be possible, freshly isolated cells can still be used in microscopy to capture images and used in single-cell PCR for genetic analysis in their characterisation and delineation. As hosts often contain diverse Symbiodinium, a further complication is performing biological tests on cells in hospite, as the result may be the outcome of a mixed response. The use of multiple genetic markers on hosts with mixed symbionts is also challenging, as determining which sequence from each gene corresponds to which symbiont in hospite is not possible. Therefore, cultures or single-cell work is required.

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35

Phylogenetically, Symbiodinium species are challenging to resolve given the multi-copy nature of the fine-scale markers currently available (Correa and Baker, 2009; Stat et al., 2011). The ITS2 is a non-coding spacer region present in each tandem repeat within eukaryotic ribosomal genes (Fig. 1.3). Although concerted evolution continuously acts to homogenise these repeats, the process is imperfect and can lead to the existence of multiple ITS2 sequences within an individual Symbiodinium cell. A variety of sequence variants associated with a single Symbiodinium ITS2 type have been reported based on cloning and sequencing of the ITS2 (Apprill and Gates, 2007; Thornhill et al., 2007; Sampayo et al., 2009; Stat et al., 2011). Using DGGE, diverse sequence variants have also been attributed to single Symbiodinium types based on complicated banding profiles containing many minor bands in conjunction with the same dominant band (e.g. Silverstein et al., 2011), the designation of paralogous sequences (based on the excision and sequencing of a given minor band), or the detection of co-migrating sequences within a single band (Apprill and Gates, 2007; Pochon et al., 2007; Sampayo et al., 2009). These issues, however, have been largely ignored and only the dominant band is typically scored when identifying the Symbiodinium ITS2 type using DGGE (e.g. LaJeunesse et al., 2003, 2004, 2010b). This approach to the DGGE technique can be subjective when multiple bands are present in a profile. Consequently, it is generally not applied in the broader literature, and it is more acceptable to score all bands in a fingerprint profile (e.g. Hedrick et al., 2000; Kvennefors et al., 2012). As long as researchers interpretate the biological meaning of the data with the limitations of the technique in mind, both cloning and DGGE have their utility and are two techniques widely used in diversity studies in the field of molecular ecology. However, neither overcomes the innate limitations of the ITS2 (intra-genomic variation) and the distribution of closely related sequences in nature that leave this marker inadequate for systematics. 4.2.6. Approaches to delineating species in basal eukaryotes Many basal eukaryotes were initially thought to be comprised of relatively few cosmopolitan species (Patterson and Lee, 2000; Finlay, 2002; Finlay and Fenchel, 2004). This perception likely stemmed from the fact that delineations were based on morphospecies and that few basal eukaryotes have successfully been brought into culture. The application of molecular tools to understanding basal eukaryotic diversity has revealed a comparatively high genetic diversity from these groups while still supporting the cosmopolitan distribution of some taxa. This suggests that allopatry is not necessarily required for speciation in basal eukaryotes. Nonetheless, the interpretation of basal eukaryotic molecular diversity data presents many of the same challenges that exist for bacteria in terms of inferring function from phylogenetic relationships and how diversity relates to species concepts (Worden and Not, 2008). The diversity and geographic distribution of many basal eukaryotes remain

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Michael Stat et al.

