Scleractinia soft tissue systematics : use of histological characters in coral taxonomy and phylogenetic reconstruction

Transcription

1 University of Iowa Iowa Research Online Theses and Dissertations Spring 2015 Scleractinia soft tissue systematics : use of histological characters in coral taxonomy and phylogenetic reconstruction David Russell Cordie University of Iowa Copyright 2015 David Russell Cordie This thesis is available at Iowa Research Online: Recommended Citation Cordie, David Russell. "Scleractinia soft tissue systematics : use of histological characters in coral taxonomy and phylogenetic reconstruction." MS (Master of Science) thesis, University of Iowa, Follow this and additional works at: Part of the Geology Commons

2 SCLERACTINIA SOFT TISSUE SYSTEMATICS: USE OF HISTOLOGICAL CHARACTERS IN CORAL TAXONOMY AND PHYLOGENETIC RECONSTRUCTION by David Russell Cordie A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in Geoscience in the Graduate College of The University of Iowa May 2015 Thesis Supervisor: Professor Ann F. Budd

3 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL MASTER'S THESIS This is to certify that the Master's thesis of David Russell Cordie has been approved by the Examining Committee for the thesis requirement for the Master of Science degree in Geoscience at the May 2015 graduation. Thesis Committee: Ann F. Budd, Thesis Supervisor Jonathan M. Adrain Bradley D. Cramer

4 To Bill and Joan Tousignant ii

5 Time is the best teacher, but unfortunately, it kills all of its students. Robin Williams iii

6 ACKNOWLEDGMENTS This work would not have been possible without the generous support from the Lerner-Gray Marine Research Grant from the American Museum of Natural History which provided funds for specimen purchases and equipment. Further aid was provided by The University of Iowa department of Earth and Environmental Sciences and Center for Global and Regional Environmental Research which aided in travel costs. Assistance with course fees for histology course and additional aid were provided by NSF grant DEB to Ann F. Budd. Slide preparation would have been impossible without the help of Kathy Walters at the Central Microscopy Research Facility and slide reading sessions with Esther Peters. Also, assistance from Tiffany Adrain in the repository and cataloging was greatly appreciated. Finally, I would like to thank all those professors and instructors that have taught me so much over the years. Special thanks to Ann F. Budd for guiding me through the world of coral taxonomy while always giving me the freedom to pursue new avenues of research within the subject. Comments and reviews from all members of my committee were greatly appreciated and insightful. And to Bart De Stasio who once told me sometimes science is hard, but that is what makes it worth exploring. iv

7 ABSTRACT Coral reefs are some of the most diverse ecosystems in the world and provide economic value as well as biodiversity stability. Yet, these ecosystems are threatened from human degradation and climate change. Phylogenetic reconstructions can help identify which species have a potential to undergo greater amounts of change in the near future and also aids in determining evolutionary distinctiveness, which are critical components of conservation management. However, traditional Scleractinia morphological characters have been shown to have limited taxonomic use. Therefore, this study attempts to discover soft tissue characters to produce more robust phylogenies. Eight coral species from the Indo-Pacific families Merulinidae and Lobophylliidae were mail ordered and prepared for histological analysis under light microscopy. A character matrix was analyzed and the results were compared to phylogenies based on skeletal and molecular data. A total of seven MPTs of length 35, C.I and R.I were found. In addition, a detailed description of the histology is included. The topology of MPTs was inconsistent, but several were broadly similar to previous phylogenies based on molecular and skeletal data. Still, using only a small number of characters, the results do promise that histological characters in conjunction with skeletal characters could better delineate species and their evolutionary history. Future results could aid in making conservation decisions based on improved phylogenies. v

8 PUBLIC ABSTRACT Human interactions with their environment can have profound negative effects and mitigation is often necessary. One area of concern is tropical coral reefs. Through anthropogenic climate change we are destabilizing coral reef ecosystems and reducing their functionality. Therefore, conservation strategies need to be implemented to reduce the negative impacts that affect these systems. Conservation methods have been developed which require well-supported evolutionary histories, but they are not currently applicable to reef-building corals. Past reconstructions of coral evolutionary history have not made use of the soft tissue due to difficulties preserving and preparing tissue. This study develops a methodology and provides a description of coral soft tissue with the goal of understanding more details about the evolutionary history of stony coral. Eight Indo-Pacific coral species were selected and prepared for analysis using equipment found in standard medical research facilities. Specifically, the morphology of stinging cells is discussed. The results show that evolutionary histories from this new source of data are broadly congruent with other studies. The addition of this new source of data to existing sources will yield results with greater utility. With these findings, conservation priorities can be developed based on evolutionary distinctiveness and coral reefs can maintain functionality in light of current environmental changes. vi

9 TABLE OF CONTENTS List of Tables..ix List of Figures.x List of Abbreviations.xi List of Museums xii Introduction 1 Materials and Methods 7 Species....7 Specimens...9 Histological Methods..10 Phylogenetic Analysis.11 Character Evolution..12 Characters..12 Results.22 Systematic Account.28 Diploastrea.28 Echinophyllia.32 Lobophyllia.35 Mycedium 38 Oulophyllia.41 Oxypora 44 Pectinia.47 Symphyllia..50 Discussion..53 Molecular vs Skeletal Phylogenies..53 Histological Phylogeny 54 Character Mapping..54 Utility of Histological Characters in Scleractinia...56 Conclusions 60 vii

10 References 62 Appendix A..68 Appendix B.. 75 Appendix C.. 76 viii

11 LIST OF TABLES Table 1. Family designations for species in study 8 2. Characters and character states for histological analysis 27 B1. Character matrix used in histology-only analysis 75 C1. List of specimens studied and repository information 76 ix

12 LIST OF FIGURES Figure 1. Strict consensus trees based on Fukami et al. (2008) molecular data (left) and Budd et al. (2012) skeletal data (right; 13 taxa and 46 characters) Strict consensus tree and character optimizations for histology-only matrix (8 taxa, 20 characters) Histological characters mapped onto MPT created from histology-only matrix Examples of histological slides, see appendix A1 for more on character coding Histological slides of Diploastrea (SUI ) Histological slides of Echinophillia (SUI ) Histological slides of Lobophyllia (SUI ) Histological slides of Mycedium (SUI ) Histological slides of Oulophyllia (SUI ) Histological slides of Oxypora (SUI ) Histological slides of Pectinia (SUI ) Histological slides of Symphyllia (SUI ) 52 A1. Histological Characters and States 68 x

13 LIST OF ABBREVIATIONS SEM scanning electron microscopy; TEM transmission electron microscopy; H&E hematoxylin and eosin staining; MPTs most parsimonious trees; C.I. consistency index; R.I. retention index; TNT tree analysis using new technology xi

14 LIST OF MUSEUMS BM(NH) The Natural History Museum, London, UK; MNHN Muséum national d Histoire naturelle de Paris, France; SUI Paleontological Repository of the University of Iowa, Iowa City, IA, USA; UF Florida Museum of Natural History, Gainesville, FL, USA; USNM The National Museum of Natural History, Washington DC, USA xii

15 Introduction Coral reefs are marine ecosystems that are critical to maintaining global biodiversity (Pandolfi et al., 2011). Whereas they occupy between 0.1% and 0.5% of the ocean floor, they comprise as much as one-third of ocean diversity (Moberg & Folke, 1999). Therefore, coral reef ecosystems are vital to maintaining high biodiversity levels that improve ocean resilience and functionality (Tilman, 1996; Carr et al., 2002; Dulvy et al., 2004). Coral reefs also offer physical protection from erosion of coast lines due to wave action (Kunkel et al., 2006), nurseries for fish stock (Adams et al., 2006) and economic value in terms of tourism attractions (Brander et al., 2007). While perhaps more visible on the islands of the Caribbean and Pacific where reefs are abundant, the global oceans depend on the health of coral reefs. It has been well documented that coral reefs are undergoing diversity declines due to modern climate change (Mumby et al., 2011; Roff & Mumby, 2012). Specifically, corals are expected to suffer due to reduced carbonate concentration resulting from ocean acidification (Ries et al., 2009; Cohen & Holcomb, 2009); reduced disease tolerance (Jones et al., 2004); bleaching due to rising sea surface temperatures (Brown, 1997); as well as degradation due to shoreline development (Rogers, 1990). All of these factors demonstrate that corals are in danger and measures need to be taken to conserve them (Pandolfi et al., 2003; De ath et al., 2009). However, due to the global nature of changes that affect coral reefs, the conservation of all corals worldwide is financially 1

16 prohibitive (Balmford & Whitten, 2003). Therefore, conservation priorities and a greater knowledge of evolutionary distinctiveness are needed. It would be prudent for conservation managers to understand which species will behave more similarly to one another, due to common adaptations, so specific conservation strategies can be implemented in the most effective manner. Furthermore, accurate biodiversity counts are critical for managers to maximize the number of conserved species with limited resources. Past management strategies have focused on protecting areas of high richness, however, these areas do not take into account evolutionary potential as well as potential future changes that could affect the site (Budd & Pandolfi, 2010). Conservation prioritization metrics exist that take into account evolutionary diversity along with the International Union for Conservation of Nature (IUCN) Red List of Threatened Species status (Huang, 2012), or include the use of phylogeography to map areas of high diversity (Carpenter et al., 2011). Critically, both of these methods hinge on using well-supported phylogenetic information. The phylogenetic history of an organism describes the evolutionary relationships between taxa. A related field, systematics, attempts to classify organisms with information gained from their phylogenetic history. By looking at which species share morphological or molecular traits, we can better determine which are more closely related, how many species are present, and how behaviors are shared amongst relatives. For example, if a monophyletic 2

17 group of corals is found to possess a trait that yields high thermal stress tolerances, this group many be placed lower on priority lists then those found to be thermal stress intolerant. But, we can only know which organisms belong to which group by looking at their phylogenetic histories. The history of coral classification is long and filled with many corrections, reorganizations and reclassifications. Originally, corals were classified based solely on broad morphological similarities such as colony shape and size. Authors such as Dana (1846) and Haeckel (1876) produced detailed drawings of corals as seen from the edges of boat decks and near shorelines in their first attempts to classify corals. However, coral colonies have been found to be highly plastic, changing shape and appearance based on environmental factors (Muko et al., 2000; Kaniewska et al., 2014). Later, Wells (1956) produced a volume in the Treatise of Invertebrate Paleontology that used more detailed morphological differences in the septal patterns and columella structure to classify corals. More recently, the use of molecular data in phylogenetics has helped to revolutionize the field of coral taxonomy. Fukami et al. (2008) used genetic material to produce one of the most robust phylogenies of corals to date. Their findings show that the group Scleractinia is monophyletic, but many of the families are not. In fact, later studies show that 14 of 24 families based on previous morphological characters are paraphyletic (Kitahara et al., 2010). Molecular phylogenetics has been immensely helpful at sorting out some of the 3

