Molecular Diversity, Phylogeny, and Biogeographic Patterns of Crustacean Copepods Associated with Scleractinian Corals of the Indo-Pacific

Home , Acropora aculeus, Asterocheridae, Clausiidae, Cryptochiridae, Cryptopontius, Ctenactis

Dissertation by Sofya Mudrova

In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy of Science

King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia

November, 2018 2

EXAMINATION COMMITTEE PAGE

The dissertation of Sofya Mudrova is approved by the examination committee.

Committee Chairperson: Dr. Michael Lee Berumen

Committee Co-Chair: Dr. Viatcheslav Ivanenko

Committee Members: Dr. James Davis Reimer, Dr. Takashi Gojobori, Dr. Manuel

Aranda Lastra

Sofya Mudrova

ABSTRACT

Molecular diversity, phylogeny and biogeographic patterns of crustacean copepods

associated with scleractinian corals of the Indo-Pacific

Sofya Mudrova

Biodiversity of coral reefs is higher than in any other marine ecosystem, and significant research has focused on studying coral taxonomy, physiology, ecology, and coral-associated fauna. Yet little is known about symbiotic copepods, abundant and numerous microscopic crustaceans inhabiting almost every living coral colony. In this thesis, I investigate the genetic diversity of different groups of copepods associated with reef-building corals in distinct parts of the Indo-Pacific; determine species boundaries; and reveal patterns of biogeography, endemism, and host-specificity in these symbiotic systems. A non-destructive method of DNA extraction allowed me to use an integrated approach to conduct a diversity assessment of different groups of copepods and to determine species boundaries using molecular and taxonomical methods. Overall, for this thesis, I processed and analyzed 1850 copepod specimens, representing 269 MOTUs collected from 125 colonies of 43 species of scleractinian corals from 11 locations in the

Indo-Pacific. The genetic assessment of the most abundant copepod morphotypes associated with hermatypic corals in Lizard Island (Great Barrier Reef) revealed a large number of species previously unknown for this region. Analyses of diversity and patterns of biogeographical distribution of copepods associated with Galaxea corals throughout the Indo-Pacific showed that the species diversity of this group is high and appears to be

5 regionally specific, an uncommon pattern in most coral reef-associated invertebrates.

Results for the symbiotic copepod fauna of Red Sea pocilloporid corals, a family of corals with a high level of morphological variability within and among its members, showed that the majority of the discovered poecilostomatoid copepods belong to the genus Spaniomolgus, which demonstrated a significant genetic diversity of morphologically-similar species. Assessment of the diversity of copepods associated with the Red Sea mushroom corals revealed several undescribed species and showed no evidence of specificity to the hosts neither on species nor on the family level, which contradicts a modern assumption of high host-specificity of copepods. Overall, this dissertation is a first study of genetic diversity of copepods associated with invertebrates, and it provides substantial insight into the diversity of coral-associated microcrustaceans and insight to patterns of their host-specificity as well as distribution around the Indo-

Pacific.

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my supervisor Dr. Michael Berumen for funding this project and providing guidance, help, and support throughout the course of this research. Very special thanks to my co-supervisor Dr. Slava Ivanenko for his academic and moral support, for sharing his collection of symbiotic copepods and his taxonomic expertise.

Many thanks to my committee members Dr. Manuel Aranda Lastra, Dr. Takashi

Gojobori, and Dr. James Davis Reimer for their feedback on the project and the dissertation.

My special appreciation goes to Mikhail Nikitin, Dr. Diego Fontaneto, and

Alejandro Martínez García for their assistance, feedback, and advice.

I am very grateful to my friends and colleagues, the department faculty and staff for making my time at King Abdullah University of Science and Technology a great experience.

Above all, I would like to express my gratitude to my husband and my parents for their support, faith, and confidence in me.

TABLE OF CONTENTS

EXAMINATION COMMITTEE PAGE ...... 2 COPYRIGHT PAGE ...... 3 ABSTRACT ...... 4 ACKNOWLEDGMENTS ...... 6 TABLE OF CONTENTS ...... 7 LIST OF ABBREVIATIONS ...... 9 LIST OF FIGURES ...... 10 LIST OF TABLES ...... 13 Chapter 1: Background and literature review ...... 15 1.1 Introduction ...... 15 1.2 Symbiotic copepods ...... 17 1.3 Diversity of copepods associated with Scleractinia and their host-specificity ...... 21 1.4 Biogeography ...... 25 1.5 Ecology and biology of the coral-associated copepods ...... 27 1.5.1 Behavior, location on the host, and movement ...... 27 1.5.2 Feeding preferences ...... 32 1.5.3 Evolutionary traits and adaptations to symbiosis and parasitism on a coral ...... 33 1.5.4 Effect on coral hosts...... 35 1.6 Molecular methods in the studies of diversity and taxonomy of copepods ...... 37 1.7 Dissertation structure and objectives ...... 39 Chapter 2: General methodology ...... 41 2.1 Sampling procedures ...... 41 2.2 Morphological analyses ...... 42 2.2.1 Light microscopy ...... 42 2.2.2 Scanning electron microscopy ...... 43 2.2.3 Confocal microscopy ...... 43 2.2.4 Coral identification ...... 44 2.3 Molecular-phylogenetic analysis ...... 44 2.3.1 DNA extraction ...... 44 2.3.2 Markers selection and DNA amplification ...... 45 2.3.3 Sequence alignments and phylogenetic analyses ...... 48 2.3.4 Species delimitation procedures ...... 50 Chapter 3: Genetic diversity of copepods associated with scleractinian corals in the Lizard Island (GBR) ...... 52 3.1 Introduction ...... 52 3.2 Samples and study sites ...... 54 3.3 Results ...... 57 3.3.1 PCR and sequencing ...... 57 3.3.2 Genetic distances and species delimitation ...... 58

3.3.3 Host specificity ...... 61 3.4 Discussion ...... 62 3.5 Conclusion ...... 67 Chapter 4: Biogeographic patterns, diversity, and host specificity of copepods living in symbiosis with Galaxea (Scleractinia)...... 69 4.1 Introduction ...... 69 4.2 Samples and study sites ...... 72 4.3 Results ...... 74 4.3.1 PCR and sequencing ...... 74 4.3.2 Phylogenetic relationships ...... 75 4.3.3 Multilocus molecular species delimitation ...... 78 4.4 Discussion ...... 83 4.4.1 Diversity of Galaxea-associated copepods ...... 83 4.4.2 Genetic diversity and biogeographic distribution of Anchimolgidae and Asterocheridae associated with Galaxea ...... 87 4.4.3 Biogeographical patterns of distribution of Anchimolgidae and Asterocheridae .... 94 4.5 Conclusions ...... 102 Chapter 5: Diversity assessment of poecilostomatoid copepods associated with Pocilloporidae corals in the Saudi Arabian Red Sea ...... 103 5.1 Introduction ...... 103 5.2 Samples and study sites ...... 107 5.3 Results ...... 109 5.3.1 PCR and sequencing ...... 109 5.3.2 Phylogenetic relationships and species delimitation ...... 109 5.3.3 Morphological analyses ...... 113 5.4 Discussion ...... 113 5.7 Conclusion ...... 117 Chapter 6: Diversity and host-specificity of copepods associated with mushroom corals in the Red Sea ...... 119 6.1 Introduction ...... 119 6.2 Samples and study sites ...... 121 6.3 Results ...... 123 6.3.1 Copepod species ...... 123 6.3.2 Symbiotic association ...... 126 6.4 Discussion ...... 129 6.5 Conclusion ...... 134 Chapter 7: Discussion ...... 135 CONCLUSIONS ...... 145 LIST OF PUBLICATIONS ...... 147 REFERENCES ...... 149 APPENDICES ...... 170

LIST OF ABBREVIATIONS

ABGD automatic barcode gap discovery BLAST the Basic Local Alignment Search Tool BOLD the Barcode of Life Data System bp base pair CCDB the Canadian Centre for DNA Barcoding CLSM confocal laser scanning microscope COI cytochrome c oxidase subunit 1 DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid GBR the Great Barrier Reef GMYC the generalized mixed Yule Coalescent GTR General Time-Reversible model ITS2 internal transcribed spacer 2 KAUST King Abdullah University of Science and Technology MAFFT Multiple Alignment using Fast Fourier Transform MCMC Markov chain Monte Carlo MOTU molecular operational taxonomic unit MT-GMYC multiple thresholds GMYC NCBI the National Center for Biotechnology Information GenBank OTU operational taxonomic unit PAUP phylogenetic analysis using parsimony PCR polymerase chain reaction PTP Poisson tree processes bPTP-ML Bayesian PTP maximum likelihood bPTP-h Bayesian PTP highest support partition RAxML Randomized Axelerated Maximum Likelihood SDS sodium dodecyl sulfate SEM scanning electron microscopy ST-GMYC single threshold GMYC ZMMU Zoological Museum of Lomonosov Moscow State University

LIST OF FIGURES

Figure 1.1 Phylogram of the copepod orders (from Khodami et al., 2017)...... 18 Figure 1.2 Global diversity and number of scleractinian coral species (Veron et al., 2009)...... 26 Figure 1.3 Photographs of galls (tubular-shaped modification of corallites) induced by a copepod Spaniomolgus sp. (A) colony of Stylophora pistillata with galls (marked with the red arrows); (B) close-up view of a modified corallite; (C) skeletal structure of a tubular corallite on SEM; (D) SEM of a gall-inducing copepod Spaniomolgus sp., female, ventrolateral view. (E) SEM of longitudinal section of the modified corallite. Illustration modified from Ivanenko et al., 2014...... 29 Figure 1.4 Photographs of gall-like formation induced by poecilostomatoid copepods Allopodion ryukyuensis (A, B) and Xenomolgus varius (C, D). (A) a dome-shaped gall induced by A. ryukyuensis on Montipora informis; (B) longitudinal section of a gall, showing a female with eggs (indicated by an arrow) of A. ryukyuensis within the gall cavity; (C) Porites sp. showing bleached spots each surrounding a crypt inhabited by X. varius; (D) magnified bleached spot, showing the aperture of a crypt formed by X. varius. Illustration from Kim & Yamashiro, 2007...... 30 Figure 1.5 Different body forms of copepods associated with scleractinian corals: (A) Asteropontopsis faviae (Asterocheridae), dorsal view of female; (B) Corallonoxia longicauda (Corallovexiidae), dorsal view of female; (C, D) Xarifia exserens (Xarifiidae), dorsal (C) and lateral (D) views of female (from Glynn & Enochs, 2011, original drawings from Stock, 1987 and Humes, 1985b)...... 34

Figure 3.1 Map showing the locations from which coral samples were obtained (shown with red circles); basemap obtained from Google Maps and http://d-maps.com/. .... 55 Figure 3.2 Bayesian phylogeny of the family Anchimolgidae based on the concatenated dataset of 18S, COI, and ITS2. Intra-specific branches are collapsed. Host specificity is coded by branch color. The columns on the right represent species delimitation based on different methods: ABGD and PTP. Numbers in the brackets represent number of specimens per each MOTU. See Appendix 3.4 for a full MrBayes tree containing all individuals...... 60 Figure 3.3 Bayesian phylogeny of the family Asterocheridae based on the concatenated dataset of 18S, COI, and ITS2. Host specificity is coded by color. The columns on the right represent species delimitation based on different methods: ABGD and PTP. Numbers in the brackets represent number of specimens per each MOTU. See Appendix 3.5 for a full MrBayes tree containing all individuals...... 61 Figure 3.4 Bayesian phylogeny of the family Xarifiidae based on the concatenated dataset of 18S, COI, and ITS2. Host specificity is coded by color. The columns on the right represent species delimitation based on different methods: ABGD and PTP. Numbers in the brackets represent number of specimens per each MOTU. See Appendix 3.6 for a full MrBayes tree containing all individuals...... 61 Figure 3.5 Comparison of the diversity of three dominant families between my samples and the data from the literature...... 63

Figure 3.6 Correlation of the copepod diversity with the host diversity in our samples (red) and in data from the literature records (black) for the seven genera present in both datasets (1 - Merulina, 2 - Galaxea, 3 - Stylophora, 4 - Seriatopora, 5 - Echinopora, 6 - Montipora, 7 - Acropora)...... 64

Figure 4.1 Map indicating collection sites of samples from Galaxea colonies used in this study. Full circles show our sampling sites; stars mark the localities where copepods from Galaxea corals were reported (see references in the text). The total number of colonies sampled at each location is indicated in parentheses. Abbreviations: RS, Red Sea; MAL, Maldives; HN, Hainan; VN, Vietnam; NR, Ningaloo Reef, Australia; LZI, Lizard Island, Australia; HEI, Heron Island, Australia...... 73 Figure 4.2 18S ML phylogenetic tree based on 992 sequences generated from my samples and additional 174 copepod sequences from GenBank (marked with black). The tree was edited using the online tool iTOL v3 (Letunic & Bork, 2016). See full tree in Appendix 4.4...... 76 Figure 4.3 Bayesian phylogeny of the family Anchimolgidae based on the concatenated dataset of 18S, COI, and ITS2. Numbers on the branches represent support values corresponding to BI posterior probabilities. The columns on the right represent species delimitation based on different methods: ABGD, PTP, and GMYC (incongruence between methods is marked with a darker color). See Appendix 4.8 for a full MrBayes tree containing all individuals...... 80 Figure 4.4 Bayesian phylogeny of the family Asterocheridae based on the concatenated dataset of 18S, COI, and ITS2. Numbers on the branches represent support values corresponding to BI posterior probabilities. The columns on the right represent species delimitation based on different methods: ABGD, PTP, and GMYC (incongruence between methods is marked with a darker color). Numbers in the brackets represent number of specimens per each MOTU. See Appendix 4.9 for a full MrBayes tree containing all individuals...... 82 Figure 4.5 The sampling coverage for the dominant taxa of copepods associated with Galaxea corals...... 94 Figure 4.6 Biogeographical regions delineated based on the distribution and delineation of copepod MOTUs from Galaxea corals in the Indo-Pacific...... 95 Figure 4.7 Map of the atolls of the Maldivian Archipelago. Sampling locations are marked with the red circles...... 97 Figure 4.8 Sampling locations in the South China Sea (marked with red circles); the star marks the records from Galaxea in the literature (Cheng & Dai, 2016). Map modified from the southchinasea.org (Ji Guoxing)...... 98 Figure 4.9 Venn diagram of the copepod entities found on Galaxea corals. Number in brackets represents the number of MOTUs in the region. Red Sea species are not included as they were not found in any other regions...... 99 Figure 4.10 Number of MOTUs identified from each sampled location for the two dominant families Asterocheridae and Anchimolgidae; the dashed line represents a number of host colonies collected in each region...... 102

Figure 5.1 Map showing the sampling localities from which Pocilloporidae corals were collected (shown with red circles); for more details see Table 5.1. Basemap for map A obtained from http://d-maps.com/, basemap for map B obtained from the British Admiralty charts...... 107 Figure 5.2 Tanglegram of the phylogenetic trees of Spaniomolgus based on the COI dataset (on the left) and ITS2 dataset (on the right). The colored bars represent combined species delimitation result obtained from ABGD, PTP, and GMYC. .... 110 Figure 5.3 Conspecificity matrix based on the results from ABGD, PTP, and GMYC. COI on the left, ITS2 on the right...... 111

Figure 6.1 Map showing the sampling localities (1 to 10) from which Fungiidae corals were collected (shown with red dots); for more details see Table 6.1. Basemap for map A obtained from http://d-maps.com/, basemap for map B obtained from the British Admiralty charts...... 122 Figure 6.2 Habitus of copepods found on mushroom corals of the Red Sea. Ventral view, confocal microscopy of animals after extraction of DNA (red: females, blue: males). (A) Prionomolgus sp. 1; (B) Prionomolgus sp. 2; (C) Schedomolgus sp. 1; (D) Schedomolgus sp. 2; (E) Paradoridicola sp. 1; (F) Asteropontius sp. 1...... 124 Figure 6.3 Phylogenetic reconstruction of the sampled copepods based on the COI analyses. The tree topology corresponds with the Bayesian analyses, whereas node values indicate support values for each clade (Bayesian posterior probabilities/maximum likelihood bootstrap). Asterisks (*) indicate maximum support values. Entities recovered by the delineation analyses are labeled with different colors indicating their distribution across different coral host coral species. See results from other phylogenetic analyses in Appendices 6.5-6.7...... 125 Figure 6.4 Phylogeny of mushroom coral species (after Gittenberger et al., 2011, Benzoni et al., 2012) and symbiotic copepods found on them in the Red Sea. Coral species marked in red have been recorded as host for species of Red Sea copepods: Sch1 = Schedomolgus sp. 1; Sch2 = Schedomolgus sp. 2; Prio1 = Prionomolgus sp. 1; Prio2 = Prionomolgus sp. 2; Par1 = Paradoridicola sp. 1; Ast1 = Asteropontius sp. 1; Ast2 = Asteropontius sp. 2; + = present, - = not observed...... 128

LIST OF TABLES

Table 2.1 List of primers used in PCR. Primers used in sequence reactions and included in analyses are marked as bold...... 47

Table 3.1 List of the coral colonies collected, with the information about the depth and location of the sampling and the number of copepods collected from each host (see full sampling information in the Appendix 3.2)...... 55 Table 3.2 Number of MOTUs obtained using different species delimitation algorithms. 58 Table 3.3 Coral hosts of symbiotic copepods reported from the Lizard Island before and found in this study...... 65 Table 3.4 List of scleractinian coral taxa reported for the first time to host symbiotic copepods...... 67

Table 4.1 List of copepod species associated with Galaxea corals (modified from Cheng & Dai, 2016) in the Indo-Pacific. Abbreviations: A - Galaxea astreata, F - G. fascicularis, H - G. horrescens, sp. - G. sp...... 70 Table 4.2 List of Galaxea corals collected, with the sampling depth, location and the total number of copepods collected from each host (see full sampling information in the Appendix 4.1)...... 73 Table 4.3 Diversity assessment of copepods found on Galaxea based on 18S gene...... 77 Table 4.4 Number of MOTUs obtained using different species delimitation algorithms (see details in the text); concat. = concatenated dataset...... 78 Table 4.5 Comparison of the discovered diversity with the estimates based on the previous records...... 83 Table 4.6 Diversity, abundance, and distribution of Anchimolgus copepods in my samples...... 88 Table 4.7 Diversity, abundance, and distribution of Clamocus copepods in my samples...... 89 Table 4.8 Diversity, abundance, and distribution of Karanges copepods in my samples...... 89 Table 4.9 Diversity, abundance, and distribution of Hetairosynopsis copepods in my samples...... 90 Table 4.10 Diversity, abundance, and distribution of Hetairosyna copepods in my samples...... 91 Table 4.11 Diversity, abundance, and distribution of Asterocheres copepods in my samples...... 91 Table 4.12 Diversity, abundance, and distribution of Asteropontius copepods in my samples...... 92 Table 4.13 Genera and the number of species of symbiotic copepods reported from the Galaxea corals before and discovered in this study...... 93

Table 5.1 List of the Pocilloporidae colonies collected, with the information about the depth and location of the sampling and the total number of copepods collected from

each host (see full sampling information in the Appendix 5.2)...... 108 Table 5.2 Diversity, host-specificity, and distribution of copepod MOTUs collected from Pocilloporidae corals; C – central Red Sea, S – southern Red Sea. Old species names are used to distinguish morphotypes of Stylophora colonies...... 112 Table 5.3 List of all species of the genus Spaniomolgus with information about the hosts and the sampling details...... 115

Table 6.1 List of the Fungiidae colonies collected, with the information about the depth and location of the sampling and the number of copepods collected from each host (see full sampling information in the Appendix 6.1)...... 122 Table 6.2 Number of copepod individuals and coral colonies, host coral species and genera for each of the most common copepod species. The results of the simulations reported for host species (and host genera) represent the proportion of the expected numbers that came out lower than the observed numbers from the resampling tests (1000 replicates) on all the occurrences of all 184 individual copepods on the host coral under a null model of no association: values smaller than 0.05 denote potentially significant associations. The other three species, found in less than 15 individuals, are not reported because no test would be meaningful with such low sample size...... 127 Table 6.3 Proportion of explained variance of genetic diversity in COI due to differences in host-coral species, and reef (nested within the Southern or the Central area, when possible) together with the p-values between parentheses. The other three species, found in less than 15 individuals, are not reported because no test would be meaningful with such low sample size...... 127

Chapter 1: Background and literature review

1.1 Introduction

Marine biodiversity of coral reefs is higher than in any other marine ecosystem (Fisher et al., 2011; Appeltans et al., 2012). Numerous and diverse marine species depend on the coral reefs. Reefs are also one of the most endangered habitats: one-third of reef-building corals are facing the risk of extinction due to rising sea surface temperatures and increasing water acidification observed in almost all regions of the World Ocean (Heron et al., 2017; Hoegh-Guldberg et al., 2017; Hughes et al., 2018b). These processes affect coral reefs causing stress, intense bleaching, and diseases and collectively reduce the rate of calcification of reef-building corals (Knowlton, 2001; Hoegh-Guldberg et al., 2007;

Carpenter et al., 2008; Van Oppen & Lough, 2008). The situation is compounded with negative anthropogenic impacts such as excessive fishing, overexploitation of marine species, and polluted coastal runoff, all of which contribute to the degradation of coastal coral reef ecosystems (Jackson et al., 2001; Hughes et al., 2003; Knowlton & Jackson,

2008; Hughes et al., 2017b).

As the primary habitat builders of shallow water coral reefs, hermatypic corals

(scleractinians) are often considered "ecosystem engineers". All reef-associated animals, by definition, depend on the presence and well-being of corals. In some cases, this is due to direct nutritional requirements or obligate sheltering behaviors (e.g., Rotjan & Lewis,

2006), while in other cases it may be indirect reliance, for example, mesopredators targeting prey species with direct reliance on corals (McMahon et al., 2016). Therefore, when corals are impacted by some external stressors, all animals dependent on the coral

16 reefs may be affected (Glynn, 1993). While coral symbionts usually depend directly on the well-being of the host, the influence they have on the coral is not always obvious. The complexity of ecological interactions among species, especially in species-rich environments, can be extremely difficult to understand. The consequences of a loss of one species on the rest of the network require an enormous amount of fundamental understanding of these interactions (Gao et al., 2016). There is no possibility to unravel such complex ecological interactions in cases where diversity of a group is not well- studied.

Traditionally, reef biodiversity assessments focus on macrofauna, while the biodiversity of cryptic meiofauna remains understudied and underestimated (Bouchet &

Duarte, 2006; Plaisance et al., 2011). Among these diverse and poorly understood groups, symbiotic copepods appear to simultaneously be among the most numerous and yet the least studied animals on the reef (Bron et al., 2011; Stella et al., 2011).

My dissertation focuses on the case of poorly studied symbiotic copepods living in association with reef-building corals. An assessment of diversity of symbiotic copepods will allow us to reveal the real level of their abundance and species composition. Which species are associated with corals is paramount information and is necessary to study copepod-coral relationships on all levels, including the patterns of copepods’ biogeographical distribution. Moreover, this knowledge will allow us to find out what has a greater impact on the species composition of copepods living in association with corals: whether it is specificity to a particular group of hosts, morphological features of the host colony, environmental factors or the geographical region.

1.2 Symbiotic copepods

Copepods are a highly diverse and numerous group of Crustacea. Currently, there are about 11,000 morphospecies described according to Walter & Boxshall (2018a).

Copepods have developed more associations with other animals than any other group of crustaceans: about 35% of them are symbiotic forms found in association with all major phyla of invertebrates, including cnidarians, sponges, polychaetes, molluscs, bryozoans, crustaceans, and echinoderms, as well as chordates such as ascidians, fishes, and cetaceans (Humes, 1985a; Ho, 2001). Due to the lack of sufficient data on biology and ecology of associated copepods, I am going to use the term "symbiosis" for both commensal and parasitic types of biological interactions between species (Boxshall &

Halsey, 2004a; Glynn & Enochs, 2011). Further, it is not always possible to determine the kind of relationship between a copepod and its host because of the difficulties in making direct observations in the field or extended observations (which might provide insight to the exact nature of the relationship). I am also not including mutualism as no copepod has been reported living in a mutualistic relationship with its host (Ho, 2001), they are usually indicated as either symbionts or parasites.

Copepods living in associations with invertebrates are in most cases poorly studied.

Estimates of the number of species associated with a particular group are rare. Moreover, most of the research on symbiotic copepods is focused on the parasites of fishes despite the fact that invertebrate hosts generally have much higher symbiont densities per individual than fish hosts (Humes, 1994; Ho, 2001). According to the most recent estimation, copepods are known from only 1.29% of 151,400 potential marine invertebrate hosts relative to 16% of the 22,000 species of fishes (Ho, 2001). Of course,

18 these estimates need to be updated as they may be misleading. The seemingly low percentages reported by Ho (2001) indicate how infrequently studied these types of symbioses are, and not the rarity of their occurrence in nature. Besides, many invertebrate taxa are understudied or incompletely described, and there are many uncertainties in the nomenclature and resulting synonymies (Bouchet & Duarte, 2006), which complicate the diversity assessment of the fauna of symbiotic copepods even further. Therefore, considering all aquatic animals to be potential hosts for copepods there are undoubtedly many more species to discover.

Figure 1.1 Phylogram of the copepod orders (from Khodami et al., 2017).

Copepods living in symbiosis are present in major copepod orders such as

Siphonostomatoida, Cyclopoida, Harpacticoida, and Calanoida (Figure 1.1). The majority of the described symbiotic species belong to poecilostomatoid Cyclopoida

(Poecilostomatoida) and Siphonostomatoida, comprising more than 3,611 (of 4,224) species associated with different hosts (Ho, 2001). The phylogenetic relationships

19 between Poecilostomatoida and Cyclopoida were in question for a long time, as it was debated whether they are two separate podoplean lineages (Huys & Boxshall, 1991) or whether poecilostomatoid copepods are part of a paraphyletic order of Cyclopoida

(Boxshall & Halsey, 2004a). According to the latest study by Khodami et al. (2017) on the copepod phylogeny, poecilostomatoid copepods were included in the order

Cyclopoida. Despite this, the position of the Poecilostomatoida in the copepod phylogeny may not yet be conclusive and is still a subject of debate (V. Ivanenko, personal communication). In this dissertation, I will refer to the order Poecilostomatoida as a separate group from Cyclopoida. There are also some species of symbiotic copepods belonging to Misophrioida and Monstrilloida, while orders Gelyelloida, Mormonilloida, and Platycopioida do not have any symbiotic copepods recorded (Ho, 2001; Boxshall &

Halsey, 2004a).

Copepods can be either specialists, inhabiting only one or a few host genera, or generalists, occurring on many different host genera. Most of the symbiotic copepods living on invertebrates are associated with a particular group of hosts. For instance, the majority of the copepods of the families Pseudanthessiidae and Synapticolidae are associates of echinoderms, almost all Sabelliphilidae are found on polychaete worms, and all species of Octopicolidae live in association only with cephalopod molluscs (Humes &

Boxshall, 1996). Sometimes closely related symbiotic genera can occupy different groups of hosts, even hosts from different phyla, due to a phenomenon known as host-switching.

For example, more than three-quarters of the species in the poecilostomatoid family

Taeniacanthidae are fish parasites, while the remainder lives with echinoids (Gotto,

1998). This suggests numerous evolutionary mechanisms of adaptation of copepods to live on or in another organism.