poorly described, as well as the mechanisms that promote their evolutionary diversification and spatial distributions (Logares et al., 2008). Nuclear ribosomal DNA (particularly the 18S and ITS regions) and mitochondrial (cytochrome b) genes have been most frequently applied to delineating species in basal eukaryotes. Most recent studies employ several lines of evidence, including multiple molecular markers (e.g. Logares et al., 2007, 2008; Gao et al., 2008, 2010). Where high-resolution markers, such as the ITS, are used to delineate species, differentiation is frequently expressed as percent sequence identity (i.e. operational taxonomic units, OTUs, although the specific threshold applied can vary, Worden and Not, 2008). Clustering of sequence micro-diversity into OTUs occurs, and organisms in distinct clusters are hypothesised to be different species. However, as has been shown in diatoms and other basal eukaryotes, the level of intra- and inter-genomic variation differs depending on genus, and therefore, the sequence variation separating species will vary (e.g. Moniz and Kaczmarska, 2010). Furthermore, single base pair differences detected between sequences using species-level markers are frequently treated as intraspecific diversity (e.g. in dinoflagellates; Adachi et al., 1996, 1997; Shao et al., 2004; Litaker et al., 2007), although there are exceptions, in certain fungi, for example, where sequence diversity of the ITS region does not have the capacity to resolve species (Stockinger et al., 2010). What is a necessity for a gene to be used as a barcode, however, is for the interspecific distances to exceed the intraspecific distances. This is not the case for ITS in Symbiodinium (Thornhill et al., 2007; Sampayo et al., 2009). Adl et al. (2007) made suggestions on how to assess the diversity of protists and delineate species. In addition to guidelines for standard molecular approaches including genetic information from multiple genes and adequate sampling of multiple individuals over broad geographic ranges to determine intra- and interspecific variation, linking phylogenetic analysis with physiological adaptations and/or other characteristics is necessary. In addition, there is a need for a publicly available repository for sequence data, phenotypic information with microscopic images, and any other biological information regarding attributes of the species. We agree with this integrative approach for delineating species in Symbiodinium and the need for a publicly available source housing this information. 4.2.7. Future directions for studying species diversity in Symbiodinium Ultimately, physiological, ecological, and molecular data should be integrated in the delineation of Symbiodinium species. These characters should be systematically tested, statistically supported, and iteratively revised as new datasets, molecular markers, and tools become available. The results of these

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37

tests must be subjected to peer review and published as formal Symbiodinium species descriptions. The development and utilisation of multiple, independent markers with a level of resolution similar to that of the ITS is critical to the designation of Symbiodinium species (Coffroth and Santos, 2005; Correa and Baker, 2009; Stat et al., 2011). The current number of molecular markers used for Symbiodinium diversity is limited, and most do not have the resolution of the ITS, and so the search for new gene candidates that are single to low copy within Symbiodinium useful for fine-scale resolution of diversity continues. As alluded to earlier, while useful for studies on diversity and the partitioning of types/sequences over spatial scales or host species, the ITS is inadequate for use in Symbiodinium systematics, and so the search for a barcode or set of genes continues. Bioinformatics, such as the screening and comparative analysis of large transcriptomic datasets, and cutting edge technologies (e.g. next generation sequencing) should be applied to Symbiodinium gene discovery. The development of theoretical models for how Symbiodinium diversity arises and is dispersed on reefs (e.g. Baskett et al., 2009; Correa and Baker, 2009; van Oppen et al., 2011) should also be a priority. These models can be used to guide future work on Symbiodinium systematics. Biological attributes of Symbiodinium should accompany molecular information. Host species range and depth are obvious characters to include and have been utilised in multiple studies on Symbiodinium diversity already. However, premature conclusions about these attributes are likely to occur if inadequate sampling of individuals and geographic scales takes place. Ideally, cultures of type organisms should be maintained and would greatly simplify characterising information from multiple genetic markers, obtaining morphological information through microscopy, as well as biochemical and physiological attributes. Unfortunately, most Symbiodinium are unculturable using current techniques and so consistent biochemical and physiological information may not be tractable. However, they should be included if cultures are available. In the absence of cultures, freshly isolated single cells from the host organism should be used for molecular genotyping and morphology. Therefore, the minimum requirement is to include genetic information from multiple markers and microscopy from freshly isolated single cells along with ecological characteristics such as host species, depth, and location. When a culture is available, further biochemical and physiological tests can be performed. Finally, it is critical that a repository for Symbiodinium sequences and associated attribute data be made publicly available, similar to the Searchable Database of Symbiodinium Diversity, Geographic and Ecological Diversity (SD2GED, Santos and LaJeunesse, 2006) and GeoSymbio (Franklin et al., 2012), and be developed and curated by a diverse consortium of researchers in the field.