18 relationships at higher taxonomic ranks, but it has not been able to resolve relationships at finer scales (Huang et al., 2014; Huang et al., 2014). Thus, better morphological characters are needed to resolve relationships within families and genera. Using the molecular data for comparison, other studies have attempted to find additional morphological characters with which to classify corals. Budd & Stolarski (2011) recognized features based on skeletogenesis of corals, such as linear vertical structures that form unique dentition patterns on septa which are taxonomically relevant (Budd & Stolarski, 2009). Differences between Atlantic and Pacific corals, previously believed to be the same family or genus, were detected with the addition of these characters (Budd & Stolarski, 2011). However, given the large number of known coral species and the general dearth of characters, still more are needed to further resolve phylogenetic relationships. All of the morphological work to this point has focused on the skeletal anatomy of corals, or hard tissue. However, there is another source of morphological characters that has not been explored, namely the soft tissue. Soft tissue has been difficult to incorporate into phylogenetic analysis due to difficulties associated with preserving undamaged tissue. A skeleton plucked from the shoreline is much easier to transport home then intact tissue samples. Moreover, due to advances in microscopy, proper microscopes are now readily available. Histology is the study of the tissue layers of an organism 4

19 and traditionally it has focused on biomedical research. Early research on the histology of corals began in the 1980s and continues today, but is more focused on the biomineralizing properties of the soft tissue (Johnston, 1980; Galloway et al., 2007; Tambutte et al., 2011; Allemand et al. 2011). In relevance to this study, researchers using modern light microscopy have shown that features such as cell organelles and cnidocytes are visible in thin section (Raz- Bahat et al., 2006). Researchers of sea anemones, also members of the phylum Cnidaria, have used similar techniques due to the lack of hard tissue within the organism (Rodriquez et al., 2012). Other exclusively soft tissue cnidarians have based their phylogenies on these characters: such as staurozoans (Miranda et al., 2013) and zoanthids (Ryland et al., 2004). Previous works focused on the structure and distribution of cnidocytes within Cnidarian, for example hydrozoans (Garcia-Arredondo et al., 2012), actiniarians (Reft & Daly, 2012), but very few have focused on scleractinians (Terron-Sigler & Lopez-Gonzalez, 2005; Martinez-Baraldes et al., 2014). The evolutionary history of cnidocytes has been covered in other studies (Fautin, 2009) and their taxonomic value was greatly increased by Östman in which terms and definitions of cnidocytes structures were clarified (2000). However, all of these studies are deficient in two critical areas. First, none of them incorporate histological characters other than cnidocytes, leaving out many characters in the tissue itself. Second, all but one of the studies focused on groups other than Scleractinia, which are the 5

20 most important reef builders in coral reefs and need more characters to reconstruct their phylogenies (Wild et al., 2011). The goals of this study are twofold: to identify characters in the soft tissue of scleractinian corals that are taxonomically relevant and use those traits to produce a new phylogeny that can then be compared and added to previous phylogenies based on skeletal and molecular data. Character maps are also created in order to compare evolutionary patterns in different characters. One could argue that using morphological characters to produce a phylogeny of organisms for which there is already molecular data is not productive. But, in many families full resolution of phylogenetic relationships has not been achieved and other types of characters need to be added for this to be accomplished (Budd et al., 2010). It can also be suggested that using other phylogenies as starting points for character identification is only going to reinforce the answer provided by the original phylogeny. However, when starting from a phylum with large amounts of labile taxa and limited morphological data - as long as care is taken not to force an agreement through haphazard character selections - using other phylogenies as a starting point does not pose harm to the results (Swain & Swain, 2014). Therefore, this study hopes to provide additional characters from a new source of morphological data within cleractinian soft tissue for future phylogenetic analysis. 6

21 Materials and Methods Species A total of 13 coral species were analyzed. Six of the scleractinian corals in this study belonged to a now obsolete family of Pacific corals called Pectiniidae. This group was once considered a family under broad morphological characters such as: laminar colony structure and long, thin striated costae, which are now shown to be taxonomically invalid. Molecular research has shown that the group belongs to two distinct clades called clade XVII and clade XIX based on phylogenetic reconstructions (Fukami et al., 2008). Their study used mitochondrial and nuclear DNA to perform a robust analysis of 127 coral species and showed the Pectiniidae was not monophyletic. Morphological studies then assigned clade XVII members to the family Merulinidae and clade XIX was assigned to a new family named Lobophylliidae (Budd et al., 2012). The Budd et al. (2012) study included the corals formally classified as Pectiniidae as well as other corals in the families Lobophylliidae and Merulinidae. Macromorphological traits were combined with micromorphological traits based on electron microscope images as well as microstructural data from thin sections. For a complete breakdown of the techniques and traits used see Budd & Stolarski, 2009; Budd & Stolarski, 2011; and Budd et al., The skeleton-only matrix presented in the present study is a subset of a larger analysis performed by Budd et al. in 2012 and the 7

22 specimens are housed at the University of Iowa Paleontological Repository or on loan from other institutions (Table C1). Table 1 Family designations for species in study. Traditional families are based on phylogenies using macromorphological skeletal features. Families from Budd et al. (2012) are based on additional micromorphological and microstructural skeletal characters and Fukami et al. (2008) families are based on molecular data which are grouped by shaded boxes. Species selected for histological study are indicated by ( ). Traditional Family from Wells, 1956 Family from Budd et al., 2012 Clade from Fukami et al., 2008 Species Montastreinae Diploastreidae XV Diploastrea heliopora * Faviidae Merulinidae XVII Oulophyllia crispa Faviidae Merulinidae XVII Oulophyllia bennettae Mussidae Lobophylliidae XIX Lobophyllia corymbosa Mussidae Lobophylliidae XIX Lobophyllia hemprichii Mussidae Lobophylliidae XIX Symphyllia recta Mussidae Lobophylliidae XIX Symphyllia radians Pectiniidae Lobophylliidae XIX Echinophyllia aspera Pectiniidae Lobophylliidae XIX Echinophyllia echinoporoides Pectiniidae Lobophylliidae XIX Oxypora lacera Pectiniidae Merulinidae XVII Mycedium elephantotus Pectiniidae Merulinidae XVII Pectinia paeonia Pectiniidae Merulinidae XVII Pectinia alcicornis * = outgroup for histological study 8

23 Species selected for histological phylogenetic analysis were chosen because of the need for further resolution within the new families and because they are the most readily available to purchase due to their popularity among salt water tank enthusiasts (Arrigoni et al., 2014, see Table 1 & C1 for species studied). All species selected for histological analysis were also present in both the Fukami et al. (2008) molecular phylogeny and Budd et al. (2012) skeletal morphological phylogeny. Specimens All specimens for histological analysis were purchased from aquaria stores based within the United States. Species identifications were confirmed visually on specimens once received. Specimens were always ordered with overnight shipping and immediately placed in a prepared tank to avoid tissue damage and dehydration. Specimens were maintained for at least hours and inspected for lesions and signs of damage during shipping before being processed. Sizes of the specimens varied depending on availability from retail stores and ranged from a diameter of 25 mm (known as frags) up to larger colonies about 24 cm in diameter. All specimens were maintained in a single tank held at 82 C, ph of 8.1, alkalinity of 10 dkh, nitrate <0.2ppm and phosphate <0.03 ppm. Water changes were performed weekly to remove wastes and tested to maintain a salinity of 35 ppt and constant calcium, magnesium and carbonate levels. A 12 hour light cycle was used as well as constant circulation across colonies. 9

24 Histological Methods Specimens chosen for histological analysis were taken from the tank and immediately placed in Z-Fix solution for 24 hours. Corals were then placed in Surgipath Decal I - formic acid - decalcification solution for 7 to 10 days until decalcification was complete. Tests were regularly run to check for complete decalcification. Specimens that completed decalcification faster than others were stored in 1x phosphate buffered saline (PBS) solution to prevent tissue dehydration. Next, specimens were cut to fit into standard tissue processing cassettes and processed using a Tissue-Tek Tissue Processer and embedded in paraffin (on the Rat No-Fix cycle). Three of the specimens, Diploastrea, Oxypora and Echinophyllia, were small enough that the entire specimen could fit into the cassette. For the other five specimens small sections needed to be selected for processing. In order not to bias the results by selecting different regions of the coral polyp two sections were taken from each specimen, one from the growing outer edge of the polyp and one from the center of the polyp. Paraffin blocks were cut using a standard microtome at a 10 degree angle in five to seven micron thick ribbons and adhered to slides. All slides were made in cross-section of the coral polyp with care taken to get a variety of depths within the polyp from acrosphere to the basal disk. In total, 312 slides of coral tissue were processed (24 per paraffin block). The slides were stained with hematoxylin and eosin (H&E) and coverslipped. All slides were analyzed and photographed with a Zeiss light microscope, at magnifications ranging from 1x to 40x, utilizing a Canon EOS Rebel T2i digital camera on the no flash setting. 10

25 Images used in this study are available at University of Iowa Digital Library (Table C1). Phylogenetic Analysis The species Mycedium elephantotus, Pectinia alcicornis, Oulophyllia crispa, Lobophyllia hemprichii, Symphyllia radians, Oxypora lacera, and Echinophyllia aspera were designated as the ingroup for the histology-only matrix (Table C1). Diploastrea heliopora was designated as the outgroup based on its basal position to all of the ingroup species in past phylogenetic analyses. Two analyses were performed with the parameters defined below, one using skeleton-only matrix and another using histology-only matrix. The histology-only matrix created in Mesquite version 2.75 included 19 binary characters and one unordered multistate character for a total of 41 states (Maddison & Maddison, 2009). All 20 characters were discrete. In instances that could not be coded due to absence of a feature, reductive coding was used designated by a? (Strong & Lipscomb, 1999). Furthermore, all characters were equally weighted. TNT version 1.1 was used to perform tree analysis based on parsimony (Goloboff et al., 2008). TNT settings were set to save a maximum of 10,000 trees and collapsing rules set to none in order to gain as many possible MPTs to investigate. Implicit enumeration was used to find the most parsimonious trees, which is based on branch and bound algorithms. TNT was also used to calculate GC scores, from 10,000 bootstrap resamplings, and Bremer support indices. Finally, WinClada version

26 was used to make and display strict consensus as well as determine tree length, C.I., and R.I. (Nixon, ). A single MPT was chosen to display ACCTRAN, DELTRAN, and unambiguous-character tree optimizations. Character Evolution Once characters were identified and coded in Mesquite, characters could be mapped on to trees to trace the evolutionary history of each character using the Parsimony Ancestral States function. Trees used to map character evolutions were based on MPTs from histological analyses performed in this study. The MPT chosen was the one most similar to previous phylogenies. Calculations of individual character C.I. and R.I. were also determined using Mesquite. Characters All Cnidaria contain stinging cells for capturing prey and defense called cnidocytes (alternatively cnidae) with harpoon-like structures within them called cnidocysts. Cnidocysts are composed of a hardened shaft connected to a flexible tubule at its base. The cnidocysts are enclosed in a capsule which is activated by a triggering mechanism called an operculum. The histomorphological characters used for this study were created using terminology defined in Östman (2000). No continuous characters were used because thin sections do not always retain the entirety of a feature. Therefore, characters such as cnidocyte size used in Terron-Sigler & Lopez-Gonzalez (2005) could not be used. An illustrated guide to identifying and coding all 12