Symbiotic copepods are widespread, occurring from pole to pole, and inhabiting all depths from the intertidal zone to the abyssal zone, including the hydrothermal vents and cold seeps, where they may occur in very large numbers (Humes, 1994; Bron et al.,

2011). The greatest abundance of symbiotic copepods, however, is observed in coral reefs (Glynn & Enochs, 2011; Stella et al., 2011). Coral reefs are complex and diverse ecosystems that provide habitat, shelter, and food for hundreds of species of invertebrates and fishes, which, in turn, serve as hosts for numerous copepods.

Among the most important components of benthic reef communities are cnidarians, such as hard corals, soft corals, sea fans, and anemones. Cnidarians harbors more species of copepods than any other group of invertebrates (Humes, 1985a; Cheng et al., 2016).

The last review of all described copepods associated with Cnidaria was conducted by

Humes in 1994. He counted 399 species living on different groups of cnidarians, though he emphasized that this number represents only a fraction of the real diversity. There is no recent assessment of the diversity of this group, but we can assert that in the last 20 years the number of known species has increased significantly. This is primarily related to the improvement of the technical capabilities of research, including the improvement of methods for collecting material, the advent of the access to the deep regions of the ocean, the possibility of sampling in remote regions, and the development of molecular methods enabling more accurate identification of the specimens.

Within Cnidaria, shallow-water reef-building hermatypic corals belonging to the

Scleractinia have the greatest number of copepod associates (Humes, 1985a; Cheng et al.,

2016). More than 360 species of copepods from different families and orders are known from scleractinian corals (Cheng et al., 2016; Ivanenko et al., 2018). This group demonstrates a high diversity due to the presence of a variety of potential habitats for the symbionts created by reef-building corals and, thus, they appear to be attractive to the copepods. Corals provide protection to symbiotic crustaceans from predators (Baeza,

2015); the range of predators threatening scleractinian corals and, accordingly, their symbiotic copepods, is limited, partially due to the fact that most of their body consists of the inedible calcareous skeleton, and partially because the living tissues are covered with numerous nematocysts containing toxins (Glynn & Enochs, 2011). However, there are more than 160 species of corallivorous invertebrates and fishes, including such groups as mucus-feeders, browsers, excavators, and scrapers (Rotjan & Lewis, 2008), and some of them could feed on copepods along with consuming the coral mucus or tissues and destroying the coral. For instance, cleaner shrimps are known to remove and eat large numbers of copepod ectoparasites from coral reef fishes (Becker & Grutter, 2004), thus, we can assume that the same can happen to copepods inhabiting the surface of corals.

1.3 Diversity of copepods associated with Scleractinia and their host-specificity

Copepods associated with corals form an important component of the cryptofauna of coral reefs (Preston & Doherty, 1994). I would like to clarify for the rest of my dissertation that under corals I mean scleractinian corals unless otherwise stated. A single colony of the coral could serve as a host for a considerable number of copepod species

(up to nine species) and a huge number of individuals (Humes, 1994). For example, on a

30cm x 30cm fragment of tabulate Acropora Oken, 1815 1250 copepods were recorded

(Humes, 1994). Another record is from one colony of Pocillopora damicornis (Linnaeus,

1758) where 668 individuals of only one species of Xarifia Humes, 1960 were found

(Humes, 1985b). Even if the numbers are not always so high, copepods can be found on almost every living and healthy colony on the reef (Humes, 1994).

Most common symbionts of the stony corals belong to poecilostomatoid

Cyclopoida (285 species) and order Siphonostomatoida (68 species), but there are also representatives in other families of Cyclopoida (three species) and Harpacticoida (seven species) (Cheng et al., 2016).

Half of the species of copepods associated with scleractinian corals (141 species) belong to Anchimolgidae, a family of poecilostomatoid copepods (Cheng et al., 2016;

Walter & Boxshall, 2018b). Further, 80 of these species belong to four genera:

Anchimolgus Humes & Stock, 1972, Odontomolgus Humes & Stock, 1972,

Schedomolgus Humes & Stock, 1972, and Scyphuliger Humes, 1991. Other numerous poecilostomatoid families associated with corals are Xarifiidae and Corallovexiidae.

These are two families of endosymbiotic (most likely parasitic) poecilostomatoid copepods known to occur exclusively on hermatypic and ahermatypic corals (Stock,

1975; Humes, 1985b; Boxshall & Halsey, 2004b). Xarifiidae comprises one-quarter of the known species of copepods associated with scleractinians (Humes, 1985b; Cheng et al., 2016). Representatives of several other families also utilize reef-building corals as hosts but do not exclusively use corals. Rhynchomolgidae Humes & Stock, 1972 is the largest family of cnidarian associates where 19 of 44 genera use scleractinians as hosts

(Boxshall & Halsey, 2004a; Cheng et al., 2016). Among other families of Cyclopoida there are from one to three species of copepods associated with hard corals.

Among siphonostomatoid copepods, Asterocheridae is the largest family associated with invertebrates (Johnsson, 2002; Boxshall & Halsey, 2004b). They inhabit a particularly wide range of hosts, and many of them are associated with shallow-water scleractinian corals (Stock, 1988). 16 of 68 genera of Asterocheridae are found on hard corals (Cheng et al., 2016). Most common coral associates from the family

Asterocheridae belong to the genera Asterocheres Boeck, 1859, Asteropontius Thompson

I.C. & Scott A., 1903, and Hetairosyna Humes, 1991 (Cheng et al., 2016). Another group of siphonostomatoids associated with madreporarian corals is Coralliomyzontidae, copepods associated exclusively with scleractinian corals. Coralliomyzontidae were distinguished as a separate family of siphonostomatoid copepods by Humes and Stock

(1991), however, Boxshall and Halsey place them back in the family Asterocheridae, marking that the whole group is pending comprehensive revision (Boxshall & Halsey,

2004b). All harpacticoid species found on the scleractinian coral hosts belong to the family Tegastidae, except for Alteuthellopsis corallina Humes, 1981 from the family

Peltidiidae (Cheng et al., 2016).

Copepods associated with scleractinian corals display different levels of host specificity. However, most of the copepod species are reported from only one or several species of the one genus/family of a coral (Cheng et al., 2016). An example of the genus- level specificity was shown in an experiment in which Goniastrea Milne Edwards &

Haime, 1848 with associated lichomolgiid copepods was kept in aquaria with specimens of two different species of Favia Milne Edwards & Haime, 1857, a genus that belongs to the same family as Goniastrea. Copepods did not move to any colonies of Favia, even after the death of their original host (Patton, 1976).

In most cases, it is hard to assess the real host-specificity on the species-level due to the inadequate amount of studies (Humes, 1985a). Therefore, we cannot claim that a copepod found on one species of a host is truly monoxenic until we have data for all potential species of hosts. Additionally, most of the coral species have not been examined for copepod associates yet (Humes, 1994). Only about 150 of 1605 species (Hoeksema &

Cairns, 2018a) of scleractinian corals currently have records of symbiotic copepods

(Cheng et al., 2016; Ivanenko et al., 2018). Given that the coral-associated copepod diversity is highly correlated with the host diversity (Humes, 1994), the whole diversity of symbiotic copepods living on stony coral is significantly underestimated and further investigations will reveal many more coral species to host copepods.

The assessment of the species-level host-specificity could also be complicated with difficulties in identification of coral host species. Classic taxonomy of the scleractinian corals is based on the specific features of the structure of the calcareous skeleton, such as the shape of the colony, the structure of the corallites and the coenosteum. However, these features could significantly differ between individuals from different locations as well as within a single colony (Veron, 1995; Todd, 2008). The high level of phenotypic plasticity and frequent hybridization make it very difficult to determine the boundaries of species and often impedes the assessment of the actual biodiversity of the coral genera and the distribution of individual species (Todd, 2008; Pfennig et al., 2010; Richards &

Hobbs, 2015).

1.4 Biogeography

Our understanding of the geographical distribution of the coral-associated copepods is based on the taxonomic data obtained from a limited number of geographic regions, and faunal lists have not been developed for the most parts of the world (Humes, 1994; Ho,

2001). The majority of scleractinian-copepod associations were reported from the Indo-

Pacific, where the coral fauna is much more diverse than in the Atlantic Ocean (Stock,

1988; Veron et al., 2009). Furthermore, fauna of copepods associated with corals significantly differs between the Indo-Pacific and Atlantic regions. In the Indo-Pacific, the majority of copepod associates belong to the families Anchimolgidae,

Rhynchomolgidae, and Xarifiidae, while families Corallovexiidae and Asterocheridae are known to dominate in Atlantic (Humes, 1985a; Stock, 1988). Moreover, members of the superfamily Lichomolgidea (currently divided to 10 families, including Anchimolgidae and Rhynchomolgidae (Humes & Boxshall, 1996)) and family Xarifiidae are absent on scleractinians in the West Indies (Humes, 1985a). There is a possibility that the low diversity of asterocherid copepods reported from the Indo-Pacific is a result of inadequate attention to this group. Researchers showed more interest in poecilostomatoid copepods for a long period of time, and new investigations could reveal higher diversity of siphonostomatoids on corals (Humes, 1985a; Ivanenko et al., 2018). Instead of endoparasitic Xarifiidae, Caribbean corals are colonized by Corallovexiidae, another family of highly transformed caterpillar-like copepods (Stock, 1975; Humes, 1985a).

Beside the differences in the taxonomic composition of these two regions, there are also disparities in the species diversity: more than 90% of reported scleractinian-copepod associates are from the Indo-Pacific, where the corals are much more diverse in

26 comparison to the Caribbean reefs. The lower diversity of coral-associated copepods and their hosts in the West Indies is presumed to be a consequence of the mass extinctions that occurred during the Oligocene-Miocene cooling of the Atlantic combined with a substantial sea-level drop of 50-90 meters (Stock, 1988; Edinger & Risk, 1994).

Figure 1.2 Global diversity and number of scleractinian coral species (from Veron et al., 2009).

The Indo-Pacific region is known to have an especially rich coral fauna with a peak of diversity in the Coral Triangle, where ranges of species from the Pacific and Indian oceans overlap (Allen, 2008; Veron et al., 2009). The areas of particular interest in my work are coral-associated copepods inhabiting geographically-separated regions located on the opposite sites of the Coral Triangle, the Red Sea, and the Great Barrier Reef

(GBR). Although the diversity of scleractinians tends to gradually decrease with increasing distance from the Coral Triangle hotspot, both Red Sea and Australian reefs have a large number of reef-building coral species (Figure 1.2) (Veron et al., 2009) and, presumably, species of copepods associated with corals.

Sampling of corals for copepods has been done at relatively few localities in the

Indo-Pacific and within these localities, there have been very different degrees of thoroughness. Therefore, the absence of records for some locations, as well as for some hosts, may not be meaningful. Instead, this may result from inadequate sampling of corals

27 or from failure to use proper sampling methods (Humes, 1985b). The most intense sampling of copepods from Scleractinia in the Indo-Pacific was conducted in New

Caledonia, northern Madagascar, Indonesia (mainly Moluccas archipelago), Taiwan,

Marshall Islands, and the Great Barrier Reef (Humes, 1985b, 1991; Cheng et al., 2016), while in the Atlantic most of the samples were collected in Caribbean islands (Stock,

1975, 1987, 1988). Nevertheless, despite the high diversity and number of corals present, the symbiotic copepod fauna remains completely unexplored in some regions, and faunal lists have not been developed for the most parts of the world (Humes, 1994; Ho, 2001).

1.5 Ecology and biology of the coral-associated copepods

Though coral-associated copepods have been studied for a considerable period of time, there remains a scarcity of data on their biology and ecology. Most of the symbiotic copepods are known to be ectosymbionts, living their entire adult lives on the surface of the host tissues, but there are also a number endosymbiotic and endoparasitic forms living in coral polyps, fish muscles, and modified tissues such as galls (Huys & Boxshall,

1991).

1.5.1 Behavior, location on the host, and movement

Due to the small size (1-3 mm) of the individuals, field observations of their behavior and feeding preferences is almost impossible. Therefore, it is often hard to understand whether a copepod should be considered as parasitic, symbiotic, or living in a loose association with coral (Baeza, 2015). There are only three records based on observations in aquaria. Ectosymbiotic copepods from the genus Lichomolgus Thorell, 1859 were reported from the faviid coral Goniastrea (Patton, 1976). The copepods were

28 distinguishable on the brown coral tissue of the coral as they had white markings on the bodies and the females carried white egg sacs, so it was possible to observe patterns of their movement behavior: they alternated between remaining motionless and swimming rapidly and erratically. Additionally, the increase in the movement of the digestive tract was observed in copepods after returning them to the host after two days of isolation.

This observation was interpreted as evidence of the copepod feeding on coral tissue

(Patton, 1976). Another interesting behavioral pattern was also discovered by Patton

(1972). Though he describes copepods inhabiting a soft coral Leptogorgia Milne

Edwards, 1857, the observed movements are so peculiar that they are worth mentioning.

Lamippidae copepods were seen “moving about within the polyps, disappearing inside the coenenchyme and appearing again in another polyp, two or three polyps down the line” (Patton, 1972). Stock (1975) in his paper describing the new family Corallovexiidae mentions that these copepods could be found between the mesenteries of the coelenteric cavity, but are mostly in the coral's mesoglea. He further claims that the whole family is endoparasitic.

The interactions between copepods and coral hosts are best studied on xarifiids, the most common parasitic copepods of corals. Xarifiidae live inside of the gastrovascular cavities of coral polyps and can crawl in and out of them periodically in a caterpillar-like way (Humes, 1960). The character of interactions between these copepods and their coral hosts was unclear for a long time, but recent studies have shed some light on their relationships. Scientists observed the process of the penetration of Xarifia into a polyp of

Stylophora pistillata Esper, 1797 (Cheng & Dai, 2009). It seems that a copepod can

29 induce the relaxation of the tentacles and secured its way inside of the polyp by releasing some unidentified chemicals (Cheng & Dai, 2009).

Figure 1.3 Photographs of galls (tubular-shaped modification of corallites) induced by a copepod Spaniomolgus sp. (A) colony of Stylophora pistillata with galls (marked with the red arrows); (B) close-up view of a modified corallite; (C) skeletal structure of a tubular corallite on SEM; (D) SEM of a gall-inducing copepod Spaniomolgus sp., female, ventrolateral view. (E) SEM of longitudinal section of the modified corallite. Illustration modified from Ivanenko et al., 2014.

Another interesting example of relationships between copepods and their coral hosts are gall-inducing copepods. These copepods might attach to the soft tissues, and by disturbing the coral with their swimming legs, they can elicit the defense mechanism of a coral to grow a calcareous barrier (Dojiri, 1988; Kim & Yamashiro, 2007; Glynn &

Enochs, 2011). The gall is a thin-walled hollow ledge on the surface of the coral, which has pores and can look like a bud of a new branch of coral (Figures 1.3A-C, 1.4A) or a discolored spot on a coral (Figure 1.4C, D) and forms a spherical cavity with a diameter of up to 2 mm (Figures 1.3E and 1.4B). The development of the gall begins with the attaching of a copepodid (free-swimming larval stage of parasitic copepods) to the coral polyp. The copepodids attach themselves to the soft tissues of the coral (with the help of second antennas and maxillipeds) and provoke a protective reaction of the host, causing

30 irritation of its tissues, so the coral creates a calcareous barrier to separate the parasite from its soft tissues. A copepod remains non-mobile long enough for the coral to form a calcareous skeleton around it (Dojiri, 1988).

Figure 1.4 Photographs of gall-like formation induced by poecilostomatoid copepods Allopodion ryukyuensis (A, B) and Xenomolgus varius (C, D). (A) a dome-shaped gall induced by A. ryukyuensis on Montipora informis; (B) longitudinal section of a gall, showing a female with eggs (indicated by an arrow) of A. ryukyuensis within the gall cavity; (C) Porites sp. showing bleached spots each surrounding a crypt inhabited by X. varius; (D) magnified bleached spot, showing the aperture of a crypt formed by X. varius. Illustration from Kim & Yamashiro, 2007.

The copepod inhabiting a gall is protected from potential predators, like crabs, shrimps, and fishes (Stella et al., 2011). The swimming legs of these copepods are not reduced: most likely they are used to maintain the current of water inside the gall (for intensive gas exchange). In addition, the water current generated by the copepod affects the formation of the gall itself; expansion of the cavity occurs due to a chemical

31 dissolution of the calcareous skeleton and/or due to the mechanical action exerted by the movement of copepods legs (Stock, 1984; Dojiri, 1988).

Although it is a very interesting case of symbiosis/parasitism, the diversity of gall- inducing species remains poorly studied. There are only six sporadic records of different genera of copepods from the family Anchimolgidae (Allopodion Humes, 1978 and

Haplomolgus Humes & Ho, 1968) and Rhynchomolgidae (Isomolgus Dojiri, 1988,

Pionomolgus Dojiri & Grygier, 1990, Spaniomolgus Humes & Stock, 1972, and

Xenomolgus Humes & Stock, 1972) described living in the galls of scleractinian corals.

Siphonostomatoid copepods are also known to induce gall growth, but only in hydrocorals (Stock, 1981; Stock, 1984). However, with the new records of the coral galls caused by symbiotic copepods, they are now considered to be much more common than originally thought (Kim & Yamashiro, 2007).

The first record of the gall-inducing copepod on Scleractinia was made in

Indonesia. Isomolgus desmotes, a new genus and species of the family Rhynchomolgidae, was found in galls of the coral Seriatopora hystrix Dana, 1846 (Dojiri, 1988). The next discovery of this kind was a copepod from the same family, Pionomolgus gallicolus

Dojiri & Grygier, 1990 (also a new species and genus), described from the scleractinian coral Echinopora lamellosa (Esper, 1795) from Australia (Dojiri & Grygier, 1990). The next record of gall-inducing species was made only 17 years later. An additional two species of poecilostomatoid copepods Allopodion ryukyuensis Kim I.H. & Yamashiro,

2007 and Xenomolgus varius Humes & Stock, 1972, were noted in the galls of scleractinian corals Montipora informis Bernard, 1897 and Porites sp., respectively, in

Okinawa, Japan (Figure 1.4) (Kim & Yamashiro, 2007). Another report is from Taiwan,

32 where two species of the gall-inducing copepods, Allopodion ryukyuensis and

Haplomolgus montiporae Humes and Ho, 1968, were recorded from Montipora aequituberculata Bernard, 1897 (Cheng et al., 2010). The most recent discovery of gall- inducing copepods in madreporarian corals were made in the Saudi Arabian Red Sea.

Copepods of the genus Spaniomolgus were found in galls on the branches of Stylophora pistillata (Figure 1.3) (Ivanenko et al., 2014).

1.5.2 Feeding preferences

For most symbiotic crustaceans, the mechanism of feeding and the feeding preferences are poorly understood (Baeza, 2015). It is generally accepted that copepods living on corals consume their secretory products (mucus and fat bodies) supplemented by small amounts of tissue and exogenous food captured by the host, including organic detritus and the bacteria that grow in it (Stock, 1975; Humes, 1985a; Dojiri, 1988; Glynn &

Enochs, 2011). However, there are very few descriptions of the feeding behavior of these copepods in the literature. For instance, Jan Stock observed copepods from the family

Corallovexiidae eating the tissue of its host coral (Stock, 1975). A Xarifia copepod was also reported tearing coral polyp tissue with its claws (Humes, 1960; von Salvini-Plawen,

1972). Furthermore, an additional source of food for xarifiid copepods was discovered recently. The guts of Xarifia fissilis were reported to contain zooxanthellae algae, which remained photosynthetically active for a long time, and, therefore, possibly fed the copepod (Cheng & Dai, 2010).

1.5.3 Evolutionary traits and adaptations to symbiosis and parasitism on a coral

Copepods have undergone a series of evolutionary changes to adapt for life in association with corals in various ecological niches. Symbiotic copepods evolved from the free-living forms, and some of them independently developed such adaptations as transformation of the body shape, development of the attachment organs in adult and copepodid stages, modification of the mouthparts, development of dwarf and parasitic males, abbreviation or loss of naupliar stages, etc. (Ho, 2001). Although copepods living on the surface of the coral tissue typically possess a relatively untransformed body shape (Figure 1.5A), they have antennae adapted to attach to the host and mouthparts adjusted for feeding on coral.

Parasitic copepods, on the contrary, have elongate (up to 3 mm), vermiform, highly modified bodies with weakly defined segmentation and reduced appendages (Figure

1.5B-D) (Glynn & Enochs, 2011). Different orders of copepods went through different modifications of the mouthparts, for example, Poecilostomatoida and Cyclopoida have a wide, open buccal cavity with mandibles adapted for biting and scraping, while

Siphonostomatoida has a conical, siphon-like mouth formed by the labrum and labium with stylet-like mandibles adapted for sucking (Ho, 1994).

Figure 1.5 Different body forms of copepods associated with scleractinian corals: (A) Asteropontopsis faviae (Asterocheridae), dorsal view of female; (B) Corallonoxia longicauda (Corallovexiidae), dorsal view of female; (C, D) Xarifia exserens (Xarifiidae), dorsal (C) and lateral (D) views of female (from Glynn & Enochs, 2011, original drawings from Stock, 1987 and Humes, 1985b).

Gall-forming copepods also have a number of specific morphological features distinguishing them from ectosymbiotic copepods. The copepod inside the gall is not attached but does not move much. Gall copepods usually have rounded, spherical body shape (Figure 1.3D), its biological advantage is not completely clear, but it is likely that this form provides freedom of movement in 3 dimensions in the gall cavity (Dojiri,

1988). Adult females are not able to leave the gall, but nauplii and males seem to be able to penetrate through the small holes in its wall. These holes allow males to enter the gall for mating and allow juveniles to come out from the gall (Kim & Yamashiro, 2007). For the stylasterine hydrocorals it is reported that both and female individuals are confined in

35 the gall, and the first arriving copepodid becomes a female, the second a male (Stock,

1984).

Apart from morphological features, copepods developed another important evolutionary adaptation for parasitizing/dwelling on corals. Apparently, they are

"immune" to coral nematocysts, though the mechanism of this is still unknown (Ho,

2001). An interesting observation was made on copepods living on sea anemones: copepods, naturally associated with Anemonia sulcata, were able to move freely among its tentacles, while other species of copepods either avoided the polyps or were paralyzed and digested (Humes, 1985a).

1.5.4 Effect on coral hosts

The majority of the endosymbiotic copepods of corals are most likely to be parasitic species. Moreover, it is conceivable that even ectosymbiotic copepods living on the surface coral tissue could cause a modest negative effect on hosts and some of them could be classified as parasites (Stock, 1975; Glynn, 1988; Glynn & Enochs, 2011).

There are reports of infestation of aquarium stony corals of genus Acropora caused by the copepod pest Tegastes acroporanus Humes, 1981, which can cause “loss of coral coloration, poor polyp extension, and loss of axial corallites resulting in the cessation of growth” (Sweet et al., 2012; Christie & Raines, 2016). There is also a hypothesis that the process of the coral bleaching could be exacerbated by the activity of parasitic copepods, but again there is not enough information about the relationship between copepod infestation and coral bleaching (Cheng et al., 2016). However, this hypothesis has not been experimentally tested for any species.

Symbiotic copepods could negatively influence the health of their coral hosts in other ways. For example, copepods could potentially transfer pathogenic agents to their hosts, however this has not been recorded to date. In a recent study, the microbiomes of the gall-inducing copepods, the gall-tissue, and the normal coral tissues were compared, and it was shown that the microbial community of the gall tissues was not directly affected by the microbiome of the gall-forming symbiotic copepods, though it was different from the bacterial community of the healthy coral tissue (Shelyakin et al.,

2018). Furthermore, cultivation of these copepods is not possible for most species without long-term maintenance of host corals, which is unfortunately still a major obstacle in many research laboratories. Even where aquaria facilities are suitable, re- creation of truly natural environments is unlikely. Rearing of symbiotic copepods through their entire life cycle has never been achieved.

Although the relationships between copepods and their coral hosts are not well known and further investigations are needed, understanding these interactions could be an important step on the way to developing an explanation of such phenomena as host- specificity. Moreover, this will allow us to estimate whether and which symbiotic copepods have a positive, neutral, or negative effect on their coral hosts. While the discussion in the preceding section is mainly about the ecological and biological component of the copepod-coral symbiosis, we need to keep in mind that our primary goal is to study species diversity and species composition of copepods associated with corals, since we will not be able to understand the interactions between these animals without knowing which species are involved in this relationship.

1.6 Molecular methods in the studies of diversity and taxonomy of copepods

Traditional taxonomic classification of symbiotic copepods has been based on various morphological features, which are highly variable from group to group, and require time- consuming, detailed observations and illustrations using microscopy (Blanco-Bercial et al., 2011). These have included anatomical morphometrics, the structure of mouth parts, segmentation and armoring of antennae, mouth parts, swimming legs, etc. Studying morphology and conducting morphological identification typically requires painstaking detailed observations and illustrations using microscopy. Nevertheless, all previous studies on copepods associated with invertebrates, including scleractinian corals, were based solely on morphological methods. In recent years, DNA-based taxonomic methods have proven to be relatively fast, reliable, and accurate tools to assess copepod biodiversity (Blanco-Bercial et al., 2014; Baek et al., 2016). New genetic approaches allow us to very efficiently identify described species and reveal potential new species in large sets of samples consisting of thousands of specimens. Molecular tools significantly facilitate the process of identification and species delimitation, can give us more options in the data processing, and provide more reliable information about the actual species diversity (Hebert et al., 2003; Hajibabaei et al., 2007; Frezal & Leblois, 2008; Fontaneto et al., 2015).

On the other hand, interpreting the validity or significance of genetic differentiation can be difficult if conducted in isolation from actual study of the specimens. Revisions and new species descriptions solely based on genetic analysis may be questioned (Will &

Rubinoff, 2004; Lajus et al., 2015). Therefore, it remains important to consider molecular and morphological work together. Thus, by far the most promising methods in copepod

38 taxonomy include a combination of molecular markers and morphological analysis

(Wyngaard et al., 2010; Cornils & Blanco-Bercial, 2013).

Molecular analyses of microscopic small-sized copepods (typically with body length less than 1-2 mm) usually involve the complete destruction of the specimens, leaving only photographs of vouchers, which cannot be a reliable source of information for species determination (particularly when detailed morphometric examination is needed). Using other specimens of the same species for extraction is not always reliable, especially in the case of cryptic or pseudocryptic species. Extraction from a body part that does not carry key identifying features, usually one of the swimming legs, typically produces an insufficient amount of DNA. Non-destructive DNA extraction methods developed recently make it possible to keep copepod exoskeletons intact and suitable for morphological investigation (Rowley et al., 2007; Cornils, 2014). When describing species, it is crucial to keep a specimen voucher, which could be accessed later (e.g., for redescription or revision of a species, genus, or even family). Plus, some morphological differences at the species level and value of these differences can be reliably determined only with the help of molecular markers. Moreover, morphological description is a rather time-consuming process and can take up to several weeks. Therefore, it is most likely that

DNA will degrade by the time a copepod will be described. Thus, for integrative analysis of the specimens, it is much more convenient to do the genetic analysis first and then conduct a morphological investigation.

1.7 Dissertation structure and objectives

The main objectives of this thesis are: (1) to explore the diversity of different groups of copepods associated with scleractinian corals in different geographical areas and variable habitats; (2) to determine species boundaries of these copepods using both molecular

(three markers: COI, ITS2, 18S) and morphological data; and (3) to reveal patterns of biogeographic distribution, endemism, and host-specificity in these symbiotic systems. In

Chapter 2, I describe the general methodology used throughout my thesis work. Each of my four data chapters contains distinct goals, original scientific contributions, and conclusions.