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4.3. The prokaryotic symbionts: Bacteria and Archaea 4.3.1. The taxonomy of prokaryotes Approximately 9000 prokaryote species have currently been isolated and validated (www.bacterio.net). Most experts agree, however, that this number vastly underestimates the true diversity of microbial species on Earth. For example, current estimates suggest that the total number of prokaryotic species in just 100 g of soil is around 11,000 (Torsvik et al., 2002) with estimates of the total number of bacterial species on the planet exceeding the number of stars in our galaxy (there are around 200–400 billion stars in the Milky Way; Whitman et al., 1998). For coral-associated bacterial species, estimates of the total number of species (across time, host, and space) remain largely unavailable. Recent counts of the number of prokaryotic cells in coral mucus from the coral host Porites lobata suggest that there are approximately 1 million microbial cells per millilitre of mucus (Garren and Azam, 2011) and in situ cell counts of bacteria and archaea are each on the order of 107 per cm2 of coral tissue (Kellogg, 2004; Wegley et al., 2004). Calculations based on 16S rDNA-based analysis of clone libraries derived from 14 samples taken from three coral species estimated that the number of total distinct bacterial ribotypes across these three corals was approximately 6000 (Rohwer et al., 2002). More recently, a 16S rDNA pyrotag sequencing study investigating associated microbial diversity of a number of coral species in the Caribbean estimated similar numbers—several thousand species associated with any given host species (Sunagawa et al., 2010). Microbial taxonomy has historically been linked with advances in technology, starting with the invention of the microscope in the 1700s and the pioneering work of Antonie van Leeuwenhoek, the father of microbiology. This was followed by early bacterial staining methods, the development of chemotaxonomy, DNA–DNA hybridisation (DDH) techniques, to the recent renaissance of microbial taxonomy via 16S rRNA gene sequence data, and the burgeoning amount of genomic sequence data that is transforming the established dogma used to describe microbial species (Klenk and Go¨ker, 2010). Like most other organisms, however, there is no clear way forward for the delineation of microbial taxa. Morphological and nutritional criteria used to describe and classify prokaryotes do not reflect evolutionary relationships (Pace, 1997), and concepts related to species definitions centred around sexual reproduction are not readily applicable to prokaryotes due to the various mechanisms that bacteria have for genetic exchange (Cohan, 2002). Some suggest that even the binomial scheme applied to eukaryotes is inappropriate for prokaryotes as it does not allow for genetic exchange such as horizontal gene transfer from distantly related strains and that a network may be more appropriate than a hierarchical scheme for prokaryote phylogeny (Rossello´-Mo´ra, 2005; Gevers et al., 2011).

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The current working standard for valid bacterial species definitions is based on the guidelines suggested by the Ad Hoc Committee in 1987 and revised in 2002 (Wayne et al., 1987; Stackebrandt et al., 2002). These guidelines propose that strains with greater than 70% DDH relatedness and within 5  C of DTm are classified as the same species. In addition, a species also needs to have a distinguishing phenotypic feature. The phenotypic description of a species is achieved via a set of biochemical tests in combination with fatty acid composition values. In addition, a near complete 16S rDNA sequence and GC content of the whole genome are required and need to be provided with the species definition (Stackebrandt et al., 2002). Phenotypic data should agree with genotype data and where strains vary genotypically but cannot be distinguished with phenotypic data, it is recommended that they are not formally named until a unique phenotypic character can be found (Wayne et al., 1987). These recommendations have been widely used and have provided microbiologists with a standardised system with which to classify prokaryotic organisms (Stackebrandt et al., 2002). The Ad Hoc Committee’s revision of the guidelines for prokaryotic species definitions in 2002 (Stackebrandt et al., 2002) acknowledged that recent advances in genomic tools and the proliferation of sequence data may be useful for re-evaluating prokaryotic taxonomy. Most recent suggestions for new standards of species definitions remain based on the assumption that bacterial species can be cultured. For example, Gevers et al. (2011) call for a sequence-based global information system of genome data that also includes biological, ecological, and phenotypic data for isolates. These and existing approaches require that strains be isolated in culture, which may not be possible for the vast majority of bacteria (Kogure et al., 1979). One of the major challenges for future microbial taxonomy is to determine how to extract whole genome sequence data from meta-genomic data of complex communities and create a standardised automated system for species identification and classification (Gevers et al., 2011). For corals, partial or complete genomes constructed from microbial meta-genomic data will likely become one of the best strategies for elucidating prokaryote species level diversity. 4.3.2. The rebirth of prokaryote taxonomy via molecular-based approaches The sequencing of the 16S rDNA has provided a phylogenetic framework within which to place metabolic and nutritional data of prokaryotes (Pace, 1997). 16S rDNA sequence similarity of >97% usually agrees with the previously designated DDH of 70% relatedness category for species, with genes showing <97% similarity generally agreeing with published taxonomic results that indicate these are distinct species. Although there are