27 characters can be found in Figure A1. The following ten cnidocytes-based characters were coded and analyzed (Table B1). 1. Cnidome density Cnidome of a coral consists of all the stinging cells possessed by a coral. There are two broad types found in Hexacorallia, spirocytes and nematocytes, which will be discussed later. In general, cnidocytes were most commonly found in cnidoglandular bands along the tips of the tentacles, but could also be found in the actinopharynx. 0 - overlapping and tightly packed cnidocytes is coded as high. 1 - sparse or spread out cnidocytes is termed low. 2. Spirocytes Spirocytes are one type of cnidocytes found in Hexacorallia used for lassoing prey. They are characterized as purple-stained (H&E), tightly coiled cylinders, sometimes mildly fanning out at the distal edge. 0 - low numbers of spirocytes (<10 per cnidoglandular band) is coded as rare. 1 - cnidoglandular band with >10 spirocytes is coded as common. 3. Spirocytes to nematocytes ratio 0 - twice as many nematocytes as spirocytes is coded as more nematocytes. 13

28 1 twice as many spirocytes as nematocytes is coded as more spirocytes. 4. Holotrichous isorhizas Two broad types of nematocytes are found in Hexacorallia; holotrichous isorhizas and what will be termed heteroneme nematocytes in this study (discussed below). Both stain pink to light red with a slightly translucent capsule containing a grey to black structure. Holotrichous isorhizas are distinct in that they have a clearly visible coiled tubule, but a poorly defined shaft. Holotrichous refers to the tubule possessing spines along the entirety of its length and isorhizas refers to its consistent diameter. 0 holotrichous isorhizas are absent 1 holotrichous isorhizas are present 5. Length of nematocytes Nematocytes were either oval, often with a length no more than two times its width, and rounded ends. Or, they had a long length relative to width and squared ends. 0 long nematocytes 1 oval nematocytes 6. Heteroneme nematocytes The second broad category of nematocytes are heteroneme nematocytes. They contained dark shafts for their nematocysts. It should be noted that 14

29 heteroneme nematocytes can still contain a coiled tubule attached to the proximal end of the shaft. 0 heteroneme nematocytes are present 1 heteroneme nematocytes are absent 7. Heteroneme nematocytes nematocyst dilation A nematocyst that had a larger diameter at its center and taper towards the distal end was termed anisorhizas. A shaft that does not show any signs of tapering is termed isorhizas. Two additional, states were also investigated, birhopaloid type I and type II, but not observed in this study. 0 nematocyst is isorhizas 1 nematocyst is anisorhizas 8. Heteroneme nematocytes nematocyst spines The nematocysts of a heteroneme nematocyte sometimes possess spines along the length of the shaft, in addition to the large main spine (stylet) at the distal end. Those without additional spines are coded as atrichous and those with spines along the entire length of the shaft are coded as holotrichous. A third state was also searched for, basitirichous for spines part way down the shaft, but was not observed in this group of corals. 0 atrichous 1 holotrichous 15

30 9. Heteroneme nematocyst size A nematocyst that is less than one and a half times the length of its capsule is termed microblastic. Nematocyst that are four times the length of the capsule is termed macroblastic and can be identified by large amounts of coiled tubule within the unfired capsule. A third state, mesoblastic for intermediate shaft lengths has been observed in Östman (2000), but was excluded for simplicity and not observed in this study. 0 microblastic 1 - macroblastic 10. Heteroneme nematocyst notch The proximal end of the nematocyst shaft in an unfired capsule can have a V-shaped notch where the tubule connects. Lacking the notch was coded as b-mastigophore and possessing a notch was coded as p- mastigophore. 0 b-mastigophore 1 p-mastigophore The phylum Cnidaria possesses only two germ layers; an ectoderm and an endoderm. A third gel-like substance separates them called the mesoglea. The subclass Hexacorallia includes organisms with six-fold symmetry such as sea anemones, zoanthids, and scleractinians. A coral colony is composed of individual colonial organisms called polyps. Polyps have a gastrovascular cavity 16

31 with a single opening called an actinopharynx, which is surrounded by tentacles to aid in prey capture. The following eight characters are derived from tissue layer characters (Table B1). Consistent trait expression across multiple slides at various depths is vital when coded. Again, these new characters were created for the purposes of this study although similar characters, adjusted for the specific organisms being studied, had been used in other anthozoans (e.g. Miranda et al., 2013). All colors are based on H&E staining. 11. Granular gland cells Granular gland cells are found in the epidermis throughout some species, but are often concentrated in cnidoglandular bands. Specifically, they are found along the basal portion of the tissue layer and taper to a dull point distally. They can be seen at low magnification levels (2.5x), but high magnification reveals that they are made up of dozens of tightly packed pink to magenta circles. Note: do not confuse purple-stained nuclei or pink-stained zooxanthellae which are more sparsely distributed and contain a darkly stained nuclei. 0 granular gland cells are present 1 granular gland cells are absent 12. Pigment cells 17

32 Pigment cells are often only seen in cnidoglandular bands. They can stain as pink or peach-toned, but also dark purple if highly concentrated. They appear to have a crusty or cracked appearance like that of dried paint beginning to flake off. 0 pigment cells are absent 1 pigment cells are present 13. Epidermis to gastrodermis ratio Epidermis contains elongated, pink cells with centrally located purple nuclei. Gastrodermis is distinct in that it possesses red- to pink-stained zooxanthellae. Zooxanthellae are distinct from other organelles because of their near-perfect circular shape, clear starch body and purple nucleus. Specimens that had relatively thicker epidermal layers are coded as thick epidermis, those with thicker gastrodermis are coded as thick gastrodermis and those with relatively equal layers were coded as even. 0 epidermis is thicker than gastrodermis 1 epidermis and gastrodermis are same thickness 2 gastrodermis is thicker than epidermis 14. Gastrodermis to calicodermis ratio Calicodermis often stains poorly, but can be identified based on elongated hazy purple nuclei, proximity to skeletal material, and opposition to gastrodermis. In addition, some well-prepared slides will contain tiny feathery 18

33 cells called desmocytes that aid polyp attachment to skeleton. Thickness of calicodermis varied from very thin, coded as thin, to roughly half that of neighboring gastrodermis. 0 calicodermis thickness roughly half of gastrodermis thickness 1 calicodermis thinner than half of gastrodermis thickness 15. Relative mesoglea thickness Separating the two dermal layers is gel-like connective tissue called the mesoglea which stains light pink or red. Species with mesoglea layers as thick as a single surrounding tissue layer are coded as even and those with larger amounts of mesoglea are coded as thick mesoglea. 0 even thickness 1 mesoglea thicker than surrounding tissue 16. Complete mesentery The internal cavity of a polyp, the gastrovascular cavity, opens to the environment through an apical opening called the actinopharynx and has dark purple, filamentous cells. Mesenteries surround the cavity and can attach to the actinopharynx, complete mesentery, or not, incomplete mesentery. Corals often have both, but how pronounced these connections are appear to vary. Thick connections that form a wagon wheel spoke pattern are coded as prominent and those lacking this structure or are severely reduced are coded as minor. 19

34 0 prominent complete mesentery 1 minor complete mesentery 17. Mucocyte size Mucocytes do not stain with H&E, but clear vacancies in the epidermis signal their presences. Mucocytes are often oval with tapered ends opening to the environment. Large mucocytes commonly reach a width greater than two times the width of an epidermal cell and will bud against one another if densely concentrated. This is coded as large and dense while narrow and sparse mucocytes concentrations are coded as small and sparse. 0 large and dense mucocyte concentrations 1 small and sparse mucocyte concentrations 18. Perforation Gaps in the tissue left from the removal of skeletal material, recognizable by surrounding calicodermis tissue, indicate perforation. Species that had skeletal material penetrate the upper most layers of sections were coded as high perforation and those without any signs of perforation were coded as low perforation. 0 high perforation 1 low perforation 20

35 The final two characters are based on macromorphological soft tissue features (Table B1). 19. Fleshiness: Large amounts of spongy soft tissue on a species is termed high fleshiness and low if not. This can be coded when still attached to skeletal material or based on amount of flesh remaining after decalcification. In thin section fleshy corals will require several sweeping views at low magnification to observe the entire slide. 0 low fleshiness 1 high fleshiness 20. Polyp size Polyps that can be easily viewed at medium magnification (10x) are coded as small. Those that must be viewed at low magnification are coded as large. 0 small polyp 1 large polyp 21

36 Results The skeleton-only phylogeny was roughly similar to past studies (Budd et al. 2012). The skeleton-only matrix used for this study was a truncated version of the Budd et al. (2012) matrix, which had 55 more taxa than this study, and therefore was not exactly duplicated. Nonetheless, the appearance of two broad clades were recovered just as in the Fukami et al. molecular phylogeny (2008; Fig. 1). A strict consensus summary yielded six MPTs of length 91 with a consistency index of 0.69 and a retention index of With the exception of a slightly higher consistency index, the analysis agrees with the findings of past studies and are used as a comparison for analysis based on histology-only matrix (Fukami et al., 2008; Budd et al., 2012). The eight species selected for histological analysis and 20 histomorphological characters were used for phylogenetic analysis based on histological characters only. Character states between center and edge sections were mostly consistent with each other. This analysis yielded seven MPTs of length 35 with a consistency index of 0.60 and a retention index of 0.58 (Fig. 2A). The seven MPTs showed no consisted clades and when summarized as a strict consensus collapsed into a single polytomy included the entire ingroup. One MPT was selected, due to its similarity to phylogenies based on molecular and skeletal character matrices, to map character optimizations (Fig. 2B-D). On this MPT characters 2 and 14, cnidome density and calicodermis thickness, 22

37 Figure 1 Strict consensus trees based on Fukami et al. (2008) molecular data (left) and Budd et al. (2012) skeletal data (right; 13 taxa and 46 characters). Numbers above the node represent GC scores and numbers below represent Bremer support values. MPTs, most parsimonious trees; C.I., consistency index; R.I., retention index. 23

38 Figure 2 Strict consensus tree and character optimizations for histologyonly matrix (8 taxa, 20 characters). Numbers above circles indicate character number and numbers below circles indicate character states. Black circles indicate synapomorphies and white circles indicate homoplasy. A Strict consensus tree summarizing seven MPTs of length 35. B Unambiguous character optimization on selected MPT. C ACCTRAN character optimization on selected MPT. D DELTRAN character optimization on selected MPT. were synapomorphies uniting Oulophyllia, Mycedium, Pectinia, and Echinophyllia. Furthermore, character 18 united Echinophyllia and Pectinia; character 17 united Mycedium and Oulophyllia; and characters six and 19 united Lobophyllia and Symphyllia (Fig. 2). 24