Chapter 3 focuses on the assessment of the genetic diversity of the copepods associated with a rich fauna of hermatypic corals in Lizard Island in the Great Barrier

Reef, an area of intense focus of marine biodiversity studies. In this chapter, I conduct an integrative study of diversity and host specificity of copepods living on 25 species from

17 genera of corals common in the GBR. Assuming that copepod diversity correlates with the host diversity, numerous species previously unknown for this region (and likely new to science) are expected to be found.

Chapter 4 focuses on the patterns of biogeographical distribution of copepods associated with Galaxea corals, a common and widespread genus throughout the Indo-

Pacific. Reducing the number of analyzed host taxa to one genus allows me to conduct a deeper analysis of the effect of host species on the distribution and diversity of the copepods, and vice-versa.

Chapter 5 focuses on the diversity and patterns of distribution of poecilostomatoid copepods (Cyclopoida) associated with corals of the family Pocilloporidae in the Saudi

Arabian Red Sea. Copepods from order Poecilostomatoida are common and well-known symbionts of scleractinian corals. Many species in this group are associated with the family Pocilloporidae, a family of corals that exhibits a high level of morphological variability within and among its members. Studying copepod fauna of these corals will allow assessment of the effect of the differences in morphotypes, environmental gradients, and spatial arrangement of the hosts on the species diversity and distribution of this group in different regions (central and southern Red Sea).

Chapter 6 focuses on a species-level diversity assessment of the copepods collected from Fungiidae corals in the Red Sea. This family of hosts is a closely related group of species with very similar ecologies. This presents an opportunity to explore co-evolution and host-specificity within a taxon which contains relatively high host species diversity but eliminates some of the potentially confounding effects of ecological variability within or among host species.

This thesis does not qualify as a published and permanent scientific record for purposes of zoological nomenclature.

Chapter 2: General methodology

2.1 Sampling procedures

The coral samples were collected by scuba diving using hammer and chisel on depths from 1 to 35 meters (detailed sampling information is provided in each data chapter). The colonies were photographed underwater (before collection) and then placed into a plastic bag underwater. After returning to the boat or lab, 96% ethanol was added to each sample until the overall solution reached a 10% concentration of ethanol. Colonies were kept in the dark in this solution for 15-20 minutes for relaxation and detachment of the symbionts from the coral surface and expulsion of the copepods from the polyps. Then all coral symbionts were removed from the host by shaking the bag. The precipitate with anesthetized symbionts was passed through a fine sieve (100 microns). Copepods were sorted out from the residue by a pipette under a dissecting microscope, preserved in 96% ethanol, and stored at -20°C for the further morphological and molecular studies.

Additionally, coral colonies were examined for the presence of modified corallites and galls potentially containing copepod, which were dissected using scalpel and tweezers, and copepods were extracted using needles. Small parts of coral tissues (1-2 cm3) were taken for genetic analysis. After all preparations, corals were bleached in sodium hypochlorite for 48 hours. Each bleached skeleton was washed, dried, labeled, and photographed with a scale for the corallite morphology. In the laboratory, all copepods were sorted, identified to putative morphotypes, numbered, photographed under a stereo microscope (Carl Zeiss™ Stemi 2000-C), and transferred individually into separate 1.5 ml tubes with 96% ethanol.

2.2 Morphological analyses

2.2.1 Light microscopy

The preliminary identification of the specimens to the order or family level was conducted under a stereomicroscope (Carl Zeiss™ Stemi 2000-C). During this process, I took photographs of every specimen and cataloged them in my database. The detailed morphological identification for most of the specimens was conducted after the results of the molecular analyses were obtained. For light microscopy, the exoskeletons were studied by applying the ‘hanging drop method' described in detail by Ivanenko and

Defaye (2004). The copepods were dissected under a Leica MZ12 microscope and stained with chlorazol black. Temporary slides were mounted using regular glass slides, and coverslips were attached with small balls of plasticine. The exuvia were placed on a cover slip in a small drop of lactic acid and studied with a Leica DMR compound microscope having bright-field and differential interference contrast optics. Then the cover slip with the exuvia and small balls of plasticine attached to the corners of the glass was turned over and mounted on a glass slide so that the exuvia did not touch the glass slide. The exuvia can be rearranged under the dissecting microscope after removing and turning over the slide. For long-term preservation, the samples were transferred on slides in glycerol.

In general, diagnostic characters assessed include anatomical morphometrics, the structure of mouth parts, and segmentation and armoring of antennae, mouth parts, and swimming legs. Taxonomic identifications were made based on published taxonomic descriptions.

2.2.2 Scanning electron microscopy

For scanning electron microscopy (SEM), copepods were dehydrated through increasing ethanol concentrations; critical point dried, mounted on aluminum stubs, coated with gold, and examined in a CamScan SEM (CamScan Electron Optics Ltd, London, UK) at the Faculty of Biology of Lomonosov Moscow State University. The bleached fragments of corals were mounted on metal stands using glue, coated with a conductive gold film and examined with the same SEM.

2.2.3 Confocal microscopy

For confocal microscopy, exoskeletons were individually transferred to distilled water and then stained with Fuchsin. The staining procedure and mounting method were adapted from Ivanenko et al. (2012) and Corgosinho et al. (2018). The copepods were inspected at Lomonosov Moscow State University on an inverted Nikon A1 confocal laser scanning microscope (CLSM, Nikon Corporation, Tokyo, Japan), using a 40× oil immersion objective and lasers with wavelengths of 532 and 640 nm. The laser power was set to 60%. The amplitude offset and detector gain were manually adjusted. CLSM image stacks were obtained throughout the whole animal, and the scanning software was adjusted to perform the optimal number of scans. Image size was set for 2000×2000 dpi, and the reconstruction of the external anatomy was obtained by maximum projection.

The final images were adjusted for contrast and brightness using the software Adobe

Photoshop CS4 (Adobe Systems, San Jose, CA, USA).

2.2.4 Coral identification

Identification of the coral species followed the descriptions and photographs of colonies in Veron (2000) and was verified by various coral experts specializing in respective taxonomic groups. Corals of the family Fungiidae were identified by Dr. Bert W.

Hoeksema, coral colonies collected during the CReefs project in 2010 were identified by

Mary Wakeford, coral colonies from the families Euphylliidae and Pocilloporidae were identified with the help of Dr. Roberto Arrigoni and Tullia Terraneo. Photos of the live coral colonies underwater and their bleached skeletons are provided in appendices of the corresponding chapters (Appendices 3.2, 4.2, 5.3, 6.3).

2.3 Molecular-phylogenetic analysis

2.3.1 DNA extraction

We used a non-destructive method of DNA extraction with proteinase K modified from the method described by Cornils (2014) and Porco et al. (2010). This method allows to dissolve and remove soft tissues of the copepod without damaging the external skeleton, which is used for the further detailed morphological analysis and preserved as a specimen voucher.

Ethanol was removed from each tube with a copepod by pipetting, and 100 µl of the lysis mix was added to each sample. Lysis mix contained lysis buffer (30 mM Tris-

HCl, 20 mM EDTA), 1% SDS (sodium dodecyl sulfate), and 0,1 mg/mL Proteinase K.

Tubes with copepods in the lysis solution were incubated at 37°C for 2-3 hours. After the incubation solution was transferred to clean 1.5 ml tubes and processed with a standard silica-based DNA extraction kit (Diatom™ DNA Prep, Isogene). An aliquot of 100 of L

45 of ethanol and glycerol in a 2:1 ratio was added to the tubes with copepod exoskeletons for the further storage.

2.3.2 Markers selection and DNA amplification

Projects assessing community assemblages or faunal diversity, where datasets include representatives from different taxonomic groups, require universal markers that provide comparable information for most of the copepod taxa present in the samples. Moreover, molecular studies dealing with small-sized copepods must use genetic markers present in many copies per cell, for example, mitochondrial or ribosomal DNA markers, as single- copy genes could be difficult to amplify due to the small amount of DNA per specimen.

In a number of recent studies on microscopic animals, a multilocus approach was used for species delineation and diversity assessment as it provides more accurate and reliable results than analysis of only one marker (Cornils & Blanco-Bercial, 2013; Fontaneto et al., 2015).

Three markers were amplified for copepod specimens: protein-coding mitochondrial gene cytochrome c oxidase subunit 1 (COI), nuclear rDNA internal transcribed spacer 2 (ITS2), and ribosomal gene small subunit 18S. The mitochondrial cytochrome oxidase c subunit I gene (COI) and the internal transcribed spacer 2 (ITS2) region located in nuclear ribosomal DNA have almost become a standard minimum combination for these kinds of analyses. COI is one of the most conserved protein-coding genes in the animal mitochondrial genome and is nearly universally used as a standard barcode region for metazoans with a few exceptions (Bucklin et al., 2011) and has been successfully used for DNA barcoding of copepods (Blanco-Bercial et al., 2014; Baek et

46 al., 2016). ITS2 is a non-coding region and, therefore, it is not exposed to the pressure of natural selection and is highly variable, which allows discrimination among closely related species. COI and ITS2 markers are widely used for species delimitation and for revealing cryptic species among different groups of arthropods, including copepods

(Rocha-Olivares et al., 2001; Bucklin & Frost, 2009), parasitoid flies (Smith et al., 2006;

Joy & Crespi, 2007), mosquitoes (Alquezar et al., 2010), amphipods (Flot et al., 2010b), beetles (Fossen et al., 2016), and others. For phylogenetic relationships analyses within

Copepoda, the 18S gene is used (Kim & Kim, 2000; Huys et al., 2006; Huys et al., 2007;

Song et al., 2008; Wu et al., 2015). The 18S rRNA gene combines highly conserved regions, used for sequences alignment, with variable and phylogenetically informative areas, which are used to determine the phylogenetic position of the group in the tree.

Copepod-specific primers were designed to improve the amplification success rate and avoid contamination with the host DNA, which is often a problem when barcoding symbiotic and parasitic organisms. Design of the copepod-specific primers were conducted by creating alignments of sequences (crustaceans and corals) from National

Center for Biotechnology Information (NCBI) GenBank and BOLD (Barcode of Life

Data System) and choosing the gene regions which are conservative for all copepods and at the same time have clear distinctions from other groups of crustaceans and especially corals. Initial amplification of copepod DNA with standard mtCOI primers (Folmer et al.,

1994) did not give the satisfactory results. These standard primers worked for only 40% of samples. After testing several of the newly-designed copepod-specific primers and optimization of the protocol, DNA was successfully amplified in about 75% of samples.

Given these results, the amplification of the Folmer fragment of the COI gene (700 bp)

47 was performed using copepod-specific forward primer LCO1490cop3 and universal reverse primer jgH2198 (Geller et al., 2013). ITS2 region (500 bp) was amplified using a pair of copepod-specific primers 58d-cop and 28r1-cop. For the amplification of the

18S ribosomal gene 18d5 (Aleshin, unpublished) and standard Q39 (Medlin et al., 1988) primers were used, with an average length of the sequenced region 650 bp. The primers used for the PCR amplifications are listed in Table 2.1 and were performed using

QIAGEN Multiplex PCR Kit in the PCR cycler Mastercycler® pro S.

Table 2.1 List of primers used in PCR. Primers used in sequence reactions and included in analyses are marked as bold.

Gene Primer name Sequence Reference LCO1490 5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′ (Folmer et al., 1994) LCO1490cop 5′-GGT CYT GYA ATC ATA AAG ATA TTG G-3′ unpublished LCO1490cop2 5′-TCI TGI AAY CAY AAA GAY ATY GG-3′ unpublished LCO1490cop3 5′-TCI TGI AAY CAY AAA GAY ATY GGI AC-3′ (Ivanenko et al., 2018) COI HCO2198 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′ (Folmer et al., 1994) jgHCO2198 5′-TGR TTY TTY GGW CAY CCW GAR GTT TA-3′ (Geller et al., 2013) mlCOIintF 5′-GGW ACW GGW TGA ACW GTW TAY CCY CC-3′ Leray et al., 2013 mlCOIintR 5′-GGR GGR TAS ACS GTT CAS CCS GTS CC-3′ Leray et al., 2013 CytB-F406 5′-CCI TGR GGI CAR ATR TCT TTY TG-3′ CytB-F424 5′-TTY TGR GGI GCI ACI GTI ATT AC-3′ CytB CytB-R827 5′-TTY TGR GGI GCI ACI GTI ATT AC-3′ CytB-R851 5′-CGY ARR ATI GCR TAI GCR AAY A-3′ 58-dcop 5′-CAG TGG ATC AYT TGG CTC GGG GG-3′ (Ivanenko et al., 2018) ITS2 28-r1cop 5′-CAT TCG CCA TTA CTA AGG GRA TCA C-3′ (Ivanenko et al., 2018) 12S-L13337 5′-TTA CTT TGC TAC GAC TTA TCT C-3′ 12S 12S-H13842 5′-AGT GCC AGC AIY CGC GG-3′ 16S-F752 5′-AAA CTG TTT ATC AAA RAC WTA G-3′ 16S-F845 5′-GTA GCN IAA TAA YTW GTT TAT TAA TTG-3′ 16S 16S-F979 5′-RGA CGA KAA GAC CCT AAR A-3′ 16S-R1278 5′-CGT CGA TYT TAA CTC AAA TCA TG-3′ 18d5 5′-AAA CTT AAA GGA ATT GAC G-3′ Aleshin, unpublished 18S Q39 5′-GAA TGA TCC WTC YGC AGG TTC ACC TAC-3′ (Medlin et al., 1988)

The following PCR conditions were used in the amplification of COI gene: initial denaturation for 15 min at 95°C; continued by 45 cycles of 94°C for 20 s, annealing at

46°C for 60 s, 72°C for 60 s; followed by a final extension at 72°C for 5 min.

Amplification of the ITS2 fragment: 95°C for 15 min; continued by 37 cycles of 94°C for

20 s, annealing at 55°C for 30 s, 72°C for 90 s; followed by a final extension at 72°C for

5 min. Amplification of 18S: 95°C for 15 min; continued by 37 cycles of 93°C for 20 s, annealing at 50°C for 30 s, 72°C for 60 s; followed by a final extension at 72°C for 5 min. PCR products were purified with 1μl of Illustra ExoStar 1-Step (GE Healthcare) and sequenced in forward and reverse directions by Sanger sequencing technology on an ABI

3130XL Genetic Analyzer.

2.3.3 Sequence alignments and phylogenetic analyses

Geneious 7.1.3 (Kearse et al., 2012) was used to automatically assemble forward and reverse sequences. After that, all contigs were inspected and edited manually. In the case of ambiguous bases and double peaks, the appropriate International Union of Pure and

Applied Chemistry (IUPAC) codes were used to rename them. Sequencher (Gene Codes) was used to automatically call secondary peaks, to manually check the ambiguous positions and to produce sequences which can be split into haplotypes in online software

Champuru (Flot, 2007) and SeqPhase (Flot, 2010). The haplotypes of length-variant ITS2 heterozygotes were reconstructed using Champuru. Phasing heterozygous ITS2 sequences of the same length with several double peaks and reconstructing haplotypes was done in SeqPhase. Both alleles of phased heterozygotes were included in the alignments and used in further analyses.

All sequences were aligned in Geneious with MAFFT (Katoh et al., 2002; Katoh &

Standley, 2013) under default settings. COI sequences were aligned as amino acids while sequences obtained for the two other markers were aligned as DNA. COI alignments were checked for potential mistakes in the reading frame and stop codons. Most of the sequences were obtained for the complete fragments. The ends of alignments were trimmed to remove low-quality parts and primers. The National Center for Biotechnology

Information’s Basic Local Alignment Search Tool (NCBI BLAST) was used to check and identify distinct sequences. PAUP (Swofford, 2003) and MrModelTest 2.3

(Nylander, 2004) with Akaike Information Criterion (AIC) were used to find and select the best-fit substitution model for use in analyses. The concatenated alignments were generated from individual alignments of each marker (COI, ITS2, and 18S) using the

SequenceMatrix 1.8 (Vaidya et al., 2011).

The analyses of the alignments were done using Bayesian inference and maximum likelihood methods. The DNA sequence of 18S of a Platycopioida copepod was used as an outgroup (from Khodami et al., 2017). Bayesian analysis was conducted using

MrBayes 3.2.2 (Ronquist & Huelsenbeck, 2003; Ronquist et al., 2012) on CIPRES

Science Gateway (Miller et al., 2010). Two runs of four Markov Chain Monte Carlo

(MCMC) chains were run and sampled every 100 generations for five million generations. The initial 25% of sampled trees were discarded as burn-in, and the remaining trees were used to construct a 50% majority-rule consensus tree. Average standard deviation of split frequencies for all analyses run was less than 0.05, therefore, the trees can be considered convergent. ML analysis was done using RAxML method

(Stamatakis, 2014) on CIPRES Science Gateway under standard settings (Miller et al.,

2010).

2.3.4 Species delimitation procedures

In the analysis of copepod diversity, we used molecular operational taxonomic units

(MOTUs) to identify species richness in the different geographical regions. To define

MOTUs in my samples, I used three species delimitation approaches.

To reveal the DNA barcoding threshold in my dataset the Automatic Barcoding

Gap Discovery tool (ABGD) was used (http://wwwabi.snv.jussieu.fr/public/abgd/)

(Puillandre et al., 2012). ABGD analyses were performed by uploading COI and ITS alignments separately to test for the existence of a barcode gap in the genetic distances and to identify groups of individuals united by shorter genetic distances than the gap.

These groups were considered as MOTUs. Since the method is based on the genetic distances calculated based on one marker, this approach was used only with the alignments of individual genes and not with the concatenated dataset. Distance matrices and barcoding gaps were calculated under the default settings, the relative gap width (X) was set to 1.0.

Poisson-Tree-Processes (PTP) analyses were conducted on the bPTP web server

(https://species.h-its.org/ptp/) (Zhang et al., 2013). The method searches for evidences on independently evolving entities by optimizing the differences in branching patterns between and within species. Each analysis was run for 500,000 MCMC generations, with a thinning every 100 generations and a burn-in of 0.25. The output from both PTP-ML and PTP-h analyses was used in the species delimitation analysis. Ultrametric trees for

Generalized Mixed Yule Coalescence (GMYC) were built using BEAST (Fujisawa &

Barraclough, 2013) on CIPRES Science Gateway (Miller et al., 2010). Input alignment files for BEAST were created using BEAUTi (Drummond et al., 2012). Different analyses with different combinations of clock and branching were run, and the tree Yule model with the strict molecular clock was chosen. The analysis was run on GTR + G + I model of molecular evolution for 100 million generations with a sampling frequency of

5,000. The output of Bayesian inference was analyzed, and the effective sample size

(ESS) was checked in Tracer v1.6.0 (Rambaut et al., 2014). TreeAnnotator v1.8.2 was used to summarize the posterior distribution and to build the maximum clade credibility tree (Rambaut & Drummond, 2015). The resulting ultrametric tree was uploaded to the

GMYC web server (https://species.h-its.org/gmyc/), and the GMYC analysis was conducted online. Results from both single threshold (ST-GMYC) and multiple threshold

(MT-GMYC) methods were evaluated (Pons et al., 2006; Monaghan et al., 2009).

Haplotypes of heterozygous specimens obtained with Champuru and SeqPhase for

ITS2 nuclear marker were used to build haplotype networks and to delineate species which were reproductively isolated (Flot et al., 2010a). Connections between haplotypes of the same individuals on the tree were added manually, and then the tree was inspected for the discrete groups of haplotypes on the tree, and, therefore, potential species boundaries.

Chapter 3: Genetic diversity of copepods associated with scleractinian corals in the Lizard Island (GBR)

3.1 Introduction

The Great Barrier Reef (GBR) of Australia, although technically lying beyond the boundary of the Coral Triangle, is nonetheless one of the diversity hotspots of marine organisms, where nearly 1500 species of reef fishes and more than 400 species of reef- building corals are encountered (compared to the 1705 species of fishes and 553 species of scleractinians in the Coral Triangle) (Allen, 2008; Veron et al., 2009). Many studies on the biodiversity of non-scleractinian invertebrates from all parts of the GBR have been conducted (Patton, 1994). However, fauna of copepods associated with scleractinian corals in the largest coral reef ecosystem on Earth still remains poorly studied (Humes,

1991).

Currently, 49 species of copepods are described living in association with hermatypic corals of the GBR (from the genera Acropora, Astrea, Echinopora, Galaxea,

Gardineroseris, Goniastrea, Hydnophora, Leptoria, Merulina, Montipora, Oulophyllia,

Pocillopora, Seriatopora, and Stylophora) (see full information in Appendix 3.1)

(Humes, 1985b, 1991, 1992; Kim, 2004a). The majority of these copepod species were reported from the Big Broadhurst Reef (500 km from Lizard Island) and belong to two poecilostomatoid families Anchimolgidae (26 species) and Xarifiidae (18 species), and

22 of them have been described as symbionts of Acropora corals (Humes, 1985b, 1991;

Kim, 2004a). There is also one species of gall-inducing poecilostomatoid copepod belonging to the family Rhynchomolgidae found on Echinopora lamellosa (Esper, 1795)

(Dojiri & Grygier, 1990). The remaining four species belong to Asterocheridae

(Siphonostomatoida), Cyclopidae (Cyclopoida), and Peltidiidae (Harpacticoida).

Although a number of copepod species are described from this area, they should be still considered as an understudied group because most of the coral species have not been checked for copepod associates yet. In the major review of copepods from scleractinians from the GBR, Humes (1991) reported symbiotic copepods for only 13 genera of hard corals, and indicated that many other copepod associates of remaining coral genera are yet to be discovered. Based on the recent review of all copepods associated with scleractinian corals conducted by Cheng (2016), only 23 species of 14 genera of scleractinian corals are currently known to be hosts of symbiotic copepods in the GBR, which is about the 6% of coral diversity in this region. It is likely that further study of additional coral genera would reveal additional copepod-coral associations.

This chapter focuses on Lizard Island, which is located in the in the northern region of the Great Barrier Reef, where 402 species of scleractinians have been recorded (Veron et al., 2009). Lizard Island includes a variety of reef environments, and it has been used as a model site in different biodiversity and ecology studies since the Lizard Island

Research Station was established in 1973 (now supported by the Lizard Island Reef

Research Foundation).

Unfortunately, the GBR was recently affected by high temperatures and consequently experienced severe bleaching events, which is considered to be a result of a particularly strong El Niño occurring on top of already increased global temperatures

(Hoegh-Guldberg & Ridgway, 2016; Hughes et al., 2018b). The northern part of the

GBR, including Lizard Island, appears to be one of the most affected areas (Heron et al.,

2017). According to recent surveys, from 60% and perhaps up to 80% of corals were severely bleached in this area (Hughes et al., 2017c; Hughes et al., 2018a; van Oppen &

Lough, 2018) and 67% did not recover (Hughes et al., 2017a). Moreover, there are great chances that the majority of corals will not recover from such severe bleaching and will die, impacting a variety of fishes and invertebrates associated with them, including many endemic species. In many cases, loss of a single coral host species leads to the disappearance of some of its associated fauna (Munday, 2004; Hoeksema et al., 2012).

Luckily, we possess a unique collection of copepods collected from 94 colonies of stony corals from Lizard Island sampled within the Census of Coral Reefs Ecosystems

(CReefs) project in 2010 (http://www.creefs.org) (stored in MSU, Moscow, Russia). In this chapter, I analyze copepod fauna of the several most common coral genera and conduct the genetic assessment of its diversity. Based on a previously observed correlation of coral-associated symbiont diversity with host diversity (Stella et al., 2011),

I expected to find a large number of species previously unknown for this region and, potentially, species and genera previously unknown to science. Thus, the symbiotic fauna of scleractinian corals from Lizard Island can serve as a starting point for describing the diversity of this group in the Great Barrier Reef and potentially for other reefs in the western Pacific. Furthermore, the obtained data will be used as a “baseline” for comparison with other regions' diversity.

3.2 Samples and study sites

For the analysis, I chose 26 coral colonies representing 25 species in 18 genera

(Acropora, Astreopora, Ctenactis, Echinopora, Fungia, Galaxea, Herpolitha,

Lobophyllia, Merulina, Montipora, Pachyseris, Pectinia, Platygyra, Polyphyllia, Porites,

Seriatopora, Stylophora, and Tubastraea). Eleven of these genera have never been assessed for copepod symbionts. See Table 3.1 and Appendix 3.2 for the sampling information and Figure 3.2 for the sampling locations. This collection enabled me to conduct a general assessment of fauna of coral-associated symbiotic copepods of the

Lizard Island.

Figure 3.1 Map showing the locations from which coral samples were obtained (shown with red circles); basemap obtained from Google Maps and http://d-maps.com/.

Table 3.1 List of the coral colonies collected, with the information about the depth and location of the sampling and the number of copepods collected from each host (see full sampling information in the Appendix 3.2). Voucher Depth # Coral family Coral species Locality number (m) 1 AU-1854 Acroporidae Acropora aculeus cf. North Direction Island 27 2 AU-1892 Acroporidae Acropora humilis Between Palfrey & South Islands 5.5 3 AU-1890 Acroporidae Acropora loripes Between Palfrey & South Islands 5.5 4 AU-1942 Acroporidae Acropora loripes cf. Mermaid Cove 7.8 5 AU-1906 Acroporidae Acropora nasuta cf. Martin Reef 5.2

6 AU-1944 Acroporidae Acropora palifera Mermaid Cove 7.8 7 AU-1851 Acroporidae Acropora pulchra North Direction Island 13.9 8 AU-1882 Acroporidae Acropora sp. Martin Reef 5.2 9 AU-1849 Acroporidae Astreopora ocellata North Direction Island 13.9 10 AU-1945 Acroporidae Montipora peltiformis Mermaid Cove 7.8 11 AU-1880 Acroporidae Montipora sp. Day Reef 0 12 AU-1889 Acroporidae Montipora sp. Linnet Reef 5.2 13 AU-1894 Acroporidae Montipora sp. Between Palfrey & South Islands 5.5 14 AU-1940 Agariciidae Pachyseris speciosa Mermaid Cove 7.8 15 AU-1901 Dendrophylliidae Tubastraea micrantha Bommie Bay 8.9 16 AU-1902 Dendrophylliidae Tubastraea micrantha Bommie Bay 8.9 17 AU-1924 Dendrophylliidae Tubastraea micrantha 14-141 14.7 18 AU-1814 Faviidae Echinopora lamellosa cf. Linnet Reef 10.4 19 AU-1929 Faviidae Echinopora mammiformis Mermaid Cove 7.8 20 AU-1903 Faviidae Platygyra daedalea cf. Martin Reef 5.2 21 AU-1910 Faviidae Platygyra pini cf. MacGillivray Reef 14.3 22 AU-1941 Fungiidae Ctenactis crassa Mermaid Cove 7.8 23 AU-1863 Fungiidae Danafungia horrida High Rock 20 24 AU-1893 Fungiidae Herpolitha limax Between Palfrey & South Islands 5.5 25 AU-1891 Fungiidae Polyphyllia talpina Between Palfrey & South Islands 5.5 26 AU-1888 Merulinidae Merulina ampliata cf. Linnet Reef 5.2 27 AU-1912 Merulinidae Merulina scabricula cf. MacGillivray Reef 14.3 28 AU-1930 Mussidae Lobophyllia flabelliformis cf. Mermaid Cove 14.3 29 AU-1899 Mussidae Lobophyllia hemprichii Bommie Bay 8.9 30 AU-1862 Oculinidae Galaxea astreata High Rock 12 31 AU-1936 Pectiniidae Pectinia alcicornis North Direction Island 20.7 32 AU-1904 Pectiniidae Pectinia lactuca Linnet Reef 5.2 33 AU-1918 Pocilloporidae Seriatopora hystrix Martin Reef 5.2 34 AU-1943 Pocilloporidae Stylophora pistillata Mermaid Cove 7.8 35 AU-1881 Poritidae Porites sp. Day Reef 15 36 AU-1946 Poritidae Porites sp. Mermaid Cove 7.8

The methodology used in this chapter follows the same protocols as for the rest of the thesis (see Chapter 2 for the methods) except for the DNA extraction, which was conducted in the Canadian Centre for DNA Barcoding (CCDB). The copepod specimens were used for DNA extraction methods testing with the following protocols: standard automated CCDB bind-wash-elute protocol (Ivanova et al., 2006) with proteinase K tissue lysis from the plate and silica-based DNA extraction and HotSHOT alkaline lysis

(Montero‐Pau et al., 2008). Both were used in combination with a voucher recovery protocol (Porco et al., 2010). For PCR amplification and sequencing of COI gene, standard LCO1490 and HCO2198 primers (Folmer et al., 1994) were used. Amplification of the COI gene was conducted under the following PCR conditions: preheat for 2 min at

94°C; continued by 38 cycles of 94°C for 20 s, annealing at 45°C for 20 s, 72°C for 60 s; followed by a final elongation at 72°C for 5 min (Ivanova & Grainger, 2007). PCR amplification and sequencing of ITS2 and 18S regions were conducted according the protocol described in chapter 2. DNA subsamples of coral hosts are deposited in the

Ocean Genome Legacy (Boston, USA) and the University of the Ryukyus (Okinawa,

Japan). All information about collected copepods and their hosts is stored in the BOLD database.