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some exceptions—some strains with > 97% 16S rDNA have <70% DDH relatedness—this gene is currently the most useful marker in prokaryotic taxonomy. There is a massive number of 16S rDNA sequences derived from bacteria deposited in publically accessible databases (e.g. NCBI). As a result, sequencing of this gene has uncovered new phyla, and it is by far the most popular molecular tool used by microbial ecologists, including coral microbial ecologists, for evaluating the species diversity in environmental samples. The 16S rDNA shows clock-like behaviour and provides a basis for molecular systematics. While the 23S rDNA is considered to be the most informative of the three rRNA genes, since it doubles the size and information of the 16S, technical and economic issues have resulted in 16S rDNA being the most widely used (Yarzaa et al., 2010). While not as informative as the larger 23S rDNA, the gene still comprises over 1500 bases, a much larger number than comparative loci such as the ITS2 region or cox1 used in eukaryotes. This gene is extremely useful for inferring phylogeny: it contains both conserved and variable regions with the conserved stem regions of the RNA element ensuring that these areas of sequences are likely to be maintained over evolutionary time. Over 91% of validly published bacteria and archaea species have good-quality 16S rDNA data associated with them (Yarzaa et al., 2008). The overwhelming majority of bacterial species from corals, and indeed all environments, are known only by their 16S rDNA, and estimates suggest that <1% of bacteria are culturable (Kogure et al., 1979). 4.3.3. Current molecular approaches and their application in identifying coral-associated prokaryotes Profiling and sequencing approaches have revolutionised our understanding of the breadth of the taxonomic diversity of prokaryotes associated with corals, primarily through use of the 16S rDNA. Early culture work indicated that a large diversity of microbes are associated with corals (Ducklow and Mitchell, 1979), but it was not until the more recent studies of Rohwer et al. (2002) that the scope of this diversity was first understood. Cloning and sequencing of the 16S rDNA and placing gene sequence data into OTUs (> 97% sequence similarity) revealed that corals were likely hosting in excess of thousands of different types of bacterial species (Rohwer et al., 2002). These results sparked a growing interest in prokaryotic species as important partners in the coral holobiont with a subsequent explosion in this field. A recent survey of 32 published studies revealed over 4500 16S rDNA sequences from prokaryotes associated with coral in the publicly available NCBI database (Mouchka et al., 2010). This is in contrast to the number of formally described strains associated with corals—the August 2011 release of the Silva living tree database (Yarzaa et al., 2008), which contains available 16S rDNA for type strains of bacteria and archaea, holds just 15 entries from coral hosts.