39 Character histories were mapped onto the new phylogeny using Mesquite. A total of six character maps are displayed on an MPT chosen for its similarity to molecular and skeletal based phylogenies. All of the mappings are unambiguous, meaning there is only one way optimize the character history, and show synapomorphies for various clades within the tree. Character consistency index values ranged from 0.33 to 1.0 with an average value of Character retention index values ranged from 0.0 to 1.0 with an average value of Nine characters showed no homoplasy: characters 2, 4, 6 8, 14, However, characters seven and eight only occurred in Mycedium making it an autapomorphy and character six was a symplesiomorphy. While these characters may not be informative in this study, addition of more taxa could make them relevant. Several synapomorphies for various parts of the MPT can be seen in the character maps and four of the characters originate at the deepest node on the tree (Fig. 3). Character one, cnidome density, grouped together Oxypora, Lobophyllia, and Symphyllia on this particular MPT. This trait is also found in Echinophyllia, which are all members of the family Lobophylliidae (Fig. 3A). The presences of spirocytes and a thin calicodermis grouped together Echinophyllia, Pectinia, Mycedium, and Oulophyllia (Fig. 3B & D). Finally, the clade grouping Echinophyllia and Pectinia had perforation as a synapomorphy while the Lobophyllia and Symphyllia clade was united by fleshiness as a synapomorphy (Fig. 3E & F). 25

40 Figure 3 Histological characters mapped onto MPT created from histology-only matrix. C.I. and R.I. statistics represent scores for individual traits. A 2:0, B 1:0, C 2:0, D 1:0, E 1:0, F 1:0 (character origination: character loss). 26

41 Table 2 Characters and character states for histological analysis. C.I. and R.I. scores represent values for each individual character. Char. # Characters States C.I. R.I. 1 Cnidome density 0 high low 2 Spirocytes 0 absent or rare common 3 Spiro. to nemato. 0 more nemato ratio 1 more spiro 4 Holotrichous 0 absent isorhizas 1 present 5 Length of hetero. 0 long nemato. 1 oval 6 Hetero. nemato. 0 present absent 7 Hetero. nemato. 0 isorhizas shaft 1 anisorhizas 8 Hetero. nemato. 0 atrichous spines 1 holotrichous 9 Hetero. nemato. 0 microblastic size 1 macroblastic 10 Hetero. nemato. 0 b-mastigophore notch 1 p-mastigophore 11 Granular gland 0 present cells 1 - absent 12 Pigment cells 0 absent present 13 Epi. to gastro. ratio 0 epi. thicker even 2 gastro. thicker 14 Gastro. to calico. 0 calico. thick ratio 1 calico. thin 15 Mesoglea thickness 0 even thick 16 Complete 0 prominent mesentery 1 minor 17 Mucocyte size 0 large/dense small/sparse 18 Perforation 0 high low 19 Fleshiness 0 low high 20 Polyp size 0 small 1 - large

42 Systematic Account The histological features of each species are described below with additional skeletal morphology information (Veron, 1977; Veron, 1980). Geographic information was taken from IUCN Red List of Threatened Species. All sections were in cross-section and colorations are specific to H&E staining. Figure 4 shows some of the features mentioned in this account, but were not used for phylogenetic analysis because of inconsistent variation within a species. However, figures 5 12 show basic histological details of each specimen. Diploastrea heliopora (Lamarck, 1816) This species is very common throughout the Indo-Pacific from the Red Sea to Samoa. It is very abundant and inhabits a diverse range of habitats from the outer barrier reef to semi-enclosed areas indicating it can tolerate both high and low wave energy environments with minimal growth variation (IUCN, 2014). Colonies typically are two to seven-meter-diameter domes. The skeleton is easily recognizable as having plocoid corallites resulting from extratentacular budding with uniform spacing. Corallites are circular with a diameter of as much as two centimeters. The calice usually is surrounded by 24 septa that thicken near the top with large dentitions and small paliform lobes in some specimens. This species has a well-developed, spongy columella and walls tend to be septothecate, but have also been found to be partially synapticulothecate (Veron, 1977). 28

43 Figure 4 - Examples of histological slides, see Figure A1 for more on character coding. A - Wide image of actinopharynx, note right side has missed structure due to oblique cut. B - Cnidoglandular band, note cross-sectional cuts of nematocytes in center and longitudinal cuts along edges. C - Projections from epidermis into mesoglea. D - Epidermis, note crust along apical boundary. E - Purple-stained mucus exiting epidermis, also note centrally clustered nuclei. F - Gastrodermis with tightly clustered zooxanthellae. G,H - Actinopharynx showing cuspate edges. Black scale bars = 100 microns, red scale bar = 1 mm. A, B, D Diploastrea (SUI ); C Oulophyllia (SUI ); E Symphyllia (SUI ); F Lobophyllia (SUI ); G, H Echinophyllia (SUI ). 29

44 Macroscopically, the species has little tissue compared to its hard, dense skeleton. Coloration is pale green with pink polyps. This specimen contains very low profile tentacles when sectioned. Extratentacular budding is readily apparent in the connections between adjacent polyps (Fig. 5A). In general, polyps are small and numerous. Sectioning of the specimen shows that it had rather shallow relief only requiring a few sections to penetrate into deeper tissue. This species has a thick epidermis with very elongated columnar cells. Large mucocytes are readily visible as gaps in the tissue layer which are often oval-shaped and taper on one or both ends (Fig. 5B). A very evident crust is visible in most slides at the apical domain of cells. This crust could be cilia, but identification is tentative without higher magnification (Fig. 4D). Furthermore, small black dots are seen at the basal domain of the epithelial cells throughout the specimen at all depths. Gastrodermis is thinner relative to epidermal tissue with a moderate level of zooxanthellate concentration. Mucocyte density is much lower in the gastrodermis compared to other layers and minimal amounts of mucus are observed. Calicodermis tissue is observed in very shallow slides of the polyp and is relatively thick compared to other species. This layer has very apparent nuclei. Mesoglea is featureless and maintained a smooth, evenly colored texture throughout (Fig. 5B). This species has a very prominent actinopharynx, highlighted with smoothly curving edges and long filamentous cells. Extremely 30

45 long and thick complete mesenteries dominate the internal structure of the polyp (Fig. 5A). However, the cnidome of the actinopharynx is poor. The cnidome of the cnidoglandular bands is extremely dense, but not very diverse (Fig. 5C). Spirocytes are very rare as are holotrichous isorhizas nematocytes. However, heteroneme nematocytes are plentiful and tightly packed throughout the bands. It should be noted that nematocytes for this species stained much more opaque than any other, having more of a flat, peach coloration. Regardless, these nematocytes are very long and narrow. Orientation Figure 5 - Histological slides of Diploastrea (SUI ). A - Wide image of a polyp. B - Mesenteries with mucocytes. C Cnidoglandular bands with nematocytes. Black scale bars = 100 microns, red scale bar = 1 mm. EP - epidermis, GA - gastrodermis, ME - mesoglea, NM - nematocytes, AC - actinopharynx, ZO - zooxanthellae, MU - mucocytes. of the nematocytes shifts from inside the band to its edges, with nematocytes being sectioned in cross-section internally and flattening to longitudinal sections along the edges (Fig. 4A & B). Occasional granular gland cells are seen in the bands as well. 31

46 Echinophyllia aspera (Ellis & Solander, 1786) The distribution of this species is poorly defined, but observations have been made from the Red Sea in the west to the Marshall Islands and Tahiti in the east (IUCN, 2014). This species prefers sheltered locations in between crevices or in the lower reef slope. Colony morphology is platy with overlapping plates that thin towards the edges of the colony. Corallites have a range of configurations from septotheca to visible corallite walls that protrude from the surface as much as five millimeters. Corallites are irregularly spaced, circular and vary in size from one to five millimeters. There can be one or two septal orders with the septa also protruding from the surface of the calice. Septa can have one to three dentitions along the upper edge and a paliform crown. The costae are very thick with clear dentition of long spines along its ridge. The columella are well-developed with either a trabecular or a compact structure. Finally, the walls tend to be septothecal with both endotheca and exotheca forming distinct blisters (Veron, 1980). The tissue is a pale green color with slightly protruding polyps. Tentacles are short and hard to distinguish. Overall, a very flat profile is noted across the colony with small polyps. The epidermis has poorly defined cells with a large spread of nuclei depths. Apical domain has an obvious crust appearance and basal domains contain a characteristic pink band of parallel protrusions. It is not clear if this was a characteristic of the species or a result of the preservation and staining 32

47 process. Large mucocytes are in the epidermis, but are spread out across the tissue layer. The epidermis also housed clearly defined granular gland cells observable at even the lowest magnification (Fig. 6A). This gave the tissue a bespeckled pattern of pink to red dots along its entire periphery. Gastrodermis is very thick, but also poorly defined at all depths (Fig. 6A). However, large mucocytes and some stained mucus are seen within the gastrovascular cavity. Zooxanthellae are small and darkly stained while being tightly clustered in certain portions of the basal region of the gastrodermis (Fig. 6E). This species appears to have a large amount of gastrovascular cavity. The calicodermis is difficult to observe as it is very faint and thin. Some regions did show that nuclei are tightly packed in regions that did preserve well (Fig. 6D). Mesoglea do not show any distinctive features and are relatively even thickness compared to surrounding layers. The actinopharynx has clearly defined complete mesenteries, however, these mesenteries are very thin in comparison to other species. These mesenteries are also very long as a result of the widely spread polyps. The actinopharynx has extremely cuspate structure with radiating, purple-stained cells and a poor cnidome (Fig. 4G & H). Cnidoglandular bands are largely devoid of cnidae (Fig. 6B). Some nematocytes are present, but are few and infrequent as well as small (Fig. 6C). This mostly consists of holotrichous isorhizas and some heteroneme 33

48 nematocysts with few spines, notches and short tubules. Spirocytes are by far the most common component of the cnidome. Figure 6 - Histological slides of Echinophillia (SUI ). A - Wide image of a polyp with granular gland cells. B - Cnidoglandular bands and connecting mesentery. C - Cnidoglandular bands with nematocytes. D Calicodermis with nuclei. E - Close-up of mesentery. Black scale bars = 100 microns, red scale bar = 1 mm. EP - wpidermis, GA - gastrodermis, ME - mesoglea, NM - nematocytes, ZO - zooxanthellae, GG - Granular gland cells, AS - acrosphere. 34