3.3 Results

3.3.1 PCR and sequencing

I studied 164 copepod specimens representing 80 morphotypes. These are the selected most abundant morphotypes of males and females that were found in my samples. For

146 of these specimens (representing 70 morphotypes), sequences of at least one marker were obtained. There was no significant difference in barcode recovery between the two methods of DNA extraction used: 89% (66/74) on plate 10753 and 82% (78/90) on plate

10754. However, HotSHOT alkaline lysis in combination with voucher recovery protocol in contrast to standard automated CCDB bind-wash-elute protocol allowed to preserve

80% of vouchers (instead of 10%) in a condition suitable for morphological studies. The

COI gene (700 bp) was successfully amplified for 108 of the 146 samples (73.9% success rate); ITS2 region (500 bp) was successfully amplified for 117 of the 146 samples (80.1% success rate); 18S rRNA (650 bp) gene was successfully amplified for

123 of the 146 samples (84.2% success rate).

Based on the examination of the external morphologies and the analysis of the 18S phylogeny, copepods of all 70 morphotypes were determined to belong to three families:

Asterocheridae (Siphonostomatoida), Anchimolgidae and Xarifiidae (Poecilostomatoida).

3.3.2 Genetic distances and species delimitation

Analyses were conducted separately for each of the three copepod families and for two genes, namely, COI and ITS2. The 18S rDNA gene fragments have shown little taxonomic resolution and were used only for phylogenetic reconstructions.

The ABGD analysis was conducted separately for alignments of each of the three families for both COI and ITS2 datasets. The estimates of the COI datasets revealed 35,

10, and 11 MOTUs of Anchimolgidae, Xarifiidae, and Asterocheridae, respectively

(Table 3.2), regardless of the prior intraspecific divergence in range 0.001 – 0.1. ITS2 datasets suggested 37, 10, and 14 MOTUs (Table 3.2) for these families and had shown higher sensitivity to prior intraspecific divergence, with values over 0.01 for

Anchimolgidae and 0.04 for other families resulting in a sharp decrease of the number of

MOTUs.

Table 3.2 Number of MOTUs obtained using different species delimitation algorithms. Algorithm ABGD ABGD ABGD PTP Morphological Dataset COI ITS2 combined concatenated identification Anchimolgidae 35 37 41 44 41 Xarifidae 10 10 10 10 12 Asterocheridae 11 14 15 15 17

The average value of COI intraspecies distances was 1.06% and varied from 0 to

1.9%. Interspecies COI distances of all copepods investigated during the project vary from 10.2% to 45.9% and are characterized by a normal distribution. ITS2 distances in

Asterocheridae and Xarifiidae are smaller, up to 0.9% intraspecies and 4-17% interspecies. ITS2 region of Anchimolgidae copepods showed less pronounced barcoding gap between intraspecies and interspecies distances (0-1% intraspecies and 2-23% interspecies). The discrepancy between COI and ITS2 estimates could be due to lack of the sequences for one of the two markers in some specimens. An output of the ABGD species delimitation analysis showed no conflicts between COI and ITS2 datasets. After combination of the COI and ITS2 estimates with the diversity assessment based on the

18S phylogenetic trees we revealed 41 MOTUs of Anchimolgidae, 15 of Asterocheridae, and 10 of Xarifiidae in my samples (Figures 3.2-3.4).

PTP species delimitation analysis showed output completely consistent with the results of the ABGD analysis for Xarifiidae and Asterocheridae (Figures 3.3-3.4). PTP analysis of the Anchimolgidae dataset revealed 44 MOTUs (three more than in ABGD) due to splitting of Anchimolgidae sp. 20, sp. 24 and sp. 40. Lower estimate (41 MOTU) will be used throughout the thesis (Figure 3.2).

The morphological assessment of species richness is consistent with the molecular analysis. However, boundaries are different between morphotypes and MOTUs for 27 out of 70 morphotypes. Some of these discrepancies occurred because male and female morphotypes were merged into a single MOTU in molecular analysis while they were distinguished morphologically. In some other cases, single morphotypes were divided into several MOTUs.

Figure 3.2 Bayesian phylogeny of the family Anchimolgidae based on the concatenated dataset of COI and ITS2. Intra-specific branches are collapsed. Host specificity is coded by branch color. The columns on the right represent species delimitation based on different methods: ABGD and PTP. Numbers in the brackets represent number of specimens per each MOTU. See Appendix 3.4 for a full MrBayes tree containing all individuals.

Figure 3.3 Bayesian phylogeny of the family Asterocheridae based on the concatenated dataset of COI and ITS2. Host specificity is coded by color. The columns on the right represent species delimitation based on different methods: ABGD and PTP. Numbers in the brackets represent number of specimens per each MOTU. See Appendix 3.5 for a full MrBayes tree containing all individuals.

Figure 3.4 Bayesian phylogeny of the family Xarifiidae based on the concatenated dataset of COI and ITS2. Host specificity is coded by color. The columns on the right represent species delimitation based on different methods: ABGD and PTP. Numbers in the brackets represent number of specimens per each MOTU. See Appendix 3.6 for a full MrBayes tree containing all individuals.

3.3.3 Host specificity

Most of the copepod MOTUs in this study were found on a single host species. The only exception was Asterocheridae sp. 13 which was found on two species of Acropora, A. loripes and A. nasuta. However, given a relatively small sampling size within each coral

62 host species, it is possible that broader host specificity was overlooked for other species.

On the higher host taxonomy levels, there is a prominent phylogenetic association between Acropora corals and anchimolgid copepods. All 14 species of Anchimolgidae collected from Acropora species form a well-supported monophyletic clade on the tree

(Figure 3.2). Three out of four species of anchimolgid copepods from Montipora colonies also fall into one monophyletic clade. However, no such patterns were found for

Asterocheridae and Xarifiidae (Figures 3.3 and 3.4).

Up to six copepod MOTUs were found on the host species Acropora loripes. A diverse copepod fauna (four MOTUs each) was also found on Montipora peltiformis,

Acropora humilis, and Danafungia horrida.

3.4 Discussion

The diversity of only abundant morphotypes of the discovered copepods is higher than the diversity of copepods reported in all previous studies for the GBR region (Table 3.3).

Copepods of the families Anchimolgidae and Xarifiidae are dominant groups in both datasets. However, I found much higher diversity of the copepods from the family

Asterocheridae (Figure 3.5). Only two species of asterocherids were previously reported for scleractinian corals in the GBR, both from genus Montipora (Humes, 1991), while in my samples I found 15 MOTUs of Asterocheridae on seven different genera of corals

(Figure 3.5). Four of these genera (Astreopora, Fungia, Pectinia, and Tubastraea) have not been checked for the associated copepods before in the GBR.

Figure 3.5 Comparison of the diversity of three dominant families between my samples and the data from the literature.

Higher diversity of the anchimolgid copepods also seem to be related to the increased number of coral taxa sampled. This group of copepods was reported as symbionts of nine genera of scleractinian corals (Ctenactis, Echinopora, Fungia,

Herpolitha, Lobophyllia, Pachyseris, Pectinia, Platygyra, Polyphyllia, and Porites) for the first time for the GBR. Xarifiidae has a slightly lower diversity in my samples compared to the literature records (Figure 3.5). This could be due to the fact that nine of

22 species of Xarifia were reported from corals Gardineroseris and Pocillopora (Humes,

1985b, 1992), which are not represented in my samples, and Pocillopora was reported as a host for 20% of described species of Xarifia (Cheng et al., 2016).

Differences in the abundance of the species of these three dominant families between my samples and the data from the literature could be also caused by the differences in the sampling: majority of the colonies used in the current study were collected on the depths from 5 to 20 m, whereas samples from the previous studies were typically collected on snorkel from shallower depths (2 to 3 m) (Humes, 1991). If we

64 compare diversity of copepods only from the host genera, which are present in both literature records and my data (Acropora, Galaxea, Echinopora, Merulina, Montipora,

Seriatopora, and Stylophora), we will see that datasets are almost equal (Figure 3.6).

However, five of these genera are presented by other species in my samples (Table 3.3).

Figure 3.6 Correlation of the copepod diversity with the host diversity in my samples (red) and in data from the literature records (black) for the seven genera present in both datasets (1 - Merulina, 2 - Galaxea, 3 - Stylophora, 4 - Seriatopora, 5 - Echinopora, 6 - Montipora, 7 - Acropora).

The visible correlation between the genus of the host and the diversity of the associated copepods (Figure 3.6) most likely represents the correlation of the copepod diversity with the number of the host colonies. However, I cannot add this parameter as there is no information about the number of colonies collected for some of the literature records. The higher number of the discovered species in my samples could also be due to a higher resolution of molecular methods in assessing copepod diversity.

Overall, symbiotic copepods have been sampled from 26 host species, 12 genera, and five families for the first time for the GBR region. Coral-associated copepods were found on all coral colonies, for the nine species of scleractinian corals copepods were recorded for the first time (Tables 3.3 and 3.4).

Table 3.3 Coral hosts of symbiotic copepods reported from the Lizard Island before and found in this study. Literature First record in this study Taxonomy records Lizard Island Worldwide Family Acroporidae + +

Acropora aculeus/carduus cf. + + Acropora gemmifera (Brook, 1892) +

Acropora humilis (Dana, 1846) + +

Acropora hyacinthus (Dana, 1846) +

Acropora intermedia (Brook, 1891) +

Acropora loripes (Brook, 1892) +

Acropora muricata (Linnaeus, 1758) +

Acropora nasuta (Dana, 1846) + +

Acropora pulchra (Brook, 1891) + +

Acropora sarmentosa (Brook, 1892) +

Acropora sp. +

Acropora squarrosa (Ehrenberg, 1834) +

Astreopora ocellata Bernard, 1896 +

Isopora palifera (Lamarck, 1816) + Montipora aequituberculata Bernard, 1897 +

Montipora peltiformis Bernard, 1897 + + Montipora sp. +

Montipora verrilli Vaughan, 1907 +

Family Agariciidae + +

Gardineroseris planulata (Dana, 1846) +

Pachyseris speciosa (Dana, 1846) +

Family Dendrophylliidae +

Tubastraea micrantha +

Family Faviidae + +

Echinopora horrida Dana, 1846 +

Echinopora lamellosa (Esper, 1791) + +

Echinopora mammiformis (Nemenzo, 1959) + + Goniastrea stelligera (Dana, 1846) +

Platygyra daedalea (Ellis & Solander, 1786) +

Platygyra pini Chevalier, 1975 + +

Family Fungiidae +

Ctenactis crassa (Dana, 1846) +

Danafungia horrida (Dana, 1846) +

Herpolitha limax (Esper, 1797) +

Polyphyllia talpina (Lamarck, 1801) +

Family Merulinidae + +

Astrea curta Dana, 1846 +

Hydnophora rigida (Dana, 1846) +

Leptoria phrygia (Ellis & Solander, 1786) +

Merulina ampliata (Ellis & Solander, 1786) +

Merulina scabricula Dana, 1846 + + Oulophyllia crispa (Lamarck, 1816) +

Family Mussidae +

Lobophyllia flabelliformis Veron, 2000 + + Lobophyllia hemprichii (Ehrenberg, 1834) +

Family Euphylliidae + +

Galaxea astreata (Lamarck, 1816) +

Galaxea horrescens (Dana, 1846) +

Family Pectiniidae +

Pectinia alcicornis (Saville-Kent, 1871) + +

Pectinia lactuca (Pallas, 1766) +

Family Pocilloporidae + +

Pocillopora damicornis (Linnaeus, 1758) +

Pocillopora grandis Dana, 1846 +

Pocillopora verrucosa (Ellis & Solander, 1786) +

Seriatopora hystrix Dana, 1846 + +

Stylophora pistillata Esper, 1797 + +

Family Poritidae +

Porites sp. +

The obtained data clearly show the effectiveness of the approach to assess the genetic diversity of large communities but also indicate that there is a significant diversity of microsymbionts living on corals that require further investigation. The material for the future work has already been collected: we possess a collection of copepods from 68 colonies of stony corals (excluding 26 colonies analyzed in this chapter), 30 of which are already processed, and copepods from these colonies sorted, photographed, and partially sequenced. These samples include ten genera of corals which were not analyzed in this chapter, eight of these genera have not been previously sampled for copepods in Australia, and two genera, Australogyra and Scolymia, have not ever been sampled for symbiotic copepods. Analysis of this extended collection is expected to give us a more or less complete knowledge of the coral-associated copepod fauna of the

Lizard Island, a model site for a variety of biodiversity studies. After the recent major bleaching event of the GBR (Hughes et al., 2017a; Hughes et al., 2017c; van Oppen &

Lough, 2018) many of these microsymbionts may be gone or be under a threat of extinction alongside their coral hosts, therefore, a comparative analysis of copepod diversity before and after bleaching should be be conducted.

Table 3.4 List of scleractinian coral taxa reported for the first time to host symbiotic copepods. New records for the GBR New records globally New families (5) New species (9) Dendrophylliidae Acropora nasuta Fungiidae A. pulchra Mussidae A. aculeus Pectiniidae Echinopora mammiformis Poritidae Lobophyllia flabelliformis Merulina scabricula New genera (12) Montipora peltiformis Astreopora Pectinia alcicornis Ctenactis Platygyra pini Echinopora Fungia Herpolitha Lobophyllia Pachyseris Pectinia Platygyra Polyphyllia Porites Tubastraea

3.5 Conclusion

In this chapter, diversity of copepods associated with corals from one of the world's biodiversity hotspots, Lizard Island on the GBR, was assessed. 26 coral colonies representing 25 species in 17 genera (Acropora, Astreopora, Ctenactis, Echinopora,

Fungia, Galaxea, Herpolitha, Lobophyllia, Merulina, Montipora, Pectinia, Platygyra,

Polyphyllia, Porites, Seriatopora, Stylophora, and Tubastraea) were collected, all copepods were removed from them, and copepods were sorted by morphotype. Analyses of three fragments of nuclear and mitochondrial DNA (18S, ITS2, COI) of only the most abundant copepod morphotypes (164 specimens), representing a third of discovered morphotypes, revealed 68 MOTUs (potential species). Most of the discovered MOTUs were found to be associated with only one coral species, whereas coral species in some

68 cases serve as hosts for multiple associations of copepods (up to 6 MOTUs on one host species).

In this chapter, I investigated the diversity of symbiotic copepods at a broad level, focusing only on one specific site, Lizard Island. Results from this chapter clearly show that copepod diversity in this region is poorly studied and much more work is needed.

Moreover, my data can serve as a starting point for describing the diversity of symbiotic copepods in the GBR and can be used as a “baseline” for comparison with other regions' diversity. My next chapter will specifically address a single host genus and expand a biodiversity assessment to a broader geographic range.

Chapter 4: Biogeographic patterns, diversity, and host specificity of copepods living in symbiosis with Galaxea (Scleractinia)

4.1 Introduction

Our understanding of the patterns of biogeographical distribution of coral-associated copepods is based on the fragmentary data obtained from a number of locations, while we have a lack of knowledge for most parts of the world including rich coral reef communities of the Red Sea, Coral Triangle, etc. (Humes, 1994; Ho, 2001). Since the overall diversity of coral-host species and their copepod symbionts numbers in the hundreds of species and, therefore, cannot be evaluated in a single project, we selected hermatypic corals of the genus Galaxea Oken, 1815 (Scleractinia: Euphylliidae) as a model group. In this chapter, we conduct the first investigation of a spatial pattern of genetic diversity among microscopic crustacean copepods living in symbiosis with two common and co-occurring scleractinian corals Galaxea astreata (Lamarck, 1816) and G. fascicularis (Linnaeus, 1767).

Galaxea is a genus of scleractinian corals widespread in the Indo-Pacific (Veron,

2000) and currently including ten species (Hoeksema & Cairns, 2018b). G. astreata and

G. fascicularis are currently considered as widespread species occurring in both Indian and Pacific oceans (Veron, 2000), however, no broad-scale genetic studies have been conducted for Galaxea corals, though such studies might change coral species boundaries and reveal cryptic species. Eighteen species from eight genera of copepods representing three orders (Siphonostomatoida, Poecilostomatoida, and Cyclopoida) have been described so far in association with three species of Galaxea: G. astreata, G. fascicularis,

70 and G. horrescens (Dana, 1846) (Humes, 1979b, 1985b, 1991, 1996b; Cheng & Dai,

2016). A list of copepod species associated with Galaxea has been published recently

(see Table 4.1 for the details) (Cheng & Dai, 2016).

TAXONOMY RECORDS FROM LITERATURE GBR Moluccas New Copepod symbiont Taiwan Madagascar (Australia) (Indonesia) Caledonia Order Poecilostomatoida Family Anchimolgidae Anchimolgus abbreviatus Humes, 1991 H 3 F 4 Anchimolgus amplius Cheng & Dai, 2016 A, F 5 Anchimolgus compressus Humes, 1996 F 4 F 4 Anchimolgus contractus Humes, 1979 F 1 F 4 A, F 5 Anchimolgus moluccanus Humes, 1996 F 4 Anchimolgus nasutus Humes, 1996 F 4 F 4 F 5 Anchimolgus tanaus Humes, 1991 H 3 F 4 A, F 5 Clamocus spinifer Humes, 1979 F 1 F 4 A, F 5 Karanges galaxeanus Humes, 1979 F 1 Karanges hypsophorus Humes, 1979 F 1 F 1 F 5 Family Xarifiidae Xarifia dongshensis Cheng & Dai, 2016 F 5 Xarifia exserens Humes, 1985 F 2 Xarifia plectrata Humes, 1985 H 3 Xarifia sp. A 2 Order Siphonostomatoida Family Asterocheridae Hetairosyna galaxeae Humes, 1996 F 3 Hetairosyna wedensis Humes, 1996 F 4 Hetairosynopsis bucculentus Humes, 1996 A, F, sp. 4 Neoasterocheres serrulatus (Humes, 1996) F 4 Order Cyclopoida Family Pterinopsyllidae Pterinopsyllus stirpipes sp. 4

1 Humes, 1979, 2 Humes, 1985, 3 Humes, 1991, 4 Humes, 1996, 5 Cheng & Dai, 2016

Copepods associated with Galaxea have been reported from only five regions:

Moluccas (Indonesia) and Madagascar in the Indian Ocean, as well as New Caledonia,

Taiwan, and Australia’s Great Barrier Reef (GBR) in the Pacific (Figure 4.1). In this study I focused on four understudied regions. No copepods have been yet reported for the

Galaxea corals in the Red Sea, which is a region with a high level of overall endemism and biodiversity (Veron et al., 2009; Bruckner & Dempsey, 2015; DiBattista et al.,

2016a). The Maldivian Archipelago reef area covers about 4500 square kilometers, with more than 2000 reefs, yet no sampling for copepods from Galaxea has previously been conducted there. The only records of copepods from corals in the Maldives were made in

1960 by Arthur Humes, who described three species of parasitic Xarifia copepods living on Pocillopora and Acropora. Copepods associated with the scleractinian corals in the

Indian Ocean were reported only for the northern Madagascar (83 species) (Humes,

1962; Humes & Frost, 1963; Humes & Ho, 1968, 1970; Humes, 1973b, 1981, 1985b;

Humes & Stock, 1991; Kim, 2009, 2010), and two species were reported for scleractinians in the Red Sea (Humes, 1960; Ivanenko et al., 2014). Fauna of copepods associated with Galaxea in the GBR, the largest coral reef ecosystem on the Earth, also remain poorly studied (Humes, 1991). Only three species of copepods have been recorded on Galaxea corals in this region: Anchimolgus abbreviatus, A. tanaus, and

Xarifia plectrata. All specimens were found on colonies of Galaxea horrescens (Dana,

1846) (=Acrhelia horrescens (Dana, 1846)) from Big Broadhurst Reef in the GBR

(Humes, 1991). Furthermore, there is no published information for the reefs on the western coast of Australia with only a few unpublished records available from Ningaloo

Reef.

Extensive sampling of Galaxea corals and their symbiotic copepods in the studied and the new regions is expected not only to increase the number of known species but,

72 what is most important, to help us answer significant biogeographical questions: whether these copepod species are cosmopolites or endemics; could species which are known to be widespread be several cryptic species instead; which region has higher diversity of copepod associates; what has a bigger effect on the species composition – biogeographical region or the species/genus of the host. By sampling from a constant host across a broad spatial range, I can assess if the composition of symbionts remains stable or, in contrast, I may find that all Indian Ocean sites have similar fauna to each other, but different from the Pacific Ocean fauna. Genetic analyses of what appear to be widespread copepod morphotypes may reveal regional endemics. Moreover, analysis of the patterns of the host-specificity, whether associated species of copepods are generalists or specific only to their host species, will help us obtain more insight in the ecology and evolution of this group.

4.2 Samples and study sites

41 coral colonies of the two species of Galaxea coral (G. astreata and G. fascicularis) were collected from 11 locations around the Indo-Pacific: Saudi Arabian Red Sea

(Farasan Islands, Al Wajh reefs, Yanbu reefs), Maldives (Addu Atoll, Foamullah Atoll,

Huvadhoo Atoll), Hainan (Sanya city), Vietnam (Hon Mun Island), Australia (Lizard

Island, Heron Island, and Ningaloo Reef) (Figure 4.1, Table 4.2, see full sampling information in the Appendix 4.1, photographs of the sampled Galaxea colonies can be found in Appendix 4.2). All corals were collected and processed using the same approach described in Chapter 2.

Table 4.2 List of Galaxea corals collected, with the sampling depth, location and the total number of copepods collected from each host (see full sampling information in the Appendix 4.1). No of # Voucher number Coral species Locality Depth (m) copepods 1 HN2009_2201 G. astreata South China Sea, Hainan Island 1-1.5 29 2 HN2009_2232 G. astreata South China Sea, Hainan Island 3-5 59 3 HN2009_06 G. astreata South China Sea, Hainan Island 3-5 21 4 HN2009_09 G. astreata South China Sea, Hainan Island 3-5 23 5 HN2009_12 G. astreata South China Sea, Hainan Island 3-5 28 6 HN2009_14 G. astreata South China Sea, Hainan Island 3-5 21 7 VN2010_33 G. fascicularis South China Sea, Nha Trang Bay 8.2 51 8 VN2010_36 G. astreata South China Sea, Nha Trang Bay 8 75 9 AU2010_1535 G. astreata Western Australia, Ningaloo Reef 19.2 43 10 AU2010_1594 G. astreata Western Australia, Ningaloo Reef 5 69 11 AU2010_1674 G. fascicularis Western Australia, Ningaloo Reef 13 7 12 AU2010_1862 G. fascicularis Australia, GBR, Lizard Island 12 24 13 AU2010_1939 G. astreata Australia, GBR, Lizard Island 20.7 16 14 AU2010_2057 G. astreata Australia, GBR, Heron Island 17.1 71 15 AU2010_2083 G. fascicularis Australia, GBR, Heron Island 17.4 94 16 SA2014_013 G. fascicularis Saudi Arabian Red Sea, Wasalayt Shoals 15.7 4

17 SA2014_029 G. fascicularis Saudi Arabian Red Sea, Farasan Islands 11.9 47 18 SA2014_036 G. fascicularis Saudi Arabian Red Sea, Farasan Islands 5.9 22 19 SA2014_037 G. fascicularis Saudi Arabian Red Sea, Farasan Islands 9.2 47 20 SA2014_054 G. fascicularis Saudi Arabian Red Sea, Farasan Islands 1.6 6 21 SA2016_009 G. fascicularis Saudi Arabian Red Sea, Al Wajh 1.5 30 22 SA2016_018 G. fascicularis Saudi Arabian Red Sea, Al Wajh 3 15 23 SA2016_019 G. fascicularis Saudi Arabian Red Sea, Al Wajh 3 30 24 SA2016_025 G. fascicularis Saudi Arabian Red Sea, Al Wajh 3 29 25 SA2016_027 G. fascicularis Saudi Arabian Red Sea, Al Wajh 4.8 10 26 SA2016_042 G. fascicularis Saudi Arabian Red Sea, Yanbu 19 42 27 SA2016_048 G. fascicularis Saudi Arabian Red Sea, Yanbu 20.5 22 28 MA2016_001 G. astreata South Maldives, Addu Atoll 18.4 10 29 MA2016_002 G. astreata South Maldives, Addu Atoll 25.6 28 30 MA2016_012 G. fascicularis South Maldives, Addu Atoll 14.4 50 31 MA2016_017 G. astreata South Maldives, Foamullah Atoll 22.4 12 32 MA2016_021 G. fascicularis South Maldives, Foamullah Atoll 14 46 33 MA2016_025 G. astreata South Maldives, Huvadhoo Atoll 21.6 34 34 MA2016_026 G. fascicularis South Maldives, Huvadhoo Atoll 19.4 10 35 MA2016_035 G. fascicularis South Maldives, Huvadhoo Atoll 27 7 36 MA2016_036 G. fascicularis South Maldives, Huvadhoo Atoll 23.5 30 37 MA2016_039 G. fascicularis South Maldives, Huvadhoo Atoll 21.5 31 38 MA2016_050 G. astreata South Maldives, Huvadhoo Atoll 20.2 7 39 MA2016_054 G. fascicularis South Maldives, Huvadhoo Atoll 14 14 40 MA2016_064 G. astreata South Maldives, Huvadhoo Atoll 15.8 15 41 MA2016_074 G. astreata South Maldives, Huvadhoo Atoll 20.2 22 Total number of copepods collected 1251

4.3 Results

4.3.1 PCR and sequencing

In total, 1251 copepod specimens in washings from 41 colonies of Galaxea were found and analyzed (see Appendix 4.3). 1988 sequences (990 contigs) of 18S gene were produced with an average read length of 650 bp, which we used for phylogeny and to define the location of genera on the overall copepod phylogenetic tree. 1286 sequences

(643 contigs) of COI gene were produced with an average read length of 700 bp; 1097 sequences of ITS2 were produced with an average read length of 500 bp. I found 224 homozygotes, 70 single-peak contigs, and 202 heterozygotes. 162 heterozygotes were split in the haplotypes using SeqPhase, and 40 contigs with phase shifts or sequences of unequal length were split in the haplotypes using Champuru. 112 contigs of ITS2 had

75 very heterogeneous regions, which were not possible to read even with SeqPhase either due to low quality of the sequences or presence of more than two copies of this region present in the genome.