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The most popular method for obtaining an estimate of microbial species diversity in corals is to clone and sequence the 16S rDNA. The advantage of cloning and sequencing over fingerprinting techniques (see subsequent section) is that full-length sequences can be used to construct phylogenies, bacteria can be classified taxonomically, and results from sequencing studies can be compared to one another (Hamady and Knight, 2009). The disadvantage is that it is still relatively expensive to sequence full-length 16S rDNA amplicons from clone libraries, which limits the number of samples and consequently the depth of coverage of the microbial community and the number of replicates that are able to be compared within a study. Fingerprinting techniques involve the amplification of a gene, usually the 16S rDNA, and separation of amplified genes based on differences in the sequence by electrophoresis or capillary. These include techniques such as terminal restriction fragment length polymorphism (TRFLP) and DGGE. The advantage of these techniques is that they are relatively cheaper than sequencing and allow for the rapid typing of many samples, including multiple replicates. TRFLP run on a capillary sequencer is advantageous over gel electrophoresis methods as the number of samples able to be compared is unlimited, whereas DGGE and fragment analyses run on gels are limited to the number of lanes available in a gel, since it is generally only acceptable to compare samples within and not among gels. Disadvantages of DGGE are that the results cannot generally be compared to one another and the number of types that are able to be distinguished from a community is limited to several hundred at best. A recent evaluation of DGGE estimated that at least six replicate coral colonies are needed to get an accurate assessment of the associated microbial community diversity (Kvennefors et al., 2010). In situ techniques have primarily been used to validate sequence, fingerprinting, and culture data regarding specific groups of bacteria. In general, they are not used to identify the specific species present within a coral sample. For example, Apprill et al. (2009) used fingerprinting techniques to reveal that the early life history stages of the coral Pocillopora meandrina harboured distinct microbial communities in comparison to the surrounding seawater, and subsequent cloning and sequence data indicated that these communities were dominated by sequences from the genus Jannaschia from the order Roseobacter. Fluorescent in situ hybridisation using a probe specific for the Roseobacter group on coral eggs and larvae confirmed these results. In situ probing techniques have also revealed that active black band disease lesions were populated with extensive microbial communities, lesions associated with white disease of Hydnophora sp. were populated with a mixed microbial community including Cytophaga–Flavobacterium species and Gammaproteobacteria including Vibrio, while the white syndrome of Acropora sp. was not associated with any bacterial penetration into diseased tissue (Ainsworth et al., 2007). Finally, use of a universal probe for bacteria on

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eggs, larvae, and juveniles of a variety of broadcast spawning corals from Belize, Florida, and Guam indicated that bacteria were acquired only after corals have settled and metamorphosed into juveniles (Sharp et al., 2010). Fluorescence in situ hybridisation techniques are superior to PCR-based techniques in that they are semi-quantitative and allow visualisation of prokaryotes in situ so the association is very clear, that is, the microbiologist can be confident that the species are really occurring in association with the coral host, and that they have not simply amplified 16S rDNA from prokaryotes in the surrounding seawater or associated debris. The disadvantage of this method is that probe design is challenging: variable regions that allow specific binding of probes to particular groups (species, genera, family, etc.) may lie on regions of the 16S rDNA that have a folded secondary structure and are difficult for probes to penetrate and bind to. This limits the sites along the gene that are available for probes and thus the potential resolution of probes. Only two or three probes can be applied to a sample simultaneously, and screening samples for probe binding can be enormously time consuming if a suitable number of samples are to be examined. There are also several practical issues relating to in situ hybridisation including high levels of background autofluorescence from both coral tissue and associated Symbiodinium cells requiring advanced laser scanning microscopy and judicious choice of fluorochromes, in addition to extensive decalcification of tissue prior to sectioning. Another technique available to coral microbial ecologists is shotgun meta-genomic sequencing, where genomic DNA is sheared and randomly sequenced. Computational comparison of 33 meta-genomic shotgun sequencing datasets from human microbe projects and comparable 16S rDNA cloning and sequencing datasets revealed similar prokaryotic diversity (Shah et al., 2011). In agreement with this, a meta-genomic analysis of microbial communities associated with Porites astreoides (Wegley et al., 2007) found strikingly similar patterns to results obtained by 16S DGGE and sequencing (Rohwer et al., 2002). Both data sets contained abundant representatives from the Proteobacteria, Firmicutes, Cyanobacteria, and Actinobacteria with the Proteobacteria dominated by Gammaproteobacteria. These results highlight that meta-genomic sequencing projects are also a valid and highly powerful method for extensively evaluating prokaryote species diversity within coral samples. A final technique that is rapidly gaining popularity is 16S rDNA 454 tag pyrosequencing. This method relies on sequencing just a very short hypervariable region of the 16S rDNA, but in extremely high numbers (usually tens of thousands per sample) (Sogin et al., 2006). Currently available data (e.g. Sunagawa et al., 2010; Barott et al., 2011) agree with earlier clone library estimates of diversity and dominant groups that make up coralassociated microbial communities. The advantage of 454 tag sequencing is that the rarer members of the community can be identified, and more