49 Lobophyllia hemprichii (Ehrenberg, 1834) Center Section The majority of the Indo-Pacific is inhabited by this species from the Red Sea to Tonga. Specifically, this species tends to live on upper slopes of reefs (IUCN, 2014). Polyps are phaceloid or hemispherical, and form large stands. This species can be polymorphic and also display a more flabellate colony shape. Corallites can grow very large, up to four centimeters, and are irregularly shaped. Clusters of three corallites are common for most branches, but can also be limited to one or two if intratentacular budding has yet to occur on a branch. Valleys have irregular spacing depending on the competition for space, but rarely are less than one centimeter. The septa are loosely packed, very thick, and create large dentitions of three or four spines along the upper margins. Six primary septa connect to the columella which is very spongy and well developed (Veron, 1980). There is a large amount of flesh along the edges of the polyp. For the specimens used in this study, the septa underneath could occasionally be seen through slightly translucent red flesh (Fig. 7A). However, the flesh extends far out from the edge of the polyp. Some slight textural variation is seen across the colony flesh in the form of tiny bumps and dimples. Tentacles are poorly defined and there are large changes in relief from the center of the polyp to the edges due to the fleshy edges. 35

50 Epidermis is slightly more prominent than any other tissue layer (Fig. 7B). Epithelial cells are tightly packed with a large spread of nuclei depths. Many narrow mucocytes are detected, but very little mucus. A thick, but faint crust could be seen along the apical domain. No basal domain features are observed. The gastrodermis is well developed and has sparse zooxanthellae density. However, the zooxanthellae that are observed appeared to be larger than other species (Figs. 4F & 7B). Very few mucocytes are seen in this layer. The internal portions of this species are mainly occupied by a large gastrovascular cavity surrounded by large, densely packed zooxanthellae. Calicodermis is easy to distinguish because of its large thickness. This tissue layer is also densely packed with many nuclei stacked on top of one another, but nuclei in this layer tend to have less distinct edges compared to epithelial nuclei. The mesoglea is exceptionally thin and occasionally shows a gradient of coloration from uneven staining. Internal sections may be more difficult to evenly stain with such thick overlying tissue layers. No entire actinopharynx is observed in this specimen but partial structures are present. There did not appear to be a prominent mesentery connection or cnidae in this structure, however, sparse granular gland cells are observed. The cnidome of this species has a poor density and diversity. Few spirocytes are seen and only sparse nematocytes are present (Fig. 7C). 36

51 Holotrichous isorhizas are stained light pink and long compared to other species. Heteroneme nematocytes are small, with short tubules and had no notch missing at their base. Edge Section Edge tissue layers display the same features as more centrally located sections with the exception of slightly thinner calicodermis. The major difference is the decreased number of cnidoglandular bands and cnidocytes in general. Figure 7 - Histological slides of Lobophyllia (SUI ). A -Wide image of a polyp. B - Mesentery with epidermis and gastrodermis. C Cnidoglandular bands with nematocytes. Black scale bars = 100 microns, red scale bar = 1 mm. EP - epidermis, GA - gastrodermis, ME - mesoglea, NM - nematocytes, GV - gastrovascular cavity, SW - seawater, SK - skeleton. 37

52 Mycedium elephantotus (Pallas, 1766) Center Section This species is common to large portions of the Indo- Pacific and is found in most environments that have low wave action (IUCN, 2014). The colony morphology is platy with overlapping plates usually oriented horizontally, but can be turned vertically if on an overhang. Colonies can reach very large sizes of up to three meters. Corallites are slightly protruding from the surface of the colony and can form clusters of three or four or be irregularly spaced. Figure 8 - Histological slides of Mycedium (SUI ). A Wide image of a polyp with actinopharynx. B - Close-up of mesentery connecting to actinopharynx. C - Cnidoglandular bands with nematocytes. D - Close-up of epidermis. E - Close-up of incomplete mesenteries tipped wth cnidoglandular bands. Black scale bars = 100 microns, red scale bar = 1 mm. EP - epidermis, GA -gastrodermis, ME - mesoglea, NM - nematocytes, MU - mucocytes, AC - actinopharynx, GV - gastrovascular cavity SK - skeleton. 38

53 Corallite diameter flares out at the top several millimeters compared to their base. Three orders of septa can be seen, each order decreasing in thickness, with the first being slightly exsert from the corallite surface. Costae tend to be very fine with long connections between widely spaced corallites and lobed dentitions with occasional ornamentation. Columella are visible, mostly spongy, but variable across the colony. The wall structure is primarily septothecal (Veron, 1980). The colony has a very thin layer of tissue stretched over the skeleton with a dull green coloration. No tentacles are obviously apparent and, with the exception of the polyps, there was very little change in relief across the colony. The epidermis is very thin in this species across the whole colony and throughout its depth. The epithelial cells is columnar and had a fairly uniform concentration of nuclei in the center of the tissue layer. Mucocytes are observed, but are low in density and small (Fig. 8D). The apical domain of this layer has some feathery appearance and is thick in most slides. There are no basal domain features. Conversely, this layer contains the lightly bespeckled marks of granular gland cells. Gastrodermis is very thick, but devoid of mucocytes. Large zooxanthellae cluster tightly along the basal portion of the tissue layer. This tissue surrounds a gastrovascular cavity that occupies a large majority of the internal space of the polyp in the deeper sections. However, some of this space also contains 39

54 calicodermis which are also prominent. In upper slides of the polyp mesoglea is more prominent but featureless. Actinopharynx is extremely large and prominent (Fig. 8A & B). It has smoothly sloping sides and very little coloration, but connects to the outer wall of the polyp by long and thick complete mesenteries. Upper slides show more incomplete mesenteries (Fig. 8E). No cnidae are observed in the actinopharynx. The cnidome of the bands of this species are very diverse and dense. Generally, bands for this species are large hemispherical structures packed with cnidocytes, little internal features and little mesoglea (Fig. 8C). Short spirocytes are seen throughout the bands and extremely long, notched heteroneme nematocytes are also observed. The longest of these nematocytes are those closest to the edge and progressively shorter ones are present more internally. Holotrichous isorhizas are also present, but in lower numbers compared to other types of cnidocytes. Edge Sections Polyps on the edge are smaller compared to internal ones, but with thicker skeletal walls. However, the histological features of the edge of the colony are roughly similar. The actinopharynx is much more cuspate and darkly stained than the inner polyps as well as containing a few cnidocytes. Cnidoglandular bands are much less common in this section although the actinopharynx contains mostly holotrichous isorhizas nematocytes. 40

55 Oulophyllia crispa (Lamarck, 1816) Center Section This species has the lowest abundance of those included in this study and is primarily found from Madagascar to the Great Barrier Reef. It has been found in most habitats, but is most common in shallow lagoons (IUCN, 2014). This species often forms dome-shaped colonies with meandroid integration. Valleys of this species are usually short with a width of millimeters and a depth of 4 14 millimeters. The septa are very thin and densely packed with two or three orders. In addition, the first order is slightly exsert, but all are continuous between valleys. The upper margin of the septa have many small dentitions that gradually decrease in size as they continue deeper into the calice. Columella has a range of forms from spongy and easily visible to laminar and difficult to distinguish. Some specimens have been observed with small paliform lobes at the base of the septa. Finally, this species has a very well-developed endotheca (Veron, 1977). The tissue of the colony is dull green and yellow in the center of the valley with ridges appearing blue with cream-colored spots. The ridges create a large amount of relief from the surface of the colony, however, this species is not particularly fleshy. In addition, the species possess many medium-length tentacles throughout the valleys. The tentacles have a much greater diameter compared to other species in this study (Fig. 9A & B). Mesoglea and 41

56 gastrodermal tissue are readily visible. Tentacles contain only a small opening of gastrovascular cavity or are severely reduced. The epidermis is thick in most areas of the tissue with elongated cells and centrally grouped nuclei (Fig. 9D). Shallower slides contain thinner epidermis layers compared to deeper slides, but the ratio of thickness to other tissue layers is maintained. No clear apical or basal domain features are observed. Mucocytes are small and thin within the epidermis. Small patches of purple and/or pink granular gland cells are present. Pigment cells are also seen as more purple stained compared to other species. Gastrodermis has very densely packed cells, but was comparatively smaller than other tissue layers. Zooxanthellae have a medium density and are relatively small in size. Mucocytes are mostly absent from gastrodermis. Calicodermis is very thin and hardly visible except for occasional desmocytes. These cells stain a distinct bright pink color compared to the purple of the calicodermis. Desmocytes resemble a loosely bound deck of cards oriented perpendicular to the tissue surface, streaking intermittently across large regions of mesoglea. The mesoglea itself is as thick as surrounding layers and featureless. Occasional long, pink projections from the epidermis intrude into the mesoglea, but this is most likely a result of non-planar epidermis punctuating overlying or underlying layers in flat projections (Fig. 4C). The actinopharynx can be hard to locate in a meandroid colony as often only part of its structure is seen in a single slide. Actinopharynx is thin and 42

57 has a cuspate edge. Complete mesenteries are rarely found and are often very thin when observed. The cnidome is very poor. Contrary to the cnidome of the actinopharynx, the cnidoglandular bands of this species are the most diverse and dense of the species studied (Fig. 9B). Large amounts of very long spirocytes are seen on all bands. Furthermore, some slight dilation at the distal ends of spirocytes is seen. Extremely long and welldefined holotrichous isorhizas are seen, often Figure 9 - Histological slides of Oulophyllia (SUI ). A Wide image of a polyp with cnidoglandular bands. B - Cnidoglandular bands and connecting mesenteries. C - Mesentery with epidermis and gastrodermis. D - Close-up of mesentery. Black scale bars = 100 microns, red scale bar = 1 mm. EP - epidermis, GA - gastrodermis, ME - mesoglea, NM - nematocytes, ZO - zooxanthellae, NU - nuclei of epidermis. with dilated distal ends as well. Other types of nematocytes are comparatively small, but well-developed and numerous. Some bands have larger 43

58 concentrations of one type of nematocytes than others, but this cannot be confirmed without more sections. Edge Section This colony is one of the largest and did show slight variations between the edge and center of the colony histologically but not in the skeleton. Epidermis appears to be slightly thicker in the center compared to the edge. Edge section also have a thin crust along the apical domain of the epidermis. However, the trademark high cnidome diversity is still present in the edge sections. Oxypora lacera (Verrill, 1864) This species is widely distributed from the Red Sea to the Marshall Islands and is especially common in the Indian Ocean. It is most often found in deeper waters under protective ledges, but it is not uncommon in shallow settings as well (IUCN, 2014). Colonies are often laminar with irregular surfaces which overlap and create void spaces within the colony. Corallites are plocoid with a wide variety of sizes depending on the age of the organism, however, they are normally elliptical in shape. Costae have two distinct forms that alternate with one another. One will fuse with the columella and the next will be aborted. All costae have an ornamentation of clusters of spines along their dentition. Septa have irregularly spaced dentition along their upper margins with a serrated pattern and are usually only present in a single order connecting to a large, spongy or sometimes absent columella. In addition, the 44