4.3.2 Phylogenetic relationships

18S sequences were analyzed and used for the general diversity assessment of copepods living in association with Galaxea corals. Copepods in my samples are represented by specimens from 6 orders: Poecilostomatoida, Siphonostomatoida, Cyclopoida,

Harpacticoida, Misophrioida, and Calanoida (Figure 4.2 and Appendix 4.4).

174 additional 18S sequences representing Platycopioida (1 species), Calanoida (18 species), Misophrioida (3 species), Cyclopoida (26 species), Poecilostomatoida (40 species), Harpacticoida (41 species), and Siphonostomatoida (45 species) were downloaded from NCBI GenBank and used in analyses. See Appendix 4.5 for a list of the

GenBank species and sequences used. These additional sequences were used to confirm the reliability of my sequence data and the accuracy of the identification.

On the two species of Galaxea, I found 185 MOTUs. The majority of the discovered copepods belong to two families: Anchimolgidae (Poecilostomatoida) representing 48% of individuals and Asterocheridae (Siphonostomatoida) comprising

23% of collected copepods (Figure 4.3). Furthermore, these two groups are known to be among the most common coral-associated families of copepods. Therefore, detailed diversity assessment based on multi-locus species delimitation methods was made for these two families, whereas for rest of the discovered copepods general diversity

76 assessment based on 18S gene was conducted (Table 4.3). Detailed information on the diversity of the families Anchimolgidae and Asterocheridae is given in section 4.3.2.

Figure 4.2 18S ML phylogenetic tree based on 992 sequences generated from my samples and additional 174 copepod sequences from GenBank (marked with black). The tree was edited using the online tool iTOL v3 (Letunic & Bork, 2016). See full tree in Appendix 4.4.

Copepods of the order Poecilostomatoida constitute more than a half (54%) of the individuals collected from the Galaxea corals. Apart from Anchimolgidae they belong to the families Clausidiidae (1 MOTU), Lichomolgidae (4 MOTU), Pseudanthessiidae (7

MOTU), Rhynchomolgidae (1 MOTU), Synapticolidae (4 MOTU), Xarifiidae (1

MOTU). The large number of Galaxea-associated copepods belong to the order

Siphonostomatoida (29%) most of which, as it was mentioned above, are species of the family Asterocheridae. The rest of the individuals belong to other families and include 14

MOTUs (Appendix 4.7). The order Calanoida is represented dominantly by the family

Pseudocyclopidae (12 of 18 MOTUs), other six MOTUs belong to Centropagidae,

Clausocalanidae, and Paracalanidae. The order Cyclopoida is represented by four families: Buproridae, Cyclopinidae, Cyclopettidae, and Cyclopidae. Diversity assessment of the Cyclopoida revealed 22-26 MOTUs based on ABGD method and the tree, and 22-

43 MOTUs were distinguished based on PTP. In my subsequent analyses, I will use the lowest estimate of 22 MOTUs to avoid overestimation. In the order Harpacticoida 43

MOTUs were revealed using the ABGD method and the tree assessment, 61-67 MOTUs were distinguished using the РТР method. The order Misophrioida is represented by only six specimens belonging to two MOTUs (based on all species delimitation methods), both from the family Misophriidae.

Table 4.3 Diversity assessment of copepods found on Galaxea based on 18S gene. Copepod taxonomy MOTUs No of specimens Order Siphonostomatoida Siphonostomatoida fam. sp. 10 62 Asterocheridae 33 224 Artotrogidae 4 5 Order Poecilostomatoida Poecilostomatoida fam. sp. 9 23 Anchimolgidae 26 480 Clausidiidae 1 2 Lichomolgidae 4 11 Pseudanthessiidae 7 8 Rhynchomolgidae 1 1 Synapticolidae 4 9 Xarifiidae 1 2 Order Harpacticoida Harpacticoida fam. sp. 37 75 Peltidiidae 6 7

Order Cyclopoida Buproridae 3 22 Cyclopettidae 6 7 Cyclopidae 9 49 Cyclopinidae 3 5 Order Calanoida Pseudocyclopidae 12 23 Paracalanidae 3 3 Centropagidae 1 1 Clausocalanidae 2 2 Family sp. 1 1 Order Misophrioida Misophriidae 2 6 Total 185 1028

4.3.3 Multilocus molecular species delimitation

As it was noted above, 71% of the discovered copepods belong to the families

Anchimolgidae and Asterocheridae, and, therefore, a detailed diversity assessment based on multilocus species delimitation methods was conducted for these two groups. The results of the species delimitation analyses conducted are summarized in Table 4.4.

Table 4.4 Number of MOTUs obtained using different species delimitation algorithms (see details in the text); concat. = concatenated dataset. Algorithm ABGD ABGD ABGD PTP-h PTP-ML GMYC-ST GMYC-MT Dataset COI ITS2 combined concat. concat. concat. concat. Anchimolgidae 30 26 33 41 41 37 44 Asterocheridae 26 35 42 48 48 46 60

The ABGD analysis was run separately for alignments of each of the two families for both COI and ITS2 datasets. However, some of the specimens only amplified for one of these two markers, hence, the estimations were based on the results combined.

The family Anchimolgidae in my samples is represented by several genera including Anchimolgus, Clamocus, and Karanges. The ABGD analyses of the COI alignments revealed 30 MOTUs, and 26 MOTUs based on the ITS2 dataset. An output of the ABGD species delimitation analysis showed no conflicts between COI and ITS2

79 datasets. After combination of the COI and ITS2 estimates with the diversity assessment based on the 18S phylogenetic trees 33 MOTUs of Anchimolgidae were revealed (ABGD bar in the Figure 4.3). Both PTP-h and PTP-ML methods gave identical partitions for the

Anchimolgidae concatenated alignment and revealed 41 MOTUs (PTP bar in the Figure

4.3). ST- and MT-GMYC analyses of the concatenated datasets were conducted, and 37 and 44 MOTUs were distinguished, respectively (ABGD bar in the Figure 4.3). All in all, a minimum of 33 MOTUs belonging to these genera were revealed using genetic analysis. Lower estimate (33 MOTU) will be used.

All four species delimitation methods gave relatively consistent delineation of the species in the genus Karanges. ABGD and ST-GMYC revealed 8 MOTUs in this group, whereas PTP and MT-GMYC divided it in 9 MOTUs by splitting Karanges sp. 6 in two

MOTUs, first from G. astreata, second from G. fascicularis, both from the Maldives. In the genus Clamocus, MOTUs sp. 2 and sp. 4 are split into two by MT-GMYC but three other methods showed them as single taxonomic units. In Clamocus sp. 2, a specimen from Heron Island was separated from the specimens from the South China Sea.

Specimens in the Clamocus sp. 4 are all from G. fascicularis collected in the Maldives. In the genus Anchimolgus, boundaries within the following MOTUs: Anchimolgus sp. 1, sp.

2, sp. 7, sp. 8, sp. 10, sp. 11, and sp. 13, were different drawn differently by all four methods (see Figure 4.3). We will use the estimation made using ABGD with 14 MOTUs revealed in the genus Anchimolgus.

Figure 4.3 Bayesian phylogeny of the family Anchimolgidae based on the concatenated dataset of 18S, COI, and ITS2. Numbers on the branches represent support values corresponding to BI posterior probabilities. The columns on the right represent species delimitation based on different methods: ABGD, PTP, and GMYC (incongruence between methods is marked with a darker color). See Appendix 4.8 for a full MrBayes tree containing all individuals.

The family Asterocheridae in my samples is represented by four genera

Asterocheres, Asteropontius, Hetairosyna, and Hetairosynopsis (Figure 4.4). The ABGD analyses revealed 26 MOTUs based on the COI alignment and 35 MOTUs based on the

ITS2 dataset. After combining the COI and ITS2 estimates with the diversity assessment based on the 18S phylogenetic trees, 42 MOTUs of Asterocheridae were revealed. PTP analysis of the Asterocheridae concatenated alignment revealed 48 MOTUs. Both PTP-h and PTP-ML methods gave identical partitions. ST- and MT-GMYC analyses of the concatenated datasets were conducted, however, the MT-GMYC method seems to overestimate the number of MOTUs, therefore, I will use the ST-GMYC results for species delimitation. ST-GMYC analysis revealed 46 MOTUs in the concatenated alignment of Asterocheridae. All methods (ABGD, PTP, and GMYC) yielded generally consistent results (42, 48, and 46 MOTUs). All in all, a minimum of 42 MOTUs belonging to these genera were revealed using genetic analysis. This lower estimate (42

MOTU) will be used.

The PTP method divided Hetairosynopsis sp. 1 in four MOTUs, whereas ABGD combined them in one, and GMYC delimitation method united two specimens from

Heron Island into one species and showed the presence of three MOTUs. According to the lowest estimate 4 MOTUs of the genus Hetairosynopsis were discovered. In the genus Hetairosyna, MOTU sp. 4 is split into two but both ABGD and GMYC showed it as a single taxonomic unit (Figure 4.4). Moreover, all specimens in sp. 4 were collected from the same geographical region. Therefore, we will use the estimation made using

ABGD and GMYC with 8 MOTUs revealed in the genus Hetairosyna. For the genera

Asterocheres and Asteropontius, results of all three methods were completely consistent,

82 except for Asterocheres sp. 4 and Asteropontius sp. 13, which were both split into two

MOTUs by PTP delimitation method. According to the ABGC and GMYC estimates, 14

MOTUs of the genus Asterocheres and 15 MOTUs of the genus Asteropontius are present in my samples.

Figure 4.4 Bayesian phylogeny of the family Asterocheridae based on the concatenated dataset of 18S, COI, and ITS2. Numbers on the branches represent support values corresponding to BI posterior probabilities. The columns on the right represent species delimitation based on different methods: ABGD, PTP, and GMYC (incongruence between methods is marked with a darker color). Numbers in the brackets represent number of specimens per each MOTU. See Appendix 4.9 for a full MrBayes tree containing all individuals.

4.4 Discussion

4.4.1 Diversity of Galaxea-associated copepods

Analysis of the genetic diversity revealed a highly diverse fauna of ectosymbiotic copepods associated with Galaxea (Table 4.5). Two co-occurring species of Galaxea corals with similar ecologies appeared to be hosts for dozens of species (185 MOTUs according to the lowest estimate). The majority of these discovered species cannot yet be linked with certainty to previously-described species (as there is no genetic information for the vouchers), therefore, I treat them as MOTUs.

Table 4.5 Comparison of the discovered diversity with the estimates based on the previous records. Copepod taxa Literature data My samples Orders 3 6 Families 4 >23 Species / MOTUs 18 185

The poecilostomatoid families Anchimolgidae, Lichomolgidae, Rhynchomolgidae, and

Xarifiidae discovered on Galaxea corals are well-known symbionts of scleractinian corals. Anchimolgidae and Xarifiidae are known to be exclusively associated with hard corals, while Lichomolgidae and Rhynchomolgidae utilize a wide range of hosts (Humes

& Boxshall, 1996). However, we need to note that the validity of these families and the genera included in these families has not yet been verified by molecular methods, and the analysis of the molecular phylogenies could change the taxonomic position of these taxa.

Diversity of Galaxea-associated copepods of the family Anchimolgidae will be discussed below in section 4.4.2. Other two poecilostomatoid families found in my samples are

Synapticolidae (Humes & Boxshall, 1996) and Pseudanthessiidae (Humes & Stock,

1972), which are typically associated with echinoderms (Humes, 1977; Humes &

Boxshall, 1996). Synapticolidae copepods are mostly associated with echinoids, holothurians (Stock et al., 1963; Humes & Boxshall, 1996), and there is one record of association with a polychaete (Bocquet & Stock, 1963). This thesis is the first report of association of Synapticolidae copepods with the scleractinian corals. Pseudanthessiidae copepods inhabit wide range of marine invertebrates but mostly occur on echinoderms

(Humes, 1977). Another family of copepods discovered in my samples from Galaxea are copepods from the family Clausidiidae (Embleton, 1901). They are commonly found in association with various marine invertebrates (e.g., sponges, polychaetes, molluscs, and crustaceans), some of them were also reported as free-living. However, there was only one record of association of copepods of this family with hard corals, Hemicyclops columnaris was reported from Porites on the Pacific coast of Panama (Humes, 1984).

The most diverse family in my samples is the siphonostomatoid family

Asterocheridae (Giesbrecht, 1899). Asterocheridae is a very big family including 68 genera (Walter & Boxshall, 2018c), reported to be symbionts and parasites of various marine invertebrates, such as sponges, cnidarians, echinoderms, molluscs, and bryozoans; and most of which utilize only one host group. The diversity of Galaxea-associated copepods of the family Asterocheridae will be discussed below in section 4.4.2. Another family known to be associated with scleractinians and other invertebrates and found in my samples is the family Artotrogidae. They are not commonly found in the samples and seem not to be strictly associated to a particular host group. There are only few records of copepods of this family from scleractinian corals: two species, Pteropontius pediculus and Bradypontius pichoni, were found on Echinopora lamellosa and Platygyra sp. on

Mauritius in the Indian Ocean (Stock, 1966), Cryptopontius sp. and Pteropontius sp. were

85 reported from Madracis in Barbados (Caribbean Sea, Atlantic Ocean) (Snelgrove &

Lewis, 1989), and the last record is species Cryptopontius tanacredii found on

Pocillopora damicornis on the Easter Island in the South Pacific Ocean (Johnsson et al.,

2002). In my samples I found three MOTUs of the family Artotrogidae on Galaxea from the Maldives and one MOTU from Heron Island (Australia).

Cyclopoid copepods (not including poecilostomatoids) found in my samples are represented by four families, two of which have not been previously reported as symbionts of hard corals. The family Cyclopinidae are mostly benthic copepods, with a few planktonic genera (Boxshall & Halsey, 2004b). Only two species in the family are found in association with other animals; and Pterinopsyllus stirpipes is the only species of this family reported as a symbiont of a scleractinian coral from Madagascar, which by coincidence is a species of Galaxea (Humes, 1996b). In my samples, I found three species of Cyclopinidae: one in the South China Sea near Hainan, one in the central Red

Sea, and one in Maldives. This study is a first report of copepods from the family

Cyclopettidae on corals, with six potential species from the Red Sea, Maldives, and

Hainan to be described, which are all putatively new for science. Another family represented in my samples is family Cyclopidae. It is extremely diverse and widespread family of planktonic and benthic copepods which are found in both fresh and sea water

(Boxshall & Halsey, 2004b). Some of these copepods are found living in sponges and, in the exotic places, like water puddles in leaf axils of tropical plants. Three species,

Coraleuryte bellatula (Humes, 1991), C. verecunda (Humes, 1992), and Euryte sp., have been reported from eight species of scleractinian corals (Montipora and Porites) (Cheng et al., 2016). Buproridae have been previously reported as a family specifically

86 associated with ascidians in Atlantic (Marchenkov & Boxshall, 2002). Three undescribed species of Buproridae discovered in my samples from the Red Sea are the first report of association of this family with scleractinian corals and the first report for the Indo-

Pacific, therefore, it can be argued that they also represent a new genus (or genera) for science. The nature of the relationship of these copepods with the corals is yet unclear.

Analysis of 18S region allowed us to determine the position of the family on the phylogenetic tree of copepods. The family Buproridae appear to be a sister group to the family Notodelphyidae (Dana, 1853) which is also known to be associated with ascidians

(Dudley, 1966) with a record from the Red Sea (Kim et al., 2016).

The order Calanoida in my samples is represented dominantly by the family

Pseudocyclopidae, which is known to be an epibenthic group of calanoids in the shallow waters of tropical seas (Boxshall & Halsey, 2004a). Calanoids are not normally considered as a taxon occurring in association with other animals, however, we assume that these copepods could be facultative symbionts of corals and occur in a loose association with them. Most of the Pseudocyclopidae I found are from the Red Sea and

Maldives, with two MOTUs from Australia. Other calanoids found in washings from

Galaxea corals belong to four other calanoid families: Centropagidae (1 MOTU),

Clausocalanidae (2 MOTUs), and Paracalanidae (3 MOTUs).

The order Harpacticoida in my samples is represented by various families. The majority of these copepods appear to be a by-catch from the dead part of the sampled corals. An effort was made to sample only living and healthy colonies, but sometimes it was unavoidable to sample a partially-dead colony. Another significant characteristic of

Galaxea corals is that they form encrusting colonies, which increases the contact surface

87 with the substrate and, therefore, increases the possibility of contamination with benthic fauna. Among all harpacticoids copepods from only two families were reported as symbionts of scleractinians. One species, Alteuthellopsis corallina Humes, 1981, from the family Peltidiidae was recorded as a species living in association with Acropora,

Astreopora, Gardineroseris, Goniastrea, Merulina, Montipora, Platygyra, Stylophora, and Pocillopora, but with no records from Galaxea (Humes, 1981). The family

Peltidiidae is the only family of copepods in my samples that was previously reported in association with corals, and it is represented in my samples by 6 MOTUs from the Red

Sea, Ningaloo, Maldives, and Hainan. The copepods from the closely related family

Tegastidae are also known to live in association with scleractinians (Humes, 1981), however, there were no representatives of this family found in my samples.

The order Misophrioida is represented in my samples by two species of the family

Misophriidae. Copepods of this family could be usually found in the shallow waters, in deep-water plankton, deep sea hydrothermal vents, and anchialine caves, with two bathypelagic species of plankton reported to feed on cnidarians and to live in a loose association with corals (Boxshall & Halsey, 2004a).

4.4.2 Genetic diversity and biogeographic distribution of Anchimolgidae and

Asterocheridae associated with Galaxea

The family Anchimolgidae is the most abundant group in my samples and is represented by three genera: Anchimolgus, Clamocus, and Karanges. These genera of copepods are all known to be exclusively associated with scleractinian corals, and all three were previously reported as symbionts of Galaxea corals.

14 putative copepod species (MOTUs) in my samples belong to the genus

Anchimolgus (Table 4.6). Seven species of this genus have been previously described from Galaxea corals from different regions of the Indo-Pacific (GBR, Indonesia, New

Caledonia, and Taiwan), with most of the species occurring in several regions (see

Appendix 4.1), e.g., A. abbreviatus has been reported from the GBR (Humes, 1991),

Moluccas (Indonesia) (Humes, 1996b), and New Caledonia (Cheng & Dai, 2016). We found species of Anchimolgus from Galaxea in the GBR (Lizard Island), Heron Island

(eastern Australia), and Ningaloo Reef (western Australia), the South China Sea

(Vietnam and Hainan), and the Maldives.

Table 4.6 Diversity, abundance, and distribution of Anchimolgus copepods in my samples.

MOTU Specimens Regions Galaxea species Anchimolgus sp. 1 6 Lizard & Heron Island A, F Anchimolgus sp. 2 33 Maldives A, F Anchimolgus sp. 3 22 South China Sea A Anchimolgus sp. 4 10 Ningaloo Reef A Anchimolgus sp. 5 1 Ningaloo Reef A Anchimolgus sp. 6 32 Heron Island A, F Anchimolgus sp. 7 15 Maldives A Anchimolgus sp. 8 7 Maldives F Anchimolgus sp. 9 2 South China Sea F Anchimolgus sp. 10 6 South China Sea F Anchimolgus sp. 11 7 South China Sea A Anchimolgus sp. 12 7 South China Sea A Anchimolgus sp. 13 10 South China Sea A, F Anchimolgus sp. 14 2 Lizard Island A

At least four putative copepod species (MOTUs) in my samples belong to the genus

Clamocus (Table 4.7). Delimitation of these species by molecular methods is confirmed

89 by results of analysis of the morphological features. In the literature, there is a record of only one species in the genus, Clamocus spinifer. It has been reported only from Galaxea corals: G. fascicularis in Moluccas (Humes, 1979b), G. fascicularis in the New

Caledonia (Humes, 1996), and G. astreata and G. fascicularis in Taiwan (Cheng & Dai,

2016).

Table 4.7 Diversity, abundance, and distribution of Clamocus copepods in my samples.

MOTU Specimens Regions Galaxea species Clamocus sp. 1 4 Vietnam & Ningaloo Reef A, F Clamocus sp. 2 38 South China Sea & Heron A, F Clamocus sp. 3 2 South China Sea (Hainan) A Clamocus sp. 4 20 Maldives F

Eight putative species (MOTUs) from two regions (Maldives and eastern Australia) in my samples belong to the genus Karanges (Table 4.8). Two species of this genus were previously described in the literature: Karanges galaxeanus from G. fascicularis in

Indonesia (Humes, 1979b) and K. hypsorophus from G. fascicularis in Indonesia

(Humes, 1979b), New Caledonia (Humes, 1996b), and Taiwan (Cheng & Dai, 2016).

Table 4.8 Diversity, abundance, and distribution of Karanges copepods in my samples.

MOTU Specimens Regions Galaxea species Karanges sp. 1 2 Heron Island A Karanges sp. 2 1 Heron Island A Karanges sp. 3 10 Heron Island A, F Karanges sp. 4 1 Maldives F Karanges sp. 5 1 Heron Island F Karanges sp. 6 7 Maldives A, F Karanges sp. 7 7 Maldives A, F Karanges sp. 8 16 Maldives A, F

The family Asterocheridae is the most diverse group in our samples and is represented by four genera: Asterocheres (Neoasterocheres), Asteropontius, Hetairosyna, and Hetairosynopsis. These genera of copepods are all known to be exclusively associated with scleractinian corals, and Asterocheres (Neoasterocheres), Hetairosyna, and Hetairosynopsis were previously reported as symbionts of Galaxea corals.

Four putative species (MOTUs) in my samples belong to the genus Hetairosynopsis

(Table 4.9). The only species of the genus is Hetairosynopsis bucculentus Humes, 1996, which has been described only from Galaxea astreata and G. fascicularis in Madagascar.

(Humes, 1996b). We report species of Hetairosynopsis to be found in both western and eastern Australia, South China Sea, and Maldives. At least three of these species are putatively new for science, and all of them are new for the sampled regions.

Table 4.9 Diversity, abundance, and distribution of Hetairosynopsis copepods in my samples.

MOTU Specimens Region Galaxea species Hetairosynopsis sp. 1 5 Heron & South China Sea A Hetairosynopsis sp. 2 6 South China Sea A, F Hetairosynopsis sp. 3 1 Maldives F Hetairosynopsis sp. 4 6 Ningaloo Reef F

Eight putative species (MOTUs) in my samples belong to the genus Hetairosyna

(Table 4.10). Two species of Hetairosyna, H. galaxeae and H. wedensis, have been described in the literature from the G. fascicularis in Moluccas (Indonesia) (Humes,

1996b). Another three species of genus Hetairosyna have been previously reported in association with 14 species of the coral Montipora Blainville, 1830 in Madagascar,

Indonesia, Marshall Islands, New Caledonia, and the GBR (Cheng et al., 2016). No other species of Hetairosyna were described. I found species of Hetairosyna in all sampled regions except Maldives.

Table 4.10 Diversity, abundance, and distribution of Hetairosyna copepods in my samples.

MOTU Specimens Region Galaxea species Hetairosyna sp. 1 1 Ningaloo Reef F Hetairosyna sp. 2 1 Ningaloo Reef F Hetairosyna sp. 3 6 Heron & Lizard Island A Hetairosyna sp. 4 33 South China Sea A Hetairosyna sp. 5 1 South China Sea A Hetairosyna sp. 6 2 Red Sea F Hetairosyna sp. 7 2 Red Sea F Hetairosyna sp. 8 58 Red Sea F

14 putative species (MOTUs) from the Red Sea, the GBR, Hainan, and Maldives in my samples belong to the genus Asterocheres (Table 4.11). The only record of this genus from Galaxea corals is the species Neoasterocheres serrulatus (Humes, 1996) from

Madagascar. It has been originally described by Humes as Madacheres serrulatus

Humes, 1996, which was later accepted as a synonym of Asterocheres serrulatus

(Humes, 1996); in the recent reassessment of the genus Asterocheres (Canario et al.,

2017), A. serrulatus was moved into the genus Neoasterocheres, and is now accepted as

Neoasterocheres serrulatus (Canario et al., 2017).

Table 4.11 Diversity, abundance, and distribution of Asterocheres copepods in my samples.

MOTU Specimens Region Galaxea species Asterocheres sp. 1 1 Lizard Island A Asterocheres sp. 2 1 Red Sea F Asterocheres sp. 3 2 Red Sea F

Asterocheres sp. 4 1 Red Sea F Asterocheres sp. 5 1 Hainan (South China Sea) A Asterocheres sp. 6 5 Maldives A Asterocheres sp. 7 3 Maldives A, F Asterocheres sp. 8 1 Red Sea F Asterocheres sp. 9 1 Heron Island A Asterocheres sp. 10 2 Red Sea F Asterocheres sp. 11 1 Heron Island F Asterocheres sp. 12 2 Heron Island F Asterocheres sp. 13 1 Red Sea F Asterocheres sp. 14 1 Lizard Island A

15 putative species (MOTUs) belong to the genus Asteropontius (Table 4.12).

Copepods of this genus were found in all sampled regions. 26 species of Asteropontius have been recorded to be associated with various families of scleractinian corals in the

Indo-Pacific and Atlantic but there were no reports of association with Galaxea corals

(Cheng et al., 2016).

Table 4.12 Diversity, abundance, and distribution of Asteropontius copepods in my samples.

MOTU Specimens Region Galaxea species Asteropontius sp. 1 2 Lizard Island F Asteropontius sp. 2 1 South China Sea A Asteropontius sp. 3 1 South China Sea A Asteropontius sp. 4 1 Heron Island F Asteropontius sp. 5 4 Heron Island A Asteropontius sp. 6 1 Heron Island A Asteropontius sp. 7 1 Ningaloo Reef F Asteropontius sp. 8 6 South China Sea A Asteropontius sp. 9 1 Ningaloo Reef A, F Asteropontius sp. 10 1 South China Sea F Asteropontius sp. 11 1 Maldives F Asteropontius sp. 12 2 Red Sea F Asteropontius sp. 13 3 South China Sea & Maldives A Asteropontius sp. 14 1 Ningaloo Reef A Asteropontius sp. 15 2 Red Sea F

Overall, according to the lower estimates, 33 MOTUs of copepods from the family

Anchimolgidae and 42 MOTUs from the family Asterocheridae were found on 41 colonies of Galaxea. Discovered diversity is significantly higher than it was known from the literature (Table 4.13). The family Asterocheridae was always considered as a group dominating in the Atlantic Ocean, with only sporadic records from the Indo-Pacific

(Humes, 1985a; Stock, 1988), however, this differentiation seem to be a result of an inadequate attention to this family (Humes, 1985a; Ivanenko et al., 2018). The sampling coverage analysis for the families Anchimolgidae and Asterocheridae shows that we did not cover the all species diversity in our sampling and there are still more species to be found, especially in the family Asterocheridae (Figure 4.5).

Table 4.13 Genera and the number of species of symbiotic copepods reported from the Galaxea corals before and discovered in this study.