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samples can be investigated since the cost is relatively cheaper than fulllength Sanger sequencing methods. New chemistry just released using the titanium 454 platform produces reads of up to 1000 bp, resulting in around 800 bp of valuable sequence data. As sequencing costs continue to reduce, tag pyrosequencing will replace clone libraries and profiling techniques over the next decade as the standard protocol for assessing coral microbial associations, because they provide increased community and phylogenetic resolution. 4.3.4. Current status of coral-associated microbiology A large number of coral microbiology studies have focused on diversitybased assessments, which have revealed some general patterns for microbial species associated with corals. A meta-analysis of the sequence data collected from 32 studies of coral-associated microbial communities revealed that sequences from both healthy and diseased coral samples in the publicly available NCBI database are largely composed of Proteobacteria groups such as the Rhodobacterales, Vibrionales, Alteromonadales, Oceanospirillales, and Bacteroides from the order Flavobacteriales and that there are substantially more Rhodobacterales and Vibrionales sequences from diseased coral samples than from healthy coral samples (Mouchka et al., 2010). Interestingly, while there are strong patterns emerging from individual studies, these do not currently result in a cohesive set of conclusive patterns regarding species diversity of coral-associated microbes. Some studies have revealed that coral-associated microbial species display site specificity, with community composition varying between location rather than coral host species (e.g. Littman et al., 2009; Barott et al., 2011) which suggests that environmental factors are predominantly responsible for coral-associated microbial community diversity. Conversely, others report that specific assemblages of microbes are associated with particular host species, even across sites located hundreds of kilometres apart (Rohwer et al., 2002; Chen et al., 2011), suggesting that the coral host somehow controls the composition of prokaryotes in the holobiont. Remarkably, one recent study found that while the microbial community associated with the tissue of each of three coral species was distinct, the microbial community associated with the coral skeleton was not (Tremblay et al., 2010). Temporal changes from the same coral across different months have also been observed (Chen et al., 2011), providing an even further layer of complexity. Extensive spatial heterogeneity appears to exist across a single coral host (Daniels et al., 2011; Sweet et al., 2011) and sampling effort may affect the outcome of some of these studies (Kvennefors et al., 2010). These results highlight that coral-associated microbial communities are both diverse and complex while strengthening the need for increased efforts investigating the partnership between corals and their prokaryotic symbionts. As outlined above, the field of microbiology in general struggles with the definition of species and,

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while extremely useful, 16S rDNA are inadequate for defining species. To fully understand the partnership between prokaryotes and corals, we need to go further than a general description of diversity and also have good species identifications to better understand their role in the coral holobiont. 4.3.5. Future directions for studying species diversity in coral-associated microbes While the 16S rDNA has been enormously useful to microbiologists for determining the number of individual OTUs within samples, species cannot reliably be determined based on a single gene since that gene will always be subject to simple stochastic variation, recombination, and horizontal gene transfer (Gevers et al., 2005). Furthermore, probes and primers designed to amplify specific or broad groups of bacteria and archaea will usually contain a degree of bias toward some members, and against others in the community (e.g. Ben-Dov et al., 2011). One option that remains relatively unexplored for defining prokaryotes associated with corals is multi-locus sequence typing (MLST) (Maiden et al., 1998). This method involves sequencing and aligning multiple housekeeping genes, such as rpoA, recA, and pyrH (Thompson et al., 2005), to create a more robust phylogeny than one based on the 16S rDNA alone. MLST is used to separate species that are closely related to one another, for example, several species within the same genus or family (Thompson et al., 2005). Current use of MLST within coral microbial ecology is limited to resolving species of cultured strains belonging to the family Vibrionaceae associated with bleaching (Thompson et al., 2005) and disease (Wilson et al., in press). Similar to 16S rDNA-based phylogenetic analysis, the main disadvantage of MLST is that it generally does not provide any information on the potential function of the organisms. Therefore, it is expected that whole genome sequencing will become widely incorporated into prokaryote taxonomy. Despite the fact that there are already several thousand bacterial whole genome sequences available in the NCBI database and that current sequencing technology enables sequencing of whole genomes in a single day at an ever-decreasing cost, whole genome sequence data are still barely being taken into account in microbial systematics/taxonomy. Microbial taxonomy is clearly on the cusp of a shift away from current methods requiring chemotaxonomy and DDH to a system incorporating genomic data, not surprisingly since the Ad Hoc Committee has encouraged efforts in this direction for the past decade (Stackebrandt et al., 2002) and there is increasing discussion within the community as to the lag in the incorporation of this powerful newly available data into taxonomic description. The benefits of whole genome sequencing to prokaryotic taxonomy will be immense: hundreds of genes to compare in phylogenomic studies, and putative functions of genes are likely to emerge by including ecological comparisons that will guide future functional analyses. The current major limitation to