59 base of the septa sometimes forms large thickened connections. Corallite walls tend to be reduced into septotheca (Veron, 1980). The colony used for this study is a vibrant green color with pink polyps. The polyps appear to have very long tentacles with large acrospheres. The irregular surface features made it difficult to determine the amount of relief on the species, but appear low. Polyps are small and not very fleshy. The epidermis consists of tightly packed and short columnar epithelial cells. However, moderate sized and frequent mucocytes are observed. These mucocytes are narrower than other species. Nuclei are somewhat centrally located, perhaps grouped slightly more distally. No obvious apical or basal domain features are observed. Gastrodermis thickness compared to epidermis in the deeper slides of the polyp is roughly equivalent (Fig. 10B). Shallower slides appear to have a higher epidermis to gastrodermis ratio, however, this was mostly limited to the tentacles of the species. The gastrovascular cavity is readily apparent as long parallel voids throughout the species as well as occasionally perforating the mesoglea in upper slides (Fig. 10A & C). Few mucocytes are seen in the gastrodermis, but there is a moderate amount of mucus present. A very high density of zooxanthellae are observed taking up much of the gastrodermis. Calicodermis is easily visible in even the uppermost slides of the polyp and had tightly packed nuclei. The mesoglea is equal to dermal tissue thickness and 45

60 display some fine protrusions originating from isolated locations of epidermis. However, it is possible that these are preservation artifacts. The actinopharynx is very heavily stained purple with a welldefined cuspate pattern (Fig. 10A). Complete mesentery are present, but are not particularly long or thick. No cnidae are observed in this region. The cnidome was characterized as very poor for this species. Few spirocytes and no heteroneme nematocytes are observed. Very large and dark pink-stained holotrichous isorhizas are present with an oval or circular shape. These Figure 10 - Histological slides of Oxypora (SUI ). A - Wide image of a polyp. B - Closeup of mesentery. C - Mesentery with epidermis, gastrodermis and gastrovascular cavity. Black scale bars = 100 microns, red scale bar = 1 mm. EP - epidermis, GA - gastrodermis, ME - mesoglea, AC - actinopharynx, GV - gastrovascular cavity, SK - skeleton. nematocytes often have very short, coiled tubules visible in cross-sections of internal portions of the bands. In addition, pigment cells are seen in the bands of this species. These tend to stain brown or peach and are found in large aggregates that are sometimes visible without magnification. The 46

61 pigment tends to obscure all other detail from the surrounding cells with the exception of occasional nuclei seen in their inner portions. Pectinia alcicornis (Saville-Kent, 1871) Center Section This species is also uncommon, but can be found in Indonesia and the Great Barrier Reef. It tends to occupy shallow habitats, often in turbid waters (IUCN, 2014). The skeleton is perhaps the most variable of those studied with a branching tapered colony shape creating a crown-of-thorns appearance. Corallites vary greatly in size, averaging one to two centimeters, and can be either circular or oval-shaped. The septa and costae are usually connected, but can vary in length depending on spacing of corallites. Primary septa can be very thick with a lobed dentition and connect with a large paliform lobe. They connect to costae, which may or may not increase in thickness as they radiate away from the corallite, and contain a variety of spiny ornamentations. The columella is usually weakly developed, but this species does have a welldefined exotheca and endotheca (Veron, 1980). The colony has a dull yellow coloration and very thin tissue stretched over its spiny skeleton. The spiny appearance creates a large amount of topography across the surface of the tissue. No clearly defined tentacles are observed. The epidermis is the same thickness as other layers of tissue and composed of short stubby cells and centrally grouped nuclei (Fig. 11B). Large and prominent mucocytes are seen mostly clustered near the base of the tissue 47

62 and tapering upward towards the surface body wall. A slightly darkened crust is visible along the apical domain, but no basal domain features are detected. Clearly defined granular gland cells bespeckle the epidermis, often more heavily in the upper slides. Both the gastrodermis and calicodermis are thin compared to other species, with the gastrodermis containing a low concentration of zooxanthellae. Gastrodermis is seen to have a large amount of mucocytes and mucus, showing as a faint purple stain across large regions of the slide. Most of the internal structure is taken up by the mesoglea. No entire actinopharynx are observed, possibly due to the uneven nature of the colony surface (Fig. 11A). Complete mesenteries are seen connecting to small regions of actinopharynx with a cuspate shape and poor cnidae density. Rare small heteroneme nematocytes are present though. Cnidoglandular bands contain mostly long holotrichous isorhizas and spirocytes (Fig. 11C). Holotrichous isorhizas have very large coils at the base of their capsule, often originating from the center of the capsule. No heteroneme nematocytes and few spirocytes are seen in the bands. In one slide, a yellowbrown cell is observed that could be a sign of melanin contained within a cell, however, this is the only observation of this feature (Fig. 11C). 48

63 Edge Section Outer edge of colony are similar in all regards except for heteroneme nematocytes being present in the cnidoglandular bands (Fig. 11D & E). Very large nematocysts have spines pointing towards the base of the nematocyst (Fig. 11E). Figure 11 - Histological slides of Pectinia (SUI ). A - Wide image of a polyp. B - Mesentery with epidermis and gastrodermis. C - Close-up of cnidoglandular bands with nematocytes. D - Close-up of nematocytes. E - Close-up of nematocytes and spirocytes. Black scale bars = 20 microns, red scale bar = 1 mm. EP - epidermis, GA - gastrodermis, ME - mesoglea, AC - actinopharynx, NM - nematocytes, SN - spines, SP - spirocytes, GG - granular gland cells, GV - gastrovascular cavity, SW - seawater, MC - melanin-containing cell(?). 49

64 Symphyllia radians Edwards & Haime, 1849 Center Section This species is widespread from the Red Sea to the Great Barrier Reef and is most often found in upper reef slopes (IUCN, 2014). Colonies form flat or hemispherical mounds and meandroid corallites. Valleys are very sinusoidal with an irregular length and a thin groove. Width of the valleys can range from millimeters and have a large depth of 12 millimeters. Corallites are about two centimeters in diameter. Up to four orders of septa can be seen with the last two often being very thin. Thicker septa have three to six large dentitions along the upper margin. Columella can be spongy or trabecular. The wall structure is usually very thin (Veron, 1980). Colony coloration is bright red throughout with very large fleshy lobes extending off the skeleton. Relief is uniform and low across skeleton surface and tentacles are minimal. The epidermis is very thin and compact. Large mucocytes occupy most of the space within the epidermal tissue and large amounts of mucus are found (Fig. 12B). The apical domain did not have any prominent features, but the basal domain contains a slightly darker purple coloration creating a bold contrast between the epidermis and mesoglea. The gastrodermis is also very thin, but the same thickness as epidermis. Large mucocytes dominate this layer and zooxanthellae density is extremely high and are often overlapping one another. Faint purple staining of mucus is 50

65 common throughout this species, producing a ghostly, filamentous appearance in many areas (Fig. 4E). Calicodermis is very prominent. The most dominant feature of this species is the large mesoglea. Mesoglea ranges from five to ten times as thick as many tissue layers and is occasionally punctured by gastrovascular cavity lying beneath it. Often only a small circle or oval section of gastrodermis appear in these breaks in the mesoglea that were filled by clusters of zooxanthellae. The actinopharynx is extremely large with thick complete mesenteries radiating off its structure (Fig. 12A). Prominent cuspate lobes emanate from the connection points of these complete mesenteries. The cnidome of the actinopharynx contains sparse and small holotrichous isorhizas. The cnidoglandular bands are very limited in density and diversity of cnidae (Fig. 12C). Often only some holotrichous isorhizas are present which are small and oval shaped. However, large amounts of purple and cracked pigment cells are present. Edge Section No differences between center and edge are observed for this species. 51

66 Figure 12 - Histological slides of Symphyllia (SUI ). A - Wide image of a polyp with actinopharynx. B - Closeup of mesentery with epidermis and gastrodermis. C - Close-up of cnidoglandular bands with nematocytes. Black scale bars = 100 microns, red scale bar = 1 mm. EP - epidermis, GA - gastrodermis, ME - mesoglea, AC - actinopharynx, ZO - zooxanthellae, MU - mucocytes, NM - nematocytes. 52

67 Discussion Molecular Phylogeny vs. Skeletal Phylogeny The revised skeleton-only matrix, analyzed herein (13 taxa, 46 characters) yielded a new phylogeny due to the exclusion of taxa. The original Budd et al. (2012) matrix included 57 taxa compared to the 13 used for this study. A reduction of taxa of this magnitude could have caused some characters to become uninformative as they previously were informative for taxa now excluded and caused the change in phylogeny. Nonetheless, the new phylogeny still maintained many characteristics of the Budd et al. (2012) results, which were very similar to the Fukami et al. (2008) results (Fig. 1). Clade XVII, recently named Merulinidae, was recovered as a monophyletic group in the present study, using skeletal characters only, with very strong support (GC score of 72 and Bremer support index of 4; Fig. 1). However, the relationships of Pectinia and Mycedium are no longer resolved in the present skeleton-only phylogeny. Another area of high support and congruence between this study and the Budd et al. (2012) study was a clade including the genera Symphyllia and Lobophyllia (GC score of 93 and Bremer support index of 6, Fig. 1). However, clade XIX, as defined in Fukami et al. (2008) was paraphyletic and the genera Oxypora and Echinophyllia were not sister to one another in the present study. Regardless, these results are encouraging for two reasons. First, two clades were recovered, just as in molecular data. Second, relationships within clade XVII and the Lobophyllia/Symphyllia clade were 53

68 similar in this study to those found in both Budd et al. (2012) and Fukami et al. (2008). The skeleton-only phylogeny can be used as a comparison for the histology-only phylogeny to test for broad-scale corroboration using a new data set. Histological Phylogeny No clades are recovered in a strict consensus tree when using the histology-only matrix (Fig. 2A). All ingroup species are grouped in a large polytomy. This is due to the seven MPTs all containing different relationships amongst the ingroup species. Fortunately, one of the MPTs does conform broadly to previous studies based on molecular and skeletal characters (Fig. 2B D). While more characters would need to be collected to maintain a consist topology that would appear in a strict consensus summary tree, the presence of at least one MPT that broadly conforms to previous studies is encouraging for the use of histomorphological characters in future studies. In fact, Symphyllia and Lobophyllia are grouped as sister in all but three of these MPTs. Oxypora is then grouped as sister to these two species or as sister to Symphyllia in the three MPTs in which Symphyllia and Lobophyllia are not sister. This suggests that some phylogenetic signal is still being sampled within the relationships of these three organisms with histomorphological characters. Character Mapping A large number of the traits in this study originate at the deepest node of the tree (Fig. 3A D). For example, two of the characters uniting the 54