Number of species (MOTUs) from Galaxea Taxonomy In the literature In our samples Family Anchimolgidae Anchimolgus 7 14 Karanges 2 8 Clamocus 1 4 Family Asterocheridae Asterocheres 1 14 Asteropontius 0 15 Hetairosyna 2 8 Hetairosynopsis 1 4

Figure 4.5 The sampling coverage for the dominant taxa of copepods associated with Galaxea corals.

4.4.3 Biogeographical patterns of distribution of Anchimolgidae and Asterocheridae

In general, I discovered high geographical fragmentation of copepods associated with

Galaxea corals. Copepods in most of the sampled regions are genetically separated, i.e., each MOTU is usually found only in one region (Tables 4.6-4.12). Six regions were delineated based on the distribution of copepod MOTUs from Galaxea corals (Figure

4.6).

The Red Sea region includes three sampled locations: the Farasan Islands in the south and Al Wajh and Yanbu reefs in the north. Overall, 12 MOTUs of copepods of the family Asterocheridae were found in the Red Sea, which include three MOTUs of

Hetairosyna, six MOTUs of Asterocheres, two MOTUs of Asteropontius, and one unidentified species of Asterocheridae. Eight MOTUs were found in the Farasan Islands in the south (Hetairosyna sp. 6, sp. 7, Asterocheres sp. 2, sp. 8, sp. 10, sp. 13,

Asteropontius sp. 15, and Asterocheridae sp. 1) and three MOTUs were found in Al Wajh in the north (Asterocheres sp. 3, sp. 4 and Asteropontius sp. 12).

Figure 4.6 Biogeographical regions delineated based on the distribution and delineation of copepod MOTUs from Galaxea corals in the Indo-Pacific.

These disparities in the species composition between the north and south parts of the Red Sea could have been explained by the presence of the latitudinal gradients. Many

MOTUs are represented by 1-2 specimens only, and the observed differences may be explained by a low sampling size. However, one MOTU, Hetairosyna sp. 8, includes copepods from both Farasan Islands and Al Wajh, with 1200 km between the most distant sampling sites. The delineation of Hetairosyna sp. 8 as a single MOTU was supported by all three species delimitation methods (ABGD, PTP, and GMYC) and was confirmed by morphological identification. Therefore, it is not really clear whether differences in the salinity, temperature, and nutrients have a direct effect on the copepod species distribution along the gradient. However, the susceptibility to these factors can

96 differ among the discovered species. To thoroughly assess these effects, further sampling of corals from different regions of the Red Sea, different depths, inner/outer parts of the reefs needs to be conducted, with analysis of these factors. MOTUs discovered in the Red

Sea were not found in any other regions, and for now they could be considered as endemic species for the Red Sea. Furthermore, despite the fact that Anchimolgidae are the most common and abundant copepod symbionts of scleractinian corals in the Indo-

Pacific (Humes, 1985a; Cheng & Dai, 2016; Walter & Boxshall, 2018b), no species of this family were found on any of the 12 colonies of Galaxea collected in the different parts of the Red Sea from the different depths (from one to 20 meters). There are also no records of the Anchimolgidae copepods from the Red Sea in the literature (Cheng et al.,

2016). However, this could be due the fact that there were almost no studies of symbiotic copepods in that region. Only two copepod species from other families were reported to be found on the Red Sea corals to date (Cheng et al., 2016): Spaniomolgus sp.

(Rhynchomolgidae) from Stylophora pistillata (Ivanenko et al., 2014) and Xarifia spp.

(Xarifiidae) from Stylophora sp. (Humes, 1960). In this dissertation I report five species of Spaniomolgus from Stylophora in the Red Sea (Conradi et al., 2018) (see Chapter 5 for the details), two species of Asteropontius, two species of Schedomolgus, two species of

Prionomolgus, and one Paradoridicola, all from the Fungiidae corals in the Red Sea

(Ivanenko et al., 2018) (see Chapter 6 for the details).

The Maldivian archipelago includes 26 atolls stretched over 800 km. In my samples, the Maldivian region is represented by three atolls in the southern Maldives:

Addu Atoll, Foamullah Atoll, and Huvadhoo Atoll (Figure 4.7). Among 288 specimens of copepods collected from 14 colonies of Galaxea, 120 copepods were attributed to the

97 family Anchimolgidae (10 MOTUs) and 12 specimens to the family Asterocheridae (5

MOTUs). The results indicate a highly diverse fauna dominated by Anchimolgidae

(about 40% of the samples). However, the diversity could have been strongly affected by the severe bleaching event which took place in the spring-summer 2016 (Marshall et al., 2017; Perry & Morgan, 2017), half a year before the sampling was conducted.

All our copepod samples appear to be new records for Maldives (or undescribed species). No new reports of copepods from scleractinians have been published for this Figure 4.7 Map of the atolls of the Maldivian Archipelago. Sampling locations are marked region since two species of Xarifia, X. with the red circles. fimbriata and X. maldivensis, were described in 1960 by Humes from Pocillopora sp.

(Humes, 1960; Cheng et al., 2016). The Maldives appear to be a poorly studied region in terms of coral-associated copepods and evidently require further investigation and sampling in the northern and central parts of the archipelago. This region could be used as a model place to study climate effects on the coral reef communities because the impact from human activity on the coral communities is not significant: people live only on some of the islands, there are almost no industrial activities, and Maldivians historically only fish for oceanic species, and rarely targeting reef fish. For the southern

98 part of Maldives, a collection of copepods from 53 more colonies of scleractinian corals has recently been obtained and is yet to be processed and analyzed.

Ten MOTUs of Anchimolgidae and ten MOTUs of Asterocheridae were discovered in the South China Sea.

Anchimolgus sp. 12, sp. 13, Clamocus sp. 2, and Hetairosyna sp. 4 were found in both Hainan (Sanya city) and

Vietnam (Hon Mun Island). These species were also the most abundant and were found on both G. fascicularis and

G. astreata. The distance between these Figure 4.8 Sampling locations in the South China Sea (marked with red circles); the star marks the sampling locations is about 650 km (750 records from Galaxea in the literature (Cheng & Dai, 2016). Map modified from the km along the coastline) (Figure 4.8), southchinasea.org (Ji Guoxing). there are no geographical barriers, and water currents, though changing throughout the year, connect these two regions (Hu et al., 2000), which could facilitate the dispersal of copepods. A series of studies of coral-associated copepods in the South China Sea have been conducted by Cheng and Ho during the last ten years (Cheng et al., 2016). All records are from the reefs around Taiwan and from Dongsha Atoll, which is located about 800 km from our sampling location in Hainan (Figure 4.8). 30 species of copepods are currently reported from this area, 20 of them belong to the family Xarifiidae, seven species belong to Anchimolgidae. There is only one report of a copepod from the family

Asterocheridae (Coralliomyzontidae) (Cheng & Dai, 2011) from Tubastraea coral. Seven

99 species of copepods have been reported from Galaxea corals in the South China Sea:

Anchimolgus contractus, A. nasutus, A. tanaus, A. amplius, Clamocus spinifer, Karanges hypsorophus, and Xarifia dongshensis (Cheng & Dai, 2016). Therefore, ten MOTUs of asterocherid copepods and at least six anchimolgids found in our samples are the new reports for the region.

In the Lizard and Heron Islands samples, despite the relative proximity of the islands (1200 km) and the more or less continuous reef line of the GBR, copepod populations seem not to be mixed, and the species composition is different. Moreover,

Heron Island is separated from the GBR by a 100-km strait. Only two species were found on both islands (Anchimolgus sp. 1 and Hetairosyna sp. 3) (Figure 4.9). This is not a unique example of the of division of the GBR region to the northern and southern part.

For instance, the reef fish Acanthochromis polyacanthus (Bleeker, 1855), which lacks a pelagic larval stage, forms morphologically and genetically distinct populations in the northern and southern parts of the GBR with a hybrid zone in the central part

(Van Herwerden & Doherty, 2006). In case of the A. polyacanthus the deep water barrier in the central Great Barrier

Reef, Hydrographers Passage, seems to play a role of a natural barrier which Figure 4.9 Venn diagram of the copepod entities prevents continuous mixing of the found on Galaxea corals. Number in brackets represents the number of MOTUs in the region. populations (Planes & Doherty, 1997). Red Sea species are not included as they were not found in any other regions.

100

Water currents could play a role of another natural barrier for mixing northern and southern copepod populations in the GBR: in the central part of the GBR the North

Caledonia Jet current bifurcates to the East Australian Current going to the south, and the

North Queensland Current going up to the north (Steinberg, 2007). Beside the differences in the species composition, the diversity of copepods found on Galaxea in the Heron

Island (17 MOTUs) is two times higher than in the Lizard Island (7 MOTUs). These differences could be also a consequence of the inadequate number of coral colonies sampled (two colonies of both species G. astreata and G. fascicularis from Heron Island and the same from Lizard Island). This pattern could probably change with an increased number of samples from both Lizard and Heron.

Another region sampled is Ningaloo Reef on the western coast of Australia. 11

MOTUs were found on the three colonies. None of these entities were found on Galaxea corals on the east coast. However, one MOTU, Clamocus sp.1, was found in Vietnam

(Figure 4.9).

Most of the discovered copepods are new for the studied regions, and all MOTUs from the Red Sea, the Maldives, and west coast of Australia (Ningaloo) are first reports for the regions. Apparently, I found no cosmopolitan species of copepods, and endemic species are present in all sampled regions (Figure 4.9). Such narrow-range endemism is relatively rare in coral reef-associated taxa. Most of the symbiotic copepods have planktonic larvae and, therefore, are expected to be able to spread with currents.

However, there is no information about their dispersal potential, and the observed geographical fragmentation needs further investigation.

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The diversity grew significantly with sampling new locations; therefore, this pattern might change with an increased number of analyzed samples. The number of samples is not equal between the sampled regions (Figure 4.10), so accurate statistical analyses and comparison of the regions cannot be conducted. However, there is no direct correlation between the number of coral colonies and the copepod MOTUs discovered. My results also suggest that copepods see no difference between species of Galaxea corals, and individuals combined in one MOTU could be often found on both species of Galaxea corals. The similar pattern was observed for the symbiotic copepods, which I discovered on the close species of Stylophora (Chapter 5) and on the Fungiidae corals (Chapter 6).

The obtained results are the first assessment of biogeographical distribution of the coral- associated fauna of copepods using genetic methods and will be used as a baseline for the future investigation of the distribution patterns of these copepods in the Indo-Pacific.

Compared to this study, Chapters 5 and 6 have higher host taxonomic resolution and a specific focus on the potential factors affecting ecological distribution.

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Figure 4.10 Number of MOTUs identified from each sampled location for the two dominant families Asterocheridae and Anchimolgidae; the dashed line represents a number of host colonies collected in each region.

4.5 Conclusions

Research conducted in this chapter revealed a highly diverse fauna of symbiotic copepods associated with Galaxea. At least 185 MOTUs of copepods of six different orders were distinguished by molecular methods, which is almost ten times more than were previously described in the literature. Representatives of the families Anchimolgidae

(poecilostomatoid Cyclopoida) and Asterocheridae (Siphonostomatoida) appear to be both most abundant and most diverse groups of copepods on the Galaxea corals. Family

Anchimolgidae comprises 48% of all individuals belonging to 33 MOTUs (lowest estimation), and family Asterocheridae constitutes 23% of individuals of 42 different

MOTUs. There were also no signs of the host-specificity on the host species level, and both species of Galaxea seem to be functionally and ecologically equivalent for the copepods. Importantly, this work is the first study addressing the patterns of biogeographical distribution of symbiotic copepods based on the genetic data. My results demonstrate a high geographical fragmentation of copepods associated with Galaxea corals, with most of the species occurring only in one region. It is not clear yet whether we can claim these species as endemics because most of the discovered copepods are new for the studied regions. This work is also the first report of copepods from Galaxea for the Red Sea, Maldives, and the west coast of Australia. Additional sampling in new regions will provide more clarity on this matter and an opportunity to find many potential new species. However, the general pattern is not likely to change, and the zoogeographical endemism seems to be inherent to the majority of the species of

103 copepods associated with Galaxea corals.

Chapter 5: Diversity assessment of poecilostomatoid copepods associated with Pocilloporidae corals in the Saudi Arabian Red Sea

5.1 Introduction

Copepods from the order Poecilostomatoida are common and well-known symbionts of scleractinian corals in the Indo-Pacific. Many species of these copepods are associated with the Pocilloporidae corals, a family that exhibits a high level of morphological variability within and among its members (Flot et al., 2011; Keshavmurthy et al., 2013).

Assessment of the diversity of the copepod species living in association with these corals and the patterns of their host-specificity allows us to reveal whether there are differences between the copepod fauna of the closely related but morphologically variable host species.

Branching and submassive corals of the family Pocilloporidae are considered to be among the main reef-framework builders and the dominant species in shallow-water reef environments exposed to strong wave action. The family includes three genera:

Pocillopora, Seriatopora, and Stylophora, widespread across all Indo-Pacific. However, unlike other scleractinian corals having the center of biodiversity in the Coral Triangle,

Stylophora is more diverse in the western Indian Ocean and the Red Sea (Scheer & Pillai,

1983; Sheppard & Sheppard, 1991; Veron, 2000).

Pocilloporidae corals are very phenotypically plastic, i.e., forming numerous morphological variations across different habitats, depths, and geographic regions (Flot et al., 2011). Species boundaries revealed with genetic methods are often not congruent

104 with the "ecomorphs" distinguished by morphological features (Shaish et al., 2007; Flot et al., 2008; Flot et al., 2011; Keshavmurthy et al., 2013). Corals of the same species may have different features appearing in response to specific environmental conditions: colonies exposed to strong wave action are often more massive with robust branch morphology, and individual colonies inhabiting deeper/less exposed parts of the reef or sites with high turbidity have thinner and longer branches (Flot et al., 2011). For instance, the latest study based on seven DNA loci demonstrates that the morphologically distinct morphotypes of Stylophora in the Red Sea, S. pistillata, S. danae Milne Edwards &

Haime, 1850, S. subseriata (Ehrenberg, 1834), and S. kuehlmanni Scheer & Pillai, 1983 should now be considered as a single species S. pistillata (Arrigoni et al., 2016).

Moreover, S. pistillata species, often considered as a cosmopolite species widespread across all Indo-Pacific, is now divided into four molecular clades with a genetically distinct Red Sea - Persian Gulf - Kenya clade (Keshavmurthy et al., 2013). According to the same study, Seriatopora hystrix/caliendrum species collected from different regions of the Indo-Pacific is a sister clade to other clades of Stylophora pistillata and seems to be embedded between the Pacific-Western Australia and the South Africa-Madagascar S. pistillata clades (Keshavmurthy et al., 2013).

Corals from the family Pocilloporidae host a great variety of copepods, including highly transformed xarifiid copepods which live in the gastrovascular cavities of the polyps. These symbiotic copepods were first noticed by Dr. Sebastian A. Gerlach during the Xarifa Expedition to the Red Sea and the Maldives Archipelago in 1957-1958

(Humes, 1985b). Since then, 34 species of copepods from order Poecilostomatoida have been found in association with Pocilloporidae corals. Among these poecilostomatoid

105 copepods, 21 species belong to the endosymbiotic family Xarifiidae (Humes, 1962;

Humes & Ho, 1968; Humes, 1985b), six species from the family Anchimolgidae (Kim,

2003, 2004b, 2007), three species (Isomolgus desmotes, Spaniomolgus crassus, S. compositus, and S. geminus) belong to the family Rhynchomolgidae (Humes & Frost,

1963; Humes & Ho, 1968; Dojiri, 1988), and three species of copepods from families

Anthessidae and Macrochironidae (Kim, 2004b, 2009; Cheng et al., 2016). Species

Isomolgus desmotes and Spaniomolgus sp. are reported from galls (tubular-shaped modification of corallites) of Seriatopora hystrix and Stylophora pistillata, respectively

(Dojiri, 1988; Ivanenko et al., 2014). Most of the species reported from Pocilloporidae corals have been described from Madagascar, Indonesia, New Caledonia, Taiwan, and the Great Barrier Reef, with only a few records from the Marshall Islands, Mauritius,

Maldives, and single records for Panama and the Red Sea (see the list of species in

Appendix 5.1).

Based on the data obtained from other groups of animals, mainly corals and fishes, it can be argued that the Red Sea is one of the regions with a high level of overall biodiversity and endemism, including approximately 1400 fish species and over 346 reef- building coral species of which 19 are endemic (5.5%) (Allen, 2008; Veron et al., 2009;

Bruckner & Dempsey, 2015; DiBattista et al., 2016b). However, there are no studies on copepods from corals in this region, except for two reports of parasitic poecilostomatoids from Stylophora (Humes, 1960; Ivanenko et al., 2014). The Red Sea appears to be an area of particular interest because of its unique environment. First of all, it is the most saline and hottest sea in the world that harbors actively growing reefs (cf. the Arabian

Gulf, with more extreme conditions but less reef growth). Such conditions are formed by

106 extremely high average temperatures in this latitude complemented with geologic activity in the deep, narrow basin and rare fresh water input (Behairy et al., 1992). High temperatures lead to high evaporation rates and increase the level of salinity. Though water exchange with Indian Ocean is limited by the narrow and relatively shallow Strait of Bab al Mandab, incoming less hot, less saline, nutrient rich water creates significant latitudinal gradients in salinity, sea-surface temperatures and nutrient concentration

(Behairy et al., 1992). The presence of major environmental gradients can create varied habitats for coral reefs and, thus, potentially increases the diversity of species associated with corals. A decrease in the sea level during the last glacial maximum resulted in almost complete isolation of the Red Sea from the Gulf of Aden and the Indian Ocean and likely contributed to the formation of the endemic fauna within the Red Sea

(DiBattista et al., 2016a). Furthermore, the Red Sea is a fairly young environment at the periphery of the world’s coral reefs distribution, which makes it an interesting region for studies on biogeography of corals and their symbiotic copepods.

The research presented in this chapter is focused on a species-level diversity assessment and host-specificity of poecilostomatoid copepods collected from one coral family, the family Pocilloporidae in the Red Sea. The variability of the host morphotypes, the differences in environmental conditions among the sampling locations together with the acknowledged high diversity of other coral-associated taxa suggest that a rich and highly diverse fauna of symbiotic copepods can be found on Pocilloporidae corals. The analysis of the species diversity of copepods from the two sampled regions will allow me to assess the effect of the environmental gradient, while samples from the different coral

107 morphotypes will give me an opportunity to analyze the effect of host morphology on the copepod species diversity.

5.2 Samples and study sites

20 colonies of three genera of Pocilloporidae (Seriatopora, Stylophora, and Pocillopora) were collected in different regions of the Red Sea: Al Wajh Bank in the north, Yanbu and reefs around KAUST in the central region, and the Farasan Islands in the south

(Figure 5.1 and Table 5.2). Samples were taken from a variety of depths (0-36 m). See

Appendix 5.2 for the detailed sampling information and the colonies collected.

Underwater photographs of the sampled Pocilloporidae colonies, with the photographs of coral skeletons with corallite structures visible are presented in Appendix 5.3. The methods used for the sampling and genetic analyses are described in detail in Chapter 2.

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Table 5.1 List of the Pocilloporidae colonies collected, with the information about the depth and location of the sampling and the total number of copepods collected from each host (see full sampling information in the Appendix 5.2).

Voucher Depth No. of # Coral species Locality number (m) copepods

1 SA2013_012 Stylophora pistillata central RS, KAUST 1 19 2 SA2013_025 Stylophora pistillata (morph. subseriata) central RS, KAUST 10.4 18 3 SA2013_031 Stylophora pistillata (morph. danae) central RS, KAUST 28 11 4 SA2013_060 Stylophora pistillata central RS, KAUST 1 43 5 SA2013_061 Stylophora pistillata central RS, KAUST 1 2 6 SA2013_072 Stylophora pistillata (morph. mordax) central RS, Al Lith 2.5 7 7 SA2014_001 Stylophora pistillata south RS, Farasan Islands 3.6 5 8 SA2014_004 Stylophora pistillata south RS, Farasan Islands 4.6 17 9 SA2014_016 Pocillopora damicornis south RS, Wasalayt Shoals 19.7 14 10 SA2014_019 Seriatopora hystrix south RS, Wasalayt Shoals 8.3 10 11 SA2014_022 Stylophora pistillata (morph. kuehlmanni) south RS, Wasalayt Shoals 35.6 4 12 SA2014_027 Seriatopora hystrix south RS, Farasan Islands 14.2 7 13 SA2014_044 Stylophora pistillata south RS, Farasan Islands 4.8 8 14 SA2014_045 Pocillopora sp. south RS, Farasan Islands 6 14 15 SA2014_069 Stylophora pistillata south RS, Farasan Islands 13.1 3 16 SA2014_075 Stylophora pistillata south RS, Farasan Islands 4.4 8 17 SA2014_087 Stylophora pistillata (morph. mamillata) central RS, KAUST 6.4 7 18 SA2014_091 Seriatopora hystrix central RS, KAUST 12.7 2 19 SA2014_092 Stylophora pistillata (morph. kuehlmanni) central RS, KAUST 25.8 12 20 SA2014_118 Stylophora pistillata (morph. wellsi) central RS, KAUST 2 12 21 SA2014_120 Stylophora pistillata (morph. wellsi) central RS, KAUST 2 19 22 SA2014_123 Seriatopora hystrix central RS, KAUST 5.6 9 Total number of copepods collected 251

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5.3 Results

5.3.1 PCR and sequencing

Overall, 251 specimens of poecilostomatoid copepods from 21 colonies were found and

244 were analyzed (see Appendix 5.4). 364 sequences (182 contigs) of COI gene were produced with an average read length of 700 bp (71.3% success rate); 396 sequences

(198 contigs) of ITS2 were produced with an average read length of 500 bp (77.6% success rate).

5.3.2 Phylogenetic relationships and species delimitation

Analyses of both COI and ITS2 markers revealed a high diversity of this group: 19

MOTUs were distinguished based on multi-locus species delimitation methods ABGD,

PTP, and GMYC. 18 of 21 MOTUs were identified as species of the genus Spaniomolgus

(Rhynchomolgidae) (Figure 5.2). Multi-locus molecular species delimitation results from

ABGD, PTP, and GMYC approaches were summarized in Figure 5.3. Additionally, analysis of the phylogenetic position of copepods of the genus Spaniomolgus showed that the family Rhynchomolgidae could be paraphyletic.

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Figure 5.2 Tanglegram of the phylogenetic trees of Spaniomolgus based on the COI dataset (on the left) and ITS2 dataset (on the right). The colored bars represent combined species delimitation result obtained from ABGD, PTP, and GMYC.

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Figure 5.3 Conspecificity matrix based on the results from ABGD, PTP, and GMYC. COI on the left, ITS2 on the right.

The morphological and genetic analyses revealed that all poecilostomatoid copepods found on the colonies of Stylophora and Seriatopora belong to only one genus,

Spaniomolgus, except only one MOTU belonging to a different family (Anchimolgidae), which was found on a Pocillopora colony (Figure 5.2). Moreover, there were no signs of preference between different morphotypes of Stylophora pistillata, except Spaniomolgus sp. 6, which was found only on two colonies belonging to Stylophora pistillata morph. wellsi (Table 5.2). Nevertheless, on the species level, most of the Spaniomolgus copepods showed preferences among the host genera: there is a clearly distinguished MOTU

Spaniomolgus sp. 1, which was found only on Seriatopora (four colonies), and MOTU

Spaniomolgus sp. 16, which was found only on Pocillopora (one colony). Twelve

MOTUs of Spaniomolgus (sp. 2-3, sp. 6-10, sp. 12-15, and sp. 17) were found only on

Stylophora (all morphotypes). Spaniomolgus sp. 4 and sp. 11 were found on both

Stylophora and Pocillopora, and Spaniomolgus sp. 5 was found on both Stylophora and

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Seriatopora. Relatively low diversity of copepods (four MOTUs) found on Pocillopora colonies is most likely related to the fact that I had samples from only two colonies of

Pocillopora included in the analysis. Six MOTUs of Spaniomolgus were found only on the Farasan Islands (southern Red Sea), seven MOTUs were found only in the central part of the Red Sea (near KAUST), and four MOTUs were found in both regions.

Table 5.2 Diversity, host-specificity, and distribution of copepod MOTUs collected from Pocilloporidae corals; C – central Red Sea, S – southern Red Sea. Old species names are used to distinguish morphotypes of Stylophora colonies.

MOTUs Host morphotypes Host colonies Depth (m) Region Spaniomolgus sp. 1 Seriatopora hystrix 19, 75, 91, 123 4.4-12.7 C-S Spaniomolgus sp. 2 Stylophora pistillata, S. subseriata 25, 44, 69 4.8-13.1 C-S Spaniomolgus sp. 3 Stylophora pistillata 1, 4, 60, 75 1-4.6 C-S Spaniomolgus sp. 4 Stylophora pistillata, Pocillopora sp. 44, 45 4.8-6 S Spaniomolgus sp. 5 Stylophora pistillata, Seriatopora hystrix 4, 19, 75 4.4-8.3 S Spaniomolgus sp. 6 Stylophora wellsi 118, 120 2 C Spaniomolgus sp. 7 Stylophora mordax 72 2.5 C Spaniomolgus sp. 8 Stylophora danae 31 28 C Spaniomolgus sp. 9 Stylophora pistillata 4 4.6 S Spaniomolgus sp. 10 Stylophora pistillata, S. wellsi 12, 118, 120 1-2 C Spaniomolgus sp. 11 Stylophora pistillata, Pocillopora sp. 44, 45 4.8-6 S Spaniomolgus sp. 12 Stylophora pistillata & wellsi 4, 60, 120 1-4.6 C-S Spaniomolgus sp. 13 Stylophora subseriata & danae 25, 31 10.4-28 C Spaniomolgus sp. 14 Stylophora pistillata 4 4.6 S Spaniomolgus sp. 15 Stylophora pistillata 12 1 C Spaniomolgus sp. 16 Pocillopora sp. 45 6 S Spaniomolgus sp. 17 Stylophora pistillata, S. wellsi 1, 12, 60, 118 1-3.6 C Anchimolgidae sp. 1 Pocillopora damicornis 16 19.7 S Anchimolgidae sp. 2 Stylophora mamillata 87 6.4 C Anchimolgidae sp. 3 Pocillopora damicornis 16 19.7 S

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5.3.3 Morphological analyses

Initial morphological examination of the discovered copepods confirmed their identification as morphologically variable species of the genus Spaniomolgus. However, after the results from genetic analysis were obtained, the subsequent thorough morphological investigation of the specimens by copepod taxonomists revealed morphological differences between the newly discovered MOTUs. Species discovered in the Red Sea differ from the previously described species of the genus and among themselves by a number of morphological features, such as shape of the prosome and the genital somite, connection of the first pedigerous somite with the cephalothorax, and armature of the antenna. Six species of the genus Spaniomolgus (one of them was previously-described species) were discovered in washings and inside the galls of the hermatypic coral Stylophora pistillata. These results point out the necessity of a revision of current morphological criteria for the identification of copepod species as some of the interspecific differences could be attributed to intraspecific variability. Five recently described species, Spaniomolgus globus, S. stylophorus, S. dentatus, S. maculatus, and S. acutus, were recorded in a paper with me as a co-author, the species descriptions with a key for the identification of all congeners are provided in Appendix 5.5. Compliance of the discovered species with the molecular MOTUs we revealed requires additional verification as described specimens were not sequenced.