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the whole genome sequencing of prokaryotes is the availability of bioinformatic tools, since sequencing itself is becoming affordable. One bioinformatic tool that has recently been developed to enhance comparisons across whole genomes is a calculation of average nucleotide identity using the software program JSpecies (Richter and Rossello´-Mo´ra, 2009). This tool has been tested against existing DDH data for several hundred type strains and mirrors the results, providing a way to compare strains, once sequence data are available. Since there is little direct funding available for prokaryote taxonomic studies, most genome sequencing projects are not done on the basis of taxonomy. Instead, genome data are acquired for strains of interest for other reasons (e.g. pathogens, the human microbe genome project, Craig Venter’s marine microbiology genome sequencing project; Klenk and Go¨ker, 2010). There has been one project developed to sequence the genomes of a variety of archaea and bacteria type strains based on their taxonomic status—the Genomic Encyclopaedia of Archaea and Bacteria project. This project aims to sequence 100 genomes a year from 2009 onward and provide the annotated data through the Integrated Microbial Genomes system. A similar scheme could also be applied to isolates from coral-associated microbial communities. Currently, however, for species to be validly described, strains need to be typed and identified based on both molecular and phenetic characterisations, and DDH is still the benchmark. Therefore, isolates are still required and they need to be characterised. Microbial taxonomy is still central to microbial ecology and coral microbial ecology is little focused upon with few resources. One idea to increase our understanding of species diversity within corals is to survey the available types currently isolated from corals and compare these to general taxonomic patterns that have emerged from molecular studies based on 16S rDNA. Strains that encompass the available taxonomic range and complement diversity as revealed by 16S rDNA typing could be submitted for whole genome amplification. Alternatively, coral tissue slurries could be filtered and single cells sorted and subjected to whole genome amplification. This sequencing effort in combination with increasingly innovative methods to isolate numerically dominant members of the community to recover isolates that more broadly reflect the known diversity would be highly valuable to the field of coral microbial ecology.

5. Concluding Remarks Corals, Symbiodinium, and prokaryotes each present distinct challenges for species delineation and do not have sufficient character states in common to utilise the same species concept. Molecular tools can aid in the description and identification of species within these taxa, but an integrative

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approach coupling molecular phylogenetics with phenetics is ultimately necessary. Molecular information should include data from multiple genes from different organelles in the case of eukaryotes, and from 16S rDNA for prokaryotes, at least until whole genomics and advanced molecular tools can be applied. Nuclear and mitochondrial genetic data should accompany morphological data for corals; nuclear, mitochondrial, and plastid genetic data should accompany environmental (host or depth) data for Symbiodinium; and 16S rDNA should accompany biochemical data for prokaryotes. With each species, it is of paramount importance that images of the organism, calcium carbonate skeleton, or isoclonal cultures be archived with DNA for future reference. These archived reference data to accompany any molecular data are required and concordant with current guidelines for species descriptions for prokaryotes, eukaryotes, and DNA barcoding and allows for future comparisons and amendments if necessary.

ACKNOWLEDGEMENTS The National Marine Sanctuary Programme (memorandum of agreement 2005-008/66882), a postdoctoral fellowship to M. S. from the AIMS-CSIRO-UWA collaborative agreement, and the U.S. National Science Foundation (NSF) grant through Biological Oceanography (OCE-0752604) to R. D. G. This is HIMB contribution number 1499 and SOEST contribution number 8683.

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