69 Symphyllia/Lobophyllia/Oxypora clade (SLO) are characters 1 and 2; cnidome density and spirocytes. The SLO clade has a very low cnidome density and lack of spirocytes. For spirocytes the plesiomorphic state is retained and a single change to low cnidome density accounts for all the members of the SLO clade. The functional use of these traits is not always well understood, but past studies have shown that spirocytes, which contain an adhesive property rather than a penetrating spine, are used to entangle fast swimming prey (Kreyesky et al., 2010). Perhaps a comparison of prey types in habitats containing these organisms will show selective pressures favoring penetrating nematocytes over spirocytes based on prey availability. Furthermore, it is also interesting to note that Echinophyllia, which is closely related to the members of the SLO clade in the molecular phylogenies, also possesses a low cnidome density (Fig. 3A). In general, investigating the histologic characteristics of the family defined as Lobophylliidae suggests an adaptation to predation that does not require a large cnidome or spirocytes and Merulinidae as adapting to more heterotrophic energy consumption. Another interesting set of characters is the granular gland cells and pigment cells which are common to the SLO clade (Fig. 3 C). Hermatypic corals are restricted to the photic zone in order to facilitate photosynthesis by their zooxanthellae. However, they must evolve protective measures to reduce damage from UV radiation. One method is to have fluorescent pigments scatter light away from the organism (Salih et al. 2000; Gittins et al., 2015). The presence of these pigment cells and granular gland cells may also serve a 55

70 photoprotective role. All of the members of the SLO clade are commonly found in shallow or upper reef slope environments where UV radiation would be highest. These traits could represent a subset of a larger suite of shallow water adaptations. Traits such as this could be instrumental in looking for appropriate locations of conservation, repopulation and preservation. Utility of Histological Characters in Scleractinia Histological characters could be a viable addition to phylogenetic studies based on Scleractinia morphology. The present small study using only 20 characters, based solely on histology, was able to identify similar relationships in MPTs - though not in a strict consensus summary - as a much larger molecular and skeletal based phylogenies. The combination of skeletal and histological characters could help to eliminate the current scarcity of characters used in morphological studies. There are a number of reasons why histological characters have not been considered until this point. First, the inability to acquire samples that could be prepared for a study such as this may limit sample collection. However, procedures for on-site fixation of coral tissue are now possible using a fixative mixed with local seawater (e.g. Sudek et al., 2012). Furthermore, the increase in salt water tank enthusiasts has made mail delivery of healthy coral, which can then be prepared in locations far from the point of collection, easier. Second, access to proper microscopy tools could have hindered previous researchers. Light microscopes have since improved greatly and electron 56

71 microscopes are becoming more common in most major institutions. Additionally, the procedure in this study used equipment common to any major university with a biomedical research facility. Finally, most coral taxonomists have focused on skeletons because they were traditionally used to classify organism. Now, interdisciplinary efforts and a greater interest in the health of corals has made histology more feasible in recent decades (e.g. Work & Meteyer, 2014). With a better understanding of coral histological variability, from studies such as this, more informative characters can be selected and used. However, several caveats should be made. Factors such as colony size and polyp selection should be standardized. Polyps on the edge of a colony tend to be smaller and less mature due competition for free space around a colony (Goffredo et al., 2011). Larger colonies may grow larger polyps at the center of the colony to increase reproductive success. Since all of the specimens used for this study were mail ordered, they were by necessity smaller fragments and less mature. This means that the entire colony was relatively homogeneous in appearance. If larger colonies are used, this small colony effect may not hold, and care needs to be taken when selecting polyps to study. It may be desirable to select polyps from both the edges and the center of a colony since they could contain more characters to code. For example, centrally located mature polyps could contain sex organs - not observed in this study, but can be coded for - while edge polyps do not. 57

72 Another consideration for histological comparisons is the effect that a two-dimensional projection of a three-dimensional object has on shape. Thin section slides may only be a few microns thick, and different orientations of a structure may appear as very different shapes. For example, a longitudinal cut on a cylindrical nematocytes may appear as an oval, but a cross-sectional cut will appear as a circle (Fig. 4 A & B). This apparent change in structure is simply an effect of cutting in different orientations. For example, distal nematocytes are in longitudinal orientation, but central nematocytes pointing in an orthogonal direction are cut in cross-section (Fig. 4B). Because of this, characters based solely on change in shape and size need to be used with care or orientation needs to be consistent. Morphometric studies would be a challenge to apply to this set of data because of this limitation. Coloration is another trait that should not be used because this can be affected by the staining process. For example, poorly filtered hematoxylin could produce artifacts of purple stain that are not related to coral histological properties. In addition, stains may fade over time making repeated coding over time variable. Future studies can improve upon these results with the inclusion of more characters from additional microscopy techniques. Different stains, such as Aniline Blue and Movat s Pentachrome, may reveal additional structures not seen with H&E. Stains for SEM can also provide more detail of cnidocytes. For example, the structure of the basal tubule is only detectable in SEM images 58

73 (Lam et al., 2013). Confocal and TEM could also be an option if procedures were developed. Furthermore, the relationships between these histological structures and the underlying skeletal structures could be investigated through techniques that maintain the tissue-skeleton interaction zone in thin section. This could help to understand potentially different selective pressures on soft and hard tissue in Cnidaria. It might also be plausible that some characters apply to certain fossilized organisms. Traits such as cnidome density and distribution could be seen in exceptionally well-preserved specimens and help understand the phylogeny of Cnidaria in deep time as well (Cartwright et al., 2007). Finally, the creation of an atlas of histology of invertebrate organisms could be of great use to future histological projects. With the application of the histomorphological characters in this study to other sources of corroborative data we can greatly improve our understanding of scleractinian phylogeny. 59

74 Conclusions This study has shown that a phylogeny based on simple histological characters is able to reconstruct phylogenies similar to other sources of data. This result suggests that histological characters are taxonomically valid and should be added to morphological matrices. Mapping these characters shows that there is a deep divergence in the phylogeny, specifically, for traits dealing with cnidocytes. Use of histological traits can lead to discoveries about the skeleton-tissue interaction zone, differential selective pressures on tissue types and, more practically, increased resolution of phylogenies. Through the increased resolution of these phylogenies we can better account for relationships among species, as well as make accurate conservation prioritization metrics for protecting these endangered and valuable organisms. 60

75 References Adams, A. J., Dahlgren, C. P., Kellison, G. T., Kendall, M. S., Layman, C. A., Ley, J. A., Nagelkerken, I., Serafy, J. E. (2006). Nursery function of tropical back-reef systems. Marine Ecology Progress Series, 318, doi: Allemand, D., Tambutté, É., Zoccola, D., & Tambutté, S. (2011). Coral Reefs: An Ecosystem in Transition. (Z. Dubinsky & N. Stambler, Eds.). Dordrecht: Springer Netherlands. doi: / Arrigoni, R., Terraneo, T. I., Galli, P., & Benzoni, F. (2014). Lobophylliidae (Cnidaria, Scleractinia) reshuffled: Pervasive non-monophyly at genus level. Molecular Phylogenetics and Evolution, 73, doi: /j.ympev Balmford, A., & Whitten, T. (2003). Who should pay for tropical conservation, and how could the cost be met? Oryx, 37(2), doi: Brander, L. M., Van Beukering, P., & Cesar, H. S. J. (2007). The recreational value of coral reefs: A meta-analysis. Ecological Economics, 63(1), doi: /j.ecolecon Brown, B. E. (1997). Coral bleaching : causes and consequences. Coral Reefs, 16, doi: /s Budd, A. F., & Stolarski, J. (2009). Searching for new morphological characters in the systematics of scleractinian reef corals: Comparison of septal teeth and granules between Atlantic and Pacific Mussidae. Acta Zoologica, 90(2), doi: /j x Budd, A. F., & Pandolfi, J. M. (2010). Evolutionary novelty is concentrated at the edge of coral species distributions. Science, 328(5985), doi: /science Budd, A. F., & Stolarski, J. (2011). Corallite wall and septal microstructure in scleractinian reef corals: comparison of molecular clades within the family Faviidae. Journal of Morphology, 272(1), doi: /jmor Budd, A. F., Romano, S. L., Smith, N. D., & Barbeitos, M. S. (2010). Rethinking the phylogeny of scleractinian corals: a review of morphological and molecular data. Integrative and Comparative Biology, 50(3), doi: /icb/icq062 61

76 Budd, A. F., Fukami, H., Smith, N. D., & Knowlton, N. (2012). Taxonomic classification of the reef coral family Mussidae (Cnidaria: Anthozoa: Scleractinia). Zoological Journal of the Linnean Society, 166(3), doi: /j x Carpenter, K. E., Barber, P. H., Crandall, E. D., Ablan-Lagman, M. C. A., Mahardika, G. N., Manjaji-Matsumoto, B. M., Juinio-Meñez, M. A., Santos, M. D., Starger, C. J., & Toha, A. H. A. (2011). Comparative Phylogeography of the Coral Triangle and Implications for Marine Management. Journal of Marine Biology, 2011, doi: /2011/ Carr, M. H., Anderson, T. W., & Hixon, M. A. (2002). Biodiversity, population regulation, and the stability of coral-reef fish communities. Proceedings of the National Academy of Sciences of the United States of America, 99(17), doi: /pnas Cartwright, P., Halgedahl, S. L., Hendricks, J. R., Jarrard, R. D., Marques, A. C., Collins, A. G., & Lieberman, B. S. (2007). Exceptionally Preserved Jellyfishes from the Middle Cambrian. PLoS One, 2(10), e1121. doi: /journal.pone Cohen, A., & Holcomb, M. (2009). Why Corals Care About Ocean Acidification: Uncovering the Mechanism. Oceanography, 22(4), doi: /oceanog Dana, J. D. (1846). Atlas of zoophytes. United States exploring expedition during the years Philadelphia: Lea and Blanchard, 1 740, 61 pls. De ath, G., Lough, J., & Fabricius, K. E. (2009). Declining Coral Calcification on the Great Barrier Reef. Science, 323(5910), doi: /science Dulvy, N. K., Freckleton, R. P., & Polunin, N. V. C. (2004). Coral reef cascades and the indirect effects of predator removal by exploitation. Ecology Letters, 7(5), doi: /j x. Fautin, D. G. (2009). Structural diversity, systematics, and evolution of cnidae. Toxicon, 54(8), doi: /j.toxicon Fukami, H., Chen, C. A., Budd, A. F., Collins, A., Wallace, C., Chuang, Y. Y., Chen, C., Dai, C., Iwao, K., Sheppard, C., & Knowlton, N. (2008). Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class anthozoa, phylum cnidaria). PLoS ONE, 3(9), e3222. doi: /journal.pone