5.4 Discussion

My results demonstrate the existence of an evolutionary radiation of symbiotic copepods that inhabit a group of closely related coral species. Sympatric speciation of the copepods

114 of the genus Spaniomolgus could occur through such mechanisms of reproductive isolation as transition to inhabiting new coral forms and/or inhabiting corals from a different depth. Therefore, presence of the high diversity of copepods could be a result of fine-scale ecological specializations. These results are consistent with the patterns previously reported for gall-inducing insects on plants (Joy & Crespi, 2007).

Up to now, the genus Spaniomolgus consisted of only three species: the type species S. compositus (Humes & Frost, 1964), S. geminus (Humes & Ho, 1968) and S. crassus (Humes & Ho, 1968). Description of this genus was originally based on three species of copepods of the genus Lichomolgus found in association with the scleractinians of the genera Acropora Oken, 1815, Seriatopora Lamarck, 1816, and Stylophora Schweigger, 1820 in only one location, Nosy Bé in northern Madagascar

(Humes & Stock, 1972). There were no records of Spaniomolgus since the revision of the lichomolgiid complex in 1973 (Humes & Stock) and until the discovery of an unidentified species of Spaniomolgus living in modified polyps (galls) of Stylophora pistillata in the Red Sea (Ivanenko et al., 2014). No other species have been described for this genus up to the publication of a paper I co-authored with a description of five species of Spaniomolgus from the Red Sea (Conradi et al., 2018). See Table 5.3 for the list of species with information about the hosts and the sampling details.

Copepod species Spaniomolgus crassus and S. geminus were reported by Humes &

Ho (1968) from washing from Stylophora mordax (Dana, 1846), which is now accepted as Stylophora pistillata. Therefore, these copepods should be now considered as co- occurring symbionts of coral host Stylophora pistillata along with the five recently described species of Spaniomolgus from the Red Sea. Thus, seven species of

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Spaniomolgus are now recorded as associates of the host species Stylophora pistillata.

There is uncertainty about the hosts of the Spaniomolgus compositus found by Humes &

Frost (1964) in washings from the Stylophora subseriata, Seriatopora octoptera, and

Seriatopora sp. We assume that it is likely that the coral indicated by Humes & Frost

(1964) as Seriatopora subseriata is actually Stylophora subseriata (Ehrenberg, 1834) as the name Seriatopora subseriata is not valid.

Symbiotic copepods have been studied from 66 genera of scleractinian corals

(Cheng et al., 2016), and only three of them, Stylophora, Seriatopora, and Acropora, are known to harbor Spaniomolgus copepods. Moreover, I have not yet analyzed copepods collected from other coral families in the Red Sea, except for copepods from Fungiidae corals but there were no Spaniomolgus discovered (see Chapter 6). Hence, for now, I assume that Spaniomolgus are specific only to these three genera of corals, and occur only in the western part of the Indian Ocean. Though, targeted sampling in other regions of the Indo-Pacific could clarify patterns of the distribution of these species.

Table 5.3 List of all species of the genus Spaniomolgus with information about the hosts and the sampling details.

Depth Species Authority Coral family Coral species Region (m) S. acutus Conradi et al., 2018 Pocilloporidae Stylophora pistillata (subseriata) Red Sea 10.4 S. acutus Conradi et al., 2018 Pocilloporidae Stylophora pistillata (danae) Red Sea 28 S. compositus (Humes & Frost, 1964) Pocilloporidae Seriatopora octoptera Madagascar NA S. compositus (Humes & Frost, 1964) Pocilloporidae Seriatopora sp. Madagascar 2 S. compositus (Humes & Frost, 1964) Pocilloporidae Stylophora subseriata Madagascar 1 S. crassus Humes & Ho, 1968 Pocilloporidae Stylophora pistillata (mordax) Madagascar 2 S. crassus Humes & Ho, 1968 Pocilloporidae Stylophora pistillata Madagascar 0.5 S. crassus Humes & Ho, 1968 Pocilloporidae Stylophora pistillata (mordax) Red Sea 2.5 S. crassus Humes & Ho, 1969 Acroporidae Acropora sp. Madagascar 2 S. dentatus Conradi et al., 2018 Pocilloporidae Stylophora pistillata (danae) Red Sea 28 S. geminus (Humes & Ho, 1968) Pocilloporidae Stylophora pistillata (mordax) Madagascar 2

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S. geminus (Humes & Ho, 1968) Pocilloporidae Stylophora pistillata Madagascar 0.5 S. geminus (Humes & Ho, 1968) Pocilloporidae Stylophora sp. Madagascar 4 S. geminus (Humes & Ho, 1968) Acroporidae Acropora sp. Madagascar 2 S. globus Conradi et al., 2018 Pocilloporidae Stylophora pistillata Red Sea 1 S. maculatus Conradi et al., 2018 Pocilloporidae Stylophora pistillata (subseriata) Red Sea 10.4 S. maculatus Conradi et al., 2018 Pocilloporidae Stylophora pistillata (danae) Red Sea 28 S. stylophorus Conradi et al., 2018 Pocilloporidae Stylophora pistillata Red Sea 1 S. stylophorus Conradi et al., 2018 Pocilloporidae Stylophora pistillata (danae) Red Sea 28

Considering the limited number of samples and only two locations studied, the revealed patterns of the host-specificity and the species distribution of Spaniomolgus copepods might change with the increased number of samples. Fortunately, in addition to the samples from 22 colonies analyzed in this chapter, I already have a collection of 39 more colonies of Pocilloporidae corals sampled in the northern, central, and southern regions of the Red Sea, and which are partially processed and waiting to be analyzed.

However, in case additional sampling is required, collection of coral host colonies of the

Spaniomolgus copepods could be challenging in some of the regions. Most of the

Spaniomolgus species are associated with Stylophora corals, which commonly inhabit shallow-water reef crests and lagoons and, therefore, are stronger affected by high temperature, water acidification, and pollution. Although, according to my observations,

Acropora species are usually occurring deeper in the reef, they are among the most susceptible species to bleaching. Copepods species associated with these corals will perish once their hosts disappear, some even before being described. For instance, reefs close to Al Lith in the central Red Sea, where some of my samples were collected, were severely affected by the major bleaching event of 2015-2016 (Monroe et al., 2018;

Osman et al., 2018). Most of the colonies of Stylophora pistillata at the Al Lith reefs and

117 about 20% of colonies at the Thuwal reefs were bleached and died (Monroe et al., 2018;

Osman et al., 2018; personal observations of Viatcheslav Ivanenko and Sofya Mudrova in May 2017). So, abundance and diversity of described copepods could have also been strongly affected, and some of the species collected from the reefs near Al Lith could be already gone.

5.7 Conclusion

In this chapter, I evaluated the diversity of the copepods associated with Pocilloporidae corals in the Red Sea. 90% of all poecilostomatoid copepods discovered both on the surface and in the galls of the Pocilloporidae corals belong to the symbiotic genus

Spaniomolgus (Rhynchomolgidae), which has been previously reported only from

Madagascar. The genetic assessment revealed an extremely high diversity of this genus:

17 MOTUs were revealed by different delimitation methods, five of which were described during this project. With these species, there are now eight species of

Spaniomolgus copepods reported living in the symbiosis with three genera of branching corals, Stylophora, Seriatopora, and Acropora, in the western Indian Ocean. Considering the number of the MOTUs distinguished herein, there are at least 12 more species of the genus waiting to be described. The process of speciation of the copepods of genus

Spaniomolgus could occur through mechanisms such as reproductive isolation as an evolutionary transition to inhabiting new coral forms and/or corals from different depths.

Future work within this study system will include processing and analyzing larger dataset with a better coverage of the different depths and host morphotypes, and which includes samples from the northern region of the Red Sea.

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In this chapter, I focused on the assessment of a species-level diversity assessment and the host-specificity of copepods collected from the corals of the family

Pocilloporidae, which are known to be forming numerous morphological variations across different habitats, depths, and geographic regions. The next chapter will, on other hand, focus on the copepods associated with mushroom coral species, which have low ecological variability.

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Chapter 6: Diversity and host-specificity of copepods associated with mushroom corals in the Red Sea1

6.1 Introduction

In this chapter, as a host model group, I selected a monophyletic group of stony corals that is well known for its associated symbiotic copepods, i.e., the scleractinian family

Fungiidae Dana, 1846, popularly known as mushroom corals, and for which the phylogeny is well studied (Wells, 1966; Cairns, 1984; Hoeksema, 1989, 1990, 1993;

Gittenberger et al., 2011; Benzoni et al., 2012; Hoeksema et al., 2012; Oku et al., 2017).

One specific trait shown by the majority of fungiids is a free-living (anthocyathus) phase, in which the whole coral becomes detached from its substrate and, therefore, is able to grow soft tissue all around its skeleton (Hoeksema & Yeemin, 2011). Due to the overall shape of mushroom corals, their lower side offers shelter to various species of invertebrates that prefer to hide, at least during daylight (Hoeksema & Fransen, 2011;

Gittenberger & Hoeksema, 2013; Hoeksema et al., 2013a; Hoeksema et al., 2013b;

Alamaru et al., 2016). However, this shelter can become temporarily lost when the corals become buried or overturned (Bongaerts et al., 2012; Hoeksema & Bongaerts, 2016).

Individuals of other associated symbiotic species, which may include many other invertebrates and various fish species, dwell on the external upper side of mushroom

1 The work presented in this chapter is published as a paper in the journal Molecular Phylogenetics and Evolution (V.N. Ivanenko, B.W. Hoeksema, S.V. Mudrova, et al., 2018). Contribution: I participated in the data collection in the field, sorted samples, extracted DNA, conducted genetic markers amplification and sequencing, conducted the sequence cleaning and initial analysis, prepared the map; took part in planning, discussion of the results and preparing of the manuscript.

120 corals (Bos, 2012; Hoeksema & Farenzena, 2012; Hoeksema & Ten Hove, 2014; Bos &

Hoeksema, 2015; Montano et al., 2015; Bos & Hoeksema, 2017). Hence, it is expected that mushroom corals may potentially offer different habitats to associated symbiotic copepods. In addition, mushroom corals species have a variety of shapes, maximum sizes, and life-history traits (Hoeksema, 1989, 1990; Gittenberger et al., 2011), creating the potential for host-shape specificity of their symbionts: about 20% of the species of

Fungiidae remain attached and do not become free-living (Hoeksema, 2009; Benzoni et al., 2012); different species show different maximum sizes depending on whether they have a single (monostomatous) or multiple mouths (polystomatous) (Hoeksema, 1990;

Gittenberger et al., 2011), with consequences for the space that is available for associated symbiotic organisms (Hoeksema et al., 2012; Hoeksema, 2014). Therefore, mushroom coral diversity makes them suitable for a study of the host specificity of the associated copepod fauna. In this chapter, I address following questions: which Red Sea fungiid species hosted symbiotic copepods, whether the symbiotic copepods were generalists or host specific, whether different symbiotic copepods with similar or closely related hosts were evolutionarily related, addressing what could be identified as “the copepod perspective” in the relationship, and whether closely related host corals shared a similar symbiotic copepod fauna, addressing “the host perspective”.

Most of the 27 species of symbiotic copepods reported from fungiid corals have been described as ectosymbionts from New Caledonia (Humes, 1973a, 1996a, 1997;

Kim, 2003) and the Moluccas in Indonesia (Humes, 1978, 1979, 1997; Humes & Dojiri,

1983; Kim, 2007); only two species of fungiid-associated copepods were found in

Madagascar (Humes & Dojiri, 1983; Kim, 2010), and none from the Red Sea until now

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(see the list of species in Appendix 6.1). According to these earlier reports, Pleuractis seychellensis (Hoeksema, 1993) has the richest copepod fauna consisting of six species followed by Ctenactis echinata (Pallas, 1766) and Sandalolitha robusta (Quelch, 1886) with five symbiotic copepod species. Twenty-six copepod species associated with

Fungiidae were restricted to this scleractinian family and were never reported from other hosts (i.e., they were never reported on non-fungiid corals). Only one copepod species,

Asteropontius latioriger, was found in Madagascar living on Fungiidae and Acroporidae.

Twenty-one copepod species are known from only a single coral species; four copepod species (Anchimolgus notatus Humes, 1978, A. punctilis Humes, 1978, Paramarda aculeata Humes, 1978, and Schedomolgus tener (Humes, 1973)) were recorded from two host species; two copepods (Anchimolgus pandus Humes, 1978 and A. latens Humes,

1978) were found on four host species. Thus, I expect to find rather strong species- specific associations between fungiid corals and their associated copepods, and I aim at providing reliable inference on the ecological and evolutionary drivers of such host- specific symbiotic relationship using a quantitative framework to address the relationship.

6.2 Samples and study sites

A total of 26 coral colonies representing 13 species of fungiid corals belonging to eight genera were collected at depths ranging from three to 34 m at ten different reefs located in the central and the southern parts (vicinity of Thuwal and Farasan Islands correspondingly) of the Saudi Arabian Red Sea (Figure 6.1, Table 6.1, for the full sampling information see Appendix 6.2, for the underwater photographs of the corals see

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Appendix 6.3). After a preliminary screening to identify putative morphotypes, up to five copepod individuals representing different morphotypes and sexes from each coral host were isolated individually in 2-ml tubes for the following analyses using morphological and molecular methods. The methods used for the genetic analysis are described in detail in Chapter 2.

Table 6.1 List of the Fungiidae colonies collected, with the information about the depth and location of the sampling and the number of copepods collected from each host (see full sampling information in the Appendix 6.1). Voucher Depth No of # Coral species Locality number (m) cop-s 1 SA2014-102 Ctenactis crassa (Dana, 1846) RS, Thuwal, Al Fahal S 2.3 2 2 SA2014-130 Ctenactis echinata (Pallas, 1766) RS, Thuwal, Fsar 24 4 3 SA2014-103 Cycloseris costulata (Ortmann, 1889) RS, Thuwal, Abu Gishaa 20 1 4 SA2014-108 Cycloseris costulata (Ortmann, 1889) RS, Thuwal, Al Asoul 34 5 5 SA2014-074 Danafungia horrida (Dana, 1846) RS, Farasan Islands 4.1 4 6 SA2014-100 Danafungia scruposa (Klunzinger, 1879) RS, Thuwal, Al Bilut 15.3 15 7 SA2014-101 Danafungia scruposa (Klunzinger, 1879) RS, Thuwal, Al Fahal N 18.5 15 8 SA2014-096 Fungia fungites (Linnaeus, 1758) RS, Thuwal, Al Bilut 6 7 9 SA2014-106 Herpolitha limax (Esper, 1797) RS, Thuwal, Abu Gishaa 8 9 10 SA2014-128 Herpolitha limax (Esper, 1797) RS, Thuwal, Fsar 11 1

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11 SA2014-131 Herpolitha limax (Esper, 1797) RS, Thuwal, Fsar 33 7 12 SA2014-015 Herpolitha limax (Esper, 1797) RS, Wasaliyat Shoals 24.9 6 13 SA2014-057 Herpolitha limax (Esper, 1797) RS, Farasan Islands 4 6 14 SA2014-061 Herpolitha limax (Esper, 1797) RS, Farasan Islands 19 11 15 SA2014-065 Herpolitha limax (Esper, 1797) RS, Farasan Islands 10 15 16 SA2014-098 Herpolitha limax (Esper, 1797) RS, Thuwal, Al Bilut 3 7 17 SA2014-105 Lithophyllon concinna (Verrill, 1864) RS, Thuwal, Al Asoul 20 6 18 SA2014-127 Lithophyllon concinna (Verrill, 1864) RS, Thuwal, Fsar 11 10 19 SA2014-093 Lithophyllon repanda (Dana, 1846) RS, Thuwal, Al Fahal S 29 3 20 SA2014-097 Pleuractis granulosa (Klunzinger, 1879) RS, Thuwal, Al Bilut 27 15 21 SA2014-099 Pleuractis taiwanensis (Hoeksema & Dai, 1991) RS, Thuwal, Al Bilut 20 1 22 SA2014-104 Pleuractis cf. seychellensis (Hoeksema, 1993) RS, Thuwal, Abu Gishaa 11 8 23 SA2014-107 Pleuractis cf. seychellensis (Hoeksema, 1993) RS, Thuwal, Abu Gishaa 20 6 24 SA2014-095 Pleuractis cf. seychellensis (Hoeksema, 1993) RS, Thuwal, Al Bilut 19 2 25 SA2014-129 Podabacia crustacea (Pallas, 1766) RS, Thuwal, Fsar 22 11 26 SA2014-094 Podabacia crustacea (Pallas, 1766) RS, Thuwal, Al Fahal N 15 7 Total number of copepods collected 184

6.3 Results

6.3.1 Copepod species

Based on the examination of the external morphologies used for copepod taxonomy, eight species were identified from the 184 individuals that were extracted from the mushroom corals: one Cyclopidae (cf. Euryte, with one individual only, unfortunately lost after extraction of DNA), one Rhynchomolgidae (one individual), two

Asterocheridae of the genus Asteropontius (35 and 50 individuals) and four

Anchimolgidae representing the genera Prionomolgus (two species with 1 and 17 individuals) and Schedomolgus (two species with 7 and 72 individuals) (Figure 6.2).

Given the systematic position of the eight potential species, the phylogenies were rooted based on the distinction between the families and classes, removing the individual

Cyclopidae for which no morphological information was available (Figure 6.3,

Appendices 6.5-6.7).

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Figure 6.2 Habitus of copepods found on mushroom corals of the Red Sea. Ventral view, confocal microscopy of animals after extraction of DNA (red: females, blue: males). (A) Prionomolgus sp. 1; (B) Prionomolgus sp. 2; (C) Schedomolgus sp. 1; (D) Schedomolgus sp. 2; (E) Paradoridicola sp. 1; (F) Asteropontius sp. 1.

The three DNA taxonomy approaches based on the three datasets (COI, ITS2, and

COI + ITS2) provided consistent estimates of seven species (excluding the Cyclopidae)

(Appendix 6.8). ABGD estimated seven entities for both COI (with prior intraspecific distances from 0.077 to 0.0215) and ITS2 markers (with prior intraspecific distances from

0.0028 to 0.0129). Lower values of prior intraspecific distances provided estimates higher than 7. This method could not be applied on the concatenated dataset. PTP estimated seven entities for both COI and COI + ITS2 datasets and nine entities for the ITS2 dataset. In the single marker cases the confidence interval of the solutions (COI: 7–33,

125 mean 16.23; ITS2: 4–20, mean 9.59) was larger than in the combined dataset (COI +

ITS2: 7–9, mean 7.14). GMYC did not provide a clear answer, and larger estimates than the other methods were obtained (Appendix 6.8).

Figure 6.3 Phylogenetic reconstruction of the sampled copepods based on the COI analyses. The tree topology corresponds with the Bayesian analyses, whereas node values indicate support values for each clade (Bayesian posterior probabilities/maximum likelihood bootstrap). Asterisks (*) indicate maximum support values. Entities recovered by the delineation analyses are labeled with different colors indicating their distribution across different coral host coral species. See results from other phylogenetic analyses in Appendices 6.5-6.7.

Overall, the morphology and DNA taxonomy on different markers and different methods collectively indicated to the presence of seven species of symbiotic copepods

(excluding the only individual of Cyclopidae representing Euryte or a closely related genus). Genetic distances within these seven species-level groups were up to 8.1% in

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COI and 2.1% in ITS2, whereas distances between species were between 16.6% and

55.7% (up to 35.1% within each copepod family) for COI and between 2.1% and 40.9%

(up to 18.5% within each family) in ITS2 (Appendix 6.9). We used these seven species to test hypotheses on species-specificity of the host association and on the effect of geographic distances in structuring genetic diversity.

6.3.2 Symbiotic association

On the 13 mushroom coral species that hosted symbiotic copepods, from one to three copepod species were found (Figure 6.4). Each copepod species occurred on as few as one and up to nine host species. The number of coral host species colonized by each copepod species correlated with the number of individuals of copepods found in our study (Pearson’s r = 0.92, p = 0.0029).

Our results did not support any specificity of the association between symbiotic copepods and their host: each species of copepod was recovered from multiple species of fungiid corals. These empirical observations were supported by the association tests, which indicated that the numbers of observed hosts for each species (except for

Prionomolgus sp. 2, with low sample size) were not significantly lower than the ones obtained from a random association between corals and copepods (Table 6.2), neither at the level of coral species nor at the level of coral genus.

None of the copepod species showed a significant value for a phylogenetic signal of the host coral phylogenetic relationships (Appendix 6.10), and no correlation was found between similarity in copepod community composition and phylogenetic similarity between host species (Mantel test: r = −0.03, p = 0.55) In addition, the genetic distances

127 between individuals within each copepod species could be explained by only 5 to 30% by the association with the host coral, and even less by geographic isolation among reefs and geographical areas in the Red Sea (Table 6.3).

Table 6.2 Number of copepod individuals and coral colonies, host coral species and genera for each of the most common copepod species. The results of the simulations reported for host species (and host genera) represent the proportion of the expected numbers that came out lower than the observed numbers from the resampling tests (1000 replicates) on all the occurrences of all 184 individual copepods on the host coral under a null model of no association: values smaller than 0.05 denote potentially significant associations. The other three species, found in less than 15 individuals, are not reported because no test would be meaningful with such low sample size.

Coral Simulation Coral Simulation Species Copepods Corals species for species genera for genera Asteropontius sp.1 50 12 7 0.145 6 0.168 Asteropontius sp.2 35 9 7 0.355 6 0.196 Prionomolgus sp.2 17 5 3 0.011 3 0.030 Schedomolgus sp.1 72 17 9 0.254 7 0.179

Table 6.3 Proportion of explained variance of genetic diversity in COI due to differences in host-coral species, and reef (nested within the Southern or the Central area, when possible) together with the p- values between parentheses. The other three species, found in less than 15 individuals, are not reported because no test would be meaningful with such low sample size.

Host coral Reef (nested Species Area Unexplained species in area) Asteropontius sp.1 0.301 0.072 0.099 0.528 (p = 0.003) (p = 0.029) (p = 0.165) Asteropontius sp.2 0.193 - 0.010 0.797 (p = 0.438) (p = 0.786) Prionomolgus sp.2 0.051 - 0.114 0.835 (p = 0.431) (p = 0.085) Schedomolgus sp.1 0.166 0.122 0.118 0.594 (p = 0.160) (p = 0.002) (p = 0.102)

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Figure 6.4 Phylogeny of mushroom coral species (after Gittenberger et al., 2011, Benzoni et al., 2012) and symbiotic copepods found on them in the Red Sea. Coral species marked in red have been recorded as host for species of Red Sea copepods: Sch1 = Schedomolgus sp. 1; Sch2 = Schedomolgus sp. 2; Prio1 = Prionomolgus sp. 1; Prio2 = Prionomolgus sp. 2; Par1 = Paradoridicola sp. 1; Ast1 = Asteropontius sp. 1; Ast2 = Asteropontius sp. 2; + = present, - = not observed.

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6.4 Discussion

Contrary to the previous assumptions and speculations on coral-copepod relationships based mostly on anecdotal evidence (Humes, 1985a, 1985b, 1994; Marchenkov &

Boxshall, 2002), the most relevant finding of our study is that the previously assumed host-specific association between symbiotic copepods and mushroom corals is unsupported. Most copepods were found on several host species and even on different genera. Moreover, the fact that the number of coral species for each copepod strictly correlated with copepod abundance suggests that also for rare copepods the diversity of host corals could be higher and that their low number of host species might simply reflect a low sample size obtained for those copepod species. Given the relatively low number of individual copepods found in each mushroom coral (Appendix 6.2), an underestimation of host diversity could be expected for the copepod-coral association; notwithstanding this potential bias in favor of supporting an artefactual species-specificity, such specificity was clearly rejected by all the tests we performed. In other words, it appears that all mushroom corals are equally suitable as habitat from the perspective of symbiotic copepods.

The previously-assumed species level host-specificity was probably due to a lack of information and to the fact that having few records for a potentially highly diverse group of symbiotic copepods and of host corals could misleadingly have provided a scenario of a species-specific association. The lack of a species-specific association radically changes the evolutionary perspective that was indicated for symbiotic copepods (Humes, 1985a,

1985b, 1994; Stella et al., 2011; Cheng et al., 2016). The assumed species-specific or at least genus-specific association meant that a co-evolutionary scenario could be

130 hypothesized (Thrall et al., 2006), as for other symbiotic relationships known between invertebrates, such as endolithic snails, boring mussels, and gall crabs living inside scleractinian coral skeletons (Gittenberger & Gittenberger, 2011; Owada & Hoeksema,

2011; Van der Meij et al., 2015), or between invertebrates and other organisms (e.g., fig wasps, yucca, etc.). However, caution is needed here because host-switching can interfere with co-evolution, even involving hosts of different phyla, subclasses, or orders, as shown by commensal shrimps (Brinkmann & Fransen, 2016; Horká et al., 2016;

Hoeksema & Fransen, 2017) and corallivorous snails (Schiaparelli et al., 2005; Potkamp et al., 2017). Given the lack of host specificity of the copepods living on mushroom corals of the Red Sea at species and genus level, the high diversity of hosts cannot be a driver of the high species diversity of symbiotic copepods, as in the case of the high diversity of pollinating insects such as bees and fig-pollinating wasps related to the diversification of flowering plants (Danforth et al., 2006; Cruaud et al., 2012). Thus, the processes related to the relatively high diversity of symbiotic copepods should be searched among other factors, maybe on differential adaptations to different body parts.

The lack of species-specific associations could be related to a general adaptation to the host defense systems and metabolites.

Different species of symbiotic copepods could still colonize different parts of the hosts and establish different associations with them: unfortunately, due to difficulties in making direct observations and the washing sampling technique, we could not clearly identify the localization of each individual copepod on the host coral, and thus we do not know whether each symbiotic copepod could be ecologically diversified in its relationship, for example by colonizing only the galls, the polyps, the intestinal cavity, or

131 the soft tissues of the hosts (Stock, 1975; Humes, 1985a, 1985b; Dojiri, 1988; Kim &

Yamashiro, 2007; Ivanenko et al., 2014). The question of the drivers of the diversification of symbiotic copepods remains open, but we can at least support that a species-specific or a genus-specific association should be ruled out. The possibility of niche-partitioning occurring at a host subcolony scale remains untested.

The finding of generalist symbionts living in association with various coral species across a range of fungiid genera is also reported for other taxa, such as coral-excavating mussels (Kleemann & Hoeksema, 2002; Owada & Hoeksema, 2011), coral barnacles

(Hoeksema et al., 2012), epitoniid snails (Gittenberger & Hoeksema, 2013), hydroids of the genus Zanclea (Montano et al., 2015), coral gall crabs (Hoeksema & Van der Meij,

2013; van der Meij et al., 2015; Hoeksema et al., 2018), sessile ctenophores (Alamaru et al., 2016; Alamaru et al., 2017), and cryptobenthic fishes (Bos, 2012; Hoeksema et al.,

2012; Bos & Hoeksema, 2015, 2017). Interestingly, some coral-dwelling polychaetes of the genus Spirobranchus, which are commonly found on large ranges of hosts

(Molodtsova et al., 2016; Hoeksema & Ten Hove, 2017; Perry et al., 2017), are scarcely recorded from fungiid corals (Hoeksema & Ten Hove, 2014). For these generalist symbiont species, one interpretation may be that the various species of fungiid corals are functionally equivalent as far as the symbionts are concerned. Thus, it could be that all symbiont copepods are strictly associated with mushroom corals only, and not with other families of stony corals, notwithstanding the wide morphological and ecological variability of mushroom corals.