77 Galloway, S. B., Work, T. M., Boschsler, V. S., Harley, R. A., Kramarsky-Winters, E., Mclaughlin, S. M., Meteyer, C. U., Morado, J. F., Nicholson, J. H., Parnell, P. G., Peters, E. C., Reynolds, T. L., Rotstein, D. S., Sileo, L., & Woodley, C. M. (2007). Coral Disease and Health Workshop: Coral Histopathology II. NOAA Technical Memorandum NOS NCCOS 56 and NOAA Technical Memorandum CRCP 4. National Oceanic and Atmospheric Administration, Silver Spring, MD. 84p. Garcia-Arredondo, A., Rojas, A., Iglesias-Prieto, R., Zepeda-Rodriguez, A., & Palma-Tirado, L. (2012). Structure of nematocytes isolated from the fire coral Millepora alcicornis and Millepora complanata (Cnidaria: Hydrozoa). The Journal of Venomous Animals and Toxins including Tropical Diseases, 18(1), doi: Gittins, J. R., D'Angelo, C., Oswald, F., Edwards, R. J., & Wiedenmann, J. (2015). Fluorescent protein-mediated colour polymorphism in reef corals: multicopy genes extend the adaptation/acclimatization potential to variable light environments. Molecular Ecology, 24(2), doi: /mec Goffredo, S., Caroselli, E., Gasparini, G., Marconi, G., Putignano, M. T., Pazzini, C. & Zaccanti, F. (2011). Colony and polyp biometry and size structure in the orange coral Astroides calycularis (Scleractinia: Dendrophylliidae). Marine Biology Research, 7(3), doi: / Goloboff, P., Farris, J., & Nixon, K. (2008). TNT, a free program for phylogenetic analysis. Cladistics, 24(5), doi: /j x Haeckel, E. (1876). Ein Ausflug nach der Korallenbänken des Rothen Meeres und ein Blick in das Leben der Korallenthiere. Berlin: Georg Reimer, Huang, D. (2012). Threatened reef corals of the world. PLoS ONE, 7(3), e doi: /journal.pone Huang, D., Benzoni, F., Arrigoni, R., Baird, A., Berumen, M., Bouwmeester, J., Chou, L. M., Fukami, H., Licuanan, W. Y., Lovell, E. R., Meier, R., Todd, P. A., & Budd, A. (2014). Towards a phylogenetic classification of reef corals: the Indo-Pacific genera Merulina, Goniastrea and Scapophyllia (Scleractinia, Merulinidae). Zoologica Scripta, 43(5), doi: /zsc

78 Huang, D., Benzoni, F., Fukami, H., Knowlton, N., Smith, N. D., & Budd, A. (2014). Taxonomic classification of the reef coral families Merulinidae, Montastraeidae, and Diploastraeidae (Cnidaria: Anthozoa: Scleractinia). Zoological Journal of the Linnean Society, 171(2), doi: /zoj Johnston, I. S. (1980). The ultrastructure of skeletogenesis in zooxanthellate corals. International Review of Cytology, 67: doi: /s (08) Jones, R., Bowyer, J., Hoegh-Guldberg, O., & Blackall, L. (2004). Dynamics of a temperature-related coral disease outbreak. Marine Ecology Progress Series, 281, doi: /meps Kaniewska, P., Anthony, K. R. N., Sampayo, E. M., Campbell, P. R., & Hoegh- Guldberg, O. (2014). Implications of geometric plasticity for maximizing photosynthesis in branching corals. Marine Biology, 161, doi: /s z Kitahara, M. V, Cairns, S. D., Stolarski, J., Blair, D., & Miller, D. J. (2010). A comprehensive phylogenetic analysis of the Scleractinia (Cnidaria, Anthozoa) based on mitochondrial CO1 sequence data. PloS One, 5(7), e doi: /journal.pone Krayesky, S. L., Mahoney, J. L., Kinler, K. M., Peltier, S., Calais, W., Allaire, K., & Watson, G. M. (2010). Regulation of spirocyst discharge in the model sea anemone, Nematostella vectensis. Marine Biology, 157(5), doi: /s x Kunkel, C. M., Hallberg, R. W., & Oppenheimer, M. (2006). Coral reefs reduce tsunami impact in model simulations. Geophysical Research Letters, 33(23), L doi: /2006gl Lam, M., Doppa, J. R., Hu, X., Todorovic, S., Dietterich, T., Reft, A., & Daly, M. (2013). Learning to Detect Basal Tubules of Nematocysts in SEM Images IEEE International Conference on Computer Vision Workshops, doi: /iccvw Maddison, W., & Maddison D. (2009). Mesquite: a modular system for evolutionary analysis. Version Published by the authors. Martinez-Baraldés, I., López-González, P. J., & Medina, C. (2014). Application of cnidae composition in phylogenetic analyses of North Atlantic and Mediterranean dendrophylliid corals (Anthozoa: Scleractinia). Invertebrate Systematics, 28, doi: 64

79 Miranda, L. S., Collins, A. G., & Marques, A. C. (2013). Internal anatomy of Haliclystus antarcticus (Cnidaria, Staurozoa) with a discussion on histological features used in Staurozoan taxonomy. Journal of Morphology, 274(12), doi: /jmor Moberg, F., & Folke, C. (1999). Ecological goods and services of coral reef ecosystems. Ecological Economics, 29(2), doi: /s (99) Muko, S., Kawasaki, K., Sakai, K., Takasu, F., & Shigesada, N. (2000). Morphological plasticity in the coral Porites sillimaniani and its adaptive significance. Bulletin of Marine Science, 66(1), Mumby, P. J., Elliott, I. A., Eakin, C. M., Skirving, W., Paris, C. B., Edwards, H. J., Enriquez, S., Iglesias-Prieto, R., Cherubin, L. M., & Stevens, J. R. (2011). Reserve design for uncertain responses of coral reefs to climate change. Ecology Letters, 14(2), doi: /j x Nixon, K.C. ( ) WinClada ver Published by the author, Ithaca, NY, USA. Östman, C. (2000). A guideline to nematocyst nomenclature and classification, and some notes on the systematic value of nematocysts. Scientia Marina 64(S1), doi: /scimar s131 Pandolfi, J. M., Bradbury, R. H., Sala, E., Hughes, T. P., Bjorndal, K. A., Cooke, R. G., McArdle, D., McCleanachan, L., Newman, M. J. H., Paredes, G., Warner, R.R., & Jackson, J. B. C. (2003). Global Trajectories of the Long- Term Decline of Coral Reef, Science 301(5635), doi: /science/ Pandolfi, J. M., Connolly, S. R., Marshall, D. J., & Cohen, A. L. (2011). Projecting coral reef futures under global warming and ocean acidification. Science, 333(6041), doi: /science Raz-Bahat, M., Erez, J., & Rinkevich, B. (2006). In vivo light-microscopic documentation for primary calcification processes in the hermatypic coral Stylophora pistillata. Cell Tissue Research, 325, doi: /s Reft, A. J., & Daly, M. (2012). Morphology, distribution, and evolution of apical structure of nematocysts in hexacorallia. Journal of Morphology, 273(2), doi: /jmor

80 Ries, J. B., Cohen, A. L., & McCorkle, D. C. (2009). Marine calcifiers exhibit mixed responses to CO2-induced ocean acidification. Geology, 37(12), doi: /g30210a.1 Rodriguez, E., Barbeitos, M., Daly, M., Gusma, L. C., & Häussermann, V. (2012). Toward a natural classification : phylogeny of acontiate sea anemones (Cnidaria, Anthozoa, Actiniaria). Cladistics, 28(4), doi: /j x Roff, G., & Mumby, P. J. (2012). Global disparity in the resilience of coral reefs. Trends in Ecology & Evolution, 27(7), doi: /j.tree Rogers, C. S. (1990). Responses of coral reefs and reef organisms to sedimentation. Marine Ecology Progress Series, 62, Ryland, J., Brasseur, M., & Lancaster, J. (2004). Use of cnidae in taxonomy: implications from a study of Acrozoanthus australiae (Hexacorallia, Zoanthidea). Journal of Natural History, 38(10), doi: / Salih, A., Larkum, A., Cox, G., Kühl, M., & Hoegh-Guldberg, O. (2000). Fluorescent pigments in corals are photoprotective. Nature, 408, doi: / Strong, E. E., & Lipscomb, D. (1999). Character coding and inapplicable data. Cladistics, 15(4), doi: /j tb00272.x Sudek, M., Work, T. M., Aeby, G. S., & Davy, S. K. (2012). Histological observations in the Hawaiian reef coral, Porites compressa, affected by Porites bleaching with tissue loss. Journal of Invertebrate Pathology, 111(2), doi: /j.jip Swain, T. D., & Swain, L. M. (2014). Molecular parataxonomy as taxon description: examples from recently named Zoanthidea (Cnidaria: Anthozoa) with revision based on serial histology of microanatomy. Zootaxa 3796(1), doi: Tambutté, S., Holcomb, M., Ferrier-Pagès, C., Reynaud, S., Tambutté, É., Zoccola, D., & Allemand, D. (2011). Coral biomineralization: From the gene to the environment. Journal of Experimental Marine Biology and Ecology, 408(1-2), doi: /j.jembe Terron-Sigler, A., & Lopez-Gonzalez, P. (2005). Cnidae variability in Balanophyllia europaea and B. regia (Scleractinia: Dendrophylliidae) in the NE Atlantic and Mediterranean Sea. Scientia Marina, 69(1),

81 The IUCN Red List of Threatened Species. Version Downloaded on 08 February, Tilman, D. (1996). Biodiversity : Population Versus Ecosystem Stability. Ecology, 77(2), doi: Veron J. E. N., & Pichon, M. (1980). Scleractinia of Eastern Australia. Part 3, Families Agariciidae, Siderastreidae, Fungiidae, Oculinidae, Merulinidae, Mussidae, Pectiniidae, Caryophylliidae, Dendrophyliidae. Australian Institute of Marine Science Monograph Series, 4, Veron, J. E. N., Pichon, M. & Wijsman-Best, M. (1977). Scleractinia of Eastern Australia. Part 2, Families Faviidae, Trachyphylliidae. Australian Institute of Marine Science Monograph Series, 3, Wells, J. W. (1956). Scleractinia p. F328 F444. In: R. C., Moore, ed. Treatise on Invertebrate Paleontology. Pt. F. Coelenterata/Cnidaria. Geological Society of America and University of Kansas Press, Lawrence. Wild, C., Hoegh-Guldberg, O., Naumann, M. S., Colombo-Pallotta, M. F., Ateweberhan, M., Fitt, W. K., Iglesias-Prieto, R., Palmer, C., Bythell, J., Ortiz, J., Loya, Y., & van Woesik, R. (2011). Climate change impedes scleractinian corals as primary reef ecosystem engineers. Marine and Freshwater Research, 62(2), doi: Work, T., & Meteyer, C. (2014). To Understand Coral Disease, Look at Coral Cells. EcoHealth. doi: /s

82 Appendix A Figure A1: Histological Characters and States 68

83 Figure A1 continued 69

84 Figure A1 continued 70

85 Figure A1 continued 71

86 Figure A1 continued 72

87 Figure A1 continued 73

88 Figure A1 continued 74