Species-specific symbionts indeed exist in association with mushroom corals and can be found among endolithic snails of the genus Leptoconchus (Gittenberger &

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Gittenberger, 2011), several epitoniid snails (Gittenberger & Hoeksema, 2013), and various taxa associated with the mushroom coral Heliofungia actiniformis (Quoy and

Gaimard, 1833), including the pipefish Siokunichthys nigrolineatus (Phillips, 1987) and the shrimp Cuapetes kororensis (Bruce, 1977) (Hoeksema et al., 2012; Hoeksema, 2017).

In other words, animals associated with Fungiidae are usually specifically associated with these corals, however, the host species and genus level host-specificity appear to be relatively rare (Hoeksema et al., 2012; Hoeksema et al., 2018).

All copepod species found in the Red Sea on mushroom corals are unknown for science and increase the overall known diversity of symbiotic copepods (Appendix 6.1).

The fact that they are still undescribed suggests that a much richer diversity may exist.

Morphologically, all of these copepods belong to unknown species, not matching any of the previously described species for their respective genera. Genetically, their identity as separate species was confirmed by various molecular taxonomic approaches (molecular phylogeny, species delimitation methods), and by genetic distances within and between species that are comparable to those already known in other copepods (e.g., Blanco-

Bercial et al., 2014; Cornils & Held, 2014; Fontaneto et al., 2015), further supporting their status as independent species. Two of the discovered copepod species belong to the genus Asteropontius (Siphonostomatoida: Asterocheridae), which also includes 36 congeneric species found in association with scleractinians, actiniarians, and corallimorpharians in other parts of the world; only three of them were previously reported from mushroom corals: A. caledoniensis, A. fungicola and A. latioriger from

New Caledonia, Madagascar, and Indonesia (Moluccas) (Kim, 2003, 2007, 2010). Two copepod species belong to the genus Schedomolgus (Poecilostomatoida: Anchimolgidae),

133 which also includes other 11 congeneric species found on scleractinian corals; only two of the species of the genus were previously found on mushroom corals: S. tener and S. dumbensis from New Caledonia (Humes, 1973a; Kim, 2003). Two other species belong to another genus of the same family (Anchimolgidae), Prionomolgus, and differ from the only known species of this genus, P. lanceolatus, which was found in association with the coral Pachyseris speciosa (Dana, 1846) (Scleractinia incertae sedis) in northern

Madagascar (Humes & Ho, 1968). The copepods of the genus Paradoridicola

(Poecilostomatoida: Rhynchomolgidae) were previously found on Indo-Pacific alcyonacean corals only and were also unknown for the Red Sea (Humes, 1990). Among the 13 mushroom coral species recorded as hosts for copepods in the present study, eight of them (Cycloseris costulata, Ctenactis cf. crassa, Danafungia horrida, Lithophyllon repanda, Pleuractis granulosa, Pleuractis cf. seychellensis, Pleuractis cf. taiwanensis,

Podabacia crustacea) were previously unknown to host symbiotic copepods, and they can now be added to the list of species reported as hosts for copepods (Hoeksema et al.,

2012).

The Red Sea copepods associated with mushroom corals belong to genera that are usually not found in association to the same coral family in other parts of the Indo-Pacific area (Humes, 1979a, 1985a, b; Cheng et al., 2016), whereas other copepod species of the same genera are found living in association with other coral families, even in the Red Sea

(Humes, 1985a). Thus, biogeographical patterns with multiple independent colonization of mushroom corals instead of host-specificity at the level of species, genus, or family could be hypothesized as relevant in the diversification of copepods associated with mushroom corals.

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6.5 Conclusion

The research presented in the chapters 5 and 6 was focused on the assessment of a species-level diversity assessment and the host-specificity of copepods collected from two coral families, Fungiidae and Pocilloporidae in the Red Sea. Though the objectives in both chapters were similar, the results show completely different patterns of species distribution and specificity to the host species. Contrary to our expectations, copepods associated with fungiid corals do not show any statistically significant evidence of host- specificity or other patterns of ecological association with their hosts on neither species nor genus level. The lack of the host-specificity may be the consequence of the low ecological variability among the mushroom coral species, which facilitates switching from one host species or genera to another.

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Chapter 7: Discussion

Cheng et al., 2016; Ivanenko et al., 2018). This research is the first study of the genetic diversity of copepods associated with invertebrates. Similar studies were conducted only for the free-living planktonic copepods and the parasitic copepods of fishes but with much smaller sample sizes (Bucklin et al., 2003; Bucklin & Frost, 2009; Chen & Hare,

2011; Morales-Serna et al., 2014). During this project, original and significant results on the diversity, biogeographical distribution, endemism, and host-specificity of copepods associated with scleractinian corals were obtained; improved methods and approaches of studying microscopic invertebrates were developed, and prospective directions for the further research were established.

Exploration of the symbiotic copepods associated with invertebrates began in the middle of the last century by Arthur Humes when scuba diving technology became readily available for researchers. Since then, more than 400 species of copepods representing 19 families and three orders have been described in association with scleractinian corals (Cheng et al., 2016). Description and identification of the new species were initially based solely on morphological methods, which are known to be extremely time-consuming and demand years of training. Thus, there are very few experts in the world able to do this kind of work. Almost all copepods discovered on corals were described by just several people from a limited number of locations (Cheng et

136 al., 2016). Thereafter, the existing diversity assessment was based on morphological observation of distinctive characters, however, this data is acknowledged to be fragmented and likely inadequate due to the scarcity of samples from most of the places and the majority of the coral hosts (Humes, 1994; Cheng et al., 2016; Ivanenko et al.,

2018).

The assessment of copepod diversity using genetic markers conducted during this project confirmed that the fauna of symbiotic copepod of corals has previously been significantly underestimated. The genetic approach allowed me to conduct efficient, fast, and accurate diversity assessment of the fauna of symbiotic copepods by estimation of the number of molecular MOTUs. Though DNA analysis is not a frequently used way to describe new species yet, applying genetic methods facilitates the identification process and also enables us to use this approach to assess the species diversity. We can thus conduct a more thorough comparison of copepod fauna from different host taxa and from different regions. Molecular methods proved to be extremely valuable in resolving phylogenetic position of copepod taxa and in defining close species boundaries in this thesis. They also enable the reliable identification of species of copepods belonging to different sexes, as well as juvenile forms, and help to recognize cases of potential hybridization. Moreover, the obtained molecular data, in contrast to the morphological identifications, create an array of digital data, which could be used for an in-depth study of the diversity and ecology of symbiotic copepods in different regions. Thus, the results obtained using the genetic analysis showed the advantage of using molecular markers in assessing the diversity of copepods in combination with morphological studies.

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In this project, I studied unique material which has been collected from various places around the Indo-Pacific: western Australia and GBR (2010), South China Sea

(2009, 2010), and Red Sea (2013, 2014). I continued the sampling during the series of expeditions to the northern, central and southern parts of the Saudi Arabian Red Sea coast

(2016a - 49 colony samples; 2016b - 9 samples; 2017 - 121 samples) and to the remote atolls of the Maldivian Archipelago in 2016 where 46 samples of corals with their copepod symbionts were collected. A huge collection of copepod specimens was obtained with all the samples accompanied by information about the location, time and depth of sampling, underwater photographs of coral hosts, as well as macroscopic photographs of bleached coral skeletons. This is a unique collection consisting of thousands of individuals and hundreds of species of symbiotic copepods, most of which are yet to be described. In addition, samples of the host tissue were collected and stored together with the rest of the collection in MSU (Moscow, Russia) for further analyses by molecular and morphological methods. Overall, during this project, I processed and analyzed 1850 copepod samples collected from 125 colonies of 43 species of scleractinian corals from 11 locations in the Indo-Pacific.

For the DNA extraction, I used an original, relatively cheap, and, most importantly, non-destructive method, which allowed me to save copepod exoskeletons for morphological analysis using light and confocal microscopy and preserve them as specimen vouchers. Furthermore, I designed copepod-specific primers for COI

(LCO1490cop3) and ITS2 regions (58dcop, 28r1cop) and optimized the amplification program, both of which significantly improved the amplification success rate. Although

DNA amplified well with ITS2 primers, in some cases up to half of the sequences were

138 heterogeneous and required additional work to determine the haplotypes of heterozygous individuals. However, this is a peculiarity of the ITS2, which is a length-variable non- coding DNA region and often has double peaks. A large amount of work was also done on optimization of the amplification protocol for the 16S, 12S, and CytB regions, which were expected to be used together with COI and ITS2 to increase the resolution and reliability of the species delimitation methods. However, these primers showed much lower success rate than COI or ITS2 and were not used in analyses. Primers for the 18S gene worked well and did not require optimization; good quality sequences were obtained for more than 90% of the samples for this region. As a result, an optimal set of three markers was defined: a fragment of 18S rDNA which allows quick and precise determination of the position of a copepod on a phylogenetic tree, and two markers, standard mitochondrial marker COI and nuclear region ITS2, an overall ideal combination for species delineation. The feasibility of using the ITS2 region as a second species delimitation marker is explained by the fact that there are no universal primers for the Folmer region that would work well for all copepod taxa. All of the above allowed me to create a large dataset in the form of barcode library of 1500+ coral-associated copepods with information including DNA sequences of mitochondria and nuclear regions together with photographs of the specimens and coral hosts (underwater and skeletal morphology). After this information will be uploaded to an online database, it will be available to the public, making it possible for them to identify MOTUs of coral- inhabiting copepods and giving an opportunity to explore and compare their diversity all around the world using genetic barcodes.

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Overall, the taxonomic status and species composition of copepods associated with scleractinian corals of different families in the GBR, with the genus Galaxea in the Indo-

Pacific, and with families Pocilloporidae and Fungiidae in the Red Sea, were defined, phylogenetic trees were built, and boundaries between species were determined.

Poecilostomatoid copepods of the families Anchimolgidae and Xarifiidae were confirmed to be among the predominant groups of copepods found in association with scleractinian corals in the Indo-Pacific. Furthermore, my findings demonstrate that copepods of the family Asterocheridae, previously reported mostly from Atlantic corals (Humes, 1985a;

Stock, 1988), appear to be numerous and diverse symbionts of the Indo-Pacific corals.

The fact that the diversity of this widespread and common group of copepods has been completely underestimated in the literature suggests that many rare and endemic species or genera remain awaiting discovery.

Taxonomic reports on copepod relationships with stony corals show that most of the copepod species are found only on a single species of host coral (70% of the 363 described scleractinian-associated copepod species) or only on coral species of the same genus (17% of scleractinian-associated copepod species); only about 5% of the copepod species are found on stony corals of two or more families (Humes, 1985a; Cheng et al.,

2016). No coral-associated species of copepods has ever been found on other invertebrate hosts; most of the coral symbiont copepods seem to be evolved to exclusively survive on coral hosts. This makes these copepods more susceptible to the loss of coral cover due to climate change and other stressors.

Though records from the literature suggest that most of the copepods are species- specific, my results, however, show evidence that in many cases this pattern may change

140 with an increased sample size. Sampling additional species of coral hosts and sampling in new regions may reveal previously-unknown associations. Additional data could alter the current paradigm of host specificity. My data from Lizard Island also shows that most of the discovered species of copepods were found in association with only one species of coral. However, most of the coral genera in my samples were represented by one

(Astreopora, Ctenactis, Fungia, Galaxea, Pachyseris, Polyphyllia, Seriatopora, and

Stylophora) or two (Echinopora, Lobophyllia, Merulina, Pectinia, Platygyra, and

Porites) coral species, which make it difficult to analyze specificity of these copepods on the host species level. Analysis of the Galaxea-associated fauna of copepods did not reveal any differences between G. astreata and G. fascicularis, therefore, these copepods do not show specificity on the species level. Spaniomolgus copepods, which I found living in big numbers on the Pocilloporidae corals, are also known to be symbionts of

Acropora, e.g., S. crassus and S. geminus. However, most of the Spaniomolgus species seem to be specific to the pocilloporid corals. The analysis of the copepod fauna of the mushroom corals did not reveal any specificity between copepods and their hosts on neither species nor genus level: each species of copepods was found on several species of corals of the family Fungiidae. The lack of the host-specificity may be the consequence of the low ecological variability among the mushroom coral species, which facilitates switching from one host species or genera to another. Since very little is known about the ecology of these copepod symbiont species, it is difficult to predict what features of the host would potentially make a difference to the copepods. For example, is the specific morphology of the coral important (e.g., corallite size or spacing, polyp tentacle length, microstructure of the coenosteum, etc.)? Are there chemical defenses of the corals that

141 lead to host-specificity in the symbionts? In other words, without knowing the functional ecology of the interaction between the symbiont and the host, it is hard to speculate on what drives the nature of the specificity or even to predict whether this specificity should extend within a genus or family of potential hosts, etc. Clearly, a large number of questions remain about these associations, but identification of these questions would not be possible without first establishing the association patterns.

The cases of transition of copepods between taxonomically distant groups (orders, classes, and phyla) of hosts are usually rare, and host-switching between closely related taxa, on the contrary, is the most frequent case (Boxshall & Halsey, 2004). My results suggest that the copepods could be specific not to a particular host but to certain micro- habitats created by the corals (seems like the shape of the colony and surface structure might play a role). I also noted that endoparasitic copepods show less evolutionary plasticity and seem to be more tightly associated with their host than ectosymbiotic forms, as the latter do not depend on the host as much as endoparasites.

Very little information is available on the biology of copepod-coral interactions, and little is known about the effect of symbiotic copepods have on their coral hosts. Some copepods from families Xarifiidae and Corallovexiidae are known to feed on coral tissue

(Humes, 1960; Stock, 1975). These corallivore copepods destroy coral tissue integrity and, therefore, open “gates” for pathogenic bacteria, viruses, and fungi. Parasitic copepods living in coral tissue could also suppress the immune response of the coral and, as a consequence, the coral microbiome could react to the stress, and the balance could shift towards pathogenic bacteria. And vice versa, the stressed corals are more susceptible

142 to the parasite infections, and they are more likely to host parasitic copepods (Cheng et al., 2016).

In addition to causing direct impacts on their host, copepods could serve as transmitters of pathogens among their hosts, like mosquitos or fleas. For instance, it has been shown that a parasitic copepod can serve as a vector of a viral disease among farmed salmons (Nylund et al., 1993; Nylund et al., 1994). For the coral hosts, there is a recent experimental study showing the possibility of transmission of a coral disease by planktonic copepods (Certner et al., 2017). However, copepods living in association with a coral are believed to remain on the same colony for their whole adult life and, therefore, there are not many opportunities for them to transfer anything from coral to coral. The only possibility for copepods to transport pathogens from one colony to another is during the copepodite stages. But, again, in the experiment with infection of the salmon, the copepodite stage was used to transfer the pathogenic agent (Nylund et al., 1994).

Clearly, the presence of symbiont copepods has the potential to affect the coral holobiont and its microbiome structure. A recent study on microbiomes of gall-inducing copepods showed that the microbiome of the galls has similar diversity but different structure than the microbiome of the normal coral tissue (Shelyakin et al., 2018). Despite an increase in amount of studies on coral diseases (Woodley et al., 2016), the actual causes of these diseases are still not well understood (Bourne et al., 2009). Therefore, studying copepod microbiomes should be one of the first steps on the way to evaluate the impact of symbiotic copepods on the coral holobiont.

At the same time, ectosymbiotic copepods are regularly found on healthy-looking coral colonies (Ho, 2001). Additionally, copepods could be found living on corals in very

143 large numbers, e.g., up to hundreds or even thousands of individuals on a single colony

(Humes, 1994), with corals not showing any signs of the stress. I presume they might have a positive impact on corals by cleaning the coral, like cleaner-shrimps (Glynn &

Enochs, 2011) and detritivorous crabs (Stewart et al., 2006) or perhaps they consume excess mucus like gall crabs (Kropp, 1986). Besides, copepods could serve as a source of the food for other macrosymbionts of corals, like crabs, shrimps, and fishes (Stella et al.,

2011), which, in turn, clean the coral and defend it from its enemies.

Crustaceans are known to use chemical cues to find and assess mates, to reveal the presence of a predator, and to find food and appropriate habitats (Hay, 2010). Therefore, it is likely that copepods also choose their coral host navigating by these chemical signals. Moreover, different levels of host-specificity could be explained by their feeding behavior: generalist consumers could be less picky to the choice of food and, therefore, can live on different hosts but species-specific copepods may be stimulated only by unique metabolites produced only by specific species of host. This is an area of future potential work. This thesis has identified some broad-scale patterns of association and host-specificity that could inform future experimental studies.

The results obtained in the course of the project raised a number of questions requiring further investigation. The biology of the copepod-coral interactions, mechanisms of host recognition and the ways to overcome the protection and the immune response of the host are among them. However, to study these processes we need to study chemical cues, metabolites, as well as sensory and immune systems, therefore, an interdisciplinary approach is required. During the work on this project, I realized that for a comprehensive study of mechanisms of the evolution of symbiosis it is necessary to

144 consider each component of the symbiotic system as a separate holobiont including the all organisms constituting it (i.e., not only copepod and the coral host but also their microbiomes, including bacteria, fungi, protists, and viruses). I had a chance to participate in the project, where we attempt to assess impact of gall-inducing copepods and their microbiomes on the coral and its own microbiome for the first time. Contrary to our expectations, the microbial community composition of the injured gall tissues was not directly affected by the microbiome of the gall-forming symbiont copepods.

However, the total microbiome composition of polyps injured by copepods was different from the microbiome of the healthy tissues (Shelyakin et al., 2018). These symbiotic systems are a complex, poorly developed area of research which requires significant resources, specialized equipment, and the possibility of solving complex bioinformatic problems. These studies could be started with comparative studies of microbial communities of animals belonging to different types of symbiotic systems.

145

CONCLUSIONS

This work makes a substantial contribution to the appreciation of biodiversity among coral reef fauna in general, as symbiotic copepods have received little attention historically, and the diversity of this group has been highly underestimated. Extensive results show that a genetic approach is more effective in the assessment of copepod diversity than traditional taxonomic methods, and this study could serve as a baseline for further exploration of copepods living in symbiosis with corals and ecological significance of this association.

My findings reveal that the real diversity of copepods living in association with corals is much higher than was previously reported. The families Anchimolgidae,

Asterocheridae, and Xarifidae are predominant groups of copepods found in association with scleractinian corals. Ecological divergence of copepods living on one species of a host as well as host-switching, migration to the new geographical areas, and geographic isolation, appear to be the primary mechanisms of speciation of symbiotic copepods and, therefore, result in high species diversity.

My data suggest that the high diversity of symbiotic copepods is a result of (1) the presence of a variety of micro-habitats (inside and outside of polyps, etc.) created by reef- building corals at various scales, (2) the process of speciation which took place due to the frequent cases of an evolutionary transition to the new host species and migration to the new geographical areas, and (3) geographic isolation, which led to formation of complexes of closely related species and high zoogeographical fragmentation. However, the question of the drivers of the diversification of symbiotic copepods remains open, and

146 the further work on the species diversity, host-specificity, and geographical distribution will give more information on this matter.

147

LIST OF PUBLICATIONS

Publications out of thesis research

1. Viatcheslav N. Ivanenko, Bert Hoeksema, Sofya V. Mudrova, Mikhail A. Nikitin, Alejandro Martínez, Nadezda N. Rimskaya-Korsakova, Michael L. Berumen, Diego Fontaneto (2018) Lack of host specificity of copepod crustaceans associated with mushroom corals in the Red Sea. Molecular Phylogenetics and Evolution, 127, 770-780.

My contributions to this paper are presented in Chapter 6, which describes fauna of copepods associated with Fungiidae corals with a focus on their host-specificity and patterns of distribution.

Publications related to thesis research

2. Viatcheslav N. Ivanenko, Sofya V. Moudrova, Jessica Bouwmeester, Michael L. Berumen (2014) First report of tubular corallites on Stylophora caused by a symbiotic copepod crustacean. Coral Reefs, 33(3), 637-637. Contribution: I took part in processing samples, discussion of the results and preparing of the manuscript.

3. Pavel V. Shelyakin, Sofya K. Garushyants, Mikhail A. Nikitin, Sofya V. Mudrova, Michael Berumen, Arjen G.C.L. Speksnijder, Bert W. Hoeksema, Diego Fontaneto, Mikhail S. Gelfand & Viatcheslav N. Ivanenko (2018) Microbiomes of gall-inducing copepod crustaceans from the corals Stylophora pistillata (Scleractinia) and Gorgonia ventalina (Alcyonacea). Scientific Reports, 8 (1), 11563. Contribution: I took part in collecting samples and field data; took part in planning, discussion of the results and preparing of the manuscript.

4. Mercedes Conradi, Eugenia Bandera, Sofya V. Mudrova, Viatcheslav N. Ivanenko (2018) Five new coexisting species of copepod crustaceans of the genus Spaniomolgus, symbionts of the stony coral Stylophora pistillata (Scleractinia) in the Red Sea. ZooKeys, in press doi: 10.3897/zookeys.@@.28775. Contribution: I created figures, took part in processing of the samples and discussion of the results and wrote a non-descriptive part of the manuscript.

Expected future publications from thesis research

5. Mikhail Nikitin, Sofya V. Mudrova, Michael L. Berumen, Diego Fontaneto, Natalia Ivanova, James Reimer, Viatcheslav N. Ivanenko (in preparation). On the neglected diversity of crustacean copepods living on stony corals (Lizard Island, Great Barrier Reef): preliminary results. Target journal: Marine Biodiversity (from results in Chapter 3).

148

6. Sofya V. Mudrova, Mikhail A. Nikitin, Michael L. Berumen, Viatcheslav N. Ivanenko. Genetic diversity of copepods associated with scleractinian corals in the Lizard Island (Great Barrier Reef). Target journals: Marine Biodiversity, Coral Reefs (from results in Chapter 3).

7. Sofya V. Mudrova, Viatcheslav N. Ivanenko, Mikhail A. Nikitin, Diego Fontaneto, Michael L. Berumen (in preparation). Biogeographic patterns, diversity, and host specificity of copepods living in symbiosis with Galaxea (Scleractinia) in the Indo- Pacific. Target journal: Journal of Biogeography (from results in Chapter 4).

8. Sofya V. Mudrova, Alejandro Martínez, Mikhail A. Nikitin, Diego Fontaneto, Viatcheslav N. Ivanenko, Michael L. Berumen (in preparation). Cryptic diversity of Poecilostomatoida (Copepoda) associated with Pocilloporidae (Scleractinia) in the Saudi Arabian Red Sea. Target journal: Marine Phylogenetics and Evolution (from results in Chapter 5).

149

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APPENDICES

Appendix 3.1 List of the copepod species associated with scleractinian corals in the Great Barrier Reef based on the literature records.

Appendix 3.2 Sampling information (table with voucher names, locations, coordinates, depths, collector names, dates) for the corals collected from the Lizard Island, GBR.

Appendix 3.3 List of the copepod specimens collected from the corals from Lizard Island, with voucher numbers, and taxonomic information.

Appendix 3.4 Bayesian phylogeny of the family Anchimolgidae based on the concatenated dataset of 18S, COI, and ITS2. Host specificity is coded by branch color.

Appendix 4.1 Sampling information (table with voucher names, locations, coordinates, depths, collector names, dates) for the Galaxea coral samples.

Appendix 4.2 Underwater photos of the sampled Galaxea colonies, with photos of coral skeletons with corallite structures visible.

Appendix 4.3 List of the copepod specimens collected from Galaxea corals, with voucher numbers and taxonomic information.

Appendix 4.4 Full 18S ML phylogenetic tree based on 992 sequences and additional 174 copepod sequences from GenBank (marked with black).

Appendix 4.5 Detailed list of 174 sequences obtained from GenBank and used in analysis of phylogenies of copepods associated with Galaxea corals.

Appendix 4.6 Phylogenetic tree (MrBayes, 30 million generations, burn-in 0.25) of the order Poecilostomatoida with all the families excluding family Anchimolgidae. Different colors highlight separate OTUs delimited based on 18S, COI, and ITS2.

Appendix 4.7 Phylogenetic tree (MrBayes, 30 million generations, burn-in 0.25) of the order Siphonostomatoida with all the families excluding family Asterocheridae. Different colors highlight separate OTUs delimited based on 18S, COI, and ITS2.

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Appendix 4.8 Full phylogenetic tree (MrBayes, 30 million generations, burn-in 0.25) of the family Anchimolgidae based on the concatenated dataset of 18S, COI, and ITS2. Numbers on the branches represent support values corresponding to BI posterior probabilities (>0.95), and ML bootstrap replicates (>50).

Appendix 4.9 Full phylogenetic tree (MrBayes, 30 million generations, burn-in 0.25) of the family Asterocheridae based on the concatenated dataset of 18S, COI, and ITS2. Numbers on the branches represent support values corresponding to BI posterior probabilities (>0.95), and ML bootstrap replicates (>50).

Appendix 5.1 List of the copepod species associated with Pocilloporidae corals based on the literature records.

Appendix 5.2 Sampling information (table with voucher names, locations, coordinates, depths, collector names, dates) for the Pocilloporidae coral samples.

Appendix 5.3 Underwater photos of the sampled Pocilloporidae colonies, with photos of coral skeletons with corallite structures visible.

Appendix 5.4 List of the copepod specimens collected from Pocilloporidae corals, with voucher numbers, and taxonomic information.

Appendix 5.5 Description of the five new species of Spaniomolgus with a key for the identification of all congeners.

Appendix 6.1 List of the copepod species associated with Fungiidae corals based on the literature records.

Appendix 6.2 Sampling information (table with voucher names, locations, coordinates, depths, collector names, dates) for the Fungiidae coral samples.

Appendix 6.3 Underwater photos of the sampled Fungiidae colonies, with photos of coral skeletons with corallite structures visible.

Appendix 6.4 List of the copepod specimens collected from Fungiidae corals, with voucher numbers, taxonomic information, and GenBank accession numbers.

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Appendix 6.5 Phylogenetic reconstruction of the sampled copepods based on the COI analyses. Tree topology corresponds to the Bayesian analyses, whereas node values indicate support values for each clade (Bayesian posterior probabilities/ maximum likelihood bootstrap).

Appendix 6.6 Phylogenetic reconstruction of the sampled copepods based on the ITS analyses. Tree topology corresponds to the Bayesian analyses, whereas node values indicate support values for each clade (Bayesian posterior probabilities/ maximum likelihood bootstrap).

Appendix 6.7 Phylogenetic reconstruction of the sampled copepods based on the combined analyses of ITS and COI. Tree topology corresponds to the Bayesian analyses, whereas node values indicate support values for each clade (Bayesian posterior probabilities/ maximum likelihood bootstrap).

Appendix 6.8 Summary of the estimates obtained from species delineation analyses performed with three methods in DNA taxonomy on the two molecular markers individually and on the combined dataset. Abbreviations: BI, Bayesian inference; LR, likelihood ratio; ML, maximum likelihood, N, number.

Appendix 6.9 Summary of the intraspecific and interspecific genetic distances, calculated assuming the seven units of diversity obtained from the morphological and DNA taxonomy analyses as species. Abbreviations: n, number of sequences; n haplo, number of haplotypes; max, maximum genetic distance; min, minimum genetic distance; mean, mean genetic distance; median, median of the genetic distances.

Appendix 6.10 Phylogenetic signal (k) and significance values (p) obtained from Blomberg’s K statistics on the distribution of the copepod species on the phylogeny of fungiid corals from Gittenberger et al. (2011).

All the appendix files can be downloaded from following public accessible Google Drive link: https://drive.google.com/open?id=12sHWOzpG5s5It1VEC4uoxAdfDGwdke52